GIO TRNH

VI SINH VT HC
Cng bn c.
Vi sinh vt (microorganisms) l nhng sinh vt nh b n mc ch c th thy c chng
di knh hin vi quang hc hay knh hin vi in t. Vi sinh vt gy ra rt nhiu bnh him ngho
cho ngi, cho gia sc, gia cm. Tuy nhin s vi sinh vt gy bnh ch chim mt phn rt nh
trong th gii vi sinh vt. T xa xa ngi ta bit ng dng cc vi sinh vt c ch (tuy cha h
bit ti s tn ti ca chng) ch bin thc phm ( nh nu ru, lm tng, mm, nc mm,
gim, sa chua, chao, mui da, mui c, ...), phn, ngm v cy ly si, xp i t, trng lun
canh vi cy h u...; hoc s dng cc bin php ngn chn tc hi ca vi sinh vt (nh p
mui tht, c, lm mt, phi kh c ci, tm, c...).
Sau vic Leeuwenhoek pht hin ra vi sinh vt v vic Louis Pasteur pht hin ra bn cht
ca cc vi sinh vt- t khai sinh ra ngnh Vi sinh vt hc (Microbiology)- th nhn loi bt u
quan tm rt nhiu n lnh vc khoa hc mi m ny.
Vi sinh vt hc tr thnh nn tng cho s pht trin ca Cng ngh sinh hc(CNSH). Ngi
ta chia s pht trin ca CNSH ra thnh 3 giai on:
* CNSH truyn thng l cc qu trnh dn d nhm ch bin , bo qun cc loi thc
phm, x l t ai, phn bn phc v nng nghip...
* CNSH cn i l qu trnh s dng cc ni ln men cng nghip sn xut quy m
ln cc sn phm sinh hc nh m chnh (bt ngt), lizin v cc axt amin khc, cc acid hu c,
cc dung mi hu c, cht khng sinh, mt s vitamin (nh vitamin B2, B12, C...), nhiu loi
enzym...
* CNSH hin i chia ra cc lnh vc nh CN di truyn (genetic engineering), cng ngh t
bo (cell engineering), cng ngh enzym v protein (enzyme/protein engineering), CN vi sinh vt/
CN ln men (microbial engineering / fermentation), CN mi trng (environmental engineering).
CNSH hin i thng gn lin vi cc c th mang gen ti t hp ( recombination gene).
Vi sinh vt hc l khoa hc nghin cu v cc c th hoc cc nhn t qu nh n mc
khng thy c bng mt thng, tc l cc vi sinh vt. Vi sinh vt hc ang c ging dy
trong rt nhiu trng i hc, Cao ng, Trung hc chuyn nghip v cng c cp n t
nhiu bc ph thng. Vi sinh vt hc l nhng kin thc lin quan n cuc sng ca mi ngi.
Bn cnh gio trnh Vi sinh vt hc c bin son t lu v c ti bn nhiu ln chng
ti mun cung cp thm cho ng o bn c, nht l cc thy c gio ang ging dy v vi sinh
vt hc tp ti liu trnh by vi nhiu tranh nh v s , biu ny. C th coi y l phn
tham kho thit k cc bi ging nhm mc tiu cp nht vi cc tin b ca ngnh khoa hc
lun lun i mi ny.
gip cc bn tr tip cn c vi sch bo nc ngoi, nht l cp nht kin thc qua
Internet chng ti c gng vit thm ting Anh sau cc thut ng khoa hc v nguyn cc ch
thch ting Anh trong hnh v. Thng qua vic gio vin hng dn sinh vin in ting Vit vo cc
hnh v s gip ch rt nhiu cho qu trnh t hc ca sinh vin.
Rt mong nhn c kin ng gp ca cc bn ng nghip v ng o bn c.
V mc ch phc v phi li nhun chng ti mong c lng th vic s dng nhng
hnh nh thu nhn qua Internet.

Bi 1 Lc s nghin cu Vi sinh vt hc

1546- Girolamo Fracastoro (1478, 1553). cho rng cc c th nh b l tc nhn gy ra
bnh tt. ng vit bi th Syphilis sive de morbo gallico (1530) v t ta ca bi th , ngi ta
dng t tn bnh
1590-1608- Zacharias Janssen ln u tin lp ghp knh hin vi.
1676- Antony van Leeuwenhoek (1632-1723) hon thin knh hin vi v khm ph ra
th gii vi sinh vt (m ng gi l anmalcules).
1688- Nh vn vt hc ngi Francisco Redi cng b nghin cu v s pht sinh t nhin
ca gii.
1765-1776- Spallanzani (1729-1799) cng kch thuyt Pht sinh t nhin
1786- Mller a ra s phn loi u tin v vi khun
1798- Edward Jenner ngh ra phng php chng m u b phong nga bnh u ma
1838-1839- Schwann v Schleiden cng b Hc thuyt t bo.
1835-1844- Basi cng b bnh ca tm do nm gy nn v nhiu bnh tt khc do vi sinh
vt gy nn.
1847-1850- Semmelweis cho rng bnh st hu sn ly truyn qua thy thuc v kin ngh
dng phng php v khun phng bnh.
1849- Snow nghin cu dch t ca bnh t vng London.
1857- Louis Pasteur (1822-1895) chng minh qu trnh ln men lactic l gy nn bi
vi sinh vt.
1858- Virchov tuyn b t bo c sinh ra t t bo.
1861- Pasteur chng minh vi sinh vt khng t pht sinh nh theo thuyt t sinh.
1867- Lister cng b cng trnh nghin cu v phu thut v khun.
1869- Miescher khm ph ra acid nucleic.
1876-1877- Robert Koch (1843-1910) chng minh bnh than do vi khun Bacillus
anthracis gy nn.
1880- Alphonse Laveran pht hin k sinh trng Plasmodium gy ra bnh st rt.
1881- Robert Koch nui cy thun khit c vi khun trn mi trng c cha gelatin.
Pasteur tm ra vaccin chng bnh than.
1882- Koch pht hin ra vi khun lao - Mycobacterium tuberculosis.
1884- Ln u tin cng b Nguyn l Koch.
Elie Metchnikoff (1845-1916) miu t hin tng thc bo (phagocytosis)
Trin khai ni kh trng cao p (autoclave)
Trin khai phng php nhum Gram.
1885- Pasteur tm ra vaccin chng bnh di.
Escherich tm ra vi khun Escherichia coli gy ra bnh tiu chy.
1886- Fraenkel pht hin thy Streptococcus pneumoniae gy ra bnh vim phi.
1887- Richard Petri phi hin ta cch dng hp lng (a Petri) nui cy vi sinh vt .
1887-1890- Winogradsky nghin cu v vi khun lu hunh v vi khun nitrat ho.
1889- Beijerink phn lp c vi khun nt sn t r u.
1890- Von Behring lm ra khng c t chng bnh un vn v bnh bch hu.
1892- Ivanowsky pht hin ra mm bnh nh hn vi khun (virus) gy ra bnh khm cy
thuc l.
1894- Kitasato v Yersin khm ph ra vi khun gy bnh dch hch (Yersina pestis).
1895- Bordet khm ph ra B th (complement)
1896- Van Ermengem tm ra mm bnh ng c tht (vi khun Clostridium botulinum).
1897- Buchner tch ra c cc men (ferments) t nm men (yeast).
Ross chng minh k sinh trng st rt ly truyn bnh qua mui.
1899- Beijerink chng minh nhng ht virus gy nn bnh khm l thuc l.
1900- Reed chng minh bnh st vng ly truyn do mui.
1902- Landsteiner khm ph ra cc nhm mu
1903- Wright v cng s khm ph ra Khng th (antibody) trong mu ca cc ng vt
min dch.
1905- Schaudinn v Hoffmann tm ra mm bnh giang mai (Treponema pallidum).
1906- Wassermann pht hin ra xt nghim c nh b th chn on giang mai.
1909- Ricketts chng minh bnh St ban ni lan truyn qua ve l do mm bnh vi khun
(Rickettsia rickettsii).
1910- Rous pht hin ra ung th gia cm.
1915-1917- DHerelle v Twort pht hin ra virus ca vi khun ( thc khun th)
1921- Fleming khm ph ra lizzim (lysozyme).
1923-Xut bn ln u cun phn loi Vi khun (Bergeys Manual)
1928- Griffith khm ph ra vic bin np (transformation) vi khun.
1929- Fleming pht hin ra penicillin.
1931- Van Niel chng minh vi khun quang hp s dng cht kh nh ngun cung cp
electron v khng sn sinh xy.
1933- Ruska lm ra chic knh hin vi in t u tin.
1935- Stanley kt tinh c virus khm thuc l (TMV).
Domag tm ra thuc sulfamide.
1937- Chatton phn chia sinh vt thnh hai nhm: Nhn s (Procaryotes) v Nhn tht
(Eucaryotes).
1941- Beadle v Tatum a ra gi thuyt mt gen- mt enzym.
1944- Avery chng minh ADN chuyn thng tin di truyn trong qu trnh bin np.
Waksman tm ra streptomycin.
1046- Lederberg v Tatum khm ph ra qu trnh tip hp (conjugation) vi khun.
1949- Enders, Weller v Robbins nui c virus Polio (Poliovirus) trn m ngi nui
cy.
1950- Lwoff xc nh c cc thc khun th tim tan (lysogenic bacteriophages).
1952- Hershey v Chase chng minh thc khun th tim ADN ca mnh vo t bo vt ch
(host).
Zinder v Lederberg khm ph ra qu trnh ti np (transduction) vi khun.
1953- Frits Zernike Lm ra knh hin vi tng phn pha (phase-contrast microscope).
Medawar khm ph ra hin tng nhn min dch (immune tolerance).
Watson v Crick khm ph ra chui xon kp ca ADN
1955- Jacob v Monod khm ph ra yu t F l mt plasmid.
Jerne v Burnet chng minh l thuyt chn lc clone (clonal selection).
1959- Yalow trin khai k thut Min dch phng x.
1961- Jacob v Monod gii thiu m hnh iu ho hot ng gen nh operon.
1961-1966- Nirenberg, Khorana v cng s gii thch m di truyn.
1962- Porter chng minh cu trc c bn ca Globulin min dch G.
Tng hp c quinolone u tin c tc dng dit khun ( acid nalidixic).
1970- Arber v Smith khm ph ra enzym gii hn (restriction endonuclease)
Temin v Baltimore khm ph ra enzym phin m ngc (reverse transcriptase)
1973- Ames trin khai phng php vi sinh vt hc khm ph ra cc yu t gy t bin
(mutagens).
Cohen, Boyer, Chang v Helling s dng vect plasmid tch dng gen vi khun.
1975- Kohler v Milstein pht trin k thut sn xut cc khng th n dng ( monoclonal
antibodies).
Pht hin ra bnh Lyme.
1977- Woese v Fox tha nhn Vi khun c (Archaea) l mt nhm vi sinh vt ring bit.
Gilbert v Sanger trin khai k thut gii trnh t ADN (DNA sequencing)
1979-Tng hp Insulin bng k thut ti t hp ADN.
Chnh thc ngn chn c bnh u ma.
1980- Pht trin knh hin vi in t qut
1982- Pht trin vaccin ti t hp chng vim gan B.
1982-1983- Cech v Altman pht minh ra ARN xc tc.
1983-1984- Gallo v Montagnier phn lp v nh loi virus gy suy gim min dch
ngi.
Mulli trin khai k thut PCR (polymerase chain reaction).
1986- Ln u tin ng dng trn ngi vaccin c sn xut bng k thut di truyn
(vaccin vim gan B).
1990- Bt u th nghim ln u tin liu php gen (gene-therapy) trn ngi.
1992- Th nghim u tin trn ngi liu php i ngha (antisense therapy).
1995- Hoa K chp thun s dng vaccin u g.
Gii trnh t h gen ca vi khun Haemophilus influenzae.
1996- Gii trnh t h gen ca vi khun Methanococcus jannaschii.
Gii trnh t h gen nm men.
1997- Pht hin ra loi vi khun ln nht Thiomargarita namibiensis
Gii trnh t h gen vi khun Escherichia coli.
2000- Pht hin ra vi khun t Vibrio cholerae c 2 nhim sc th ring bit.


Janssen
Leeuwenhoek (1632-
1723)
Pasteur (1822-1895)

Knh hin vi ca Leeuwenhoek



Bt tch miu t vi sinh vt ca
Leeuwenhoek
Th nghim bnh c cong
phn i thuyt t sinh
(Pasteur)



Robert Koch (1843-
1910)
Vi khun lao chp qua
knh hin vi
Elie Metchnikoff
(1845-1916)


Alexander Fleming (1881-1955) Nm Penicillium sn
sinh penicillin


Bi 2 Nhng c im chung ca Vi Sinh Vt
1-Vi sinh vt thuc gii sinh vt no?

Vi sinh vt khng phi l mt nhm phn loi trong sinh gii m l bao gm tt c cc sinh
vt c kch thc hin vi, khng thy r c bng mt thng, do phi s dng knh hin vi
thng hoc knh hin vi in t. Ngoi ra mun nghin cu vi sinh vt ngi ta phi s dng ti
phng php nui cy v khun.
T trc n nay c rt nhiu h thng phn loi sinh vt. Cc n v phn loi sinh vt ni
chung v vi sinh vt ni ring i t thp ln cao l Loi (Species), Chi (Genus), H (Family), B
(Order), Lp (Class), Ngnh (Phylum), v Gii (Kingdom). Hin nay trn gii cn c mt mc
phn loi na gi l lnh gii (Domain). y l cha k n cc mc phn loi trung gian nh Loi
ph (Subspecies), Chi ph (Subgenus), H ph (Subfamily), B ph (Suborder),Lp ph
(Subclass), Ngnh ph (Subphylum).


John Ray Carl Von Linnaeus
Xa kia John Ray (1627-1705) v Carl Von Linnaeus (1707-1778) ch chia ra 2 gii l Thc
vt v ng vt. Nm 1866 E. H. Haeckel (1834-1919) b sung thm gii Nguyn sinh (Protista).
Nm 1969 R. H. Whitaker (1921-1981) xut h thng phn loi 5 gii : Khi sinh
(Monera), Nguyn sinh (Protista), Nm (Fungi), Thc vt (Plantae) v ng vt (Animalia).
Khi sinh bao gm Vi khun (Bacteria) v Vi khun lam (Cyanobacteria).
Nguyn sinh bao gm ng vt nguyn sinh (Protzoa),
To (Algae) v cc Nm si sng trong nc (Water molds).

Gn y hn c h thng phn loi 6 gii- nh 5 gii trn nhng thm gii C vi khun
(Archaebacteria),
gii Khi sinh i thnh gii Vi khun tht (Eubacteria) (P. H. Raven, G. B. Johnson,
2002).
C vi khun v Vi khun tht thuc Cn

T. Cavalier-Smith (1993) th li xut h thng phn loi 8 gii:
Vi khun tht (Eubacteria),
C vi khun (Archaebacteria),
C trng (Archezoa),
Sc khun (Chromista),
Nm (Fungi),
Thc vt (Plantae) v
ng vt (Animalia).

Theo R. Cavalier-Smith th
C trng (nh Giardia) bao gm cc c th n bo nguyn thu c nhn tht, c ribosom
70S, cha c b my Golgi, cha c ty th (mitochondria) cha c th dip lc (Chloroplast), cha
c peroxisome.
Sc khun bao gm phn ln cc c th quang hp cha th dip lc trong cc phin
(lumen) ca mng li ni cht nhn (rough endpplasmic reticulum) ch khng phi trong t bo
cht (cytoplasm), chng hn nh To silic , To nu, Cryptomonas, Nm non.

Nm 1980, Carl R. Woese da trn nhng nghin cu sinh hc phn t pht hin thy C
khun c s sai khc ln trong trt t nucleotid ARN ca ribosom 16S v 18S. ng a ra h
thng phn loi ba lnh gii (Domain) bao gm
C khun (Archae),
Vi khun (Bacteria) v
Sinh vt nhn thc (Eucarya).
C khun l nhm vi sinh vt c ngun gc c xa. Chng bao gm cc nhm vi khun c
th pht trin c trong cc mi trng cc oan (extra), chng hn nh nhm a mn
(Halobacteriales), nhm a nhit (Thermococcales, Thermoproteus, Thermoplasmatales), nhm k
kh sinh mtan (Methanococcales, Methanobacteriales, Methanomicrobiales), nhm vi khun lu
hunh a nhit (Sulfobales, Desulfurococcales).
Monera trong h thng 5 gii tng ng vi Vi khun v C khun trong h thng 8 gii
v trong h thng 3 lnh gii. Nguyn sinh trong h thng 5 gii tng ng vi 3 gii C trng
(Archaezoa), Nguyn sinh (Protista-Protozoa) v Sc khun (Chromista) trong h thng 8 gii v
tng ng vi 5 nhm sau y trong h thng 3 lnh gii (domain): Archaezoa, Euglenozoa,
Alveolata, Stramenopila v Rhodophyta.
Theo h thng 3 lnh gii th Archaezoa bao gm Diplomonad, Trichomonad v
Microsporidian. Euglenozoa ao gm Euglenoid v Kinetoplastid. Alveolata bao gm
Dinoflagellate, Apicomplexan, v Ciliate. Strmenopila bao gm To silic (Diatoms) , To vng
(Golden algae), To nu (Brown algae) v Nm si sng trong nc (Water mold) . Rhodophyta
gm cc To (Red algae). Ring To lc (Green algae) th mt phn thuc Nguyn sinh
(Protista) mt phn thuc Thc vt (Plantae)

H thng phn loi 5 gii sinh vt


H thng phn loi 6 gii sinh vt


H thng phn loi 8 gii sinh vt


H thng 3 lnh gii (domain)
Monera hay 2 lnh gii Vi khun v C khun thuc nhm Sinh vt nhn s (Prokaryote),
cn cc sinh vt khc u thuc nhm Sinh vt nhn tht (Eukaryote). Sai khc gia 3 lnh gii
Bacteria, Archaea v Eukarya c trnh by trn bng di y:

***- So snh ba lnh gii Bacteria, Archaea v Eukarya

c im Bacteria Archaea Eukarya
Nhn c mng nhn v hch Khng Khng C
nhn
Phc hp bo quan c mng Khng Khng C
Thnh t bo
Hu ht c peptidoglycan
cha acid muramic
Nhiu loi khc nhau, khng
cha acid muramic
Khng cha acid
muramic
Mng lipid
Cha lin kt este, cc acid
bo mch thng
Cha lin kt ete, cc chui
aliphatic phn nhnh
Cha lin kt este, cc
acid bo mch thng
Ti kh C C Khng
ARN vn chuyn
Thymine c trong phn ln
tARN

tARN m u cha N-
formylmethionine
Khng c thymine trong
nhnh T hoc TyC ca tARN

tARN m u cha
methionine
C thymine

tARN m u cha
methionine
mARN a cistron C C Khng
Intron trong mARN Khng Khng C
Ghp ni, gn m v gn
ui polyA vo mARN
Khng Khng C
Ribosom
Kch thc 70S 70S 80S (ribosom t bo cht)
Yu t ko di EF2
Khng phn ng vi c t
bch hu
C phn ng C phn ng
Mn cm vi
cloramphenicol v
kanamycin
Mn cm Khng Khng
Mn cm vi anisomycin Khng Mn cm Mn cm
ARN polymerase ph thuc ADN
S lng enzym Mt Mt s Ba
Cu trc 4 tiu n v 8-12 tiu n v 12-14 tiu n v
Mn cm vi rifampicin Mn cm Khng Khng
Promoter typ Polymerase II Khng C C
Trao i cht
Tng t ATPase Khng C C
Sinh methane Khng C Khng
C nh N
2
C C Khng
Quang hp vi dip lc C Khng C
Ho dng v c C C Khng

hiu c chi tit ni dung ghi trong bng ni trn gio vin cn gii thch cho sinh vin
nhng kin thc c bn thuc gio trnh T bo hc v Di truyn hc
Phn ln vi sinh vt thuc v ba nhm C khun, Vi khun v Nguyn sinh. Trong gii
Nm, th nm men (yeast), nm si (filamentous Fungi) v dng si (mycelia) ca mi nm ln u
c coi l vi sinh vt. Nh vy l vi sinh vt khng c mt trong hai gii ng vt v Thc vt.
Ngi ta c tnh trong s 1,5 triu loi sinh vt c khong 200 000 loi vi sinh vt (100 000 loi
ng vt nguyn sinh v to, 90 000 loi nm, 2500 loi vi khun lam v 1500 loi vi khun). Tuy
nhin hng nm, c thm hng nghn loi sinh vt mi c pht hin, trong c khng t loi vi
sinh vt.
Virus l mt dng c bit cha c cu trc c th cho nn cha c k n trong s 200
000 loi vi sinh vt ni trn. S virus c t tn l khong 4000 loi.

Poliovirus Virus cm g H5N1 Virus HIV/AIDS
Trong thc t, s loi vi sinh vt phi ti hng triu loi. Bo tng ging chun vi sinh vt
(VTCC) thuc TT Cng ngh Sinh hc, HQG H Ni hp tc vi cc nh khoa hc Nht bn v
da trn cc k thut sinh hc phn t bc u pht hin c kh nhiu loi vi sinh vt mi
c th gii cng nhn.

2-Cc c im chung ca vi sinh vt :
Vi sinh vt c cc c im chung sau y :
1)-Kch thc nh b :
Vi sinh vt thng c o kch thc bng n v micromet (1mm= 1/1000mm hay
1/1000 000m). virus c o kch thc n v bng nanomet (1nn=1/1000
000mm hay 1/1000 000 000m).
Kch thc cng b th din tch b mt ca vi sinh vt trong 1 n v th
tch cng ln. Chng hn ng knh ca 1 cu khun (Coccus) ch c 1mm,
nhng nu xp y chng thnh 1 khi lp nhng c th lch l 1cm
3
th
chng c din tch b mt rng ti ...6 m
2
!


Light microscope : KHV quang hc
Electron microscope : KHV in t
Most bacteria: Phn ln vi khun

Kch thc vi khun so vi u kim khu


Ba dng ch yu vi khun : trc khun, cu khun v xon khun.

2)-Hp thu nhiu, chuyn ho nhanh :
Tuy vi sinh vt c kch thc rt nh b nhng chng li c nng lc hp
thu v chuyn ho vt xa cc sinh vt khc. Chng hn 1 vi khun lctic
(Lactobacillus) trong 1 gi c th phn gii c mt lng ng lactose ln
hn 100-10 000 ln so vi khi lng ca chng. tc tng hp protein ca
nm men cao gp 1000 ln so vi u tng v gp 100 000 ln so vi tru
b.


Lactobacillus qua KHV in t

3) Sinh trng nhanh, pht trin mnh :
Chng hn, 1 trc khun i trng (Escherichia coli ) trong cc iu kin
thch hp ch sau 12-20 pht li phn ct mt ln. Nu ly thi gian th h l
20 pht th mi gi phn ct 3 ln, sau 24 gi phn ct 72 ln v to ra 4 722
366 500 000 000 000 000 000 t bo (4 722 366. 10
17
), tng ng vi 1
khi lng ... 4722 tn. Tt nhin trong t nhin khng c c cc iu kin
ti u nh vy ( v thiu thc n, thiu oxy, d tha cc sn phm trao i
cht c hi...). Trong ni ln men vi cc iu kin nui cy thch hp t 1 t
bo c th to ra sau 24 gi khong 100 000 000- 1 000 000 000 t bo. Thi
gian th h ca nm men di hn, v d vi men ru (Saccharomyces
cerevisiae) l 120 pht. Vi nhiu vi sinh vt khc cn di hn na, v d vi
to Tiu cu ( Chlorella ) l 7 gi, vi vi khun lam Nostoc l 23 gi...C th
ni khng c sinh vt no c tc sinh si ny n nhanh nh vi sinh vt.

Vi kun Escherichia
coli
Nm men
Saccharomyces
cerevisiae
Nm si
Alternaria
Vi to Chlorella

4) C nng lc thch ng mnh v d dng pht sinh
bin d :
Trong qu trnh tin ho lu di vi sinh vt to cho mnh nhng c ch
iu ho trao i cht thch ng c vi nhng iu kin sng rt khc
nhau, k c nhng iu kin ht sc bt li m cc sinh vt khc tgng khng
th tn ti c. C vi sinh vt sng c mi trng nng n 130
0
C, lnh
n 0-5
0
C, mn n nng 32% mui n, ngt n nng mt ong, pH
thp n 0,5 hoc cao n 10,7, p sut cao n trn 1103 at. hay c
phng x cao n 750 000 rad. Nhiu vi sinh vt c th pht trin tt trong
iu kin tuyt i k kh, c noi nm si c th pht trin dy c trong b
ngm t thi vi nng Formol rt cao...
Vi sinh vt a s l n bo, n bi, sinh sn nhanh, s lng nhiu,
tip xc trc tip vi mi trng sng ... do rt d dng pht sinh bin d.
Tn s bin d thng mc 10
-5
-10
-10
. Ch sau mt thi gian ngn c th
to ra mt s lng rt ln cc c th bin d cc h h sau. Nhng bin d
c ch s a li hiu qu rt ln trong sn xut. Nu nh khi mi pht hin ra
penicillin hot tnh ch t 20 n v/ml dch ln men (1943) th nay c th
t trn 100 000 n v/ml. Khi mi pht hin ra acid glutamic ch t 1-2g/l
th nay t n 150g/ml dch ln men (VEDAN-Vit Nam).


Nh my Vedan-Vit Nam

5) Phn b rng, chng loi nhiu :
Vi sinh vt c mt khp mi ni trn Tri t, trong khng kh, trong
t, trn ni cao, di bin su, trn c th, ngi, ng vt, thc vt, trong
thc phm, trn mi vt...
Vi sinh vt tham gia tch cc vo vic thc hin cc vng tun hon sinh-
a-ho hc (biogeochemical cycles) nh vng tun hon C, vng tun hon n,
vng tun hon P, vng tun hon S, vng tun hon Fe...
Trong nc vi sinh vt c nhiu vng duyn hi (littoral zone), vng
nc nng (limnetic zone) v ngay c vng nc su (profundal zone), vng
y ao h (benthic zone).
Trong khng kh th cng ln cao s lng vi sinh vt cng t. S lng vi
sinh vt trong khng kh cc khu dn c ng c cao hn rt nhiu so vi
khng kh trn mt bin v nht l trong khng kh Bc cc, Nam cc...
Hu nh khng c hp cht carbon no (tr kim cng, grapht...) m
khng l thc n ca nhng nhm vi sinh vt no (k c du m, kh thin
nhin, formol. dioxin...). Vi sinh vt c rt phong ph cc kiu dinh dng khc
nhau : quang t dng (photoautotrophy), quang d dng
(photoheterotrophy), ho t dng (chemoautotrophy), ho d dng
(chemoheterotrophy).t dng cht sinh trng (auxoautotroph), d dng
cht sinh trng (auxoheterotroph)...

6)- L sinh vt xut hin u tin trn tri t :
Tri t hnh thnh cch y 4,6 t nm nhng cho n nay mi ch tm
thy du vt ca s sng t cch y 3,5 t nm. l cc vi sinh vt ho
thch cn li vt tch trong cc tng c. Vi sinh vt ho thch ca nht
c pht hin l nhngdng rt ging vi Vi khun lam ngy nay. Chng
c J.William Schopf tm thy ti cc tng c min Ty Australia. Chng
c dng a bo n gin, ni thnh si di n vi chc mm vi ng knh
khong 1-2 mm v c thnh t bo kh dy. Trc cc nh khoa hc cng
tm thy vt tch ca chi Gloeodiniopsis c nin i cch y 1,5 t nm v
vt tch ca chi Palaeolyngbya c nin i cch y 950 triu nm.




Vt tch vi khun
lam cch y 3,5 t
nm
Vt tch Gloeodiniopsis cch
y 1,5 t nm
Vt tch Palaeolyngbya cch
y 950 triu nm


Bi 3 Cu trc t bo vi khun
1. Thnh t bo :
Thnh t bo (cell wall) gip duy tr hnh thi ca t bo, h tr s chuyn ng ca tin
mao (flagellum) , gip t bo khng vi p sut thm thu, h tr qu trnh phn ct t bo , cn
tr s xm nhp ca mt s cht c phn t ln, lin quan n tnh khng nguyn , tnh gy bnh,
tnh mn cm vi Thc khun th (bacteriophage).
Nm 1884 H.Christian Gram ngh ra phng php nhum phn bit phn chia vi khun
thnh 2 nhm khc nhau : vi khun Gram dng (G+) v vi khun Gram m (G-). Phng php
nhum Gram v sau c s dng rng ri khi nh loi vi sinh vt. Thnh phn ho hc ca 2
nhm ny khc nhau ch yu nh sau :

Gram dng Gram m
Thnh phn T l % i vi khi lng kh ca thnh t bo
Peptidoglycan 30-95 5-20
Acid teicoic (Teichoic acid) Cao 0
Lipid Hu nh khng c 20
Protein Khng c hoc c t Cao

Mng sinh cht (plasma membrane); Mng ngoi (outer membrane); Chu cht
(Periplasmic space)

Peptidoglycan l loi polyme xp, kh bn vng, cu to bi 3 thnh phn:
-N-Acetylglucosamin ( N-Acetylglucosamine, NAG)
-Acid N-Acetylmuramic (N-Acetylmuramic acid, NAM)
-Tetrapeptid cha c D- v L- acid amin





Thnh t bo vi khun Gram dng


Thnh t bo vi khun Gram m
2- Mng sinh cht:
Mng sinh cht hay Mng t bo cht (Cytoplasmic membrane, CM) vi
khun cng tng t nh cc sinh vt khc. Chng cu to bi 2 lp
phospholipid (PL), chim 30-40% khi lng ca mng, v cc protein (nm
trong, ngoi hay xen gia mng), chim 60-70% khi lng ca mng. u
phosphat ca PL tch in, phn cc, a nc ; ui hydrocarbon khng tch
in, khng phn cc, k nc.
CM c cc chc nng ch yu sau y:
- Khng ch s qua li ca cc cht dinh dng, cc sn
phm trao i cht
- Duy tr p sut thm thu bnh thng trong t bo.
- L ni sinh tng hp cc thnh phn ca thnh t bo v
cc polyme ca bao nhy (capsule).
- L ni tin hnh qu trnh phosphoryl oxy ho v qu trnh
phosphoryl quang hp ( vi khun quang t dng)
- L ni tng hp nhiu enzym, cc protein ca chui h
hp.
- Cung cp nng lng cho s hot ng ca tin mao

Sinh vin in ch thch theo hng dn ca gio vin


Cu trc ca u v ui ca phospholipid

2. T bo cht :
T bo cht (TBC-Cytoplasm) l phn vt cht dng keo nm bn trong mng sinh cht,
cha ti 80% l nc. Trong t bo cht c protein, acid nucleic, hydrat carbon, lipid, cc
ion v c v nhiu nhiu cht khc c khi lng phn t thp. Bo quan ng lu trong
TBC l ribosom (ribosome). Ribosom nm t do trong t bo cht v chim ti 70% trng
lng kh ca TBC. Ribosom gm 2 tiu phn (50S v 30S), hai tiu phn ny kt hp vi
nhau to thnh ribosom 70S. S l n v Svedberg- i lng o tc lng khi ly tm cao
tc. Cu trc ca ribosom vi khun so vi ribosom 80S cc sinh vt nhn tht (nm, thc
vt, ng vt) c trnh by trong bng sau y (Gio vin ging sinh vin ch thch vo
hnh bng ting Vit)

Ribosom vi khun


So snh Ribosom Vi khun v cc Sinh vt nhn tht (Eukaryotic ribosome)

Trong t bo cht ca vi khun cn c th gp cc cht d tr nh cc
ht glycogen, ht PHB (Poly--hydroxybutyrat), Cyanophycin, Phycocyanin,
cc ht d nhim sc (metachromatic body), cc git lu hunh...
loi vi khun dit cn trng Bacillus thuringiensis v Bacillus
sphaericus cn gp tinh th c (parasoral body) hnh qu trm, c bn cht
protein v cha nhng c t c th git hi trn 100 loi su hi (tinh th c
ch gii phng c t trong mi trng kim do cc vi khun ny hon ton
v hi vi ngi, gia sc, gia cm, thu hi sn- c hi i vi tm). Bacillus
sphaericus c th dit cung qung ca cc loi mui.


Bo t (spore) v Tinh th c (Crystal) Bacillus thuringiensis (tri) v
Bacillus sphaericus (phi).

3. Th nhn:
Th nhn ( Nuclear body) vi khun l dng nhn nguyn thu, cha c mng nhn nn
khng c hnh dng c nh, v v vy cn c gi l vng nhn. Khi nhum mu t bo bng
thuc nhum Feulgen c th thy th nhn hin mu tm. l 1 nhim sc th (NST,
chromosome) duy nht dng vng cha 1 si ADN xon kp ( X khun Streptomyces c th gp
nhim sc th dng thng). NST vi khun Escherichia coli di ti 1mm (!), c khi lng phn
t l 3.10
9
, cha 4,6.10
6
cp base nit. Th nhn l b phn cha ng thng tin di truyn ca vi
khun.
*
Th nhn trong t bo vi khun Escherichia coli.

Ngoi NST, trong t bo nhiu vi khun cn gp nhng ADN ngoi NST.
l nhng ADN xon kp c dng vng khp kn, c kh nng sao chp c
lp, chng c tn l Plasmid.
4. Bao nhy:
Bao nhy hay Gip mc (Capsule) gp mt s loi vi khun vi cc
mc khc nhau:
-Bao nhy mng ( Vi gip mc, Microcapssule)
-Bao nhy (Gip mc, Capsule)
-Khi nhy ( Zooglea)
Mun quan st bao nhy thng ln tiu bn vi mc tu, bao nhy c
mu trng hin ln trn nn ti.
Thnh phn ch yu ca bao nhy l polysaccarid, ngoi ra cng c
polypeptid v protein. Trong thnh phn polysaccarid ngoi glucose cn c
glucozamin, ramnose, acid 2-keto-3-deoxygalacturonic, acid uronic, acid
pyruvic, acid axetic...
ngha sinh hc ca bao nhy l:
-Bo v vi khun trong iu kin kh hn, bo v vi khun trnh b thc
bo (trng hp Ph cu khun-Diplococcus pneumoniae)
-Cung cp cht dinh dng cho vi khun khi thiu thc n
-L ni tch lu mt s sn phm trao i cht (dextran, xantan...)
-Gip vi khun bm vo gi th ( trng hp cc vi khun gy su rng
nh Streptococcus salivarrius, Streptococcus mutans...)


Vi khun Acetobacter xylinum c bao nhy cu to bi cellulose. Ngi ta dng
vi khun ny nui cy trn nc da ch to ra Thch da (Nata de coco).

da (Nata de coco)

Vi khun Leuconostoc mesenteroides c bao nhy dy cha hp cht polyme l
Dextran c tc dng thay huyt tng khi cp cu m thiu huyt tng. Sn
phm ny rt quan trng khi c chin tranh. Vi khun ny thng gp cc
nh my ng v gy tn tht ng trong cc b cha nc p ma. Nh
enzym dextransuccrase m ng saccarose b chuyn thnh dextran v
fructose.

Vi khun Leuconostoc mesenteroides

Mt s bao nhy ca vi khun cn c dng sn xut Xantan
(Xanthane) dng lm cht ph gia trong cng nghip du m.

5. Tin mao v khun mao :
Tin mao (Lng roi, flagella) khng phi c mt mi vi khun, chng quyt nh kh nng
v phng thc di ng ca vi khun. Tin mao l nhng si lng di, di knh hin vi quang hc
ch c th thy r khi nhum theo phng php ring. Di knh hin vi in t c th thy rt r
cu trc ca tng si tin mao. xc nh xem vi khun c tin mao hay khng cn c cch th
gin tip nhm bit kh nng di ng ca chng. Cy bng que cy nhn u vo mi trng thch
ng cha 0.4% thch (agar-agar), cn gi l mi trng thch mm. Nu thy vt cy lan nhanh
ra xung quanh th chng t l vi khun c tin mao, c kh nng di ng.
Tin mao c th gc (basal body), gm 1 tr nh c gn vi 4 a trn (vi khun G
-
) c
dng vng nhn (ring), k hiu l cc vng L,P,S v M. Vng L nm ngoi cng, tng ng vi lp
liposaccarid ca mng ngoi ; vng P tng ng vi lp peptidoglycan, vng S tng ng vi lp
khng gian chu cht ; vng M nm trong cng. Vi khun G
+
ch c 2 vng : 1 vng nm ngoi
tng ng vi thnh t bo v 1 vng trong tng ng vi mng sinh cht. Xuyn gia cc vng l
1 tr nh (rod) c ng knh 7nm. Bao bc tin mao phn pha ngoi l mt bao ngn c hnh
mc (hook). Si tin mao (filament) di khong 10-20m v c ng knh khong 13-20nm.
ng knh ca bao hnh mc l 17nm. Khong cch gia vng S v vng M l 3mm, gia vng P
v vng L l 9nm, gia vng P v vng S l 12nm. ng knh ca cc vng l 22nm, ng knh
cc l cc vng l 10nm. Khong cch t mt ngoi ca vng L n mt trong ca vng M l
27nm. Si tin mao cu to bi loi protein c tn l flagellin, c trng lng phn t l 30 000-60
000. Mt s vi khun c bao lng (sheath) bao bc sut chiu di si, nh trng hp chi
Bdellovibrio hay vi khun t Vibrio cholera.


Tin mao v khun mao vi khun



Tin mao VK Gram dng Tin mao VK Gram m



Tin mao vi khun G
+



Tin mao vi khun G
-

Cc tiu phn (subunit) ca flagellin c tng hp t cc ht ribosom nm gn mng sinh
chy tng hp nn v i qua li m to dn thnh si tin mao

Tin mao ca vi khun c cc loi khc nhau tu tng loi :
-Khng c tin mao (v mao, atrichia)
-C 1 tin mao mc cc ( n mao, monotricha)
-C 1 chm tin mao mc cc ( chm mao, lophotricha)
-C 2 chm tin mao mc 2 cc ( song chm mao, amphitricha)
-C nhiu tin mao mc khp quanh t bo (chu mao, peritricha)
C loi tin mao mc gia t bo nh trng hp vi khun
Selenomonas ruminantium.

Cc loi tin mao vi khun
Kiu sp xp tin mao lin quan n hnh thc di ng ca vi khun. Tin mao mc cc
gip vi khun di ng theo kiu tin- li. Chng o ngc hng bng cch o ngc hng
quay ca tin mao. Vi khun chu mao di ng theo hng no th cc tin mao chuyn ng theo
hng ngc li. Khi tin mao khng t li v mt hng th vi khun chuyn ng theo kiu nho
ln. Tc di chuyn ca vi khun c tin mao thng vo khong 20-80m/giy, ngha l trong 1
giy chuyn ng c mt khong cch ln hn gp 20-80 ln so vi chiu di ca c th chng.
Cc chi vi khun thng c tin mao l Vibrio, Spirillum, Pseudomonas, Escherichia,
Shigella, Salmonella, Proteus... cc chi Clostridium, Bacterium,Bacillus, ...c loi c tin mao c
loi khng. cu khun ch c 1 chi (Planococcus) l c tin mao
Xon th c mt dng tin mao c bit gi l tin mao chu cht (periplasmic flagella), hay
cn gi l si trc ( axial fibrils), xut pht t cc t bo v qun quang c th. Chng gip xon
th chuyn ng c nh s un vn t bo theo kiu vn nt chai.

Xon th (Spirochete) quan st di knh hin vi nn en.





AF: Si trc
PC: ng nguyn sinh cht
OS: V ngoi
IP: L ni

C ch chuyn ng un vn t bo Xon th ( OS, AF, PC- xem ch thch
hnh trn).

6. Khun mao v Khun mao gii:
Khun mao (hay Tim mao, Nhung mao , Fimbriae) l nhng si lng rt
mnh, rt ngn mc quanh b mt t bo nhiu vi khun Gram m. Chng c
ng knh khong 7-9nm, rng rut (ng knh trong l 2-2,5nm), s lng
khong 250-300 si/ vi khun. Kt cu ca khun mao gin n hn nhiu so
vi tin mao. Chng c tc dng gip vi khun bm vo gi th ( nhiu vi
khun gy bnh dng khun mao bm cht vo mng nhy ca ng h
hp, ng tiu ho, ng tit niu ca ngi v ng vt).

Khun mao vi khun E.coli
C mt loi khun mao t bit gi l Khun mao gii (Sex pili, Sex pilus-s
nhiu) c th gp mt s vi khun vi s lng ch c 1-10/ vi khun. N c
cu to ging khun mao , ng knh khong 9-10nm nhng c th rt di.
Chng c th ni lin gia hai vi khun v lm cu ni chuyn vt cht di
truyn (ADN) t th cho (donor) sang th nhn (recipient). Qu trnh ny c
gi l qu trnh giao phi (mating) hay tip hp (conjugation). Mt s thc
khun th (bacteriophage) bm vo cc th th (receptors) khun mao gii
v bt u chu trnh pht trin ca chng.
Bi 4 Cc nhm vi khun ch yu
Theo quan im hin i (NCBI- National Center for Biotechnology Information, 2005) th vi
khun bao gm cc ngnh sau y :
1. -Aquificae
2. -Thermotogae
3. -Thermodesulfobacteria
4. -Deinococcus-Thermus
5. -Chrysiogenetes
6. -Chloroflexi
7. -Nitrospirae
8. -Defferribacteres
9. -Cyanobacteria
10. -Proteobacteria
11. -Firmicutes
12. -Actinobacteria
13. -Planctomycetes
14. -Chlamydiae/Nhm Verrucomicrobia
15. -Spirochaetes
16. -Fibrobacteres /Nhm Acidobacteria
17. -Bacteroidetes/Nhm Chlorobia
18. -Fusobacteria
19. -Dictyoglomi
Vic phn ngnh da trn cc c im hnh thi, sinh l, sinh ha, sinh thi...

Cn c vo t l G + C trong ADN ngi ta xy dng c cy pht sinh chng loi
(Phylogenetic tree) v chia vi khun thnh 11 nhm sau y :
1. -Nhm Oxy ho Hydrogen
2. -Nhm Chu nhit
3. -Nhm Vi khun khng lu hunh mu lc
4. -Nhm Deinococcus
5. -Nhm Vi khun lam
6. -Nhm Proteobacteria
7. -Nhm Chlamydia
8. -Nhm Planctomyces
9. -Nhm Spirochaetes (Xon th)
10. -Nhm Vi khun lu hunh mu lc
11. -Nhm Cytophaga
12. --Nhm Vi khun Gram dng
























S v cc loi in hnh
c khi nim v cc chi vi khun thng gp chng ta lm quen vi mt s kho phn loi
n gin, da trn cc c im v hnh thi, sinh l , sinh ho. Trong thc tin vi cc loi vi
khun gy bnh ngi ta thng chn on thm bng phn ng huyt thanh (vi cc khng
th c hiu)

1.Vi khun quang hp (Phototrophic bacteria) :
1.1.Vi khun lu hunh mu ta (Purple sulfur bacteria):
Thuc nhm ny l cc vi khun k kh bt buc, c kh nng quang t dng v c
(photolithoautotroph), t bo c cha chlorophyll a hoc b , h thng quang hp cha cc mng
hnh cu hay hnh phin (lamellar) gn vi mng sinh cht. dng lm ngun cho in t
(electron donors) trong quang hp thng s dng H
2,
H
2
S hay S . C kh nng di ng vi tin
mao mc cc, c loi chu mao, t l G+C l 45-70%.
a- H Chromatiaceae:
1.1.1- Chi Thiospirium
1.1.2. Chi Chromatium
1.1.3. Chi Thiocapsa
1.1.4. Chi Thiocystis
1.1.5. Chi Thiospirillum
1.1.6. Chi Thiorhodovibrio
1.1.7. Chi Amoebobacter
1.1.8. Chi Lamprobacter
1.1.9. Chi Lamprocystis
1.1.10.Chi Thiodyction
1.1.11.Chi Thiopedia
1.1.12. Chi Rhabdochromatium
1.1.13. Chi Thiorhodococcus



Chromatium Thiocapsa


Thiocystis Thiospirillum



Lamprocystis Thiopedia

b- H Ectothiorhodospiraceae:
1.1.14- Chi Ectothiorhodospirace
1.1.15- Chi Halorhodospira
1.2-Vi khun khng lu hunh mu ta (Nonsulfure purple
bacteria)
Vi khun khng lu hunh mu ta l nhm vi khun quang d dng hu c
(photoorganoheterotrophs) thng k kh bt buc, mt s loi l quang t dng v c khng bt
buc (trong ti l ho d dng hu c- chemoorganoheterotrophs). T bo cha chlorophyl a hoc
b, h thng quang hp cha cc mng hnh cu hay hnh phin (lamellar) gn vi mng sinh cht.
dng lm ngun cho in t (electron donors) trong quang hp thng s dng cht hu c, i
khi s dng hp cht lu hunh dng kh hoc H
2
. C kh nng di ng vi tin mao mc cc,
hoc khng di ng, mt s loi c ti kh (gas vesicles), t l G+C l 61-72%.
1.2.1- Chi Blastochloris
1.2.2- Chi Phaeospirillum
1.2.3- Chi Rhodobacter
1.2.4- Chi Rhodobium
1.2.5- Chi Rhodocista
1.2.6- Chi Rhodocyclus
1.2.7- Chi Rhooferax
1.2.8- Chi Rhodomicrobium
1.2.9- Chi Rhodoplanes
1.2.10-Chi Rhodopila
1.2.11- Chi Rhodopseudomonas
1.2.12- Chi Rhodospira
1.2.13- Chi Rhodospirillum
1.2.14- Chi Rhodothalassium
1.2.15- Chi Rhodovibrio
1.2.16-Chi Rhodovulum
1.2.17- Chi Rosespira
1.2.18- Chi Rubiviva




Rhodospirillum Rhodospirillum di KHV int


Rhodopseudomonas Rhodopseudomonas di KHV in t


Rhodobacter Rhodopila


Rhodocyclus purpureus Rhomicrobium

1.3.Vi khun lu hunh mu lc (Green sulfure bacteria)
Thuc nhm ny l cc vi khun k kh bt buc, c kh nng quang t dng v c
(photolithoautotroph), t bo c cha chlorophyll a cng vi b , c hoc e, cha caroten nhm 5, h
thng quang hp lin quan n cc lc th (chlorosom) v c lp i vi mng sinh cht. dng
lm ngun cho in t (electron donors) trong quang hp thng s dng H
2,
H
2
S hay S . Ht lu
hunh tch lu bn ngoi t bo Khng c kh nng di ng , mt s loi c ti kh; t l G+C l
48-58%.
1.3.1- Chi Chlorobium
1.3.2- Chi Prosthecochloris
1.3.3- Chi Pelodictyon
1.3.4- Chi Ancalichliris
1.3.5- Chi Chloroherpeton



Chlorobium Pelodictyon


Prosthecochloris

1.4.Vi khun khng lu hunh mu lc (Green nonsulfur
bacteria)
Thuc nhm ny l cc vi khun a bo, dng si,thng k kh khng bt buc ,thng l
quang d dng (photoheterotroph), c loi quang t dng hoc ho d dng. T bo c cha
chlorophyll a v c, trong iu kin k kh thy c chlorosom. dng lm ngun cho in t
(electron donors) trong quang d dng l glucose, axit amin, axit hu c; trong quang t dng l
H
2,
H
2
S. Di ng bng phng thc trn (gliding) , t l G+C l 53-55%. Chi in hnh l
Chloroflexus., Chloronema

Chloronema Chloroflexus


1.5- Vi khun lam (Ngnh Cyanobacteria)
Trc y thng nhm ln l To lam (Cyanophyta). Thc ra y l nhng c th nhn
nguyn thu, khng lin quan g n to , ngoi kh nng quang hp hiu kh (quang t dng v
c) v dng H
2
O lm cht cho in t trong qu trnh quang hp. Vi khun lam cha chlorophyll a
v phycocyanin- phycobiliprotein. Mt s loi c sc t phycoerythrin. Chng phi hp vi sc
t lc to nn mu nu. Mng lin kt vi phycobilisom. n bo hoc a bo dng si. Khng di
ng hoc di ng bng cch trn (gliding), mt s loi c ti kh (gas vesicles).Nhiu loi c d t
bo (heterocysts) v c kh nng c nh nit. Vi khun lam c mt khp mi ni, trong t, trn
, trong sui nc nng, trong nc ngt v nc mn. Chng c nng lc chng chu cao hn so
vi thc vt i vi cc iu kin bt li nh nhit cao, pH thp. Mt s loi c kh nng sng
cng sinh vi cc c th khc nh Ru, Dng x, Tu...Nhiu loi cng sinh vi nm to ra a
y (Lichen). Vi khun lam c th l sinh vt xut hin sm nht trn Tri t
Vi khun lam c chia thnh 5 nhm (subsection) nh sau:
a- Nhm I (c tc gi gi l b Chroococcales):
Hnh que hoc hnh cu n bo, khng c dng si hay dng kt khi (aggregate); phn
i hoc ny chi; khng c d t bo (heterocytes). Hu ht khng di ng. T l G+C l 31-71% .
Cc chi tiu biu l:
-Chamaesiphon
-Chroococcus
-Gloeothece
-Gleocapsa
-Prochloron

Chamaesiphon Chroococcus



Glooeothece Gleocapsa


Prochloron

b-Nhm II (c tc gi gi l b Pleurocapsales):
Hnh que hoc hnh cu n bo. c th to dng kt khi (aggregate); phn ct nhiu ln to
ra cc baeocytes; khng c d t bo.Ch c cc baeocytes l c di ng. T l G+C l 40-46% . Cc
chi tiu biu l:
-Pleurocapsa
-Dermocapsa
-Chroococcidiopsis


Pleurocapsa Dermocapsa Chroococcidiopsis


c-Nhm III (c tc gi gi l b Oscillatorriales):
Dng si (filamentous) ; dng lng (trichome) khng phn nhnh ch c cc t bo dinh
dng; phn i trn mt phng, c kiu t on (fragmentation); khng c d t bo; thng di
ng. T l G+C l 34-67%. Cc chi tiu biu l:
-Lyngbya
-Osscillatoria
-Prochlorothrix
-Spirulina
-Pseudanabaena

Lyngbya Oscillatoria Prochlorothrix


Spirulina Pseudanabaena

d-Nhm IV (c tc gi gi l b Nostocales) :
Dng si ; dng lng (trichome) khng phn nhnh c th cha cc t bo bit ho
(specialized cell) ; phn i trn mt phng, c kiu t on to thnh on sinh sn
(hormogonia) ; c t bo d hnh ; thng di ng c th sn sinh bo t mng dy (akinetes). T l
G+C l 38-47%. Cc chi tiu biu l :
-Anabaena
-Cylindrospermum
-Aphanizomenon
-Nostoc
-Scytonema
-Calothrix

Anabaena Anabaena trong Bo hoa du


Cylindrospermum Calothrix



Nostoc Scytonema

e-Nhm V (c tc gi gi l b Stigonematales) :
Lng (trichome) dng si, phn nhnh hoc do cc t bo nhiu hn mt chui to thnh ;
phn i theo nhiu mt phng, hnh thnh on sinh sn (hormogonia) ; c t bo d hnh ; c th
sn sinh bo t mng dy ( alkinetes), c hnh thi phc tp v bit ha (differentiation). T l G+C
l 42-44%. Cc chi tiu biu l :
-Fischerella
-Stigonema
-Geitlerinema

Fischerella


Geitlerinema

Theo NCBT (2005) th Vi khun lam bao gm nhng b sau y:
-Chlorococcales
-Gloeobacteria
-Nostocales
-Oscillatoriales
-Pleurocapsales
-Prochlorales


2- Vi khun sinh ni bo t (Endospore-forming bacteria):
A-Vi khun hnh cu

2.1- Chi Sporosarcina
AA-Vi khun hnh que

B-K kh bt buc
C-Sinh trng c nng 3-12% NaCl
2,2- Chi Sporohalobacter
CC-Khng sinh trng c nng 3-12% NaCl
D-Kh sulfat
2.3- Chi Desulfotomaculum
DD-Khng kh sulfat
E-Phn gii Axit 3-hydroxybenzoic
2.4- Chi Sporomaculum
EE- Khng phn gii Axit 3-hydroxybenzoic
F- Chiu rng ca t bo > 2,5mm
2.5- Chi Oscillosporia
FF- Chiu rng ca t bo < 2,5mm
G-S dng axit bo bo ho v axit butyric
2.6- Chi Syntrophospora
GG- Khng s dng axit bo bo ho
2.7- Clostridium

BB- Vi hiu kh
2.8-Sporolactobacillus

BBB- Hiu kh v k kh khng bt buc
C- Phn gii lignin trn mi trng kim
2.9- Amphibacillus
CC- Khng phn gii lignin trn mi trng kim
D-Sinh trng trn mi trng > 10% NaCl
2.10- Halobacillus
DD-Khng sinh trng trn mi trng > 10% NaCl
E-C th phn gii Thiamin
2.11- Aneurinibacillus
EE- Khng c th phn gii Thiamin
F-C cha acid bo vng w trong lipid
2.12- Alicycolobacillus
FF- Khng cha acid bo vng w
G- C th tp giao vi 515F (chy PCR)
2.13- Paenibacillus
GG- Khng th tp giao vi 515F
H- C th tp giao vi 1741F (chy PCR)
2.14- Brevibacillus
HH- Khng th to giao vi 1741F
I-Phn gii qung pyrit
2.15- Sulfidobacillus
II- Khng phn gii qung pyrit
1.16- Bacillus

Sporosarcina Desulfotomaculum Clostridium


Paenibacillus Bacillus

3- Trc khun Gram m, ln men , hiu kh hoc k
kh khng bt buc

A- Catalase dng tnh, khng di ng hay di ng nh tin mao cc
B- Thng di ng nh tin mao cc, khng k sinh ng vt c xng sng
H Vibrionaceae
3.1- Chi Aeromonas
3.2- Chi Enhydrobacter
3.3- Chi Photobacterium
3.4- Chi Pleisiomonas
3.5- Chi Vibrio
BB- Khng di ng, k sinh ng vt c xng sng
H Pasteurellaceae
3.6- Chi Actinobacillus
3.7- Chi Haemophilus
3.8-Chi Pasteurella
AA- Catalase m tnh, khng di ng hay di ng nh chu mao
H Enterobacteriaceae
3.9- Chi Proteus
3.10-Chi Enterobacter
3.11-Chi Pantoea
3.12-Chi Rhanella
` 3.13-Chi Providencia
3.14-Chi Morganella
3.15-Chi Tatumella
3.16-Chi Salmonella
3.17-Chi Edwardisiella
3.18-Chi Citrobacter
3.19-Chi Budvicia
3.20-Chi Pragia
3.21-Chi Leminorella
3.22-Chi Serratia
3.23-Chi Xenorhabdus
3.24-Chi Klebsiella
3.25-Chi Kluyvera
3.26-Chi Yersinia
3.27-Chi Cedecea
3.28-Chi Ewingella
3.29-Chi Buttiauxella
3.30-Chi Moellerlla
3.31-Chi Leclecia
3.32-Chi Escherichia
3.33-Chi Yokenlla
3.34-Chi Hafnia
3.35-Chi Tatumella


Aeromonas Photobacterium Vibrio



Vibrio Actinobacillus Proteus


Pasteurella Pasteurella Enterobacter


Morganella Salmonella Salmonella Yersinia

Citrobacter Serratia Serratia

Klebsiella Klebsiella Escherichia


4-Trc khun Gram m , khng ln men , hiu kh hoc k
kh khng bt buc

A- Sinh trng c 60

C
B-T bo ln, chiu rng 1,3-1,8mm
4.1- Chi Thermomicrobium
BB-T bo nh, chiu rng 0,4-0,8mm
C-C th dng glucose lm ngun carbon
D-Sinh trng c pH 4,5
4.2- Chi Acidothermus
DD-Khng sinh trng c pH 4,5
4.3- Chi Thermus
CC-Khng th dng glucose lm ngun carbon duy nht
4.4- Chi Thermoleophilum
AA- Khng sinh trng c 60

C
B-C th oxy ho etanol thnh axit axetic
C-C th oxy ho etanol ti CO
2
v H
2
O
D-C th oxy ho acid DL-lactic ti CO
2
v H
2
O
4.5- Chi Acetobacter
DD-Khng oxy ho acid DL-lactic ti CO
2
v H
2
O
4.6- Chi Acidomonas
CC-Khng th oxy ho ethanol ti CO
2
v H
2
O
D-C th oxy ho acid DL-lactic ti CO
2
v H
2
O
4.7- Chi Frateuria
DD-Khng oxy ho acid DL-lactic ti CO
2
v H
2
O
4.8- Chi Gluconobacter
BB-Khng oxy ho etanol thnh acid acetic
C-C th c nh Nit ngoi c th
D-C th c nh Nit trong iu kin hiu kh
E-C th sinh bo xc (cysts)
4.9-Chi Azotobacter
EE-Khng sinh bo xc
F-C lipoid trong phn cc ca t bo
4.10-Chi Beijerinckia
FF-Khng c lipoid trong phn cc t bo
G-T bo rng > 2,0mm
4.11- Chi Azomonas
GG- T bo rng < 2,0mm
4.12- Chi Derxia
DD- C th c nh nit trong iu kin vi hiu kh
E-Tin mao thng mc cc
F-Trong t, c th trao i carbohydrat
4.13- Chi Agromonas
FF-Trong nc, khng trao i carbohydrat
4.14-Loi Aquaspirillum fasciculus
EE-Chu mao
F-Sinh trng v c nh nit pH < 3,0
4.5- Loi Acetobacter diazotrophicus
FF-Khng sinh trng v c nh nit pH < 3,0
4.15- Chi Xanthobacter
CC- Khng th c nh nit ngoi c th
D-Hnh thnh nt sn trong r, trong thn thc vt, c th c nh nit t
khng kh.
E-Sinh acid trn mi trng Thch-Cao nm men-Mannit
F-C th dng DL-Arginin, L-Histidin lm ngun nit duy
nht.
4.16- Chi Rhizobium
FF- Khng th dng DL-Arginin , L-Histidin lm ngun nit
duy nht
4.17- Chi Sinorhizobium
EE-Khng sinh acid trn mi trng Thch- Cao nm men- Mannit
F-C Dihydrolase arginin v Decarbocylase lysin
4.18- Chi Azorhizobium
FF-Khng c Dihydrolase arginin v Decarboxylase lysin
4.19-Chi Bradyrhizobium
DD- Khng sinh nt sn trong c th c nh nit
E- Sinh khi u r thc vt
4.20- Chi Agrobacterium
EE- Khng sinh khi u r thc vt
F-C nh nit mt l
4.21- Chi Phyllobacterium
FF-Khng c nh nit mt l
G-C th dng hp cht 1 carbon lm ngun carbon duy nht
H-C tch lu PHB trong t bo
I-Gy bnh thc vt
4.22-Chi Rhizobacter
II- Khng gy bnh thc vt
J-Khng di ng
4.23- Loi Paracoccus denitrificans/ Paracoccus
alcaliphalus
JJ-Di ng
K-Nhum Gram dng tnh
hoc kh bin
4.15- Chi Xanthobacter
KK- Khng nh trn
4.24- Chi Methylobacter
HH-Khng tch lu PHB trong t bo
I-C th dng methane lm
ngun carbon duy nht
K-Di ng
4.25- Chi Methylomonas
KK-Khng di ng
4.26-Chi Methylococcus
II- Khng th dng methane lm ngun C
duy nht
J-Catalase dng tnh
K-Sinh trng cn NaCl
4.27- Chi Methylophaga
KK- Sinh trng khng cn NaCl
4.28- Chi Methylophilus
JJ-Catalase m tnh
4.29- Chi Methylobacillus
GG- Khng th dng hp cht 1 carbon lm ngun carbon duy nht
H-Sinh trng cn NaCl hay nc bin
I- Khng di ng
4.30- Chi Mesophilobacter
II- Di ng
J-C gelatinase
4.31- Chi Alteromonas
JJ-Khng c gelatinase
K-C tch lu PHB
L-S dng glucose
M-C esterase
4.32- Chi Deleya
MM-Khng c esterase
4.33- Loi Oceanospirillum kriegii
LL- Khng s dng glucose
4.33- Loi Oceanospirillum jannaschii
KK- Khng tch lu PHB
L- Sinh axt t mannit
4.34- Chi Marinomonas
LL- Khng sinh axt t mannit
4.36- Chi Pseudomonas (P.stanieri, P.perfectomarina,
P.doudoroffii, P.nautica)
HH- Sinh trng khng cn NaCl hay nc bin
I- C t dng hydrogen
J-C tin mao cc hay gn cc
K-Khun lc mu vng
L-Khng sinh trng 52
0
C
4.35- Chi Hydrogenophaga
LL- Sinh trng 52
0
C
4.36- Loi Ps. hydrogenothermophila
KK- Khun lc khng mu vng
4.36- Chi Pseudomonas (P. saccharophila, P.facillis,
P.hydrogenovora)
JJ- C chu mao tha
4.50- Chi Alcaligenes (A.paradoxus, biovar, A.eutrophus,
A.latus, A.denitrificans subsp. xylosoxidans)
II- Khng c t dng hydrogen
J- Di ng
K- Tin mao mc cc
L-Catalase dng tnh
4.36~47 Chi Pseudomonas
LL-Catalase m tnh
4.48- Chi Xanthomonas
KK- Chu mao
L-Sinh acid t ng
4.49- Chi Ochrobactrum
LL-Khng sinh acid t ng
4.50- Chi Alcaligenes
JJ- Khng di ng
K-Khun lc mu vng
L-Phospholipid cha sphingosin
4.51- Chi Sphingobacterium
LL-Phospholipid khng cha sphingosin
4.52- Flavobacterium
KK- Khun lc khng c mu vng
L- Yu cu cht ch v dinh dng
M-Lng cha G+C cao (66-70mol%)
4.53- Chi Bordetella
MM- Lng cha G+C thp (40-47mol%)
N-Sinh trng mnh
4.54- Chi Moraxella
NN- Sinh trng yu
4.55- Chi Oligella
LL-Khng yu cu cht ch v dinh dng
M- Khun lc mu tm
4.56- Chi Chromobacterium
MM- Khun lc khng c mu tm
N-Catalase dng tnh
O-Hnh que c lc bin hnh cu
4.57- Chi Acinetobacter
OO- Khng bin thnh hnh cu
4.58- Chi Weeksella
NN-Catalase m tnh
O- Sinh indol
4.59- Chi Suttonella
OO- Khng sinh indol
4.60- Chi Kingella

Thermomicrobium Thermoleophilum Acetobacter

Thermus



Gluconobacter Azotobacter Methylobacterium



Bordetella Rhizobium v nt sn trn r


Sinorhizobium v nt sn trn r


Methylococcus Bradyrhizobium v nt sn trn r



Agrobacterium v nt sn Pseudomonas



Alcaligenes Flavobacterium v khun lc



Moraxella Chromobacterium Acinetobacter
5- Cu khun Gram m, k kh bt buc
A- ng knh t bo > 1,3m
5.1- Chi Megasphaera
AA- ng knh t bo < 1,3m
B- Khun lc di tia t ngoi (360nm) c mu hunh quang, ch sinh acid bo 2
carbon
5.2- Chi Syntrophococcus
BB- Khun lc di tia t ngoi (360nm) khng c mu hunh quang, c th sinh
c cc acid bo khc
C- Aminoacid l ngun nng lng ch yu, khng ln men lactic
5.3- Chi Acidaminococcus
CC- Aminoacid khng phi l ngun nng lng ch yu, ln men lactic
5.4- Chi Veillonella

Megasphaera Acidaminococcus Veillonella

6-Trc khun, phy khun, xon khun Gram m, k kh :

I- Sinh trng nhit 55
0
C
A-Khng ln men v ng ho carbohydrat , s dng peptid, aminoacid, cao nm men...
B- T bo c bao, khng kh Fe
3+
.
6.1- Chi Thermosipho
BB- T bo khng c bao
C-S dng cc loi acid hu c v H
2


kh Fe
3+

6.2- Chi Deferribacter
CC-Khng kh Fe
3+

6.3- Chi Thermosyntropha
AA-Ln men v ng ho carbohydrat
B-a mn. sinh trng nng NaCl 4%
C-Di ng nh chu mao, sn phm ln men ch yu l acid acetic, ethanol,
H
2
/CO
2

6.4- Chi Halothermothrix
CC- Di ng nh tin mao mc bn cnh hay gn cc t bo, sn phm ln
men ch yu l acid acetc, acid lactic, acid succinic, H
2
/CO
2

6.5- Chi Thermotoga
BB- Khng a mn, khng sinh trng nng NaCl 4%
C-Sn phm ln men ch l acid acetic
D-Thnh t bo in hnh Gram m, cn vitamin B
12

6.6- Chi Acetothermus
DD-Thnh t bo khng in hnh Gram m, cn H
2
S hoc Cystein
6.7- Chi Acetogenium
CC-Sn phm ln men l hn hp acid acetic v cc acid hu c khc
D-Di ng
E-Phy khun, di ng nh tin mao mc bn hay gn cc
6.8- Chi Acetomicrobium
EE-Hnh que hoc bin i,khng di ng hoc di ng nh chu mao
6.9- Chi Fervidobacterium
DD-Khng di ng
E-Sn phm ln men gm H
2
/CO
2
v hn hp acid
F-Sn phm ln men gm acid acetc, ethanol, H
2
/CO
2

6.10- Chi Thermohydrogenum
FF-Sn phm ln men gm acid acetic v ethanol, hoc acid acetic
v H
2
/CO
2

6.11- Chi Coprothermobacter
EE-Khng sinh kh, sn phm ln men gm acid acetic, acid
propionic, acid isopentanoic
6.12- Chi Anaerobaculum
II- Khng sinh trng nhit 55
0
C, a m
A- Khng ng ha v ln men carbohydrat
B-Khng nui dng c thun khit, phi nui chung vi mt vi khun khc,
phn gii cc on axt bo ngn
C-T bo hnh que hay hnh si di, sn phm phn gii acid bo l acid acetic v
H
2
/CO
2

6.13- Chi Syntrophobacter
CC-Phy khun, sn phm phn gii acid bo l acid acetic, acid propionic, acid
isobutiric, acid isopentanoic v H
2/
/CO
2

6.14- Chi Syntrophomonas
BB-C th nui cy thun khit
C-Cha sc t t bo, vi hiu kh
6.15- Chi Wolinella
CC- Khng cha sc t t bo, k kh bt buc
D-Ch s dng acid succinic v ch sn sinh acid propionic
E-Phy khun, di ng nh tin mao mc bn
6.16- Chi Schuartzia
EE- Trc khun, khng di ng
6.17- Chi Succinoclastium
DD- ng ho cc loi protein
E-Di ng nh chu mao, khng ln men hoc ln men
yu glucose, sn phm l ethanol v acid butyric
6.18- Chi Tissierella
EE- Khng di ng
F- Trn a thch mu khun lc c mu t nu n en
6.19- Chi Porphyromonas
FF- Trn a thch mu khun lc khng c mu t nu n en, ln
men pyridin v arginin
6.20- Chi Synergistes
AA-C th ng ho v ln men hydrat carbon, sn sinh cc loi axit hi c
1. Sn phm ch yu ln men hydrat carbon l acid acetic
hoc acid acetic vi ethanol v H
2
/CO
2

B-Acid acetic l sn phm ln men duy nht
C- Di ng nh chu mao
6.21- Chi Acetoanaerobium
CC- Khng di ng
D-C th ln men aminoacid, sinh indol t tryptophan
6.22- Chi Acidaminobacter
DD- Khng ln men aminoacid, ln men carbohydrat v acid hu c
E- Sn phm ln men ngoi acid acetic cn c mt t acid formic v acid lactic
6.23- Chi Catonella
EE-Sn phm ln men ngoi acid acetic cn c mt t acid isopentanoic, acid
lactic, acid succinic, acid isobutyric v acid butyric
6.24- Chi Johngonella
BB- Sn phm ln men gm c acid acetic, ethanol v H
2
/CO
2

C-Trc khun hoc phy khun, di ng
D- Di ng nh tin mao mc cc, khng a mn
6.25- Chi Acetovibrio
DD- Di ng nh chu mao, a mn
6.26- Chi Haloanaerobacter
CC- T bo dng si, khng di ng
6.27- Chi Acetofilamentum
2- Sn phm ch yu ln men carbohydrat l acid lactic hoc
acid lactic , acid acetic v cc acid khc
B-Di ng, a mn
6.28- Chi Halocella
BB- Khng di ng, khng a mn
C-Sn phm ln men ch c acid lactic
6.29- Chi Leptotrichia
CC- Sn phm ln men gm acid lactic v cc acid khc
D-Sn phm ln men l acid lactic v acid acetic
E- T bo phnh to gia, khng sinh kh
6.30- Chi Sebaldella
EE- T bo hnh cong, sinh kh
6.31- Chi Lachnospira
DD- Sn phm ln men l acid lactic, acid acetic v acid succinic
E- T bo ln, ng knh ti 3mm
6.32- Chi Megamonas
EE- T bo < 1,5mm
6.33-Chi Mitsuokella
6.34- Chi Halella
3-Sn phm ch yu ln men carbohydrat l acid propionic
hoc acid propionic v cc acid khc
B-Sn sinh acid hu c v H
2
/CO
2

C-Khng di ng, a mn, cn NaCl 1%
D- Ch sn sinh acid propionic v CO
2

6.35- Chi Propionigenium
DD- Ngoi acid propionic v CO
2
cn c cc acid khc
E- Sn sinh acid propionic, CO
2
,acid acetic v ethanol
6.36- Chi Pelobacter
EE- Sn sinh acid propionic, CO
2 ,
acid aceic v acid butyric
6.37- Chi Haloanaerobium
CC- Di ng, khng a mn
D-T bo hnh cong, di ng nh tin mao cc hay bn
E- ng knh t bo 0.9mm
6.38- Chi Selenomonas
EE- ng knh t bo 0,5mm
6.39- Chi Anaerovibrio
DD-T bo hnh cong hay hnh xon, di ng nh chu mao
6.40- Chi Propionispira
BB- Sn sinh hn hp acid, acid propionic l chnh, khng sinh kh
C-Ch yu sn sinh acid propionic
D-T bo hnh que
6.41- Chi Oribaculum
DD-T bo cong hnh thn rn
6.42- Chi Centipeda
CC-Sn sinh acid propionic, acid acetic v acid succinic
D-Di ng
E- Tin mao dng lc mc bn, t bo hnh cong
6.43- Chi Pectinatus
EE-T bo dng cong hoc dng xon
6.44- Chi Zymophilus
DD- Khng di ng
E-ng knh t bo 3mm
6.32- Chi Megamonas
EE- ng knh t bo < 3m
F-T bo hnh que, ch sn sinh acid propionic v acid acetic
6.45 Chi Anaerorhabdus
FF- T bo hnh bin i, sn sinh acid propionic v cc acid khc
G-T bo nh, u nhn, sn sinh acid propionic v acid succinic
6.46- Chi Rikenella
GG- Kch thc t bo thay i, sn sinh acid propionic, acid
formic, acid acetic, acid lactic, v acid succinic
6.47- Chi Bacteroides
4- Sn phm ch yu ln men carbohydrat l acid butyric
hoc acid butyric v cc acid khc
B-Di ng nh tin mao mc cc hay gn cc, t bo hnh cong
C- Sn phm ln men ch l acid butyric
D- Di ng
6.48- Chi Pseudobutyrivibrio
DD- Khng di ng
6.49- Chi Fusobacterium
CC-Ngoi acid butyric cn c cc sn phm ln men khc
D-Sn sinh acid butyric v CO
2

6.50- Chi Roseburia
DD-Sn sinh acid butyric, acid acetic hoc acid lactic, khng sinh kh
6.51- Chi Butyrivibrio
BB-Di ng nh chu mao hoc khng di ng
C- Sn sinh acid butyric, acid acetic v ethanol, khng sinh kh, cn 1% NaCl
6.52- Chi Iliobacter
CC-Sn sinh acid butyric, acid acetic...v c sinh kh, cn 13% NaCl
6.37- Chi Haloanaerobium
5-Sn phm ln men carbohydrat l hn hp acid c cha
acid succinic hoc acid formic
B- Sn sinh hn hp acid c cha acid formic
C-Sn sinh axit formic v CO
2

6.53- Chi Oxalobacter
CC-Sn sinh acid formic v cc acid khc nhng khng sinh CO
2

D-T bo hnh que, sn sinh acid formic, acid acetic v acid succinic
6.54- Chi Ruminobacter
DD- T bo hnh dng thay i, sn sinh acid formic, acid acetic, acid succinic, acid
lactic v acid propionic
6.47- Chi Bacteroides
BB-Sn sinh hn hp acid c cha acid succinic
C- Di ng
D-T bo hnh que ngn, ch sn sinh acid succinic
6.55- Chi Succinimonas
DD- T bo hnh cong, sn sinh hn hp acid c cha acid succinic
E-Sn sinh acid succinic, acid acetic v c sinh CO
2

6.56- Chi Malonomonas
EE- Sn sinh acid succinic, acid acetic, khng sinh CO
2

6.57- Chi Succinovibrio
CC- Khng di ng
D-Sn sinh acid acetic, acid lactic v acid succinic
6.33- Chi Mitsuokella
DD-Trong hn hp acid khng c acid lactic
E-Sn sinh acid propionic v acid succinic
6.46- Chi Rikenella
EE- Sn sinh acid acetic v acid succinic
F- T bo hnh xon, di ng
6.58- Chi Anaerobiospirrillum
FF- Khng di ng
G-T bo hnh que ngn, ch yu sng d c v manh trng
6.59- Chi Fibrobacter
GG- T bo a hnh thi, ch yu sng khoang ming
6.60- Prevotella





Thermosipho
Syntrophobacter nui cy chung vi
Methanobrevibacte




Wolinella Porphyromonas gingivalis vt cy trn thch mu



Leptotrichia Bacteroides Bacteroides



Fusobacterium Fusobacterium




Mitsuokella

7- Cu khun Gram dng hiu kh v k kh khng bt buc
A- Hiu kh
B-Di ng
C-a mn bt buc, cn NaCl 7,5%
7.1- Chi Marinococcus
CC-Khng nh trn
7.2- Chi Planococcus
BB-Khng di ng
C-a mn, cn NaCl 7,5%
D- Lng cha GC > 50mol %
7.3- Chi Salinicoccus
DD- Lng cha GC < 50mol %
7.1- Chi Marinococcus
CC-Khng nh trn
D-Sinh acid t glucose
7.4- Chi Micrococcus
DD-Khng sinh hay sinh t acid t glucose
7.5- Chi Deinococcus
AA- K kh khng bt buc
B-T l G + C trong ADN < 30mol %
7.6- Chi Melissococcus
BB- T l G + C trong ADN > 50mol %
7.7- Chi Stomatococcus
BBB- T l G + C trong ADN > 30 mol % , nhng < 50mol %
C- Catalase (+)
D- Thnh t bo cha acid teichoic
7.8- Chi Staphylococcus
DD- Thnh t bo khngch acid teichoic
7.9- Chi Saccharococcus
CC- Catalase (-)
D- Ngoi xp thnh i cn c dng xp thnh bn
E- khng vi Vancomycin (30mg)
7.10- Chi Pediococcus
EE- Khng khng vi Vancomycin
7.11- Chi Aerococcus
DD- Khng c dng xp thnh bn
E-Xp thnh chui rt di
7.12- Chi Trichococcus
EE- Khng xp thnh chui di
F-Sinh trng 10

C
G- Sinh kh t glucose
7.13- Chi Leuconostoc
GG- Khng sinh kh t glucose
H- Sinh trng 45

C
7.14- Chi Enterococcus
HH- Khng sinh trng 45

C
I-Di ng, sng trong nc
7.15- Chi Vagococcus
II- Khng di ng
7.16- Chi Lactococcus
FF- Khng sinh trng 10

C
G-Lng cha G + C ca ADN>35mol %
7.17- Chi Streptococcus
GG-Lng cha G + C ca ADN< 35mol %
7.18- Chi Gemella


Micrococcus Deinococcus Deinococcus


Melissococcus Melissococcus Khun lc Stomatococcus


Staphylococcus Staphylococcus Pediococcus





Aerococcus Leuconostoc Leuconostoc



Enterococcus Lactococcus Lactococcus



Streptococcus Streptococcus Gemella

8- Trc khun Gram dng hiu kh hoc k kh khng bt buc

A-Hiu kh
B-C chu k bin ho hnh cu, hnh que
C-Thnh t bo c cha DAP (acid diaminopimelic)
D- C cha LL-DAP
E-Di ng
8.1- Chi Pimelobacter
EE- Khng di ng
8.2- Chi Terrabacter
DD- Khng cha dng LL- m cha dng Meso-DAP
E-Loi quinone l MK-8 (H
2
), MK-7 (H
2
)
8.3- Chi Brevibacterium
EE-Khng nh trn
8.4- Chi Brachybacterium
CC- Peptidoglycan khng cha DAP
D-Hnh que khng quy tc
8.5- Chi Arthrobacter
DD-Hnh que c quy tc
8.6- Chi Kurthia
BB- Khng c bin ho hnh cu, hnh que
C-C cha acid mycolic
8.7- Chi Caseobacter
CC-Khng cha axt mycolic
D-Sp xp thnh hnh ch V
E- Peptidoglycan c cha D-ornitin, quinone l MK-11,MK-12
8.8- Chi Aureobacterium
EE- Peptidoglycan cha L-ornitin, quinone l MK-8
8.9- Chi Sphaerobacter
DD-T bo khng sp xp thnh hnh ch V
E- Hnh que khng quy tc
F-Khng c dng t bo hnh cu hay hnh que ngn
G- Quinone l MK-8
8.10- Chi Rubrobacter
GG- Khng nh trn
H- Peptidoglycan cha D-ornitin
8.11- Chi Curtobacterium
HH- Khng nh trn
8.12- Chi Clavibacter
FF-C dng t bo hnh cu hay hnh que ngn
G-Peptidoglycan cha DAP
8.13- Chi Aeromicrobium
GG-Peptidoglycan khng cha DAP
8.14- Chi Microbacterium
EE- Hnh que c quy tc
8.15- Chi Renibacterium
AA- K kh khng bt buc
B-Catalase (+)
C- C cha DAP
D-C acid mycolic
8.16- Chi Corynebacterium
DD- Khng c acid mycolic
E-Sn sinh acid propionic
8.17- Chi Propionibacterium
EE- Khng sn sinh acid propionic
F-Hnh que khng quy tc
8.18- Chi Dermabacter
FF-Hnh que c quy tc
G-Chiu rng vi khun > 1,0mm
8.19- Chi Caryophanon
GG- Chiu rng vi khun < 1,0mm
H-Gy bnh ng vt
8.20- Chi Listeria
HH- Khng gy bnh ng vt
8.21- Chi Brochothrix
CC-Khng cha DAP
D-Peptidoglycan cha lysin
E- Di ng
F- Quinone l MK-7
8.22- Chi Exiguobacterium
FF- Quinone l MK-9
8.23 Chi Jonesia
EE- Khng di ng
8.24- Chi Rothia
DD Peptidoglycan khng cha lysin
E- Phn gii cellulose
8.25- Chi Cellulomonas
EE- Khng phn gii cellulose
8.26- Chi Rarobacter
BB- Catalase (-)
C-Peptidoglycan thuc nhm B (theo Schleiter v Kandier,1972)
D- Hnh que khng quy tc
8.27- Chi Agromyces
DD- Hnh que c quy tc
E- Thng c dng hnh si
8.28- Chi Erysipelothrix
EE-Khng c dng hnh si
8.29- Chi Carnobacterium
CC-Peptidoglycan khng thuc nhm B m thuc nhm A
D-Hnh que khng quy tc
E- Peptidoglycan cha LL-DAP
8.30- Chi Arachnia
EE- Peptidoglycan khng cha LL-DAP
F-Khng c menaquinone
8.31- Chi Gardnerella
FF- C menaquinone
G-C menaquinone MK-10 (H4)
8.32- Chi Actinomyces
GG- C menaquinone MK-9 (H4)
8.33 - Chi Arcanobacterrium
DD- Hnh que c quy tc
8.34- Chi Lactobacillus

Brevibacterium Arthrobacter Arthrobacter


Rubrobacter Aeromicrobium Renibacterium


Corynebacterium Propionibacterium Listeria



Cellulomonas Cellulomonas Agromyces


Actinomyces Lactobacillus Lactobacillus


9- Trc khun khng quy tc, khng bo t
A- Gram (+)
B- a nhit
C- T bo c phn nhnh
D-a kim
9.1- Chi Anaerobranca
DD- Khng a kim
9.2- Chi Thermobrachicum
CC- T bo khng phn nhnh
D-Di ng
9.3- Chi Thermoanaerobacter
DD-Khng di ng
9.4- Chi Thermoanaerobium
BB-a m
C-T bo hnh b dc, hnh que ngn hay hnh cong
D- T bo nh nhn hnh con, sp xp tng i hnh li lim
9.5- Chi Falcivibrio
DD- T bo hnh bu dc, hnh que ngn, khng sp xp thnh hnh li lim
E-Ln men hydrat carbon ch sn sinh acid acetic, cn c th tng hp ra acid
acetic t H
2
v CO
2

F- Thch hp sinh trng 30

C
9.6- Chi Acetobacterium
FF-Thch hp sinh trng 38

C
9.7- Chi Acetitomaculum
EE- Ln men hydrat carbon sinh acid lactic, khng tng hp ra acid acetic t H
2

v CO
2

9.8- Chi Atopobium
CC-T bo hnh que khng quy tc
D-T bo c phn thnh hnh na (inh ba), ln men hydrat
carbon khng sinh kh
9.9- Chi Bifidobacterium
DD- T bo khng phn thnh hnh na, ln men hydrat carbom c
sinh kh
E-Sn phm ln men ch yu l acid butyric, acid acetic hoc acid formic.
9.10- Chi Eubacterium
EE- Sn phm ln men ch yu l acid acetic, acid lactic v etanol
9.11- Chi Coribacterium
AA- Gram (-)
A- T bo hnh que, a nhit, khng di ng
9.12- Chi Acetogenium
BB- T bo dng cong hay dng si, di ng
C-Ln men hydrat carbon sn phm ch yu l acid butyric
9.13- Chi Butyrivibrio
CC-Ln men hydrat carbon sn phm ch yu khng l acid butyric
D-Ln men glucose, sn phm ch yu l acid acetic v acid lactic, khng sinh
kh
9.14- Chi Mobiluncus
DD- Ln men glucose, sn sinh acid formic, acid acetic, acid lactic v etanol, c
sinh kh
9.15- Chi Lachnospira

Thermoanaerobacter
di knh hin vi
in t
Bifidobacterium Eubacterium


Mobiluncus

10- Cu khun Gram dng k kh bt buc
A- T bo phn ct theo 3 mt phng thng gc, sp xp thnh hnh lp th
10.1- Chi Sarcina
AA- T bo phn ct theo 1 hoc 2 mt phng thng gc , sp xp thnh tng i
hay tng 4 t bo
B-Khng ln men hydrat carbon, khng sinh kh
C- T l G + C mol % trong ADN 50
10.2- Chi Peptococcus
CC- T l G + C mol % trong ADN 45
10.3- Chi Peptostreptococcus
BB- Ln men hydrat carbon sinh acid
C-Trong sn phm ln men ngoi cc acid hu c khc cn c acid butyric v
aceton (acetone)
10.4- Chi Coprococcus
CC- Trong sn phm ln men khng c acid butyric v aceton
10.5- Chi Ruminococcus




Peptostreptococcus Peptostreptococcus

Ruminococcus
11- Phn loi x khun
X khun l nhm vi khun c bit. Chng c khun lc kh v a s c
dng hnh phng x (actino-) nhng khun th li c dng si phn nhnh nh
nm (myces). V x khun c cu trc nhn nguyn thu nh mi vi khun
khc, chiu ngang ca si cng nh nh vi khun, cho nn c ti liu gi chng
l nm tia l khng hp l. X khun phn b rng ri trong t nhin. S lng
n v sinh khun lc (CFU- colony-forming unit) x khun trong 1g t thng
t ti hng triu. Trn mi trng c a s x khun c hai koi khun ty:
khun ty kh sinh (aerial mycelium) v khun ty c cht (substrate mycelium).
Nhiu loi ch c khun ty c cht nhng cng c loi (nh chi Sporichthya) li
ch c khun ty kh sinh. Gia khun lc thng thy c nhiu bo t mng
mng gi l bo t trn (conidia hay conidiospores). Nu bo t nm trong bo
nang (sporangium) th c gi l nang bo t hay bo t kn
(sporangiospores). Bo t x khun c sinh ra u mt s khun ty theo
kiu hnh thnh cc vch ngn (septa). Cc chui bo t trn c th ch l 1
bo t (nh Thermoactinomyces, Saccharomonospora, Promicromonospora,
Micromonospora, Thermomonosspora...),c th c 2 bo t (nh
Microbispora), c th l chui ngn (nh Nocardia, Pseudonocardia,
Streptoverticillium, Sporichthya, Actinomadura, Microtetraspora,
Streptoalloteichus, Glycomyces, Amycolata, Amycolatopsis, Catellatospora,
Microellobosporia...), c th l chui di (nh Streptomyces,
Saccharopolyspora, Actinopolyspora, Kibdelosporangium, Kitasatosporia,
Saccharothrix, nhiu loi Nocardia, Nocardioides, Pseudonocardia,
Amycolatopsis, Streptoverticillium...), c th cc bo t trn nm trn b si
(synnema), tng t b si ca nm (nh Actinosynnema, Actinomadura...).
Cc chui bo t c th thng, c th xon, c th dng ln sng, c th
mc n hay mc vng... Cc cung sinh bo t (sporophore) v cung sinh
nang bo t (sporangiophorres) c th ring r, c th phn nhnh. Cc c
im hnh thi ny rt quan trng khi tin hnh nh tn x khun.
X khun thuc nhm vi khun Gram dng, thng c t l GC trong
ADN cao hn 55%. Trong s khong 1000 chi v 5000 loi sinh vt nhn s
cng b c khong 100 chi v 1000 loi x khun. X khun phn b ch yu
trong t v ng vai tr rt quan trng trong chu trnh tun hon vt cht
trong t nhin. Chng s dng acid humic v cc cht hu c kh phn gii
khc trong t. Mc d x khun thuc nhm sinh vt nhn s nhng chng
thng sinh trng di dng si v thng to nhiu bo t. Thm ch mt s
loi x khun cn hnh thnh ti bo t nh chi Streptosporangium,
Micromonospora v bo t di ng nh chi Actinoplanes, Kineosporia.
Trc y, v tr phn loi ca X khun lun l cu hi gy nhiu tranh
lun gia cc nh Vi sinh vt hc ,do n c nhng c im va ging Vi
khun va ging Nm. Tuy nhin, n nay, X khun c chng minh l
Vi khun vi nhng bng chng sau y:
1. Mt s x khun nh cc loi thuc chi Actinomyces v Nocardia rt
ging vi cc loi vi khun thuc chi Lactobacillus v Corynebacterium.
2. X khun ging vi khun ch khng c nhn tht, chng ch cha
nhim sc cht phn b dc theo cc si hoc cc t bo.
3. ng knh ca si x khun v bo t ging vi vi khun. ng
thi si x khun thng khng cha vch ngn.
4. X khun l ch tn cng ca cc thc khun th ging nh vi khun,
trong khi , nm khng b tn cng bi thc khun th.
5. X khun thng nhy cm vi cc khng sinh c tc dng ln vi
khun, nhng li thng khng vi nhng khng sinh tc dng ln nm
nh cc polyen.
6. X khun khng cha chitin, cht c mt trong si v bo t ca nhiu
nm, m khng c vi khun. ng thi ging nh phn ln vi khun,
x khun khng cha cellulose.
7. Tng t vi vi khun, x khun nhy cm vi phn ng acid ca mi
trng, c im ny khng c nm.
8. Cc c im v si v nang bo t kn(sporangium) ca chi
Actinoplanes cho thy c th chi ny l cu ni gia vi khun v cc nm
bc thp.
X khun thuc v lp Actinobacteria, b Actinomycetales, bao gm 10
di b, 35 h, 110 chi v 1000 loi. Hin nay, 478 loi c cng b thuc
chi Streptomyces v hn 500 loi thuc tt c cc chi cn li v c xp vo
nhm x khun him.







Khun lc x khun Khun ty x khun v bo t


Chui bo t trn cung sinh bo t dng xon


Bo t x khun


S hnh thnh hai loi khun ty sau khi bo t x khun ny mm


Si bo t v chui bo t trn

Mt s dng bo t x khun (SV t ch thch tng hnh)

Mt s dng nang bo v nang bo t x khun


Mt s dng chui bo t x khun (SV t ch thch tng hnh)

c im ca khun ty kh sinh v khun ty c cht c trnh by trong s
sau y:







phn loi x khun ngi ta s dng cc tiu chun nh trnh t
rADN 16S, lai ADN, hnh thi, sinh l sinh ha v ha phn loi. Hin nay, i
a s cc nh khoa hc ng vi quan nim hai chng c coi l hai loi
ring bit nu chng ging nhau di 70% khi tin hnh lai ADN. Keswani v
cng s chng minh rng nu s tng ng gia hai trnh t rADN 16S l
98.6% th xc sut mc ging nhau trong php lai ADN thp hn 70% s
l 99%. V th gi tr tng ng 98.6% ca trnh t rADN 16S c coi l
ngng phn bit hai loi khc nhau. Tuy nhin, cng c nhiu nh khoa
hc ly gi tr ny l 98%.
c bit ha phn loi l rt quan trng trong vic phn loi x khun.
Chng rt c ch trong phn loi mc n chi. l nhng c im sau:
ng, loi acetyl, acid mycolic trong thnh t bo, menaquinone,
phospholipid, acid bo v t l GC trong ADN.
1= Chi; 2=Loi thnh t bo ; 3=Thnh phn ng ; 4=Loi peptido glycan ;
5=Acid mycolic
6=Thnh phn acid bo ; 7=Mena quinon ch yu ; 8=Loi phospho-lipid ; 9=T l
mol GC trong ADN (%)
1 2 3
4 5
6
7
8 9
H Corynebacteriaceae v mt s h lin quan
Corynebacterium IV A
A1
+ 1a 8(H
2
), 9(H
2
) PI
51-
67
Turicella IV A - 1b 10,11
65-
72
Gordonia IV A A1 + 1b 9(H
2
) PII
63-
69
Williamsia IV A + 1b 9(H
2
) PII
64-
65
Skermania IV A + 1b 8(H
4
) PII 68
Dietzia IV A + 8(H
2
) PII 73
Mycobacterium IV A A1 + 9(H
2
) PII
62-
70
Nocardia IV A A1 + 1b 8(H
4
) PII
64-
72
Rhodococcus IV A A1 + 1b 8(H
2
), 9(H
2
) PII
60-
69
Tsukamurella IV A A1 + 9 PII
66-
73
H Micromonosporaceae
Micromonospora II D A1 - 3b
9(H
4.6
),
10(H
4,6
)
PII
71-
72
Actinoplanes II D A1 - 3b
9(H
4,6
),
10(H
4,6
)
PII
72-
73
Cattelatospora II D -
9(H
4,6
),
10(H
8,6
)
PII
71-
73
Catenuloplanes VI D A3 - 10(H
4
), 11(H
4
) PIII
71-
73
Couchioplanes VI D A3 - 9(H
4
) PII
69-
72
Dactylosporangium II D A1 - 3b 9(H
4,6,8
) PII
69-
73
Pilimelia II D A1 - 2d 9(H
4,2
) PII 71
Spirilliplanes II - 2d 10(H
4
) PII 69
Verrucosispora II A1 - 2b 9(H
4
) PII 70
H Propionibacteriaceae v Nocardia
Propionibacterium I A3 - 9(H
4
)
57-
63
Friedmaniella I A3 - 9(H
4
) PI 73
Luteococcus I A3 - 1a 9(H
4
) PI
66-
68
Microlunatus I A3 - 2d 9(H
4
) PII 68
Propioniferax I A3 - 9(H
4
), 7(H
4
) PI
59-
63
Tessaracoccus I A3 - 8(H
4
) PI 74
Nocardioides I A3 - 3a 9(H
4
) PI
67-
72
Aeromicrobium I A3 - 3a 9(H
4
) PII
69-
72
Hongia I A3 - 2d 9(H
4
) PI 71
Kribbella I A3 - 9(H
4
) PIII
68-
70
Marmoricola I A3 - 1a 8(H
4
) PI 72
H Pseudonocardiaceae
Pseudonocardia III A A1 - 1 8(H
4
)
PIII/
PII
68-
79
Actinobispora IV A A1 - 7(H
2
), 9(H
2
) PIV 71
Actinopolyspora IV A A1 - 2c 9(H
4
), 10(H
4
) PIII
64-
68
Amycolatopsis III A A1 - 9(H
2,4,6
) PII
66-
69
Kibdelosporangium III A - 3c 9(H
4
) PII 66
Prauserella IV A - 3f 9(H
2,4
) PII
67-
69
Saccharomonospora III A A1 - 2a 9(H
4
) PII
69-
74
Saccharopolyspora IV A A1 - 2c 9(H
4
) PIII
70-
77
Thermocrispum III C - 9(H
4
) PII
69-
73
H Actinosynnemataceae
Actinosynnema III C A1 - 9(H
4,6
) PII
69-
71
Actinokineospora III C A1 - 9(H
4
) PII 73
Kutzneria III C - 9(H
4
) PII
70-
71
Saccharothrix III C A1 - 9(H
4
), 10(H
4
) PII 70-
76
H Streptosporangiaceae
Streptosporangium III B A1 - 3c 9(H
0,2,4
) PIV
69-
71
Acrocarpospora III B A1 - 9(H
2,4
)
PIV/
PII
68-
69
Herbidospora III B - 10(H
4,6
) PIV 71
Microbispora III B A1 - 3c 9(H
0,2,4
) PIV
67-
74
Microtetraspora III B A1 - 3c 9(H
0,2,4
) PIV 66
Nonomuraea III B A1 - 3c 9(H
0,2,4
) PIV
64-
69
Planobispora III B A1 - 3c 9(H
0,2,4
) PIV
70-
71
Planomonospora III B A1 - 3c 9(H
0,2,4
) PIV 72
Planotetrespora III B - 3c PIV
H Nocardiopsaceae
Nocardiopsis III C A1 - 3d 10(H
2,4,6
) PIII
64-
69
Thermobifida III C - 3c 10(H
6,8
) PII
H Thermomonosporaceae
Thermomonospora III B A1 - 3e 9(H
6,8
) PI
Actinocollaria III - 3e 9(H
4,6
) PII 73
Actinomadura III B A1 - 3a 9(H
6,8
) PI
66-
70
Spirillospora III B A1 - 3e 9(H
4,6
) PI
69-
71
H Glycomycetaceae
Glycomyces II - 2c
10(H
4
),
11(H
4
)
PI
71-
73
H Streptomycetaceae
Streptomyces
I
A3 - 2c 9(H
6,8
) PII
69-
78
Kitasatospora I/III A3 - 9(H
6,8
) PII
66-
73
H Frankiaceae v mt s h lin quan
Frankia III - 1 9(H
4,6,8
) PI
66-
71
Geodermatophilus III - 2b 9(H
4
) PII
73-
76
Microsphaera III A1 - 8(H
4
) 68
Cryptosporangium II - 9(H
6,8
) PII 70
Sporichthya I A3 - 9(H
6,8
) PI 70
Cc h khc
Kineosporia I/III A3 9(H
4
) PIII
64-
79
Thermobispora III - 9(H
0,2
) PIV 71
Thermoactinomyces III - 7,9
53-
55

Cc s liu trn c ly t sch Bergeys manual systematic of bacteriology v sch
Identification manual of actinomycetes.
Thnh t bo: Theo Lechevalier v Lechevalier, thnh t bo x khun c chia thnh 8
loi da trn cc c im v thnh phn acid amin, c bit l acid diaminopimelic, lysine v thnh
phn ng trong thnh t bo, c th l
Loi thnh
t bo
Acid
diamino
Cc acid amin khc ng
I LL-A
2
pm Gly*
II
meso-
A
2
pm
Gly**
III
meso-
A
2
pm

IV
meso-
A
2
pm
Ara, Gal***
V Lys, Orn
VI Lys Asp
VII Lys, A
2
bu Asp
VIII Asp

Tt c cc loi thnh t bo u cha alanine, acid glutamic, glucosamine v acid muramic.
A
2
pm: Acid diamino pimelic c th gm cc ng phn quang hc LL- A
2
pm, DL- A
2
pm
(meso- A
2
pm), DD- A
2
pm. Ngoi ra cn c OH- A
2
pm khi c thm nhm OH gn vo v tr C th
4.
Lys: Lysine
Orn: Ornitin
A
2
bu: acid diamino butyric
Gly: glycin
*Glycin nm trong mi lin kt gia hai mch tetrapeptid
** Glycin thay th L- alanin v tr u tin trong mch tetrapeptid
Asp: Asparagin
Ara: Arabinose
Gal: Galactose
*** Trong x khun cha acid mycolic nh Nocardia v Mycobacterium, thnh t bo cha
arabinogalactan, l hp cht c tng hp t arabinose v galactose.
Thnh phn ng:
Thnh phn
ng
Cc loi ng ch yu
A
Arabinose, Galactose (khng cha
Xylose)
B
Madurose (khng cha Arabinose hay
Xylose)
C Khng c ng c trng
D Arabinose, Xylose

Peptidoglycan: Theo Schleifer v Kandler (1972), peptidoglycan x
khun c chia thnh cc nhm nh sau:
K
hiu
V tr
ca mi
lin kt
K
hiu
Cu ni gia hai mch
tetrapeptid
K
hiu
Acid amin
v tr th
3
A 3-4 1 Trc tip L-Lys
L-Orn
meso-
A
2
pm
2 Tiu n v peptid L-Lys
3
L-acid amin cha mt nhm
carboxyl (gly) hoc chui peptid
ngn ca acid amin
L-Lys
L-Orn

meso-
A
2
pm
4
D-acid amin cha hai nhm
carboxyl (D-Asp hay D-Glu)
L-Lys
L-Orn

meso-
A
2
pm
L-A
2
bu
B 2-4 1 L-acid amin (Gly
n
-L-Lys) L-Lys
L-Hsr
L-Glu
L-Ala
2
D-acid amin (D-Orn hay D-
A
2
bu)
L-Orn
L-Hsr
L-A
2
bu
Du phy (): alanine v tr th nht trong mch tetrapeptid c
thay th bi glycine.
V d: Loi peptidoglycan ca chi Actinoplanes l A1 c ngha l: Hai
mch tetrapeptid lin kt vi nhau v tr acid amin s 3 v 4, mi lin kt ny
l trc tip v acid amin v tr s 3 l meso- A
2
pm.
Thnh phn acid bo:
K
hiu

Bo
ha
Khng
bo
ha
Iso-
14/16/18
Iso-
15/17
Anteiso-
15/17
10-
Methyl-
17
10-
Methyl-
18
Cyclopropane
1 a +++ +++ - - - - - -
b +++ +++ - - - - ++ -
c +++ +++ - - - - - ++
2 a ++ + +++ + (+) - - -
b (+) + ++ +++ + - - -
c + (v) +++ + +++ - - -
d + + +++ +++ +++ - - -
3 a +++ ++ +++ (+) (+) (+) +++ -
b + + +++ +++ ++ ++ (+) -
c + + ++ + + +++ (+) -
d + + +++ ++ +++ (+) +++ -
(v): t hn 5%

Thnh phn menaquinone:
X khun ch cha menaquinone, khng cha ubiquinone. K hiu 8(H
2
) hay MK-8(H
2
) ch
menaquinone cha mt trong s tm n v isoprene b hydro ha.
Thnh phn phospholipid:
K
hiu
Thnh phn phospholipid c trng
PI Phosphatidylglycerol(v)
PII Phosphatidylethanolamine
PIII
Phosphatidylcholine, phosphatidylethanolamine(v),
phosphatidylmethyl-ethanolamine(v),
phosphatidylglycerol(v), khng cha phospholipid cha
glucosamine
PIV
Phospholipid cha glucosamine,
phosphatidylethanolamine(v),
phosphatidylmethylethanolamine(v)
PV Phospholipid cha glucosamine, phosphatidylglycerol
Tt c cc loi u cha phosphatidylinositol

V tr phn loi ca X khun:
Lp Actinobacteria
Di lp Actinobacteridae
B Actinomycetales
Di b Actinomycineae
H Actinomycetaceae
Cc chi: Actinomyces, Actinobaculum,
Arcanobacterium, Mobiluncus
Di b Micrococcineae
H Micrococcaceae
Cc chi: Micrococcus, Arthrobacter, Kocuria,
Nesterenkonia, Renibacterium, Rothia
H Bogoriellaceae
Chi: Bogoriella
H Brevibacteriaceae
Chi: Brevibacterium
H Cellulomonadaceae
Chi: Cellulomonas
H Dermabacteraceae
Cc chi: Dermabacter, Brachybacterium
H Dermacoccaceae
Cc chi: Dermacoccus, Demetria, Kytococcus
H Dermatophilaceae
Chi: Dermatophilus
H Intrasporangiaceae
Cc chi: Intrasporangium, Janibacter, Terrabacter,
Terracoccus, Tetraphaera
H Jonesiaceae
Chi: Jonesia
H Microbacteriaceae
Cc chi: Microbacterium, Agrococcus, Agromyces,
Clavibacter, Cryobacterium, Curtobacterium,
Frigoribacterium, Leifsonia, Leucobacter, Rathayibacter
H Promicromonosporaceae
Chi: Promicromonospora
H Rarobacteraceae
Chi: Rarobacter
H Sanguibacteraceae
Chi: Sanguibacter
Incertae sedis (cha xc nh)
Cc chi : Beutenbergia, Ornithinicoccus, Kineococcus
Di b Corynebacterineae
H Corynebacteriaceae
Cc chi: Corynebacterium, Turicella
H Dietziaceae
Chi: Dietzia
H Gordoniaceae
Chi: Gordonia
H Mycobacteriaceae
Chi: Mycobacterium
H Nocardiaceae
Cc chi: Nocardia, Rhodococcus
H Tsukamurellaceae
Chi: Tsukamurella
Incertae sedis (cha xc nh)
Cc chi : Skermania, Williamsia
Di b Micromonosporineae
H Micromonosporaceae
Cc chi: Micromonospora, Actinoplanes, Catellatospora,
Catenuloplanes, Couchioplanes, Dactylosporangium,
Pilimelia, Spirilliplanes, Verrucosispora
Di b Propionibacterineae
H Propionibacteriaceae
Cc chi: Propionibacterium, Friedmaniella, Luteococcus,
Microlunatus, Propioniferax, Tessaracoccus
H Nocardioidaceae
Cc chi: Nocardioides, Aeromicrobium, Hongia,
Kribbella, Marmoricola
Di b Pseudonocardineae
H Pseudonocardiaceae
Cc chi: Pseudonocardia, Actinobispora,
Actinopolyspora, Amycolatopsis, Kibdelosporangium,
Prauserella, Saccharomonospora, Saccharopolyspora,
Thermocrispum
H Actinosynnemataceae
Cc chi: Actinosynnema, Actinokineospora,
Saccharothrix
Incertae sedis (cha xc nh)
Cc chi: Kutzneria, Streptoalloteichus
Di b Streptosporangineae
H Streptosporangiaceae
Cc chi: Streptosporangium, Acrocarpospora,
Herbidospora, Microbispora, Microtetraspora,
Nonomuraea, Planomonospora, Planotetraspora
H Nocardiopsaceae
Cc chi: Nocardiopsis, Thermobifida
H Thermomonosporaceae
Cc chi: Thermomonospora, Actinocorallia,
Actinomadura, Spirillospora
Di b Glycomycineae
H Glycomycetaceae
Chi: Glycomyces
Di b Streptomycineae
H Streptomycetaceae
Cc chi: Streptomyces, Kitasatospora
Di b Frankineae
H Frankiaceae
Chi: Frankia
H Acidothermaceae
Chi: Acidothermus
H Geodermatophilaceae
Cc chi: Geodermatophilus, Blastococcus
H Microsphaeraceae
Chi: Microsphaera
H Sporichthyaceae
Chi: Sporichthya
Incertae sedis (cha xc nh)
Chi: Cryptosporangium
B Bifidobacteriales
H Bifidobacteriaceae
Cc chi: Bifidobacterium, Gardnerella
Di lp Acidimicrobidae
B Acidimicrobiales
H Acidimicrobiaceae
Chi: Acidimicrobium
Di lp Coriobacteridae
B Coriobacteriales
H Coriabacteriaceae
Cc chi: Coriobacter, Atopobium, Collinsella,
Cryptobacterium, Denitrobacterium, Eggerthella,
Slackia
Di lp Rubrobacteridae
B Rubrobacteriales
H Rubrobacteriaceae
Chi: Rubrobacter
Di lp Sphaerobacteridae
B Sphaerobacteriales
H Sphaerobacteriaceae
Chi: Sphaerobacter

M t mt s chi thng gp:
Chi Rhodococcus:
Pht trin di dng que hoc khun ty c cht phn nhnh. tt c cc chng, chu trnh sng
(morphogenetic) u bt ngun t giai on hnh cu hoc que ngn. Bng cch phn on, cc t
bo hnh cu s to thnh dng que ri dng si, si phn nhnh v h si. Mt s chng cn to
khun ty kh sinh phn nhnh hoc b si (synnemata). Chng khng c kh nng chuyn ng
cng nh khng hnh thnh bo t hay ni bo t.
Gram dng. Acid-alcohol fast (nhum khng acid-cn) mt s giai on pht trin. Hiu kh.
Ha d dng hu c. Catalase dng tnh. Hu ht cc chng u mc tt trn cc mi trng tiu
chun 30
o
C, s khc cn thiamin cho sinh trng. Khun lc c th sn si hoc trn nhn, c
mu vng sm, kem, vng, vng da cam, hoc khng mu. Arylsulfatase m tnh, nhy cm vi
lysozyme, khng phn hy c casein, cellulose, chitin, elastin hay xylan. C th s dng c rt
nhiu loi hp cht hu c lm ngun cacbon v ngun nng lng.
Thnh t bo cha lng ln acid meso- diaminopimelic (meso-DAP), arabinose v galactose. Cha
diphosphatidylglycerol, phosphatidylethanolamin, phosphatidylinositol mannoside. Thnh phn
menaquinone chnh l MK-8(H
2
) v MK-9(H
2
). Cha lng ln acid tuberculostearic mch thng,
khng bo ha v acid mycolic vi 32-66 cacbon, vi nhiu nht bn lin kt i. T l mol G+C
trong ADN l 63-72%.
C nhiu trong t v phn gia sc. Mt s chng gy bnh cho ngi v ng vt.
Loi chun: Rhodococcus rhodochrous

Chi Nocardioides:
Si s cp phn nhnh trn b mt, sau xm nhp vp trong thch ri t on thnh dng que
hay cu hay khng u. Si kh sinh c th tha tht, phn nhnh hoc khng sau cng t thnh
nhng mu ngn hoc dng que. Nhng mu ny s l ngun gc ca nhng si mi. Khng c t
bo di ng. Khun lc nho. Gram dng. Khng nhum khng acid ( Non-acid fast ). Catalase
dng tnh. Hiu kh bt buc. Ha d dng hu c. Mc d dng trn cc mi trng tiu chun.
Nhy cm vi thc khun th c hiu. Acid amin chnh trong thnh t bo l L-DAP v glycine.
Khng cha acid mycolic. Phospholipid ch yu l phosphatidylglycerol v
acylphosphatidlyglycerol. Acid bo chim u th l 14-methylpentadecanoic. Thnh phn
menaquinone chnh l MK-8(H
4
). T l mol G+C trong ADN t 66,1-72,7%. C nhiu trong t.
Loi chun: Nocardioides albus

Chi Pseudonocardia:
Si kh sinh v si c cht u sinh bo t dng chui. Si phn on, thng c dng zich-zac vi
xu hng phng ln ngn hoc gia. Si ko di bng cch ny chi. Cc on si c chc
nng ca bo t hoc bin i thnh bo t. Thnh si c hai lp. Gram dng. Khng c giai on
di ng. Hiu kh. Sinh trng trn nhiu loi mi trng hu c hoc tng hp. a m hoc a
nhit.
Loi chun: Pseudonocardia thermophila

Chi Saccharopolyspora:
Si c cht pht trin mnh, phn nhnh, t thnh cc on dng que vi kch thc 1x5m, ch
yu nhng phn gi hn ca khun lc. Khun ty kh sinh phn on to cc chui bo t. Gram
dng. Khng nhum khng acid. Hiu kh. Khun lc mng, nh ln, hi nhn, si kh sinh t,
thng to thnh chm, ch yu nhng phn gi. C th s dng nhiu loi cht hu c nh
ngun cacbon v nng lng duy nht, c th phn gii adenine, khng nhiu loi cht khng sinh
nhng nhy cm vi lysozym. T l mol GC trong ADN l 77%.
Loi chun: Saccharopolyspora hirsute

Chi Intrasporangium:
Si phn nhnh, c xu hng t thnh nhiu on c kch thc v hnh dng khc nhau. Khng
bao gi c si kh sinh. Cc ti bo t hnh oval hay hnh qu chanh c to thnh gia hoc/v
u si. Cc bo t khng di ng. Gram dng. Khng nhum khng acid. Ha d dng hu
c. Catalase dng tnh. Hiu kh. Mc tt nht 28-37
o
C, khng mc c 45
o
C. Sinh trng
tt hn trn cc mi trng cha peptone v cao tht. T l mol GC trong ADN l 68,2%. Thnh t
bo cha L-DAP v glycine. Thnh phn phospholipid ch yu l mt loi phospholipid cha
glucosamin cha bit. Thnh phn acid bo ch yu l cc acid bo mch thng bo ha v khng
bo ha. Menaquinone chnh l MK-8.
Loi chun: Intrasporangium calvum

Chi Actinopolyspora:
H si phn nhnh, hnh thnh rt nhiu si kh sinh c ng knh khong 1 m. Si c cht hu
ht khng t on. Cung sinh bo t cha 20 hoc hn bo t dng que ngn hoc dng cu vi
v nhn hnh thnh trn si kh sinh theo chiu hng gc. Si c cht khng sinh bo t. Gram
dng. Nhum khng acid. Thnh t bo cha meso-DAP, arabinose v galactose. Khng cha acid
mycolic. Hiu kh. Ha d dng hu c. T l mol GC trong ADN l 64,2%.
Loi chun: Actinopolyspora halophila

Chi Saccharomonospora:
Ch yu sinh bo t n trn si kh sinh. Bo t khng bn nhit, khng c kh nng di ng,
c hnh thnh trn cc cung sinh bo t n gin, khng phn nhnh, di ngn khc nhau. Trn
mi trng thch, h si dinh dng phn nhnh, bao ph bi lp si kh sinh vi cc bo t mc
dy c dc trn cc si. Si kh sinh ban u c mu trng ri chuyn thnh xm xanh v xanh
sm. Sc t xanh cng c si dinh dng v khuch tn ra mi trng. Thnh t bo cha meso-
DAP, arabinose v galactose. Khng cha acid mycolic. Cha lng ln iso- v anteiso- acid bo,
phosphatidylethanolamin v MK-9(H
4
). Hiu kh. Ha d dng hu c. Nhit thch hp cho
sinh trng l 35-50
o
C, pH 7-10. Sinh catalase, deaminase v phosphatase. Phn hy c casein,
gelatin, tinh bt. xylan v tyrosin. c bit c th s dng glycerol lm ngun cacbon.
Phn lp c trong t, cht lng cn h, than bn, thy nhiu trong phn bn, phn compt, v
c kh. T l mol GC trong ADN l 69-74%.
Loi chun: Saccharomonospora viridis

Chi Frankia:
Khng sinh si kh sinh. Ti bo t thng sinh trn cung sinh bo t. Bo t khng c kh nng
di ng vi hnh dng khng c nh, t khng mu n mu en. Trong iu kin kh khn nh
khng c qu trnh c nh m, cc ti (vesicle) c th c hnh thnh. Gram dng- hoc Gram
khng c nh (variable). Hiu kh hoc vi hiu kh. Catalase dng tnh. a m. Ha d dng hu
c. Thng mc rt chm (thi gian gia hai ln phn i t bo l 1-7 ngy). Hu ht cc chng
u c kh nng c nh nit khng kh invitro v in planta. Thnh t bo cha meso-DAP, acid
glutamic, alanin, acid muramic, v glucosamine. Thnh phn phospholipid gm
phosphatidylinositiol mannoside, phosphatidylinositol v diphosphatidylglycerol. Acid bo dng
thng, mch thng, v khng bo ha. Thnh phn ng gm xylose, madurose hoc fucose,
hoc ch gm glucose hoc galactose. Nhiu chng gm 2-O-methyl-D-mannose, hu ht cc chng
cha rhamnose. Hu ht cng sinh vi mt s thc vt ht kn nht nh, to cc nt sn trn r
vt ch thch hp. C th tm trong t. T l mol GC trong ADN l 66-71%.
Loi chun: Frankia alni

Chi Actinoplanes:
Pht trin di dng si phn nhnh, khng t on. Gram dng, nhng mt phn si dinh
dng c th l Gram m. Khng nhum khng acid. Rt t si kh sinh hoc khng c. To nhiu
loi sc t c kh nng khuch tn. Bo t cha trong ti bo t, sinh trn cung sinh bo t hoc
khng cung, t khi trong thch. Di iu kin nht nh, nhiu chng c h si sp xp dng que
(palisade). Khi , ti bo t ch yu c sinh u cc si. Bo t hnh cu hoc que ngn, sp
xp theo nhiu cch khc nhau bn trong ti bo t, c hnh thnh bng cch t on si bn
trong ti bo t trc tip hoc sau vi ln phn nhnh. Sau khi ngm trong nc, bo t di ng
c gii phng ra t ti bo t, trong mt s trng hp, kh nng di ng xut hin sau khi bo
t c gii phng. Bo t di ng cha tin mao cc. Thnh t bo cha meso-DAP v glycin.
Thnh phn ng cha D-xylose v L-arabinose. Thnh phn phospholipid chnh l
phosphatidylethanolamin. Hiu kh, ha d dng hu c, a m hoc a nhit va phi. Hu ht
cc chng u khng cn cc nhn t sinh trng hu c. T l mol GC trong ADN l 72-73%.
Loi chun: Actinoplanes philippinensis

Chi Pilimelia:
Ti bo t c sinh ra trn b mt ca c cht t cung sinh bo t. Ti bo t c hnh cu, hnh
trng, hnh qu l, hnh chung hay hnh tr, c kch thc xp x 10-15 m. Ti bo t cha rt
nhiu bo t xp thnh chui song song hoc cc hng cun khng u. Gram dng. Khun ty c
cht c ng knh 0,2-0,8 m, phn nhnh v c vch ngn. Khun ty kh sinh tht s khng c
hnh thnh. Thnh t bo cha meso-DAP v glycin. Thnh phn ng cha xylose v arabinose.
Ch mc c trn mi trng hn hp. Khun lc nh, c hoc mm. Khun ty c cht c mu
vng chanh, vng, da cam hoc xanh xm, chuyn sang nu n nu sm khi gi. Hiu kh, ha t
dng hu c, iu kin sinh trng ti u l pH 6,5-7,5 v 20-30
o
C. C kh nng phn hy c
cht cheratin (lng tc ca ng vt c v). T l mol GC trong ADN l 72-73%.
Loi chun: Pilimelia terevasa

Chi Dactylosporangium:
Ti bo t hnh ngn tay hoc hnh chy c hnh thnh t cung sinh bo t ngn ca khun ty
c cht. Cc ti bo t pht trin n l hoc t thnh tng m trn b mt c cht. Mi ti bo t
cha mt dy gm ba n bn bo t. Bo t c hnh ch nht, hnh elip, hnh trng hoc hi tr,
c kh nng di ng nh mt tm tin mao cc. Khng c khun ty kh sinh thc s. Khun ty c
cht c ng knh 0,5-1,0 m, phn nhnh v t khi c vch ngn. Gram dng , Khng nhum
khng acid. Thnh t bo cha meso-DAP v glycin. Thnh phn ng cha xylose v arabinose.
C kh nng sinh trng trn nhiu loi mi trng. Khun lc c, hi th, thng phng, i khi
nh ln mt b mt trn hoc hi nhn. Khun ty c cht c mu vng xanh, da cam, hay nu.
Hiu kh, ha t dng hu c, iu kin sinh trng ti u l pH 6,0-7,0 v 25-37
o
C. T l mol
GC trong ADN l 71-73%.
Loi chun: Dactylosporangium aurantiacum

Chi Micromonospora:
C h si sinh trng mnh, phn nhnh, c vch ngn, ng knh trung bnh 0,5 m. Bo t
khng c kh nng di ng mc trc tip hoc t cung sinh bo t. Khng c khun ty kh sinh.
Gram dng, khng nhum khng acid. Thnh t bo bo cha meso-DAP, v/ hoc acid 3-
hydroxy diaminopimelic, glycin. Thnh phn ng cha xylose v arabinose. Thnh phn
phospholipid gm phosphatidylethanolamin, phosphatidylinositol v phosphatidylinositol
mannoside. Thnh phn menaquinone chnh l MK-9(H
4
), MK-10(H
4
), MK-10(H
6
) v MK-12(H
6
).
Hiu kh n vi hiu kh. Ha d dng hu c. Nhy cm vi pH di 6,0. C kh nng sinh
trng nhit t 20-40
o
C. T l mol GC trong ADN l 71-73%.
Loi chun: Micromonospora chalcea

Chi Streptomyces:
H si sinh dng phn nhnh nhiu ln, t khi t on, ng knh 0,5-2,0 m. Khun ty kh sinh
giai on trng thnh to chui t ba n nhiu bo t. Mt s t loi hnh thnh chui bo t
ngn trn khun ty c cht. Bo t khng c kh nng di ng. Cc khun lc ban u thng trn
nhn nhng sau khun ty kh sinh s pht trin rt mnh m. Sinh nhiu loi sc t khc nhau
cng nh sc t c kh nng khuch tn ra mi trng. Rt nhiu chng sn sinh ra mt hoc nhiu
loi cht khng sinh. Hiu kh, Gram dng, khng nhum khng acid-cn, ha d dng hu c,
catalase dng tnh. Thng c kh nng kh nitrate thnh nitrit, phn hy adenine, esculin, casein,
gelatin, hypoxanthine, tinh bt v L-tyrosine. C kh nng s dng nhiu hp cht hu c lm
ngun cacbon duy nht. Nhit sinh trng ti u l 25-35
o
C, pH ti u l 6,5-8,0. Thnh t bo
cha L-DAP, khng cha acid mycolic. Thnh phn acid bo gm phn ln acid bo bo ha, iso-
v anteiso-. Thnh phn menaquinone chnh l MK-9(H
6
), MK-9(H
8
). Thnh phn phospholipid
chnh l diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol v
phosphatidylinositol mannoside. C nhiu trong t, phn compt. Mt s loi gy bnh cho ngi
v ng vt, mt s loi gy bnh thc vt. T l mol GC trong ADN l 69-78%.
Loi chun: Streptomyces albus

Chi Actinomadura:
H si sinh dng phn nhnh, rt pht trin. Khun ty c cht khng t on, c hoc khng c
khun ty kh sinh. Khun ty kh sinh giai on trng thnh s hnh thnh chui ngn, i khi di
ca cc bo t t (arthrospore). Chui bo t c th dng thng, un cong hoc xon khng u
(1-4 vng). B mt bo t nhn hoc c cc nt. Khun ty kh sinh khi hnh thnh bo t c mu
trng, xm hay nu, vng, , xanh lc, xanh lam hay tm. Hiu kh, ha d dng hu c. Nhit
sinh trng t 20 n 45
o
C. Mt s loi a nhit. Gram dng. Thnh t bo cha meso-DAP,
khng cha acid mycolic. T bo cha madurose. Thnh phn menaquinone chnh l MK-9(H
4
) v
MK-9(H
6
). T l mol GC trong ADN l 65-69%.
Loi chun: Actinomadura madurae

Chi Microbispora:
Khun ty kh sinh phn nhnh, hnh thnh tng cp hai bao t gn vi nhau. Bo t mc trc tip
hoc trn cung sinh bo t ngn, hnh cu hoc oval, ng knh trung bnh 1,2-1,6m v khng
c kh nng di ng. Thnh t bo cha acid muramic, meso-DAP nhng khng cha ng c
trng. Thnh phn ng ca ton t bo cha madurose. Thnh phn menaquinone chnh l MK-
9(H
4
). Thnh phn phospholipid chnh cha phosphatidylcholine v phospholipid cha
glucosamine. Gram dng, hiu kh, ha d dng hu c. a m v a nhit. Hu ht cc loi
sinh trng u cn cc vitamin nhm B, c bit l thiamin. Trong t nhin thng tn ti trong
du. T l mol GC trong ADN l 71-73%.
Loi chun: Microbispora rosea

Chi Streptosporangium:
Ti bo t hnh cu ng knh khong 10m c hnh thnh trn khun ty kh sinh. Cc bo t
c hnh thnh bng cch hnh thnh vch ngn ca si cun xon khng phn nhnh bn trong ti
bo t. Bo t hnh cu, hnh oval hoc hnh que, khng c kh nng di ng. Thnh t bo cha
acid muramic, meso-DAP nhng khng cha ng c trng. Thnh phn ng ca ton t bo
cha madurose. Thnh phn phospholipid chnh cha phosphatidylcholine v phospholipid cha
glucosamine. Gram dng, hiu kh, ha d dng hu c. a m, mt s loi c kh nng chu
c nhit cao. Mt s loi cn cc vitamin nhm B cho sinh trng. T l mol GC trong ADN
l 69-71%.
Loi chun: Streptosporangium roseum

Chi Thermomonospora:
Khun ty kh sinh sn sinh ra cc bo t n l, bn nhit, khng c kh nng di ng. Bo t c th
sinh trc tip nhng thng l trn u ca cc cung sinh bo t c hoc khng phn nhnh. Bo
t cng c h c sinh ra t khun ty c cht. Gram dng. Thnh t bo cha meso-DAP nhng
khng cha cc loi acid amin hay ng c trng. Hiu kh, ha d dng hu c. Cn cung cp
ngun acid amin v cc vitamin cho sinh trng nh cao nm men. Tt c cc chng u c th
sinh trng c nhit 40-48
o
C, pH 7,0-9,0. pH > 8,0 thch hp cho s sn sinh khun ty kh
sinh v hnh thnh bo t. Sn sinh catalase, deaminase, -glucosidase v -galactosidase. C kh
nng phn gii esculin, xylan, casein, gelatin v cacboxymetylcellulose. Bo t cht 90
o
C trong
30 pht trong nc. Tt c cc chng u nhy cm vi novobiocin (50 g/ml). C th phn lp
c t t nhng tn ti nhiu trong phn bn, phn compt v c kh sy.
Loi chun: Thermomonospora curvata

Chi Actinosynnema:
Khun ty gm hai loi l khun ty c cht hnh thnh b si (synnemata) trn b mt thch v
khun ty kh sinh hnh thnh t b si. Khun ty sinh ra cc chui bo t. Cc bo t c kh nng
hnh thnh tin mao trong mi trng nc. Thnh t bo cha meso-DAP, acid glutamic, alanin,
glucosamin v acid muramic. T bo khng cha thnh phn ng c trng. Thnh phn
phospholipid chnh l phosphatidylinositol mannoside, phosphatidylinositol,
phosphatidylethanolamine v phosphatidylglycerol. Acid bo gm loi mch thng v mch nhnh.
Thnh phn menaquinon chnh l MK-9(H
4
) v MK-9(H
6
). Gram dng, khng acid fast, sinh
catalase. Hiu kh. a m. Ha d dng hu c. Hu ht cc chng c phn lp trc tip t m
thc vt nh l c bn b sng. T l mol GC trong ADN l 73%.
Loi chun: Actinosynnema mirum

Chi Nocardiopsis:
Khun ty c cht pht trin mnh, phn nhnh nhiu, c th t on thnh cc th hnh cu v hnh
que. Khun ty kh sinh di, phn nhnh khng u, thng, un hoc hnh zch zc, t on thnh
cc bo t vi chiu di khc nhau. Bo t hnh oval hoc ko di, b mt nhn. Gram dng, hiu
kh, ha d dng hu c, khng nhum khng axt. Thnh t bo cha meso-DAP, khng cha
ng c trng, khng c acid mycolic. Thnh phn menaquinon chnh l MK-10(H
2,4,6
) hoc
MK-9(H
4,6
). T l mol GC trong ADN l 64-69%.
Loi chun: Nocardiopsis dassonvillei
Di y l hnh nh mt s chi x khun thng gp:


Frankia cng sinh r cy
Phi lao
Nang bo


Nocardia- Khun lc v khun ty t on


Mt s loi Streptomyces


Mt s chi x khun: A-Microtetraspora; B- Planomonospora; C- Actinosynnema; D-
Actinobispora; E- Saccharothrix; F- Crosiella

Chi Actinomadura


Cc chi: A- Micromonospora;
B- Dactylosporangium; C- Verrucosispora
D- Pilimelia; E- Catenuplanes


Chi Actinoplanes


Loi Actinomyces viscosus


Trn y l quan h pht sinh chng loi ca khong 90 chi x khun khc nhau
Mt s phng php phn lp x khun:

Mi trng HV (Humic acid-vitamin agar)
Thnh phn:
Acid humic 1 g
CaCO
3
0,02 g
FeSO
4
.7H
2
O 0,01 g
KCl 1,71 g
MgSO
4
.7H
2
O 0,05 g
Na
2
HPO
4
0,5 g
Hn hp vitamin nhm B* 5 ml
Cycloheximide 500 mg
Thch 18 g
Nc ct 1 L
pH 7,2

*Hn hp vitamin nhm B
Thiamine-HCl 10 mg
Riboflavin 10 mg
Niacin 10 mg
Pyridoxin-HCl 10 mg
Inositol 10 mg
Ca-Pantothenate 10 mg
Acid p-Aminobenzoic 10 mg
Biotin 10 mg
Nc ct 10 ml

Mi trng YS (Yeast extract-Soluble starch agar)
Cao nm men 2 g
Tinh bt tan 10 g
Thch 18 g
Nc ct 1 L
pH 7,3

Thng s dng mi trng YS pha long 5 ln phn lp x khun v pha long 2 ln nghin
cu.

Mi trng Maltose-Bennette
Cao nm men 1 g
Cao tht 1 g
NZ amine 2 g
Maltose monohydrat 10 g
Thch 18 g
Nc ct 1 L
pH 7,3

Mi trng ISP-2

Cao nm men 4 g
Cao malt 10 g
Glucose 4 g
Thch 18 g
Nc ct 1 L
pH 7,2


Mi trng dch th YG (Yeast extract-Glucose)
Yeast extract 1%
Glucose 1%

Phng php RC (Rehydration- Centrifugation method):
dng phn lp x khun c kh nng di ng
- mu kh t nhin trong 3-5 ngy, nghin nh mu
- Ly 0,5 g mu cho vo 50 ml m phosphat 10 mM- 5% cao nm men (pH 7)
- Loi b phn ni trn b mt
- Gi 28
o
C trong 1,5 gi
- Chuyn 8 ml lp dch pha trn sang ng ly tm
- Ly tm vi tc 3000 rpm (1500 x g) trong 20 pht
- Gi nhit phng trong 30 pht
- Chuyn 3 ml lp dch trn sang ng khc.
- Ly 1 ml pha long vi nc ct (10
-3
, 10
-4
)
- Tri trn mi trng HV vi pha long 10
-2
, 10
-3
, 10
-4

- Nui cy nhit 28
o
C trong 14-21 ngy

Phng php SDS-YE (Sodium dodecyl sulfate Yeast
extract method): dng phn lp tt c cc loi x khun
- mu kh t nhin trong 3-5 ngy, nghin nh mu
- Ly 1 g mu cho vo 10 ml nc ct v trng (10
-1
)
- Ly 1 ml dch trn cho vo 9 ml SDS-YE trong m phosphat (10
-2
) (SDS 0,05%, cao
nm men 6%, m phosphat 5mM, pH 7,0)
- Sc nhit 40
o
C trong 20 pht
- Pha long bng nc ct v trng
- Tri trn mi trng HV
- Nui cy 28
o
C trong 14-21 ngy

Phng php DH (Dry heating method): dng phn lp x
khun ch yu thuc chi Streptomyces
- mu kh t nhin trong 3-5 ngy, nghin nh mu
- Sy mu 100
o
C trong 30 pht
- Rc mt lng rt nh mu ln mi trng HV
- Nui cy 28
o
C trong 7-14 ngy

Mt s phng php ha phn loi x khun:
Chun b thnh t bo
1. T bo t (1-2 ml) + 5ml nc ct v trng + ht thy tinh (loi ln v nh, ng
knh 0.11-0.12mm). Ph t bo bng sng siu m ca.180 trong 1- 1.5 gi cho n khi
dung dch tr nn trong. Chuyn sang ng falcon 50ml. Nu ngng cng vic y, gi
mu 4
o
C.
2. Ly tm vi tc 3000g trong 30 pht. Chuyn dch trn sang ng ly tm tc cao
(c np y bn trong). Ly tm vi tc 17000rpm trong 30 pht. B dch trn.
3. Thm 3ml SDS 4% (SDS c tc dng loi lipid v protein). Nu dng cng vic y,
gi mu nhit phng.
4. Gi 100
o
C trong 40 pht, nh vn cht np.
5. Ly tm vi tc 17000 rpm trong 30 pht 30
o
C (nu gi nhit thp, SDS s b
kt tinh). Loi b dch trn.
6. Ra vi nc t nht 3 ln vi iu kin ging trn (17000rpm, 30 pht)
7. Nu cn thit, x l bng RNase v DNase.
8. ng kh qua m.

Phn tch thnh phn acid amin trong thnh t bo
1. 2-3mg thnh t bo + 200l HCl 4N, vn cht np, gi 100
o
C qua m
2. Lm kh bng ht chn khng (khong 1 ngy 1 m)
3. Thm 200 l nc ct, trn u, chuyn sang ng eppendorf, ly tm vi tc 12000 rpm
trong 5 pht. Lc mu.
4. Chy sc k bn mng TLC:
- Dung mi:
Methanol 80ml
Nc ct 17.5ml
6N HCl 4ml
Pyridine 10ml
- Cellulose TLC
- Chun (1l): DAP, Ala, Gly, Glu, Lys
- Mu: 3-5l
- Nhn bit bng phun ninhydrin, gi 100
o
C trong 5 pht. Cc acid amin s hin
ln di dng cc chm mu tm. lu, cc chm ny dn dn bin mt, nhng
ring chm ca DAP s chuyn sang mu vng.
5. Chun b mu chy HPLC: chun b cho c mu v chun
- 10-20 l (25nmol) mu, lm kh bng ht chn khng (khong 2h)
- Thm 20 l Ethanol/DW/Triethylamine 2/2/1, trn u, lm kh bng ht chn khng
(khong 2h)
- Thm 20 l Ethanol/DW/Triethylamine/Phenylisothiocyanate 7/1/1/1, trn u, gi
nhit phng trong 20 pht, lm kh bng ht chn khng (khong 2h)
- Thm 0.5ml dung dch A(dich chy HPLC), lc mu dng lm mu chy HPLC

iu kin chy HPLC
- hp th (absorbance) 0.01
- S dng UV detector bc sng 254nm
- Dung dch A: 6% CH
3
CN, dung dch B: 60% CH
3
CN
- Tc dng chy: 1ml/pht
Chng trnh
Thi gian
(pht)
Nng dung dch
B(%)
0 0
15 70
15.1 100
25 100
25.1 0
30 Dng


Phn tch thnh phn menaquinone
1. 100-500mg t bo kh + 10-15 ml Chloroform: methanol (2:1), trn bng khuy t chm
trong 2-3 gi.
2. Chuyn sang ng falcon, ly tm 3000 rpm trong 5-10 pht, ly dch trn, lm kh bng c
quay
3. Ha tan bng 1,5 ml acetone, chuyn sang ng eppendorf, lm kh bng ht chn khng
4. Thm 50 l ethanol, chm ton b mu ln bn TLC silicagel, trnh nh sng v nhit
cao( khng lm kh mu bng my sy).
5. Dung mi chy TLC: Toluen. Sau , kim tra menaquinone bng tia UV (thng nm
di vch mu vng c th nhn thy bng mt thng). Chun: Vitamin K. Vch ngang vi
vitamin K l menaquinone.
6. Co vch c nhn bit bng UV, cho vo ng eppendorf, thm 1,5ml acetone, trn
u.
7. Ly tm vi tc 3000 rpm trong 5 pht, ly dch trn, lm kh bng Nit lng.
8. Thm 50-100 l ethanol, lc v dng lm mu chy HPLC v Mass spectrometer (MS)
9. iu kin chy HPLC
- Dung mi: Methanol: Propanol (2:1)
- Tc dng chy: 0.2ml/min
- Nhit ct: 40
o
C
- Nhn bit bng UV detector vi bc sng 275 nm

Sau khi phn tch, ng vi mi pick trn LC, s c pick trn MS. Ch s ca MS chnh l ch s m/z,
sau tr i 1(v MS thm vo 1 proton), ri so vi gi tr trong bng s bit c l loi
menaquinone no. Quan trng nht l pick cui cng v cao nht (v menaquinone rt d b t
gy). VD: MK-9 (H
4
) c ngha l menaquinone c cha 9 n v isoprene, trong c hai n v
isoprene bo ha. Menaquinone ny c th b t gy d dng nn xut hin cc pick nh hn. X
khun c MK 7,8,9,10,11. Mt vi loi c MK12.

Phn tch thnh phn ng trong t bo
1. 30-50mg t bo kh + 1ml H
2
SO
4
1N, lc nh, vn cht nt, gi 100
o
C trong 2 gi.
2. ngui, chuyn sang cc thy tinh, chnh pH 5.2-5.5 bng dung dch Ba(OH)
4
bo ha.
3. Chuyn sang ng eppendorf. Ly tm vi tc 3000 rpm trong 5 pht, chuyn dch trn
sang cc thy tinh, lm kh bng ht chn khng
4. Ha tan bng 300 l DW, chuyn sang ng eppendorf, ly tm vi tc 3000rpm trong 5
pht, ly dch trn, lc mu.
5. Chy sc k bn mng TLC:
- TLC cellulose
- 3 l mu
- 1 l chun
Chun 1: galactose, arabinose, xylose 0.1% (w/v) ha tan trong nc
Chun 2: rhamnose, mannose, glucose, ribose 0.1% (w/v) ha tan trong nc
Chun 3: madurose (3-O-methyl-D-glucose) 0.1% (w/v) ha tan trong nc
- Dung mi chy TLC: n-buthanol:water:pyridine:toluene= 10:6:6:1 (v/v)
- kh, phun Acidity phthalate aniline (phtalic acid 3.25g ha tan trong 100 ml dung dch
n-buthanol bo ha, thm 2ml aniline). Gi 100
o
C trong 4 pht
- Chun b dung dch n-buthanol bo ha: DW + n-buthanol (1:1), cho vo bnh tch mu,
lc trn u, loi lp di

Chun 1: Glucose: vng
Mannose: vng
Ribose:
Rhamnose: vng
Chun 2: Galactose: vng
Arabinose: Arabinose:
Xylose:
Chun 3: Madurose: vng

6. iu kin chy HPLC:
- Ct: ct trao i anion
- Dung dch A: Acid boric 0.1 M
+ Ha tan 6,2g acid boric trong 1L DW
+ Thm KOH 4M n pH 8
- Dung dch B: Acid boric 0.4M
- + Ha tan 24,8g acid boric trong 1L DW
+ Thm KOH 4M n pH 9
- Tc dng chy: 0,5ml/min
- Nhit : 65
o
C
- Phn ng nhn bit:
Dung dch C: 5g arginine + 15g acid boric trong 500ml DW
Tc dng chy: 0,5ml/min
Nhit : 150
o
C
Bc sng: Ex=320nm, Em=430nm
Chng trnh
Thi gian (pht) Nng Sol B
0 0 Dung dch C: 0.2ml/min
50 100 57
100 57.1 0
65 dng

Phn tch thnh phn acid bo
1. 5mg t bo kh + dung dch kim (R1) 1ml, vortex 5- 10 sec, gi 100
o
C trong 5min,
vortex 5-10 sec, gi tip 100
o
C trong 25min, ngui xung nhit phng, c th t
trc tip di vi nc sau khi h xung 80
o
C
2. Thm dung dch methyl ha (R2): 2ml, gi 80
o
C trong 10min, ngui xung nhit
phng. Qu trnh ny thy xut hin khi trng
3. Thm dung dch tch (R3) 3ml, o ln u ui nh nhng trong10min, loi b lp di
(lp nc)
4. Thm dch ra (R4) 3ml, o ln u ui nh nhng trong 5 min, ly tm 2000rpm trong
3min, chuyn 2/3 lp trn (lp dung mi) sang ng ng mu (mu thng khng mu)
5. Nu lp pha trn cha trong, thm 1-3 git dung dch R5, o ln u ui nh nhng
trong 5min, ly tm li ln na.
Ch : Cc bc cn thc hin lin tc, trc khi tin hnh cn chun b heatblock 100 and
80
o
C.

R1: NaOH 15g, Methanol (HPLC grade) 50 mg, MQ 50ml
R2: 6N HCl 65ml, Methanol (HPLC grade) 55ml, pH 1.5
R3: n-hexane (HPLC grade or n-hexane 1000) 50ml, methyl-ter-butyl ethel (HPLC grade) 50ml
R4: NaOH 10.8g, MQ 900ml
R5: 100ml MQ + 40g NaCl

6. Mu c phn tch bng sc k kh (h thng MIDI)

Phn tch thnh phn phospholipid
1. 100mg t bo kh + dung dch chlorofolm: methanol: 0,3% NaCl (50:100:40) = 2ml:
4ml:1,6ml . Trn bng khuy t trong 4 gi. Chuyn sang ng ng falcon, ly tm 3000 rpm
trong 5 pht.
2. Ly dch trn. Thm 1,75ml NaCl 0.3% v 1,75ml chloroform. Lc u, ly tm 5000 rpm
trong 5 pht. Loi lp trn cng v lp gia.
3. Lm kh bng Nit lng. Nu ngng cng vic, gi -20
o
C.
4. Ha tan bng 100 l ethanol.
5. Chm mu ln 4 bn HP-TLC (high performance)
6. Dung mi chy TLC chiu 1:
Chloroform: Methanol: Water = 65:25:4
Dung mi chy TLC chiu 2:
Trc khi chy chiu 2, chm Standard
Chloroform: Acetic acid: Methanol: Water = 80:18:12:5
7. Nhn bit cc loi phospholipid:
Bn 1: Nhn bit cc glycolipit:
Chun b thuc th anisaldehyde:
Acetic acid 1ml
p-anisaldehyde 5ml
H
2
SO
4
5ml
Ethanol 90ml
Phun thuc th, gi 110
o
C trong 15 pht
Cc phospholipid cha ng nh GlcNU cha NPG v PIMs, glycolipit s hin ln di dng
cc chm mu vng xanh.

Bn 2: Nhn bit cc amino lipit
- Phun ninhydrin ln bn TLC. Gi 120
o
C trong vi pht.
- Cc phospholipid cha nhm amino c nhn bit di dng cc chm mu tm nh
PE, OH-PE, lyso-PE, PME, PS, NPG
Nhn bit tt c phospholipid bng thuc th Dittmer Lester
- Chun b thuc th Ditmer Lester:
Sol I 25N H
2
SO
4
100mg
MoO
3
4,011g
Ha tan bng cch un 90
o
C n khi dung dch tr nn trong
SolII Iot 50mg
Mo 0,178g
un trong 15 pht, ngui, lc
Trc khi dng, trn Sol I + Sol II (1:1) + 1 V nc ri pha long 3 ln. Mu ca dung dch s
chuyn t vng sang xanh
- Phun thuc th ln bn TLC, tt c cc phospholipid s hin ln

Bn 3: Nhn bit cc choline lipit
- Thuc th Dragendorff:
Sol A: Basic bismuth nitrate [Bi(OH)
2
NO
3
] 1,7g
Acetic acid 20ml
Water 80ml
Sol B: KI 10g
Nc 25ml
Trc khi dng, trn 20ml Sol A v 5ml Sol B vo 70ml nc.
- PC v sphingomyelin c nhn bit bng chm vng da cam

Bn 4:
- Gi bn TLC trong hp cha tinh th Iot trong 3-5 pht. Cc chm vng v nu vng s
xut hin. Phn ng dng nhn bit tt c cc loi phospholipid.
- Nhng chm vng s dn dn bin mt, c th s dng bn ny cho th nghim tip theo.
Nhn bit cc glycolipit cha nhm OH
- Thuc th Periodic acid: 1% natri metaperiodic
- Phun thuc t periodic acid ln bn TLC. Gi trong 5-10 pht nhm glycol tch khi
lipit bi oxi ha
- Phun nc (loi mui periodate)
- t bn TLC vo b cha acid sulfuric cho n khi cc chm vng nhn bit bng Iot
bin mt.
- Phun thuc th Schiff
- PI hin ln mu vng, PG hin ln mu n tm, Cc lipit cha ng hin ln mu
xanh

Phn tch thnh phn G+C trong ADN
1. 25l ADN (2-25g), gi 60
o
C trong 1 gi. Gi 100
o
C trong 2 pht. Lm lnh lp tc
trong chu , spin down.
2. Mu ADN 25l + m acetate (40mM, pH 5,3) cha 2mM ZnSO
4
25l + dung dch
nuclease P1 (20U/ml) 25l. Spin down. Gi 37
o
C trong 1 gi.
3. Glycine buffer (0,1 M, pH 10,4) 25 l + alkaline phosphatase 2l (0.35U/l). Spin down.
Gi 37
o
C trong 1-2 gi. S dng lm mu chy HPLC
4. iu kin HPLC:
- Ct Cosmosil5 C18
- Nhit 35
o
C
- Dung mi: 0,2M ammonium phosphat: acetonenitril (40:1)
- Tc dng chy: 1 ml/min
- Bc sng: 275nm

Phng php tch ADN x khun

- Nui cy lc trong 5 ml mi trng dch th YG nhit 28
o
C
- Ly 1,5 ml dch nui cy, ly tm vi tc 16000g trong 5 pht 4
o
C, thu sinh khi
- Ra bng TE
- Nghin mu
- Thm 500 l EDTA 5mM (pH 8,0), trn u
- Thm 50 l lysozym (0,75 mg trong 1 ml Tris-HCl 10mM pH 8,0), trn, gi 37
o
C trong
1 gi
- Thm 50 l SDS 20%, 50 l proteinase K (4 mg trong 1 ml Tris-HCl 50mM pH 7,5),
trn, gi 55
o
C trong 30 pht
- Thm 400 l phenol: chloroform: isoamylalcohol (25:24:1), trn u, ly tm vi tc d
16000g trong 10 pht 4
o
C, thu lp trn. Thc hin 3 ln.
- Thm 1/10 th tch Natriacetate 3M, 1 th tch propanol, trn u
- Thu ADN bng a thy tinh
- Ra trong 500 l ethanol 70% trong 30 giy, kh t nhin
- Thm 500 l TE

Nu s dng mu ADN tin hnh th nghim lai ADN, cn lm tip cc bc sau:

- Thm 50 l RNase A (1 mg trong 1 ml Tris-HCl 50mM pH 7,5), 0,5 l RNase T
1
, gi
37
o
C trong 20 pht
- Thm 50 l proteinase K, gi 37
o
C trong 1 gi
- Thm 400 l phenol: chloroform: isoamylalcohol (25:24:1), trn u, ly tm vi tc d
16000g trong 10 pht 4
o
C, thu lp trn. Thc hin 3-4 ln.
- Thm 1/10 th tch Natriacetate 3M, 1 th tch propanol, trn u
- Thu ADN bng a thy tinh
- Ra trong 500 l ethanol 70% trong 30 giy, kh t nhin
- Thm 100-200 l TE
Cc s liu trn c ly t sch Bergeys manual systematic of bacteriology v sch
Identification manual of actinomycetes.
Ti liu tham kho:
1. Miyadoh S. (ed.). 1997. Atlas of actinomycetes. Asakura Publishing Co. Ltd, Japan.
2. Miyadoh S. (ed.). 2001. Identification manual of actinomycetes. Business Center for
Academic Societies Japan.
3. Williams S. T.,M.E. Sharpe , J.G. Holt (ed.). 1989. Bergeys manual of systematic
bacteriology. Williams & Wilkins, Baltimore, USA.
Holt J.G, N.R.Krieg, P.H.A.Sneath, J.T.Staley, S.T.Williams.2000. Bergeys Manual of
Determinative Bacteriology (9
th
edition). Lippincott Williams & Wilkins

Bi 5 C khun(Archaea)
Phn loi c khun
M u
Nhng vi sinh vt c kh nng sinh methane (mtan), mn cm vi
oxygen v c cu trc mng t bo c bit c bit n t lu
nhng mi n cui nhng nm 1970 chng mi c nhn nhn nh
i din ca mt dng sng th ba trn tri t bn cnh vi khun v
sinh vt nhn tht, l c khun. Carl R. Woese v cng s (1977)
sau khi xem xt trnh t 16S rARN nhn thy rng cc sinh vt nhn
nguyn thu (Prokaryote) cn c chia thnh hai nhm khc bit
nhau hon ton l Vi khun (Eubacteria hay Bacteria) v C khun
(Archaeabacteria hay Archaea), v cng vi cc Sinh vt nhn tht
(Eukarya) lm thnh ba lnh gii (Domains) sinh vt (Hnh 1). Cc
nghin cu su hn v ph h v c im sinh l sinh ho cho thy
rng c khun c tch ra t rt sm trong qu trnh tin ho, chng
khng gn vi khun nhiu hn so vi sinh vt nhn tht, do vy tn
gi Archaea c xut thay cho Archaeabacteria. Hin nay c hai
tn gi Archaea v Archaeabacteria u c s dng trong cc ti liu vi sinh vt, tuy nhin thut
ng Archaea chnh xc hn v r rng c khun khng phi vi khun m l mt nhm vi sinh vt
ring bit.

Hnh 1. Ba lnh gii ca sinh vt: Vi khun (Bacteria), C khun (Archaea) v Sinh vt nhn tht
(Eukarya).
C khun l mt nhm vi sinh vt c bit
C khun (Archaea) bt ngun t ting La tinh Archaios c ngha l c, l mt nhm vi sinh vt c
nhiu c im rt khc bit (Bng 1).
Bng 1. Nhng c im khc bit ca c khun so vi vi khun v sinh vt nhn tht
c im
Vi khun
(Bacteria)
C khun(Archaea)
Sinh vt nhn tht
(Eukarya)
Thnh t bo Peptidoglycan
Pseudo-peptidoglycan,
protein, polysaccharid,
glycoprotein
cellulose, carbonat,
silicat, chitin
Mng t bo Este-lipid Ete-lipid Este-lipid
ARN polymeraza (trn
khun ADN)
Ch c mt loi
4 n v 2o||
C nhiu loi
7 12 n v
C ba loi
7 12 n v
Ribosom 70 S 70 S 80 S
Phn ng ca ribosom
vi c t bch hu
khng Mn cm Mn cm

Cng nh t bo vi khun, t bo c khun (ngoi tr chi Thermoplasma) c thnh t bo
bn ngoi gi chc nng bo v. Tuy nhin, khng nh vi khun, thnh t bo ca c khun
khng cha peptidoglycan v v th khng b ph hu di tc dng ca lysozym. C khun c rt
nhiu dng cu trc thnh t bo khc nhau. Mt s c khun (nh cc loi sinh methane) c thnh
t bo cu to bi mt loi polysaccharid rt ging vi peptidoglycan c gi l pseudo-
peptidoglycan (pseudomurein). Chui pseudo-peptidoglycan gm cc n nguyn N-acetyl-
glucosamin v N-acetyl-alosamin-uronic acid (thay cho N-acetyl-muramic acid trong
peptidoglycan). Ngoi ra, y cu ni glycosid |13 thay th cho cu ni glycosid |14
peptidoglycan. Mt s c khun khc li hon ton khng c c peptidoglycan v pseudo-
peptidoglycan trong thnh t bo m thay vo l hn hp gm polysaccharid, glycoprotein hoc
protein. V d nh cc loi Methanosarcina (c khun sinh methane) c thnh t bo l mt lp
polysaccharid dy cu to t glucoza, glucuronic acid, galactosamin v acetat. Cc loi c khun a
mn cc oan (extreme halophiles) nh l Halococcus c thnh t bo tng t nh
Methanosarcina nhng cha nhiu hp cht c nhm sulfat ging nh chondroitin sulfat t chc
lin kt ca ng vt. Dng cu trc thnh t bo ph bin nht c khun l lp paracrystallin b
mt (S-layer) gm protein hay glycoprotein. Cu trc ny c tm thy cc i din thuc tt c
cc nhm c khun, t a mn cc oan (extremely halophilic), a nhit cc oan (extremely
thermophilic) v c cc loi sinh methane. c bit cc chi Methanospirillum v Methanothrix (c
khun sinh methane) c cu trc thnh t bo v cng phc tp. Cc loi thuc hai chi ny mc
thnh chui di gm nhiu t bo, gia mi cp t bo c mt lp m dy v ton b cu trc
chui li c bc kn trong mt lp paracrystallin b mt.

Thnh phn v cu trc lipid ca mng t
bo l mt trong nhng c im ni bt phn bit
c khun v hai nhm cn li. Trong khi vi khun
v sinh vt nhn tht cu ni acid boglycerol
trong lipid mng t bo l lin kt este (ester) th
c khun li l lin kt ete (ether) (Hnh 2). Acid
bo trong este-lipid thng l cc phn t ngn,
mch thng. Tri li, acid bo trong ete-lipid l cc
phn t mch di, phn nhnh, thuc c hai dng
phytanyl (C
20
cacbuahydro tng hp t isopren) v
biphytanyl (C
40
). Do ch c c khun v khng b
bin i di nhit cao nn isopren-lipid c
ly lm cht ch th ca c khun ho thch.
Enzyme polymeraza thc hin qu trnh sao
m trn khun ADN (DNA-dependent RNA
polymerase) ba lnh gii sinh vt cng c nhiu
im khc nhau. Vi khun ch c mt loi ARN-
polymeraza c cu trc khng gian n gin, gm
bn chui polypeptid 2o, 1|, 1| v mt nhn t o
khng c nh. C khun c nhiu loi ARN-
polymeraza, cu trc mi loi li phc tp hn
nhiu so vi ARN-polymeraza vi khun. ARN-
polymeraza ca c khun sinh methane v cc loi
a mn (halophilic) gm tm chui polypeptid (5
chui di v 3 chui ngn). ARN-polymeraza c
khun a nhit cao (hyper-thermophilic) li phc
tp hn, gm t nht 10 chui peptid. Polymeraza
thc hin qu trnh tng hp ARN thng tin (mARN) sinh vt nhn tht gm 10-12 chui
polypeptid c kch thc tng t nh ARN-polymeraza ca c khun a nhit cao. Ngoi ra,
sinh vt nhn tht cn c hai loi ARN-polymeraza khc na c hiu cho qu trnh tng hp ARN
ca ribosom (rARN) v ARN vn chuyn (tARN). Nh vy cht khng sinh rifampicin c tc dng
c ch n v | ca polymeraza ch c hiu qu i vi vi khun v c khun v sinh vt nhn tht
khng c loi polymeraza ny.
Vi nhng im khc bit trong trnh t 16S rARN cng nh cu trc ARN-polymeraza,
hin nhin b my sinh tng hp protein ca ba lnh gii sinh vt cng s khng ng nht. Tuy c
kch thc ca ribosom ging vi vi khun (70S) nhng c khun li c nhiu bc trong qu trnh
sinh tng hp protein rt ging vi sinh vt nhn tht (80S ribosom). Nhiu cht khng sinh c ch
qu trnh sinh tng hp protein vi khun li khng c hiu lc i vi c khun v sinh vt nhn
tht (Bng 2). Ngoi ra, tng t nh sinh vt nhn tht, nhn t ko di EF-2 trong ribosom c
khun c phn ng vi c t bch hu, mt loi c t v hi i vi vi khun. Tuy nhin nhn t
EF-2 c khun mang tnh c hiu cao, nhn t ny hon ton khng hot ng trong mi trng
ribosom ca vi khun hoc sinh vt nhn tht. Cc th nghim lai ribosom in vitro cho thy ribosom
ghp gia n v ln (50S) ca c khun v n v nh (40S) ca sinh vt nhn tht vn thc hin
chc nng gii m mt cch bnh thng, trong khi vic ghp tng t gia vi khun v sinh vt
nhn tht li hon ton khng tng thch. Nh vy cu trc b my sinh tng hp protein ca c
khun c nhiu im tng ng vi sinh vt nhn tht hn l vi vi khun.

Hnh 2. Lipid trong mng t bo ca c
khun (ete-lipid) khc vi ca vi khun v
sinh vt nhn tht (este-lipid)
Ging nh vi khun, c khun c mt nhim sc th dng vng, tuy nhin genom ca c
khun thng nh hn nhiu so vi genom ca vi khun. Chng hn ADN ca Escherichia coli l
2,5 x 10
9
Da, trong khi ADN ca Thermoplasma acidophilum l 0,8 x 10
9
Da, hay ca
Methanobacterium l 1,1 x 10
9
Da. Ngoi ra thnh phn GC (mol%) ca ADN c khun dao ng
trong phm vi rt ln, t 21 n 68 %, chng t tnh a dng ca c khun. So snh trnh t y
ca genom c khun Methanococcus jannaschi vi genom ca vi khun v sinh vt nhn tht cho
thy 56% trong 1738 gen khng tng ng.

Bng 2. Tnh mn cm ca i din ba lnh gii sinh vt i vi cc cht c ch qu trnh
sinh tng hp protein
Cht khng
sinh
Tc dng c ch
C khun Vi khun Sinh vt nhn
tht
Methano-
bacterium
Sulfo-
lobus
Escheri-
chia coli
Saccharomyces
cerevisae
Cycloheximid c ch bc khi
u

+
Virginiamycin,
pulvomycin
c ch bc ko
di
+

+

Neomycin,
puromycin
Dng tng hp
sm
+ + + +
Rifamycin
c ch enzyme
ARN polymeraza

+

Erythromycin,
streptomycin,
chloramfenicol
Tng tn s mc
li v mt s hiu
ng khc

+


Cc hnh thc dinh dng c khun
C khun c nhiu hnh thc dinh dng: ho dng hu c (chemoorganotrophy), ho dng v
c (chemolithotrophy), t dng (autotrophy), hay quang hp (phototrophy). Ho dng hu c l
hnh thc dinh dng ca nhiu loi c khun, tuy nhin cc chu trnh phn gii cht hu c thng
c mt s im khc bit so vi vi khun. C khun a mn (halophiles) v a nhit cc oan
(extreme thermophiles) phn gii glucoza theo mt dng ci bin ca con ng Entner-Doudoroff
(E-D). Nhiu loi c khun li c kh nng sn sinh ra glucoza t cc cht ban u khng phi l
hydratcarbo (gluconeogenesis) thng qua cc bc o ngc ca qu trnh glycolysis (con ng
Embden-Meyerhof). Oxygen ho acetat thnh CO
2
c thc hin qua chu trnh TCA (i khi vi
mt s thay i trong cc bc phn ng), hoc qua con ng acetyl-CoA (Ljungdahl-Wood). Cc
thnh phn ca chui vn chuyn in t nh vi khun u c tm thy c khun, trong
cytochroma, b v c c cc loi a mn cc i, cytochroma c mt s loi a nhit cao.
M phng da trn chui chuyn in t phn ln c khun cho thy chng thu np in t t
cht cho vo chui nc thang NADH, oxygen ho cht nhn in t cui cng l O
2
, S
0
hay mt
s cht khc, ng thi to ra lc y proton (proton motiv force) tng hp ATP nh b my
ATPaza kh tr trong mng t bo. Ho dng v c kh ph bin c khun, trong hydro
thng c s dng lm cht cho in t.
T dng c bit ph bin c khun v din ra di nhiu hnh thc khc nhau. c
khun sinh methane v c khun ho dng v c a nhit cao CO
2
c chuyn ho thnh cc hp
cht hu c qua con ng acetyl-CoA, trong mt s loi c ci bin cc bc phn ng khc
nhau. Mt s loi c khun khc (nh Thermoproteus) c nh CO
2
theo chu trnh citric acid o
ngc, tng t nh vi khun lam lu hunh. Mc d cc loi c khun a nhit cc oan u
thc hin hnh thc dinh dng hu c nhng nhiu loi vn c kh nng c nh CO
2
v thc hin
qu trnh ny theo chu trnh Calvin, tng t nh vi khun v sinh vt nhn tht.
Kh nng quang hp c mt s loi c khun a mn cc oan, tuy nhin khc vi vi khun,
qu trnh ny c thc hin hon ton khng c s tham gia ca chlorophill hay
bacteriochlorophill m nh mt loi protein mng t bo l bacteriorhodopsin kt gn vi phn t
tng t nh carotenoid c kh nng hp ph nh sng, xc tc cho qu trnh chuyn proton qua
mng nguyn sinh cht v s dng tng hp ATP. Tuy nhin, bng hnh thc quang hp ny c
khun a mn cc oan ch c th sinh trng vi tc thp trong iu kin k kh, khi mi
trng thiu cht dinh dng hu c.

Mi trng sng ca c khun v gi thuyt v hnh thnh s
sng trn tri t
C khun c bit n nh nhng vi sinh vt thch nghi vi cc mi trng c iu kin cc oan
(extreme) nh nhit cao (thermophilic), ni lnh gi (psychrophilic), nng mui cao
(halophilic) hay acid cao (acidophilic) v.v. cng l mt l do gii thch ti sao c khun li
kh c phn lp v nui cy trong iu kin phng th nghim. Trong gii sinh vt, c khun c
cc i din c tr cc iu kin nhit cao hn c (Bng 3, Hnh 3), nhiu loi c th sng
nhit trn 100 C di p sut cao nh cc ming ni la di y i dng. C ch thch
nghi ca t bo vi sinh vt vi nhit cao nh vy cn ang c nghin cu. c khun, mt s
phng thc thch nghi vi nhit cao c bit n nh tc dng ca enzyme gyraza trong vic
bo v cu trc xon ca ADN di tc ng ca nhit, hay ete-lipid, nht l C
40
-lipid trong mng
t bo ca c khun, gip lm tng bn vng ca mng. Tuy nhin c khun khng ch sng
cc mi trng cc oan. Ngoi i dng c khun tn ti vi mt s lng ln. Trn t lin cc
loi c khun sinh methane a m c mt nhiu mi trng khc nhau, nh cc b ln men cht
thi hu c, cc chn rung la ngp nc, ng tiu ho ca ng vt v.v.

Bng 3. Nhit pht trin cao nht ca cc i din sinh vt trn tri t
C 38 C
Cn trng 50
ng vt n bo 50
To 56
Nm 60
Vi khun thng 90
C khun 113
Kh nng thch nghi i vi cc iu kin sng cc oan ca c khun l c s gi thuyt
rng chng l nhng sinh vt sng u tin xut hin trn tri t. Tri t ca chng ta trong thi
k u c nhit rt cao, khong 100 C tr ln, cha nhiu ammon v kh methane trong kh
quyn, do vy nhng dng sng u tin phi l cc sinh vt ym kh v a nhit cao (hyper-
thermophiles). Vi cc c im sinh l nh tnh a nhit, sng k kh, s dng cc cht hu c v
v c l ngun nng lng, cc loi c khun a nhit cao c l ph hp vi dng sng nguyn thu
m phng theo iu kin ca tri t trong thi k u. Trong thc t, cht ch th mch isoprene-
lipid thnh phn mng t bo ca c khun c tm thy trong cc lp trm tch c tui l 3,8 t
nm. Cc nghin cu da trn trnh t 16S rARN cho thy c khun, c bit l nhm c khun a
nhit cao, tin ho chm hn ng k so vi vi khun v sinh vt nhn tht. Tuy nhin tc tin
ho chm ca c khun so vi hai lnh gii cn li c th do mi trng sng khc nghit ca chng
to ra. Cho n nay cu hi v ngun gc s sng v vai tr ca c khun trong vn cn ang
tip tc c tranh lun.

Hnh 3. Mt trong nhng ni u tin c khun c tm thy: sui nc nng trong cng vin
Quc gia Yellowstone (M).

Ph h c khun da trn trnh t 16S rARN
Da trn so snh trnh t 16S rARN cc i din c khun phn lp c chia thnh hai nhm
chnh l Euryarchaeota v Crenarchaeota (Hnh 4,5). Euryarchaeota l nhm c khun c bit
r nht, bao gm nhiu loi sinh methane, c khun a mn, kh sulfat (Archaeoglobales),
Thermoplasmalates v Thermococcales. Nhm Crenarchaeota gm ba lp Desulfococcales,
Sulfolobales v Thermoproteales. Sau ny nhm c khun Korarchaeota c xut thm (Hnh
6), tuy nhin ch da trn cc trnh t 16S rADN c c t cc mu ADN tch trc tip t mi
trng ch cha c i din no c phn lp v nui cy trong phng th nghim.



Hnh 4. Cc i din ca hai nhm c khun Crenarchaeota v Euryarchaeota.

Hnh 5. Hnh thi mt s i din ca hai nhm c khun Euryarchaeota v Crenarchaeota



Hnh 6 Mi lin quan ph h ca ba nhm c
khun Euryarchaeota v Crenarchaeota v
Korarchaeota
Hnh 7 Nanoarchaeum equitans (cu
khun nh) trn b mt Ignicoccus sp.
(cu khun ln)

Gn y (2002), nhm nghin cu ca gio s Stetter, mt trong nhng nh nghin cu c
khun hng u th gii, cng b s hin din ca nhm c khun th t, Nanoarchaeota, gm
nhng c khun c kch thc rt nh vi mt i din duy nht c tm thy l Nanoarchaeum
equitans (Hnh 7). Loi c khun ny c t bo hnh cu, ng knh 400 nm, sng bm trn b mt
t bo ca mt loi c khun mi Ignicoccus sp., phn lp t mu nc nng su 106 m di
y bin. y l mt loi a nhit cc oan, pht trin nhit ti u 75-98 C. Nhiu trnh t
16S rARDN trc tip c c t mi trng c nhit cao cng khng nh s tn ti v khc bit
ca nhm Nanoarchaeota so vi cc nhm c khun cn li.

a dng v cc nhm c khun i din
Xt v cc c im sinh l, c khun c th phn thnh bn nhm chnh l sinh methane
(methanogens), c khun a nhit cao (hyper-therrmophiles), c khun a mn (halophiles) v c
khun a acid (acidophiles) thuc lp Thermoplasmatales vi nhiu i din c phn lp v
nghin cu trong phng th nghim (Hnh 8).


Hnh 8. i din cc nhm c khun chnh

C khun sinh methane (methanogens)
Trong c khun, cc loi sinh methane lm thnh mt nhm ln v a dng vi cc c im chung
l (1) to kh methane nh sn phm cui cng ca chu trnh trao i nng lng v (2) sng k kh
bt buc. C khun sinh methane thu nng lng cho qu trnh sinh trng t vic chuyn ho mt
s cht thnh kh methane. Ngun c cht ch yu ca cc vi sinh vt ny l hydro, format v
acetat. Ngoi ra, mt s hp cht C
1
nh metanol, trimethylamin, dimethylsulfid v ru nh
isopropanol, isobutanol, cyclopentanol, etanol cng c s dng lm c cht (Bng 4).
Qu trnh sinh methane c khun c th coi nh l qu trnh h hp k kh, trong cht
nhn in t l CO
2
hoc nhm methyl trong cc hp cht C
1
v acetat. Tuy nhin, nh ta thy
trong bng 4, nng lng c gii phng ra trong cc phn ng to methane u rt nh. so
snh ta c th ly nng lng gii phng t phn ng oxygen ho glucoza bng oxygen C
6
H
12
O
6
+
6 O
2
6 CO
2
+ 6 H
2
O (G
0
= 2870 kJ/mol). L nhng sinh vt duy nht c kh nng to ra kh
methane, c khun sinh methane c nhng enzyme v coenzyme thit yu cho qu trnh tng hp
methane v ng vai tr ch th cho nhm, v d nh coenzyme F
420
v coenzyme M. S hin din
ca coenzyme F
420
khin cho cc t bo ca c khun sinh methane c tnh t pht sng di nh
n hunh quang (bc sng 350420 nm). Mc d hin tng t pht sng ny c th mnh, yu,
hay i khi mt hn, tu thuc vo cc pha sinh trng ca t bo, nhng vn l mt c im
n gin v tin li nhn bit c khun sinh methane di knh hin vi. Bn cnh , trnh t
acid amin trong cc chui peptid ca coenzyme M cng c dng phn loi c khun sinh
methane. Nhng nghin cu trong lnh vc ny cho thy s tng ng gia cy phn loi da trn
trnh t 16S rARN v cy phn loi da trn trnh t acid amin ca cc n v o v | trong
coenzyme M.

Bng 4. Phn ng to methane trn cc c cht khc nhau v nng lng c gii phng t

Phn ng sinh methane
Nng lng c gii
phng AG
0
(kJ/ methane)
4H
2
+ CO
2
CH
4
+ 2H
2
O 135,6
4 Format CH
4
+ 3CO
2
+ 2H
2
O 130,1
4 Isopropanol + CO
2
CH
4
+ 4 Aceton + 2H
2
O 36,5
2 Etanol + CO
2
CH
4
+ 2 Acetat 116,3
Metanol + H
2
CH
4
+ H
2
O 112,5
4 Metanol 3CH
4
+ CO
2
+ 2H
2
O 104,9
4 Methylamin + 2H
2
O 3CH
4
+ CO
2
+ 4NH
4
+
75,0
2 Dimethylamin + 2H
2
O 3CH
4
+ CO
2
+ 2 NH
4
+
73,2
4 Trimethylamin + 6H
2
O 9 CH
4
+ 3CO
2
+ 4 NH
4
+
74,3
2 Dimethylsulfid + 2H
2
O 3CH
4
+ CO
2
+ H
2
S 73,8
Acetat CH
4
+ CO
2
31,0
Nhng ni thng thng c th tm thy c khun sinh methane l cc b ln men hu c k
kh, cc lp trm tch thiu oxygen, t ngp ng v h ng rut ca ng vt. Khi dng chng
n c khun sinh methane rt nhy cm vi oxygen, tuy vy trong t nhin chng c th tn ti
mi trng hiu kh nh c bao bc v bo v bi cc vi sinh vt hiu kh v k kh khc. Trong
mi trng k kh, c khun sinh methane phi cnh tranh v c cht, c bit l hydro v acetat,
vi cc nhm vi sinh vt s dng cht nhn in t c hiu in th kh dng tnh hn so vi CO
2

nh l nitrat, sulfat v xit st III. Nh vy c khun sinh methane s chim lnh cc mi trng
ni khng c nhiu cc loi cht nhn in t tim nng ny. Do khng c kh nng s dng rng
ri cc loi c cht khc nhau, trong t nhin c khun sinh methane thng phi ph thuc vo cc
loi vi khun ln men v chng chuyn ho a dng cht hu c thnh cc acid hu c, hydro,
format v acetate, trong hydro, format v acetate l ngun thc n trc tip cho c khun sinh
methane, cn cc acid hu c sn phm ca qu trnh ln men nh propyonat, butyrate th cn phi
c mt nhm vi khun khc chuyn ho thnh c cht thch hp ri mi n lt c khun
chuyn thnh kh methane. C hai hnh thc cng sinh: bt buc v khng bt buc. Trong hnh
thc cng sinh gia c khun sinh methane v vi khun ln men, ch c c khun ph thuc vo
mi lin h ny do nhu cu v thc n cn cc hot ng trao i cht ca chng hon ton khng
c nh hng g ti cc vi khun ln men, v th hnh thc ny c gi l cng sinh khng bt
buc.
Cng sinh bt buc din ra gia c khun
sinh methane v mt nhm vi khun cng
sinh bt buc, trong c i bn cng cn
n nhau, v d nh hin tng cng sinh
gia Methanobrevibacter v
Synthrophobacter (Hnh 9). Nhm vi khun
cng sinh trong mi lin kt ny oxygen
ho acid hu c nh propionate, cc acid c
mch carbon di hn, cc hp cht thm v
chuyn in t sang proton hay CO
2
, to
thnh hydro hay format tng ng. Nhm
vi khun cng sinh ny ch c th thc hin
c trao i cht khi nng hydro v
format trong mi trng xung quanh c
gi mc rt thp, v nhim v do c
khun sinh methane m nhim. Cc loi
sinh methane thng c mt trong mi lin
kt cng sinh bt buc l Methanoplanus
endosymbiosus, cc loi
Methanobrevibacter, v Methanobacterium
formicicum.
mt s mi trng c bit nh cc sui nc nng hay cc tng nham thch ni la c
khun sinh methane thng c mt vi s lng ln. Trong trng hp ny chng sng t do,
khng ph thuc vo cc vi sinh vt khc v ngun c cht ch yu c s dng y l hydro,
sn phm c c t con ng l ho ch khng qua con ng sinh hc.
Hin nay tng s c khun sinh methane c bit n l 50 loi thuc 19 chi, su h v ba
lp (Bng 5 v 6). S phn loi ny da trn trnh t 16S rARN hin c, tuy nhin chng ta c th
hnh dung c rng theo thi gian khi nhng loi mi c bit thm th c th bng phn loi ny
s cn phi thay i.

Bng 5. Cc nhm phn loi chnh ca c khun sinh methane
Lp H Chi
Methanobacteriales Methanobacteriaceae (T) Methanobacterium (T)

Hnh 9 : Cng sinh gia Methanobrevibacter (t
bo trc khun) v Synthrophobacter (t bo hnh
oval).
-nt- -nt- Methanobrevibacter
-nt- -nt- Methanosphaera
-nt- Methanothermaceae Methanothermus (T)
Methanococcales Methanococcaeae (T) Methanococcus (T)
Methanomicrobiales Methanosarcinaceae Halomethanococcus
-nt- -nt- Methanococcoides
-nt- -nt- Methanohalobium
-nt- -nt- Methanohalophilus
-nt- -nt- Methanolobus
-nt- -nt- Methanosarcina (T)
-nt- -nt- Methanosaeta
(Methanothrix)
-nt- Methanomicrobiacaea (T) Methanoculleus
-nt- -nt- Methanogenium
-nt- -nt- Methanolacinia
-nt- -nt- Methanomicrobium (T)
-nt- -nt- Methanoplanus
-nt- -nt- Methanospirillum
-nt- Methanocorpusculaceae Methanocorpusculum (T)
T = h/ chi chun; -nt- nh trn
V hnh thi, c khun sinh methane rt a dng, trong mt s loi c hnh dng c
trng d nhn bit di knh hin vi nh Methanosarcina, Methanospirillum hay Methanosaeta
(Hnh 10). Ngoi ra, mt trong nhng c im quan trng dng phn loi nhm c khun ny l
ngun c cht c th s dng sinh methane (Bng 6).
H Methanobacteriaceae c thnh t bo cu to t pseudomurein, v th bt mu Gram (+). H
Methanobacteriaceae gm c ba chi l Methanobacterium, Methanobrevibacter v
Methanosphaera. Cc loi thuc chi Methanobacterium c t bo hnh que hoc hnh si, i khi
to nhm gm nhiu t bo. Tt c cc loi thuc chi ny u c kh nng sinh methane t H
2
+
CO
2
, mt s loi s dng.
Bi 6 Phng php thc nghim dng nh tn cc loi vi
khun
1. C IM HNH THI:
1.1. Nhum Gram (phng php Hucker ci tin)
Vt liu, ho cht:
- Dung dch Tm kt tinh (Crystal violet):
2g tm kt tinh ho tan trong 20 ml etanol 95%
0,8 g ammon oxalat ho tan trong 80 ml nc ct
Trn hai dch ni trn li vi nhau, gi 48 gi ri lc. Bo qun trong l ti, s dng
vi thng.
- Dung dch Iod:
Ho tan 1 g Iod (Iodine) trong 3-5ml nc ct, thm 2g KI (Kali iodide), khuy cho
tan ht, thm nc ct cho 300ml. Bo qun trong l ti.
- Dung dch ty mu:
Etanol 95% hoc trn hn hp 70ml etanol 95% vi 30ml aceton.
- Dung dch nhum b sung:
Chun b sn dung dch Safranin O 2,5%, trc khi dng pha vi nc ct theo t l
1:5 (vol/vol) c dung dch 0,5%.
Cc bc tin hnh:
- Chun b vt bi: dng que cy v trng ly mt t vi khun t thch (sau khi cy 24
gi) ho vo 1 git nc ct gia phin knh, lm kh trong khng kh.
- C nh t bo: h nhanh vt bi trn ngn la n cn 2-3 ln.
- Nhum bng dung dch Tm kt tinh trong 1 pht, ra nc, thm kh.
- Nhum li bng dung dch Iod trong 1 pht, ra nc, thm kh.
- Nh dch ty mu, gi khong 30 giy (cho n khi va thy mt mu), ra nc,
thm kh.
- Nhum b sung bng dung dch Safranin trong 2-3 pht, ra nc, kh trong
khng kh.
- Soi knh: dng vt knh du 100.
Kt qu:
Vi khun Gram (+) bt mu tm, Gram () bt mu .


Hnh 1.1. Cc bc tin hnh nhum Gram v v d minh ho kt qu.
1.2. Nhum tin mao
Vt liu, ho cht:
- Dung dch A: Acid tannic 5 g
FeCl
3
1,5 g
Formalin 2 ml
NaOH 1% 1 ml
Nc ct 100 ml
- Dung dch B: 2 g AgNO
3
ho tan trong 100 ml nc ct, ly 10 ml d ring. Nh
dung dch NH
4
OH m c vo 90 ml

cn li, thy hnh thnh ta rt c, tip tc
nh NH
4
OH vo cho n khi tan ht ta. Ly 10ml AgNO
3
b ra ban u nh t
t vo dung dch, thy xut hin vng mng, tip tc nh vo cho n khi va tan
ht vng th thi.
- Chun b phin knh: ra tht sch, ngm trong cn, t chy ht cn ri mi s
dng.
Cc bc tin hnh:
- Hot ho vi khun 2-3 ln trc khi tin hnh nhum.
- Dng que cy v trng ly vi khun t mt thch (mi cy 18-24 gi) ho vo 1
git nc ct t gia phin knh, nghing cho chy v mt pha, lm kh trong
khng kh.
- C nh t bo: h nhanh trn ngn la n cn 2-3 ln.
- Nh dch A ln vt bi, gi 10 pht, ra bng nc ct.
- Dng dch B cho chy qua loi ht nc. Nhum bng dch B trong 30-60 giy.
H nng, ngui ri ra li bng nc ct.
- Soi knh: dng vt knh du 100.
Kt qu:
T bo vi khun bt mu nu thm, tin mao bt mu nu.


Hnh 1.2. V d minh ho kt qu nhum t bo vi khun v tim mao
1.3. Kim tra kh nng di ng
Vt liu, ho cht:
- Chun b ng nghim cha mi trng thch bn lng (0,3-0,6% thch).
Cc bc tin hnh:
- Dng que cy c u nhn cy vi khun theo kiu chch su vo mi trng thch bn
lng.
- t ng nghim thng ng, nhit thch hp v quan st sau 1-3 ngy, c khi
lu hn.
Kt qu:
Vi khun mc lan rng quanh vt cy tc l chng c kh nng di ng.
Vi khun ch mc theo vt cy tc l chng khng c kh nng di ng
Ch : vi vi khun hiu kh ch quan st phn trn ca vt cy.
1.4. Nhum bo t
C hai phng php nhum
1.4.1. Nhum Lc Malachit (phng php Schaeffer-Fulton)
Vt liu, ho cht:
- Dung dch Lc Malachite (Malachite green) bo ho (khong 7,6%)
- Dung dch Safranin 0,5% (xem nhum Gram)
Cc bc tin hnh:
- Lm vt bi v c nh t bo nh i vi nhum Gram.
- Nhum bng dung dch Lc Malachite trong 10 pht, ra nc.
- Nhum li bng dung dch Safranine trong 30 giy, ra nc, thm kh.
- Soi knh: dng vt knh du 100
Kt qu:
Bo t c mu lc, t bo c mu .
Ch : phn bit bo t vi cc ht d nhim cng bt mu lc
1.4.2. Nhum Carbolic Fuchsin
Vt liu, ho cht:
- Dung dch A:
10 ml dung dch Fuchsin kim bo ho trong etanol (khong 10%)
100 ml dung dch acid carbolic (phenol) 5% (trong nc).
Trn u vi nhau (chun b trc khi dng)
- Dung dch B:
100 ml Etanol 95%
3 ml HCl m c
- Dung dch C:
30 ml dung dch Xanh metylen (Methylene blue) bo ho trong etanol (khong 2%)
100 ml dung dch KOH 0,01% trong nc.
Trn u vi nhau, cng lu cng tt.
Cc bc tin hnh:
- Lm vt bi trn phin knh, c nh t bo.
- Nh dung dch A ln vt bi, h nng nh bn di bay hi, trnh si. Thm
dn dn thuc nhum khng b kh cn, gi trong 5 pht. i ngui, thuc nhum
i.
- Dng dch B ra cho n khi va thy va ht mu , ra nc.
- Nhum li bng dung dch B trong 2-3 pht, ra nc, thm kh.
- Soi knh: dng vt knh du.
Kt qu:
Bo t bt mu , t bo bt mu xanh

Hnh 1.3. Cc bc tin hnh nhum Carbolic Fuchsin v v d minh ho kt qu.

1.5. Nhum v nhy (Capsule)
C hai phng php
1.5.1. Phng php nhum m bn
Vt liu, ho cht:
- Mc tu
- Metanol
- Dung dch safranin 0,5%
Cc bc tin hnh:
- Ly vi khun ho vo git nc trn phin knh.
- Nh thm 1 git mc tu, trn u. Dng cnh lamen dn mng vt bi, lm kh trong
khng kh.
- C nh vt bi bng cch nh metanol ln vt bi, gi trong 1 pht.
- Thm dn dn dung dch Safranin 0,5% ln vt bi ra metanol, sau gi trong
30 giy nhum li, ra nc, thm kh.
- Soi knh: dng vt knh du 100.
Kt qu:
Nn vi trng mu en, t bo mu , v nhy mu hng.
1.5.2. Phng php Congo
Vt liu, ho cht:
- Dung dch Congo (Congo red) 2% trong nc
- Dung dch gelatin 0,01-0,1% trong nc
- Dung dch HCl 1%
- Hn hp 30 ml dung dch Xanh metylen bo ho (khong 2%) trn vi 100 ml dung
dch KOH 0,01%.
Cc bc tin hnh:
- Nh 1 git dung dch Congo v 1 git dung dch gelatin ln phin knh sch.
- Ly vi khun trn u vi 2 git ni trn lm vt bi, kh trong khng kh
- Nh dung dch HCl ln ra, phin knh c mu xanh.
- Ra bng nc loi b dung dch HCl.
- Nhum li bng Xanh metylen trong 1 pht, ra nc, hong kh.
- Soi knh: dng vt knh du 100.
Kt qu:
Nn vi trng mu xanh, t bo mu , v nhy khng mu
1.5.3. Phng php nhum m bn n gin (Hnh 1.4):
- Nh 1 git Nigrosin vo u mt phin knh sch.
- Ly 1 vng que cy vi khun trn vi git nigrosin.
- Ly mt phin knh khc nghing 45
0
v ko git nigrosin trn vi khun v pha
phi mt cht sau ko nh v pha tri dn mng thnh mt vt bi. kh t
nhin (khng h la).
- Quan st di knh hin vi s thy v nhy mu sng ni ln trn mt nn mu en.


Hnh 1.4. Nhum m bn dng Nigrosin v v d minh ho kt qu.

1.5.4. Phng php dng Tm kt tinh (Crystal violet)
Vt liu, ho cht:
- Dung dch Tm kt tinh 1% trong nc
- Dung dch Sulfat ng CuSO
4
20% trong nc
Cc bc tin hnh:
- Nh vi git dung dch tm kt tinh ln phin knh sch
- Ly vi khun t ng thch nghing (48 gi) trn vi dung dch tm kt tinh, dng cnh
lam knh dn u trn ton b phin knh lm vt bi, gi trong khng kh 5-7 pht
lm kh, khng c h nng.
- Ra vt bi bng dung dch sulfat ng 20%, thm kh.
- Soi knh: dng vt knh du 100.
Kt qu:
- T bo c mu sm, v nhy mu nht xung quanh.
1.6. Nhum thnh t bo
Vt liu, ho cht:
- Dung dch acid tannic 5%
- Dung dch Tm kt tinh 0,2%
Cc bc tin hnh:
- Lm vt bi vi khun
- Nhum bng dung dch acid tannic trong 5 pht, ra nc.
- Nhum bng dung dch Tm kt tinh trong 3-5 pht, ra nc, thm kh.
- Soi knh: dng vt knh du 100
Kt qu:
- Thnh t bo bt mu tm, t bo cht mu tm nht.
1.7. Nhum ht d nhim
Vt liu, ho cht:
- Dung dch A: Xanh Toluidin (Toluidine blue) 0,15 g
Lc malachite 0,2 g
Acid acetic (glacial) 1 ml
Ethanol 95% 2 ml
Nc ct 100 ml
- Dung dch B: Iod (I) 2 g
Iodid kali (IK) 3 g
Nc ct 300 ml
Cc bc tin hnh:
- Lm vt bi, c nh vt bi.
- Nhum bng dch A trong 5 pht, i
- Trng bng dch B, nhum thm trong 1 pht, ra nc, thm kh.
- Soi knh: dng vt knh du 100.
Kt qu:
Cc ht d nhim c mu en.
Cc thnh phn khc ca t bo bt mu lc hay lc nht
1.8. Nhum ht PHB (Poly--hydroxybutyric acid)
Vt liu, ho cht:
- Dung dch A: en Sudan B (Sudan black B) 0,3 g
Etanol 70% 100 ml.
Trn u, lc mnh, qua m mi s dng.
- Dung dch B: Xylene
- Dung dch C: Dung dch Safranin 0,5% trong nc.
Cc bc tin hnh:
- Lm vt bi, c nh vt bi.
- Nhum bng dch A trong 10 pht, ra nc, thm kh.
- Dng dch B ra cho n khi mt mu.
- Nhum bng dch C trong 1-2 pht, ra nc, thm kh.
- Soi knh: dng vt knh du 100
Kt qu:
Ht PHB bt mu lam en. T bo v cc b phn khc c mu .
1.9. Nhum tinh th protein ( Bacillus thuringiensis)
Vt liu, ho cht:
- Dung dch nhum:
1 ml dung dch Fuchsin bo ho bo ho (xem nhum carbolic fuchsin) trn vi 100 ml
dung dch acid carbolic 5% trong nc.
Khi dng pha long 10 ln.
Cc bc tin hnh:
- Lm vt bi, c nh vt bi
- Nhum trong 1 pht, ra nc, hong kh
- Soi knh: dng vt knh du
Kt qu:
Tinh th bt mu . Bo t tch ri c vng mu
1.10. Nhum vi khun khng acid
Vt liu, ho cht:
- Dung dch A:
Dung dch Fuchsin kim bo ho trong nc (~ 10%) 10 ml
Dung dch acid carbolic 5% trong nc 100
ml
Trn u 2 dung dch vi nhau.
- Dung dch B:
Etanol 95% 100 ml
HCl c 3 ml
- Dung dch C:
Dung dch Xanh metylen bo ho trong etanol (~ 2%) 30 ml
Dung dch KOH 0,01% trong nc 100 ml
Cc bc tin hnh:
- Lm vt bi, c nh
- Nh dch A ln vt bi, un nh di phin knh cho bay hi nhng khng si.
Thng xuyn b sung thm dch A trnh kh vt bi. Gi trong 5 pht. i ngui,
dch A i.
- Dng dch B ra cho n khi thy va mt mu . Ra nc k.
- Nhum bng dch C trong 2-3 pht, ra nc, thm kh.
- Soi knh: dng vt knh 40
Kt qu:
Vi khun khng acid bt mu .
Vi khun khng khng acid bt mu xanh.

Hnh 1.5. V d minh ho kt qu xc nh tnh khng acid ca vi khun.

2. IU KIN NUI CY V C IM SINH L:
2.1. Hnh thi khun lc
- Ly 15-20ml mi trng thch v trng, ngui n 50
0
C ri vo a Petri (thao tc
v trng). Nu c nc ngng t trn np hp cn p ngc xung vo t m 30-37
0
C
lm kh mt thch.
- Ly mt vng que cy gt 3-4 ln trn mt thch mt gc. Quay a thch sang hng
khc v ria cy t mt vch thnh 3-4 ng khc sao cho khng trng vi cc ng trc.
Lp li theo mt hng th ba pha long hn na phn vi khun dnh trn que cy. Ch
khng nhc tay ln v khng thay i hng ca vng que cy.
- t vo nhit thch hp trong 1-2 ngy chn ra cc khun lc mc ring r.
- Tin hnh quan st cc khun lc ny t cc pha (t trn xung, t bn cnh), ch v
kch thc, hnh dng khun lc, hnh dng mp, b mt, dy, c nm hay khng,
trong, mu sc (trn, di, c khuch tn ra mi trng hay khng)


Hnh 2.1. Cy ria t bo tch khun lc n v cc dng khun lc thng gp.
2.2. Nhit sinh trng v tnh bn nhit
- Ly mt vng que cy sinh khi cy vo cc ng nghim c mi trng dinh dng thch
hp (mi trng thch hoc dch th).
- t cc iu kin nhit khc nhau (3 ng trong mt nhit ). i vi cc loi a m
(mesophiles), nhit sinh trng v tnh bn nhit thng c kim tra vi thang nhit
4, 20, 30, 37, 41, 45 v 65
0
C. T 37
0
C tr ln c th dng cc ni cch thy n nhit.
- Quan st kh nng sinh trng ca vi khun (to sinh khi trn mi trng thch, hoc o
OD nu th nghim c tin hnh trn mi trng dch th)
c bit, tnh bn nhit cn xc nh khi phn loi cc Lin cu khun, cch lm nh sau:
Cy 1 git dch nui cy 24 gi vo ng nghim ng mi trng dch th thch hp.
Gi ni cch thy 60
0
C trong 30 pht sau t vo t m 35-37
0
C, nui trong 48 gi.
Nu vi khun pht trin c l c tnh bn nhit. Dng chng vi khun Enterococcus faecalis
lm i chng dng tnh.

2.3. Nhu cu v O
2
v CO
2

Cn c vo nhu cu i vi xy, vi khun c thnh cc nhm hiu kh, k kh, k kh khng bt
buc v vi hiu kh.

2.3.1. Nhu cu i vi xy
- Vi khun hot ho cy vo mi trng dch th thch hp.
- t cc iu kin: - khng kh cha 5% CO
2
v 10% O
2

- khng kh bnh thng.
Theo di s pht trin ca vi khun.

2.3.2. Tnh k kh ca vi khun sinh bo t
- Chun b mi trng thch k kh:
Casein thy phn 20 g
NaCl 5 g
Na-mercaptoacetat 2 g
Na-formaldehyd sulfoxylate 1 g
Thch 15 g
Nc ct 1000 ml.
- Cy vi khun theo kiu chch su 1 vng que cy (ng knh 1,5 mm) vo mi
trng ni trn.
- Nui 30
0
C, kim tra kt qu sau 3 ngy v 7 ngy. Nu vi khun mc pha trn l
thuc loi hiu kh; nu mc dc ng cy l k kh khng bt buc; nu ch mc bn
di l k kh bt buc.

2.4. Kh nng ng ha cc ngun carbon
- Mi trng khong c bn (g/l):

(NH
4
)
2
SO
4
2 g
MgSO
4
.7H
2
O 0,2 g
NaH
2
PO
4
.H
2
O 0,5 g
CaCl
2
.2H
2
O 0,1 g
K
2
HPO
4
0,5 g
Nc ct 1000 ml
- Ngun carbon (ng, polysaccarid, ru, axit bo, axit amin, axit hu c, hydroxy
axit (alcohol axit, dicarboxylic axit) c kh trng qua mng lc.
- B sung ngun carbon vo mi trng khong c bn (ng v ru nng 0,5-
1%, cc loi khc nng 0,1-0,2%), chia mi trng vo cc ng nghim tit
trng.
- Cy vi khun vo mi trng, s dng 3 ng nghim i vi mi loi ngun carbon.
- Theo di s pht trin nh gi kh nng ng ha ngun carbon.
Cch khc l chun b mi trng khong c s c thch v a Petri. Vi khun c cy dn
u trn a bng que gt. Ngun carbon dng tinh th c a ln a (khong bng ht go)
cho khuych tn dn ra xung quanh. Nu vi khun ng ha c ngun carbon no s mc thnh
vng xung quanh ch c t ngun carbon .

Cc ngun carbon thng c dng trong th nghim:
ng 5C Arabinoza, Riboza, Xyloza, Fucoza, Rhamnoza
ng 6C Glucoza hay Dextroza, Mannoza, Sorboza, Fructoza hay Levuloza, Galactoza
ng kp Saccharoza hay Sucroza-ng knh, Maltoza, Sorboza, Lactoza, Trehaloza,
Cellobioza, Melibioza
ng tam Raffinoza, Melizitoza
ng a Tinh bt (Starch), Dextrin, Inulin, Glycogen
Ru bc 3 Glycerol
Ru bc 4 Erythritol
Ru bc 5 Adonitol, Arabitol, Xylitol
Ru bc 6 Mannitol, Sorbitol, Dulcitol
Ru bc 6 mch vng Inozitol
Glucoside Salicin, Coniferrin, Aesculin, Arbutyl, Amygdalin, alpha-Methylglucosid

2.5. Kh nng ng ha cc ngun nit
- Mi trng c s:
KH
2
PO
4
1,36 g
CaCl
2
5 ml
NaHPO
4.
2,13 g
MgSO
4
.7H
2
O 0,2 g
FeSO
4
.7H
2
O 0,5 ml
Glucoza 10 g
Nc ct 1000 ml
Glucoza c th c thay th bng ngun carbon thch hp khc nh Citrat, Acetat,
Mannit vi nng 0,2-0,5%. Cc ngun nit khc nhau (mui ammon, mui nitrat)
c a vo mi trng vi nng 0,05-0,1%. i chng l mi trng hon ton khng
b sung ngun nit. Chnh pH ti 7,0-7,2.
Chia mi trng vo cc ng nghim (4-5 ml/ ng), kh trng 112
0
C trong 20-30 pht.
- Cy vi khun hot ho 18-24 gi (3 ng cho mi loi ngun nit).
- t nhit thch hp trong 3 ngy v 7 ngy. So snh s pht trin vi i chng
(o c ca dch t bo) xc nh kh nng ng ha ngun nit.

2.6. Kh nng sinh sc t hunh quang
- Mi trng lm ng thch nghing:
Pepton 20 g
Glyxerin 10 g
K
2
HPO
4
1,5 g
MgSO
4
.7H
2
O 1,5 g
Thch 15 g
Nc ct 1000 ml
pH = 7,2.
Kh trng 121
0
C trong 20 pht, t thch nghing.
- Cy vi khun hot ha 24 gi ln mt thch, a vo t m v theo di sau 1, 3, 5
ngy.
- Quan st ng nghim di n t ngoi xem c sn sinh sc t hunh quang hay
khng?

2.7. Tnh mn cm i vi cht khng khun
- Chun b mi trng thch a thch ng vi tng loi vi khun.
- Cy gt vi khun ln mt thch a (c th trn vi khun vo mi trng thch lm
ngui ti 50
0
C ri a).
- t ln mt thch cc khoanh giy (t ch hoc mua sn) tm cht khng khun cc
nng khc nhau.
- Nui trong t m 30
0
C trong 24-48 gi.
- Quan st cc vng v khun to thnh xung quanh cc khoanh giy cha cht khng
khun. Vng v khun cng ln tc l vi khun cng mn cm.


Hnh 2.2. Vng v khun to thnh quanh cc khoanh giy thm cht khng khun.


2.8. ng ha Malonat

- Mi trng dch th:
Cao men 1 g
(NH
4
)
2
SO
4
2 g
K
2
HPO
4
0,6 g
KH
2
PO
4
0,4 g
Natri malonat 3 g
Bromophenol blue (BPB) 0,025 cg
Nc ct 1000ml
pH= 7,0-7,4
- Chia mi trng vo ng nghim, kh trng 121
0
C trong 15 pht.
i chng l mi trng khng c Natri-malonat.
- Cy vi khun hot ha, t nhit thch hp trong 1-2 ngy.
- Quan st s i mu ca mi trng: nu chuyn mu t lc sang lam l c ng ha
malonat (dng tnh), nu khng l m tnh.

Hnh 2.3. S i mu ca mi trng khi vi khun ng ho malonat.

2.9. Xt nghim ng ha Citrat

Mc ch: phn bit cc nhm vi khun ng rut da trn kh nng ng ho citrat.
- Mi trng Simmons:
NaCl 5 g
MgSO
4
.7H
2
O 0,2 g
NH
4
H
2
PO
4
1 g
K
2
HPO
4
.3H
2
O 1 g
Natri xitrat 2 g
BPB 1% trong nc 10 ml
Thch ( ra nc) 12 g
Nc ct 990 ml.
un tan thch, chnh pH n 7,0, thm ch th mu BPB v phn vo ng nghim lm
thch nghing. Kh trng 121
0
C trong 15 pht.
- Cy vi khun vo ng thch, t nhit thch hp trong 3-7 ngy.
- Mi trng bin mu t lam sang o tc l vi khun c kh nng ng ha citrat
(dng tnh).
- Vi cc loi Bacillus cn s dng mi trng sau:
NaCl 1 g
MgSO
4
.7H
2
O 0,2 g
NH
4
H
2
PO
4
0,5 g
Natri xitrat 2 g
Nc ct 1000 ml
phenol (Phenol red) 0,04% 20 ml.

2.10. Nhu cu mui v tnh chu mui
- Chun b mi trng dch th thch hp i vi tng loi vi khun.
- B sung NaCl cc nng 2, 5, 7, 10%, mi trng cn trong sut.
- Cy vi khun hot ha, trong 3-7 ngy.
- Theo di mc sinh trng ca vi khun (mi trng khng cy vi khun lm i
chng).

2.11. Kh nng ng ha Tartrat
- Mi trng dch th kim tra:
Pepton 10 g
NaCl 5 g
Nc ct 1000 ml
BPB (0,2%) 12,5 ml
Kali tartrat 10 g
Chnh pH = 7,4.
Phn mi trng vo ng nghim, kh trng 115
0
C trong 20 pht, nn dng ngay. Nu
mi trng qu 14 ngy th cn kh trng li bng un cch thy 10 pht.
- Cy vi khun vo mi trng, hng ngy theo di s i mu.
- Sau 14 ngy thm mt lng dung dch ch acetat bo ha trung tnh bng th tch mi
trng (vol/vol). i chng l mi trng khng cy vi khun.
- Nu c chuyn sang mu lc vng v c t ch acetat kt ta l c kh nng ng ha
tartrat (dng tnh). Nu mu vng hay mu lc v c nhiu ch acetat kt ta l xt
nghim m tnh.
- Phn ng ny dng phn bit Salmonella zava (dng tnh) v Salmonella
paratyphi B (m tnh).

2.12. Kh nng sinh trng vi KCN
KCN c tc dng c ch Escherichia coli nhng khng c ch Citrobacter freundi, v th thng
c dng phn bit 2 loi ny.
- Mi trng c s:
Pepton 3 g
NaCl 5 g
KH
2
PO
4
0,22 g
K
2
HPO
4
5,64 g
Nc ct 1000 ml.
pH = 7,6.
Kh trng 115
0
C trong 20 pht. mi trng vo t lnh cho ngui n 4
0
C, thm
15ml dung dch KCN 0,5%, phn vo mi ng nghim 1ml (thao tc v trng); c th bo
qun c trong 2 tun. Mi trng i chng khng thm dung dch KCN.
- Cy vi khun t mi hot ho (24 gi) vo cc mi trng chun b trn, t
nhit thch hp trong 4 ngy v quan st s chuyn mu ca mi trng.
- Nu mi trng chuyn mu l sinh trng dng tnh (Citrobacter freundii), nu
mi trng vn khng mu l sinh trng m tnh (Escherichia coli).

3. CC C IM SINH HA:
3.1. Xt nghim oxidaza
Mc ch: phn bit cc nhm vi khun da trn hot tnh cytochrom oxidaza
- Pha dung dch Tetramethyl-p-phenylen diamin dihydrochlorid (TPPDD) 1% trong
nc, bo qun trong l mu ti 4 C, s dng trong 2 tun.
- t mt ming giy lc trong np hp Petri sch, nh dung dch TPPDD 1% ln trn
ming giy lc sao cho va m, khng qu t.
- Dng que cy c u que lm bng si platin hay dng a thy tinh (khng dng u
que cy bng si kim loi st, niken...) ly mt t vi khun hot ho (18-24 gi) bi
ln ming giy lc.
- Sau 10 giy nu vi khun chuyn sang mu hng tc l c oxydaza dng tnh; nu
sau 60 giy mi chuyn mu l oxydaza m tnh.
- Ch : nu dung dch t chuyn sang mu hng ri th khng c s dng. Nu
giy lc qu t s cn tr khun lc tip xc vi khng kh nn chuyn mu chm, to
nn m tnh gi.

3.2. Xt nghim catalaza
Mc ch: kim tra kh nng phn hu H
2
O
2
ca vi sinh vt nh sn sinh ra enzyme
catalaza.
- Chun b dung dch H
2
O
2
nng 3-10%, nh mt git ln phin knh.
- Dng u que cy platin ly mt t vi khun mi hot ho (24 gi) trn vo git H
2
O
2

trn phin knh.
- Nu thy si bt l dng tnh, khng si bt l m tnh.
- C th nh trc tip dung dch H
2
O
2
ln khun lc trn thch a cng cho kt qu
tng t.

Hnh 3.1. Phn ng si bt khi tip xc vi dung dch H
2
O
2
ca vi khun c catalaza dng
tnh.

3.3. Kh nng ln men/xy ha glucoza
C th dng mt trong hai mi trng sau lm th nghim
- Mi trng I:
Pepton 2 g
NaCl 5 g
K
2
HPO
4
0,2 g
Glucoza 10 g
Thch 6 g
Dung dch BTB 1% 3 ml (pha trong mt t cn 95%, sau mi thm nc
thnh dung dch 1%)
Nc ct 1000 ml.
pH = 7,0 - 7,2.
Phn mi trng vo cc ng nghim (4-5 ml), kh trng 115
0
C trong 20 pht.
- Mi trng II:
NH
4
H
2
PO
4
0,5 g
K
2
HPO
4
0,5 g
Cao men 0,5 g
Glucoza 10 g
Thch 5-6 g
Dung dch BTB 1% 3 ml (pha nh trn)
Nc ct 1000 ml
pH = 7,0 - 7,2.
Phn mi trng vo ng nghim v kh trng nh trn.
- Ly vi khun mi hot ho (18-24 h) cy chch su vo mi trng bng que cy
thng (mi chng vi khun cy vo 4 ng). Bt kn 2 ng nt bng bng vaselin-paraffin
(ly vaselin lm chy ra, thm 1/3 du paraffin) cch ly vi khng kh. Ngoi ra ly
thm 2 ng khng cy vi khun lm i chng. Theo di kt qu sau 1,2,3,7 v 14 ngy.
- Kt qu: - Nu ch c ng khng bt kn sinh axit (chuyn mu vng)
tc l vi khun thuc dng xy ha.
- Nu c ng khng bt v ng bt kn u sinh axit (chuyn mu vng) tc
l vi khun thuc dng ln men.

3.4. Kh nng ln men ng, ru
- Mi trng:
Cao tht 3 g
Pepton 10 g
NaCl 5 g
Cht ch th mu Andrade* 10 ml
(hay dung dch Xanh bromophenol 0,2%)
Nc ct thm n 1000 ml
- B sung ng vi nng 0,5%. Phn mi trng vo cc ng nghim, mi ng
1ml.
- t vo mi ng nghim 1 ng nh (ng Durham) ln ngc u hng kh CO
2

sinh ra nu vi khun c kh nng ln men ng. Kh trng trong 15 pht 121
0
C.
ng arabinoza, xyloza, v cc ng kp cn kh trng ring bng mng lc ri mi
b sung vo mi trng.
* Cch pha cht ch th mu Andrade:
Fuchsin axit 0,5 g
NaOH 1M 16 ml
Nc ct 100 ml
Nu dung dch c mu hng, dng NaOH 0,1 M (1-2 ml) trung ha cho n khi mt
mu.
Xanh bromophenol (BPB):
2 g BPB, b sung dn 5 ml NaOH 0,1M, nghin trong ci s, ha vi nc
ct cho 100 ml.
- Cy vi khun mi hot ho vo cc ng nghim, t 36
0
C, theo di hin tng sinh
axit sau 1-3 ngy. C trng hp cn dng paraffin bt kn nt bng v theo di trong
14-30 ngy
- Nu vi khun c kh nng ln men ng (sinh axit) th cht ch th Andrade s
chuyn mu , cht ch th BPB s chuyn mu vng lc.
C th lm cch khc nh sau:
- Vi vi khun ni chung dng mi trng I (xem mc 3.3), thay glucoza cc ng
khc hay ru (nng 1).
- Vi vi khun sinh bo t dng mi trng sau:
(NH
4
)
2
HPO
4
1 g
KCl 0,2 g
MgSO
4
0,2 g
Cao men 0,2 g
Thch 5-6 g
ng hay ru 10 g
Nc ct 1000 ml
Dung dch BTB 0,04% 15 ml
pH = 7,0-7,2.
Phn mi trng vo cc ng nghim (4-5 ml), kh trng 112
0
C trong 30 pht.
- Vi vi khun lactic dng mi trng sau:
Pepton 5 g
Cao tht 5 g
Cao men 5 g
Tween 80 0,5 ml
Thch 5-6 g
Nc ct (hay nc my) 1000 ml
Dung dch BTB 1,6% 1,4 ml
pH = 6,8-7,0.
Phn mi trng vo ng nghim, kh trng ti 112
0
C trong 30 pht.
- Ly vi khun mi hot ho (18-24 gi) cy trch su vo mi trng thch, t nhit
thch hp v quan st sau 1,3,5 ngy.
- Nu ch th mu bin vng l vi khun c kh nng ln men sinh axit (phn ng dng
tnh); nu vn gi mu lam l phn ng m tnh.

3.5. Phn ng M.R. ( Methyl - Methyl Red)
- Mi trng:
Pepton 5 g
Glucoza 5 g
K
2
HPO
4
hoc

NaCl 5 g
Nc ct 1000 ml.
pH = 7,0 - 7,2.
Phn mi trng vo ng nghim (4-5 ml), kh trng 115
0
C trong 30 pht.
- Thuc th:
Methyl 0,1 g
Etanol 95% 300 ml
Nc ct 200 ml.
- Cy vi khun vo mi trng (lp li 2 ln), t nhit thch hp trong 2-6 ngy
(nu kt qu m tnh cn ko di thm thi gian). Vi Vi khun ng rut thuc h
Enterobacteriaceae, t ng nui cy 37
0
C v kim tra sau 4 ngy.
- Nh 1 git thuc th vo dch nui cy, nu chuyn mu l phn ng dng tnh,
mu vng l m tnh (mu pH 4,4; mu vng pH 6,0).

Hnh 3.2. Cht ch th methyl chuyn mu khi tip xc
vi dch nui cy vi khun ln men ng.

3.6. Phn ng V.P. (Voges-Proskauer)
- Mi trng: xem phn 3.5.
- Thuc th:
Creatin 0,3% (hoc nguyn dng tinh th)
NaOH 40%
- Cy vi khun mi hot ho, t nhit thch hp trong 2-6 ngy.
- B sung NaOH 40 (bng th tch dch nui cy), sau nh mt t dung dch creatin
(hoc thm mt t tinh th), i khong 10 pht (c khi lu hn). Nu dch th chuyn
mu l phn ng dng tnh.
Cch khc:
- Mi trng Clark-Lubs:
Pepton 3 g
K
2
HPO
4
5 g
Glucoza 5 g
pH = 7,5.
- Phn ng V.P: nh 5 git dung dch alpha naphtol 6% (trong cn 90%, gi 4 C
trc khi dng) v 5 git NaOH 16% (trong nc), lc nh. Nu dch th chuyn sang
mu nu l phn ng dng tnh, mu vng nht l m tnh.
- Phn ng M.R: nh 2-3 git dung dch methyl 0,5% (trong cn 60%), lc nh. Nu
dch th chuyn mu l phn ng dng tnh, mu vng nht hay khng mu l m
tnh.

Hnh 3.3. V d minh ho kt qu phn ng V.P. v M.R.

3.7. Phn ng ONPG-aza (O-nitrophenyl--D-
galactopyranosidase)
- Mi trng:
ONPG 0,6 g
m phosphat 0,01M pH 7,5 100 ml
Dung dch pepton 1% (pH 7,5) 300 ml.
(Ho 0,6 g ONPG trong 100 ml dung dch m, kh trng bng mng lc, sau trn vi
300 ml dung dch pepton kh trng).
Bng thao tc v khun phn mi trng vo cc ng nghim nh, bo qun 4 C trong
vng 1 nm.
- Ct nhng khoanh giy lc, kh trng 112 C, 30 pht.
- Nh vo mi khoanh giy lc mt git dung dch sau:
ONPG 0,06 g
Na
2
HPO
4
.2H
2
O 0,017 g
Nc ct 10 ml
Lm kh 37
0
C trong 24 gi, bo qun trong cc ng nghim c nt xoy nhit
phng.
- Cy vi khun mi hot ho (1 vng que cy) vo mi trng chun b nh trn, t
nhit thch hp trong 24 gi.
- Ly tm dch nui cy thu t bo, lm dch huyn ph m c trong 0,5 ml nc mui
sinh l.
- a khoanh giy ONPG vo dch huyn ph, gi 35-37
0
C trong 24 gi.
Quan st kt qu: mu vng l phn ng dng tnh (Escherichia coli); khng mu l
m tnh (Salmonella paratyphi B).

Proteus mirabilis- ONPG (ng th 8) m tnh


Serratia marcescens- ONPG (ng th 8) dng tnh
Hnh 3.4. V d minh ho kt qu phn ng OPNG-aza

3.8. Kh nng thy phn tinh bt
- Mi trng: B sung tinh bt tan (0,2%) vo mi trng nc tht pepton, kh trng
121
0
C trong 20 pht, a Petri.
- Ly vi khun mi hot ho cy vch hay cy chm ln a thch. Sau 2-5 ngy nh
thuc th Lugol (xem phn nhum Gram) ln vt cy quan st kh nng phn gii
tinh bt. Nu thuc th Lugol khng bt mu quanh vt cy tc l vi khun c kh nng
phn gii tinh bt.

Hnh 3.5. Phn ng vi dch Lugol kim tra kh nng phn gii tinh bt ca vi khun.
3.9. Kh nng to tinh th Dextrin
- Mi trng: ho 50 g bt go vo 200 ml nc, quy k, thm 20 g CaCO
3
, sau b
sung dn dn 750 ml nc si, ng thi quy u ri un si 10 pht. Phn mi trng
vo cc ng nghim (15 ml/ng), kh trng 121
0
C trong 30 pht.
- Cy vi khun mi hot ho (18-24 gi) vo mi trng trn, t 30
0
C trong 5-10
ngy.
- B sung 1ml dung dch tinh bt 3%, gi 40
0
C trong 15 pht.
- Ly 3 git dch trong pha trn ho vi 1 git dung dch Lugol v dn ln phin knh,
lm kh trong khng kh v quan st di knh hin vi.
- Nu c sn sinh ra, nhng tinh th dextrin hnh lc gic bt mu lam c th quan st
c st mp vt bi.
3.10. Kh nng phn gii celluloza
- Mi trng khong:
NH
4
NO
3
1 g
K
2
HPO
4
0,5 g
KH
2
PO
4
0,5 g
MgSO
4
. 7H
2
O 0,5 g
NaCl 1 g
CaCl
2
0,1 g
FeCl
3
0,02 g
Cao men 0,05 g
Nc ct 1000 ml
pH = 7,0- 7,2.
Phn mi trng vo ng nghim, kh trng 121
0
C trong 20 pht.
- Mi trng Pepton:
Pepton 5 g
NaCl 5 g
Nc my 1000 ml
pH = 7,0-7,2
Phn mi trng vo ng nghim, kh trng 121
0
C trong 20 pht.
- Cho vo ng nghim mt bng giy lc di 5-7 cm (vi vi khun hiu kh mt phn
bng giy lc nh ln khi mi trng; vi vi khun k kh th bng giy lc ngp
trong mi trng).
- Cy vi khun mi hot ho, t nhit thch hp, quan st nh hng ca vi khun
ti bng giy lc sau 1-4 tun. Nu vi khun pht trin v lm nt giy lc tc l chng
c kh nng phn gii celluloza (phn ng dng tnh); m tnh l khng lm bin i
giy lc.
Cch khc:
- vo a Petri mt lp thch 2% (15 ml thch cho mt a C 9 cm).
- B sung bt celluloza (0,8%) v thch (1,5%) vo mi trng ghi trn, 5 ml ln
trn lp thch 2% chun b trong a Petri.
- Cy vi khun mi hot ho thnh im trn mi trng, t nhit thch hp trong
1-4 tun v quan st vng phn gii celluloza c to ra quanh vt cy.

3.11. Kh nng thy phn pectin
- Mi trng:
Cao men 5 g
CaCl
2
.2H
2
O 0,5 g
Thch 8 g
Na-polypectat 10 g
Nc ct 1000 ml
NaOH 1N 9 ml
Dung dch BTB 0,2% 12,5 ml.
ho tan Na-polypectat v cc thnh phn khc cn khuy mnh v lm nng mi trng
trong ni cch thy. Kh trng 121
0
C khng qu 5 pht ri a Petri.
- Cy vi khun mi hot ho thnh 8 chm trn thch a, t nhit thch hp 3
ngy ri quan st. Nu quanh vt cy c vt lm xung l dng tnh (Erwinia
carotova); khng c vt lm xung l m tnh (Erwinia herbicola).

3.12. Kh nng thy phn Esculin
- Mi trng:
B sung Esculin (0,1%) v citrat st (0,05%) vo mi trng nc tht pepton. Phn mi
trng vo cc ng nghim lm thch nghing. Kh trng 121
0
C trong 20 pht.
- Cy vi khun mi hot ho (18-24 gi), t nhit thch hp sau 3,7 v 14 ngy
ri ly ra quan st.
- Kt qu: xut hin sc t mu en nu l phn ng dng tnh, khng c l m tnh

3.13. Kh nng to Dextran v Levan
- Mi trng
Casein thy phn 15 g
Pepton 5 g
ng knh 50 g
K
2
HPO
4
4 g
Thch 10 g
Nc ct 1000 ml
Dung dch Xanh Trypan (Tripan blue) 1% trong nc 7,5 ml
Dung dch Tm kt tinh 1% trong nc 0,1 ml
pH 7,0
Kh trng 115
0
C trong 20 pht. ngui n 50
0
C, thm 1ml dung dch Kali-tellurit 1%
( kh trng bng mng lc) ri a Petri.
- Cy ria to khun lc n. t 37
0
C trong 24 gi, sau gi thm nhit
phng trong 24 gi.
- Vi khun sinh dextran s c khun lc nh, mu lam ti, b mt nhy v mc lm vo
thch (loi Streptococcus sanguis).
Vi khun sinh levutan c khun lc nhy mu phn hng (Streptococcus salivarius).
Nu khng sinh dextran v levan th vi khun c mu lam nht hoc ti, kch thc nh,
d ha sa (Streptococcus mitis).

3.14. Xc nh 3-Ketolactoza
- Mi trng:
Lactoza 10 g
Cao men 1 g
Thch 20 g
Nc ct 1000 ml
pH = 7,0-7,2
Kh trng 115
0
C trong 20-30 pht, a Petri.
- Ly vi khun mi hot ho (18-24 gi) cy im ln thch a, t nhit thch
hp trong 2 ngy to khun lc r rt.
- Pha thuc th Benedict:
CuSO
4
.5H
2
O 17,3 g
Na
2
CO
3
(khan) 100 g
Na-Citrat 173 g
Nc ct thm ti 1000 ml
Cch pha: ho Na
2
CO
3
v Na-Citrat trong 600 ml nc ct, lc trong, sau thm nc ti
850ml. Ho tan CuSO
4
trong 100 ml nc, b sung nc cho ti 150 ml. Cui cng trn
dung dch CuSO
4
vo dung dch u, va va khuy.
- Nh thuc th Benedict ln khun lc trn mt a thch, t 30 pht tr ln nhit
phng.
- Kt qu: nu quanh khun lc xut hin nhng kt ta mu nu th l phn ng dng
tnh, nu khng th l m tnh.

3.15. Kh nng kh Nitrat
- Mi trng:
Nc tht pepton 1000 ml
KNO
3
1 g
pH = 7,0-7,6
Phn mi trng vo cc ng nghim (4-5 ml/ng), kh trng 121
0
C trong 15-20 pht.
- Chun b thuc th Griess:
Dung dch A: Acid sulfanilic 0,5 g
Acid acetic long (khong 10%) 150 ml.
Dung dch B: Alpha Naphtylamin 0,1 g
Nc ct 20 ml
Acid acetic long (khong 10%) 150 ml.
- Chun b thuc th Diphenylamin: 0,5 g Diphenylamin ha vo 100 ml H
2
SO
4
c,
thm 20ml nc ct.
- Cy vi khun mi hot ho vo mi trng (mi chng cy 2 ng), t nhit
thch hp trong 1,3,5 ngy. Chn 2 ng khng cy vi khun lm i chng.
- Ly ng nghim sch v b sung ln lt cc dung dch nh sau:
Dch nui cy vi khun (hoc mi trng ng i chng)
1 git dung dch A
1 git dung dch B
- Kt qu:
- Nu dch nui cy chuyn mu (, hng, da cam hay nu) l biu th c nitrit, tc
l vi khun c kh nng kh Nitrat.
- Nu dch nui cy khng chuyn mu, thm 1-2 git thuc th Diphenylamin
kim tra s c mt ca Nitrat (chuyn mu xanh lam l c Nitrat chng t vi khun
khng kh Nitrat; khng chuyn mu tc l Nitrat c kh ht v nitrit c
kh tip tc thnh cc cht khc nh N
2
).
- Ch : phn ng kh Nitrat thc hin trong iu kin k kh, v vy khng c phn
vo ng nghim qu t mi trng.
i vi cc vi khun khc nhau nitrit c th l sn phm cui cng ca qu trnh kh
Nitrat, nhng cngc th ch l sn phm trung gian. Ngoi ra, tc kh ca cc loi
cng khc nhau, v th cn theo di thng xuyn mu sc ca mi trng. Trong mi
trng hp cn phi lm thm phn ng vi cht ch th diphenylamin.

3.16. Kh nng kh Nitrit
- Mi trng:
Peptone 5 g
NaNO
2
1 g
Nc ct 1000 ml
pH = 7,3-7,4
Phn mi trng vo cc ng nghim, kh trng 121
0
C trong 15 pht.
- Chun b thuc th Griess: ging nh phn kh Nitrat.
- Cy vi khun, t 30
0
C trong 1,3,7 ngy ri lm phn ng xc nh.
- Nh vo dch nui cy 1 git dung dch A v 1 git dung dch B (xem phn kh
Nitrat), lc nh. Nu mt mu v sinh ra NH
3
l kt qu dng tnh (Alcaligenes
odorans); nu vn gi mu l phn ng m tnh, khng kh nitrit (Acinetobacter
calcoaceticus).

3.17. Kh nng phn nitrat ha (Denitrification)
- Mi trng:
Nc tht pepton 100 ml
KNO
3
1 g
pH = 7,2-7,4
Phn mi trng vo cc ng nghim (4-5 ml), kh trng 121
0
C trong 30 pht.
- Cy vi khun mi hot ho. Dng vaselin bt kn nt ngn xy, t nhit thch
hp trong 1-7 ngy v quan st s pht trin ca vi khun (tng c ca dch nui
cy, sinh kh NH
3
). Vi khun c pht trin l phn ng dng tnh, khng pht trin l
m tnh.

3.18. Kh nng sinh amonia
- Mi trng:
Pepton 5 g
Nc ct 1000 ml
pH= 7,2
Phn mi trng vo cc ng nghim, kh trng 121
0
C trong 15-20 pht.
- Chun b thuc th Nessler:
Ho tan 20 g IK trong 50 ml nc; b sung I
2
Hg cho n khi bo ha (khong 32g), sau
thm 460 ml nc. Cui cng b sung 134 g KOH. Bo qun trong l ti nhit
phng.
- Cy vi khun mi hot ho (18-24 gi), t nhit thch hp trong 1,3,5 ngy.
- Ly vo ng nghim sch mt t dch nui cy, nh vi git thuc th Nessler. Nu
xut hin kt ta mu vng nu l phn ng dng tnh.

3.19. Xt nghim Ureaza
Mc ch: kim tra kh nng phn hu ur nh enzyme ureaza
- Mi trng:
Pepton 1 g
NaCl 5 g
Glucoza 1 g
KH
2
PO
4
2 g
Dung dch phenol 0,2% trong nc 6 ml
Thch 20 g
Nc ct 1000 ml
Kh trng xong chnh pH n 6,8-6,9, mi trng c mu vng hi nh l c. Phn
mi trng vo cc ng nghim lm thch nghing. Kh trng li 115
0
C trong 30 pht.
- Chun b dung dch Ur 20%, kh trng bng mng lc, b sung vo cc ng nghim
khi ngui n 50-55
0
C (t nng Ur 2%), t thch nghing.
- Cy vi khun mi hot ho, t nhit thch hp, sau 2-4 gi ly ra quan st. Kt
qu m tnh cn tip tc quan st sau 4 ngy.
- Kt qu: mi trng chuyn mu cnh o l phn ng dng tnh, mu sc khng
thay i l m tnh.
- Ch : cn lm i chng m tnh (khng b sung Ur), nht l khi xc nh cc loi
Pseudomonas, v i chng dng tnh (so snh vi 1 chng bit c hot tnh
ureaza).

Lm cch khc:
- Cy vi khun vo mi trng thch nghing ni trn v xc nh hot tnh ureaza sau
3 ngy v 7 ngy.
- Ly vi khun t thch nghing lm dch huyn ph m c trong ng nghim sch.
- Nh 1 git phenol vo dch huyn ph, chnh pH n 7 ( phenol chuyn t vng
sang da cam).
- Chia dch huyn ph vo 2 ng nghim sch. Trong ng 1 thm vi tinh th Ur
(khong 0,05-0,1 g), ng th 2 gi nguyn lm i chng. Sau vi pht nu dch
trong ng 1 (c Ur) chuyn sang kim ( phenol chuyn mu ), biu th vi khun c
hot tnh Ureaza; nu khng th l m tnh.

3.20. Xt nghim sinh Indol
- Mi trng:
Dung dch Pepton 1% trong nc
pH n 7,2- 7,6.
Phn mi trng vo cc ng nghim (1/3-1/4 th tch ng), kh trng 115
0
C trong 30
pht.
- Chun b thuc th:
Para-dimethyl-amino-benzaldehyde 8g
Etanol 95% 760 ml
HCl c 160 ml
- Cy vi khun mi hot ho (18-24 gi), t nhit thch hp v lm php th ti
cc thi im 1, 2, 4, 7 ngy.
- Nh thuc th theo mp ng nghim (to thnh lp dy 3-5 mm). Gia hai lp thuc
th v dch nui cy nu c mu l phn ng dng tnh. Nu mu sc khng r rt
th thm 4-5 git eter vo dch nui cy, lc nh lm cho eter khuch tn vo lp dch,
yn mt lt khi eter ni ln b mt th li thm thuc th ni trn. Nu trong mi trng
c indol th s xut hin mu trong lp eter.

Hnh 3.5. Thuc th chuyn mu khi trong dch nui cy c indol.

3.21. Xt nghim Phenylalanin desaminaza
(kim tra kh nng chuyn ho nhm amin (NH
2
) trong acid amin)
- Mi trng:
Cao men 3 g
Na
2
HPO
4
1 g
DL-Phenylalanin (hoc L-Phenylalanin) 1 g
NaCl 5 g
Thch 12 g
Nc ct 1000 ml
pH = 7,0
Phn mi trng vo cc ng nghim, kh trng 121
0
C trong 10 pht, t thch
nghing.
- Thuc th: dung dch FeCl
3
10% (W/V)
- Cy vi khun mi hot ho, t 37
0
C, lm php th sau 4 gi hoc 8-24 gi.
- Nh 4-5 git thuc th ln b mt thch nghing c vi khun pht trin, nu xut
hin mu lc l phn ng dng tnh (do sn sinh ra acid Phenylpyruvic), nu khng
i mu th l phn ng m tnh.

Hnh 3.6. Th nghim kim tra phenylalanin desaminaza.

3.22. Tryptophan desaminaza
C hai cch kim tra:
Cch th nht:
xc nh ng thi Tryptophan desaminaza, Ureaza v kh nng sinh Indol.
- Mi trng:
L-Tryptophan 3 g
KH
2
PO
4
1 g
K
2
HPO
4
1 g
NaCl 5 g
Etanol 95% 10 ml
Nc ct 900 ml.
Thm phenol (khong 25- 30mg)
pH = 6,8-6,9
Phn mi trng vo cc bnh tam gic, kh trng 121
0
C trong 20 pht.
- Ho 20 g Ur vo 100ml nc, kh trng bng mng lc, b sung vo mi trng
chun b trn (thao tc v trng). Phn mi trng vo cc ng nghim v khun (3-4
ml).
- Cy vi khun mi hot ho (18-24 gi), nui nhit thch hp trong 24 gi.
- Ly ra 2-4 git dch nui cy, thm 1 git dung dch FeCl
3
(khong 33%). Nu hin
mu nu l phn ng Tryptophan desaminaza dng tnh, khng hin mu l phn
ng m tnh. Dng vi khun Proteus lm i chng dng tnh.
- Nu mi trng sau khi nui cy vi khun chuyn t mu vng sang l biu hin
c hot tnh Ureaza. Dng thuc th para-dimethylaminobenzaldehyd kim tra s
hnh thnh indol. Nu khng nh kim tra ureaza th khng cn b sung phenol v
Ur vo mi trng.

Cch 2 th hai:
- Chun b ho cht:
L-Tryptophan 0,2-0,5%
Nc mui sinh l hoc dung dch m phosphat pH 6,8
Dch A: 50 ml KH
2
PO
4
0,2 M (27,2g/L)
Dch B: 23,6 ml Na
2
CO
3
0,2 M (8g/L)
Trn dch A v B vi nhau
FeCl
3
33%
- Cy vi khun vo mi trng thch nghing nc tht pepton, t nhit thch
hp trong 24 gi.
- Ly 4 ng nghim sch, thm vo mi ng 4 git dung dch L- Tryptophan 0,2-0,5%
v 4 git nc mui sinh l (hay dung dch m phosphat pH 6,8).
- Ly vi khun t thch nghing lm thnh dch huyn ph m c trong 2 ng, li
2 ng lm i chng, gi nhit phng trong 15-20 pht.
- Thm vo mi ng 1 git dung dch FeCl
3
(33%). Nu hin mu nu l phn ng
dng tnh, khng i mu l m tnh. C th dng vi khun Proteus lm i chng
dng tnh.

3.23. Carboxylaza i vi Ornithin, Lysin, v Arginin
- Mi trng:
Pepton 5 g
Cao tht 5 g
D-Glucoza 0,5 g
Bromocresol purple (BCP) 1,6% 0,625 ml
Cresol 0,2% 2,5 ml
Thch 3-6 g
Nc ct 1000 ml
Ha tan cc thnh phn trn trong ni cch thy, chnh n pH 6, thm ch th mu.
- Chia mi trng thnh 4 phn u nhau, b sung tng cht L-Ornithin, L-Lysin, L-
Arginin vi nng 1% (nu dng DL-acid amin th ly nng 2%), sau chnh pH
n 6,0-6,3. Mt phn khng thm acid amin dng lm i chng. Phn vo cc ng
nghim nh (mi ng 3-4 ml), kh trng 121
0
C trong 10 pht. Mi trng cha
Ornithin c th to mt t kt ta nhng khng nh hng n kt qu th nghim.
- Cy vi khun mi hot ho (18-24 gi), sau vaselin bt kn nt, nui iu
kin thch hp. Vi khun ng rut thuc h Enterobacterobacteriaceae cn nui 37
0
C trong 4 ngy v theo di kt qu hng ngy. Cc vi khun phi lm sng nui 30
0
C, quan st trong 7 ngy. Nu ch th mu chuyn sang mu ta hay mu ta c nh
l dng tnh, nu mu vng (nh ng i chng) l m tnh. Vi khun ng rut
thng biu hin phn ng dng tnh sau 1-2 ngy, nhng cng c khi chm hn, cn
theo di qua 3-4 ngy.

3.24. Arginin dihydrolaza
- Mi trng Thornley:
Pepton 1 g
NaCl 5 g
K
2
HPO
4
0,3 g
Thch 6 g
Phenol 0,01 g
L-Arginat 10 g
Nc ct 1000 ml
pH = 7,0-7,2.
Phn mi trng vo cc ng nghim (4-5 ml), kh trng 121
0
C trong 15 pht. Ch
lm ng i chng khng c Arginat.
- Cy vi khun mi hot ho, dng vaselin bt kn nt ng nghim, nui nhit
thch hp trong 3, 7, 14 ngy quan st. Mi trng chuyn sang mu l dng
tnh, khng chuyn mu l m tnh.

3.25. Acetylamin
- Dung dch Acetylamin:
Acetylamin 2 g
Nc ct 20 ml
(khng cn kh trng)
- Dung dch m:
K
2
HPO
4
0,4 g
KH
2
PO
4
0,1 g
KCl 8 g
Nc ct 1000 ml
Kh trng 115
0
C trong 20 pht.
- Dung dch lm th nghim: Pha long dung dch Acetylamin trong dung dch m
theo t l 1:99 vol/vol.

- Thuc th Nessler:
KI 5 g
Nc ct 5 ml
Thm dch HgCl
2
bo ho, lnh cho n khi lc mnh m vn cn mt t kt ta th
dng. Thm 40ml NaOH 9N ri b sung nc n 100 ml.
Ly 1 vng que cy vi khun trn vi dch th nghim ni trn, t nhit thch hp
trong 24 gi. Thm 1 git thuc th Nessler. Phn ng l dng tnh khi to kt ta
mu nu hay nu (vi khun Comamonas acidovorans), phn ng m tnh khi thy
mu vng (Pseudomonas stutzeri).
3.26. Thy phn Hippurat ; Phng php Yong &
Thompson
(dng khi nh tn Streptococcus, Campylobacter v Gardnerella vaginalis):
- Dung dch c cht:
Na-Hippurat 0,25 g
Nc ct 25 ml
Kh trng bng mng lc
- Thuc th :
Ninhydrin 3,5 g
Aceton-butanol (1:1 vol/vol) 100 ml
Bo qun trong l ti
- Cy 2 git huyn ph vi khun vo dung dich c cht, gi 37
0
C trong 1 gi. Thm 2
git thuc th, gi 15 pht.
- Phn ng l dng tnh nu xut hin mu ta (Campylobacter jejuni, Gardnerella
vaginlis, Streptococcus agalactiae), phn ng l m tnh nu sau 15 pht cha i mu
(Campylobacter coli, Streptococcus agalactiae).
- Ch : lng vi khun cy phi tha ng, thi gian trc v sau khi thm thuc th
phi chun xc. Trnh nh sng khi gi thuc th.
Phng php Baird-Parker:
- Mi trng:
Pepton ty tng 10 g
Cao tht 1 g
Glucoza 1 g
NaH
2
PO
4
5 g
Na-Hyppurat 10 g
Nc ct 1000 ml
Phn mi trng vo cc ng nghim, kh trng 121
0
C trong 30 pht.
- Thuc

th:
H
2
SO
4
c 50 ml
Nc ct 50 ml
t t H
2
SO
4
c vo nc ct
- Cy vi khun mi hot ho (18-24 gi) vo mi trng trn, nui nhit thch hp
trong thi gian 4-6 tun.
- Phn ng xt nghim: trn 1 ml dch nui cy vi 1,5 ml thuc th. Phn ng l dng
tnh khi xut hin tinh th (do Hyppyrat c chuyn ho thnh Benzoin); khng xut
hin tinh th l m tnh.

3.27. Hot tnh ADN-aza
Mi trng:
Pepton casein
Pepton u tng
NaCl
ADN
Toluid-Blue
Thch

Nc ct

10 g
5 g
5 g
2 g
0,1 g (c th pha thnh dung dch ri
cho vo)
15 g
1000 ml

Ho tan cc thnh phn ca mi trng bng nhit, sau b sung ADN v Toluid-blue,
trn u ri phn vo bnh. Kh trng 121
0
C trong 30 pht, thch a.
- Cy vi khun mi hot ho (18-24 gi) thnh im trn a thch, nui iu kin
thch hp trong 2 ngy.
- Phn ng l dng tnh trong trng hp quanh cm cy c vng mu (dng
chng Salmonella lm i chng dng tnh).

3.28. Hot tnh Phosphataza
- Mi trng:
Lm nng chy mi trng thch-nc tht-pepton ( kh trng)
Thm 1% Phenolphthalein diphosphat (kh trng bng mng lc).
thch a.
- Cy vi khun mi hot ho thnh im trn thch a, nui iu kin thch hp
trong 2 ngy.
- Phn ng xt nghim: ly 0,1ml nc amonia ph ln mt thch, sau 20 pht xem
kt qu. Phn ng l dng tnh nu khun lc chuyn mu phn (Staphylococcus
aureus); khng chuyn mu l m tnh.

3.29. Kh nng lm dch ha Gelatin
- Mi trng:
Pepton 5 g
Gelatin 100-150 g
Nc ct 1000 ml
pH = 7,2-7,4
Phn mi trng vo cc ng nghim (4-5 ml), kh trng 115
0
C trong 20 pht.
- Cy vi khun mi hot ho (18-24 gi) chch su vo mi trng gelatin, gi 2 ng
khng cy lm i chng, nui 20
0
C trong thi gian 2,7,10,14,30 ngy.
- Quan st kh nng lm dch ho gelatin ti nhit phng t 20
0
C tr xung. Nu
b mt mi trng gelatin khng lm xung, gelatin vn trng thi n nh l phn
ng m tnh (khng sinh gelatinaza); nu mt phn hay ton b gelatin ha lng th l
phn ng dng tnh. Nu so vi i chng m tnh thy vi khun mc, gelatin cha
ha lng nhng b mt lm xung th cng vn coi l dng tnh (mc dch ha
thp). Nu vi khun hon ton khng sinh trng th c th l khng mc c trn
gelatin hoc mi trng c s cha thch hp.
- Ch :
- Nu vi khun ch sinh trng nhit trn 20
0
C, lc quan st gelatin ho lng cn
t ng nui cy mt lc vo nc lnh ri so snh vi i chng m tnh.
- Kh trng nhit qu cao hay qu thp u nh hng n kt qu, nn kh trng
115
0
C trong 15 pht.
- Gelatin cht lng khng u nhau, lng dng kh thng nht, nn chn nng to
ng tt 20
0
C l c. Nn dng thng nht mt loi gelatin cho ton b th nghim.

Hnh 3.7. V d minh ho kt qu kim tra kh nng lm dch ho gelatin (sinh gelatinaza),
Escherichia coli- m tnh, Pseudomonas aeruginosa- dng tnh.

3.30. Hot tnh Lipaza (vi Tween 80)
- Mi trng:
Pepton 10 g
NaCl 5 g
CaCl
2
. 2H
2
O 0,1 g
Thch 9 g
Nc ct 1000 ml
pH = 7,4.
Kh trng 121
0
C trong 20 pht, ngui n 50
0
C ri thm Tween 80 n nng 1%,
thch a (c th thay Tween 80 bng du tributyrin).
- Cy vi khun mi hot ho (18-24 gi) thnh vch, nui trong 7 ngy, hng ngy ly ra
quan st.
- Phn ng l dng tnh nu quanh vt cy c vch trong, nu khng c th l m tnh.

Hnh 3.8. V d minh ho kt qu phn ng th hot tnh Lipaza: m tnh - Salmonella
typhimurium (bn tri), dng tnh - P. aeruginossa (bn phi).

3.31. Hot tnh Lipaza (vi du ng)
- Mi trng:
Pepton 10 g
Cao 3 g
NaCl 3 g
Thch 20 g
Xanh Victoria (Victoria Blue)
dung dch 1:5000 trong nc 100 ml
Du ng 50 ml
Nc ct 900 ml.
Ho tan cc thnh phn ca mi trng (tr du ng) bng un nng, sau b sung du ng,
khuy u bng my khuy t, chnh n pH 7,8. Phn mi trng vo cc ng nghim, kh
trng 115
0
C trong 30 pht.

t thch nghing hoc thch a, mi trng c mu
nht.
- Cy vi khun mi hot ho (18-24 gi), nui nhit thch hp trong 24 gi.
- Quan st: phn ng l dng tnh khi mi trng chuyn sang mu lam, khng chuyn
mu l m tnh.


Hnh 3.9. V d minh ho kt qu phn ng th hot tnh lipaza vi du ng.

3.32. Hot tnh Lecithinaza
- Trn lng trng vi cng trng lng nc mui sinh l (thao tc v trng) to
thnh dch huyn ph.
- Ly ra 10 ml dch huyn ph trn ha tan vo mi trng thch-nc tht-pepton va
kh trng, ngui n 50-55
0
C ri a Petri.
- Cy vi khun mi hot ho (18-24 gi) thnh im trn a thch, mi im ng knh
khong 2-3mm. Cy 5-7 chng trn mt a. Vi vi khun k kh c th y l knh mng
(lamelle) ln vt cy, tuy nhin tt nht l a vo t nui k kh.
- t nhit thch hp trong 18-24 gi, mt s chng (nh chi Bacillus) cn thi gian
lu hn (48 gi) v quan st bin i ca mi trng thch.
- Nu xung quang v di vt cy c vch trong l phn ng dng tnh (Lecithin c
chuyn ho thnh lipid do vi khun sinh men lecithinaza).
- Ch : khi trn dch huyn ph lng trng vo mi trng thch khng nn tin
hnh nhit qu cao v s lm ngng kt lecithin c trong lng trng.

Hnh 3.10. Kh nng phn hy Lecithin ca Clostridium

3.33. Kh nng sn sinh H
2
S
Phng php gii giy
- Mi trng:
Pepton 10 g
NaCl 5 g
Cao tht 10 g
Cystein 0,5 g
Nc ct 1000 ml
pH = 7,0-7,4
Phn mi trng vo cc ng nghim (4-5 ml), kh trng 112
0
C trong 20-30 pht.
- Ct giy lc thnh di rng 0,5-1cm, di ty thuc vo ng nghim v cao ca
mi trng. Tm vo giy dung dch Ch-acetat, sy kh giy trong t sy t trong hp
Petri v kh trng.
- Cy vi khun mi hot ho (18-24 gi). Dng panh v khun gp giy tm ch-acetat
a vo tng ng nghim, di n nt bng nhng khng chm vo mi trng. Nui vi
khun nhit thch hp, sau 3,7,14 ngy th ly ra quan st. Nu giy bin en l phn
ng dng tnh, nu khng i mu th l m tnh.
- Ch : phng php ny rt mn cm, khng thch hp i vi trc khun ng rut.
Khng t giy lc tm ch-acetat gn mt mi trng qu trnh b ht m, nhng cng
khng nn t cch xa qu. Ngoi ng i chng khng cy vi khun nn ly chng vi
khun bit l m tnh lm i chng.
Phng php i vi Trc khun ng rut
- Mi trng:
Cao tht 7,5 g
Pepton 10 g
NaCl 5 g
Gelatin 100-120 g (hay thch 15 g)
Dung dch FeCl
2
10% 5 ml (kh trng ring bng mng lc)
Nc ct 1000 ml
pH = 7,0
Kh trng 112
0
C trong 20 pht, b sung dung dch FeCl
2
( kh trng) vo khi thch hay
gelatin cha ng. Phn vo cc ng nghim v khun (4-5 ml), ngay lp tc nhng vo nc
lnh cho ng li.
- Cy chch su vi khun vo cc ng, nui 30
0
C trong 1,3,7 ngy. Nu mi trng
chuyn thnh mu en l phn ng dng tnh, nu khng i mu l m tnh.
- Ch : phng php ny dng khi cn nh tn vi khun thuc h Enterobacteriaceae.
C th dng FeSO
4
thay th cho FeCl
2
. Nu nui cy 20
0
C c th dng kt hp xc
nh gelatinaza.

3.34. Kh nng phn gii sa (Litmus Milk Reaction)
- Mi trng: sa ti un si, lnh qua m, ly tm v ht b b lp trn, phn
di l sa khng cha lipid. C th dng sa bt loi cht bo (ho tan 100 g sa bt
vi 1000 ml nc).
- Thuc th Litmus:
ho tan 2,5 g Litmus trong 100 ml nc ct, lc bng giy lc, qua m mi s dng. C
th bo qun lu.
- Mi trng sa Litmus:
Dung dch Litmus 2,5% 4 ml
Sa loi b 1000 ml
Mi trng c mu ta.
Phn mi trng vo cc ng nghim (4 ml), kh trng gin on hay kh trng 112
0
C trong
20-30 pht.
- Cy vi khun mi hot ho (18-24 gi), nui nhit thch hp, sau 1,3,5,7,14 ngy
ly ra quan st.
- Da vo bin i ca mi trng m c nhng kt lun nh sau:
Mi trng chuyn thnh mu trng phn ng kh Litmus
Mi trng tr nn trong phn ng peptn ho
Mi trng chuyn mu phn ng sinh acid
Mi trng chuyn mu xanh lam phn ng sinh kim
Mi trng chuyn mu , sa ngng kt sinh acid v ngng kt
Ngng kt do men: khng chuyn mu hoc c mu lam, sa vn cc v ngng kt
- Ch : tt nht nn dng sa ti, khng cn iu chnh pH.

3.35. Kh nng oxy ha Gluconat
- m Kali phosphat 1/15 mol/L, pH 7,2:
A) KH
2
PO
4
9,078 g/L
B) K
2
HPO
4
.12H
2
O 23,876g/L
Trn 3 ml dung dch A vi 7 ml dung dch B c dung dch m phosphat 1/15 mol/L,
pH 7,2
- Thuc th Feling:
Dung dch A: CuSO
4
tinh th 34,64 g
Nc ct thm ti 500 ml
Dung dch B: Na-Tartrat 173 g
KOH 125 g
Nc ct thm ti 500 ml
Trc khi dng trn hai dung dch A v B theo t l 1:1 (vol/vol), s dng trong ngy.
- Chun b dung dch gluconat 1% trong m phosphat pH 7,2, phn vo cc ng nghim,
mi ng 2ml. Kh trng 112
0
C trong 30 pht.
- Ly vi khun mi hot ho (18-24 gi) to dch huyn ph m c trong m
phosphat, gi 30
0
C qua m.
- Thm vo mi ng 0,5 ml thuc th Feling, t trong bnh cch thy si 10 pht.
- Kt qu: nu dch huyn ph chuyn t mu lam sang mu vng lc, lc da cam hoc
c kt ta l phn ng dng tnh; nu khng i mu l m tnh.
- Ch : nu dng gluconat canxi th d to kt ta vi gc phosphat, tuy nhin khng
nh hng n kt qu th nghim.

3.36. Kh nng oxy ha Etanol
- Vi vi khun Acetobacter dng mi trng sau:
Cao men 10 g
Nc my 1000 ml
Xanh Bromophenol (BPB) 0,04% 20 ml.
pH = 6,8-7,0
Phn mi trng vo cc ng nghim (4-5 ml), kh trng 121
0
C trong 20 pht. Lc s
dng thm ethanol vo mi ng nng khong 2-10% (vol/vol)
- Vi cc vi khun khc: dng mi trng nh trong th nghim oxy ha/ln men, thay
ng bng etanol vi nng 1%. C th khng cn thch.
- Cy vi khun mi hot ho, nui 1,3,7,14 ngy trong iu kin thch hp
- Kt qu: nu mi trng chuyn mu vng (do sinh acid) th l dng tnh, khng i
mu l m tnh.
Phng php khc (dng phn lp v kim nh Acetobacter):
- Thm vo cc mi trng ni trn 2% thch v 1% CaCO
3
; nng etanol cui cng l
2%; khng thm ch th mu. thch a.
- Cy vi khun trn thch a, nui 3, 7, 14 ngy iu kin thch hp.
- Kt qu: nu xung quanh khun lc c vng phn gii trong l kt qu dng tnh, nu
khng l m tnh (Acetobacter lc u phn gii CaCO
3
nn to vng trong, nhng sau
acetat canxi to ra b oxy ha tip chuyn thnh CaCO
3
vng trong chuyn mu trng sa
sng do acid acetic b oxy ha).

3.37. Kh nng oxy ha acid acetic
- Mi trng:
Cao men 10 g
Ca-Acetat 10 g
Thch 20 g
Nc my 1000 ml
pH 7,0-7,2
Phn mi trng vo bnh tam gic hoc cc ng nghim ln kh trng, thch a hoc
lm ng thch nghing.
- Cy vi khun mi hot ho (18-24 gi), nui 3-5 ngy iu kin thch hp
- Kt qu: nu xung quanh khun lc c vng trng sa l phn ng dng tnh (acetat
b oxy ha, canxi gii phng ra to mu trng sa), nu khng th l m tnh.

3.38. Xc nh Indol-Pyruvic acid (IPA)
- Mi trng SIM:
Cao tht 3 g
Pepton 30 g
Na
2
S
2
O
3
.5H
2
O 0,05 g
Cystin hydrochlorid 0,2 g
Ammonium-ferric-citrat 0,5 g
Thch 4 g
Nc ct 1000 ml
Ho tan cc thnh phn mi trng trong ni cch thy, chnh pH n 7,4, phn vo cc ng
nghim nh, kh trng 121
0
C trong 15 pht, t ng thch ng.
- Thuc th Kovac (c th mua sn hoc t pha):
p-dimethyl aminobenzaldehyd 8 g
Etanol 760 ml
HCl c 160 ml
- Cy vi khun mi hot ho (18-24 gi) vo mi trng SIM, nui 30
0
C trong 24 gi.
- Kt qu: nu phn trn ca mi trng chuyn mu nu l phn ng IPA dng tnh,
nu khng l phn ng m tnh.
- Sau nh thm thuc th Kovac vo v quan st. Nu trn mt thch xut hin mu
o l phn ng Indol dng tnh.


Hnh 3.11. V d minh ho phn ng Indol dng tnh.

Ti liu tham kho :
1. James G. Cappuccino & Natalie Sherman, 2002. Microbiology: a Laboratory Manual, 6
th
ed.
Pearson Education, Inc., Sanfransisco, CA.
2. Hans Trueper & Karl-Heinz Schleifer, 2000. Prokaryote Characterization and Identification. In:
Dworkin W., Falkow S., Rosenberg E., Schleifer K.-H. & Stackebrandt E. (ed.). The Prokaryotes:
an Evolving electronic resource for the microbiological community. Springer-Verlag, New York.
3. V Th Minh c, 1998. Thc tp Vi sinh vt. NXB GD, HTH H Ni.
4. Krieg N.R. & Holt J.G. (ed.) 1984. Bergeys manual of systematic bacteriology. Williams &
Wilkins, Baltimore, USA.
5. ng T Chu et al.,2001, Thng kin t khun h thng gim nh th sch, Khoa hc xut
bn x (Trung vn).

Bi 7 Virus
1. Vi nt lch s nghin cu ca virus hc

Ngay t nm 1883 nh khoa hc ngi c Adolf Mayer khi nghin cu bnh
khm cy thuc l nhn thy bnh ny c th ly nu phun dch p l cy b
bnh sang cy lnh, tuy nhin ng khng pht hin c
tc nhn gy bnh.
Nm 1884 Charles Chamberland sng ch ra mng
lc bng s tch cc vi khun nh nht v
vo nm 1892 nh thc vt hc ngi Nga Dimitri
Ivanovski dng mng lc ny nghin cu bnh
khm thuc l. ng nhn thy dch p l cy b bnh
cho qua mng lc vn c kh nng nhim bnh cho cy
lnh v cho rng tc nhn gy bnh c l l vi khun c
kch thc nh b n mc c th i qua mng lc, hoc
c th l c t do vi khun tit ra. Gi thuyt v c t
qua mng lc b bc b
vo nm 1898 khi nh khoa hc ngi H Lan Martinus Beijerinck chng minh
c rng tc nhn ly nhim l cht c sng (Contagium vivum fluidum) v
c th nhn ln c. ng tin hnh phun dch p l cy bnh cho qua lc ri
phun ln cy v khi cy b bnh li ly dch p cho qua lc phun vo cc cy
khc. Qua nhiu ln phun u gy c bnh cho cy. iu chng t tc
nhn gy bnh phi nhn ln c v nu l c t th nng lc gy bnh s
phi dn mt i.
Nm 1901 Walter Reed v cng s Cuba pht hin tc nhn gy bnh st
vng, cng qua lc. Tip sau cc nh khoa hc khc pht hin ra tc nhn
gy bnh di v u ma. Tc nhn gy bnh u ma c kch thc ln,
khng d qua mng lc, do cc tc nhn gy bnh ch n gin gi l virus.


Dimitri Ivanovski
Martinus
Beijerinck
Walter Reed



Felix d'Hrelle Frederick Twort Wendell Stanley

Nm 1915 nh vi khun hc ngi Anh Frederick Twort v nm 1917 nh khoa
hc ngi Php Felix d'Hrelle pht hin ra virus ca vi khun v t tn l
Bacteriophage gi tt l phage.
Nm 1935 nh khoa hc ngi M Wendell Stanley kt tinh c cc ht
virus gy bnh m thuc l (TMV). Ri sau TMV v nhiu loi virus khc
u c th quan st c di knh hin vi in t.
Nh vy nh c k thut mng lc em li khi nim ban u v virus v
sau nh c knh hin vi in t c th quan st c hnh dng ca
virus, tm hiu c bn cht v chc nng ca chng.
Ngy nay virus c coi l thc th cha c cu to t bo, c kch thc siu
nh v c cu to rt n gin, ch gm mt loi acid nucleic, c bao bi v
protein. Mun nhn ln virus phi nh b my tng hp ca t bo, v th
chng l k sinh ni bo bt buc.
Virus c kh nng gy bnh mi c th sng t vi khun n con ngi, l
th phm gy thit hi nng n cho ngnh chn nui, gy tht bt ma mng
v cn tr i vi ngnh cng nghip vi sinh vt.
T nhng thp k cui ca th k XX tr li y ngy cng xut hin cc dng
virus mi l ngi, ng vt m trc y hc cha h bit ti, e do mng
sng ca con ngi. Sau HIV, SARS, Ebola, cm A H5N1 s cn bao nhiu loi
na s xut hin gy tai ho cho con ngi.
Mt khc, do c cu to n gin v c genom nhiu kiu vi c ch sao chp
khc hn cc c th khc nn virus c chn l m hnh l tng nghin
cu nhiu c ch sinh hc mc phn t dn n cuc cch mng sinh hc
cn i: Sinh hc phn t, di truyn hc phn t. V nhng l do trn vic
nghin cu virus c y mnh v tr thnh mt ngnh khoa hc c lp
rt pht trin.

2. Hnh thi v cu trc ca virus

2.1. Cu to c bn:

Tt c cc virus u c cu to gm hai thnh phn c bn: li l acid nucleic
(tc genom) v v l protein gi l capsid, bao bc bn ngoi bo v acid
nucleic. Phc hp bao gm acid nucleic v v capsid gi l nucleocapsid hay
xt v thnh phn ho hc th gi l nucleoprotein. i vi virus ARN th cn
gi l ribonucleoprotein
Genom ca virus c th l ADN hoc ARN, chui n hoc chui kp, trong khi
genom ca t bo lun l ADN chui kp, v trong t bo lun cha hai loi
acid nucleic, ADN v ARN.

2.2. V capsid:

Capsid l v protein c cu to bi cc n v hnh thi gi l capsome.
Capsome li c cu to t 5 hoc 6 n v cu trc gi l protome. Protome
c th l monome (ch c mt phn t protein) hoc polyme (c nhiu phn t
protein)
- Pentame (penton) c 5 protome nm trn cc nh ca khi a din, cn
hexame (hexon) to thnh cc cnh v b mt hnh tam gic.
- Capsid c kh nng chu nhit, pH v cc yu t ngoi cnh nn c chc nng
bo v li acid nucleic
- Trn mt capsid cha cc th th c hiu, hay l cc gai glicoprotein, gip
cho virus bm vo cc th th trn b mt t bo. y cng chnh l cc
khng nguyn (KN) kch thch c th to p ng min dch (MD).
- V capsid c kch thc v cch sp xp khc nhau khin cho virus c hnh
dng khc nhau. C th chia ra ba loi cu trc: i xng xon, i xng hnh
khi v cu trc phc tp (Hnh 1).

Hnh 1. Kch thc v hnh thi ca mt s virus in hnh .Theo Presscott L.
M. et al. , Microbiology. 6th ed. Intern. Ed. 2005.

2.2.1 Cu trc i xng xon:

S d cc virus c cu trc ny l do capsome sp xp theo
chiu xon ca acid nucleic. Tu loi m c chiu di, ng
knh v chu k lp li ca cc nucleocapsid khc nhau. Cu
trc xon thng lm cho virus c dng hnh que hay hnh si
v d virus m thuc l (MTV), di (rhabdo), quai b, si
(paramyxo), cm (orthomyxo). virus cm cc nucleocapsid
c bao bi v ngoi nn khi quan st di knh hin virus
in t thy chng c dng cu.






2.2.2 Cu trc i xng dng khi a din 20 mt

Hnh ca phng th nghim Robert M Bock i hc University of Wisconsin-
Madison.

cc virus loi ny, capsome sp xp to v capsid hnh khi a din vi 20
mt tam gic u, c 30 cnh v 12 nh. nh l ni gp nhau ca 5 cnh
thuc loi ny gm cc virus adeno, reo, herpes v picorna. Gi l i xng v
khi so snh s sp xp ca capsome theo trc. V d i xng bc 2, bc 3,
bc 5, v khi ta xoay vi 1 gc 1800 (bc 2), 1200 (bc 3) v 720 (bc 5) th
thy vn nh c.

Cc virus khc nhau c s lng capsome khc nhau. Virus cng ln, s lng
capsome cng nhiu. Da vo s lng capsome trn mi cnh c th tnh
c tng s capsome ca v capsid theo cng thc sau:
N= 10(n-1)2+2
Trong N- tng s capsome ca v capsid, n-s capsome trn mi cnh.


Hnh 2.
A S virus hnh que vi cu trc i xng xon (virus khm thuc l).
Capsome sp xp theo chiu xon ca acid nucleic.
B- S virus a din n gin nht. Mi mt l mt tam gic u. nh do 5
cnh hp li. Mi cnh cha 3 capsome.
C- S i xng ca hnh a din th hin khi quay theo trc bc 2 (1800), bc
3 (1200) v bc 5 (720). Theo J. Nicklin et al., Instant Notes in Microbiology,
Bios Scientific Publisher, 1999.


2.2.3 Virus c cu to phc tp

Mt s virus c cu to phc tp, in hnh l phage v virus u ma. Phage
c cu to gm u hnh khi a din, gn vi ui c cu to i xng xon.
Phage T chn (T2, T4, T6) c ui di trng ging nh tinh trng, cn phage T
l (T3,T7) c ui ngn, thm ch c loi khng c ui (?6, ?X174).
Virus u ma c kch thc rt ln, hnh vin gch. gia l li lm hai pha
trng nh qu t. i din vi hai mt lm l hai cu trc dng thu knh gi l
th bn. Bao bc li v hai th bn l v ngoi.

2.3 V ngoi:

Mt s virus c v ngoi (envelope) bao bc v capsid. V ngoi c ngun gc
t mng sinh cht ca t bo c virus cun theo khi ny chi. V ngoi c
cu to gm 2 lp lipid v protein.
Lipid gm phospholipid v glycolipid, hu ht bt ngun t mng sinh cht (tr
virus pox t mng Golgi) vi chc nng chnh l n nh cu trc ca virus.
Protein v ngoi thng l glycoprotein cng c ngun gc t mng sinh cht,
tuy nhin trn mt v ngoi cng c cc glycoprotein do virus m ha c
gn trc vo cc v tr chuyn bit trn mng sinh cht ca t bo, ri v sau
tr thnh cu trc b mt ca virus. V d cc gai gp 120 ca HIV hay
hemaglutinin ca virus cm, chng tng tc vi receptor ca t bo m
u s xm nhp ca virus vo t bo.
V ngoi cng c ngun gc t mng nhn do virus lp rp v ny chi qua
mng nhn (virus herpes)
Di tc ng ca mt s yu t nh dung mi ho tan lipid, enzym, v ngoi
c th b bin tnh v khi virus khng cn kh nng gy nhim na.

2.4 Protein ca virus :

2.3.1 Cc phng php nghin cu protein virus

Trc ht cn phi tch chng khi t bo. iu ny c th thc hin c nh
hng lot cc bc ly tm tch, tip l ly tm theo gradient nng
saccaroza.
Ly tm gradient nng saccaroza thng cho kt qu th hin cc bng
(band) rt r nt ti cc v tr c th trn gradient. Cc bng ny c dng
cho cc nghin cu tip theo. Thng thng nghin cu cc virion nh du
ng v phng x, ngi ta dng hng lot k thut nh in di trn gel
polyacrylamit, western Blotting (phn ng vi khng th).
V tr protein ca virus trong t bo c th xc nh c nh k thut nhum
phn bit v min dch hunh quang, cho khng th n dng tng tc vi
epitop c hiu ca protein sau dch m th dng cc cht c ch proteaza v
c ch qu trnh glycosyl ho.
Vic xc nh trnh t gen v vic d on acid amin s gip hiu c cu trc
v chc nng ca chng.

2.4.2 Cc loi protein virus

Protein virus c tng hp nh mARN ca virus trn riboxom ca t bo. Tu
theo thi im tng hp m c chia thnh protein sm v protein mun.
Protein sm do gen sm m ho, thng l enzym (protein khng cu trc)
cn protein mun do gen mun m ho, thng l protein cu trc to, nn v
capsid v v ngoi.

Protein khng cu trc

Protein khng cu trc c th c gi vo trong virion, nhng khng phi l
thnh phn cu to virion. y l cc enzym tham gia vo qu trnh nhn ln
ca virus, v d enzym phin m ngc, proteaza v integraza ca virus retro,
timidinkinaza v ADN polymeraza ca HSV.
Protein khng cu trc khc ch c mt trong t bo nhim m khng c a
vo virion, bao gm cc protein tham gia vo qu trnh iu ho sao chp,
phin m, dch m (v d Tat ca HIV, Protein mng trong ca HSV, helicaza,
protein gn ADN...); protein c ch qu trnh tng hp acid nucleic v protein
ca t bo ch. Ngoi ra thuc loi ny cn c cc protein gy ung th do cc
oncogen m ha; cc protein gy chuyn dng t bo, nh khng nguyn T ln
ca SV-40 hoc protein EBNA ca virus Epstein.Barr. mt s virus c protein
khng cu trc lin quan n hot tnh anti-apoptosis v anti-cytokin...

Protein cu trc

Capsid ca rhinovirus 14 c
dng hnh cu ng knh 4
angstrom gm 4 protein l
VP1, VP2, VP3 v VP4 (VP4
khng thy trn hnh). VP1 c
hnh ng gic , i xng bc
5
Trong hnh ny, khi g 2 VP1 hnh
ng gic, s thy c bn trong VP4
mu vng
Hnh ca phng th nghim Robert M Bock i hc University of Wisconsin-
Madison.

Protein cu trc tham gia vo cu to ht virus, lm cho chng c hnh dng,
kch thc nht nh v bo v genom ca virus khi cc iu kin bt li.
Protein cu trc bao gm protein ca nucleocapsid, protein nn (matrix),
protein v ngoi (Hnh 3). Protein nucleocapsid c th t lp rp (v d TMV,
polio) hay lp rp vi s tr gip ca mt khung protein tm thi, lm nhim
v dn gio to u phage hoc cu trc khi a din, protein ny ch tn
ti khi lp rp nucleocapsid v s b mt i virus trng thnh. V d capsid
ca virus polio c cu to tng i n gin, gm 4 protein l VP1, VP2, VP3
v VP4. Cc protein ny tham gia lp rp to capsid thng qua mt cu trc
tin cht (procapsid), bao gm VPO (mt protein tin cht) v VP1, VP3.
Protein VPO li c ct thnh VP2 v VP4 khi v capsid tin hnh lp rp vi
acid nucleic ca n. Capsid ca virus reo phc tp hn nhiu. y l capsid
trn c hai lp v. Protein capsid ngoi chc nng bo v acid nucleic genom
v lp rp hnh thnh virion cn phi tng tc vi acid nucleic genom trong
sut qu trnh lp rp. S bao gi phn t acid nucleic vo trong vng xc
nh, cn phi c s sp xp v nn genom li v bo hiu s kt hp vi
protein capsid la chn, thng qua lin kt ha hc. V d protein N ca
virus rhabdo v protein NP ca virus cm l cc protein c acid amin mang
in tch dng s tng tc vi acid nucleic mang in tch m, nn chng s
ht nhau, to thun li cho vic lp rp. Vic bc gi s tr nn phc tp hn
khi virus c genom nhiu on, v d virus cm c 8 on ARN cn phi bc
gi trong cng mt v capsid.
V ngoi bao quanh nucleocapsid c hnh thnh t mng nhn, mng sinh
cht hoc mng li ni cht khi virus ny chi. Pha trong ca v ngoi l
protein glycolipid.
Protein nn l protein nm pha trong, gia v
capsid v v ngoi, gi mi lin kt gia hai v
ny. Chng thng khng c glycosyl ha v
c th cha cc protein xuyn mng lm
neo, hoc c th lin kt vi mng nh cc
vng k nc nm trn b mt hoc nh mi
tng tc gia protein ca chng vi
glycoprotein v ngoi. HSV, khong khng
gia v ngoi v capsid l lp v nh hnh
c xem nh mt lp mng (tegument).
Glycoprotein ngoi ca virus c neo vo v
nh cc protein xuyn mng. Phn ln chng
nm nh ra pha ngoi v vi mt ci ui ngn
pha trong. Nhiu glycoprotein l monome,
chng ghp li vi nhau to thnh nhng chic
gai c th quan st c di knh hin vi in
t. cc virus c v ngoi, cc gai ny c chc
nng khng nguyn, v d gai G virus di, gai gp 120 HIV v gai HA virus
cm.
c virus trn (v d polio) v virus c v ngoi cc protien cu trc ny s
tng tc vi receptor trn b mt t bo ch m u cho qu trnh gy
nhim. C th chng tr li thng qua p ng min dch. Da vo mi tng
tc ny thit k vacxin chng virus.



1 : Nucleocapsid
2: Protein nn
3: V ngoi
4: Cu disulfur
5: ui trong gn protein nn
6: Knh vn chuyn
7: Glycoprotein (gai ph)
8: Protein vn chuyn mng
9: Glycoprotein v ngoi





Hnh 3. Mt s lp protein lin kt vi v ngoi virus. Cc protein nn ni v
ngoi vi nucleocapsid. Glycoprotein do virus m ha c gn sn vo v
ngoi m nhim nhiu chc nng. Cc gai glycoprotein chu trch nhim
nhn bit th th b mt ca t bo v gn vo trong khi cc protein xuyn
mng hot ng nh l nhng knh vn chuyn qua v ngoi. Cc protein bt
ngun t t bo ch i khi cng lin kt vi v ngoi nhng vi lng nh.
(Cann.A, Principle of Molecular Virology,1993, Academic press).

Tt c cc protein ca virus u c dch m t mARN ca virus. Cc mARN
ny c th
a)- c phin m t genom ADN ca virus (v d HSV), hoc
b)- t mch b sung vi genom ARN m (v d virus cm) hoc
c)-chnh l genom ca virus ARN dng (v d virus bi lit) Protein ca virus
c th c x l sau dch m. Lc u tng hp mt protein ln (polyprotein)
sau nh proteaza phn ct to cc phn t nh (v d polyprotein ca
virus polio, phc hp gag-polymeraza ca virus HIV). Chng c th c
phosphoryl ha. Mc phosphoryl ha thng xc nh mc chc nng
ca protein (v d protein N v NS ca virus rhabdo). S gn gc ng vo
protien (glycosyl ho) l gn c vi N v O. i vi nhiu protein virus (thng
l glycoprotein v ngoi) gc ng c th chim 70% trng lng protein.
Cc bin i sau dch m khc nh myristyl ha, acyl ho v palmitoyl ha
cng c tin hnh.




Hnh 4. S cc h v chi ca phage. Theo Prescott L. M. et al., Microbiology
6th ed. Intern. Ed., 2005






Hnh 5. S m t cc h v chi ca virus thc vt. Theo Prescott L. M. et
al. , Microbiology 6th ed. Intern ed. , 2005






Hnh 6. S miu t cc h v chi ca virus k sinh ng vt khng xng
sng (RT ch virus cha enzym phin m ngc). Theo Prescott L. M. et al.,
Microbiology 6th ed. Intern ed., 2005

2.4 Acid nucleic ca virus

2.5.1 Cc loi genom ca virus

Nh trn ni, genom ca virus rt a dng v cu trc, kch thc v thnh
phn nucleotid. Chng c th l ADN hoc ARN, chui n hoc kp, thng
hoc khp vng. Kch thc genom c th t 3500 nucleotid ( phage nh) n
560.000 nucleotid ( virus herpes). Cc trnh t genom virus phi c c m
bi t bo ch, cho nn cc tn hiu iu khin phi c cc yu t ca t bo
ch nhn bit. Cc yu t ny thng lin kt vi protein virus. Do c kch
thc nh nn genom virus tin ho s dng ti a tim nng m ha
ca mnh. V th hin tng gen chng lp v hin tng ct ni (splicing)
mARN virus l rt ph bin.




Hnh 7. S genom ca virus ARN cho thy s phn b ca cc gen m ho
cho protein cu trc, protein khng cu trc, cng nh cc vng khng dch
m UTR (unstranslated region). Theo J. Nicklin et al., Instant Notes in
Microbiology, Bios Scientific Publisher, 1999.

Genom ca virus c xc nh da theo cc thng s sau:
* Thnh phn acid nucleic (ADN hay ARN).
* Kch thc genom, chui n hay kp.
* Cu trc u chui
* Trnh t nucleotid
* Kh nng m ho
* Cc yu t iu ho, promoter, enhancer v terminater

Mt s c im ca genom virus cn lu :

* Genom ADN kp (v d virus pox, herpes v adeno) thng c kch thc
ln nht.
* Genom ADN kp khp vng (siu xon hoc khng siu xon) thng thy
phage
* Genom ADN kp virus vaccinia c hai u khp kn
ADN n dng thng (v d virus parvo) c kch thc rt nh.
Cc ADN dng thng thng c trnh t lp li u.
* Tt c genom ARN kp u phn on (cha mt s on khng ging nhau,
mang thng tin di truyn tch bit).
* Genom ARN n c phn thnh ARN dng (genom +) v ARN m (genom
-) da vo trnh t nucleotid ca mARN.
Phn ln genom ARN n u khng phn on tr virus orthomyxo (virus
cm).
* Virus retro c genom l hai phn t ARN n ging nhau, ni vi nhau u
5 nh cu ni hydro.
* Virus m cyAlfalfa (AMV) c genom gm 4 on ARN n, dng, dng
thng, c gi vo 4 v capsid khc nhau nn cn gi l virus d capsid
(hetero-capsidic) phn bit vi virus m tt c cc on u c gi trong
mt ht-virus ng capsid (isocapsidic).

2.5.2 Phng php nghin cu

Nhng tin b v sinh hc phn t trong vi thp nin gn y gip cho
vic nghin cu acid nucleic tr nn d dng v nhanh chng hn. H gen ca
cc i din ca hu ht cc h virus u c gii trnh t, cc khung c m
ca chng c bit r, cc sn phm ca h gen c xc nh tnh
cht. iu cho php c th so snh cc trnh t bit trong ngn hng
gen vi cc trnh t ang nghin cu v so snh vi cc trnh t ca cc sinh
vt khc, nhn s v nhn tht, qua c th thy s tng ng cng nh s
tin ho trong sinh gii.
Gen virus cng c th c tch dng vo cc vect khc nhau v c phn
tch nh k thut pht sinh t bin im nh hng (site-directed
mutagenesis) v k thut pht sinh t bin im c hiu (site-specific
mutagenesis) nghin cu vai tr ca cc acid amin ring bit trong vic xc
nh cu trc v chc nng ca protein.
Virus ADN thng c biu hin trn s l mt phn t dng thng vi cc
v tr enzym gii hn nm ri rc khp genom. C hng chc enzym gii hn
c dng phn ct ADN thnh cc on nh vi trnh t nucleotid c th.
Mi genom ADN c mt bn enzym ct gii hn c trng cho chng. iu
ny khng th c vi genom ARN, tr phi nh enzym phin m ngc tin
hnh tng hp cADN t ARN khun. Lc cADN s b enzym gii hn ct.
Acid nucleic ca virus c th c c trng bi nhit nng chy (Tm), mt
ni trong gradient nng xesi clorua (CsCl), gi tr S trong gradient nng
saccaroza, c hoc khng c kh nng gy nhim, s mn cm vi nucleaza
v s xut hin di knh hin vi in t

Loi acid
nucleic
Cu trc V d
ADN n
Chui n, dng thng
Chui n, khp vng
Virus parvo
Phage jX174, M13, fd
Herpes, adeno, coliphage T, phage l.
ADN
kp
Chui kp, dng thng
Chui kp, dng thng, trn mt
mch c nhng ch t cu ni
phosphodieste.
Chui kp vi hai u khp kn
Chui kp khp vng kn
Coliphage T5
Vaccinia, Smallpox
Polioma (SV40), papiloma, phage
PM2, virus m hoa l

ARN n

Chui n, dng dng thng
Chui n, m, dng thng
Chui n, dng, dng thng, nhiu
on.
Chui n, dng dng thng gm hai
on gn vi nhau.
Chui n, m dng thng, phn on

Picorna (polio, rhino), toga, phage
ARN, MTV v hu ht virus thc
vt.
Rhabdo, paramyxo, (si, quai b)
Virus m cy tc mch (Bromus)
(cc on c bao gi trong cc
virion tch bit).
Retro (HIV, Sarcoma Rous)
ARN
kp
Chui kp, dng thng, phn
on
Orthomyxo (cm)

Reo (rota), mt s virus gy u
thc vt, NPV cn trng, phage
j6 v nhiu virus nm
(mycovirus).

Bng cc loi acid nucleic ca virus


Kch thc genom thay i rt nhiu cc virus khc nhau. Cc genom nh
nht (v d Bactariophage MS2, Q) c kch thc 1x10
6
Da m ha cho
3-4 protein. Mt s virus khc tn dng ti a khng gian ca genom bng
cch s dng cc gen chng lp, tc l cc gen gi ln nhau trn cng khung
c, ch khc nhau dim khi u hoc kt thc.
Cc genom ca coliphage T chn, herpes, vaccinia c kch thc 1,6x10
8
Da c
th m ha cho 100 protein.

Genom ADN

* Cc virus ADN c kch thc rt nh (nh x 174, M13 hay parvo) thng c
genom l ADN chui n. Mt s l ADN n, dng thng, song mt s khc li
khp vng.
* Hu ht virus ADN s dng ADN kp lm vt liu di truyn. Mt s cha
genom ADN kp dng thng nhng s khc li cha ADN kp dng vng.
Phage lamda cha ADN kp dng thng nhng c hai u dnh l on n b
sung di 12 nucleotid nn c th bt cp khp vng.
* Ngoi cc nucleotid thng thng, nhiu virus cn c cc base c bit, v
d phage T chn k sinh E.coli mang 5 hydroxymetyl cytosin thay v cytosin.
Glucoza thng gn vo nhm hydroxymetyl.
* virus ADN kp c kch thc ln (v d virus h herpes) genom c cu to
kh phc tp. Kch thc genom thay i, t virus herpes simplex v varicella
zoster (120 180kbp) n virus cytomegalo v HHV-6 (180 230 kbp). ADN m
cho hn 40 protein cu trc v hn 40 protein khng cu trc. Cu trc genom
t thay i gia cc thnh vin trong h nhng chng l nhm duy nht cha
cc ng phn (isomer) ca cng mt phn t ADN. Mi ht cha mt ng
phn gm hai on ni vi nhau bng lin kt cng ha tr, on di duy nht
(UL) v on ngn duy nht (US). hai u mi on li c cc on ngn lp
li tri chiu. Cc on ny khc nhau cc ht virus khc nhau, do lm
cho genom thay i t nhiu (ln hoc nh hn kch thc trung bnh).
* Genom ca virus adeno l dng thng c kch thc 30 - 38 kbp nh hn
genom ca virus herpes. Mi virus cha 30 40 gen. Genom c hai u lp li
tri chiu di 100-180 kbp. on 50 base u tin kh ging nhau cc virus
khc nhau v thng cha nhiu cp A-T. im ni bt ca on u phn t
ADN virus adeno l khi genom tch khi virion mt mch s to vng
"panhandle" v oligome. iu ny lin tng n phage , nhng khc ch
ADN ca adeno khng c u n. mi u 5 ca genom c gn mt protein
55 kDa. Protein ny ng vai tr quan trng trong sao chp.

Genom ARN

Virus ARN thng c genom nh hn genom ca virus ADN
* Cc phn t ARN c chia lm hai loi: ARN (+) v ARN (-)
ARN (+) c trnh t nucleotid trng vi trnh t nucleotid ca mARN, nn c
th dng thay cho mARN trong qu trnh dch m.
ARN (-) c trnh t b sung vi mARN
* C ch tng hp mARN l c im quan trng phn bit cc virus ARN
* Hu ht cc phn t mARN eukaryota l n gen (monocistronic), ch m
ha cho mt protein, trong khi tt c cc virus ARN bit u l a gen
(Polycistronic), m ha cho nhiu protein
* Genom ARN khng dng lm khun trc tip tng hp ARN ca virion m
phi qua mch trung gian
* a s ARN (+) u c m u 5 bo v khi tc ng ca phosphataza
v nucleaza. virus picorna m c thay th bi protein VPg (protein gn vi
genom).
* u 3 ca a s genom ARN (+) c gn ui poly (A) ging nh mARN
ca eukaryota
* Virus ARN (-) thng c genom ln hn virus ARN (+)
Mt s virus ARN c genom phn on. V d virus cm c 8 on ARN (-),
virus reo c 10 12 on ARN kp. Cc on ny khng ging nhau v m ha
cho cc protein khc nhau, trong khi 2 phn t ARN (+) virus retro th ging
ht nhau.

3. Phn loi virus:

Virus hc c lch s trn 100 nm, tuy nhin cc nguyn tc phn loi virus
th vn cn rt mi m. Vo nhng nm u ca th k trc cc virus u
tin c phn loi ch bng mt cch n gin l cho chng i qua mng lc
vi khun. Nhng khi s lng virus tng ln th lc phi phn bit chng
da vo kch thc, vo vt ch v vo cc triu trng bnh do chng gy ra.
V d tt c cc virus ng vt c kh nng gy vim gan u xp thnh mt
nhm gi l virus vim gan hay tt c cc virus thc vt c kh nng gy m
trn l cy u xp vo mt nhm gi l virus m. V sau, vo nhng nm
30, nh s bng n v k thut, gip ngi ta m t c cc c im vt
l ca nhiu loi virus, cung cp nhiu c im mi c th phn bit c
cc virus khc nhau. Cc k thut ny bao gm phng php phn lp, tinh
sch virus, xc nh c im ho sinh ca cc virion, cc phng php huyt
thanh hc v c bit l s ra i ca knh hin vi in t gip m t c
hnh thi ca nhiu loi virus khc nhau. n nhng nm 50 da trn cc c
im ny ngi ta phn bit c ba nhm virus quan trng ng vt l
Myxovirus, Herpesvirus v Poxvirus.
Vo nhng nm 60, cc kin thc v d liu v virus rt phong ph, i hi
phi cho ra i mt t chc ca cc nh virus hc thng nht v h thng
phn loi vi cc qui tc cht ch v cch t tn... l U ban quc t v
phn loi virus, gi tt l ICTV (International Committee on Nomenclature of
Viruses), c thnh lp nm 1966. Chc nng ca ICTV l tho lun i n
thng nht v cc qui tc phn loi, cch t tn, xy dng th mc v cung
cp thng tin cho cc nh virus hc trn khp th gii. Cc thng tin ca ICTV
c th truy cp theo website <http://www.ncbi.nlm.gov/ICTV/>.
ICTV a ra cc tn chun phn loi da trn cc c im hnh thi, bao gm
kch thc, hnh dng, kiu i xng ca capsid, c hay khng v ngoi; cc
c im vt l bao gm cu trc genom, s mn cm i vi cc tc nhn l,
ho; cc c im ca lipid, cacbohidrat, cc protein cu trc v khng cu
trc; c trng khng nguyn v cc c im sinh hc khc nh phng thc
nhn ln, loi vt ch, phng thc ly truyn v kh nng gy bnh.
Tuy nhin ngi ta c on rng mun xc nh chnh xc mt loi virus mi
cn phi c ti khong 500 c im. ICTV ang to dng c s d liu
(database) cho php lit k cc loi virus n mc chng.

U ban cng thng nht a ra cc khi nim v cc th bc trong phn
loi, bao gm:

B virus (order).
B l i din cho cc nhm ghp ca cc h, c cc c im chung khc bit
vi cc b v h khc. Cc b c k hiu bi nhng v t (suffixe) -virales.
C mt b c ICTV chp thun l Mononegavirales bao gm cc h
Paramyxoviridae, Rhabdoviridae v Filoviridae. l cc virus ARN n, m,
khng phn on v c v ngoi.

H virus (family).
H l i din cho cc nhm ghp ca cc chi, c cc c im chung khc vi
cc thnh vin ca cc h khc. H c v t -viridae, ng vai tr trung tm
v thng c tip u ng mang c im c trng. V d Picornaviridae l t
ghp pico/rna/viridae (pico ting l nh) gm cc virus c kch thc nh;
Flavoviridae - tin Latinh flavo l vng (v trong c virus gy bnh st vng)
mt s h (v d Herpesviridae) c s khc nhau gia cc thnh vin trong
h, dn n s hnh thnh cc h ph, c k hiu vi v t -virinae. Nh
vy h Herpesviridae cn c phn tip thnh cc h ph Alphaherpesvirinae,
(virus, Herpes simplex), Betaherpesvirinae (virus cytomegalo) v
Gammaherpesvirinae (virus Epstein-Barr).

Chi virus.
Chi l i din cho cc nhm ghp ca cc loi, c cc c im chung v khc
vi cc thnh vin ca cc chi khc. Tn chi thng c v t -virus. Cng nh
tn h, tn chi thng c tip u ng mang c im c trng. V d
Rhinovirus (Rhino ting Hy Lp l mi, m ch virus gy bnh s mi. Cc tiu
chun phn nh cc chi thay i gia h ny vi h khc nhng chng vn
bao gm s khc nhau v cc c im di truyn, cu trc v cc c im
khc.

Loi virus.
Loi virus c nh ngha nh l mt lp phn loi da trn mt s lng ln
cc c im lp (polythetic class), to lp mi lin h vi nhau v sao chp v
chim mt sinh thi ring bit. ICTV hin ang xem xt mt cch cn trng
cc c im thit yu cn phi c xc nh loi. S phn chia gia loi v
chng vn cn l mt vn kh khn.

Mt s tnh cht c ICTV s dng trong phn loi loi Tnh cht ca
virion

c im hnh thi.

* Kch thc virion
* Hnh dng virion
* C hay khng c peplome (liproprotein v ngoi) v bn cht ca peplome
* C hay khng c v ngoi (envelope)

* Kiu i xng v cu trc ca capsid

Tnh cht ho l, vt l.

Khi lng phn t ca virion (M2).
Mt ni (buoyant density) ca virion (trong CSCl, sacaroza vv).
H s lng ca virion.
Tnh n nh pH.
Tnh n nh nhit.
Tnh n nh vi cc cation (Mg2+, Mn2+).
Tnh n nh vi cc dung mi.
Tnh n nh vi cc cht ty ra.
Tnh n nh vi chiu x.

Genom

Loi acid nucleic (ADN hay ARN).
Kch thc genom, tnh theo Kb hoc Kbp.
Acid nucleic si n (ss) hay si kp (ds).
Chui thng hay khp kn.
Phn cc (ngha - dng, m hay lng cc).
S lng v kch thc cc on (segment).
Trnh t nucleotid.
C cc trnh t lp li.
C cc ng phn.
T l G+C.
C hay khng m u 5.
C hay khng protein lin kt u 5.
C hay khng c ui poly (A) u 3.

Cc protein.

S lng, kch thc v hot ng chc nng ca cc protein cu trc.
S lng, kch thc v hot ng chc nng ca cc protein khng cu trc.
Nhng chi tit v hot ng chc nng ca cc protein, c bit l
transcriptaza phin m ngc, hemaglutinin, neuraminidaza v cc protein
dung hp.
Trnh t ton phn hoc tng phn acid amin ca protein.
Tnh cht glycosyl ho, phosphoryl ha, myristyl ho ca protein.
Lp bn ca quyt nh khng nguyn (epitop)

Cc lipid

Hm lng, c im.

Hydrat cacbon

Hm lng, c im

Cu trc genom v phng thc sao chp.

Cu trc genom.
Phng thc sao chp.
S lng v v tr khung c.
c im phin m.
V tr tch lu cc protein ca virion.
V tr lp rp virion.
V tr v bn cht ca s hon thin v gii phng virion.

Tnh cht ca khng nguyn

S ging nhau v typ huyt thanh, c bit nhn c t cc trung tm lin
quan.

Cc c im sinh hc.

Phm vi vt ch t nhin.
Phng thc ly truyn trong t nhin.
Mi quan h vi vect truyn bnh.
S phn b a l.
Kh nng gy bnh v bnh lin quan.
Tnh hng m, bnh l hc v bnh l hc m.

H thng phn loi Baltimore.

Nm 1971, David Baltimore a ra h thng phn loi virus da trn mi quan
h gia genom virus v mARN. Theo tt c cc virus u c chia ra lm 6
nhm hay 6 lp, bt k chng l virus ca ng vt, thc vt hay vi sinh vt.

Lp I:
Virus c genom l ADN kp, mARN c tng hp ging nh t bo, tc l
dng si ADN(-) lm khun.

Lp II:
Virus c genom l ADN n. thi im a ra h thng phn loi, khoa hc
mi ch bit n genom ADN n, dng, nn khi pht hin ra genom ADN m
th lp II c tch ra l IIa v IIb. i vi genom ADN n, mun tng hp
mARN phi qua giai on tng hp ADN kp trung gian, gi l dng sao chp
(RF - replicative form).

Lp III:
Virus c genom ARN kp. Mt trong hai si tng ng vi mARN.

Lp IV:
Virus c genom ARN n, (+). Do c trnh t nucleotid trng vi trnh t
nucleotid ca mARN nn c th dng trc tip lm mARN. Lp IV li chia thnh
IVa v IVb da trn s khc bit v c ch biu hin v sao chp genom.

Lp V:
Virus c genom ARN n, (-). Do c trnh t nucleotid ngc vi trnh t
nucleotid ca mARN, nn khng th dng trc tip lm mARN. Lp V cng
c chia thnh Va v Vb da trn s khc bit v c ch biu hin v sao
chp genom.

Lp VI:
Virus c genom l ARN. Trong qu trnh biu hin v sao chp cn phi c giai
on tng hp phn t ADN kp. Virus retro thuc lp ny.

Bng 1. Cc nhm virus chnh phn theo Baltimore.
Nhm Vt ch Hnh thi Cc virus i din
Lp I. Virus c genom ADN kp
Myoviridae
Siphoviridae
Vi khun
Vi khun
Phc tp
Phc tp
T4

Rodoviridae
Papovaviridae
Adenoviridae
Herpesviridae
Poxviridae
Baculoviridae
Hepadnaviridae
Caulimoviridae
Vi khun
ng
vt
ng
vt
ng
vt
ng
vt
Cn
trng
ng
vt
Thc vt
Phc tp
Khi, khng v ngoi
Khi, khng v ngoi
Khi, c v ngoi
Phc tp
Xon, c v ngoi
Khi, c v ngoi
Khi, khng v ngoi
T7
Polyoma , SV40
Adeno
Herpes simplex, varicella-
zoster
u ma, u b
Virus nhn a din (NPV)
Virus vim gan B (HBV)
Khm hoa l
Lp II. Virus c genom ADN n
Microviridae
Parvoviridae
Geminiviridae
Vi khun
ng vt
Thc vt
Khi, khng v ngoi
Khi, khng v ngoi
Khi, hai ht gn thnh i
X174
Virut parvo, virut lin quan Adeno
Sc ng
Lp III. Virus c genom ARN kp
Reoviridae ng vt Khi, khng c v ngoi Rota, reo
Lp IV. Virus c genom ARN n, dng
Leviviridae
Picornaviridae
Caliciviridae
Togaviridae
Flaviviridae
Coronaviridae
Potyviridae
Tymovirus
Tobamoviridae
Cormoviridae
Vi khun
ng vt
ng vt
ng vt
ng vt
ng vt
Thc vt
Thc vt
Thc vt
Thc vt
Khi, khng v ngoi.
Khi, khng v ngoi.
Khi, c v ngoi.
Xon, c v ngoi.
Xon, khng v ngoi.
Khi, khng v ngoi.
Xon, khng v ngoi.
Khi, khng v ngoi.
Xon, khng v ngoi.
Khi, khng v ngoi.
MS2, QB.
Polio, rhino, HAV, Coxsackie.
Norwark.
Rubella, Sindbis.
HCV, st vng, vim no Nht Bn,
Denge.
SARS, vim gan chut.
Khoai ty typ Y
Khm vng Tulyp.
Khm thuc l.
Khm u a.
Lp V. Virus c genom ARN n, m.
Rhabdoviridae
Paramyxoviridae
Bunyaviridae
Arenaviridae
ng, thc vt.
ng vt
ng vt
ng vt
Xon, c v ngoi
Xon, c v ngoi
Xon, c v ngoi
Xon, c v ngoi
Di, hoi t vng rau dip.
Quai b, si, cm.
Hanta, Phelebovirus (St thung lng Rift).
Lassa, Junin, Machupo.
Filoviridae ng vt Xon, c v ngoi Ebola, Marburg
Lp VI. Virus c genom ARN dng, nhn ln cn ADN kp trung gian.
Retroviridae ng vt
Khi, c v
ngoi
AIDS, HTLV-I, HTLV-II


Hnh 8. Nguyn tc phn loi virus theo Baltimore

S phn chia genom ca virus da trn mARN. acid nucleic (genom) c trnh
t nucleotid ging vi trnh t ca mARN, c quy c l (+) hay genom (+).
Ngc li nu c trnh t b sung vi mARN c quy c l (-) hay genom (-
). Genom ADN n mun to thnh mARN phi qua dng trung gian (RF). Tt
c virus ARN (+) lp IV mun to mARN. u phi phin m t khun ARN
(-).

Bng tm tt cc c im chnh ca cc nhm virus theo Baltimore (Bruce
A.Voyles, 2002).

I. Lp I: Virus c genom ADN kp

A- Bacteriophage

1. Myoviridae (nhm phage T chn- T2, T4, T6)

a. Genom: Dng thng hai u dnh, kch thc 172 kbp, khi lng phn t
20x106 dalton.
b. Cu to: u 110x8nm, ui co c, di 110nm
c. Hp ph v xm nhp: Nh lng ui; xm nhp nh sp xp li protein bao
ui.
d. Biu hin genom: ARN polymeraza ca vt ch c ci bin nhn din
promoter cu phage
e. Sao chp genom: Hnh thnh concateme dng thng nh cc u dnh (to
chui gm cc on trng lp)
f. Lp rp: Ngu nhin, gii phng: lm tan bo.

2. Siphoviridae (nhm phage l).

a. Genom: Dng thng vi hai u dnh, kch thc 48 Kbp, khi lng phn t
33x106 dalton.
b. Cu to: u c ng knh 60nm. ui di n 500nm, khng co c.
c. Hp ph v xm nhp: Nh ui
d. Biu hin genom: Dng ARN polymeraza ca t bo
e. Sao chp genom: Khp vng nh c 2 u dnh. Lc u sao chp dng mt
(theta), sau theo c ch vng trn xoay.
f. Lp rp/ gii phng: Cc thnh phn c lp rp nhu nhin; gii phng
nh lm tan bo.
g. Dng khc: Genom gn xen vo nhim sc th ca t bo v dng tim
tan.

3. Podoviridae (nhm phage T3, T7)

a. Genom: Thng, hai u dnh, kch thc 40 kbp, khi lng phn t 25x106
dalton
b. Cu to: u c ng knh 65nm, ui di 20nm
c. Hp ph v xm nhp: Nh ui
d. Biu hin genom: Lc u dng ARN popymeraza ph thuc ADN ca t bo
phin m cho 3 gen tin sm. Sn phm ca cc gen ny tham gia vo iu
ha t bo ch v chun b sao chp v biu hin gen virus.
e. Sao chp genom: To thnh concateme dng thng nh cc u dnh bt
cp
f. Lp rp v gii phng: Lm tan bo.

B- Virus ng vt

1. Papovaviridae

a. Genom: Khp vng, 5-8 Kbp, khi lng phn t 3-5x106 dalton
b. Cu to: Capsid dng khi, khng c v ngoi, ng knh 40-55nm
c. Hp ph v xm nhp: Nhp bo, ci o trong nhn.
d. Biu hin genom: Dng ARN polymeraza ca vt ch to ARN, sau
nh ct ni (splicing) to cc mARN khc nhau.
e. Sao chp genom: Dng ARN polymeraza ca vt ch. Sao chp theo c ch
theta
f. Lp rp trong nhn. Gii phng nh tan bo.
g. Gy bnh: HPV (mn cc, ung th c t cung), SV40.

2. Adenoviridae

a. Genom: Dng thng, 36-38 Kbp, khi lng phn t 30x106 dalton vi hai
u lp li tri chiu. u 5 c gn protein.
b. Cu to: Hnh khi vi cc si glycoprotein nh, khng c v ngoi, ng
knh 40-50nm.
c. Hp ph v xm nhp: Theo c ch nhp bo, ci v trong nhn.
d. Biu hin genom: Dng ARN polymeraza ca vt ch, lp rp, ct ni bit
ho (differential splicing) to ra cc bn phin m (mARN) khc nhau.
e. Sao chp genom: ADN polymeraza ca virus s dng protein thay cho mi
ARN. Sao chp AND c mi bi lin kt hydro ca dCMP gn vo protein 80
Kdalton bt cp vi dGTP u 3 ca genom m khng cn to cc on
okazaki.
f. Lp rp: Trong nhn, gii phng: Lm tan bo.
g. Gy bnh: Virus adeno gy vim hng, phi, kt mc, bng quang xut
huyt.

3. Herpesviridae

a. Genom: Dng thng, 124-235 Kbp, khi lng phn t 80-150x106 dalton.
b. Cu to: Nuclecapsid dng khi, ng knh khong 30nm, c v ngoi.
c. Hp ph v xm nhp: Dung hp v ngoi vi mng sinh cht. Ci v trong
nhn.
d. Biu hin genom: ARN polymeraza ca vt ch c iu ho bi cc yu t
ca virus.
e. Sao chp genom: S dng ADN polymeraza ca virus, khp vng, sao chp
theo c ch vng trn xoay.
f. Lp rp: Trong nhn, gii phng: ny chi qua mng nhn vo mng li ni
cht ri ra ngoi.
g. Gy bnh: Thu u, zona, c bo, EBV, roseola.

4. Poxviridae

a. Genom: Dng thng vi hai u lp li tri chiu 130-375 Kbp, khi lng
phn t 85-240x106 dalton.
b. Cu to: Capsid phc tp (gm nhiu lp v thng c v ngoi), c dng
hnh thoi hoc hnh vin gch, di 200-900nm
c. Hp ph v xm nhp: Dung hp trc tip gia v ngoi vi mng sinh cht.
d. Biu hin genom: S dng ARN polymeraza ca virus.
e. Sao chp genom: Dng AND polymeraza ca virus tng hp thay th to
cc concateme.
f. Lp rp trong t bo cht, gii phng: ny chi.
g. Gy bnh: u ma, u b.

5. Hepadnaviridae

a. Genom: Khp vng nhng khng khp kn gm hai si: si m (L) di3,2
Kb, khi lng phn t 1,6x106 dalton bt cp vi si m, ngn (S). Chiu di
si m thay i cc virion.
b. Cu to: Dng khi, c v ngoi, ng knh 42nm.
c. Hp ph v xm nhp: Virus gn vo th th dnh cho IgA trn b mt t
bo gan.
d. Biu hin genom: Sau khi genom chuyn thnh phn t AND kp, khp
vng, siu xon (cADN), ARN polymeraza ca vt ch tin hnh phin m to
4 loi mARN, trng nhau u 3 (3,5; 2,4; 2,1 v 0,7 Kb) si di nht c kch
thc 3,5 Kb di hn genom 200 base, dnglm khun tng hp genom
(si L), nn gi l tin genom.
e. Sao chp genom: S sao chp ngc mARN di nht to si L xy ra
trong t bo cht. S tng hp si th hai (S) xy ra trong virion.
f. Lp rp: Trong t bo cht, gii phng: ny chi vo mng li ni cht to
ra bng ri dung hp vi mng t bo ra ngoi.
g. Gy bnh: Virus HBV gy vim gan B ngi, DHBV gy vim gan vt

C. Virus thc vt

1. Caulimovirus (MaMV- Cauliflower mosaic virus)

a. Genom: Khp vng nhng khng si no khp kn, 8Kbp, khi lng phn t
4-5x106 dalton. Si m c mt ch t qung, si dng c 2 ch t qung.
b. Cu to: Capsid dng khi, ng knh 50nm.
c. Gn v xm nhp: Nh vectr v rp.
d. Biu hin genom: ARN polymeraza ca vt ch to ra 2mARN, trng nhau
u 3. Si di nht di hn genom.
e. Sao chp genom: Phin m ngc si mARN di hn to AND(-), ri si
m lm khun tng hp si (+).
f. Lp rp trong t b cht, gii phng nh vectr l rp.
g. Gy bnh khm hoa l, bnh khm cc cy h ci.

II. Lp II. Virus c genom ADN n

A. Bacteriophage

1. Microviridae (nhm ?X174)

a. Genom: Khp vng, 5,4 Kb, khi lng phn t 1,7x106 dalton, si dng.
b. Cu to: Capsid hnh khi, ng knh 27nm
c. Hp ph c cc gai gn vo trong lipoprotein thnh t bo. Xm nhp nh
protein pilot gn u AND gip a n vo trong t bo cht.
d. Biu hin genom: ADN polymeraza cu k ch xc tc tng hp 3mARN a
gen (polycistronic). Protein khng cu trc c tng hp t khung c chng
lp hoc t cc v tr khi u khc nhau ca cng khung c.
e. Sao chp genom: Tng hp atingenome (i genom), to dng sao chp
(RF), sau , lc u sao chp to nhiu RF theo c ch theta, tip theo l sao
chp theo c ch vng trn xoay.
f. Lp rp v ra khi t bo nh lm tan bo.

B. Cc virus ng vt

1. Parvoviridae (erythro-B19, cc bnh ng vt)

a. Genom: Khp vng, 5-8Kbp, khi lng phn t 3-8x106 kdal
b. Cu to: Capsid hnh khi, ng knh 40-55nm
c. Xm nhp: Theo c ch nhp bo, ci v trong nhn
d. Biu hin genom: Dng ARN polymeraza ca k ch, phin m nh ct ni
(splicing) bit ha.
e. Sao chp genom: Dung ARN polymeraza ca k ch sao chp kiu theta.
f. Lp rp trong nhn, gii phng khi lm tan bo
g. C hai chi: Mt chi l virus t ch (autonomous virus) c th t nhn ln
trong t bo ch, mt chi l virus khuyt tt. Mun nhn ln cn h tr ca
virus khc. V d virus khuyt tt ph thuc virus adeno (AAV-defective adeno-
associated virus), cn gi l virus km adeno.

C. Virus thc vt

1. Geminivirus

a. Genom: Khp vng, 2,7-2,9. Khi lng phn t 1x106 dalton. Mi virion
cha 1-2 phn t, ADN n, dng.
b. Cu to: Capsid dng khi a din gn vi nhau thnh i.
e. Xm nhp: Nh cn trng b cnh trng v b n l
d. Biu hin genom: Dng ARN polymeraza ca k ch. Phin m t genom v
t si b sung ca genom
e. Biu hin genom: C th theo c ch vng trn xoay thng qua RF trung
gian
f. Lp rp trong nhn. Lan truyn nh cn trng b cnh trng v b n l.
g. i din: Virus gy bnh sc l ng, khm vng u.

III. Lp III: Virus ARN kp

A. Virus ng vt

1. Reoviridae

a. Genom: Cha 10-12 on, tng cng 16-23Kbp. Khi lng phn t 12-
20x106 dalton
b. Cu to: Capsid hnhkhi a din c v kp, khng v ngoi, ng knh 60-
80nm, vi cc gai ngn.
c. Xm nhp theo c ch nhp bo, to endosom, sau loi b v capsid
ngoi. Khi vo t bo cht vn cn v capsid trong
d. Biu hin genom: ARN polymeraza do virus mang theo tin hnh phin m
si m to mARN monocistronic ri ra khi nucleocapsid
e. Sao chp genom: mARN c ng gi trong v capsid to thnh
nucleocapsid sau tng hp cc si trong nucleocapsid.
f. V capsid ngoi lp rp xung quanh nucleocapsid trong t bo cht, sau
c gii phng khi lm tan bo.
g. i din: Virus rota gy bnh tiu chy tr em.

IV. Virus ARN n, dng

A. Bacteriophage

1. Leviviridae

a. Genom: Dng thng, 3,5-4,7 Kb. Khi lng phn t 1,2x10
6
dalton
b. Cu to: Capsid c dng gn nh khi a din, ng knh 23 nm
c. Hp ph: Gn vo th th t bo nh si lng F
d. Biu hin genom: Cc protein c m ho t khung c chng lp hoc t
cng mt khung c s dng cch c qua im dng yu (read-through of
weak terminatior)
e. Sao chp genom: Replicaza ca virus tin hnh tng hp antigenom lm
khun tng hp cc genom cho virus mi.
f. Lp rp ngu nhin ri chui ra khi t bo tan
g. i din: Ch c 1 chi l Levivirus. Cc phage: MS2, QB, f2, fr, R17....

B. Virus ng vt

1. Picornaviridae

a. Genom: Thng, 7,2-8,4 Kb, khi lng phn t 2,4x10
6
dalton. u 5 c
protein VPg, u 3 c ui poly (A).
b. Cu to: Capsid dng khi a din, khng v ngoi, ng knh 27-30 nm
c. Xm nhp: Theo c ch nhp bo, ci v trong t bo cht
d. Biu hin genom: genom m ho cho mt polyprotein duy nht sau c
proteaza phn ct thnh cc protein vi chc nng khc nhau.
e. Sao chp genom: ARN polymeraza ca virus xc tc tng hp antigenom
lm khun tng hp cc genom ca virus mi.
f. Lp rp trong t bo cht. Lm tan bo ra ngoi
g. i din: 5 chi, trong c cc virus gy bnh l mm long mng, bi lit,
Coxsackie, ECHO, vim gan A, x mi.

2. Togaviridae

a. Genom: Dng thng, 9,7-11,8 Kb, khi lng phn t 4x10
6
dalton. u 5
c m, u 3 c ui poly A
b. Cu to: Capsid dng khi a din, ngknh 30-42 nm, c v ngoi vi
cc gai (ng knh v ngoi 60-65 nm).
c. Xm nhp theo c ch nhp bo v dung hp gia v ngoi vi mng
endosom. Ci v trong t bo cht.
d. Biu hin genom: Mt polyprotein c tng hp trc tip t pha u 5 ca
genom (v genom lm nhim v mARN) cn protein th hai c tng hp t
mARN c phin m t antigenom (si m) nm pha u 3 ca genom.
e. Sao chp genom: ARN polymeraza ca virus xc tc tng hp antigenom
dng lm khun tng hp genom mi.
f. Lp rp trong t bo cht. Ny chi ra ngoi.
g. i din: Virus gy bnh Rubella.

3. Coronaviridae

a. Genom: Thng, 20-30 Kb, khi lng phn t 9-11x10
6
dalton. u 5 gn
m, u 3 gn ui poly(A).
b. Cu to: Nucleocapsid dng xon, c v ngoi vi cc gai, ng knh 60-
120 nm.
c. Xm nhp: Dung hp trc tip gia v ngoi vi mng sinh cht ca t bo
hoc theo li nhp bo to endosom ri dung hp gia v ngoi virus mi vi
mng endosom. Ci v trong t bo cht.
d. Biu hin genom: S dch m u tin xy ra on pha u 5 ca genom
to polyprotein, sau dch m t cc mARN (nested mARN) c
phin m t antigenom pha u 3. y l nhm mARN c chung u 3.
e. Sao chp genom: ARN polymeraza ca virus xc tc tng hp antigenom
dng lm khun tng hp genom mi.
f. Lp rp: Ny chi vo mng li ni cht ht, gii phng nh tan bo hoc
dunghp gia mng bng vi mng sinh cht.
g. Virus gy bnh cho ngi (SARS) v ng vt.

C. Virus thc vt

1. Potyvirus (nhm virus Y gy bnh khoai ty)

a. Genom: Thng, 9,5 Kb, khi lng phn t 3-3,5x10
6
dalton. u 5 gn
protein VPg, u 3 gn poly-A
b. Cu to: Capsid xon, mm do, ng knh 11nm, di 700nm.
c. Xm nhp nh cn trng (rp).
d. Biu hin genom: Genom c m ho cho mt polyprotein duy nht, sau
phn ct thnh cc protein chc nng.
e. Sao chp genom: ARN polymeraza ca virus xc tc tng hp antigenom
dng lm khun tng hp cc genom mi.
f. Lp rp trong t bo cht, gii phng nh rp.
g. Gm cc virus gy bnh khoai ty nhm Y.

2. Tymovirus (nhm virus khm vng cy ci c).

a. Genom: Dng thng, 6,3Kb. Khi lng phn t 2x10
6
dalton. u 5 gn
m, u 3 gn tARN thay cho ui poly(A).
b. Cu to: Capsid dng khi a din, ng knh 29nm.
c. Xm nhp nh ong
d. Biu hin genom: u tin mt polyprotein c to thnh t on pha u
5 ca genom, sau protein th hai c tng hp t mARN do phin m t
antigenom nm pha u 3.
e. Sao chp genom: ARN polymeraza ca virus xc tc tng hp antigenom
dng lm khun tng hp genom mi.
f. Lp rp trong t bo cht, gii phng nh ong.

3. Tobamovirus (nhm virus khm thuc l)

a. Genom: Dng thng, 4,8kb. Khi lng phn t 2x10
6
dalton. u 5 gn
m, u 3 gn tARN thay cho ui poly(A).
b. Cu to: Capsid dng xon, cng, ng knh 18nm, di 300nm
c. Xm nhp: Thng qua cc vt try xc hoc qua ht
d. Biu hin genom: Dch m lc u tin hnh on nm pha u 5 ca
genom to hai polyprotein nh vic c m vt qua im kt thc yu
(weak terminator) sau l dch m nhm cc mARN c tng hp t
antigenom pha u 3.
e. Sao chp genom: ARN polymeraza ca virus tng hp antigenom dng lm
khun cho genom mi.
f. Lp rp trong t bo cht. Xm nhp qua cc vt try xc hoc qua ht.

4. Comovirus (nhm virus khm u a)

a. Genom: Gm hai phn t dng thng, 5,9-3,5 Kb, khi lng phn t
2,4x10
6
dalton. u 5 gn VPg, u 3 gn ui poly(A).
b. Cu to: Capsid dng lng ht, khi a din (gm hai ht lun i km
nhau), ng knh 28 nm.
c. Xm nhp: Nh ong.
d. Biu hin genom: Mi ARN m ho cho mt polyprotein.
e. Sao chp genom: ARN polymeraza ca virus tng hp antigenom dng lm
khun cho genom mi.
f. Gii phng v khuch tn nh ong.

V. Lp V: Virus c genom ARN n, m.

A. Virus ng vt.

1. Rhabdoviridae

a. Genom: Dng thng, 13-16 Kb. Khi lng phn t 4,6x10
6
dalton. Hai u
c trnh t lp li, b sung cho nhau.
b. Cu tao: Nucleocapsid dng xon, mm mi, cun li thnh hnh vin n,
ng knh 45-100 nm, di 100-430 nm. C v ngoi vi cc gai b mt.
c. Biu hin genom: mARN n gen (monocistron) c phin m t genom
nh ARN polymeraza do virus mang theo.
e. Sao chp genom: ARN polymeraza ca virus tng hp antigenom sau
tng hp genom.
f. Lp rp: V tr lp rp v ny chi thay i tu theo tng loi.
g. Gm 2 chi: Mt chi gy nhim c ng vt c v khng xng sng, mt
chi gy nhim thc vt. i din: Virus gy bnh di.

2. Paramyxoviridae.

a. Genom: Dng thng 16-20 Kb, khi lng phn t 5-7x10
6
dalton vi hai
u lp li tri chiu.
b. Cu to: Nucleocapsid a hnh thi, c v ngoi vi cc gai, ng knh 150
nm.
c. Xm nhp: Dung hp gia v ngoi vi mng t cht, ci v trong t bo
cht.
d. Biu hin genom: Cc mARN n gen (monocistron) c phin m t
genom nh ARN polymeraza do virus mang theo, ARN editing.
e. Sao chp genom : ARN polymeraza ca virus tnghp antigenom dng lm
khun tng hp genom mi.
f. Lp rp trong t bo cht. Ny chi qua mng sinh cht.
g. Cc virus i din: Cc virus gy bnh si, cm, quai b, Newcastle,
Nipal...

3. Filoviridae

a. Genom: Dng thng, 19,1 Kb. Khi lng phn t 4,2x10
6
dalton, vi hai
u lp li tri chiu.
b. Cu to: Nucleocapsid a hnh thi, dng xon, ng knh 50nm, c v
ngoi vi cc gai, chiu di rt thay i, i khi phn nhnh hoc c dng hnh
s 6.
c. Xm nhp: Cha r
d. Biu hin genom: ARN polymeraza ca virus xc tc tng hp cc mARN,
ARN editing v dch m theo kiu dch khung (translational fraeshifting) xy ra
trong tng hp Gp (glycoprotein).
e. Sao chp genom : ARN polymeraza ca virus xc tc tng hp antigenom
dng lm khun tng hp genom mi.
f. Lp rp trong t bo cht, ny chi qua mng sinh cht.
g. Virus i din: Ebola, Marburg gy bnh st xut huyt.

4. Orthomyxoviridae

a. Genom: Gm 8 on ARN dng xon, tng chiu di 10-13,6 Kb. Khi lng
phn t 4,5x10
6
dalton. Tt c cc on u c cng on u ging nhau.
b. Cu to: Nucleocapsid a hnh thi dng xon, mm mi, c v ngoi vi
cc gai, ng knh virion khong 100 nm.
c. Xm nhp theo cc cch nhp bo, sau tin hnh dung hp gia v ngoi
vi mng endosom. Ci v trong t bo cht.
d. Biu hin genom: u 5gn m. C t 8 n 15 base ca mARN ca k ch
c s dng lm mi cho ARN polymeraza ca virus tng hp cc mARN
ca mi on genom. Hai phn t mARN nh nht c to thnh nh ct
ni(splicing) bit ho.
e. Sao chp: ARN polymeraza ca virus tng hp antigenom dng lm khun
tng hp genom mi ca virus con trong nhn t bo ch.
f. Lp rp: Trong t bo cht, ny chi qua mng sinh cht.
g. i din:virus cm A, B, C, cm gia cm.

5. Bunyaviridae.

a. Genom: gm ba on ARN thng, tng cng 11-12 kb. Khi lng phn t
6x106 dalton. on nh nht c trong mt chi l genom lng cc.
b. Cu to: Nucleocapsid xon, mm mi. C v ngoi vi cc gai. Virion c
ng knh 100nm.
c. Xm nhp theo li nhp bo, sau tin hnh dung hp gia v ngoi virus
vi mng endosom. Ci v trong t bo cht.
d. Biu hin genom: ARN polymeraza va virus tng hp antigenom dng lm
khun tng hp genom mi.
f. Lp rp trong t bo cht, ny chi qua mng b my golgi. Lan truyn nh
bn chn khp (tr chi Hantavirus).
g. Hantavirus (st xut huyt Triu Tin), virus ho m c chua...

6. Arenaviridae

a. Genom: L hai phn t ARN n, thng, 10-14 kb, khi lng phn t, on
ln 3x10
6
dalton. C hai u l genom lng cc (trn genom ARN n c c
on dng v m).
b. Cu to: Nucleocapsid xon, mm mi, c v ngoi vi cc gai b mt,
ng knh virion 60-300 nm.
c. Xm nhp theo li nhp bo sau tin hnh dung hp gia v ngoi virus
vi mng endosom. Ci v trong t bo cht
d. Biu hin genom : mARN th nht c tng hp t mi on genom nh
ARN polymeraza ca virus. mARN th hai c tng hp t antigenom ca
chnh on .
e. Sao chp genom: ARN polymeraza ca virus tng hp antigenom dng lm
khun tng hp genom mi.
f. Lp rp trong t bo cht, ny chi qua mng sinh cht.
g. Virion cha riboxom ca k ch. i din: Virus gy bnh LASSA, Junin,
Machupo, vim mng no, mng m, lympho bo.

VI. Lp VI:
Virus c genom ARN n, dng vi enzym phin m ngc (virus Retro).

a. Genom: Thng, 7,2-11 kb. Khi lng phn t 2-3,5x10
6
dalton. u 5 gn
m, u 3 gn ui poly (A). Mi virion cha 2 phn t ARN ging nhau.
b. Cu to: Nucleocapsid dng khi a din, c v ngoi vi cc gai b mt,
ng knh virion 100nm.
c. Xm nhp theo li nhp bo sau l dung hp gia v ngoi vi mng
endosom. Ring HIV tin hnh dung hp trc tip gia v ngoi vi mng sinh
cht.
d. Biu hin genom: Genom ARN c sao thnh ADN kp nh enzym phin
m ngc (RT) do virus mang theo, sau ADN virus gn vo nhim sc th
ca t bo di dng provirus. Provirus phin m nh ARN polymeraza ca t
bo to mARN a gen (polycistron) sau c x l (splicing) thnh
mARN m ha cho polyprotein.
e. Sao chp genom : Provirus phin m to genom.
f. Lp rp trong t bo cht, ny chi qua mng sinh cht.
g. i din: Virus gy suy gim min dch ngi (HIV), virus gy ung th
bch cu t bo T ngi ( HTLV-I, HTLV-II), virus Sarcoma Rous.



Bng phn loi cc h chnh ca virus ADN ng vt c xng sng


Bng phn loi cc h chnh ca virus ARN ng vt c xng sng

4. Cc virus chnh gy bnh cho ngi.

1. Cc virus chnh gy bnh cho da, nim mc, mt


Tn virus C quan nhim Tn bnh ng ly truyn
u ma
(smallpox
viriola) H
Poxviridae
Da u ma
Qua sol kh, vy mn
cha virus
Molluscipox H
Poxviridae
Da U mm ly
Ly ngi sang ngi
qua tip xc
Herpes simplex-
1 H
Herpesviridae
Mi, da Herpes mi, mn rp.
Qua nim mc, vt
xc da
Herpes simplex-
2 H
Herpesviridae
C quan sinh
dc
Herpes sinh dc, mn
rp
ng tnh dc.

Varicella-Zoster
H
Herpesviridae
ng h hp,
mt, mu, da
Thy u, Zona Ngi sang ngi
Herpes 6 (HHV-
6) H
Herpesviridae
Da, hch
lympho
Ban o (roseola) Qua sol kh, nc bt
Papiloma
ngi (HPV) H
Papovaviridae
Biu m da,
nim mc
Mn cm
Tip xc trc tip,
ng sinh dc
Parvo B19 H
Parvoviridae
Da (m, tay,
i, mng,
mnh)
Ban truyn nhim,
bnh th 5
Qua sol kh cha virus
Coxsackie A H
Picornaviridae
Ming, hng,
da
Vim hng,mn nc
(tay-chn-ming)
vim kt mc, xut
huyt, vim mng
no v khun.
ng phn, ming
Rubella H
Togaviridae
Da, ng h
hp, hch
lympho, mu
Rubella ng h hp
Si (measles)
H
Paramyxoviridae
ng h hp,
h bch huyt,
tun hon,
no, niu, thn
kinh trung
ng.
Si ( Rubeola)
ng h hp (sol
kh).


2. Virus gy bnh h thn kinh.
Tn virus C quan nhim Tn bnh ng ly truyn
Polyoma JC H
Popovaviridae
Cc t bo thn,
mu, no, h thn
kinh trung ng.
Bnh no bch cu
nhiu tin trin
(PML)
Qua ng h hp
Polio H
Picornaviridae
T bo rut, hng,
no h thn kinh
trung ng tu
sng
Bi lit tr em
ng tiu ha,
phn, ming
Flavi H Mu i khi no, Vim no St. Louis, Truyn qua bn
Flaviviridae gan, da v mch
mu
Ty sng Nile, Xun
h Nga, vim no
Nht Bn, st Dengue,
st vng.
chn khp.
Toga H
Togaviridae
Mu i khi no,
gan, da v mch
mu
Vim no nga min
ty (WEE), vim no
nga min ng
(EEE), vim no nga
Venezuela
Truyn qua bn
chn khp
Di (Rabies) H
Rhabdoviridae
C vn, nron, tu
sng, no tuyn
nc bt.
Di
Qua nc bt
ng vt di qua
vt cn da.
Bunya H
Bunyaviridae
Mu, no, thn,
gan, mch mu
Vim no california,
st thung lng Rift

Truyn qua bn
chn khp.
HIV H
Retroviridae
T bo TH, i
thc bo, t bo c
trn, t bo tua
AIDS
Qua ng tnh
dc, tim chch
ma ty, tip vi
dch c th ngi
b nhim HIV
Priron No
Bnh xp no ( JD),
Kuru
Ph hu cc m b
nhim.

3. Virus gy bnh tim mch v h bch huyt

Tn virus C quan nhim Tn bnh ng ly truyn
Epstein Barr
(EVB) H
Herpesviridae
T bo biu m ca
hng, tuyn nc bt
mang tai, mu v
lympho B
U lympho
Burkit, bnh
bch cu n
nhn.
Nc bt
Cytomegalo
(CMV) H
Herpesviridae
Gan, hch lympho
D tt bm
sinh
Qua dch tuyt ca c
th nh nc bt, nc
tiu c t cung, truyn
qua ng tnh dc,
sinh n, truyn mu,
ghp tng, tim chch.
Coxsackie B H
Picornaviridae
C tim, da
Vim c tim,
vim mng
ngoi tim,
vim no v
trng, chng
au nhi ngc.
Qua ng phn ming
HTLV-1, HTLV-
2 H
Retroviridae
T bo lympho T
Ung th bch
cu t bo T
ngi, ung th
bch cu t
bo lng.
Qua ng tnh dc,
truyn mu, tim chch
Marburg, Ebola
H Filoviridae
Nhiu loi t bo, c
bit l t bo gan v
i thc bo
St xut huyt
do virus
Ly trc tip qua dch c
th, trc ht l mu.
Hanta H
Bunyaviridae
Mu, phi
Hi chng
bnh phi do
virus Hanta
km sc tim
Ht phi virion t bi
phn v nc tiu ca
chut.

4. Virus gy bnh ng h hp


Tn virus
C quan
nhim
Tn bnh ng ly truyn
Adeno H
Adenoviridae
ng h
hp
Cm lnh thng Qua sol kh ng h hp
Rhino H
Picornaviridae
ng h
hp trn
Cm lnh (s mi)
Qua sol kh ng h hp,
vt b nhim hoc tip
xc trc tip vi ngi
bnh.
Corona ( Hcov)
H Coronaviridae
ng h
hp
Hi chng vim
ng h hp cp
nng (SARS), cm
lnh thng
Qua sol kh ng h hp.
Quai b (mumps)
H
Paramyxoviridae
Cc t bo
ng h
hp trn
Quai b
Dch tit ng h hp
(qua ho, ht hi), nc
bt.
Hp bo h hp
(RSV)
ng h
hp, phi
Bnh hp bo h
hp
Qua bi nn, chn tay b
nhim, nht l vn tr
Cm H
orthomyxoviridae

Phi,
ng h
hp
Cm Qua git tit ng h hp

5. Virus gy bnh ng tiu ho
Tn virus C quan nhim Tn bnh ng ly truyn
Vim gan B
(HBV) H H
Hepadnaviridae
T bo gan
Vim gan huyt
thanh (vim gan B),
ung th gan
Qua ng tnh dc, qua
dch c th b nhim,
tim chch ma ty.
Vim gan A
(HAV) H
Picoraviridae
T bo gan
Vim gan nhim
trng (vim gan A)
Qua ng tiu ha.

Vim gan C
(HCV) H
Flaviridae
T bo gan Vim gan mn tnh
Qua ng tnh dc,
truyn mu, ghp tng,
tim chch.
Vim gan D
(HDV) Cha
phn loi
T bo gan Vim gan Delta
Qua ng tnh dc, bm
tim b nhim
Vim gan E
(HEV) H
Hepeviridae
T bo gan Vim gan E Qua ng phn-ming
Rota H
Reoviridae
ng h hp
v ng tiu
ha
Tiu chy, vim d
dy rut tr em
Qua ng phn-ming
C bo (CMV)
(Cytomegalo)
H
Herpesviridae
Hch lympho,
gan
D tt bm sinh
Qua dch tit ca c th
(nc bt, dch nhn,
sa, nc tiu, phn,
dch t cung, qua quan
h tnh dc, truyn mu.

6. Virus gy bnh ng niu v sinh dc
Tn virus C quan nhim Tn bnh ng ly truyn
Herpes
simplex-2 H
Herpesviridae
C quan sinh
dc
Herpes sinh dc,
mn rp.
Qua quan h tnh dc
Papilloma
(HPV) H
Papovaviridae
Biu m da,
nim mc.
Mn cm
Qua tip xc trc tip, qua
dng c b nhim, qua
quan h tnh dc.
Vim gan B
(HBV) H
Hepadnaviridae
Gan
Vim gan huyt
thanh ung th
Qua tip xc trc tip vi
dch c th b nhim. Qua
quan h tnh dc, bm tim
b nhim.
HIV H
Retroviridae
T bo T, i
thc bo, t
bo c trn,
Hi chng suy
gim min dch
mc phi (AIDS).
Qua quan h tnh dc, tim
chch ma ty, truyn mu,
tip xc vi dch c th b
t bo tua. nhim, ghp tng.


5. Hnh nh mt s virus thng thng:


Kch thc Virus (1000nm= 1m)





H Orthomyxoviridae


Virus cm (Influenzavirus)






Virus cm gia cm H5N1 (HPAV- Highly pathogenic avian influenza)




H Paramyxoviridae




Virus quai b (Mumps virus)






Virus si (Measles virus)








H Togaviridae
Virus si c (Rubella virus)




Virus hp bo h hp (RSV- Respiratory syncytial virus)


H Coranoviridae






Virus SARS (Severe Acute Respiratory Syndrome)




Virus gy bnh di ( Rhabdovirus)




H Hepadnaviridae
Virus vim gan B (HBV- Hepatitis B virus)



H Picornaviridae (HAV- Hepatitis A virus)




H Flaviviridae
Virus vim gan C (HCV- Hepatitis C virus)




Virus vim no Nht Bn B (Japanese B Encephalitis virus)






Virus vim gan D (HDV- Hepatitis D virus)






H Togaviridae
Virus vim gan E ( HEV- Hepatitis E virus)




Virus st xut huyt Dengue (Dengue fever virus)







Virus HIV (Human immunodeficiency virus)




Virus bi lit (Poliomyelitis virus)




H Poxviridae
Virus u ma (Pox virus)



H Filoviridae
Virus Ebola (Ebola hemorrhagic fever virus)
80 x 14 000 nm



H Picornaviridae
Virus st l mm long mng (Foot and mouth disease virus)







Virus hi l cy trng





Virus hi c qu cy trng


Ti liu tham kho

David Greenwood, Richard C., B. Slack, 1998 Medical Microbiology. Churchill
Levingstone.
Lansing M. Prescott, john P. Harley, onal A. Klein, 2005 Microbiology. Mc
Graw Hill
Robert W. Bauman, 2004 Microbiology Benjamin Cummings
Harper D. R., 1998 Molecular Virology. Springer and Bios Scientific Publisher
Bruce A. Voyles, 2002 The biology of Viruses. Mc Graw Hill.
Bi 8 Vi nm (Microfungi)
I CNG V VI NM



Nm (Fungi, s t l Fungus) l mt gii ring- Gii nm (Fungi), khoa hc nghin cu v nm
c gi l Nm hc (Mycology).
Ngi ta bit n nm v s dng nm t thi c xa. Theo Quch Mt Nhc, tc gi B Trung
quc s co th ngi Trung Quc bit n nm t cch y 6000-7000 nm . Ngh nu ru c
s dng nm men v nm si xut hin Trung Quc t cch y 7000-8000 nm. Vic s dng
nm lm dc liu (Thn khc) c Trung Quc t cch y 2550 nm. Cc nm dng lm
thuc nh Phc linh, Ch linh, Linh chi , T linh, Li hon, M bt, Thin hoa, Trng tho, Mc
nh c ghi trong schThn nng bn tho kinh trong thi gian khong nm 100-200 sau
Cng nguyn.
phng Ty , Ray ( 1684-1704) ngi Anh, cn c vo c im sinh thi l chnh phn 94
loi nm thnh 4 nhm khc nhau trong sch Lch s thc vt. Sau l cc nghin cu phn loi
nm ln cn c vo hnh thi ca Magnol (1689), Tournefort (1694).
Khi Leewenhoek (1632-1723) lm ra chic knh hin vi phng i c 200-300 ln th ngi ta bt
u ch n cc nm nh hay gi l vi nm.Nh khoa hc Italia P.A.Micheli (1679-1737) l ngi
u tin dng knh hin vi nghin cu nm. trong tc phm Cc chi thc vt mi (Nova
Plantarum Genera) xut bn nm 1729 ng nu ln cc bng phn loi cc chi nm nh Mucor,
Tuber, Aspergillus. Hc gi H Lan D.C.H. Persoon (1761-1836) trong cc sch Synopsis
Methodica Fungarum v Mycologia Europeae t c s cho phng php v h thng phn loi
nm . Hc gi Thy in E.M. Fries (1794-1878) c nhiu cng hin trong vic phn loi cc
nm ln. Khong 100 nm sau vic phn loi a s nm ln u da trn nghin cu ca Fries.
Ngi u tin vn dng thuyt tin ha ca Darwin vao vic phn loi nm l nh khoa hc c
H.A.De Bary (1831-1888). ng xut bn sch Hnh thi hc v sinh l hc nm vo nm 1866
vi c s phn loi da trn trt t tin ha. ng cn nghin cu ngun gc v tin ha ca nm,
sng to nn gi thuyt n nguyn lun.
V sinh l hc, nm 1869 J.Raulin pht hin nguyn t vi lng Zn rt cn cho s sinh trng ca
Asperrgillus niger; nm 1901 E.Wilders cho bit sinh trng nm cn cn cc nhn t nh
Biotin, Thiamin, Inositol
V Di truyn hc Blakeslee (1904) pht hin ra s phi hp ca cc si nm khc gii tnh nm
Mucor. Sau l cc pht hin tng t ca Kniep (1920 vi nhiu loi nm m, Dodge (1928)
vi nm Neurospora. V sau vi nm Neurosporra ngi ta nghin cu su v di truyn hc v
trn c s cc nghin cu ny m Beadles (1945) mi xut c hc thuyt Mt gen-mt enzym.
Nh nm hc Italia P. A. Saccardo (1845-1920) chnh l cc nghin cu v nm v bin son
bng ting La Tinh 25 tp K yu Nm.
Tin b ca Sinh hc phn t v k thut knh hin vi in t em li mt din mo mi cho
vic nghin cu phn loi hc v sinh l hc nm. Cc thnh tu nghin cu c tng kt kh
y trong 5 tp sch Gii Nm (The Fungi) ca G. C. Ainsworth v cng s (Vol 1, 2.3.4A.4B.
New York and London: Academic Press, 1963-1973). Nm 1995 ti bn ln th 8 cun T in
v nm (Dictionary of the Fungi) ca Ainsworth v Bisby. Nm c chia thnh 4 ngnh (Division,
Phylum):
- Ngnh Chytridiomycota
- Ngnh Zygomycota
- Ngnh Ascomycota
- Ngnh Basidiomycota
Theo thut ng Latinh tn cc taxon trong phn loi nm l nh sau: Ngnh-mycota; Ngnh ph-
mycotina; Lp- mycetes; Lp ph- mycetidae; B-ales; B ph- ineae;H-aceae; H ph- oideae.
Hin nay tn ti cc h thng phn loi nm khng thng nht vi nhau, ch yu l cc h thng
phn loi theo Ainsworth v cng s (1973), V.Arx (1981), Ainsworth & Bisby (1983), Kendrick
(1992), Ainsworth & Bisby (1995), Alexopoulos & Mins (1996).
Chng ti s dng h thng phn loi theo Gio trnh nm hc CBS. ( CBS Course of Mycology ),
ln xut bn th 4, Baarn, Delft, 1998:

Ngnh Lp Lp ph hoc nhm
Labyrinthomorpha
Labyrinthulea
Thraustochytriacea

Pseudofungi
Hyphochytriomycetes
Oomycetes

Chytridiomycota Chytridiomycetes
Zygomycota
Zygomycetes
Trichomycetes

Ascomycota
Archiascomycetes
Saccharomycetes
Ascomycetes











discomycetes
Major licheneized orders
plectomycetes
pyrenomycetes
loculoascomycetes
powdery mildews
Laboulbeniomycetes
conidial ascomycetes
(Hyphomycetes,
Coelomycetes)
Basidiomycota
Urediniomycetes

Platyglomycetidae
Urediniomycetidae
Ustilaginomycetes
Hymenomycetes
Tremellomycetidae
Dacrymycetidae
Auriculariomycetidae
Hymenomycetidae

Cc loi nm khng tm thy (ng ra l cha tm thy) dng sinh sn hu tnh c xp
chung vo nhm Nm bt ton Fungi imperfecti. Theo h thng phn loi ca Saccardo
(1880,1886) th cc nm ny c xp thnh mt lp- Lp Deuteromycetes. Khi pht hin thy c
quan sinh sn hu tnh th ngi ta i tn loi v xp sang cc lp khc. V d nm la von trc
kia c gi l Fusarium moniliforme, nhng sau khi tm thy c quan sinh sn hu tnh th li
chuyn thnh loi Gibberella fujikuroi. Cc nm bt ton hin c xp trong cc nhm conidial
Ascomycetes hay conidial Basidiomycetes.
Hin nay ngi ta cho rng trong t nhin c khong 1 triu n 1,5 triu loi nm nhng
mi nh tn c khong 10 000 chi v 70 000 loi, Trung Quc iu tra c 40 000 loi.
Ring cc loi nm thuc Nm bt ton nc ta hin mi ch pht hin c 338 loi thuc 306
chi khc nhau (Bi Xun ng, 2004). Bo tng ging chun vi sinh vt (VTCC) thuc Trung tm
Cng ngh sinh hc i hc Quc gia H Ni ang hp tc vi Vin NITE ca Nht Bn iu tra
nghin cu khu h vi nm Vit Nam v c nhiu kh nng tm thy nhng loi mi trong khoa
hc.
Vy nhng loi nm no l Vi nm (Microfungi)? l tt c cc nm khng c m nm
(qu th, fruit-body) c th thy r bng mt thng. Ngi ta gi nhng nm c m nm l cc
nm bc cao. Tuy nhin khi nui cy si nm ca cc nm bc cao nghin cu hoc sn xut
sinh khi th chng cng c coi nh vi nm v l i tng nghin cu ca ngnh Vi sinh vt (
ging nh cc loi vi nm khc). nghin cu vi nm bt buc phi quan st di knh hin vi v
phi nui cy trong cc iu kin v khun nh i vi vi khun.
Cn c vo hnh thi ngi ta chia vi nm thnh hai nhm khc nhau: nhm Nm men (
Yeast ) v nhm Nm si (Filamentous fungi). Chng ch khc nhau v hnh thi ch khng phi l
nhng taxon phn loi ring bit. Nhiu nm men cng c dng si v rt kh phn bit vi nm
si.

I- CU TO CHUNG CA SI NM
1. Hnh thi v cu to ca si nm
Si nm (hypha) c dng hnh ng phn nhnh bn trong cha cht nguyn sinh c th lu
ng. V chiu di chng c s sinh trng v hn nhng v ng knh th thopng ch thay i
trong phm vi 1-30m (thng thng l 5-10 m).
u si nm c hnh vin tr, phn u gi l vng ko di (extension zone). Lc si nm sinh
trng mnh m y l vng thnh t bo pht trin nhanh chng, vng ny c th di n 30 m.
Di phn ny thnh t bo dy ln v khng sinh trng thm c na. Mng nguyn sinh cht
thng bm st vo thnh t bo. Trn mng nguyn sinh cht c mt s phn c k cu go np
hay xo li, ngi ta gi l bin th mng (plasmalemmasome) hay bin th (lomasome). Nhiu khi
chng c tc dng tit xut cc cht no .
Thnh t bo (cell wall) ca nm c thnh phn ha hc khc nhau. y l mt tiu ch quan
trng khi nh loi nm. sau y l cc thnh phn chnh ca thnh t bo mt s nm:
Chi nm: 1 2 3 4 5 6
Thnh phn thnh t bo:
Glucan 16 54 0 43 29 6
Cellulose - 36 0 0 0 0
Chitine 58 0 9 19 1 10
Chitosan - 10 33 - 0 -
Mannan - <1 2 2 31 <3
Protein 10 5 6 11 13 7
Lipid - 3 8 5 9 3

Ch thch: 1- Allomyces
2- Phytophthora
3- Mucor
4- Aspergillus
5- Saccharomyces
6- Schizophyllum
Thnh phn chnh trong thnh t bo ca mt s nhm nm l nh sau:
Acrasiales Cellulose, Glycogen
Oomycetes Cellulose, Glucan
Hyphochytriomycetes Cellulose. Chitine
Zygomycetes Chitin, Chitosan
Chytridiomycetes, Ascomycota (dng si), Basidiomycota (dng si),
Fungi Imperfecti Chitine, Glucane
Ring vi Saccharomycetales v Cryptomycocolacales l Glucan, mannan
Vi Rhodotorula v Sporobolomycetales l Chitine, mannan
Vi mng nguyn sinh cht (protoplasmic membrane) th thnh phn t thay i cc loi nm si,
c khc nhau vi dng nm men. Sau y l mt s v d:
Loi nm 1 2 3 4 5
Thnh phn mng NSC
Protein 52,0 38,5 46,5 45,5 25,5
Lipid 43,0 40,4 41,5 31,0 40,0
Polysaccharide 9,0 5,2 3,2 25,0 30,0
Acid nucleic 0,3 1,1 7,5 0,5 -

Ch thch: 1- Candida albicans (dng nm men)
2- Candida utilis
3- Saccharomyces cerevisiae
4- Candida albicans (dng nm si)
5- Fusarium culmorum

Nhn t bo c bao bc bi mng nhn, trn mng nhn c nhiu l thng, trong nhn c
hch nhn (nucleolus). Thng c nhiu nhn tp trung phn ngn ca si nm. Trong cc t bo
pha sau ngn thng ch c 1-2 nhn.
Nhn ca nm thng nh, kh thy r di knh hin vi quang hc. Nhim sc th trong
nhn thng khng d nhum mu, s lng tng i nh. S lng ny l 6 cc nm
Magnaporthe grisea, Paecilomyces fumosoroseus, Trichoderma reesei; l 7 cc nm
Histoplasma capsulatum, Neurospora crassa, Phenaerchateae chrysosporium, Podospora anserina,
l 8 cc nm Aspergillus nidulans. Aspergillus niger,Acremonium chrysogenum, Beauveria
basiana, Lentinus edodes, l 10 nm Penicillium janthinellum,l 11 nm Schizophyllum
commune, l 12 nm Curvularia lunata, l 13 nm Agaricus bisporus, l 15 nm
Cyanidioschyzon merolae, l 20 nm Ustilago maydis.
Trong t bo nm cn c cc c quan ging nh trong t bo cc sinh vt c nhn thc
(Eukaryote) khc. l ty th (mitochondrion), mng ni cht (endoplasmic reticulum), dch bo
hay khng bo (vacuolus), th rib (ribosome), bo nang (vesicle) , th Golgi sinh bo nang (Golgi
body, Golgi apparatus, dictyosome), cc git lipid (lipid droplet), cc tinh th (chrystal) v cc vi
th ng knh 0,5-1,5 nm (microbody), cc th Vrnin ng knh 0,2m (Woronin body), th
Chit ng knh 40-70nm(chitosome) Ngoi ra trong t bo cht cn c cc vi qun rng rut,
ng knh 25nm (microtubule), cc vi si ng knh 5-8nm( microfilament), cc th mng bin (
plasmalemmasome)

Ribosome ca nm thuc loi 80S ( S l n v h s lng Svedberg) c ng knh khong
20-25nm, gm c 2 bn n v ( subunit); bn n v ln (large subunit ) 60S (gm 3 loi ARN-
25S; 5,8S v 5S cng vi 30-40 loi protein). Bn n v nh (small subunit) 40S (gm loi ARN
18S v 21-24 loi protein)
Phn ngn c th tch vi phn bn di bng mt khng bo, lc u nh nhng v sau kt
hp li vi nhau ln dn, to n p lc dn t bo cht v pha nh ngn si nm. Ti phn gi
nht ca si nm thng xy ra hin tng t tan (autolysis) hoc b tan r di tc dng ca cc
men phn ct (lytic enzyme) do cc vi sinh vt khc sinh ra. Cng c nhng phn si nm gi phn
lipid tch t nhiu v kt hp vi thnh t bo to nn mt mng dy, to thnh nhng bo t o
(chlamydospore). Loi bo t ny c th gip si nm tn ti c qua nhng iu kin mi trng
khc nghit. Trng hp ny rt ging vi cc bo t ni sinh (endospore).
Si nm khng ngng phn nhnh v v vy khi mt bo t ny mm trn mt mi trng
c s pht trin thnh mt h si nm (mycelium, s nhiu- mycelia), sau 3-5 ngy c th to thnh
mt m nhn thy c gi l khun lc (colony). Vo giai on cui ca s pht trin khun lc
s xy ra s kt mangh (anastomosis) gia cc khun ty vi nhau, lm cho c khun lc l mt h
thng lin thng mt thit vi nhau, thun tin cho vic vn chuyn cht dinh dng n ton b h
si nm. Hin tng kt mng thng gp nm bc cao nhng li t gp cc si nm dinh
dng ca nm bc thp. Hnh thi, kch thc mu sc, b mt ca khun lcc ngha nht
nh trong vic nh tn nm.
Phn ln si nm c dng trong sut, mt s nm si nm mang sc t to nn mu ti hay
mu sc s. Sc t ca mt s nm cn tit ra ngoi moi trng v lm i mu khu vc c nm
pht trin. Mt s nm cn tit ra cc cht hu c to nn cc tinh th trn b mt khun lc. V bo
t ca nm thng cng c mu nn c khun lc thng c mu

2-Vch ngn si nm:
Si nm mt s nm c vch ngn ngang (septa, cross wall), l cc si nm c vch
ngn (septate hyphae). Cng c nhiu nm si nm khng c vch ngn ngang- si nm khng vch
ngn (aseptate hyphae). Cc nm bc thp thng c si nm khng vch ngn, ngc li cc nm
bc cao thng mang si nm c vch ngn. Si nm khng vch ngn mang nhiu nhn nhng vn
c th gi l n bo. cc nm khng vch ngn th vch ngn vn c th hnh thnh khi sn sinh
th sinh sn hay c quan sinh sn. ti cc b phn b thng tno si nm cng c th hnh thnh
vch ngn. l loi vch ngn lin, khng c l thng, c tc dng bo v c th.
Si nm c vch ngn l c th a bo. Mi t bo c 1 nhn hoc nhiu nhn. Tuy nhin
si nm vn khng phi do nhiu t bo hp thnh m l do cc vch ngn tch si nm ra thnh
nhiu t bo.
Vch ngn ngang c hnh thnh t thnh t bo. Lc u l mt g nh hnh khuyn sao
tin dn vo trong v lp kn li. Tuy nhin vch ngn thbf c 1 hay nhiu l thng. cc l c
kch thc bng nhau hay c 1 l ln nht gia v nhiu l nh xung quanh. Vch ngn in
hnh Nm ti (Ascomycota) v Nm bt ton (Deuteromycetes) l loi c mt l chnh gia,
ng knh khong 0,05-0,5m. nm Geotrichum candidum v nm Fusarium vch ngn thng
c nhiu l nh. nm Fusarium l chnh gia vch ngn ln hn so vi cc l khc. Mt s
Nm m (Basidiomycota) c l hnh thng trn vch ngn ngang, bc bn ngoi bi mt cu trc
mng gi l mng l thng (perenthesome). Trong iu kin kh hn vic hnh thnh vch ngn c
th gip si nm khng c tt hn i vi mi trng.
Nm ti v Nm bt ton nhiu khi trong t bo cn thy c cc th Vrnin (Woronin
body) v cc tinh th protein.


Cu to ca phn ngn si nm



Cu trc ca si nm



Si nm c vch ngn Si nm khng vch ngn


H si nm


S pht trin ca h si nm


Khun lc nm


Mt v d v cu trc ca Ribosome Eukaryote

Th Golgi v Mng li ni
cht

Th Woronin t tp l thng ca vch ngn (nm Penicillium)


Ty th (mitochondrion)

3-M (tissue) ca h si nm:
M hay t chc ca h si nm thng gp cc nm bc cao. Chng xut hin trong mt
giai on ca vng i nm. Loi m ny c gi l t chc si dy (plectenchyma). T chc ny
li chia ra thnh hai loi: loi t chc si xp hay t chc t bo hnh thoi (prosenchyma) v t chc
mng mng hay t chc nhu m gi ( pseudoparenchyma). T chc si dy gp thn nm v m
nm cc nm bc cao, nht l ngnh Nm ti v Nm m. Hch nm (scleorotium) xut hin
khi nui cy nm si (nm bc thp) trn cc mi trng gi (nui cy lu ngy). Hch nm l hnh
thc bo v nm qua giai on bt li. cn l ni d tr cc cht dinh dng cho nm. Kt cu
t chc ca hch nm gm lp ngoi cng c thnh t bo mu en. Di l mt lp biu b
rng. Bn trong l t chc si dy vi cc si nm c mng dy. Trong cng l khi t chc si
xp. Th m hay m nm (stroma) cng l khi t chc si nm dnh nhau theo nhiu hng.
Trn hoc trong th m c cc c quan sinh sn ca nm.Th m ch gp Nm ti v Nm m.
Thng nm hay khun sch (rhizomorph) cng c t chc si nm bn li theo kiu t chc si dy.


Hch nm (Sclerotium)


T chc si xp (prosenchyma)

T chc mng mng (Pseudoparenchyma)-hnh pha trong

Th m ( Stroma )


Thng nm (Rhizomorph)

4-Cc dng bin i ca h si nm:
4.1-Bo t o, bo t, t bo phnh ln:
i khi gp nhng t bo si nm phnh ln mt cch v quy tc. Ni chung khi gp iu
kin ngoi cnh bt li nguyn sinh cht co li, vo trn li, pha ngoi to thnh lp mng dy, bn
ngoi thng c gai hay cc mu li. Kt cu ny c gi l bo t o hay bo t mng dy
(Chlamydospore). Loi bo t ny khng phi loi bo t sinh sn (v tnh hoc hu tnh) gip pht
tn nm ra mi trng xung quanh. Cc loi bo t ny ta s xem xt ti phn di.
4.2-Si ht (haustorium):
Si nm k sinh trn b mt t bo cy ch ri mc ra si nm m su vo pha trong v
to nn cc si ht m vo cc t bo ht cht dinh dng ca cy ch. Si ht c th c cc
hnh dng khc nhau : hnh si, hnh ngn tay, hnh cuNi chung cc nm k sinh bt buc u
l nhng loi c si ht.
Khi xuyn vo t bo cy ch mt s si ht khng lm rch c mng nguyn sinh cht
m ch lm cho mng ny lm vo. Nhiu khi si ht phn ra kh nhiu nhnh bn trong t bo
tng din tch hp thu cht dinh dng.


Si ht

4.3- Si p (appressorium) v m xm nhim (infection cushion):
T mt bo t nm ny mm trn b mt vt ch (thng l l cy) s mc ra mt si nm
v tit ra cht nhy p st b mt ny. u si nm phnh to ln nh chic a v to thnh si
p. T si p mc ra si ht m su vo t bo vt ch ht cht dinh dng. M vt ch b
tiu hy dn di tc dng ca cc enzym thy phn ca si p. m xm nhim l mt dng mng
si phn nhnh v gn vi nhau nh mt tm m t bo vt ch, u si nm thng phnh to
ln. Vic hnh thnh m xm nhim thng do c ch khng ch ni sinh v nh hng kch thch
ca mi trng.




4.4- Si nm bt mi (traps):

Mt s nm sng trong t c c ch bt mi (giun trn-nematode, amip-amoeba..). Chng hnh
thnh nn nhng cu trc c dng nhng ci by v v vy c gi l nm by mi (trapping
fungi). C th c my kiu si nm by mi sau y:
- Bng dnh (adhesive knobs): S nm pht sinh nhng nhnh ngn ,thng thng
gc vi si nm chnh. nh cc nhnh ny phnh to ln thnh nhng bng hnh cu.
cc bng ny tit ra cht dnh c th gi cht ly con mi. Sau t bng mc ra si
nm xuyn qua v con mi. Cui si ny phng ln thnh bng nh bn trong con
mi v tip tc phn nhnh thnh cc si ht
- Li dnh (sticky nets): Cc nhnh mc ra c ni mng vi nhau thnh dng
li. Bn ngoi li cng ph y cht dnh by mi. Sau mt t bo ca li
s pht trin thnh bng nh bn trong con mi v t bng mc ra cc si ht
- Vng tht ( constricting rings): Si nm mc ra cc nhnh c bit vi cc vng
tht cu to bi 3 t bo. Khi mt trong ca cc t bo ny tip sc vi con mi th
khng bo s phnh to ln v tht cht con mi li. Sau s mc ra cc nhnh
xuyn vo con mi, ri mc ra cc si ht ni trn
- Bo t dnh (Sticky spores): C cc bo t ph y cht dnh v c tc dng by
mi tng t nh cc trng hp ni trn.
Cc chi nm thng c si nm by mi l Arthrobotrrys, Daxtylaria, Daxtylella,
Trichothecium Mt s loi nm nh Zoopage phanera, Arthrobotrys anomala tit ra
cht dnh trn ton b mt ngoi si nm v cng c tc dng by mi nh cc trng
hp ni trn.












4.5- R gi (rhizoid) v Si b (stolon):
Si b l on si nm kh sinh khng phn nhnh pht sinh t cc si nm mc ln t c cht,
chng c dng thng hay hnh cung. Phn chm vo c cht pht trin thnh nhng r gi ngn cm
vo c cht. Do s pht trin ca si b m cc nm ny lan rng ra xung quanh, v c mi pha, k
c trn thnh ca hp lng (hp Petri). R gi v thn b c trng cho cc nm thuc chi Rhizopus
(r gi mc ngay ch cm si nm kh sinh), Abssidia (r gi khng mc di ch cm si nm kh
sinh) . R gi cn gp nhiu loi nm thuc cc chi Rhizidiomyces, Sclerotinia




Nm Rhizopus





Nm Absidia
Bi 9 Nm si (Filamentous Fungi)
NM SI (FILAMENTOUS FUNGI)

PHNG PHP LY MU :
I. Mu t.
II. L v cnh cy kh:
III. L ti v v cy:
IV. Phn.
V. Cn trng.
VI. Nc ngt v cc vt liu c trong nc ngt.
VII. Nc mn v cc vt liu c trong nc mn.

PHNG PHP PHN LP:
1. Phng php dng kim nhn:
2. Phng php phn lp bo t n c:
3. Phng php dng vi thao tc Skerman:
4. Phng php pha long:
5. Phng php pha long kt hp vi x l tia cc tm:
6. Phng php phn lp nm m v nm ti (Basidiomycetes v Ascomycetes)
7. Phng php ra b mt:

PHNG PHP NH LOI NM SI
I Yu cu:
II. Quy trnh nh loi mt chng nm si:
III. Tin hnh nh loi

KHO PHN LOI N LP
I. LP NM TIP HP (ZYGOMYCETES)
II. LP NM BT TON (DEUTEROMYCETES)
1. c im c trng
2. Phn lp ca nm bt ton:
3. Nhng b phn sinh sn v tnh.
KHO 1:
KHO 2
I. Amerocodidium
II. Didymoconidium
III. Phragmoconidium
IV. Dictyoconidium
V. Scolecoconidium
VI. Helicoconidium
VII. Stauroconidium
VIII. Miscellaneous fungi
IX. Synnematous fungi:

PHNG PHP LY MU :
Chng ta c th phn lp vi nm t bt k mt c cht t nhin no v mi c cht
trong t nhin u c vi sinh vt bn trong. Tuy nhin nu chng ta chn ly mu mt cch
ngu nhin, khng c mc ch th rt mt thi gian v tin bc.
Nhng vt liu khc nhau s c coi nh nhng c cht khc nhau
phn lp nm. Vt liu chung nht thng l: l cy ti, l cy rng, l cy
mc, phn ng vt, cn trng, nc ngt, nc bin,....
Trc khi i ly mu phn lp nm si chng ta phi cn nhc v suy ngh v
nhng iu cn thit sau:
1, S chn loi vt liu phn lp cho loi nm no.
2, Dng bn kim tra li v tr ly mu.
3, S dng phng php phn lp no cho mu .
4, V cui cng l s mang mu nh th no v phng th nghim.
Sau s quyt nh ni ly mu, chun b cho chuyn i ly mu ,
mang theo nhng dng c cn thit nh: Ti ni lng, tha cn, ti ng mu
lm bng giy, chai l, n gas xch tay, ti cha, ... Khi ti ni ly mu
chng ta quyt nh s lng mu s ly.
Mt iu cng rt quan trng l phn lp nm sau khi ly mu cng sm
cng tt.
I. Mu t.
Mu t l mu c hiu qu nht v chng ta c th phn lp nm trn
mu t vi s lng ln. Chng ta c th ly mu t nhiu ni chng hn
nh rung la, ni trng cy ly ht hoc cnh ng trng rau, di rng
tng hay di rng tn l rng, vng ni cao, ven b sui,... Thng thng
nm phn lp c t mu t gi l nm t, nhng mt s nm phn lp
c y h ao, sng sui hay y bin li gi l nm a nc hay nm a
mn.
phn lp c nhiu loi nm khc nhau, chng ta nn ly mu t
trn b mt di lp l cy mc bi v qun th nm tp trung trn lp t b
mt nhiu hn l phn di t su. Khi ly mu ta gt b lp l mc l b
mt lp t v dng tha sch ly phn t b mt cho vo ti ni lng.
ng thi ghi cc thng s cn thit v ni ly mu: a ch, kinh , v ,
ngy ly mu, tnh trng ly mu, nhit lc ly mu, ngi ly mu.
II. L v cnh cy kh:
Nm Si phn lp t l v cnh cy kh gi l nm rc (litter fungi).
phn lp nm rc c hiu qu chng ta phi ly mu l v cnh cy tt, trong
trng hp nm rc, chng ta phn lp c cc loi nm khc nhau khi ly
mu su khc nhau trong cng mt mu.
Mu l cy kh ly c nn trong ti bng giy v nn x l ngay
phn lp, v nu lu theo thi gian mu kh dn, nm rc trong mu s
pht trin km i v cc vi sinh vt bn ngoi s xm nhim vo.
V th phi ghi r tn thc vt ly mu, phn hu ca l cy rng, ni
ly mu (kinh d, v , a ch), ngy thng ly mu, ngi ly mu.
III. L ti v v cy:
C t nht 3 nhm nm phn lp t l cy ti, nhm th nht l nm b
mt l (phyloplane fungi). Nhm th 2 thuc v nm gy bnh, bao gm k
sinh bt buc v khng bt buc. Nhm th 3 l nm thc vt hoi sinh,
nhng s khc nhau gia nhm th 3 ny v nhm k sinh khng bt buc
khng phi lc no cng r rng.
Trong khi ly mu l cy ti v v cy, lun phi ghi nh 6 iu sau:
1, Qu trnh phn hu c th din ra, mc d trng b ngoi l vn nh
ang cn ti (c bit iu ny hay din ra vng nhit i).
2, H sinh thi nm trn l thc vt sau khi ngt c th khc i so vi khi
chng cn trn cy.
3, Tnh a dng sinh hc ca nm l ti cn non s km hn l trng
thnh.
4, H sinh thi nm c th khc nhau gia b mt l v mt sau ca l.
5, Trong cng mt cy cao ly mu ca chng s cho h sinh thi nm khc
nhau.
6, Mi loi thc vt khc nhau s phn lp c cc chng nm c hiu, v d
nh loi nm Trochophora simplex ch phn lp c t cy Daphniphyllum
macropodium.
c bit khi phn lp nm t mu l v v cy thc vt chng ta rt kh
c th phn lp c loi nm trong thc vt gi l nm nc trn cn
(terrestrial aquatic fungi). Bi v nhm nm ny sinh trng rt chm v khng
d dng sinh bo t nh nhng nm trn b mt, nm gy bnh hay ccnm
hoi sinh khc. V th kh c th phn lp cc loi nm ny t l cy hay t v
cy nu ch s dng nhng k thut phn lp thng thng.
Nm nc trn cn sinh bo t nhanh chng nu c nc kch thch,
chng hn gp ma, sng. Chng ta ly mu sau khi tri ma hoc ly mu
t nhng git sng ng trn cnh l.
IV. Phn.
Thnh phn hu c trong phn l rt ln, do khu h vi sinh vt trong
v cng a dng. Nhng loi nm c th tn ti c trn phn ng vt
c gi l nm phn. S thay i ca nm phn xut hin trn mt mu phn
ng vt l mt v d in hnh v tnh k tha nm. Xut hin u tin l
ngnh nm tip hp Zygomycota (v d Basidiobolus), tip n l
Pyrenomycetes, Plectomycetes, v Discomycetes ca ngnh nm ti
Ascomycota (v d Arthroderma, Gymnoascus,...) ri n cc nm hoi sinh (V
d Arthrobotrys, Oedocephalum, Scopulariopsis,...) v cui cng l nm m
Basidiomycota (v d Corprinus) [5].
Ngi ta thng phn lp nm phn t phn ng vt n c chng hn
nh phn nga, cu, d, th, gia sc, v thm ch l chut cng c cng
b. [5]
V. Cn trng.
Nhng cn trng nh b, chng hn nh amip, giun t, ve rn, su
b,.... l nhng mu tt dng cho phn lp nm. c bit l nm hoi sinh
giun trn (nematode-trapping fungi).
VI. Nc ngt v cc vt liu c trong nc ngt.
T mu nc ngt ch yu phn lp c 2 nhm nm: nhm th nht
l Chytridiomycota, nhm th 2 l cc loi nm a nc, a nc hiu kh.
phn lp nhm nm ny, nn ly mu nc ngt v l ri nhng sinh thi
nc, chng hn nh sng, sui, ao h. c bit nm a nc d dng b
mc vo nhng khc g mc di lng sui hay ng sau cc hc c dng
nc chy to bt.
VII. Nc mn v cc vt liu c trong nc mn.
Nm si sng nc mn gi l nm si a mn, nhng c im hnh
thi v sinh l ca chng gip chng thch nghi c vi mi trng mn. C 2
nhm nm a mn: mt loi l a mn bt buc (ton b vng i ca chng
tri qua trong mi trng mn); loi kia l a mn khng bt buc (phn ln
c th phn lp c chng trong mi trng t v c trong mi trng
mn).
phn lp nm a mn, ta c th ly mu t cc cnh ci mc, thc
vt a mn, thc vt ven bin, t ct bi bin v t nc bin, c bit l
bt bin l mu rt tt phn lp nm a mn.
Tm li: ly mu phn lp nm th bt k loi mu no cng u tt,
u quan trng, iu ch yu vn da vo mc ch phn lp nhm nm no
cn thit cho cng tc nghin cu s la chn mu v phng php ly
mu.
Trn cng mt c cht, c th c nhiu nhm nm tn ti mi nhm li
c mt chu k sng khc nhau, nn phn lp nhiu thi im khc nhau v
bng cc phng php khc nhau th c th phn lp c loi mi hay tm ra
mt h sinh thi mi ca nm.
PHNG PHP PHN LP:
K thut phn lp tm ra loi nm mi hay nm hu ch l rt quan
trng trong qu trnh nghin cu. Khng c mt quy lut chung no dng cho
phn lp vi sinh vt. Phng php phn lp tt nht s l phng php m ta
la chn phn lp vi sinh vt m ta cn. Suy ngh mt cch sng to cho
cng vic v thnh tho vi cc k thut phn lp mi l yu cu chung ca
bt k mt nh vi sinh vt no.
Cc nh nm hc Nht Bn thng dng cc phng php sau phn
lp nm si: S dng kim nhn nht bo t (Dr. Chiharu Nakashima); Phn lp
bo t n c (Dr. Chiharu Nakashima); S dng vi thao tc Skerman (Dr.
Katsuhiko Ando); Phng php pha long (Dr. Misa Otoguro); Phn lp t bo
t m (Dr. Chiharu Nakashima); Phng php ra b mt (Dr. Katsuhiko
Ando); Phng php st khun b mt (Dr. Ju-Young Park); Phng php
dng bung kch m (Dr. Katsuhiko Ando); Phng php kch thch s ny
mm ca nm(Dr. Ju-Young Park); Phng php sc kh (Dr. Katsuhiko Ando).
Di y chng ti xin php trnh by mt s phng php hay dng nht,
p dng phn lp nm trong t, trong l cy rng v nm ni sinh endophyte
ti Bo tng Ging chun Vi sinh vt- Trung tm cng ngh Sinh hc- i hc
Quc gia H ni trong chng trnh hp tc v nghin cu a dng sinh hc vi
Vin Cng ngh v o lng Quc gia Nht Bn (NITE- Japan).
1. Phng php dng kim nhn:
S dng phng php ny phn lp tt c mi loi nm pht trin trn bt
k mt c cht no.
Cch lm:
1) t c cht cn phn lp (l, cnh cy mc,...) ln knh hin vi vt knh
X10 ( phng i 10 ln) hoc ln knh lp.
2) Chnh tiu c quan st c bo t, cung sinh bo t t c cht.
3) Kh trng que nhn bng ngn la n cn.
4) a que nhn vo khong gia vt knh v c cht, nht bo t chuyn
sang mi trng phn lp.
5) Nui cy bo t nhit 25
0
C, quan st s ny mm ca bo t hng
ngy n khi hnh thnh khun lc th chuyn sang mi trng thch nghing.
2. Phng php phn lp bo t n c:

S dng phng php ny phn lp nm gy bnh thc vt.
Cch lm:
1) Chun b dch huyn ph bo t:
Dng kim nhn nh bt 1 u, gn vo mt ci tay cm chc chn, dng que
co vo ch trn l cy bnh c xut hin m nm, t sang lam knh
c sn mt git nc v trng, khuy .
2) Dng u kim ly mt git nc c ho tan bo t v mt hnh tam gic
ln mi trng thch nc (Water agar). 20
0
C trong 24 gi.
3) Quan st bng knh hin vi, tm bo t ny mm trn mi trng thch a
theo ng vin tam gic trn. nh du im c bo t ny mm bng bt
vit knh mt di a thch.
4) Chuyn a mi trng thch c nui cy cc bo t trn sang knh lp.
Quan st v tr nh du bo t ny mm. Thanh trng que cy, dng que cy
ly bo t ny mm ra, chuyn sang ng nghim mi trng thch nghing
nui cy.

3. Phng php dng vi thao tc Skerman:
Chun b dng c:
B vi thao tc Skerma gm 5 phn (hnh bn).

1) Ly mt ng pipet past h trn ngn la n cn, ko thnh ng rt nh,
di khong 4cm, gn vo ng st trn cc nam chm D ( c nh bng parafin
nng chy).
2) C nh phn dng c B vo vt knh 10
3) Gn nam chm c ng thu tinh vo rnh kim loi ca dng c B.
4) Di chuyn phn thit b c gn cc nam chm ln xung cho n khi nhn
thy u ng thu tinh gia vi trng.
5) Gn u kim hn E vo bin th A v t vo v tr t tiu bn ca knh
hin vi.
6) Chnh knh, tm vi trng, quan st u kim hn, iu chnh sao cho u
que hn v ng thu tinh st nhau v u quan st c di knh hin vi.
7) Bt bin th A nc cao u que hn nng v tin v pha ng thu
tinh, n que hn chm vo ng thu tinh ng thu tinh bt u nng chy,
ko nhanh ng thu tinh khi kim hn to thnh que thu tinh thun nhn.
8) H nhit que hn bng cch gim bin th, dng u que hn chm
nh vo u que thu tinh ko nh to thnh hnh ch L.
Gi y ta c th bt u thc hnh ly tng bo t n c trn a thch.
Cc bc tin hnh:
1) Chun b mu: l cy kh, cho vo hp nha hoc ti ni lng c cha nc
v trng sn lm m, gi 2-3 ngy nhit phng, kch thch bo t
ny mm.
2) Ly mu l trn cho vo cc thu tinh, cho thm nc ct, khuy nh nhng
cho bo t ni ln mt nc.
3) Thu thp bo t bng cch ly lam knh nghing 45
0
so vi mt nc
ly nc trn b mt cc. Dng lam knh tri mt ng thng trn mt a
mi trng phn lp nm LCA (Low cacbon agar), MEA (Malt extract agar).
4) Quan st a thch trn di knh hin vi, tm bo t c hnh dng c bit.
Khi thy bo t cn tm th gi nguyn v tr.
5) Gn phn dng c hnh ch V (hnh C) ca b vi thao tc vo vt knh 10
ca knh hin vi. Tip n gn phn nam chm c que thu tinh hnh ch L va
lm xong vo phn dng c ch V .
iu chnh ln xung sao cho c th quan st c u ch L ca que thu
tinh.
6) Nng knh ln sao cho b mt thch st vi u que thu tinh, lc ny ta c
th quan st c c bo t nm cn tm v c u L ca que thu tinh.
7) Dng que L ko bo t cn ly ra phn mi trng sch ca a thch.
Dng chnh kim phn ch L nh du im t bo t.
8) H a mi trng thp xung v dng que cy v trng ly bo t ti im
nh du chuyn sang a thch mi trng MEA. Nui cy nhit phng
v quan st s ny mm ca bo t hng ngy. Nu bo t ny mm th c
th chuyn sang thch nghing gi ging sau khi thch a 2-4 tun.
4. Phng php pha long:
Phng php ny dng cho cc mu t.
1. Trc ht cc mu t phi c x l trc khi phn lp bng cch phi
kh nhit phng 2-3 ngy, sau dng ry qua sng c kch thc 2-
3mm.
2. Cn 1g t x l cho vo 9ml nc ct kh trng, dng vortex ho tan
t.
3. Pha long mu cc nng pha long khc nhau: 10
-4
, 10
-5
l 2 nng
thch hp phn lp nm trong t Vit Nam.
4. Ht 0,5 ml dch pha long 2 nng trn nh ln a Peptri cha mi
trng phn lp ( mi trng Martin, hoc mi trng c s). Dng que gt
trang u dch trn b mt thch.
5. t a thch trong t 25
0
C, phn lp nm si t nhng khun lc ring r
trn a thch sau khong thi gian 4- 10 ngy.
5. Phng php pha long kt hp vi x l tia cc tm:
Tng t nh 4 bc ban u ca phng php pha long. Thm mt
bc tip l t a Peptri cha mi trng phn lp v gt dch pha
long u trn b mt thch vo t cy v bt n tm 20 pht.
Cc bc khc tng t nh phng php pha long thng thng.
6. Phng php phn lp nm m v nm ti
(Basidiomycetes v Ascomycetes)
1. Qu th nm sau khi thu hi, ra sch, ct ming nh khong 5cm
phn bn trong.
2. Ra sch bng nc ct v trng (2-3 pht), sau dng bng dnh 2
mt dnh 3-4 ming ct qu th gn vo np trn ca a thch nc. nh
du v tr gn ming nm bng bt vit knh y a thch nc.
3. 20
0
C trong vng 24 gi.
4. Soi di knh lp ti v tr nh du xem ny mm ca bo t nm.
5. Dng que cy hnh vng trn ly m bo t va ny mm chuyn
sang a mi trng thch cao malt.
7. Phng php ra b mt:
Phng php ny dng phn lp nm t l ti hoc l rng.
Cch lm:
1) Ct mu l cy (ti v kh), r cy, cnh cy,... ra thnh cc ming
nh, sau cho vo ng nghim.
2)Thm 20ml dung dch 0.005% Aerosol OT (di-iso-octyl sodium
sulfosuccinate) hoc dung dch 0.005% Tween 80, lc mnh bng my vortex
khong 10 pht loi tr vi sinh vt bm trn b mt mu.
3) Bc lm kh mu: loi b nc trong ng nghim, dng kp v trng
ly mu ra t ln b mt giy lc qua m trong t cy v trng, bc ny
c tc dng loi b s nhim khun.
4) Dng kp v trng t mu lm kh ln b mt mi trng thch
LCA ln mt vng mu l ( loi tp nhim (nu c) mt ln na), mi a
thch c th chia lm 8 phn kim tra.
5) Sau chuyn mu sang a thch mi trng LCA v trng khc, mi
a nn t mt mu, mi mu 5 ming ct. nhit 25
0
C trong vng mt
thng.
6) Trong khong thi gian hng ngy phi kim tra s ny mm ca
cc vi sinh vt di knh lp, phn lp nm si nu c.

PHNG PHP NH LOI NM SI
I Yu cu:
- C c cc chng nm si tht thun khit (khng c ln tp nm
hoc cc vi sinh vt khc).
- Cc chng cn nh loi phi c nui cy trn cc mi trng, nhit
, thi gian nui cy theo ng quy nh ca cc kho phn loi i vi tng
chi nm mc.
II. Quy trnh nh loi mt chng nm si:
1. Quan st c im phn loi ca chng nm si cn nh
loi.
1.1. Quan st c im khun lc trn thch
- Hnh dng
- Kch thc (ng knh, chiu dy)
- Dng mt (nhung mt, mn, len xp, dng ht, li lm, c kha hay
khng)
- Mu sc khun lc mt trn v mt di
- Dng mp khun lc (mng, dy, phng, nhn nheo)
- Git tit nu c (nhiu, t, mu sc)
- Mi khun lc (c, khng mi)
- Sc t ho tan (mu ca mi trng xung quanh khun lc) nu c.
- Cc cu trc khc: b si, b gi (Synnematous or sporodochial
conidiomata) cc cu trc mang bo t trn (Fruit body - conidiomata) nh a
gi (acervuli) hoc ti gi (Pycnidia), m nm (Stroma), hch nm (sclerotia)
vv..
1.2. Quan st cc c im vi hc
- Si nm: (hyphae) c vch ngn, khng c vch ngn, c mu.
- Bo t trn(conidia): kiu pht sinh bo t trn ( nm bt ton), hnh
dng, kch thc, mu sc, b mt (nhn, c gai, g gh) cch sp xp n
c chui gc non (basipetal) chui gc gi (acropetal) khi cu vv
- B my mang bo t (spore apparatus): nang bo t v nang bo t
nh (nu c) (Sporangia & Sporangiola) hnh dng, kch thc, mu sc, b
mt ca nang, cung nang, khng hoc c nhnh (nhnh mc vng, mc cch,
hp trc vv); bo t nang ( hnh dng, kch thc, b mt, ) nm tip
hp.
- B my mang bo t trn (conidiogenous apparatus): gi bo t trn
(conidiophore) - kch thc, ng knh, chiu di, b mt (nhn, c gai, c
nt sn, vv) mu sc, c vch ngang hoc khng, c hoc khng c cu trc
c bit nh t bo chn (foot cell) si cng (setae) tng trng gc hoc
ngn c hoc khng c dng hnh thi c bit nh b gi, m gi. Cc
nhnh (ca bo t trn) s lng nhnh, kch thc b mt, mu sc, cch sp
xp (i xng, khng i xng st nhau hoc t rng, v tr dc gi bo t trn
hoc tp trung ngn gi)vv
- T bo sinh bo t trn (conidiogenous cell)
Kiu t bo sinh bo t trn (th bnh, dng c khuyn nh, dng sinh bo
t trn ng thi, nh dng (dng bnh- phialo type; dng phn t, Annello
type). Dng sinh bo t trn khng ng thi (Aleurio- type), bo t nh kiu
ny chi (Blasto - type), dng sinh bo t trn qua l li so (Poro - type)
vv.. hnh dng, kch thc, mu sc, cch sp xp (n dc hoc thnh cm,
thnh vng), v tr (trn si nm, dc theo gi bo t trn, cc nhnh hoc
nh gi) vv
- Th qu ti (ascocarp) nh cleistothecium, cn kho st v tr, s lng,
hnh dng, kch thc, mu sc, b mt ca th qu, quan st ti bo t
(ascus) bo t ti (ascospore) v hnh dng, kch thc b mt.
III. Tin hnh nh loi
Cn c vo kt qu quan st y , chnh xc cc c im ca khun lc v
cc c im vi hc tin hnh xc nh tn loi (hoc th nu c) chng nm
mc nh sau:
- Dng kho phn loi ca cc nhm phn loi bc cao (ngnh, ngnh
ph, lp, b, h, chi) xc nh ln lt xem nhng nm mc thuc chi nm
mc no. Tip theo dng kho phn loi ca chi xc nh n loi (th)
bng cch so snh tt c cc c im quan st c ca chng nm mc
vi cc c im tng ng ca loi no trong kho phn loi. Nu cc
c im so snh ph hp vi c im ca loi m t trong kho, ta xc nh
c tn loi ca chng nm mc cn nh loi.
- Nu c chng nm mu ca loi (hoc th) va xc nh th tin hnh so
snh cc c im hnh thi khun lc, c im vi hc ca chng mu vi cc
c im tng ng ca chng cn nh loi. Nu cc c im ca 2 chng
ph hp vi nhau th c th khng nh chc chn loi (hoc th) ca chng
nm mc cn nh loi.
- Nu c chng nm mu ca loi (hoc th) va xc nh th tin hnh so
snh cc c im hnh thi khun lc, c im vi hc ca chng mu vi cc
c im tng ng ca chng cn nh loi. Nu cc c im ca 2 chng
ph hp vi nhau th c th khng nh chc chn loi (hoc th) ca chng
nm mc cn nh loi.

KHO PHN LOI N LP( Robert A. samson, 1984)

1a. Khun lc gm cc t bo ny chi, khng c h si NM MEN ( YEAST)
1b. Khun lc vi h si sinh dng pht trin,
bo t trn hoc bo t sinh ra trong hoc trn cc bo t c bit 2
2a. Bo t sinh ra trong ti bo t NM TI (ASCOMYCETES)
2b. Bo t hoc bo t trn khng sinh ra trong ti bo t 3
3a. H si khng c hoc c rt t vch ngn, thng rng;
bo t kn sinh ra trong cc nang bo t kn NM TIP HP (ZYGOMYCETES)
3b. Si nm thng c vch ngn, bo t trn khng sinh
ra trong cc nang bo t kn. NM BT TON (DEUTEROMYCETES)

I. LP NM TIP HP (ZYGOMYCETES)
i vi nm mc thuc lp nm tip hp c th dng cc chuyn lun
phn loi sau:
- Kerry L.Odonnell, 1979 Zygomycetes in culture Department of Botany,
University of Georgia.
- M.A.A. Schipper, 1973 A study on variability in Mucorhemalis and
related spicies. Studies in Mycology, No. 4
- M.A.A. Schipper, 1978 On certain species of Mucor with a key to all
accepter species. Studies in Mycology, No. 17
- M.A.A. Schipper, 1984 A revision of the genus Rhizopus Studies
Mycology, No. 25

II. LP NM BT TON (DEUTEROMYCETES)
* Nm bt ton l g:
- Nm bt ton l giai on v tnh ( Anamorph) ca nm ti hoc nm
m.
1. c im c trng:
Gm cc loi nm mc gy nhim thc phm, c nhiu loi sinh c t
(mycotoxin), cc chi, loi ca lp nm ny c phn loi theo giai on sinh
sn v tnh (anamorph) tuy vy mt s loi ngi ta pht hin thy c giai
on sinh sn hu tnh (Teleomorph- bo t ti hoc bo t m).
Theo h thng phn loi cn c vo c im pht sinh ca bo t trn
ca Hughes (1953). Lp nm bt ton c chia thnh 3 nhm:
- Nhm Hyphomycetes: Gm cc nm bt ton khng c ti gi v a gi
(gi sinh bo t trn trn cc si nm hoc cc si nm kt li thnh b si,
b gi).
- Nhm Coelomycetes: Gm cc nm bt ton c ti gi hoc a gi, gi
bo t trn trong cc th qu (Fruit - body) gi l cc conidiomata.
- Nhm Agonomycetes: Gm cc nm bt ton khng c bo t trn.
y chng ti gii thiu 2 kho phn loi n chi ca lp nm bt ton.

2. Phn lp ca nm bt ton:
2.1. Hyphomycetes (1700 chi, 11000 loi)
- Cc dng h si nm mang cc bo t trn si ring r hoc trn cm
si nm (nh cc m nm, cc b gi), nhng khng trong cc conidiomata
(fruit body) ring bit.


2.2. Coelomycetes (700 chi, 9000 spp) ring r
- Bo t trn sinh ra trong cc ti gi, a gi, ti gi dng cc hoc m gi.




1. Bo t trn, 2. T bo sinh bo t trn,
3. Cung sinh bo t trn, 4. Si nm
C quan sinh sn v tnh ca chi Penicillium

3. Nhng b phn sinh sn v tnh.
3.1. Hypha (si nm) s nhiu ca Hyphae.
- Mt trong cc si trong h nm l cc tn ca mt si nm. Cc tn
khc bit vi cc phn si sinh dng hp ph cht dinh dng.
3.2. Bo t trn:
- Mt loi bo t v tnh, khng chuyn ng (Zoospose) c bit. Mt
loi pht tn.
3.3. T bo sinh bo t trn:
- Mt t bo bt k m t mt bo t c sinh ra trc tip.
3.3.1. Cc kiu pht sinh bo t trn.
a. Tn: - Tn ton b
- Tn bn trong
b. Ny mm: - Ny mm ton th
- Ny mm t trong



a-b. Dng tn. a- To chui. b- n c






a-f. Dng pht sinh kiu ny chi (blastic) a. Ny chi n c, b. To thnh
chui, c. Cc Bo t c to thnh cng mt lc, d. Cc bo t c y hp,
e. y bng, f. Cc bo t chui qua l

3.3.2. Cc kiu pht trin bo t trn trong s pht sinh bo
t dng ny chi (blastic),(Theo Katsuhiko Ando, 2002).
* S pht sinh bo t phn ct hon ton (ngoi sinh)
a. Kiu bo t nh (Aleurio - type).
- Mt im trn t bo sinh
bo t trn, bo t n c
b. Kiu ny chi (Blasto - type)
- Cu trc mng nh v tr trn
t bo sinh bo t trn v chui bo t
vi mt v tr to thnh m chui
lin tip khng phn nhnh (chui gc gi)

* Pht sinh bo t ni sinh.
c. Dng bnh (Phialo - type)
- Cc chui bo t ra khi t bo
sinh bo t trn cng mt v tr,
i khi trong cc chui ri t bo c thay i.
d. Dng phn t (Annello - type)
- Cc chui bo t ra khi t
bo sinh bo t trn cc v tr cao hn,
thnh thong trong cc chui ri,
t bo c thay i.

3.4. V tr sinh bo t trn t bo sinh bo
t trn:
a. Mt v tr
- Bo t trn pht trin mt
v tr trn t bo sinh bo t trn.
b. Nhiu v tr
- Bo t trn pht trin cc v tr
khc nhau trn t bo sinh bo t trn.


3.5. S pht trin ca cc t bo sinh bo
t trn:
a. n nh
- Kch thc ca t bo sinh bo t trn l n nh
b. Ko di
- T bo sinh bo t trn pht trin
cng vi s sn sinh cc bo t trn
c. Gim dn
- di cc t bo sinh bo t trn
gim i khi mi bo t trn c sinh ra


3.6. Cung sinh bo t trn:
- Mt si nm phn nhnh hoc n gin
(mt si nm sinh sn) mang hoc gm c cc t
bo sinh bo t trn m t cc bo t trn
c sinh ra. i khi c dng khi m t nhng
cu trc bin i i vi cc t bo sinh bo t
trn.
3.6.1. Cung sinh bo t trn khng c, t bo
sinh bo t trn gm cc t bo si nm.
a. Cung sinh bo t trn khng c,
t bo sinh bo t trn nm trn si nm.
b. Khng c cung sinh bo t trn
hoc khng r.
c. Cung sinh bo t trn c hoc
khng c vch ngn,
n gin khng phn nhnh.
d. Cung sinh bo t trn ko di
cng vi vic sinh bo t trn.
e. Cung c vch ngn, pht trin
n c hoc phn nhnh
f. B cung bo t trn (Synemata).
Gm mt nhm cc cung sinh bo t trn
b cht hoc lng mang cc bo t trn
nh hoc c nh v bn cnh cung.

3.7.Bo t trn (conidia)
3.7.1. Hnh dng ca bo t trn
a. Hnh dng n gin:
V d: Cu, gn cu, elip, hnh qu thn.
b. Bo t si:
- T l chiu di v rng ca bo t = 15:1.
c. Bo t vch li
- Bo t c chia nh ra bi cc vch ngn cho nhau.
d. Bo t xon c
- Trc bo t un cong hn 180
0
.
e. Bo t dng ch thp
- Bo t c nhiu trc, c mu, c lng mm chim 1/4 chiu di ca bo t.


3.7.2. S cc t bo ca bo t trn:
a. Bo t khng vch:
Bo t khng c vch ngn.
b. Bo t kp (dnh i):
Bo t mt vch ngn.
c. Bo t vch:
Bo t c hai vch ngn tr ln.
3.7.3. Mu ca bo t trn:
- Trong sut hoc c mu (nu, nu en).
3.7.4. B mt ca bo t trn:
- Gai, ht, c mu, nhn...
3.7.5. Kch thc ca bo t trn:
(35-)50-200(-300) x (2,0-)2,5-3,5(-4,5)m.
3.7.6. Cc hnh dng khc nhau ca bo t.


1. Hnh cu, 2. Gn cu, 3. Hnh bnh quy, 4. Hnh git l, 5. Hnh trng,
6. Hnh trng ngc, 7. Qu l, 8. Qu l ngc, 9. Elp, 10. Hnh elp ch
nht, 11. Hnh thoi, 12. Hnh thoi mo, 13. Elp nhn u, 14. Hnh thoi trn
u,
15. Dng thuyn, 16. Hnh qu thn, 17. Dng xc xch



1. Dng kim, 2. Dng qun, 3. Dng ch S, 4. Dng ch, 5. Dng que, 6. Hnh
tr, 7. Hnh ch nht thun, 8. Hnh thng, 9. Hnh m, 10. Hnh c vnh,
11. Hnh chui, 12. Hnh c c mu, 13. Hnh gc cnh, 14. Hnh qu du,
15. 16. Dng x x, 17. Dng gai.
KHO 1:
KHO 2
I. Amerocodidium
II. Didymoconidium
III. Phragmoconidium
IV. Dictyoconidium
V. Scolecoconidium
VI. Helicoconidium
VII. Stauroconidium
VIII. Miscellaneous fungi
IX. Synnematous fungi

Chng ti xin gii thiu hai kho phn loi n chi ca lp Nm Bt ton.
Kho 1 ca Robert A. Samson, (1984). Kho 2 ca Katsuhiko Ando, (2002)

KHO 1:
Phoma
1a. Bo t sinh ra trong ti gi

1b. Bo t khng sinh ra trong ti gi m trn si nm, gi sinh bo t trn
trn cm gi hoc b gi.
2a. Bo t sinh ra trong chui hng gc t cc t bo sinh bo t trn c
bit (th bnh, phn t vv) hoc to thnh cc git nhy u.
2b. Bo t sinh ra theo chui hng ngn hoc bng cch t on ca cc si
hu th, hoc n c
3a. Bo t trn trong cc chui kh
3b. Bo t trn trong cc git nhy u.
4a. Bo t trn lun 2 t bo,
sinh ra trn nhng t bo sinh
bo t trn dng si, hi cho
nhau xp sp nh bng la,
khun lc mu hng.

Trichothecium

4b. Bo t trn lun 1 t bo, sinh ra trn cc t bo sinh bo t trn hnh
bnh trong cc chui thng, khun lc nhiu
mu.
5a. Khun lc rt nh, mu nu .
Bo t trn hnh thnh (trong mt
nhm 4 bo t trn) bng cch
phn chia mt si hu th sn si
hnh tr, hnh khi sau tr thnh
hnh cu, gn cu.


Wallemia

5b. Khun lc lun khng nh (tr cc
loi Aspergillus a thm thu) khng
c mu nu . Bo t trn khng hnh
thnh sau s phn chia ca si hu
th

6
Aspergillus
6a. Gi sinh bo t trn vi nh phng to
6b. Gi sinh bo t trn khng c nh phng to
7a. T bo sinh bo t trn phn t.
Bo t trn c y bng, rng.


Scopulariopsis

7b. T bo sinh bo t trn hnh bnh. Bo t trn khng bng y.
8a. Khun lc mu vng n nu. Th
bnh c c di.


Paecilomyces

8b. Khun lc thng c mu lc (mt s loi mu trng) th bnh c ngn.
9a. Th bnh di, hnh ci di, n c

Penicillium

9b. Th bnh n c hoc phn nhnh nhiu t bo, t nhiu c hnh bnh.
10a. Th bnh n c hoc trn cc
gi sinh bo t trn phn nhnh, phn
nhnh ch gn y, thng khng to
thnh vng t bo


Acremonium


10b. Th bnh r rt, gi
sinh bo t trn phn
nhnh vng



Verticillium Trichoderma

11a. Khun lc thng mu lc
11b. Khun lc mu trng, vng, tm nht, hng nu hoc en.

12a. Khun lc mu trng, hng vng,
tm thnh thong c mu lc.
Bo t trn hnh qu chui c vch
lun c mt.


Fusarium
12b. Khun lc mu en, thnh thong
c mu hng. Bo t trn khng ngn vch

13a. Th bnh n c hoc trong cc
vng lng lo hnh bnh vi c r rt,
gi sinh bo t trn khng r rng.


Phialophora

13b. Th bnh nhiu, dy trn nh gi, thon rng, gn nh rng nht, khng
c c. Gi sinh bo t trn phn bit r, khng c cung.


Stachybotrys Chrysonilia

14a. Khun lc mc rt nhanh, ph kn a Petri trong mt t ngy,lng lo,
xp bng, mu da cam.
14b. Khun lc khng c mu da cam, khng ph kn a Petri trong mt t
ngy
15a. Bo t trn ch t on

Geotrichum


15b. Bo t trn pht sinh kiu t on si nm, pht sinh kiu blastic hoc
ch theo kiu blastic
16a. Bo t trn to thnh trong mt nhm 4 bo t do s phn chia ca 1 si
nm hu th hnh tr.
16b. Bo t trn khng to thnh trong nhm
17a. Cu trc bo t trn
gm bo t trn t on
v bo t trn kiu blastic
(so snh vi
Trichosporon trong nm
men v cc t bo si
nm mng dy, mu nu
ging bo t trn t
on trong chi


Aureobasidium).

Wallemia Moniliella

17b. Cu trc bo t trn ch c cc bo t trn kiu blastic
18a. Bo t trn blastic sinh ra trn cc si nm hoc t cc t bo phng to
hoc t cc nhnh
18b. Bo t trn blastic khng hnh thnh nhiu trn cc si nm, cc t bo
phng to hoc nhnh.
19a. Bo t trn sinh ra t cc
mu rng trn u cc t bo sinh
bo t trn phng to, gi sinh bo t
trn thng, phn nhnh nh (ging
ci cy) khun lc mng, nu xm.


Botrytis

19b. Bo t trn sinh ra trn
si nm
hoc sinh ra trn cc nhnh
phng to.
Khun lc ging nm men,
mu vng kem n nu sng,
da cam hng
hoc lc en.


Aureobasidium

20a. Bo t trn sinh ra n c trn
cc gia sinh bo t trn khng r rt,
to thnh tng m nhn thy bng
mt nh cc chm mu
en.


Epicoccum

20b. Bo t trn sinh ra n c hoc thnh chui, gi bo t trn r rt,
khng to thnh m.
21a. Bo t trn thnh chui, nhn.
Khun lc mu kem lc u sau gi
mu ti.


Moniliella


21b. Bo t trn thnh chui hay n c, rp. Khun lc mu xanh lc en
hoc nu lc.
22a. Bo t trn mng kh mng,
hu ht mt t bo. Bo t trn gc
thng
ngn vch vi vch ngn ngang.


Cladosporium

22b. Bo t trn c vch ngang v dc
23a. Bo t trn non trn y.Bo t trn trng thnh to thnh chui, c
hoc khng c mu.
23b.
Bo t
trn
non tht
y,
bo t
trn
trng
thnh
n c
hoc
to
chui
ngn
(cn gi
l chui
gi).



Alternaria Ulocladium




3.8.Cc kiu pht sinh bo t trn ca nhm Hyphomycetes (Lp nm
bt ton Deuteromycetes), (Theo Robert A. Samson, 1984).


Hnh : a j; c im ca cung sinh bo t trn, t bo sinh bo t trn v
bo t trn. a. cung n gin hoc to thnh b trn si nm; b. a gi; c.
ti gi; d. t bo sinh bo t trn n c; e. bo t trn pht sinh ng thi;
f. to chui; g. to git nhy; h. chui hng ngn; i. chui hng gc; j.
chui i xng hp trc.

*C 4 kiu pht sinh bo t trn
3.8.1. Athroconidi (Thallic development): Bo t c sinh ra n c hoc
thnh chui bng s ct on cc si nm. V d: Geotrichum. mt s chi
bo t trn dng phn on v blastic cng c to ra. V d: Moniliella.
3.8.2. Blastoconidi (Blastic development- kiu ny chi): Bo t trn c
to ra do cc si nm v c ca bn thn cc bo t trn to thnh. V nguyn
tc, bo t trn va c chc nng ca mt t bo sinh bo t trn va c chc
nng pht tn. Mng ca t bo sinh bo t trn gin ra v phng ln to
thnh mng bo t trn. Bo t trn c to ra n c hoc ng thi nhiu
bo t trn mt lc (v d: Botrytis, Aureobasidium) hoc to chui gc gi
(Acropetal chains) v d: Cladosporium. Chng c th c y hp (nh
Botrytis) hoc y rng nhu Epicoccum. Mt s chi c c trng l cc bo t
trn to thnh qua l nh trn vch cc t bo sinh bo t trn. Kiu pht sinh
bo t trn ny ging kiu Blastic, nhng c khc l cc t bo sinh bo t
trn mu ti v c mt nh vch dy, nhiu sc t m qua nh ny cc bo
t trn c chui ra. (v d: Alternaria, Ulocladium)
3.8.3. Pht sinh kiu th bnh (Phialidic development): Bo t trn pht
sinh trong chui gc non (Basipetal chains) t ming ca mt t bo c bit
gi l phialide- th bnh. Cc th bnh c hnh di, hnh bnh, thng c c
(cu to hnh cc nh). Bo t trn hnh thnh chui nh Penicillium,
Aspergillus, Paecilomyces hoc kt li thnh git nhy nh Trichoderma,
Phialophora, Stachybotrys, Acremonium, Verticillium.
3.8.4. Pht sinh dng phn t (Annellidie development): Cc bo t trn
to thnh t mt lot cc t tng trng ko di hoc mt t bo sinh bo t
trn phn t. Cc phn t thng kh nhn thy di knh hin vi quang hc
bnh thng, nhng c th nhn bit bng s tng chiu di ca nh t bo
sinh bo t trn (vng phn t trong lc sinh bo t trn). Mt c tnh vi hc
c trng na l bo t trn c y bng rng nh Scopulariopsis, t bo sinh
bo t trn ko di thm mt ngn sau khi bo t trn c sinh ra.

KHO 2 (Katsuhiko Ando, 2002)
1. To thnh dng b gi.Synnematous fungi (IX)
1. Khng to thnh dng b gi.2
2. Trc bo t xon hn 180.Helicoconidium (VI)
2. Trc bo t xon di 180.................3
3. nh bo t to thnh nhiu trc; cc mu li, ngoi lng t, c mt 1/4
chiu di thn bo tStauroconidium (VII)
3. nh bo t ch c mt trc; bt k mu li no ngoi lng t nu c th
khng qu 1/4 thn bo t..................4
3. Dng nh bo t khng nh trn..Miscellaneous fungi (VIII)
4. T l chiu di/ chiu rng ca thn nh bo t vt qu 15:1
Scolecoconidium (V).
4. T l chiu di/ chiu rng ca thn nh bo t t hn 15:1. 5
5. nh bo t thiu vch ngn...........Ameroconidium (I).
5. nh bo t c 1 hoc nhiu vch ngn..6
6. nh bo t c 1 vch ngn...........Didymoconidium (II)
6. nh bo t nhiu hn 1 vch ngn.7
7. nh bo t ch c vch ngn ngang......Phragmoconidium (III)
7.Thn nh bo t b chia nh bi cc vch ngn giao nhau nhiu hn 1 mt
phng........................................................................Dictycoconiium
(IV)
7. Dng nh bo t khng nh trn........................Miscellaneous (VIII)
I. Amerocodidium
A. Khng sinh cung nh bo t, t bo sinh nh bo t khng l ra
trong t bo si.
1. nh bo t phn ct hon ton
- Bo t c sinh ra n c.Aureobasidium ( s 15), Chryrosporium
(9), Humicola* (11, 154), Mammaria (8), Endocalyx, Trichosporiella.
- Bo t c sinh ra nhiu, to thnh chui.Torula* (22)
2. nh bo t ni sinh
- nh bo t ngn th bnh.......Cladorrhium.
- nh bo t phn t.Coniosypha* (72), Exophiala, Honmonema.
3. nh bo t tnGeotrichum (1), Scytalidium (6), Sporendonema.,
Trichosporon.
B. Khng sinh cung nh bo t hoc c nhng khng r rng.
1. nh bo t phn ct hon ton.
- Bo t c sinh ra n c
+ nh bo t sinh ra mt v tr ca t bo sinh bo t ..Gilmaniella*
(13), Humicola* (11, 154), Mycogone* (18), Nigrospora (10), Sepedonium
(16), Thermomyces.
+ nh bo t sinh ra nhiu v tr ca t bo sinh bo t. Beauveria
(30), Idriella (25), Rhinocladiella* (29), Torula* (22) Ramichloridium,
Isthmolongispora, Acrodontium.
+ nh bo t ko di im pht trin c s (Basauxic
conidium)..Arthrinium (12), Gilmaniella* (13), Pleroconium, Cordella.
- nh bo t c sinh ra nhiu, to thnh chui.
+ T bo sinh nh bo t khng nghch tin..Torula* (22),
Hormomyces.
+ T bo sinh nh bo t nghch tin.Basipetospora (14).
2. nh bo t ni sinh.
- nh bo t ngn th bnh.Acremonium (40), Chalara* (43),
Gliomastix (41), Monocillium (42), Phialophora (44), Torulomyces* (48),
Myrioconium.
- nh bo t tCircinotrichum (75), Conioscypha* (72), Gyrothrix
(77), Scopulariopsis* (73).
3. nh bo t tn..Malbranchea, Ovandendron, Bahuakala,
Arthrographis* (2), Mauginiela.
C. Cung bo t nh c hoc khng c vch ngn, pht trin n c,
khng phn nhnh.
1. nh bo t phn ct hon ton
- Bo t c sinh ra n c
+ nh bo t sinh ra mt v tr ca t bo sinh bo t
Acrogenospora, Domingoella.
+ nh bo t sinh ra nhiu v tr ca t bo sinh bo t.
- Nhng t bo sinh bo t khng tng nhanh.Beltrania (38), Beltraniella
(37), Beltranioposis (39), Calcarisporium* (32), Oedocephalum (21),
Asteromyces (158), Dactylaria* (36), Verticicladiella, Gonatobotrys.
- Nhng t bo sinh bo t tng nhanh..Sporothrix (28), Verticicladium*
(27), Rhinocladiclla* (19), Sublispora* (33), Leptodontidium, Solosympodiella.
- nh bo t c sinh ra nhiu, to thnh chui.
+ T bo sinh nh bo t khng nghch tin..Cladosporium (17),
Hyalodendron (19), Periconia, Goonatobotryum.
+ T bo sinh nh bo t nghch tin.Cladobotryum* (96).
2. nh bo t ni sinh
- nh bo t ngn th bnh
a. Mt th bnh
+ Cung nh bo t ko di theo dnh bo t c sinh
raChloridium (47), Exochalara, Bisporomyces.
+ Cung nh bo t khng ko di.Torulomyces* (48), Condinaea (46),
Zakatoshia, Condinaeopsis, Menispora, Pleurocatena.
b. Nhiu th bnh..), Acrophialophora* (59), Chaetopsina (68),
Gliocephalotrichum (67), Graphium* , Gonytrichum (45), Aspergillus (53),
Penicillium* (54), Phialocephala (51), ), Phialomyces (60), Stachybotrys (61),
Stachylidium* (50), Memmoniella (62), Thysanophora* (52), Verticillium*
(64), Tolypocladium, Mariannaea* (58), Eladia (49), Paecilomyces* (56),
Gliocladium* (55), Stachybotryna (70), Zygosporium.
- nh bo t phn t Graphium*, Scopularopsis* (73),
Leptographium*, Ceratocladium.
3. nh bo t tnSympodiclla* (7), Wallcmia (5).
D. Cung bo t nh c vch ngn pht trin n c hoc phn
nhnh.
1. nh bo t phn ct hon ton
- nh bo t c sinh ra n c
+ nh bo t sinh ra mt v tr ca t bo sinh bo t
Staphylotricum, Botryotricum.
+ nh bo t sinh ra nhiu v tr ca t bo sinh bo t.
- T bo sinh bo t khng ko diBotrytis (24), Chromclosporium
(23), Costantinclla (34), Calcarisporium* (32), Hanstordia (35), Ditangium.
- T bo sinh bo t ko di.Selenosporella (31), Tritirachium (26),
Verticicladium* (27), Virgaria, Rhinotrichella, Puciola, Geniculosporium,
Geniculodendron, Nodulisporium.
- nh bo t c sinh ra nhiu to thnh chui.
+ T bo sinh bo t khng nghch tin.Dimorphospora (20),
Alysidiopsis.
+ T bo sinh bo t nghch tinCladobotryum* (96).
2. nh bo t ni sinh.
- nh bo t ngn th bnh.Acrophialophora* (59), Clonostachys (57),
Gliocladium* (55), Mariannaea* (58), Myrothecium (66), Paecilomyces* (56),
Penicillium* (54), Stachylidium* (50), Thysanophora*(52), Trichoderma (63),
Verticillium* (64), Metarhizium (71), Sesquicillium (65), Phaeostalagmus,
Volutella, Craspedodidyum.
- nh bo t phn t.Doratomyces* (74,76), Scopulariopsis (73),
Leptographium*.
3. nh bo t tn..Geomyces (3), Sympod, Oidiodendron (4),
Parasympodiella* (79), Arthrographis* (2), Oncocladium, Briosia.
II. Didymoconidium
A. Khng sinh cung nh bo t, t bo sinh nh bo t khng l ra
trong t bo si.
1. nh bo t phn ct hon ton..Trichocladium* (161).
2. nh bo t ni sinh
- nh bo t ngn th bnh..Cyphellophora.
- nh bo t phn t.Rhynchosporium.
B. Khng sinh cung nh bo t hoc c nhng khng r rng.
1. nh bo t phn ct hon ton
- nh bo t c sinh ra n c
+ nh bo t sinh ra mt v tr ca t bo sinh bo t Mycogone*
(18), Trichocladium* (161).
+ nh bo t sinh ra nhiu v tr ca t bo sinh bo t..
Cephaliophora* (98), Leptodiscella, Ochroconis.
- nh bo t c sinh ra nhiu to thnh chui.
+ T bo sinh bo t khng nghch tin.Ramularia, Arxiella.
+ T bo sinh bo t nghch tin.............Trichocthecium* (83)
2. nh bo t ni sinh
- nh bo t ngn th bnhChalara* (43), Cephalosporiopsis,
Cylindrodendrum.
- nh bo t phn t..............................Oedothea.
C. Cung bo t nh c hoc khng c vch ngn, pht trin n c, khng
phn nhnh.
1. nh bo t phn ct hon ton.
- Bo t c sinh ra n c
+ nh bo t sinh ra mt v tr ca t bo sinh bo t
Endophragmiella.
+ nh bo t sinh ra nhiu v tr ca t bo sinh bo t Chaetopsis,
Wardomyces.
- nh bo t c sinh ra nhiu to thnh chui.
+ T bo sinh bo t khng nghch tin.Bispora* (81
+ T bo sinh bo t nghch tinCladobotryum*(96
2. nh bo t ni sinh
- nh bo t ngn th bnhPenicillifer, Sibirina, Septomyrothecium.
- nh bo t phn t..Gymnodochium, Exophiala.
3. nh bo t tn..Parasympodiella* (79).
III. Phragmoconidium
A. Khng sinh cung nh bo t, t bo sinh nh bo t khng l ra
trong t bo si.
1. nh bo t phn ct hon tonCeratophorum, Arthrocladium,
Microsporium*, Trichophyton*, Trichocladium* (161).
2. nh bo t ni sinh..Cha thy.
B Khng sinh cung nh bo t hoc c khng r rng.
1. nh bo t phn ct hon ton
- nh bo t c sinh ra n c
+ nh bo t sinh ra mt v tr ca t bo sinh bo t Bactridium
(95) Microsporium*, Pithomyces* (111), Trichophyton*, Trichocladium*
(161), Tretospora.
+ nh bo t sinh ra nhiu v tr ca t bo sinh bo t..
Cephaliophora* (98), Dactylaria* (80), Scolecobasidium* (102),
Wicsnoriomyces (99).
- nh bo t c sinh ra nhiu to thnh chuiLylea.
2. nh bo t ni sinh
- nh bo t ngn th bnhFusarium* (89), Stigmina
- nh bo t phn t..Toxosporium.
C. Cung bo t nh c hoc khng c vch ngn, pht trin n c,
khng phn nhnh.
1. nh bo t phn ct hon ton
- nh bo t c sinh ra n c
+ nh bo t sinh ra mt v tr ca t bo sinh bo t Dactylella*(87),
Deightoniella* (92), Moncrosporium (86), Sporidesmium (85,101),
Corynespora, Endropharagmia (100), Kramasamuha.
+ nh bo t sinh ra nhiu v tr ca t bo sinh bo tBrachysporium
(93), Camposporium (90), Dendryphiella (155), Pyricularia (94), Curvularia
(103), Dactylaria* (80), Drechslera (105), Endophragmiclla, Subulispora*
(33), Helminthosporium (106), Dactyrella* (87), Scolecobasidium* (102),
Cercospora, Exerohilum.
- Bo t c sinh ra nhiu, to thnh chui.Septonema* (91).
2. nh bo t ni sinh
- nh bo t ngn th bnh.Cacumisporium, Fusarium* (89),
Fusichalara.
- nh bo t phn tAnnellophora.
D. Cung nh bo t c vch ngn pht trin n c hoc phn
nhnh.
1. nh bo t phn ct hon ton
- nh bo t c sinh ra n c
+ nh bo t sinh ra mt v tr ca t bo sinh bo t Brachysporeilla.
+ nh bo t sinh ra nhiu v tr ca t bo sinh bo tSterigmatobotrys.
- Bo t c sinh ra nhiu, to thnh chui.Septonema* (91).
2. nh bo t ni sinh
- nh bo t ngn th bnh.Cylindrocarpon (88), Cylindrocladium (97),
Fusarium* (89)
- nh bo t phn tArthrobotryum.
3. nh bo t tnParasympodiella* (79).
IV. Dictyoconidium
A. Khng sinh cung nh bo t, t bo sinh nh bo t khng l ra
trong t bo si.
1. nh bo t phn ct hon tonDictyosporium* (113), Piricauda,
Bioconiosporium.
2. nh bo t ni sinhKonodospora*
B. Khng sinh cung nh bo t hoc c khng r rng
1. nh bo t phn ct hon ton. Diheterospora (114), Pithomyces* (111),
Scutisporus (115), Spogazzinia (112), Epicoccum (110), Sarcinclla, Acrospeira,
Stemphyliopsis, Dictyosporium* (113), Monodictys, Hanstordiellopsis,
Pseudopetrakia, Dictyodesmium, Dictyoarthrinium.
2. nh bo t ni sinh
- nh bo t ngn th bnh.Ascoconidium
- nh bo t phn tAnnellophorella, Stigmina.
C. Cung bo t nh c hoc khng c vch ngn, pht trin n c,
khng phn nhnh.
1. nh bo t phn ct hon ton
- nh bo t c sinh ra n c
+ nh bo t sinh ra mt v tr ca t bo sinh bo t Stemphylium (104),
Septosporium, Actodictys, Tretospeira.
+ nh bo t sinh ra nhiu v tr ca t bo sinh bo tUlocladium (108),
Dactylosporium, Dactyospiropes.
- Bo t c sinh ra nhiu, to thnh chui.Alternaria (107),
Coniosporium.
2. nh bo t ni sinh..Cha bit.
D. Cung nh bo t c vch ngn pht trin n c hoc phn
nhnh.
1. nh bo t phn ct hon ton.Pseudodictyosporium.
2. nh bo t ni sinh..Cha bit
V. Scolecoconidium
A. Khng sinh cung nh bo t, t bo sinh nh bo t khng l ra
trong t bo si.
1. nh bo t phn ct hon tonSigmoidea* (162), Irpicomyces
2. nh bo t ni sinhCha thy.
B. Khng sinh cung nh bo t hoc c khng r rng
1. nh bo t phn ct hon ton. Sigmoidea* (162), Microdochium,
Pseudoanguillospora, Cercosporella.
2. nh bo t ni sinhPseudomicrodochium.
C. Cung bo t nh c hoc khng c vch ngn, pht trin n c,
khng phn nhnh.
1. nh bo t phn ct hon ton
+ nh bo t sinh ra mt v tr ca t bo sinh bo t Anguillospora
(141,142)
+ nh bo t sinh ra nhiu v tr ca t bo sinh bo tMirandina
2. nh bo t ni sinh..Cryptophiale* (69).
D. Cung nh bo t c vch ngn pht trin n c hoc phn
nhnh.
1. nh bo t phn ct hon ton.Harpographium.
2. nh bo t ni sinh
- nh bo t ngn th bnh.Flagellospora
- nh bo t phn tRogersia.
VI. Helicoconidium
A. Khng sinh cung nh bo t, t bo sinh nh bo t khng l ra
trong t bo si.
1. nh bo t phn ct hon tonHelicoon
2. nh bo t ni sinhCha thy
B Khng sinh cung nh bo t hoc c khng r rng.
1. nh bo t phn ct hon ton
- nh bo t c sinh ra n c
+ nh bo t sinh ra mt v tr ca t bo sinh bo t Zalerion
(156), Cirrenelia (157), Troposporella.
+ nh bo t sinh ra nhiu v tr ca t bo sinh bo t..Everhartia*
(116), Helicomyces* (120), Trocophora*, Dicotomophthoropsis.
- nh bo t c sinh ra nhiu to thnh chuiHelocodendron* (121).
2. nh bo t ni sinh.Cha thy.
C. Cung bo t nh c hoc khng c vch ngn, pht trin n c,
khng phn nhnh.
1. nh bo t phn ct hon ton
- nh bo t c sinh ra n c
+ nh bo t sinh ra mt v tr ca t bo sinh bo t Everhartia*
(118), Xenosporium (122).
+ nh bo t sinh ra nhiu v tr ca t bo sinh bo t..Helicomyces*
(120), Helicosporium (119), Trocophora*.
- nh bo t c sinh ra nhiu to thnh chuiHelocodendron* (121).
2. nh bo t ni sinh.Cha thy.
D. Cung nh bo t c vch ngn pht trin n c hoc phn
nhnh.
1. nh bo t phn ct hon ton.Helicomyces* (120).
2. nh bo t ni sinh.Cha thy.
VII. Stauroconidium
A. Khng sinh cung nh bo t, t bo sinh nh bo t khng l ra
trong t bo si.
1. nh bo t phn ct hon tonAlatosessillispora (136), Amallospora*
(129), Ordus (139), Tretraploa (128), Trisulcosporium (131).
2. nh bo t ni sinh..Kodonospora*
B Khng sinh cung nh bo t hoc c khng r rng.
1. nh bo t phn ct hon ton..Amallospora* (129), Diplocladiella (130),
Dwayaangum (137), Lateriramulosa (138), Stephanoma (127), Tricellula
(135), Trifurcospora (133), Varicosporina (159), Titaeela, Dendrosporium*
(134), Scolecobasidium, Kazulia.
2. nh bo t ni sinh.Cha thy.
C. Cung bo t nh c hoc khng c vch ngn, pht trin n c,
khng phn nhnh.
1. nh bo t phn ct hon ton..Speiropsis (132), Dicradinion* (116),
Articulospora (144), Clavariopsis (145), Culicidospora (146), Tetrachaetum
(149), Dendrospora (150), Tricladium (152), Varicosporium (153), Orbimyces
(163), Dendrosporium* (134), Asterosporium (109), Pyramidospora,
Arachnophora, Tridentaria.
2. nh bo t ni sinh.Alatospora (140), Helicus (143), Lemonniera
(147,148), Clavatospora (160).
D. Cung nh bo t c vch ngn pht trin n c hoc phn
nhnh.
1. nh bo t phn ct hon ton.Dicranidion* (116), Tetraladium (151).
2. nh bo t ni sinh.Cha thy.
VIII. Miscellaneous fungi
Aegerita (126), Canceilidium (125), Candelabrum (123), Clathosphaerina
(124), Strumella (117), Beverwkella.
IX. Synnematous fungi: Dratomyces* (74,76), Graphium*, Stibella,
Tubercularia, Phaeoisaria, Podosporium, Didymobotrium.



Hnh 1: 1. Geotrichum candidum, 2. Arthrographis sp., 3. Geomyces
pannorum, 4. Oidiodendron flavum, 5. Wallemia sebi, 6. Scytalidium lignicola,
7. Sympodiella acicola.


Hnh 2: 8. Mammaria echinobotryoides, 9. Chrysosporium sp., 10. Nigrospora
oryzae, 11. Humicola grisea, 12. Arthrinium phaeospermum, 13. Gilmaniella
humicola


Hnh 3: 14. Basipetospora rubra, 15. Aureobasidium pullulans, 16. Sepedonium
chysospermum, 17. Cladosporium sp., 18. Mycogone perniciosa,
19. Hyalodendron.,



Hnh 4: 20. Dimorphospora foliicola, 21. Oedosephalum glomerulosum, 22.
Torula herbarum, 23. Chrimelosporium sp., 24. Botrytis cinerea

Hnh 5: 25. Idriella lunata, 26. Tritirachium oryzae, 27. Verticicladium trifidum,
28. Sporothrix sp., 29. Rhinocladiella sp., 30. Beauveria bassiana.


Hnh 6: 31. Selenosporella curvispora., 32. Calcarisporium arbuscula,
33. Subulispora procurvata, 34. Costantinella micheneri, 35. Hansfordia
pulvinata.







Hnh 7: 36. Dactylaria sp., 37. Beltraniella odinae, 38. Beltrania rhombica,
39. Beltraniopsis esenbeckiae.







Hnh 8: 40. Acremonium butyli, 41. Gliomastix murorum var. felina.
42. Monocillium indicum, 43. Chalara sp., 44. Phialophora sp., 45.
Gonytrichum macrocladum




Hnh 9: 46. Codinaea sp., 47. Chloridium chlamydosporis, 48. Torulomycrs
lagena, 49. Eladia saccula, 50. Stachylidium bicolor, 51. Phialocephala
bactrospora.


Hnh 12: 63. Trichoderma harzianum & T.polysporum, 64. Verticillicum sp, 65.
Sesquicillium candelabrum, 66. Myrothecium sp, 67. Gliocephalotrichum
simplex.


Hnh 13: 68. Chaetopsina fulva, 69. Cryptophiale udagawae, 70.
Stachybotryna columare, 71. Metarhizium anisopliae, 72. Conioscypha sp.


Hnh 14: 73. Scopulariopsis brevicaulis, 74. & 76. Doratomyces spp,
75. Ciricinotrichum maculiforme, 77. Gyrothrix circinata.


Hnh 15: 78. Pseudobotrytis terrestris, 79. Parasympodiella longispora, 80.
Dactylaria obtriangulata, 81. Bispora betulina, 82. Arthrobotrys oliospora.


Hnh 16: 83. Trichothecium roseum, 84. Menisporopsis novae-zelandiae, 85.
Sporidesmium goidanichii, 86. Monacrosporium sp, 87. Dactylella atractoides,
88. Cylindrocarpon destructans, 89. Fusarium sp.

Hnh 17: 90. Camposporium antennatum, 91. Septonema scedens, 92.
Deightniella elegans, 93. Brachysporium nigrum, 94. Pyricularia oryzae


Hnh 18: 95. Bactridium flavum, 96. Cladobotryum mycophilum,
97. Cylindrocladium ilicicola, 98. Cephaliophora irrgularis, 99. Wiesneriomyces javanicus.


Hnh 19: 100. Endophragmia hyalosperma, 101. Sporidesmium niligirense, 102.
Scolecobasidium variabile, 103. Curvularia oryzae, 104. Stemphylium botryosum.

Hnh 20: 105. Drechslera sp., 106. Helminthosporium solani, 107. Alternaria alternata, 108.
Ulocladium atrum.

Hnh 21: 109. Asterosporium orientale, 110. Epicoccum purpurascens, 111. Pithomyces
chartarum, 112. Spegazzinia tessarthra.


Hnh 22: 114. Diheterospora catenulata, 115. Scutisporus brunneus,
116. Dicranidion fragile, 117. Strumella griseola, 118. Everhartia lignatilis.


Hnh 23: 119. Helicosporium panacheum, 120. Helicomyces ambiguus, 121. Helicodendron
conglomeratum, 122. Xenosporium berkeleyi.


Hnh 24: 123. Candelabrum japonense, 124. Clathrosphaerina zalewskii, 125. Cancellidium
applanatum, 126. Aegerita candida, 127. Stephanoma strigosum.



Hnh 25: 128. Tetraploa aristata, 129. Amallospora dacrydion, 130. Diplocaladiella scalaroides,
131. Tripospermum acerinum, 132. Speiropsis pedatospora.


Hnh 26: 133. Trifurcospora irregularis, 134. Dendrosporium lobatum, 135. Tricellula curvatis,
136. Alatosessilispora bibrachiata, 137. Dwayaangam yakuensis, 138. Lateriramulosa
uniinflata, 139. Ordus chiayiensis.

Hnh 27: (140-153: Aquatic, 154-163. Marine) 140. Alatospora acuminata,
141. Anguillospora longissima, 142. Anguillospora crassa, 143. Heliscus lugdunensis, 144.
Articulospora tetracladia, 145. Clavariopsis aquatica, 146. Culicidospora gravida, 147.
Lemonniella aquatica, 148. Lemonniera cornuta, 149. Tetrachaetum elegans,
150. Dendrosporium erecta, 151. Tetracladium setigerum, 152. Tricladium splendens, 153.
Varicosporium elodeae, 154. Humicola alopallonella, 155. Dendryphiella salina, 156. Zalerion
maritimum, 157. Cirrenalia macrocephala, 158. Asteromyces cruciatus, 159. Varicosporina
ramulosa, 160. Clavatospora bulbosa, 161. Trichocladium achrasporum, 162. Sigmoidea
luteola, 163. Orbimyces spectabilis.

LP NM TI (Ascomycetes)

Giao t c



1. c tnh c trng:
Cc si sinh trng ngn vch v n bi. Giai on hu tnh bng s hnh thnh ca cc ti
bo t (Asci) do si tip hp nhn ca hai giao t khc nhau (giao t c gi l Antheridium,
giao t ci l ascogonium). Sau phn chia gim phn, trong cc ti bo t thng c 8 bo t
ti (cng c th l 2 hoc 4). Cc ti bo t lun c nm trong cc ascomata (ascocarps) l
cc th qu sinh ra n c hoc kt li trong hoc trn mt m nm (mt m cc si sinh
dng). Hnh dng ca cc th qu rt quan trng phn loi. Th qu c cc dng: Hnh cc
(Apothecia), hnh cu, gn cu khng c ming (cleistothecia), hnh bnh (Perithecia). Ti bo
t cng c th pht trin trong cc ngn ca cc m nm (Stroma) hoc pht sinh t cc cu
trc dng gi bn nguyt (Pseudothecia). Giai on sinh sn hu tnh (Teleomorph) thng
km theo cc giai on sinh sn v tnh (anamorph). Hu ht cc loi nm bt ton l dng
sinh sn v tnh trong vng i ca lp nm ti, cc loi nm ti thng gp nhng sn
phm sy kh bo qun.
C th dng mt s chuyn lun sau: v nm ti, th qu ti hnh cu zeemvon Arx
(1981), cc loi nm ti cng b chi Byssochlamys ca Stolk & Samson (1971) v
Samson (1974), Eurotium of Blaser (1975) v kho nm ti dng sinh sn v tnh thuc chi
Aspergillus ca Raper & Fennell (1965).

KHO N CHI ( Robert A. samson, 1984)
1a. Th qu ti (ascomata) c cung r rt,
cung l mt si nm n c,
dng sinh sn v tnh l Basipetospora, Monascus.
1b. Th qu ti khng c cung,
dng v tnh Paecilomyces
hoc Aspergillus 2
2a. Th qu ti khng c vch r rng,
dng v tnh l Paecilomyces, Byssochlamys.
2b. Th qu ti c vch dng v tnh Aspergillus 3
3a. Vch ca th qu ti c bao quanh
bi cc t bo Hiille vch dy,
dng v tnh nhm Asp.nidulans. Emericella
3b. Vch ca th qu ti khng c
bao quanh bng cc t bo Hiille 4
4a. Th qu ti mu vng, vch gm
mt lp cc t bo dt, dng v tnh
thuc nhm Asp.glaucus Eurotium
4b. Th qu ti trng hoc mu kem,
vch gm vi lp t bo dt,
dng v tnh thuc nhm Asp.fumigatus Neosartorya

LP NM TIP HP
c im dc trng:
Lp ny c h si a nhn, ch mt vch ngn c to thnh phn cch cc c quan
c bit nh nang bo t kn (Sporangia) v cc bo t tip hp vi si nm sinh sn vch
ngn thnh thong xut hin cc phn trng thnh ca h si nm.
Sinh sn v tnh bng cc bo t nang (Sporangiaspores Bo t bt ng n c)
c to ra trong cc nang bo t kn hnh cu hay qu l (Sporangia) c tr hoc khng c
tr (columella phn bng hoc phn gia bn trong nang bo t ni tip vi cung sinh nang
bo t) hoc trong cc ti chui bo t (cc nang bo t hnh tr chia thnh mt dy cc bo
t). Mt nang bo t chui khng c tr, nang bo t kn nh (Sporangiola l cc nang bo t
kn nh, hnh cu vi mt hoc mt t bo t v tr rt nh) thy c h Thamnidiaceae
Cunninghamellaceae & Choanephoraceae. Mt s chi c apophysis (ch phng ca gi sinh
nang bo t ngay di nang bo t) c th c si b ngang (stolons): chng l cc si nm
c bit chy khp b mt v mang cc gi sinh nang bo t. Cc si ny c th bm cht vo
c cht bng cc Rhizoids (r). Mt s loi sinh bo t o (cc t bo vch dy) hoc cc bo
t phn (cc t bo vch mng, hnh cu), chng c th u, gia si nm n c hoc
thnh chui. Cc bo t nang, bo t o, bo t phn ny mm to thnh h si nm mi.
Sinh sn hu tnh bng hai giao t a nhn to ra bo t tip hp mng dy mu en,
nu, vng, thng c bao ph mt lp gai hoc cc si ru. Hai on si nm mi u bo
t tip hp (Zygospose) gi l si treo (suspensor). Cc si treo c th c kch thc hoc hnh
dng khc nhau, thnh thong c si treo hnh thnh cc mu ph (v d: mt s loi ca
Absidia). Hu ht cc loi thuc lp ny l hoi sinh, mt s loi k sinh trn cc nm khc
cng nh trn ng vt, thc vt. Mt s loi ca chi Rhizopus v Absidia phn b rng trn
cc ng cc bo qun, rau, qu, trong khng kh v phn xanh. Mt s loi ca chi Mucor v
Rhizopus c tm quan trng trong thc phm ln men v sn xut cc axt hu c, nhng
cng gy bnh thi rau hoa qu thu hoch.
Cc ti liu c th dng: i vi lp Zygomycetes l kho Zycha et al (1969) Hanlin
(1973), Hesseltine & Ellis (1973), ODonnell (1979) & von Arx (1981). Kho n loi ca chi
Mucor ca Schipper (1978), chi Rhizopus ca
Inui et al (1965), Scholer & Miiler (1971) & Schipper (1984).

KHO PHN LOI N CHI ( Robert A. samson, 1984)
1a. Bo t nang c to thnh trong cc
ti nang bo t dy bao ph b mt
bng cung sinh bo t nang Syncephalastrum
1b. Cc bo t nang c to thnh
trong cc nang bo t hnh cu, qu l vi tr 2
2a. Nang bo t v cung sinh bo t nang
lun c mu ti, cung sinh bo t nang
thng khng phn nhnh,
thng sinh trong nhm. Nang bo t
kch thc thay i t 50 - 360m.
Bo t nang c vch sc
Rhizopus
2b. Nang bo t v cung sinh bo t nang
khng mu hoc mu nht, thng phn nhnh.
Nang bo t khng qu 100m.
bo t khng c vch sc 3
Apophysis
3a. Nang bo t hnh qu l vi c r rt,
d = 10 - 40m. Cc nang cui
cung sinh bo t chnh ln hn 80m. Absidia
3b. Nang bo t kn hnh cu
khng c c, hu ht ln hn 40m Mucor




Cu trc hnh thi lp Nm Tip hp

TI LIU THAM KHO
1. Bi Xun ng, 2004, Nguyn l phng chng Nm mc v mycotoxin, Nh xut bn
khoa hc v k thut H Ni.
2. Katsuhiko Ando, 2002, Identification of Fungi Imperfecti, NITE Biological Resource
Center National Intitute of Technology and Evaluation.
3. Katsuhiko Ando at al, 2004, Sampling and Isolation Methods of Fungi, Department of
Biotechnology, National Institute of Technology and Evaluation (NITE), Japan.
4. Robert A. Samson at al, 1984, Introduction Food Borne Fungi, CBS, Institute of
the Royal Netherlands Academy of Arts and Sciences.
5. W. Gam, E.S. Hoekstra and A. Aptroot, 1998, CBS Course of Mycology, CBS, P.O.
Box 273, 3740 AG Baarn, the Netherlands.

Bi 10 Nm men
A. PHN LOI NM MEN
B - CC PHNG PHP THC NGHIM DNG NH TN NM MEN

1. Quan st hnh thi t bo nm men v o kch thc
2. Nhum mu t bo nm men:
- Thuc nhum soudan III:
- Thuc nhum en Soudan B (theo Burdon):
- Thuc nhum safranin:
- Dung dch nhum nhn t bo:
- Dung dch lc malachit:
3. Quan st qu trnh ny chi ca t bo nm men
- Mi trng mch nha - cao nm men - glucoza - pepton:
4. Quan st khun ty gi:
- Mi trng khoai ty - glucoza:
- Mi trng ng:
5. Quan st bo t bn (Ballistoconidium, Ballistospore):
- Mi trng bt ng:
6. Quan st bo t ti (ascospore):
a. Mi trng ming thch cao:
b. Mi trng ming thch cao ci tin:
c. Mi trng Gorodkowa (1908)
d. X l vi tia t ngoi:
e. Mi trng thch nc
f. Mi trng Amano (1950)
g. Mi trng dch tinh bt khoai ty 0,5% (Almeida v Lacaza)
h. Mi trng Kleyn:
6. Quan st c tnh nui cy
7. Th nghim xc nh kh nng ln men cc loi ng
8. Th nghim xc nh kh nng ng ho cc hp cht cacbon khc nhau:
8.1. Phng php nh gi kh nng sinh trng trn mi trng dch th:
8.2. Sinh trng trn mi trng thch
8.3. Phng php dng con du
9. Th nghim xc nh kh nng ng ho cc ngun nit
10. Th nghim xc nh kh nng hnh thnh hp cht loi tinh bt:
11. Th nghim xc nh nhu cu vitamin cho sinh trng ca nm men:
12. nh gi s sinh trng trn mi trng c nng ng cao
13. nh gi s pht trin khi c mt Cycloheximit
14. Xc nh hot tnh phn gii Urea (hay hot tnh Ureaza)
15. Th nghim lm i mu Diazonium blue B (DBB test)
1. Mi trng Acetat (g/l) (M.C. Clary et al., 1959)
2. Mi trng thch Gorodkowa (Dodder v Kreger - van Rij, 1952) (g/l)
3. Mi trng cao ng (Lodder v Kreger - van Rij, 1952)
4. Mi trng thch V-8 (Wicketam v cng s, 1946)
5. Mi trng pepton - cao men - glucoza (Vander Walt v Codder, 1970)
6. Thnh phn mi trng tng hp (tinh khit v thnh phn ho hc)
7. Mi trng quan st hnh thi t bo nm men:
8. Mi trng nit c s:
9. Mi trng carbon c s:
10. Mi trng khng c vitamin

A. PHN LOI NM MEN
Thut ng Nm men (yeast, levure) ch l tn chung ch nhm vi nm thng c cu to
n bo v thng sinh si ny n bng phng php ny chi (budding). Nm men khng
thuc v mt taxon phn loi no nht nh, chng c th thuc ngnh Nm ti (Ascomycota)
hoc ngnh Nm m (Basidiomycota).
Ny chi l cch sinh sn v tnh in hnh ca nm men. Khi thnh t bo m ra
to ra mt chi (bud). Chi pht trin thnh t bo con v c th tch khi t bo m ngay t
khi cn nh hoc cng c th vn khng tch ra ngay c khi ln bng t bo m. Nhiu khi
nhiu th h vn dnh vo mt t bo u tin ny chi v to thnh mt cnh nhiu nhnh t
bo trong ging nh cy xng rng. Chi c th mc ra theo bt k hng no (ny chi a
cc- multilateral budding) hoc ch ny chi hai cc (ny chi theo hai cc- Bipolar budding)
hoc ch ny chi mt cc nht nh (ny chi theo mt cc monopolar budding). Nm
men cn c hnh thc sinh sn phn ct nh vi khun. C th hnh thnh mt hay vi vch
ngn phn ct t bo m thnh nhng t bo phn ct (fission cells). in hnh cho kiu
phn ct ny l cc nm men thuc chi Schizosaccharomyces. mt s nm men thuc ngnh
Nm m, c th sinh ra dng bo t c cung nh (sterigmatoconidia) hoc bo t bn
(ballistoconidia hay ballistospore). Bo t c cung nh thng gp cc chi nm men
Fellomyces, Kockovaella v Sterigmatomyces, khi chi sinh ra trn mt nhnh nh v tch
ra khi nhnh b gy. Bo t bn c sinh ra trn mt gai nhn ca t bo nm men v b bn
ra ph i din khi thnh thc. Nu cy cc nm men sinh bo t bn thnh hnh zich zc trn
thch nghing hoc trn a Petri th sau mt thi gian nui cy s thy xut hin trn thnh
ng nghim hoc np a Petri c mt hnh zch zc khc c hnh thnh bi cc bo t bn
ln. Bo t bn l c im ca nm men thuc cc chi Bensingtonia, Bullera, Deoszegia,
Kockovaella, Sporobolomyces.... Mt s nm men cn c mt hnh thc sinh sn v tnh na,
l vic hnh thnh cc bo t t (arthroconidia hay arthrospore). Khi s hnh thnh cc
vch ngn u cc nm men dng si, sau tch ra thnh cc bo t t. Loi ny gp
cc nm men thuc c hai ngnh: Nm ti v Nm m. Thng gp nht l cc chi nm
men Galactomyces, Dipodascus (dng v tnh l Geotrichum) v Trichosporon. Nm men cn
c th to thnh dng tn (thallus) di dng khun ty (si nm- hyphae) hay khun ty gi
(gi si nm pseudohyphae).
Dng sinh sn hu tnh nm men l dng cc bo t ti (ascospore) c sinh ra t
cc ti (asci). C th xy ra s tip hp (conjugation) gia hai t bo nm men tch ri hoc
gia t bo m v chi. Cn c c s bin np trc tip trong 1 t bo sinh dng (vegetative
cell), t bo ny bin thnh ti khng qua tip hp (unconjugated ascus). Thng trong mi
ti c 4 hay i khi c 8 bo t ti. Trong mt s trng hp li ch c 1-2 bo t ti. Bo t
ti chi Saccharomyces c dng hnh cu, hnh bu dc; chi Hanseniaspora v loi
Hansenula anomala c dng hnh m ; loi Hansenula saturnus bo t ti c dng qu xoi
gia c vnh ai nh dng Sao Th. Mt s bo t ti c dng ko di hay hnh xonB mt
bo t ti c th nhn nhi, c th x x hoc c gai Bo t mng dy (hay bo t o-
chlamydospore) l dng bo t gip nm men vt qua c iu kin kh khn ca ngoi
cnh, ch khng phi l hnh thc sinh sn. Mt s nm men cn c th sinh v nhy.
Bn cnh rt nhiu nm men c ch nh l cc loi nm men dng sn xut ru
trng, ru vang, bia, lm n bt m, to sinh khi giu protein v vitamin, sn xut enzym,
sn xut acid citric t kh thin nhin, sn xut riboflavin (vitamin B2) cn c nhng loi nm
men c th gy bnh.





N= nhn; M= ty th; Va= khng bo; ER= mng li ni cht; Ves= bo nang










Bo t bn Phn ct t bo




Ny chi


Bo t ti Bo t mng dy Bo t t


V nhy nm men

Mt vi loi nm men gy bnh ngi:


Candida albicans Cryptococcus neoformans
phn loi nm men ngi ta phi tin hnh nghin cu cc c im sau y:
*c im hnh thi: t bo, khun lc, kiu ny chi, cc dng bo t v tnh v hu
tnh, khun ty v khun ty gi...
*c im sinh l v sinh ho:
- Ln men 13 loi ng
- ng ha 46 ngun carbon. C th dng b kt chn on nhanh ID 32C (Bio
Mrieux SA, Marchy-ltoile)
- Tnh chng chu vi 0,01% hoc 0,1% cycloheximide (c th bao gm trong b
kit ID 32C).
- ng ho 6 ngun nit: nitrate, nitrite, ethylnamine hydrochloride, L-lyzine,
cadaverine dihydrochloride, creatine
- Sinh trng khi thiu ht mt s vitamin (myo-Inositol, calcium pantothenate,
biotin, thiamine hydrochloride, pyridoxin hydrochloride, niacin, folic acid, p-aminobenzoic acid.
- Sinh trng ti cc nhit khc nhau: 25, 30, 35, 37, 42
0
C.
- To thnh tinh bt.
- Sn sinh acid t glucoz
- Thy phn Ur
- Phn gii Arbutin
- Phn gii lipid
- Nng lc sn sinh sc t
- Sinh trng trn mi trng cha 50% v 60% glucoza
- Ha lng gelatine
-Phn ng vi Diazonium Blue B
- Pht trin trn mi trng cha acid acetic 1%
xc nh loi mi cn cn phn tch thnh phn acid bo ca t bo, thnh phn
ng trong t bo, phn tch h coenzyme Q, t l G+C, c tnh huyt thanh min dch, gii
trnh t ADN v lai ADN...
B - CC PHNG PHP THC NGHIM DNG NH TN NM MEN

1. Quan st hnh thi t bo nm men v o kch thc
Khi xc nh hnh thi v kch thc t bo nm men ngi ta thng nui cy nm men trong
mi trng thch - mch nha v mi trng mch nha dch th. Nu s dng cc mi trng
khc th hnh thi v kch thc t bo nm men c th thay i, khng ph hp vi hnh thi
v kch thc tiu chun c ghi trong bng phn loi.
- Mi trng mch nha: Ly la i mch cho ny mm (loi nhp khu dng
lm bia), em phi kh ri xay nh thnh bt. Cn 1kg bt ny, thm 3 lt nc, gi 60
0
C
ng ho cho n khi ht tinh bt (th vi dch Lugol khng thy c mu xanh lam). Lc ly
dch trong c th thm 3 lng trng trng ri trn u, un si ri lc ly dch trong. iu
chnh bng nc c nng ng t 6
o
Baume.
Phn vo cc dng c thu tinh kh trng. Nu lm mi trng c th thm 2% thch. Tt
nht l dng mm i mch, nu khng th c th dng mm la. Nng thch hp nui
cy nm men dng khi phn loi l 5,7
o
Baume. C ti liu li s dng nng 5-8
0
Baume.
Nm men c nui cy trong cc ng nghim thch nghing hoc cc ng nghim ng 3 ml
mi trng dch th. Nui cy 25-30
0
C trong 3 ngy, sau ly ra lm tiu bn v quan st.
Mun o kch thc t bo nm men ngi ta thng s dng trc vi th knh). S t bo nm
men c o khng t hn 20. Ch l phi o cc t bo trng thnh ch khng o cc chi
mi ny sinh. T bo nm men c hnh thi v kch thc khc nhau tu loi, tu chi. Chng c
th c hnh cu, hnh bu dc, hnh trng, hnh qu chanh chu u, hnh ng v.v... Khi quan
st t bo nm men di knh hin vi c th phn bit c thnh t bo, t bo cht, khng
bo (vacuole) v cc ht d nhim (metachromatic granules). Thnh t bo nm men thm
hn so vi nguyn sinh cht, cn khng bo thng c hnh trn, mu nht hn. Cc ht d
nhim thng bt nh sng mnh hn, chng lc l trong nguyn sinh cht theo chuyn ng
Brown. Kch thc ca t bo nm men khc nhau rt nhiu tu loi thu chi, tu iu kin
sinh trng v c th thay i trong khong 1-5 x 5-30m hay c khi di hn na. Kch thc
t bo ca cc loi nm men thng thng vo khong 4-5m.

2. Nhum mu t bo nm men:
Mun quan st t bo nm men mt cch t m hn ngi ta thng s dng cc loi thuc
nhum nhum c t bo hoc mt s phn t bo nm men. C th dng mt trong nhng
loi dung dch thuc nhum sau y:
- Dung dch Lugol:
Iot 2g
Iodua Kali 4g
Nc ct 100ml
(Nghin nh I v KI trong ci s ri sau dng nc ho tan dn).
- Dung dch xanh methylen (methylene blue)
Xanh methylen 1g
Nc ct 1000ml
(C th pha thnh dung dch 1% sau lc ri dng nc ct pha long thm 10 ln na).
- Dung dch fuchsin cacbolic:
Fuchsin kim (basic fuchsin) 0,1g
Cn 90
0
10ml
Dung dch phenol 3% 90ml
(Ho tan fuchsin trong cn, sau trn u vo dung dch phenol).
C th dng que cy, pht dch nui cy nm men thnh mt lp mng trn phin knh
sau lm kh, c nh v nhum n bng xanh methylen hay fuchsin cacbolic nh khi
nhum tiu bn vi khun. Thng ngi ta dng lamelle (l knh mng) quan st t bo
nm men. Ly mt phin knh sch nh ln mt git thuc nhum (xanh methylen chng
hn). Git thuc nhum khng nn to qu (v sau s trn khi lamelle), cng khng nn nh
qu (to thnh nhiu bt kh khi y lamelle). Ly mt t nm men nui cy 2-3 ngy ho
vo git thuc nhum. t mt cnh ca lamelle st vo pha ngoi git mu ri h t t
lamelle xung cho git mu trn u khp lamelle. Nu trn ra ngoi th dng giy lc thm
bt. Soi vt knh nh trc, sau chuyn sang vt knh ln. C th cn c vo mc bt
mu m nht phn bit c t bo sng v t bo cht. Mun quan st cc ht glycogen
trong t bo nm men th nhum bng dung dch Lugol. T bo s c mu vng nht cn cc
ht glicogen c mu nu. Mun quan st cc git m trong t bo nm men c th lm tiu
bn nh sau:
R mt git formalin ln phin knh, dng que cy ly mt t nm men nui cy 48-
72 gi ho vo git formalin. yn 5 pht sau thm mt git dung dch thuc nhum
xanh methylen, li thm mt git thuc nhum soudan III. t lamelle di knh hin vi ri
quan st ta s thy nguyn sinh cht ca t bo nm men bt mu lam nht, khng bo
khng bt mu cn cc git m c mu hng.
- Thuc nhum soudan III:
Soudan III 0,05g
Cn 90% 100ml
Cng c th nhum cc git m bng phng php sau y: Ly thuc nhum en
Soudan B (Soudan black B) cho vo ng nghim. Dng que cy ly mt t nm men ho vo
dch thuc nhum ny. Gi 20 pht. Ly khong 2 vng que cy dch thuc nhum c nm
men pht ln phin knh. Lm kh t nhin. Nhum tiu bn trong 30 giy bng dch thuc
nhum safranin. Ra nc, i kh ri soi knh. Nguyn sinh cht ca t bo nm men s c
mu nht cn cc git m c mu en lam.
- Thuc nhum en Soudan B (theo Burdon):
Soudan black B 0,3g
Cn 70% 100ml
(Trn u, 24 gi ri mi s dng. Dng trong vng mt thng).
- Thuc nhum safranin:
Dch safranin 2,5% (trong cn 95%) 10ml
Nc ct 100ml
Mun nhum nhn ca t bo nm men c th s dng phng php sau y:
Ly mt git nc t ln 1 phin knh. Dng que cy ly mt t nm men ho vo git nc
ri dn thnh vt mng. Lm kh t nhin. Thm vi git dung dch picroformol, gi vi
pht sau ra bng cn 70%. Ngm tiu bn vo trong dung dch FeNH
4
(SO
4
).12H
2
O 3%
trong 4-7 gi. Ly tiu bn ra dng nc ra sch sau li ngm vo dch thuc nhum
hematoxilin 10% trong 24 gi. Ly ra ra nc ri li ngm vo dch FeNH
4
(SO
4
).12H
2
O cho
n khi va mt mu th ly ra ra sch bng nc, lm kh ri soi knh. Nguyn sinh cht ca
t bo nm men c mu tro cn nhn t bo c mu en.
- Dung dch nhum nhn t bo:
+ Dung dch picroformol:
Dung dch acid picric bo ho 75 phn
Axetit acetic glacial 5 phn
Dung dch fomol long 20 phn
+ Dung dch ngm FeNH
4
(SO
4
)
2
.12H
2
O
FeNH
4
(SO
4
)
2
.12H
2
O 3g
Nc 100ml
+ Dung dch thuc nhum hematoxilin
Hematoxilin 1g
Cn 10ml
Nc 90ml
Ho tan hematoxilin trong cn, sau thm nc, y nt bng ri 1 thng sau mi s
dng.
quan st t bo nm men c th dng dung dch nigrozin 5%. Khi t bo s khng bt
mu, c th phn bit r trn mt nn mu lam en.
quan st bo t ti (trnh lm vi cc khng bo) c th s dng mt s cc phng php
nhum bo t c gii thiu trong chng IV (phng php nghin cu vi sinh vt hc tp
1 NXB KHKT 1971). Cng c th nhum bo t ti bng phng php sau y: R 1 git nc
ln phin knh. Dng que cy ly mt t nm men trn vo git nc, dn thnh vt mng,
kh t nhin.
C nh trn ngn la bng n cn, nhum bng dung dch lc malachit. Nhum 2 pht,
thng xuyn h trn ngn la cho bc hi (khng c un si). Ra nc nh, nhum thm
30 giy bng dung dch safranin 0,5% trong nc. Nang bo t s bt mu xanh lc cn t
bo dinh dng c mu hng.
- Dung dch lc malachit:
Lc malachit (malachite green) 1g
Nc 100ml (khng un nng)

3. Quan st qu trnh ny chi ca t bo nm men
(s dng cho t bo nm men dinh dng khng c dng si - non filamelletous vegetative
cells).
Cy mt vng que cy t bo nm men mt ngy tui vo bnh nn loi 100ml cha
30ml mi trng dch th (mi trng nc chit mch nha, mi trng cao nm men-pepton-
glucoza hay mi trng mch nha - cao nm men - glucoza - pepton).
- Mi trng mch nha - cao nm men - glucoza - pepton:
Cao mch nha (malt extract) 3g
Cao nm men (yeast extract) 3g
Glucoza 10g
Pepton 5g
Nc 1000ml
Nui cy trong bnh nn 25-28
0
C sau hai n ba ngy tin hnh ly mu quan st. Khi
quan st di knh hin vi cn phn bit c l nm men sinh sn theo cch ny chi hay
phn ct hoc c hai.
- Nu ny chi th chi xut hin u? c hai u (hai cc) hay v tr bt k no
trn t bo? S lng chi trn t bo m?
- Chi con sau khi pht trin c ri khi t bo m hay khng?
- Dng v kch thc ca t bo? Ch : phng php ny phi dng vi cc mi
trng xc nh v pha sinh trng logarit ca t bo.

4. Quan st khun ty gi:
C mt s nm men khi pht trin trong nhng mi trng nui cy lu hay trong
nhng iu kin thiu oxy c th to thnh nhng t bo di, xp ni tip nhau, c gi l
khun ty (mycelium). Ngi ta phn bit hai loi khun ty: khun ty gi v khun ty tht.
Khun ty tht l cc t bo dng si c vch ngn, khun ty gi l cc t bo dng si khng
c vch ngn. Vic to thnh khun ty l mt c im quan trng trong phn loi nm men.
Cng c mt t loi nm men khi pht trin bnh thng cng to thnh khun ty gi
(pseudomycelium).
Mun kim tra vic to thnh khun ty ngi ta thng nui cy nm men trn mi
trng pepton - glucoza, mi trng khoai ty - glucoza hay mi trng ng.
- Mi trng thch - pepton - glucoza:
Pepton 10g
Glucoza 20g
Thch 20g
Nc 1000ml
- Mi trng khoai ty - glucoza:
Nc chit khoai ty 10% 1000ml
Glucoza 20g
Thch 20g
Cch lm nc chit khoai ty: cn 100g khoai ty gt v, ra sch v thi nh,
thm 300ml nc, hp p lc 1at trong 1 gi sau b sung nc cho 1000ml.
- Mi trng ng:
cn 12,5g ng, thm 300ml nc un cch thu 60
0
C trong 1 gi, lc ly nc trong.
Thm nc cho 300ml. Sau thm 3,8g thch. Hp p lc 1at trong 15 pht. Lc nng
qua bng thm nc ri phn vo cc ng nghim v kh trng p lc 1at trong 15 pht.
mi trng vo hp Petri. Dng que cy, cy nm men thnh 3 cp ng song
song ngn, 3 ch. Dng panh ly l knh mng (thng xuyn ngm trong cn 70%) t nh
ht cn, ngui mt cht ri cn thn t nh nhng ln vt cy. Phi cy th no hai
ng cy song song mi ch c chiu ngang nm gn gia l knh mng, hai u di hn l
knh mng mt cht ( sau ny d quan st). Cn ch l b mt thch phi tht kh, khi
y l knh mng, phi trnh bt kh, y xong phi trnh di chuyn lm x lch l knh mng.
Cng c th tin hnh theo phng php sau y: mi trng vo mt hp Petri, i
ngui 60
0
C, dng panh ly cc phin knh (lamelle) t nh vo sao cho c mt lp mi
trng bm vo to thnh lp mng trn mt mt ca phin knh. Lp ba phin knh ph
mi trng nh vy t vo hp Petri khc. Trong hp Petri ny c ng mt t nc v trng
v mt gi thu tinh hnh ch U (cc phin knh t thng gc so vi gi thu tinh). Cy nm
men thnh ba vt trn mi phin knh. Phi cy ba vt ny cch nhau nh th no trn mi
vt c th t va mt l knh mng (cc vt cy song song vi chiu rng ca phin knh).
Sau khi t l knh mt cch nh nhng v cn thn ta y hp Petri li v nui cy 25-30
0
C
trong 4-5 ngy. Ly ra v quan st cc vt cy di knh hin vi.
Mt vi phng th nghim lm theo cch sau: nh mt t mi trng thch nng ln
trn b mt phin knh, lng u to thnh mt lp tht mng. Sau khi kh b mt, cy
mt hoc 2 ng dc theo lam. Ly l knh mng t ln trn mi ng cy. t phin knh
vo a Petri v cho mt t nc v trng trnh kh mi trng. Quan st trn knh hin vi
trong vi ngy.
Vi cc phng php trn rt d dng quan st thy vic to thnh khun ty mt s
loi nm men.
5. Quan st bo t bn (Ballistoconidium, Ballistospore):
Ly 10-15ml mi trng nc chit mch nha, mi trng khoai ty - glucoza hay mi
trng bt ng a vo mt a Petri khi thch ng (nh lm kh v trng mt thch) ly que
cy cy nm men theo hai ng vung gc gia sau p ngc ln mt a Petri khc
cha cng mi trng nhng khng cy nm men. Trong a Petri ny cha 1 lamelle v trng,
20
0
C sau 3 tun bo t bn s to thnh cc khun lc trn a Petri cha mi trng
pha di v ly phn lamelle mang cc bo t bn a i quan st di knh hin vi.
- Mi trng bt ng:
Cn 60 gam bt ng ho vo trong 500ml nc si. un si tip 1 gi. Lc qua vi
mn. Thm nc cho 1000ml, thm 20g thch. un cho tan thch ri phn vo cc dng c
thu tinh. Kh trng ni p lc 120 pht trong 30 pht.
Cng c th pht hin bo t bn theo cc cch khc nh sau:
Cy cc loi nm men nghi ng c hnh thnh bo t bn ln mi trng thch - mch
nha (trn a Petri hay ng nghim thch nghing). Sau my ngy nui cy trn mt thy tinh
i din vi vt cy s c mt hnh nh m ging ht vi hnh dng vt cy. l cc bo t
bn bn ra lu li trn pha i din vt cy.
Hoc cy nm men theo ng thng hoc zich zc vo a Petri cha mi trng bt
ng, 20
0
C sau tng thi im 3, 5,7,10, 15 ngy. p ngc a Petri ln mt phin knh
sch, qua m. Quan st bo t bn trn phin knh trn knh hin vi.

6. Quan st bo t ti (ascospore):
Mt s nm men c kh nng hnh thnh bo t hu tnh gi l bo t ti (ascospore
hay asconidium). Bo t ti c kh nng bo v nm men chng li vi nhiu nh hng c hi
ca iu kin ngoi cnh. Thng quan st thy bo t ti ca nm men trong nhng mi
trng nui c?y lu C th l do vic tch lu mt s sn phm trao i cht kch thch qu
trnh to thnh bo t ti. Trong t bo ca mi loi nm men sinh bo t ti thng to
thnh mt s lng bo t ti nht nh. Khi cha bo t ti th t bo c gi l ti (asci, s
t - ascus).
Thng mi ti c 4 bo t, mt s loi ch c 1-2 bo t, mt s rt t loi li c ti 8
bo t. Bo t ti nm men c hnh dng rt khc nhau, y cng l mt c im thng
dng khi phn loi nm men. Saccharomyces cerevisiae v rt nhiu loi nm men khc c
bo t ti hnh cu hay hnh trng. Hansenula anommala, Hanseniaspora c bo t ti hnh
bn cu, pha di c mp nh vnh m, Pichia membranaefaciens c bo t ti v quy tc (c
th c hnh trng, di, tam gic, bu dc, bn cu...), Hansenula saturnus c hnh bo t ti
hnh qu xoi, gia c mt vnh ai nh. Bo t ti Schawanniomyces occidentalis cng c
hnh dng tng t nh vy nhng b mt c gai. Mt s loi nm men li c bo t ti di, c
khi hnh xon. Thng thng nm men to thnh bo t ti sau 5-10 ngy nui cy trn mi
trng thch mch nha. Mun quan st ch vic ly mt t nm men lm tiu bn soi ti
khng cn nhum mu. Mc ch vic quan st bo t ti phi tr li ba cu hi sau y:
1. Nm men c hnh thnh bo t ti hay khng.
2. Bo t ti hnh thnh t cc t bo dinh dng khng xy ra s tip hp trc
hay l sau khi c s tip hp gia hai t bo dinh dng; cng c th l xy ra sau khi c
s tip hp gia t bo m v t bo con (t bo ny chi) ca n.
3. Nghin cu hnh dng bo t v s lng ca bo t ti
Cch tin hnh:
Nm men tr sau khi nui cy qua m c a vo mi trng malt - cao nm men
- glucoza - pepton v 2-3 ngy sau a chuyn vo mi trng sinh bo t. Gi 25
0
C
trong 3 ngy v quan st di knh hin vi. Nu khng quan st thy bo t th li tip tc gi
v quan st tng tun cho n 6 tun lin. Cc mi trng hnh thnh bo t c th c s
dng l mi trng: V-8-agar, Gorodkowa-aga, acetat-agar, malt-yeast-glucoza, pepton-agar,
malt-acetat-agar. Tuy nhin thng s dng cc mi trng sau:
a. Mi trng ming thch cao:
Ly hai phn bt thch cao trn vi mt phn nc lm thnh bt nho sau vo
nhng ci khun lm bng giy da b hay giy thic (ng knh 1-1,5cm, cao 1,5-2cm).
Dng dao lm cho nhn b mt. Sau khi thch cao ng ta loi b khun giy ri cho vo
nhng hp thy tinh c bit gi l hp Koch. Cng c th lm nhng ming thch cao hnh
trn sau cho vo hp Petri. nc ngp 2/3 chiu dy ca ming thch cao sau a i
kh trng (120
0
C trong 30 pht). Cy nm men ti (t thch nghing mi nui cy 48 gi
tui). Gi 25
0
C trong vi ngy, ly ra lm tiu bn quan st bo t ti. Tsetlin (1913) ngh
trc khi cy nm men sang mi trng ming thch cao nn chun b nm men ti trn mi
trng c thnh phn sau:
Nc ct: 100 ml
Glucoza: 5 g
KH
2
PO
4
: 0,2g
CaCl
2
: 0,05g
MgSO
4
: 0,05g
FeSO
4
: 0,001g
(NH4)
2
SO
4
: 0,5g
b. Mi trng ming thch cao ci tin:
Cch tin hnh nh trn nhng b mt ming thch cao c lm m bng nc mch
nha long hoc dung dch c cha 2% manitol v 0,5% KH
2
PO
4
.
c. Mi trng Gorodkowa (1908)
Nc tht: 1 g
Pepton: 1 g
NaCl: 0,5 g
Glucoza: 0,25 g
Thch: 2 g
Nc: 100ml
d. X l vi tia t ngoi:
Cy nm men ln mi trng Gorodkowa (mi trng c) ri chiu tia t ngoi theo thi
gian khc nhau: 5, 10, 15 pht. Sau tip tc nui cy v quan st bo t ti.
e. Mi trng thch nc:
Mi trng ny l mi trng ngho ch c nc v thch, khng b sung thm cc cht
dinh dng khc.
f. Mi trng Amano (1950)
Amano ngh dng phng php sau: Cy truyn hai ln nm men trn mi trng
thch tht pepton, ly nm men nui cy 24 gi cy sang mi trng sau y:
Glucoza: 0,4 g
Axetat Na khng ngm nc: 1,4 g
Thch: 20 g
Nc: 1000 ml
g. Mi trng dch tinh bt khoai ty 0,5% (Almeida v Lacaza)
Cy nm men ln mi trng trn ri nui cy 37
0
C, sau quan st bo t ti theo
tng thi im thch hp.
h. Mi trng Kleyn:
Natri glutamat (hay asparagin, pepton, glixin): 0,25g
Glucoza: 0,062g
NaCl: 0,062g
Natri axeta: 0,5g
KH
2
PO
4
: 0,012 g
K
2
HPO
4
: 0,02g
Biotin (c th khng cn): 2
Dch hn hp I: 1 ml
Thch: 2 g
Nc: 100 ml
Cch pha dch hn hp I: MgSO
4
- 0,4g; CuSO
4
- 0,002g; FeSO
4
- 0,2g; MnSO
4
- 0,2g; NaCl -
0,4g; nc ct - 100ml).
Ch : Quan st bo t ti l c im quan trng trong phn loi, tuy nhin trong mt s
trng hp kh c th pht hin thy bo t ti. iu ny c th do cc nguyn nhn sau:
- Mi trng sinh bo t ti l khng thch hp.
- Chng nghin cu l d tn (heterothalic) hay ng tn (homothalic).
- Bo t ti kh pht hin v do ngi lm phn loi cn thiu kinh nghim.
- Nm men nghin cu thuc loi khng sinh bo t ti (anascoporogenous).

6. Quan st c tnh nui cy
quan st cc c tnh nui cy ca nm men ngi ta thng cy nm men ln mi
trng dch th, mi thng thch nghing v mi trng thch a (hoc dng chai Roux).
Cy nm men vo cc ng nghim ng 3ml mi trng mch nha 10-15
0
Baling (xem phn
Quan st hnh thi t bo nm men v o kch thc). Nui cy 25
0
C ri sau 24 gi, 48 gi,
72 gi v 198 gi ly ra quan st. Cn quan st xem nm men c pht trin b mt dch th
hay khng. Nu c th chng to thnh vng ph kn, vng ph khng kn hay to thnh mt
vng quanh thnh ng nghim. Nm men c lng xung th quan st xem cn c dng xp,
dng nhy hay dng rn chc. Quan st xem cht dch vn trong hay tr nn c. Dng tay
p kh vo y ng xem cn c ni ln hay khng (nu cn xp th d ni ln, nu cn rn
chc th kh ni ln). Dng que cy khu cn, nu cn nhy th d khu ln c tng, nu
khng th kh khu. Nu c vng th cn quan st xem b mt vng nh th no, kh hay t,
phng mn hay nhn nheo v cng cn quan st xem vng dy hay mng. quan st s pht
trin ca nm men trn mi trng thch nghing ta cy nm men ln trng thch - mch
nha 12-15
0
Baling sau t m 25-30
0
C. Quan st ngy nui cy th 3 v th 14. Ch
quan st xem b mt ca vt cy l trn nhn hay x x, t bng hay kh, c np nhn hay
khng, mu sc th no, mp thng hay mp rng ca v.v... quan st khun lc ta em
mi trng thch - mch nha hoc mi trng gelatin - mch nha phn vo cc hp Petri hay
bnh Roux, bnh Kolle. Dng que cy c u nhn cy nm men thnh mt chm chnh gia.
Trc khi cy cn lm kh b mt (lp thch nn dy khong 5mm). Nui cy 25
0
C trong
14 n 30 ngy. Ly ra quan st khun lc ln. Cn ch miu t:
- Kch thc khun lc (v hnh).
- Chiu dy khun lc (phi v hnh ct ngang khun lc).
- C to thnh nhng vng ng tm hay khng?
- C nhng hnh tia phng x hay khng? (t tm n gia hay t gia n mp?)
- Gia khun lc li ln, lm xung hay phng?
- B mt trn, nhn, bng, t hay x x, rp, c cu to nhung hay gai, c np nhn
hay khng?
- Mu sc khun lc.

7. Th nghim xc nh kh nng ln men cc loi ng
Kh nng ln men cc loi ng l mt trong nhng ch tiu quan trng c s dng
phn loi nm men. Trong th nghim ny ngi ta thng s dng mi trng nc chit
gi u hay mi trng chit nm men 0,5%.
- Cch lm mi trng nc chit gi u: cn 200g gi u thm 1000ml nc. un
si 30 pht. Lc ly dch trong ri thm nc cho 1000ml.
- Cch lm mi trng nc chit nm men: cn 200g men bia p (hay 100g men bia
kh) thm 1000ml nc. Hp bng ni hp p lc trong 15 pht nhit 120
0
C. Lc nng
qua nhiu lp giy lc. Li lc ngui ti trong. Thm nc cho 1000ml. Cng c th dng
cao nm men vi nng s dng l 0,5%.
Cch tin hnh:
- S dng ng nghim c kch thc 180 x16 mm. t ngc mt ng Durham
(50x6mm) (ming ca ng Durham chm y ng nghim).
- Phn 10-15ml mi trng c s (0,5% cao nm men) v cha tng ngun ng khc
nhau, nng 50mM (tng ng 1%) ring vi rafinoza 100mM (tng ng 2%). Ring
ng kim tra l khng c mt ngun ng no. ng i chng l D-glucoza. Cc ng nghim
cha mi trng c kh trng sau cy vo 100l (khong 2 git) dung dch huyn ph
nm men c mt 10
7
t bo/ml, 25
0
C sau mt tun.
- Quan st vic to CO
2
y ng Durham bit nm men c hay khng c kh nng
ln men tng ngun ng. C nm men ch c th pht trin b mt dch huyn ph v
chng c kh nng ng ho cc ngun ng ny.
- Ch : Theo phng php ny mt s trng hp CO
2
to ra thp phi xc nh bng
in cc CO
2
hay dng p k Warburg. Trong iu kin c qu t lng ng dng lm th
nghim cn c th ht mi trng cha ng (v cy nm men) vo nhng micropipet (ht
n 1/10ml). u pipet sau c gn bng vaselin ( trn thm vi mt t parafin). Nui
cy 25-30
0
C v hng ngy quan st xem c bt kh sinh ra hay khng, mc mi trng b
y ra xa nhiu hay t. Cc th nghim c tin hnh u tin bng glucoza. Nu nm men c
kh nng ln men glucoza th hy lm tip th nghim vi cc loi ng khc. Mi ngy quan
st kt qu ln men mt ln, quan st trong 10 ngy lin.

8. Th nghim xc nh kh nng ng ho cc hp
cht carbon khc nhau:
y l c im sinh l quan trng dng trong phn loi. C 2 phng php ch yu
c dng l phng php nh gi kh nng sinh trng trn mi trng dch th v mi
trng c. Tuy nhin cn c th s dng phng php dng con du trn mi trng c.
8.1. Phng php nh gi kh nng sinh trng trn mi trng dch th:
- S dng ng nghim c kch thc 100x15mm. Cc ng nghim cha 1,8 ml mi
trng nit c s (Nitrogen base) khng c carbon v 0,2 ml ngun carbon 10X. Cc ng th
nghim cha 1% ngun carbon nghin cu khc nhau (tng ng 50mM), ng thi lm
mt ng nghim kim tra m (khng c ngun carbon no) v mt ng kim tra dng dng
ngun carbon l D-glucoza.
- 46 ngun carbon gm: glucoza, galactoza, L-sorboza, sucroza, maltoza, xenlobioza,
trelaloza, lactoza, melibioza, raffinoza, melezitoza, inulin, tinh bt tan, D-xyloza, L-arabinoza,
D- arabinoza, D-riboza, L-rhamnoza, D-glucosamin, N-acetyl-D-glucosamin, methanol,
ethanol, glycerol, erythritol, ribitol, galactitol, D-mannitol, D-glucitol, a- metyl-D-glucosid,
salicin, glucono-d-lacton, D-gluconat, 2-ketogluconat, 5-ketogluconat, DL-lactat, succinat,
citrat, inositol, hexandecan, saccharat, xylitol, L-arabinitol, propan 1,2 diol, butan 2,3 diol, D-
glucuronic acid, D-galacturonic acid.
- Ging gc c chun b trn mi trng thch - pepton - glucoza - cao men - malt
qua m. Sau t bo c ly ra v pha trong mi trng nit c s t ti mt t
bo l 25x10
6
/ml (hay mt A
640
= 1,0).
- Sau ly ra 100l
cy vo cc ng mi trng chun b sn, sau tnh hay lc tay tng lc (hng
ngy). Cng c th lc nghing bng my lc ngang (gc lch 15-40
0
) hay lc trn vi cc nm
men c lng cao.
nh gi s sinh trng thng l dng mt so vi hai ng dng v m bng cch t
mt ming ba trng vch mt ng en v t ng sau cc ng nghim so snh. Tuy
nhin c th dng phng php so mu so snh vi cc ng chun c v t sinh khi
kh (cch ny thng t c dng vi cc t bo nm men c cc t bo kt dnh vi nhau).
Th nghim c th ko di trong mt tun hoc c th n 4 tun.
8.2. Sinh trng trn mi trng thch
ng nghim c cha 15ml mi trng thch nit c s 45
0
C sau thm 0,5 ml dch
huyn ph t bo nm men c chun b nh m t trn v lc cho u (trnh to bt).
Cc bc phi thao tc nhanh thch khng b ng v nm men khng b cht. Sau
ton b ra a Petri v trng 37
0
C sau 30 pht cho ng thch. C th sau p ngc
37
0
C trong 90 pht kh mt thch. Sau t t 2-5mg tng loi ng (ngun carbon)
nghin cu ln trn b mt thch gn mp a Petri. Thng thng trong mt a dng 3 loi
ng khc nhau nh: a thch c chia lm 4 gc, 1 gc khng cho ngun carbon no (tuy
nhin phi lm th nghim kim tra c vi D-glucoza). Theo di kt qu sau 48 gi n 1 tun.
8.3. Phng php dng con du
a Petri gc cha mi trng malt - cao men - glucoza - pepton - thch cha khong
25 khun lc nm men nghin cu khc nhau. Sau dng con du nhung v trng in ln cc
a thch c ngun carbon khc nhau (0,5% = 25 mM) (ch nh du v tr ca cc a
khi b nhm ln). Mt ln in t a gc c th in ln 10 a khc nhau bt u l a i chng
m (khng cha ngun carbon no) v cui cng l a i chng dng (cha D-glucoza).
Ch thch s dng phi l loi thch tt khng cha mt thnh phn carbon d b ng ho
no. Theo di th nghim t 48 gi n 1 tun.

9. Th nghim xc nh kh nng ng ho cc ngun nit
C khong 1/4 cc chng nm men c kh nng s dng nitrat v vy y l c im
cn cho phn loi. Tuy nhin cc thnh phn khc nhau nh nitrite, ethylamine, L-lysine,
sunfat amn, cadaverine cng cn cho cc th nghim phn loi. Phng php tin hnh th
nghim y tng t nh cc nghin cu vi vic s dng cc hp cht carbon trn vi c
mi trng dch th v mi trng c nhng y dng mi trng carbon c s (carbon
base) v phi nui nm men trn mi trng carbon c s trong 2 ngy trc khi cy vo cc
ngun nit.
Do nitrit c cho nm men v n d c hnh thnh trong mi trng acid do pH
mi trng phi c iu chnh n 6,5. Dng phng php nh gi s sinh trng l ph
hp cho vic nghin cu kh nng s dng nitrit v ethylamin. Trong lng nitrogen c
dng nn l 2-5mM (0,05-0,1%). Tuy nhin c th quan st thy s sinh trng mc
thp cc ng kim tra (khng c nit) do lng NH
3
kh quyn ho tan vo mi trng.

10. Th nghim xc nh kh nng hnh thnh hp cht
loi tinh bt:
- Chun b dch Lugol nh sau:
5g I
2
v 10g KI c pha trong 100ml nc ct. Pha long 5 ln trc khi dng.
Lodder v Kreger - van Rij s dng th nghim ny phn bit hai ging nm men
Torulopsis (khng hnh thnh hp cht loi tinh bt) v Cryptococcus (hnh thnh hp cht loi
tinh bt).
Mi trng :
(NH
4
)
2
SO
4
: 1 g
KH
2
PO
4
: 1 g
MgSO
4
.7H
2
O: 0,5 g
Glucoza: 10 g
Thch: 25 g
Nc ct: 1000 ml
pH : 4,5
Kh trng mi trng nhit 110
0
C (trong 15 pht) sau phn phi vo cc hp
Petri. Trong mi hp Petri cho sn mt git hn dch vitamin hoc dch t phn nm men v
trng. Cy nm men v nui cy 25
0
C trong 1-2 tun. Sau dng thuc th Lugol kim
tra xem c hnh thnh hp cht loi tinh bt hay khng. Nu c th vt cy s xut hin mu
xanh.
Phng Tm Phng (Trung Quc) ngh s dng mi trng dch th sau y:
(NH
4
)
2
SO
4
: 5 g
KH
2
PO
4
: 1 g
MgSO
4
.7H
2
O: 0,5 g
CaCl
2
.2H
2
O: 0,1 g
NaCl: 0,1 g
Cao nm men: 1 g
Glucoza: 30 g
Nc: 1000 g
Phn vo cc bnh tam gic mt lp mi trng cao khong 0,5cm ri kh trng. Cy
nm men v nui cy 25-30
0
C trong ba tun, sau dng thuc th Lugol kim tra kh
nng sinh tinh bt.

11. Th nghim xc nh nhu cu vitamin cho sinh trng ca nm men:
Cc nm men khc nhau c nhu cu khc nhau v vitamin sinh trng. Cc th
nghim y nhm kim tra s sinh trng ca chng nm men vng mt tt c hay tng loi
vitamin khc nhau.
Cch tin hnh nh sau:
Th nghim c tin hnh trn mi trng dch th. Mi trng c y cc thnh
phn dinh dng tr vitamin (mi trng khng cha ngun vitamin no). Ton b ng
nghim th nghim c chia thnh hai l. Mt l khng c mt bt k mt loi vitamin no v
mt l thiu tng vitamin ln lt theo th t di y (lng vitamin cho 1 lt mi trng).
Acid para-amino benzoic acid: 200 mg
Biotin: 20 mg
Acid folic: 2 mg
Myo - inositol: 10 mg
Acid nicotinic: 400 mg
Pantotenat (Ca): 2 mg
Pyridoxin (HCl): 400 mg
Riboflavin: 200 mg
Tiamin HCl: 400 mg
Myo - inositol cng c dng cho sinh trng hiu kh nh l ngun carbon).

12. nh gi s sinh trng trn mi trng c nng
ng cao
Mt s nm men c kh nng sinh trng trn mi trng c nng ng cao so vi
cc chng khc.
Th nghim c tin hnh nh sau: ng thch nghing c chun b vi cao nm men
v thch c lng ng D-glucoza t 50% (W/W). Sau ging nm men c cy vo v
kim tra s sinh trng 25
0
C trong 4 tun. Ch trnh cho mi trng b kh bng cch bc
bng giy nn (wax-paper).

13. nh gi s pht trin khi c mt Cycloheximit
Th nghim tin hnh trong mi trng c cao nm men, ngun nit v D-glucoza nhm
nh gi kh nng s dng D-glucoza khi b sung xycloheximit ( c lc v trng) vi
nng 0,1% hay 0,01%.
Xycloheximit c ch s sinh trng ca Eukaryota bng cc c ch qu trnh sinh tng hp
protein riboxom loi 80S. Cc nm men c kh nng chng li c xycloheximit c th
c thay i v loi riboxom ny.

14. Xc nh hot tnh phn gii Urea (hay hot tnh Ureaza)
Mi trng Christensen:
pepton : 1g
NaCl: 5g
KH
2
PO
4
: 2g
glucoza : 5g
thch: 20g
nc: 1000ml
un tan mi trng, thm 6ml dung dch phenol (phenol red) c nng 0,2%
trong cn. Kh trng mi trng ni hp (115
0
C/15 pht). i ngui n 50
0
C thm 100ml
dung dch urea (dung dch 20% kh trng ring qua mng lc). Phn vo ng nghim thy
tinh v trng, lm thch nghing. Sau cy nm men v gi 26
0
C trong 7 ngy. Nu nm
men c kh nng sinh ureaza phn gii urea th mi trng s chuyn mu xm. Cng
c th tin hnh th nghim vi mi trng dch th.

15. Th nghim lm i mu Diazonium blue B (DBB test)
Mt ng ging c cy trn mi trng pp ton - cao men - glucoza - malt - thch 10
ngy tui v gi 5
0
C trong vng vi gi. Sau khi dung dch DBB lnh (ice - cold) ln trn.
Nu mi trng chuyn sang mu sm trong 2 pht nhit phng, kt qu c coi l
dng tnh.
Dung dch DBB c gi trong lnh bng (ice - cold) v c dng trong t pht trc
khi n mt mu. Chun b dung dch ny bng cch ho tan mui DBB (Brentamine Blue B ca
hng ICI Ltd., hay Hoechst AG) trong m Tris HCl 0,1M, trong lnh, pH = 7,0; nng
1mg/ml.
Cc mi trng th nghim cha c ghi phn trn
1. Mi trng Acetat (g/l) (M.C. Clary et al., 1959)
Acetat natri: 9,80
D-Glucoza: 1,00
NaCl: 1,20
MgSO
4
.7H
2
O: 0,70
Cao nm men: 2,50
Thch: 20
Kh trng: 120
0
C/15 pht
2. Mi trng thch Gorodkowa (Dodder v Kreger - van Rij, 1952) (g/l)
D-glucoza: 1,0
Pepton: 10,0
NaCl: 5,0
Thch: 20,0
Kh trng: 120
0
C/15 pht
3. Mi trng cao ng (Lodder v Kreger - van Rij, 1952)
Ho tan 12,5g cao ng maize extract) vo 300ml nc 60
0
C sau 1 gi v lc thu ly
dch trong. Lng dch thu c thm nc n 300ml. Thm vo 3,8g thch v kh trng
120
0
C/15 pht (Mi trng ny c sn xut v bn rng ri).
4. Mi trng thch V-8 (Wicketam v cng s, 1946)
y l mi trng c chun b t hn dch chit ca mt s loi rau v men bnh m.
Bnh A cha 14g thch v 340 ml nc. Bnh B cha 350 ml dch cha V-8 (sn xut t cng ty
Campbeoo soup, Camden, N.J. USA) c trn u vi 5g men p c lm tan trong 10ml
nc.
Bnh B c un si trong 10 pht v ngui, iu chnh pH n pH = 6,8 ti 20
0
C.
Bnh A c lm tan thch v trn u vi bnh B v phn vo cc ng nghim kh trng
120
0
C trong 15 pht.
5. Mi trng pepton - cao men - glucoza (Vander Walt v Codder, 1970)
Mi trng dch th c chun b t 5g- cao nm men, 20g- D-glucoza, 10g- pepton
trong 1000ml nc. Khng cn iu chnh pH, thanh trng 120
0
C trong 15 pht.
6. Thnh phn mi trng tng hp (tinh khit v thnh phn ho hc)
(Wikerlam v Duta, 1948; Wikerlam, 1951; Barnett v Ingram, 1955; Difco manual of
Dehydroted culture media and Keagents).
Ngun nit:
(NH
4
)
2
SO
4
: 3,5 g
L-Asparagin: 1,5g
Ngun carbon
D-glucoza: 10 g
Aminoacid
L-Histidin: 10 mg
DL-Methionin: 20 mg
DL-Triptophan: 20 mg
Cht sinh trng
Acid P-aminobenzoic: 200 mg
Biotin: 20 mg
Acid folic: 2 mg
Myo-inositol: 10 mg
Acid nicotinic: 400 mg
Pantotenat (Ca): 2 mg
Pyridoxin HCl: 400 mg
Riboflavin: 200mg
Tiamin HCl: 400 mg
Vi lng
H
3
BO
3
: 500 mg
CuSO
4
.5H
2
O: 40 mg
KI: 100 mg
FeCl
3
.6H
2
O: 200 mg
MnSO
4
.4H
2
O: 400 mg
Na
2
MoO
4
.H
2
O: 200 mg
ZnSO
4
.7H
2
O: 400 mg
Mui khong
KH
2
PO
4
: 850 mg
K
2
HPO
4
: 150 mg
MgSO
4
.7H
2
O: 500 mg
NaCl: 100 mg
CaCl
2
.6H
2
O: 100mg
7. Mi trng quan st hnh thi t bo nm men:
Cng c th dng mi trng 6 nhng thm 2% thch (W/W)
8. Mi trng nit c s:
Nh trn nhng khng c ngun nit (5g (NH
4
)
2
SO
4
), khng c L-asparagin v D-glucoza.
9. Mi trng carbon c s:
Nh trn nhng khng c ngun nit, thm 1mg L-Histidin, 2mg DL-metionin, 2mg DL-
tryptophan.
10. Mi trng khng c vitamin
Nh trn nhng khng c 5g (NH
2
)
2
SO
4
, L-asparagin v cc cht sinh trng.
Phn loi cc chi nm men

Phn loi cc chi nm men da trn cc ch tiu ch yu l: c im t bo dinh dng, c
im sinh sn hu tnh v c im sinh l, nui cy. Sau y l cc c im ch yu ca tng chi
c th:

I. Cc chi nm men thuc ngnh nm ti.
Bo t ti l bo t hu tnh c trng cho Ngnh Nm ti (Ascomycota) .
Bo t m l bo t hu tnh c trng cc nm thuc ngnh nm m (Basidiomycota).

A. Dng sinh sn hu tnh ( Teleomorph)

1. Ambrosiozyma (5 loi)

Sinh sn sinh dng: T bo ny chi a cc, khun ty gi v khun ty tht vi bo t trn ny
chi (blastoconidia) , khun ty tht c cc l trn vch ngn, c th nhn thy cc chm nh khi
quan st trn knh hin vi.
Sinh sn hu tnh: Cc ti bo t cm thnh tng m. Ti thng to thnh trn si. Mi ti
thng c 1-4 bo t ti dng m.
c im sinh l: Ln men : Yu v chm
ng ho nitrat : - (1 loi c)
Mng trn mi trng dch th : -
C cht ging tinh bt : -
ng ha inositol : -
Hot ho Ureaza : -
Ha lng gelatin : -
Phn ng DBB : -

2. Arxyozyma (1 loi)
Sinh sn sinh dng: T bo ny chi a cc, ch hnh thnh khun ty gi
Sinh sn hu tnh: Cc ti (asci) cha 1-2 bo t ti hnh cu, x x.
c im sinh l: Ln men : +
ng ho nitrat : -
Mng trn mi trng dch th : -
C cht ging tinh bt : -
ng ha inositol : -
Hot ho Ureaza : -
Ho lng gelatin : -/+
Phn ng DBB : -

3. Ascoidea (4 loi)
Sinh sn sinh dng: Thng c t bo ny chi v khun ty gi, khun ty tht, bo t
chi c th c sinh ra trn cc gai hoc khng
Sinh sn hu tnh: Cc ti (asci) sinh chm hoc tn cng ca khun ty tht. Ti c hnh elip
hay hnh kim. Ti mi c hnh thnh bn trong ti c. Mi ti c 16-160 bo t ti hnh m hay
elip.
c im sinh l: Ln men : -
ng ho nitrat : -/+
Mng trn mi trng dch th : -
C cht ging tinh bt : -
ng ha inositol : -/+
Hot ho Ureaza : rt yu
Ho lng gelatin : -
Phn ng DBB : -

4. Babjevia (1 loi)
Sinh sn sinh dng: T bo ny chi a cc, khun ty tht him khi to thnh, khun ty
gi s ng
Sinh sn hu tnh: Chi pht trin trong cc ti bo t v vn gn vi t bo m. C 4-30 bo t
ti trong sut, hnh cu hay elip.
c im sinh l: Ln men : -
ng ho nitrat : -
Mng trn mi trng dch th : -
C cht ging tinh bt : -/+
ng ha inositol : -
Hot ho Ureaza : -
Ho lng gelatin : -

5. Cephaloascus (2 loi)

Sinh sn sinh dng: T bo ny chi a cc, c c khun ty gi v khun ty tht, c bo
t trn ny chi (blastoconidia) c sinh ra trn cc gai khc nhau
Sinh sn hu tnh: Cc bo t ti thng ng c th nhn hoc x x v trong sut, mu hi
nu. Cc ti bo t cm thnh tng m, mi ti cha 4 bo t ti hnh m.
c im sinh l: Ln men : -/+
ng ho nitrat : -
Mng trn mi trng dch th : +
C cht ging tinh bt : -
ng ha inositol : -
Hot ho Ureaza : -
Ho lng gelatin : -


6. Citeromyces (1 loi)
Sinh sn sinh dng: T bo ny chi a cc, khng c khun ty tht v khun ty gi.
Sinh sn hu tnh: Ti c mt bo t (him khi 2), bo t ti hnh cu, x x
c im sinh l: Ln men : +
ng ho nitrat : +
Mng trn mi trng dch th : -
C cht ging tinh bt : -
ng ha inositol : -
Hot ho Ureaza : -
Ho lng gelatin : -
Phn ng DBB : -

7. Clavispora (2 loi)
Sinh sn sinh dng: T bo ny chi a cc, i khi c khun ty gi. Khng c khun ty
tht.
Sinh sn hu tnh: Ti cha 1-4 bo t ti hnh chu
c im sinh l: Ln men : +
ng ho nitrat : -
Mng trn mi trng dch th : -
C cht ging tinh bt : -
ng ha inositol : -
Hot ho Ureaza : -
Ho lng gelatin : -
Phn ng DBB : -

8. Coccidiascus (1 loi)
Sinh sn sinh dng: T bo hnh cu hay ovan, khng to khun ty tht v khun ty gi.
Sinh sn hu tnh: Cc ti bo t c vch dy, cha 2 bo t ti hnh thon di qun cht dng
xon c.
c im sinh l: Cha bit, taxon ny mi ch c phn lp t m rui gim Drosophilla.

9. Cyniclomyces (1 loi)

Sinh sn sinh dng: T bo hnh ovan di hay hnh tr, thng ny chi nh hay gn
nh. i khi c dng khun ty gi. Khng c khun ty tht.
Sinh sn hu tnh: Mi ti cha 1-4 bo t ti dng t ovan ti hnh tr.
c im sinh l: Nhu cu dinh dng phc tp. Ch sinh trng trong khong nhit t 30 -
40
0
C.
Ln men : yu.
ng ho nitrat : -
Mng trn mi trng dch th : -
C cht ging tinh bt : -
ng ha inositol : -
Hot ho Ureaza : -
Ho lng gelatin : -

10. Debaryomyces (15 loi)
Sinh sn sinh dng: T bo ny chi a cc, i khi c khun ty gi. Khng c khun ty tht.
Sinh sn hu tnh: Mi ti cha 1-4 bo t ti x x hoc nhn, hnh trn hay ovan. C s
tip hp gia t bo m v t bo chi.
c im sinh l: Ln men : +/-
ng ho nitrat : -
Mng trn mi trng dch th : -
C cht ging tinh bt : -
ng ha inositol : -
Hot ho Ureaza : -
Ha lng gelatin : -
Phn ng DBB : -

11. Dekkera (2 loi)

Sinh sn sinh dng: T bo ny chi a cc, thng c dng t bo hnh cu hay elip, i khi c
hnh cung. Khun ty gi c th c nhng khng c khun ty tht.
Sinh sn hu tnh: Ti khng tip hp v tr nn d chy, c 1-4 bo t ti dng m hoc
dng cu.
c im sinh l: Ln men : +
ng ho nitrat : +/-
Mng trn mi trng dch th : +/-
C cht ging tinh bt : -
ng ha inositol : -
Hot ho Ureaza : -
Phn ng DBB : -
Sinh axit axetic hiu kh t D-glucoza.
Sinh trng chm , vng i ngn.

12. Dipodascopsis (2 loi)

Sinh sn sinh dng: C th c t bo ny chi, ch c khun ty tht. Sinh sn hu tnh: Ti
hnh kim hay tr. Mi ti cha 32-128 bo t ti trong sut, hnh elip hoc hnh thn
c im sinh l: Ln men : -
ng ho nitrat : -
Mng trn mi trng dch th : -
C cht ging tinh bt : +
ng ha inositol : +
Hot ho Ureaza : -
Ha lng gelatin : -
Phn ng DBB : -

13. Dipodascus (13 loi)

Sinh sn sinh dng: Hnh thnh bo t t, c khun ty tht.
Sinh sn hu tnh: Ti hnh kim, tr, elip hay cu. Mi ti cha 4-128 bo t ti trong sut,
hnh elip.
c im sinh l: Ln men : -/yu
ng ho nitrat : -
Mng trn mi trng dch th : -
C cht ging tinh bt : -
ng ha inositol : -
Hot ho Ureaza : -
Ha lng gelatin : -

14. Endomyces (1 loi)

Sinh sn sinh dng: Khng c khun ty, c th c cc bo t trn ny chi (blastoconidia)
Sinh sn hu tnh: Ti c th hnh thnh trn si lng bi (c tip hp). Ti hnh gn cu hay
hnh chu. Mi ti cha 4-6 bo t ti hnh m.
c im sinh l: Ln men : +/-
ng ho nitrat : -
Hot ho Ureaza : -
Phn ng DBB : -


15. Cc chi nm k sinh ging Endomyces

15.1. Helicogonium W. L.White
Chi Helicogonium l nm k sinh. Khun lc c dng si phn nhnh. Ti c cha phn ho,
th sinh ti l cun lng, phnh theo mt pha sinh ra ti n. Ti dng chu cha 8 bo t
ti. Bo t ti trong sut, vch mng, hnh thoi c 1-2 t bo v c chi bn trong ti.

15.2. Myriogonium Cain
Chi Myriogonium l nm k sinh. Khun lc dng si phn nhnh. Ti giao t cha phn ho.
Ti c sinh ra do cc t bo cui si hp li. Ti dng chu, cha 8 bo t ti, chng tch
bit bi vch ngn. Bo t ti trong sut, vch mng, thun di v 1 t bo.

15.3. Phialoascus Redhead & Malloch
L chi nm k sinh. Si trong sut , phn nhnh. Ti dng n, bo t ti trong sut, 1 t bo.


15.4. Trichomonacus H.S. Jackson
L chi nm k sinh. Ti nm trong m ca vt ch, c sinh ra t cui ca si cha phn ho.


16. Eremothecium (5 loi)

Sinh sn sinh dng: Ny chi a cc. C khun ty tht. C hoc khng xut hin khun ty gi.
Sinh sn hu tnh: Cc ti khng tip hp. Ti bo t hnh thoi ti hnh tr. Mi ti cha 8-
32 bo t ti cng hnh kim hay hnh thoi. Bo t ti c th c vch ngn gia.
c im sinh l: Ln men : -/+
ng ho nitrat : -
Mng trn mi trng dch th : -/+
C cht ging tinh bt : -
ng ha inositol : -
Ha lng gelatin : -/+

17. Galactomyces (3 loi)
Sinh sn sinh dng: Hnh thnh bo t t, c khun ty tht.
Sinh sn hu tnh: Ti bo t dng gn cu, trong cha 1 bo t hnh elip.
c im sinh l: Ln men : -/yu
ng ho nitrat : -
Mng trn mi trng dch th : -
C cht ging tinh bt : -
ng ha inositol : -
Hot ho Ureaza : - Ha lng gelatin
: -

18. Hanseniaspora (6 loi)
Sinh sn sinh dng: Ny chi hai cc. C th c khun ty gi. Khng to khun ty tht
Sinh sn hu tnh: Cc ti khng tip hp. Mi ti cha 1-4 bo t ti hnh m hay hnh cu,
nhn hay x x, c hoc khng c g gia.
c im sinh l: Ln men : +
ng ho nitrat : -
Mng trn mi trng dch th : -
C cht ging tinh bt : -
ng ha inositol : -
Hot ho Ureaza : - Ha lng gelatin
: -
Phn ng DBB : -

19. Issatchenkia (4 loi)
Sinh sn sinh dng: Cc t bo ny chi a cc, c khun ty gi, khng c khun ty tht.
Sinh sn hu tnh: Mi ti cha 1-4 bo t ti hnh trn, b mt c th x x
c im sinh l: Ln men : +
ng ho nitrat : -
Mng trn mi trng dch th : +
C cht ging tinh bt : -
ng ha inositol : -
Hot ho Ureaza : - Ha lng gelatin
: -/+
Phn ng DBB : -

20. Kluyveromyces (15 loi)

Sinh sn sinh dng: Cc t bo ny chi a cc, i khi c khun ty gi.
Sinh sn hu tnh: Cc ti bo t c th hoc khng c s tip hp. Mi ti c 1-4 (hoc > 4)
bo t ti nhn, hnh van, trn, que hay hnh thn. Bo t ti c phng ra khi chn.
c im sinh l: Ln men : +
ng ho nitrat : -
Mng trn mi trng dch th : : -/+
C cht ging tinh bt : -
ng ha inositol : -
Hot ho Ureaza : -
Ha lng gelatin : -
Phn ng DBB : -

21. Lipomyces (5 loi)

Sinh sn sinh dng: T bo ny chi a cc, khng c khun ty hoc ch c khun ty gi rt s
ng.
Sinh sn hu tnh: Mi ti c 4-20 bo t ti mu nu hoc h phch, nhn hoc x x, hnh
elip.
c im sinh l: Ln men : -
ng ho nitrat : -
Mng trn mi trng dch th : +/-
To c cht ging tinh bt : +
ng ha inositol : +/-
Hot ho Ureaza : -
Ha lng gelatin : -
Phn ng DBB : -
Dng nui cy : nht

22. Lodderomyces (1 loi)

Sinh sn sinh dng: T bo ny chi a cc, ch c khun ty gi.
Sinh sn hu tnh: Cc ti khng tip hp, thun di, bn, mi ti c 1-2 bo t hnh van
di.
c im sinh l: Ln men : +
ng ho nitrat : -
Mng trn mi trng dch th : -
C cht ging tinh bt : -
ng ha inositol : -
Hot ho Ureaza : -
Ha lng gelatin : -
Phn ng DBB : -

23. Metschnikowia (10 loi)

Sinh sn sinh dng: T bo ny chi a cc, i khi c dng trng khuyt hay li lim. C to
khun ty gi.
Sinh sn hu tnh: Ti bo t c dng thun di, mi ti c 1-2 bo t hnh kim.
c im sinh l: Ln men : +
ng ho nitrat : -
Mng trn mi trng dch th : +/-
C cht ging tinh bt : -
ng ha inositol : -
Hot ho Ureaza : -
Ha lng gelatin : -
Phn ng DBB : -

24. Nadsonia (2 loi)

Sinh sn sinh dng: T bo ny chi 2 cc. T bo to, hnh qu chanh chu u hay hnh van.
Khng to khun ty.
Sinh sn hu tnh: Ti c pht trin t s tip hp ca t bo v chi ca n, Mi ti cha
1-2 bo t ti dng trn, x x.
c im sinh l: Ln men : +/-
ng ho nitrat : -
Mng trn mi trng dch th : +
C cht ging tinh bt : -
ng ha inositol : -
Hot ho Ureaza : -
Ha lng gelatin : -
Phn ng DBB : -

25. Pachysolen (1 loi)

Sinh sn sinh dng: T bo ny chi a cc, rt him khi c khun ty gi
Sinh sn hu tnh: Mi ti hnh thnh t u tn cng ng ph ca mt t bo sinh dng.
Mi ti c cha 4 bo t ti dng m.
c im sinh l: Ln men : + (gluco, D-xylose)
ng ho nitrat : +
Mng trn mi trng dch th : +/-
C cht ging tinh bt : -
ng ha inositol : -
Hot ho Ureaza : -
Ha lng gelatin : -
Phn ng DBB : -

26. Pichia (91 loi)

Sinh sn sinh dng: T bo ny chi, c th c khun ty gi v khun ty tht.
Sinh sn hu tnh: Ti c cha 1-4 (t khi 8) bo t ti nhn trn, hnh m hay sao Th, c
hoc khng c ra ng xch o.
c im sinh l: Ln men : +/-
ng ho nitrat : +/-
Mng trn mi trng dch th : +/-
C cht ging tinh bt : -
ng ha inositol : -/+ ( 1 loi)
Hot ho Ureaza : -
Ha lng gelatin : -/+
Phn ng DBB : -

27. Protomyces (6 loi)

Sinh sn sinh dng: T bo ny a phn cc, Khun ty tht c to thnh trong cc m thc
vt m n k sinh.
Sinh sn hu tnh: Ti bo t cng to bn trong m thc vt ch, cha hng trm cc bo t
ti c kh nng bn ra mnh m.
c im sinh l: Ln men : -
ng ho nitrat : +/-
Mng trn mi trng dch th : +/-
C cht ging tinh bt : +
ng ha inositol : -
Hot ho Ureaza : -
Ha lng gelatin : -/+( rt yu)
Phn ng DBB : -

28. Saccharomyces (14 loi)

Sinh sn sinh dng: T bo ny chi a cc, i khi c khun ty gi.
Sinh sn hu tnh: Ti kh bn v hnh thnh trc tip t mt t bo lng bi. Mi ti c
cha 1-4 (t khi nhiu hn) bo t ti hnh van, trn nhn.
c im sinh l: Ln men : +
ng ho nitrat : -
Mng trn mi trng dch th : -
C cht ging tinh bt : -
ng ha inositol : -
Hot ho Ureaza : -
Ha lng gelatin : -
Phn ng DBB : -

29. Saccharomycodes (1 loi)

Sinh sn sinh dng: T bo ny chi ti hai cc. T bo dng di hay hnh qu chanh chu u.
i khi c khun ty gi.
Sinh sn hu tnh: Mi ti c 1-4 bo t ti trn nhn, mi bo t c mt rnh hp.
c im sinh l: Ln men : +
ng ho nitrat : -
Mng trn mi trng dch th : -
C cht ging tinh bt : -
ng ha inositol : -
Hot ho Ureaza : -
Ha lng gelatin : -/+(yu)
Phn ng DBB : -

30. Saccharomycopsis (10 loi)

Sinh sn sinh dng: T bo ny chi a cc, c bo t t, c khun ty gi, khun ty tht nhiu
v thng sinh bo t trn dng chi.
Sinh sn hu tnh: Mi ti c 2-4, him khi l 8 bo t ti, dng m, thn,ovan hay dng hnh
cu v c th c mt hay nhiu rnh. B mt c th nhn hoc x x.
c im sinh l: Ln men : +/-
ng ho nitrat : -
Mng trn mi trng dch th : +/-
C cht ging tinh bt : -
ng ha inositol : +/-
Hot ho Ureaza : -
Ha lng gelatin : + ( khng mnh)
Phn ng DBB : -

31. Saturnispora (4 loi)

Sinh sn sinh dng: T bo ny chi a cc. C th c khun ty gi.
Sinh sn hu tnh: Mi ti c 1-2, him khi l 8 bo t ti c hnh cu. B mt nhn, c rnh
trn ng xch o.
c im sinh l: Ln men : + (glucose)
ng ho nitrat : -
Mng trn mi trng dch th : +/-
C cht ging tinh bt : -
ng ha inositol : -
Hot ho Ureaza : -
Ha lng gelatin : +/- ( yu)
Phn ng DBB : -
ng ho D-xyloza : -

32. Schizosaccharomyces (3 loi)

Sinh sn sinh dng: Cc t bo khng ny chi m phn ct. i khi c khun ty tht v thng
ngt ra khi gii phng bo t t.
Sinh sn hu tnh: Ti c 2-8 bo t ti dng trn, van hay thn
c im sinh l: Ln men : +
ng ho nitrat : -
Mng trn mi trng dch th : -
C cht ging tinh bt : +
ng ha inositol : -
Hot ho Ureaza : -
Ha lng gelatin : -
Lu : Pht trin chm trn mi trng sinh trng tiu chun v vy dng cc th nghim ln men
l chnh xc hn khi nh tn.


33. Sporopachydermia (3 loi)

Sinh sn sinh dng: T bo ny chi a cc, khng c khun ty, hai loi t bo c dng lp
xng.
Sinh sn hu tnh: Mi ti c cha 1-4 bo t ti thnh dy, hnh t trn, elp hoc gy.
c im sinh l: Ln men : -/+ (glucose, yu)
ng ho nitrat : -
Mng trn mi trng dch th : -
C cht ging tinh bt : -
ng ha inositol : + (ngun cacbon duy nht)
Hot ho Ureaza : -
Ha lng gelatin : -
Phn ng DBB : -

34. Stephanoascus (3 loi)

Sinh sn sinh dng: Ny chi a cc. C khun ty gi v khun ty tht cc bo t chi c th
c to thnh trn khun ty tht .
Sinh sn hu tnh: Cc ti hnh thnh sau khi c s tip hp gia cc t bo t hai khun ty.
Mi ti cha 2-4 bo t ti dng dt n dng m.
c im sinh l: Ln men : -
ng ho nitrat : -
Mng trn mi trng dch th : -
C cht ging tinh bt : -
ng ha inositol : +/-
Hot ho Ureaza : -
Ha lng gelatin : -
Phn ng DBB : -

35. Torulaspora (3 loi)

Sinh sn sinh dng: T bo ny chi, c th c khun ty gi.
Sinh sn hu tnh: Cc ti kh bn, mi ti c cha 1-4 bo t ti, hnh hi trn, nhn hoc
x x. Thng c cu ni gia t bo m v t bo chi.
c im sinh l: Ln men : +
ng ho nitrat : -
Mng trn mi trng dch th : -
C cht ging tinh bt : -
ng ha inositol : -
Hot ho Ureaza : -
Ha lng gelatin : -
Phn ng DBB : -

36. Wickerhamia (1 loi)

Sinh sn sinh dng: T bo ny chi ti hai cc, cc t bo hnh ong hay van hoc hnh ko
di. Khng c khun ty.
Sinh sn hu tnh: Mi ti c 1-2, him khi l 16 bo t ti, dng m bng chy.
c im sinh l: Ln men : +
ng ho nitrat : -
Mng trn mi trng dch th : -
C cht ging tinh bt : -
ng ha inositol : -
Hot ho Ureaza : -
Ha lng gelatin : -
Phn ng DBB : -

37. Wickerhamiella (1 loi)

Sinh sn sinh dng: T bo ny chi a cc, khng c khun ty.
Sinh sn hu tnh: Cc ti bo t c tip hp to ra 1 bo t ti c dng di, khng i
xng v x x.
c im sinh l: Ln men : -
ng ho nitrat : +
Mng trn mi trng dch th : -
C cht ging tinh bt : -
ng ha inositol : -
Ha lng gelatin : -
Hot ho Ureaza : -
Phn ng DBB : -

38. Williopsis (5 loi)

Sinh sn sinh dng: T bo ny chi a cc, i khi c khun ty gi.
Sinh sn hu tnh: Mi ti c 2-4 bo t ti nhn, dng cu, ovan, c ng rnh gia.
c im sinh l: Ln men : +(glucoza)
ng ho nitrat : +/-
Mng trn mi trng dch th : +
C cht ging tinh bt : -
ng ha inositol : -
Hot ho Ureaza : -
Ha lng gelatin : +/ -
Phn ng DBB : -

39. Yarrowia (1 loi)

Sinh sn sinh dng: Ny chi a cc, thng c bo t t. Khun ty gi, khun ty tht c
to thnh
Sinh sn hu tnh: Cc ti khng tip hp v tan ra khi trng thnh. Mi ti c 1-4 bo t ti
dng m hay hnh bn cu, cu hoc hnh sao Th
c im sinh l: Ln men ng : -
ng ho nitrat : -
Mng trn mi trng dch th : +
C cht ging tinh bt : -
ng ha inositol : -
Hot ho Ureaza : +
Ha lng gelatin : +
Hot ha lipaza : +

40. Zygoascus (1 loi)

Sinh sn sinh dng: T bo ny chi a cc. C c khun ty tht v khun ty gi.
Sinh sn hu tnh: Cc ti bo t hnh cu, bn. Xut hin sau khi c s tip hp gia cc si.
Trong ti c t 1-4 bo t , dng gn cu hay m.
c im sinh l: Ln men : +
ng ho nitrat : -
Mng trn mi trng dch th : +/-
C cht ging tinh bt : -
ng ha inositol : +
Hot ho Ureaza : -
Ha lng gelatin : -

41. Zygosaccharomyces (9 loi)

Sinh sn sinh dng: T bo ny chi a cc, i khi c khun ty gi. Khng c khun ty tht.
Sinh sn hu tnh: Ti thng bn, c th khng tip hp hoc ch tip hp gia t bo v
chi ca n. Mi ti c 1-4 bo t hnh trn hay hnh van, nhn
c im sinh l: Ln men : +
ng ho nitrat : -
Mng trn mi trng dch th : -
C cht ging tinh bt : -
ng ha inositol : -
Hot ho Ureaza : -
Ha lng gelatin : -
Phn ng DBB : -


42. Zygozyma(4 loi)

Sinh sn sinh dng: Ny chi a cc, khng to khun ty tht v khun ty gi .
Sinh sn hu tnh: Cc ti b tan ra do s ln ca cc t bo n hoc t cu ni ca cc t
bo tip hp. C 4 bo t hoc hn trong mt ti, dng di, nhn v mu h phch.
c im sinh l: Ln men : -
ng ho nitrat : -
Mng trn mi trng dch th : -
C cht ging tinh bt : +
ng ha inositol : +/-
Hot ho Ureaza : -
Ha lng gelatin : -

B. Dng sinh sn v tnh (Anamorph)

43. Aciculoconidium (1 loi)
T bo dinh dng: T bo ny chi a cc, khun ty tht v gi c to thnh vi cc chui bo
t trn dng chi. Cc bo t trn hnh kim ti cc u tn cng si.
c im sinh l: Ln men : +
ng ho nitrat : -
ng ha inositol : -
C cht ging tinh bt : -
Hot ho Ureaza : -
Phn ng DBB : -
44. Arxula (2 loi)
Sinh sn sinh dng: Cc t bo chi l bo t trn dng t. C c khun ty tht v khun ty
gi

c im sinh l: Ln men : -
ng ho nitrat : +
Mng trn mi trng dch th : +/-
ng ha inositol : +

45. Blastobotrys (7 loi)
Sinh sn sinh dng: Ny chi a cc, khun ty tht v gi rt nhiu. Chi ny c nhn bit nh
s to thnh cc bo t trn dng chi trn cung hoc rng nh.
c im sinh l: Ln men : +/-
ng ho nitrat : -
ng ha inositol : +/-

46. Botryozyma (1 loi)
Sinh sn sinh dng: Ny chi a cc. Ch sinh khun ty gi. c trng ca chi l cc t bo khun
ty gi ch i on cui.
c im sinh l: Ln men : -
ng ho nitrat : -
Mng trn mi trng dch th : -
C cht ging tinh bt : -
ng ha inositol : -

47. Brettanomyces (5 loi)
Sinh sn sinh dng: Ny chi a cc; cc t bo hnh cu n hnh elip. Ch sinh khun ty gi.

c im sinh l: Ln men : +
ng ho nitrat : +/-
Mng trn mi trng dch th : +/-
C cht ging tinh bt : -
ng ha inositol : -
Hot ho Ureaza : -
Sinh axit axetic : +
Sinh trng chm, vng i ngn

47. Candida (163 loi)
Sinh sn sinh dng: Ny chi a cc, t bo hnh cu, elip hoc hnh tr, i khi c hnh cung
nhn, tam gic hoc hnh li lim. i khi c khun ty gi v khun ty tht.
c im sinh l: Ln men : +/-
ng ho nitrat : +/-
Mng trn mi trng dch th : +/-
C cht ging tinh bt : -
ng ha inositol : +/-
Ha lng gelatin : +/-
Hot ho Ureaza : -

48. Geotrichum(11 loi)
Sinh sn sinh dng: Khng c t bo ny chi.C bo t t (arthroconidia). C khun ty tht ri
ra thnh tng t.
c im sinh l: Ln men : +/-
ng ho nitrat : -
ng ha inositol : -
49. Kloeckera (7 loi)
Sinh sn sinh dng: T bo ny chi 2 cc, cc t bo hnh van hay dng qu chanh chu u, t
khi c dng khun ty gi.
c im sinh l: Ln men : +
ng ho nitrat : -
Mng trn mi trng dch th : -
C cht ging tinh bt : -
ng ha inositol : -
Hot ho Ureaza : -

50. Lalaria (23 loi)
Sinh sn sinh dng: Ny chi pha trn hoc gn nh. Mu hi hng, i khi hi vng.

c im sinh l: Ln men : -
ng ho nitrat : -
C cht ging tinh bt : +

51. Myxozyma(8 loi)
Sinh sn sinh dng: Ny chi a cc. i khi c khun ty gi s ng c im sinh l:
Ln men : -
ng ho nitrat : -
Mng trn mi trng dch th : -
C cht ging tinh bt : +
ng ha inositol : +/-
Ha lng gelatin : +/-
Nui cy dng nhy

52. Oosporidium (1 loi)
Sinh sn sinh dng: Ny chi a cc. C khi tn ti dng khun ty gi v khun ty tht. c trng
ca chi ny l sinh ni bo t. Mu hng n cam c im sinh l: Ln men
: -
ng ho nitrat : +
Mng trn mi trng dch th : -
C cht ging tinh bt : +
ng ha inositol : -

53. Saitoella (1 loi)
Sinh sn sinh dng: Ny chi a cc. Khng c khun ty. C mu cam n mu .
c im sinh l: Ln men : -
ng ho nitrat : +
Mng trn mi trng dch th : -
C cht ging tinh bt : -
ng ha inositol : -
Hot ho Ureaza : +

54. Schizoblastosporion (1 loi)
Sinh sn sinh dng: T bo ny chi hai cc t cc t bo hnh van hay hnh tr. i khi c
khun ty gi s ng.
c im sinh l: Ln men : -
ng ho nitrat : -
Mng trn mi trng dch th : -
C cht ging tinh bt : -
ng ha inositol : -
Hot ho Ureaza : -
Phn ng DBB : -


55. Sympodiomyces (1 loi)
Sinh sn sinh dng: Ny chi a cc. C khun ty tht v khun ty gi. c trng ca chi l bo t
chi trn cung ngn c sinh trc tip t cc t bo nm men.
c im sinh l: Ln men : -
ng ho nitrat : -
Mng trn mi trng dch th : -
C cht ging tinh bt : -
ng ha inositol : +
Ho lng gelatin : -
Hot ho Ureaza : -
Phn ng DBB : -
56. Trigonopsis (1 loi)
Sinh sn sinh dng: Cc t bo c hnh tam gic l ch yu, t cc gc mc ra cc chi. Tuy nhin
cng c cc t bo hnh van ny chi nhiu pha.
c im sinh l: Ln men : -
ng ho nitrat : -
Mng trn mi trng dch th : -
C cht ging tinh bt : -
ng ha inositol : -
Hot ho Ureaza : -
Phn ng DBB : -

II. Cc chi nm men thuc ngnh nm m.

Bo t m l bo t hu tnh c trng cc nm thuc ngnh nm m (Basidiomycota).
A. Dng sinh sn hu tnh ( Teleomorph)

57. Agaricostibum (1 loi)
Sinh sn sinh dng: Khun lc mu cam hi vng, dng b. T bo hnh ellip, c khi dnh vo
nhau thnh chui ny chi nh.
Sinh sn hu tnh: Qa m synnemata-like. Cc si thiu mu ni. Cc m hnh tr c 4
bo t, vch ngang. L vch n gin.
c im sinh l: Ln men : -
ng ho D-glucuronat: +
ng ha inositol : -
C cht ging tinh bt : -
ng ho nitrat : -
Hot ho Ureaza : +
Phn ng DBB : +
Xyloza trong t bo : -
58. Bulleromyces (1 loi)
Sinh sn sinh dng: Khun lc mu kem, dng b n nhy. T bo hnh ellip, hnh gn cu n
hnh tr, ny chi cc. i khi c khun ty gi. Bo t m dng i xng trn.
Sinh sn hu tnh: Khng c qu m, cc si c mu ni . m dng keo cha 2-4 t
bo.
c im sinh l: Ln men : -
D-Glucuronate : +
Myo-inositol : +
C cht ging tinh bt : +
ng ho nitrat : -
Phn ng DBB : +
Hot ho Ureaza : +
Coenzym : Q10
Xylose : +

59. Chionosphaera (1 loi)
Sinh sn sinh dng: Khun lc mu kem, dng b n nhy. T bo hnh ellip hoc dng xc xch,
ny chi cc.
Sinh sn hu tnh: Qu m dng stilbelloid, sp kh hoc hi sn st, cc si khng c mu ni.
m cha 1 t bo hnh chu. L vch n s .
c im sinh l: Ln men : -
D-Glucuronate : -
Myo-inositol : -
C cht ging tinh bt : +
ng ho nitrat : -
Phn ng DBB : +
Hot ho Ureaza : +
Coenzym : Q10

60. Cystofilobasidium (4 loi)
Sinh sn sinh dng: Khun lc mu cam, c hi . T bo hnh trng, hnh trng di. i khi c
khun ty gi.
Sinh sn hu tnh: Khng c qu m, i khi si c mu ni. i khi c ni bo t. m cha 1 t
bo hnh chu hoc hnh u. C l vch.
c im sinh l: Ln men : -/+
D-Glucuronate : +
Myo-inositol : +
C cht ging tinh bt : +
ng ho nitrat : +
Phn ng DBB : +
Hot ho Ureaza : +
Coenzym : Q8
Xylose : +

61. Erythrobasidium (1 loi)
Sinh sn sinh dng: Khun lc mu cam-. T bo hnh trng, hnh trng di. Chi a cc.

Sinh sn hu tnh: Khng c qu m, si c gi mu ni. m cha 1 t bo. C l vch n gin
c im sinh l: Ln men : -
D-Glucuronate : +
Myo-inositol : -
C cht ging tinh bt : -
ng ho nitrat : +
Phn ng DBB : +
Hot ho Ureaza : +
Coenzym : Q10(H
2
)
Xylose : -
62. Fibulobasidium (1 loi)
Sinh sn sinh dng: Khun lc mu vng xm n vng nu, dng b. T bo hnh gn cu n
hnh cu. Bo t bn i xng trn.
Sinh sn hu tnh: Qu m hnh gi, sn st, si c mu ni. m dng keo, thnh chui do pht
trin t cc mu ni.
c im sinh l: Ln men : -
D-Glucuronate : +
Myo-inositol : +
C cht ging tinh bt : +
ng ho nitrat : -/+(yu)
Phn ng DBB : +
Hot ho Ureaza : +
Coenzym : Q9
63. Filobasidiella (1 loi)
Sinh sn sinh dng: Khun lc mu kem trng n kem vng, dng nhy. T bo hnh cu, hnh
trng, hnh trng nhn, ny chi mt cc hoc a cc. Sinh sn hu tnh: Khng c qu m, si c
mu ni. m 1 t bo vi chui bo t hnh u c cung. C l vch.
c im sinh l: Ln men : -
D-Glucuronate : +
Myo-inositol : +
C cht ging tinh bt : +
ng ho nitrat : -
Phn ng DBB : +
Hot ho Ureaza : +
Coenzym : Q10
Xylose : +

64. Filobasidium (5 loi)
Sinh sn sinh dng: Khun lc mu trng, kem hoc hi hng, dng nhy. T bo hnh cu, hnh
trng, hnh elip, hoc hnh trng ko di. i khi c si gi.
Sinh sn hu tnh: Khng c qu m, si c mu ni. m 1 t bo hnh chu c cc bo t m
khng cung. C l vch.
c im sinh l: Ln men : -/+
D-Glucuronate : -/+
Myo-inositol : -/+
C cht ging tinh bt : +
ng ho nitrat : -/+
Phn ng DBB : +
Hot ho Ureaza : +
Coenzym : Q9 hoc Q10
Xylose : +

65. Holtermannia (1 loi)
Sinh sn sinh dng: Khun lc mu trng hi vng, dng nhy. T bo hnh elip, ny chi mt cc.
Sinh sn hu tnh: Qu m dng san h thng ng, dng sn st v thng to thnh m dy c.
Si c mu ni. m dng keo
c im sinh l: Ln men : -
D-Glucuronate : +
Myo-inositol : +
C cht ging tinh bt : +
ng ho nitrat : -
Phn ng DBB : +
Hot ho Ureaza : +
Xylose : +

66. Leucosporidium (3 loi)
Sinh sn sinh dng: Khun lc mu kem, dng nhy. T bo hnh trng, hnh trng ko di. i
khi c si gi.
Sinh sn hu tnh: Khng c qu m, si c mu ni. C bo t (teliospore) Cc m c vch
ngang. C l vch th s.
c im sinh l: Ln men : -/+
D-Glucuronate : +/-
Myo-inositol : -
C cht ging tinh bt : -
ng ho nitrat : +
Phn ng DBB : +
Hot ho Ureaza : +
Coenzym : Q9 hoc Q10
Xylose : -

67. Mrakia (1 loi)
Sinh sn sinh dng: Khun lc mu trng hoc mu kem, dng nhy. T bo hnh trng, hnh trng
ko di. i khi c si gi.
Sinh sn hu tnh: Khng c qu m, si khng c mu ni. C bo t (teliospore) Cc m l
mt t bo hoc c vch ngang. C l vch.
c im sinh l: Ln men : -/+(glucoza hoc galactoza)
D-Glucuronate : +
Myo-inositol : -/+
C cht ging tinh bt : +
ng ho nitrat : +
Phn ng DBB : +
Hot ho Ureaza : +
Coenzym : Q9
Xylose : +
Nhit sinh trng : di 20
0
C
68. Rhodosporidium (10 loi)
Sinh sn sinh dng: Khun lc mu trng hoc mu cam, hoc hi vng, dng b n nhy. T
bo hnh trng, hnh trng ko di. i khi c si gi.
Sinh sn hu tnh: Khng c qu m, i khi si c mu ni. C bo t (teliospore) Cc m c
vch ngang. C l vch dng th s.
c im sinh l: Ln men : -
D-Glucuronate : -/+
Myo-inositol : -
C cht ging tinh bt : -
ng ho nitrat : -/+
Phn ng DBB : +
Hot ho Ureaza : +
Coenzym : Q9 hoc Q10
Xylose : -

69. Sirobasidium (2 loi)
Sinh sn sinh dng: Khun lc hi vng n vng kem, dng nhy. T bo hnh elip n gn cu,
ny chi nh. Bo t bn i xng i. i khi c si gi.
Sinh sn hu tnh: Qu m hnh gi hoc dng thu, dng l khng u, si c mu ni. Cc m
dng keo, thng thnh chui. C l vch.
c im sinh l: Ln men : -
D-Glucuronate : +
Myo-inositol : +
C cht ging tinh bt : +
ng ho nitrat : -
Phn ng DBB : +
Hot ho Ureaza : +


70. Sporidiobolus (4 loi)
Sinh sn sinh dng: Khun lc mu hng n mu hi . T bo t hnh elip n hnh trng ko
di v ny chi nh. Bo t bn dng i xng i. i khi c si gi.
Sinh sn hu tnh: Khng c qu m, si thng c mu ni. C bo t (teliospore) Cc m
thng c vch ngang. Cc l vch dng th s.
c im sinh l: Ln men : -
D-Glucuronate : -
Myo-inositol : -/+
C cht ging tinh bt : -
ng ho nitrat : -/+
Phn ng DBB : +
Hot ho Ureaza : +
Coenzym : Q10
Xylose : -
71. Sterigmatosporidium (1 loi)
Sinh sn sinh dng: Khun lc mu kem, dng b. T bo t hnh elip n hnh trng ko di v
ny chi nh, c cc cung vi vch ngn cui. C si gi.
Sinh sn hu tnh: Khng c qu m, si c mu ni. Bo t o dikaryotic. Dng m l 1 t bo.
c im sinh l: Ln men : -
D-Glucuronate : +
Myo-inositol : +
C cht ging tinh bt : +
ng ho nitrat : -
Phn ng DBB : +
Hot ho Ureaza : +
Coenzym : Q10
Xylose : +
72. Tilletiaria (1 loi)
Sinh sn sinh dng: Khun lc mu nu hi xm v dai. Si khng c cc mu ni. C bo t o.
Bo t bn dng i xng 2 bn.
Sinh sn hu tnh: Khng c qu m. Bo t (teliospore) l cc mu nh mu nu. Cc m
thng c vch ngang. Cc l vch nguyn thu.
c im sinh l: Ln men : -
D-Glucuronate : +
Myo-inositol : -
C cht ging tinh bt : -
ng ho nitrat : -
Phn ng DBB : +
Hot ho Ureaza : +
Coenzym : Q10
Xylose : -
73. Tremella (11 loi)
Sinh sn sinh dng: Khun lc mu kem n mu nu nht, dng nhy. T bo t hnh elip , gn
cu n hnh cu v ny chi nh hoc a cc. i khi c si gi.
Sinh sn hu tnh: Qu m thng dng nh cc l hnh gi to ra. Si thng c mu ni. Cc
m thng dng keo. C l vch.
c im sinh l: Ln men : -
D-Glucuronate : -/+
Myo-inositol : -/+
C cht ging tinh bt : -/+
ng ho nitrat : -
Phn ng DBB : +
Hot ho Ureaza : +
Coenzym : Q10
Xylose : +
74. Trimorphomyces (1 loi)
Sinh sn sinh dng: Cc khun lc n bi mu hng nht n mu hng nu, dng b. T bo t
hnh gn cu, elip, tam gic hoc hnh trng ko di v ny chi nh hoc a cc. Cc khun lc
dikaryotic cng tng t nhng t bo nm men c hnh ch H hu ht cc chng. Cc t bo
hnh ch H pht trin (synchronously as closely situated) ng b nh dng ng, chng tip hp
trc khi thnh thc v gii phng cc t bo ch H mi
Sinh sn hu tnh: Qu m nh, hnh gi v thng kt hp vi Arthrinium trn cy tre hoc cc
loi c. Si c cc mu ni. Cc m thng dng keo. C l vch.
c im sinh l: Ln men : -
D-Glucuronate : +
Myo-inositol : -/+
C cht ging tinh bt : -/+
ng ho nitrat : -
Phn ng DBB : +
Hot ho Ureaza : +
Xylose : +
75. Xanthophyllomyces (1 loi)
Sinh sn sinh dng: Khun lc mu cam n mu c hi, dng b. T bo t hnh elip. i khi
c si gi v bo t o.
Sinh sn hu tnh: Khng c qu m. Khng c si. Cc m thng c to thnh do s tip
hp gia 1 t bo v chi ca n.
c im sinh l: Ln men : +
D-Glucuronate : +
Myo-inositol : -
C cht ging tinh bt : +
ng ho nitrat : -
Phn ng DBB : +
Hot ho Ureaza : +
Coenzym : Q10
Xylose : +
B. Dng sinh sn v tnh ( Anamorph)

60. Bensingtonia ( 9 loi)
Sinh sn sinh dng: Khun lc mu trng, hi hng, son, nu n mu xm, dng b. T
bo hnh elip, hnh trng, n hnh trng ko di, thng ny chi nh. Bo t bn i xng 2
bn. C khi xut hin bo t o, si tht v si gi. i khi c cc l vch dng s ng.
c im sinh l: Ln men : -
D-Glucuronate : -/+
Myo-inositol : -
C cht ging tinh bt : -
ng ho nitrat : -/+
Phn ng DBB : +
Hot ho Ureaza : +
Coenzym : Q9
Xylose : -

61. Bullera (13 loi)

Sinh sn sinh dng: Khun lc t mu trng cam n mu nu, dng b n nhy. T bo
hnh gn cu, hnh elip, hnh trng, n hnh trng ko di, thng ny chi nh. Bo t bn i
xng trn. C khi xut hin si tht v si gi. i khi c cc l vch.
c im sinh l: Ln men : -
D-Glucuronate : +
Myo-inositol : -/+
C cht ging tinh bt : -/+
ng ho nitrat : -/+
Phn ng DBB : +
Hot ho Ureaza : +
Coenzym : Q9
Xylose : +

62. Cryptococcus ( 34 loi)

Sinh sn sinh dng: Khun lc t mu trng cam n mu nu, thng
dng nhy. T bo hnh gn cu, hnh cu, hnh elip, hnh trng, n hnh trng ko di, thng
ny 1 chi nh hoc a chi. C khi xut hin si tht v si gi. i khi c cc t bo ging
dng bo t o v cc mu ni.
i khi c cc l vch.
c im sinh l: Ln men : -
D-Glucuronate : +
Myo-inositol : +/-
C cht ging tinh bt : +/-
ng ho nitrat : -/+
Phn ng DBB : +
Hot ho Ureaza : +
Coenzym : Q8, Q9 hoc Q10
Xylose : +

63. Fellomyces( 4 loi)
Sinh sn sinh dng: Khun lc mu kem, dng b.
T bo hnh gn cu, hnh cu, hnh elip, chi c cung. C khi xut hin si tht v si gi.
c im sinh l: Ln men : -
D-Glucuronate : +
Myo-inositol : +
C cht ging tinh bt : +
ng ho nitrat : -
Phn ng DBB : +
Hot ho Ureaza : +
Coenzym : Q10
Xylose : +

64. Hyalodendron (1 loi)

Sinh sn sinh dng: Khun lc mu trng n mu kem, b mt nh c rc bt mn. Bo t nh
dng chi (blastoconidia) hnh chp ct thnh chui hng ngn. i khi xut hin cc t bo ging
nm men hoc ging bo t o. Co cc l vch.
c im sinh l: Ln men : -
Myo-inositol : +
ng ho nitrat : -
Phn ng DBB : +
Hot ho Ureaza : +
Coenzym : Q9
Xylose : +

65. Itersonilia ( 1 loi)

Sinh sn sinh dng: Khun lc dng si mu trng, c lp lng mn bao ph. Si c cc t bo
ging bo t o v cc mu ni. Bo t bn i xng 2 bn. Khun lc mu kem vng, dng b. T
bo hnh elip n hnh trng ko di, thng c chi nh. C khi xut hin si tht v si gi. C
cc l vch.
c im sinh l: Ln men : -
D-Glucuronate : +
Myo-inositol : -/+
C cht ging tinh bt : -
ng ho nitrat : +/-
Phn ng DBB : +
Hot ho Ureaza : +
Coenzym : Q9
Xylose : +

66. Kockovaella ( 2 loi)

Sinh sn sinh dng: Khun lc mu cam xm n mu vng, b mt t dng kh, b n nhy.
T bo hnh gn cu, hnh cu, hnh elip cho n hnh thn, chi c cung. Bo t bn i xng
trn. Khng c si tht v si gi.
c im sinh l: Ln men : -
D-Glucuronate : +
Myo-inositol : +
C cht ging tinh bt : -
ng ho nitrat : -
Phn ng DBB : +
Hot ho Ureaza : +
Coenzym : Q10
Xylose : +

67. Kurtzmanomyces (2 loi)
Sinh sn sinh dng: Khun lc mu hng nht, dng b.
T bo hnh gn cu, hnh cu, hnh trng n hnh tr, chi c cung. C khi xut hin si tht. C
cc l vch.
c im sinh l: Ln men : -
D-Glucuronate : -
Myo-inositol : -
C cht ging tinh bt : -
ng ho nitrat : +
Phn ng DBB : +
Hot ho Ureaza : +
Coenzym : Q10
Xylose : -

68. Malassezia ( 3 loi)
Sinh trng sinh dng: Khun lc mu nu hi vng, b mt t dng b n kh.
T bo hnh gn cu, hnh cu, hnh elip, thng c chi 1 cc. thng khng c cc l vch
c im sinh l: Ln men : -
D-Glucuronate : +
Myo-inositol : +
ng ho nitrat : +
Phn ng DBB : +
Hot ho Ureaza : +
Coenzym : Q9
Xylose : -

69. Moniliella ( 4 loi)
Sinh trng sinh dng: Khun lc lc u mu kem, sau chuyn sang mu xm en hoc mu en
oliu, b mt nhn mt. Cc chui hng ngn ca cc bo t nh dng chi c to thnh t
cc gai nh. i khi xut hin bo t o hnh chu, vch dy.C khi xut hin si gi.
T bo hnh elip n hi tr. C l vch.
c im sinh l: Ln men : +
Myo-inositol : -
ng ho nitrat : +
Phn ng DBB : +
Hot ho Ureaza : +
Coenzym : Q9
Xylose : -

70. Phaffia (1 loi)
Sinh trng sinh dng: Khun lc mu cam n mu c hi, dng b .
T bo hnh elip. C th xut hin si gi v bo t o.
c im sinh l: Ln men : +
D-Glucuronate : +
Myo-inositol : -
ng ho nitrat : -
C cht ging tinh bt : +
Phn ng DBB : +
Hot ho Ureaza : +
Coenzym : Q10
Xylose : +

71. Pseudozyma ( 8 loi)
Sinh trng sinh dng: Khun lc mu cam hng, cam nht hoc nu. C si, khng c cc mu
ni. Bo t nh dng chi hnh thoi to chui hng ngn. i khi c bo t o. T bo hnh elip,
hnh trng n hnh tr, thng ny chi nh. C th xut hin si gi. C cc l vch siu nh.
c im sinh l: Ln men : -
D-Glucuronate : +
Myo-inositol : +
ng ho nitrat : +
C cht ging tinh bt : -
Phn ng DBB : +
Hot ho Ureaza : +
Coenzym : Q10
Xylose : -

72. Reniforma( 1 loi)
Sinh trng sinh dng: Khun lc mu trng n mu kem, b mt x x hoc kh. T bo hnh
thn, thng c cc cung. Khng c si gi v si tht.
c im sinh l: Ln men : -
Myo-inositol : -
ng ho nitrat : -
C cht ging tinh bt : -
Phn ng DBB : +
Hot ho Ureaza : +
Coenzym : Q7
Xylose : -

73. Rhodotorula (34 loi)

Sinh trng sinh dng: Khun lc mu cam n mu , vng, dng b hoc nhy.
T bo hnh cu, gn cu, elip, trng hoc trng ko di, thng ny chi nh. C th xut hin
si gi v si tht.
c im sinh l: Ln men : -
D-Glucuronate : +/-
Myo-inositol : -
ng ho nitrat : +/-
C cht ging tinh bt : -
Phn ng DBB : +
Hot ho Ureaza : +
Coenzym : Q9 hoc Q10
Xylose : -

74. Sporobolomyces ( 21 loi)

Sinh trng sinh dng: Khun lc mu cam n mu , hng, vng, dng b n nhy.
T bo hnh cu, gn cu, elip, thn, trng hoc hnh tr, thng ny chi nh hoc him khi a
cc. Bo t bn i xng 2 bn. C th xut hin si gi v si tht.
c im sinh l: Ln men : -
D-Glucuronate : -/+
Myo-inositol : -
ng ho nitrat : -/+
C cht ging tinh bt : -
Phn ng DBB : +
Hot ho Ureaza : +
Coenzym : Q10 hoc Q10(H
2
)
Xylose : -

75. Sterigmatomyces (2 loi)

Sinh trng sinh dng: Khun lc mu trng hoc mu kem, dng b .
T bo hnh cu, gn cu hoc trng, ny chi trn cc cung c vch gia. C th xut hin si
gi v si tht.
c im sinh l: Ln men : -
D-Glucuronate : +/-
Myo-inositol : -
ng ho nitrat :+/ -
C cht ging tinh bt : -
Phn ng DBB : +
Hot ho Ureaza : +
Coenzym : Q9
Xylose : -

76. Sympodiomycopsis (1 loi)
Sinh trng sinh dng: Khun lc mu kem, dng b n nhy .
T bo hnh chu ngc, trng hoc trng ko di, With holoblastic-annellidic or holoblastic-
sympodial budding. Bo t nh dng chi c to thnh theo hng ngn ngn v thnh chui
phn nhnh hoc n l. C khi gp bo t o. Xut hin si gi v si tht. C cc l vch n.
c im sinh l: Ln men : -
Myo-inositol : +
ng ho nitrat : +
C cht ging tinh bt : -
Phn ng DBB : +
Hot ho Ureaza : +
Coenzym : Q10
Xylose : +

77. Tilletiopsis (6 loi)

Sinh trng sinh dng: Khun lc mu trng, mu kem, hng, vng n mu nu, c gn hoc
lng nhung .
T bo t hnh elip n trng ko di, ny chi nh Si khng c cc mu ni. i khi xut hin
bo t o. Bo t bn i xng 2 bn. Cc l vch rt nh.
c im sinh l: Ln men : -
D-Glucuronate : -/+
Myo-inositol : -/+
ng ho nitrat : -
C cht ging tinh bt : -
Phn ng DBB : +
Hot ho Ureaza : +
Coenzym : Q10
Xylose : -

78. Trichosporon (19 loi)

Sinh trng sinh dng: Khun lc mu kem, b mt t t n kh .
T bo t hnh elip n hnh trng ko di, ny chi nh. Si thng ct di thnh bo t t.
i khi c ni bo t hoc bo t o. C cc l vch. c im sinh l: Ln men
: -
D-Glucuronate : +/-
Myo-inositol : +/-
ng ho nitrat : -/+
C cht ging tinh bt : +/-
Phn ng DBB : +
Hot ho Ureaza : +
Coenzym : Q9 hoc Q10
Xylose : +

79. Trichosporonoides ( 5 loi)
Sinh trng sinh dng: Khun lc mu kem sau chuyn thnh mu nu oliu, b mt nhn hoc
cerebriform. T bo hnh elip. i khi si b phn ct. Bo t nh dng chi pht trin theo hng
ngn. C th xut hin si gi . C cc l vch.
c im sinh l: Ln men : +
Myo-inositol : -
ng ho nitrat : +
Phn ng DBB : +
Hot ho Ureaza : +
Coenzym : Q9
Xylose : -

80. Tsuchiyaea (1 loi)
Sinh trng sinh dng: Khun lc mu ng, dng b .
T bo hnh cu, gn cu, trng hoc hnh tr , ny chi trn cc cung c vch ngn gia.
c im sinh l: Ln men : -
D-Glucuronate : +
Myo-inositol : +
ng ho nitrat : -
C cht ging tinh bt : -/+(yu)
Phn ng DBB : +
Hot ho Ureaza : +
Coenzym : Q9
Xylose : +

Hnh nh mt s loi nm men mi c pht hin Vit Nam




Bo t bn v t bo sinh dng ca loi Kockovaella litseae




Bo t bn v t bo sinh dng ca loi Kockovaella calophylli



Bo t bn v t bo sinh dng ca loi Kockovaella cucphuongensis



Bo t bn v t bo sinh dng ca loi Kockovaella vietnamensis



Bo t bn v t bo sinh dng ca loi Bullera hoabinhensis
Nm men 1
Nm men 2
Nm men 3

Ti liu tham kho

o Th Lng, Phm Vn Ty, Nguyn Ln Dng, Masako Takashima, Takashi Nakase. Bullera
ninhbinhensis sp. nov., Mt loi nm men mi sinh bo t bn c phn lp Vit nam.
Chuyn san Cng ngh Sinh hc. Tp ch Di truyn v ng dng. 2002. 35-43.
Barnett J.A., Payne R.W., Yarrow D.Y.,1990, Yeasts: Characteristics and identification, Cambridge
Univ. Press.
Kurtzman, C. P. and Fell, J. W., (1998). The Yeasts, a Taxonomic Study, 4
th
ed., ed. by Elsevier,
Amsterdam
Luong, D. T., Takashima, M., Ty, P.V., Dung, N. L.,and Nakase, T. (2000) Four new species of
Kockovaella isolated from plant leaves collected in Vietnam. J. Gen. Appl. Microbiol., 46, 297-
301.
Luong, D. T., Takashima, M., Ty, P.V., Dung, N. L.,and Nakase, T. (2005) Bullera hoabinhensis sp.
nov., a new ballistoconidiogenous yeast isolated from a plant leaf collected in Vietnam. J. Gen.
Appl. Microbiol., 51, 335-342.

Bi 11 Vi to (Microalgae)
Vi to (Microalgae) l tt c cc to (Algae) c kch thc hin vi. Mun
quan st chng phi s dng ti knh hin vi.Trong s khong 50 000 loi to
trn th gii th vi to chim n khong 2/3.
Nm 1969 R.H. Whitake a ra h thng phn loi 5 gii, trong ton
b To c xp trong gii Nguyn sinh. Sau khi xut vic phn chia sinh
gii thnh 3 lnh gii (domain) Carl R. Woese xut h thng phn loi 6 gii
( Vi khun, C khun, Nguyn sinh, Nm, Thc vt, ng vt) th ton b To
vn c xp trong gii Nguyn sinh.
Gn y , theo P.H. Raven v G.B. Johnson (2002) cn c h thng phn
loi chia lnh gii Sinh vt nhn tht (Eukarya hay Eukaryotic Kingdoms) ra
thnh 6 gii, gm c:
-Gii Archezoa: gm cc Nguyn sinh cha c ty th, bao gm Pelomyxa,
Giardia.
-Gii Protozoa (ng vt nguyn sinh): bao gm 14 ngnh Nguyn sinh-
trong c Hypermastigotes, Euglenoides, Slime molds (Nm nhy),
Choanoflagellates, Dinoglagellates, Ciliates, Apicomplexans, Rhizopods,
Heliozoans, Foraminiferans, v Radiolarians.
-Gii Chromista: gm 10 ngnh Nguyn sinh, trong c To nu
(Phaeophyta) v To silic (Diatoms )
-Gii Fungi (Nm): Bao gm nm v 1 ngnh Nguyn sinh sng hoi sinh
l ngnh Chytridiomycota.
-Gii Plantae (Thc vt) : bao gm Thc vt v 5 ngnh Nguyn sinh
(nhiu To lc nh Volvox, Ulva, Spirogyra v To (Rhodophyta).
-Gii Animalia (ng vt) .
Nh vy chng t vic gp rt nhiu nhm sinh vt khc nhau rt xa vo
gii Nguyn sinh l cha hp l.

C nhiu h thng phn loi to rt khc nhau. Chng ti gii thiu h
thng cc ngnh To (bao gm c Vi khun lam- Cyanophyta) v cc lp , b
ch yu theo Peter Pancik ( http://www.thallobionta.czm.sk ) nh sau:

Cyanophyta - Vi khun lam
1.1 Chroococcales
1.2 Oscillatoriales
Prochlorophyta - Ngnh To tin lc
Rhodophyta- Ngnh To
1 Bangiophycidae
2 Florideophycidae
Heterokontophyta- Ngnh To lng roi lch
1 Chrysophyceae- To vng nh
1.1 Chrysomonadales
1.2 Rhizochrysidales
1.3 Chrysocapsales
1.4 Chrysosphaerales
1.5 Phaeothamniales
2 Xantophyceae- To vng lc
2.1 Heterochloridales
2.2 Rhizochloridales
2.3 Heterogloeales
2.4 Mischococcales
2.5 Heterotrichales
2.6 Botrydiales
3 Bacillariophyceae- To silic
3.1 Coscinodiscales
3.2 Naviculales
4 Phaeophyceae- To nu
4.1 Isogeneratae
4.2 Heterogeneratae
4.3 Cyclosporae
5 Raphidophyceae
Haptophyta- Ngnh To lng roi bm
Eustigmatophyta- Ngnh To ht
Cryptophyta- Ngnh To hai lng roi
Dinophyta- Ngnh To hai rnh
Euglenophyta- To mt
Chlorophyta- Ngnh To lc
1 Prasinophyceae
2 Chlorophyceae- Lp to lc
2.1 Volvocales
2.2 Tetrasporales
2.3 Chlorococcales
2.4 Ulotrichales
2.5 Bryopsidales
2.6 Siphonocladales
3 Conjugatophyceae- Lp To tip hp
3.1 Zygnematales
3.2 Mesotaeniales
3.3 Desmidiales
4 Charophyceae- Lp To vng

Vi to ch yu thuc v cc chi trong cc ngnh sau y:
1-Ngnh To lc (Chlorophyta):
Cc chi Closterium, Coelastrum, Dyctyosphaerium, Scenedesmus,
Pediastrum, Staurastrum, Dunaliella, Chlamydomonas, Haematococcus,
Tetraselmis, Chlorella,...
2- Ngnh To lng roi lch (Heterokontophyta)
Cc chi Melosira, Asterionella, Cymatopleurra, Somphonema, Fragilaria,
Stephanodiscus, Navicula, Malomonas, Dinobryon, Peridinium, Isochrysis,
Chaetoceros, Phaeodactylum, Skeletonema, Nitzschia......
3- Ngnh To mt (Euglenophyta):
Cc chi Phacus, Trachelomonas, Ceratium...
4- Ngnh To (Rhodophyta):
Cc chi Porphyridium, Rhodella...
Vai tr ca vi to trong t nhin v trong i sng nhn
loi:
To ni chung v vi to ni ring c vai tr rt quang trng trong t
nhin v trong i sng nhn loi. Chng ta bit rng i dng chim 71%
din tch b mt Tri t. Mt s tc gi Hoa K cho rng hng nmg to c
th tng hp ra trong i dng 70-280 t tn cht hu c. Trong cc thy vc
nc ngt to cung cp xy v hu ht thc n s cp cho c v cc ng vt
thy sinh khc. To gp phn bo v mi trng nui thy sn bng cch tiu
th bt lng mui khong d tha. Canh tc bin l nhm trng v thu hoch
cc to sinh khi ln v c hm lng dinh dng cao. Nhiu to bin cn khai
thc sn xut thch (agar), alginate, sn phm giu iod... Nhiu to n
bo c nui trng cng nghip to ra nhng ngun thc n cho ngnh
nui tm hay thuc b tr giu protein , vitamin v vi khong dng cho ngi.
Mt s vi to c dng sn xut carotenoid, astaxanthin, cc acid bo
khng bo ha... To silic to ra cc m diatomid, l loi nguyn liu xp,
nh, mn c dng trong nhiu ngnh cng nghip.
To phn b ht sc rng ri khp mi ni, t nh ni cao n y bin
su. Nhng to sng lp nc pha trn c gi l To ph du
(Phytoplankton) cn nhng to sng bm di y thy vc, bm trn cc vt
sng hay thnh tu thuyn c gi l To y (Phytobentos).
Dng to cng sinh vi nm thnh a y cng l dng phn b rt rng
ri v nhiu loi c khai thc dng lm dc phm, nc hoa, phm
nhum v cc mc ch kinh t khc (hin bit ti 20 000 loi a y thuc
400 chi khc nhau).

Hnh thi v cu to t bo ca To:
To c hnh thi c th rt a dng. C th chia thnh 8 kiu hnh thi
nh sau:
1) Kiu Monad: To n bo, sng n c hay thnh tp on, chuyn
ng nh lng roi
2) Kiu Pamella: To n bo, khng c lng roi, cng sng chung trong
bc cht keo thnh tp on dng khi c hnh dng nht nh hoc khng.
Cc t bo trong tp on khng c lin h ph thuc nhau
3) Kiu Ht: To n bo , khng c lng roi, sng n c.
4) Kiu Tp on: Cc t bo sng thnh tp on v gia cc t bo c
lin h vi nhau nh tip xc trc tip hay thng qua cc si sinh cht
5) Kiu Si: Cu to thnh tn (thallus) a bo do t bo ch phn i
theo cng mt mt phng ngang, si c phn nhnh hoc khng.
6) Kiu Bn: Tn a bo hnh l do t bo sinh trng nh hay gc
phn i theo cc mtphng c ngang ln dc. Bn cu to bi mt hay nhiu
lp t bo.
7) Kiu ng: Tn l mt ng cha nhiu nhn, c dng siphn nhnh
hay dng cy c thn , l v r gi (rhizoid). cc t bo thng vi nhau v tuy
phn chia nhng khng hnh thnh vch ngn
8) Kiu Cy: Tn dng si hay dng bn phn nhnh, hoc c dng thn-
l- r gi. Thng mang c quan sinh sn c mc phn ha cao.

T bo ca to c nhiu c im chung ca cc sinh vt c nhn tht
(Eukarya).
Thnh t bo ca to cu to bi polysaccharide. Thnh t bo gm cc
si cellulose lin kt thnh b xng (skeleton) nhm bo v v duy tr hnh
dng n nh cho t bo. Mt s to c mannan hay xylan thay th cho
cellulose. Ngoi ra cn c phn v nh hnh to nn cht nn ca thnh t
bo. Bn ngoi thnh t bo mt s to c mng keo cha cc
polysaccharide c gi tr thc tin nh alginate, fucoidine, agar, carragenan,
porphyrane, furcelleran, funoran... Nhiu to n bo thnh t bo ch l cht
nguyn sinh m c hay chu cht (periplast). Thnh t bo ca to silic cu
to bi cht silic. Mt s to c lp mui oxyd st calcium carbonat bn ngoi
thnh t bo.
T bo ca nhiu to vn ng c l nh Lng roi (flagella). Roi cu
to bi 9 cp vi ng bao quanh 2 vi ng gia v c bao bc bi mng sinh
cht. Hai vi ng gia xut pht t a gc (dense plates) v th gc (basal
body).
Mng sinh cht cng ging nh cc sinh vt khc. Trong t bo cht c
nhiu bo quan khc nhau. Sc lp (chromoplast) ca to c cu to nh
thc vt, gm hai lp mng bao bc, bn trong c cht nn (stroma) cng vi
h thng cc ti dt gi l thylakoid. Cc thylakoid xp chng ln nhau to
thnh loi cu trc ging nh grana thc vt. Trn mng ca thylakoid c
nhiu cht dip lc (chlorophyll) v cc enzim tham gia vo qu trnh quang
hp. Ngoi cht dip lc (a,b,c,d) cn c th c cc sc t carotenoid, ph bin
nht l b-caroten. Nhiu to cha sc t xanthophyll, phycobiliprotein...Trong
cht nn ca sc lp cn c ADN dng vng v ribsom. i khi cc lp c mt
vng m c protein lin kt vi cc sn phm d tr to thnh mt cu trc
gi l nhn tinh bt hay nhn protein (pyranoid). Sc lp cn c cha cc git
lipid nh nm gia cc thylakoid. Mt s to cn c thm mt hai lp mng
li ni cht lc lp (CER- chloroplast endoplasmic reticulum). Cn c cc v
sc lp gm leucoplast v amyloplast. Chng lm nhim v tch ly cht d
tr. Ty th ca to cng tng t nh ty th ca cc sinh vt khc. l bo
quan c hai lp mng bao bc, mng ngoi trn nhn cn mng trong n su
vo pha trong cht nn v to thnh nhng mo (crista) trn mang nhiu
loi enzim h hp. Cht nn ca ty th c cha ADN v ribosm. T bo ca
to cng c th Golgi (Golgi body) nh t bo nhiu sinh vt khc. l cc
ti dp xp hu nh song song vi nhau v c hnh vng cung, pha li gi l
mt trans cn pha lm gi l mt cis.Th Golgi to lm nhim v tng hp
v tit ra polysaccharide. T bo cht (cytoplasm) ca to c cha ribosom
80S v cc git lipid. Mt s to di ng c cc nhm ht lipid mu vng cam
cu to nn cc im mt (stigma). Cht d tr trong t bo thuc v nhiu
dng khc nhau: tinh bt to lc, floridean To , laminarian To nu,
leucosin To roi Prymnesiophyta, fructosan To lc Acetabularia... Ngoi ra
cn c cc cht d tr phn t thp nh ng, glycoside, polyol...To c
khng bo co rt (contractile vacuoles) gip cho vic duy tr nc trong t bo
v lai b cht thi ra khi t bo.
Nhn t bo to cng khng khc my so vi cc t bo nhn thc
khc nhng hu ht l nhn n bi. To silic v cc pha bo t th To nu,
To lc v mt s To c nhn lng bi. Nhn c mng kp bao bc,
trong nhn c ADN.

To n bo thuc To lc (Chlorophyta):
To lc n bo c cha chlorophyl a v b, xanthophyll. Hnh thi rt khc
nhau, c loi n bo, c loi thnh nhm (nh hnh hay phi nh hnh), c
loi dng si, c loi dng mng, c loi dng ng...Phn ln c mu lc nh
c. Sc lp (chromoplast) c th c hnh phin, hnh di, hnh li, hnh tr,
hnh cc, hnh sao...Thng c 2-6 thylakoid xp chng ln nhau. Phn ln c
1 hay nhiu pyrenoid nm trong sc lp. Nhim v ch yu ca pyranoid l
tng hp tinh bt. Trn sc lp ca lc to n bo hay t bo sinh sn di ng
thng c im mt (stigma hay redeyespot) mu . Phn ln t bo di ng
ca lc to c si lng roi (tin mao) di bng nhau v trn nhn (gi l
Isokontan). Mt s loi lng roi rp v c lng nh trn mt. C loi trn b
mt lng roi c 1 hay vi tng vy nh (scale). Lng roi ca t bo di ng
to lc thng c 2 si, mt s t c 4 si, 8 si hay nhiu hn. Cng c khi ch
c 1 si lng roi. Phn ln t bo to lc c 1 nhn. Mt s t c nhiu nhn
(coenocytic).Thnh t bo ca to lc ch yu cha cellulose, mt s t cha
xylan hoc mannan.

To lc c 3 loi phng thc sinh sn :
- Sinh sn sinh dng: phn ct t bo, phn ct tng on to
- Sinh sn v tnh: hnh thnh cc loi bo t v tnh, nh Bo t tnh
(Aplanospore), Bo t ng (Zoospore), Bo t t thn (Autosporre), Bo t
mng dy (Akinet)
- Sinh sn hu tnh: c ng giao (homogamy), D giao (heterogamy) v
non giao (ogamy).

Theo ti liu phn loi ca H.C.Bold , M.J. Wynne (Introduction to Algae,
Prentice Hall Inc., 1985) th ngnh To lc (Chlorophyta) ch gm 1 lp l
Chlorophyceae, trong c 16 b.
Tin ha ca cc b trong ngnh To lc theo Prescott (1968) c th
trnh by nh sau:


Vi to thng thuc v 2 b l Volvocales v Chlorococcales:
B Volvocales gm cc vi to c lng roi , n bo hay thnh nhm ,
c dng phn ct bc cu (desmoschisis)
B Chlorococcales gm cc vi to khng c tin mao, n bo hay
thnh nhm , c dng phn ct tch ri (eleutheroschisis)

Vi to trong b Volvocales l nhng n bo di ng hay nhng nhm di
ng a bo c hnh dng nht nh. Qun th t bo l bi s ca 2. T bo
dinh dng c lng roi, di ng t do. T bo hnh cu, hnh trng, hnh tim,
hnh bu dc, hnh vin tr, hnh thoi... cng c loi c hnh v quy tc. Mt s
loi khng c thnh t bo, ch l khi nguyn sinh cht trn. Phn ln c
thnh t bo vng chi- tng trong l cellulose, tng ngoi l pectin. Mt s
loi c bao keo lin kt cc t bo thnh qun th. T bo thng c 2 lng roi
di bng nhau, mt s t c 4 lng roi, mt s rt t c 1, 6 hay 8 lng roi. T
bo c 1 hay nhiu sc lp, thng c hnh chn, cng c th c hnh phin,
hnh a , hnh sao. Rt t loi v mu. Sc lp c 1 hay vi pyrenoid. Thng
c im mt mt pha phn trn ca t bo, mt s t c im mt gia
hay cui t bo. T bo dinh dng c nhn n bi.
Khi sinh sn v tnh mi t bo mt i lng roi, nguyn sinh cht trong
t bo bt u phn ct to ra 2,4,8,19 t bo. Trong iu kin mi trng bt
li lng roi mt i hay co li, nh ch di ng. t bo tit ra mt tng keo sau
phn ct lin tip to ra mt qun th keo, a bo, v nh hnh, l giai
on qun th keo (palmella stage). Khi mi trng thch hp tr li th mc ra
lng roi, chuyn sang giai on di ng. Cc loi nguyn thy th mi t bo
u c th sinh ra qun th con. cc loi phn ha thnh t bo dinh
dng v t bo sinh sn th ch c t bo sinh sn mi c th sinh ra qun th
con.
Khi sinh sn hu tnh c loi ng giao, d giao hay non giao. Sau khi
giao t kt hp s hnh thnh hp t. Hp t ny mm s sinh ra t bo con
hay qun th con.

Sinh sn hu tnh vi to Chlamydomonas
Trong b Volvales c c thy 6 h, u l vi to. ng ch l cc chi
Dunaliella, Tetraselmis, Haematococcus, Chlamydomonas...

To thuc b Chlorococcales l cc to lc n bo hay qun th khng
di ng. T bo c th c hnh cu, hnh thoi, hnh a gic. Sc lp ch c 1
hay nhiu, hnh chn, hnh phin,a hay hnh li. C 1, nhiu hay khng c
pyranoid., T bo 1 nhn , c lc c nhiu nhn. Cc chi c nhiu ng dng
thc tin l Chlorella,Scenedesmus,...

Di y l hnh v mt s i din vi to trong To lc
(theo http://www.thallobionta.szm.sk ):



1 Dunaliella salina, 2 Chlamydomonas debaryana, 3 Chloromonas tatrae,
4 Chlorogonium elongatum,5 Phacotus lenticularis, 6 Polytoma uvella,
7 Carteria turfosa, 8 Haematococcus pluvialis, 9 Coccomonas orbicularis


1 Gonium sociale, 2 Eudorina elegans, 3 Volvox aureus, 4 Pandorina morum
1 Schizochlamys gelatinosa, 2 Asterococcus superbus,
3 Tetraspora lemmermannii

1 Ankyra ancora, 2 Hydrodictyon reticulatum , 3 Trebouxia arboricola,
4 Characium acuminatum, 5 Chlorococcus multinucleatum,
6 Pediastrum boryanum

1 Monoraphidium griffithii, 2 Ankistrodesmus falcatus, 3 Kirchneriella lunaris, 4
Siderocelis ornata,
5 Lagerheimia wratislawiensis, 6 Oocystis solitaria

1 Crucigenia fenestrata, 2 Tetrastrum staurogeniaeforme, 3 Scenedesmus
quadricauda

1 Ulothrix zonata, 2 Raphidonema nivale, 3 Enteromorpha intestinalis, 4
Pleurococcus vulgaris, 5 Stichococcus bacillaris, 6 Ulva lactuca

1 Draparnaldia glomerata, 2 Fritschiella tuberosa, 3 Coleochaete divergens,
4 Trentepohlia

1 Oedogonium pringsheimii, 2 Bulbochaete elatior

Di y l nh chp mt s chi tiu biu trong ngnh To lc ( theo
http://botanika.biologija.org) :



Nhng nh chp chi tit hn c th thy trong cc hnh sau y :



Dysmorphococcus Platydorina



Gonium Volvox


Eremosphaera Oedogonium


Kircheneriella Tetraselmis Chlorella


Hydrodiction Pediastrum


Scenedesmus Dunaliella


Coelastrum Haematococcus



Botryococcus Oocystis




Staurastrum Ulothrix


Spirogyra Stigeoclonium


Closterium Codium Chlorococcum


Asterococcus Bryopsis


Pyrobotrys


Sinh sn hu tnh d phi Caulerpa

Cosmarium Micrasterias



Collodiction Pteromonas


Chlorogonium


Spondylomorum Polytoma

Pandorina



Eudorina Pleodorina Lobomonas




Carteria Sphaerellopsis




Chlorobrachys Spermatozopsis Platymonas




Dysmorphococcus Coccomonas



Phacotus Pedinomonas



Pyramidomonas Golenkinia Chodatella



Schroederia Tetraedron



Ankistrodesmus



Micractinium Characium


Selenastrum

Dictyosphaerium Actinastrumm Crucigenia


Westella
Coelastrum

Cosmarium
Elakatothrix

Sphaerocystis Acanthosphaera Polyedriopsis


Treubaria Echinosphaerella Trochiscia

Sorastrum

Errerella Tetrastrum Chaetophora



Sirogonium Triploceras Xanthidium


Onychonema Sphaerozosma Gloeocystis


Scotiella Tetradesmus Gloeotaenium
Ngnh To lng roi lch (Heterokontophyta
hay Chromophyta)
y l mt ngnh ln bao gm nhiu nhm trc y gi l ngnh nh
To vng nh, To vng lc, To slic, To nu. Cc lp trong ngnh l lp
Chrysophyceae, lp Xanthophyceae, lp Bacillariophyceae, lp Phaeophyceae
1) Lp To vng nh (Chrysophyceae):
Lp ny gm nhiu loi c hnh thi a dng (cc hnh amp, monad,
ht, tp on palmella, si, bn, cy...). Dng chuyn ng thng c 1 hay
2 lng roi ( khng u nhau). Sc t trong t bo l chlorophyl a v c,
carotenoid v xantophin. Mu to thay i t vng kim, vng xanh hay nu
xanh. Sn phm to thnh khng phi l tinh bt m l leucosin . Mt s
khng c thnh t bo. Nhiu loi c thnh t bo v v gip. Thnh t bo v
v gip l cellulose v pectin, c th c thm hay khng thm silic. Phn ln
phn b ch yu cc thy vc nc ngt cha b nhim c mc dinh dng
trung bnh hay ngho, c kh hu lnh hay mt. Phn ln c i sng t dng,
ph du, mt s loi d dng. t gp cc loi sng trong t m hay y nc
. Sinh sn bng cch phn chia t bo, sinh sn v tnh bng ng bo t. Ch
rt t loi c sinh sn hu tnh ng giao. Hp t hnh thnh thng c dng
ti, thnh ti nhim silic vng chi nn c th gip chng vt c qua cc
iu kin bt li.
Nhiu loi to vng nh l thc n cho cc ng vt ph du. Khi nc
nhiu cht hu c hay giu m to vng nh c th gy ra hin tng nc
n hoa (algal bloom), gy mi tanh thi.
Di y l hnh nh cc chi To vng nh thng gp
(theo http://www.thallobionta.szm.sk ):

1 Ochromonas fragilis, 2 Monas elongata, 3 Uroglena americana, 4 Ochromonas
ludibunda, 5 Chrysococcus rufescens, 6 Stenokalyx monilifera, 7 Chromulina rosanoffii, 8
Synura uvella, 9 Mallomonas fastigata, 10 Dinobryon divergens, 11 Kephyrion ovum, 12
Pseudokephyrion pulcherrimum,
13 Rhipidodendron splendinum, 14 Anthophysa vegetans

1 Rhizochrysis Scherffelii, 2 Chrysidiastrum catenatum

1 Hydrurus foetidus, 2 Bitrichia danubiensis, 3 Bitrichia longispina,
4 Lagynion Scherffelii

1 Chrysosphaera paludosa, 2 Stichogloea olivacea

1 Phaeodermatium rivulare, 2 Phaeothamnion confervicola

Di y l nh chp mt s chi To vng nh thng gp:

Dinobryon Uroglena

Synura Uroglenopsis


Chrysostephanosphaera Chromulina

Ochromonas

Mallomonas Distephanus


Pleurasiga Pseudopedinella

Pedinella Proterospongia


Oikomonas Dendromonas Anthophysis


Chrysamoeba Chrysosphaerella Chrysidiastrum

Chrysocapsa Phaeoplaca


2) Lp To vng lc (Xanthophyceae)

To vng lc khc vi To lc ch khng c chlorophyll b v sn phm ng ha CO
2

khng phi l tinh bt m l leucosin v lipid. To vng lc khc vi To vng nh v To silic
ch khng c sc t Fucoxanthin v nhiu c im khc na. Hnh thi to vng lc rt a
dng: hnh monad, hnh amp, hnh ht... Sng n c hay thnh tp on. Mt s c dng
si n hay phn nhnh, dng ng thng sut cha nhiu nhn. Thnh t bo cu to bi
cellulose. Cc dng monad v ng bo t ca cc dng khc thng c 2 lng roi khng u
nhau, cng c khi c 1 hay nhiu lng roi (xp thnh tng i khng u, nh pha cc t
bo). Lng roi di thng c lng v di gp 4-6 ln lng roi ngn . Lng roi di hng v pha
trc cn lng roi ngn trn nhn hng xin so vi trc dc hay hng hn v pha sau.
Thnh t bo nguyn vn, tr chi Tribonema thnh t bo gm hai mnh. Sc lp c t 2 dn
6 trong mi t bo, c hnh khay. Thnh phn sc t gm c chlorophyll a, c, carotenoid,
xanthophyll. Tn thng c mu vng lc.
Sinh sn sinh dng theo cch phn i t bo hay t mt phn ca tp on. Sinh sn
v tnh bng ng bo t. ng bo t c hai lng roi lch nhau, c khi ch c 1 lng roi.
Thng ng bo t c sinh ra t nang ng bo t (zoosporangium). C loi sinh sn v
tnh bng bo t bt ng. C loi sinh sn v tnh bng t thn bo t (autospore) hay bng
bo t mng dy. Sinh sn hu tnh rt t khi gp To vng lc. Tribonema c hai loi giao
t- bt ng v di ng. Botrydium c giao t chuyn ng, ng giao hay d giao.

Sinh sn hu tnh (non giao) Vaucheria
Autheridium- Ti c; Oogonium- Ti ci; Eggs- Cc non cu
To vng lc thng gp trong cc thy vc nc ngt c dinh dng trung bnh hay
ngho. Chng c pi sng ph du hay sng bm. Mt s loi sng trn t hay trn thn cy
m t. To vng lc c cc chi ph bin sau y:


Vaucheria (non giao)

Tribonema Botrydium


Olisthodiscus Vacuolaria

Characiopsis Ophiocytium

Goniochloris Botrydiopsis

Chlorothecium Gloeobotrys

Gonyostomum
Merotrichia

3) Lp To silic (Bacillariophyceae)

To silic c cu to n bo sng n c hay thnh tp on dng palmella, dng
si, dng chui, dng zic-zc, dng di, dng sao, dng ng, dng cy... Kch thc thay i t
vi mm n 1 mm. T bo c nhn lng bi. c im ca lp to ny l c thnh t bo
gm hai mnh v. Lp trong l pectin, lp ngoi l oxyd silic . Hai mnh v (np y v y)
nh hai ci np ca mt ci hp nh lp kht vo nhau, bn trong cha t bo cht. Nhiu to
silic c cu trc hoa vn trn mt v. Hoa vn cu to bi cc l nh hay cc rnh nh. C khi
c cc khe h. Mt s c kh nng di ng nh ni cht chuyn ng trong cc khe trn thnh
t bo C th phn bit hai loi hnh thi c bn:
- Hnh phng x i xng: a s c hnh trn, mt s c hnh tam gic, hnh a gic,
hnh bu dc , hnh trng...
- Hnh di, hai bn i xng nhau: c hnh si, hnh kim, hnh bu dc, hnh trng, hnh
trng non, hnh cung, hnh ch S, hnh tr, hnh c u, hnh thuyn, hnh v cm...
T bo cht trong sut ,to thnh lp mng nm bn di thnh t bo hay to thnh
khi nh trung tm vi nhiu si sinh cht ni vi thnh t bo..
To si lic c mu vng lc hay vng nu. Loi to silic trung tm c sc lp hnh ht,
hnh a nh, gm nhiu a. Loi to silic lng chim c sc lp ln hnh phin ch H hay hnh
sao, c 1-2 ci. Mt s t c nhiu a nh. Khng c ht pyrenoid, mt s t c ht penoid
trn khng c bao tinh bt. Sn phm ng ha t CO
2
l lipid v chrysolaminaran, thng t
li thnh cc git cht d tr mu da cam. Ngoi ra cn c cc git volutin mu xanh da tri.
Trong t bo to silic cn thy c ty th, b my Golgi, cc tm thylakoid quang hp, lc lp
(chloroplast)...

Cu trc t bo To silic (theo http://www.thallobionta.szm.sk )

To silic sinh sn bng cc hnh thc sau y:
- Phn ct t bo: y l phng php ph bin nht. Khi ni cht phn i, hai
mnh v tch ta km theo mt na ni cht, sau t tng hp nn v th hai. Cc t bo
cc th h sau nh hi hai th h u.
- Sinh sn bng bo t t thn: Khi cc th h sau c mnh v qu nh chng s hnh
thnh nn v tm thi (perizonium). trong lp v t bo ln ln v to thnh bo t t thn
(autospore). Bo t t n kch thc chun s tng hp nn hai np v mi.
- Gp iu kin bt li to silic hnh thnh bo t ngh bng cch cht t bo mt nc,
co li v to ra lp v tm thi kh dy, nhiu khi c gai nhng vn nm trong np c.. Khi
gp iu kin thn li tr li th np v ngoi tan i v ny mm thnh t bo sinh trng bnh
thng.
- Rt t gp sinh sn hu tnh to silic. Mt s loi sinh sng nc ngt c th tin
n gn nhau, ni cht thot ra khi np v v to nn bao nhy, sau phn chia gim
nhim to ra 4 nhn con. Hai nhn con v sau thoi ha i, hai nhn cn li s bin thnh 2
giao t. Chng kt hp vi 2 giao t ca 2 t bo bn cnh v to thnh 2 hp t. Hp t s
pht trin 2 np v mi ca t bo. Mt s to si lic trung tm sng nc mn hnh thnh
nn 2 hay 4 giao t (ty loi) khng c lng roi nm trong t bo m hay c tung ra ngoi.
C loi li sinh ra 16 hay 64 giao t nh chuyn ng nh 1 lng roi. Lng roi cu to bi 9 i
si ngoi bin nhng khng c i trung tm. Khi hai giao t kt hp vi nhau s to thnh
hp t. Hp t v sau s to ra hai np v pht trin tip.
To silic c s loi nhiu th hai sau To lc. Chng phn b ht sc rng ri trn Tri
t: trn thn cy nh ni cao, trn t, m, mi thy vc nc ngt, nc l, nc
mn. C th gp to silic c y bin su ti hng nghn mt. Trong nc thnh phn to
silic l phong ph nht su 5-30m, nhng sinh khi li thng t mc cao nht su
20-50m. Khi sng bm to silic to nn mt lp trn mu nu. Trong cc thy vc nc ngt
to silic lng chim chim u th v thnh phn loi . Trong cc thy vc nc ngt t l to
silic lng chim gim so vi to silic trung tm khi nng mui tng. Lc nng mui tng
co th hu nh to silic trung tm hon ton chim u th.
Trong cc thy vc nc ngt to silic l thnh phn chnh ca nng sut s cp. Trong
cc bin v i dng to silic chim u th c v sinh khi ln thnh phn loi. Hng nm
ythc vt ph du (m ch yu l to silic to ra ti 19 t tn cht hu c, nui sng c ti 5
t tn ng vt khng xng sng (!) Nhiu tnh ton cho kt qu cn cao hn na. To silic
ng vai tr ch o trong vic to nn cc nng sut s cp trong h sinh thi bin, nht l
nhng vng khng c tip nhn ngun thc n hu c mang ti t cc dng lc a.
Qua nhiu th k x ca to silic to nn cc cc m diatomid ln do cu trc silic ca
np v xc to silic khng b phn hy. Diatomid c tnh cht nh, xp, cch in, tr vi
acid... cho nn c ng dng rng ri ch to cc sn phm cch in, cch nhit, cht
m trong thuc nhum, trong x phng, sn xut thy tinh lng, lm nn khi in di chit
tch dc phm...Cc tng diatomid cn l c s xc nh tui ca cc a tng v lch s
ca v Tri t t cui k Jura cho n nay. To silic cng gp phn to nn hin tng nc
n hoa lm h hng ngun nc sch
To silic trc y c coi l mt ngnh ring ( ngnh Bacillariophyta) v bao gm hai
lp l lp To silic trung tm (Centriophyceae) v lp To silic lng chim (Pennatophyceae).
Nay to si lic ch c coi l mt lp (Bacillariophyceae) trong ngnh Heterokontophyta v
c phn thnh hai b l b Coscinodiscales v b Naviculales.
Cc chi thng gp trong lp to silic l cc chi c hnh dng nh sau (theo
http://www.thallobionta.szm.sk ):

1 Stephanodiscus astrea, 2 Triceratium distinctum, 3 Chaetoceros muelleri, 4
Biddulphia aurita, 5 Attheya zachariasii, 6 Melosira distans

1 Tabellaria fenestrata, 2 Meridion circulare, 3 Diatoma vulgare,
4 Achnanthes lanceolata, 5 Surirella elegans, 6 Asterionella formosa,
7 Eunotia lunaris, 8 Pleurosigma elongatum, 9 Nitzschia linearis,
10 Synedra acus, 11 Cymbella lanceolata, 12 Navicula radiosa,
13 Pinnularia viridis, 14 Fragilaria virescens

Di y l nh chp mt s chi to silic thng gp :

Achnanthes Asterionella Attheya

Biddulphia Chaetoceros

Cocconeis Coscinodiscus

Cyclotella Cymbella

Epithemia Eunotia

Diatoma Fragilaria Frustulia









Gomphoneis



Gomphonema Navicula Hemidiscus


Gyrosigma Hydrosera

Melosira Nitzschia Planktoniella

Pinnularia Pinnularia

Pleurosigma Rhizosolenia

Skeletonema

Stauroneis Stephanopyxis Surirella

Synedra Thalassiosira

Tabellaria


Triceratium


Ngnh To mt (Euglenophyta)
To mt sng ring r, t bo kiu monad c 1 hay 2 lng roi. Thnh t bo ch l cht
nguyn sinh m c li do hnh dng c th thay i.Mt s chi c thnh t bo l th chu
cht (periplasst) th c hnh thi t bo n nh. Mt s chi c thnh t bo l v CaCO
3
thm

mui st 3 nn c mu nu. V cancium lm cho nhng t bo ny c hnh damngj n
nh. Lng roi nm u trc, xut pht t im gc (nm trong nguyn sinh cht hay
trong khng bo) v i qua mt phn lm di gi l hng. Hng thng vi mt khng bo d
tr ln, xung quanh thng vi mt s khng bo co bp (contractile vacuole). Khng bo co
bp lm nhim v thi nc v cc cht bi tit, iu ha p sut thm thu. im mt
(stigma, eye spot) mu lm nhim v cm quang. Nhn nm phn sau ca t bo. Lc
lp (chloroplast) hnh khay hay hnh phin nm ri rc hay tp trung, c khi xp thnh hnh
sao. Sc t c chlorophyll a v b, cn c c carotenoid. Sn phm ng ha CO
2
l paramylon
v lipid. Thng thy c c ty th v cc ht pyranoid. To mt thng sinh sn bng phng
php phn i hay bng cch to ti c thnh dy hay bao dy. Cha pht hin thy sinh sn
hu tnh to mt.






To mt ch
yu phn b cc
thy vc nc ngt,
chng a mi trng
giu dinh dng,
giu cht hu c.
Mt t loi sng c
mi trng nc l
c nng mui
di 0,5%. Phn ln
to mt c i sng
t dng nhng
cng c loi d
dng (khng c sc
t quang hp) Cc
vng mu xanh,
vng, , nu trong
cc ao t thng l
vng to mt.
Di y l hnh dng mt s chi to mt thng gp ( theo
http://www.thallobionta.szm.sk) :












1 Euglena viridis, 2 Euglena gracilis, 3 Euglena polymorpha, 4 Menoidium tortuosum, 5
Phacus tortus, 6 Colacium cyclopicola,
7 Trachelomonas volvocina, 8 Urceolus cyclostomus



Euglena

Phacus


Menoidium

Colacium


Eutreptia

Eutreptiella


Trachelomonas

Urceolus

Astasia


Lepocinclis

Peranema

Hyalophacus




Rhabdomonas

Peranema


Ceratium

Petalomonas

Anisonema





Vi to thuc ngnh To (Rhodophyta)



To ch c vi chi l vi to. Trong chu k sng to khng c giai on di ng. c
im ca to l s a dng ca sc t quang hp : chlorophyll a v d, carotin a v b, xanthophyll,
lutein, phycocyanin, phycoerythrin. Mu ca tn quyt nh bi phycocyanin v phycoerythrin
(thuc nhm biliprotein). Thng tn c mu t hng, n tm thm hay xanh lam. Hai chi vi to
thng gp l Porphyridium v Rhodella :

Porphyridium Rhodella
Ti liu tham kho
1- Nguyn Ngha Thn, ng Th Sy, H thng hc thc vt, Gio trnh Khoa Sinh hc , H
Khoa hc t nhin , HQG H Ni, 1998
2-Trn Phong ch bin, Vi to sinh vt k thut, Trung Quc khinh cng nghip xut bn
x,1999.
3- Chu Phng H, Trn Kim Hng, m thy vi hnh sinh vt ph, Ha hc cng
nghip xut bn x, 2005.
4-Borowitzka M.A. et al., Micro-algal Biotechnology, Cambridge Univ.Press, 1988
5- Bold H.C. , M.J. Wynne, Introduction to Algae, Prentice Hall Inc., 1985.
6-Penter Panoik, www.thallobionta.szm.sk
7-Amos Richmond (Ed.) Handbook of Microalgal Culture, Cambridge Univ. Press, 2004

Bi 12 Phn loi vi sinh bng Sinh Hc Phn T
1. Khi qut v cc phng php phn loi vi sinh vt
truyn thng:
Trc y cng tc phn loi vi sinh vt vn da cn bn trn cc c tnh
hnh thi, sinh l v ha vi sinh vt: nhum, hnh dng t bo khun lc, kh
nng di ng, nhu cu dinh dng, kh nng sinh acid trong mi trng cng
nh sc t to thnh v.v..Cc c trng ny i khi cng bc l nhng hn ch
do cc c tnh c dng cho nhm vi sinh vt ny (Enterobacteriaceae)
nhng li khng c ngha i vi nhm khc (vi khun gram m - gram
negative bacteria). Hn ch ca cc phng php phn loi truyn thng dn
n nhiu trng hp phi xc nh li tn phn loi ca mt s vi sinh vt. T
trc n nay, n v c bn ca nh tn vi sinh vt l loi, bao gm nhm
cc c th c mc tng ng cao v cc c im hnh thi.
Cc phng php da trn cc phn ng sinh ha:
T nhng hn ch ca vic xc nh cc c tnh hnh thi dn n nhiu
nghin cu tp trung vo cc phn ng sinh ha c trng cho cc vi sinh vt
ring bit. S khc bit ca cc phn ng c ngha cho phn loi cc vi sinh
vt.
- API20E KIT,
nguyn tc: da vo 20 phn ng khc nhau. Ni chung hin nay c nhiu
ni vn dng k thut ny nhng nhn chung kt qu cng cn nhiu
sai s do nhiu nguyn nhn khc nhau: c th trong cc trng hp
gene quyt nh phn ng sinh ha nm trong plasmid li b mt do
nhiu nguyn nhn khc nhau nh tui t bo, lng ging cy hay
thay i trong qu trnh nui cy dn n sai khc v lm sai kt qu.
- Phn bit bng thc khun th:
Cc vi khun c mn cm vi thc khun th khc nhau. C thc khun
th xm nhim lm tan t bo ngay lp tc sau thc khun th
nhn ln thnh cc ht trong t bo ch, trong khi vi mt s vi
khun th thc khun th xm nhim nhng li khng lm tan t bo vi
khun v chng cng tn ti vi t bo vt ch. Da vo s khc bit
ny m ngi ta dng cc thc khun th khc nhau phn bit cc
i tng vi khun nghin cu.
Tuy nhin, phng php ny cng bc l mt s nhc im kh gii
quyt l cc c tnh mn cm ca vi khun vi thc khun th li thay
i do iu kin ngoi cnh hoc l vi khun li c mc mn cm
khc nhau i vi cc thc khun th khc nhau. Mt khc na, thc
khun th rt d thay i cc c tnh do cng lm thay i c ch
xm nhim vo vi khun ch.
- Phn bit theo Typ huyt thanh:
y l phng php c dng kh lu nhng rt hiu qu v hin vn
ang c s dng (v d nhm vi khun Bt). Nguyn tc l da vo
nhm quyt nh khng nguyn trn t bo vi sinh vt (b mt t bo
tin mao hoc protein v). u th ca phng php ny l cc khng
huyt thanh c dng bit ha nhiu chi khc nhau, trong nhiu
trng hp c trng cho loi. Ni chung y l phng php kh n
nh nhng hn ch ch yu ca phng php ny ch: yu cu k
thut sn xut khng huyt thanh v tiu chun ha phn ng khng
huyt thanh khng ng nht ti cc phng th nghim v tnh n nh
gia cc ln lp li.

- Phn bit bng loi hot cht khng khun
(Bacteriocin):
Bacteriocin bn cht l peptid khng khun sinh ra bi vi khun chng
li vi khun khc. Nh vy, loi vi khun to ra loi bacteriocin no th c
kh nng khng li chnh bacteriocin . Bi vy c nhiu loi vi khun
c phn loi da vo kiu bacteriocin.
Kt lun: C nhiu phng php truyn thng c s dng trong nhiu
nghin cu phn loi nhng khng c phng php no t ra vn nng thch
hp cho mi i tng vi sinh vt, tnh chnh xc ch c th t c khi kt
hp nhiu phng php khc nhau.

2. Cch tip cn phn loi hc vi k thut sinh hc
phn t:
Ngy nay nhng tin b trong sinh hc phn t m ra kh nng ng
dng hu hiu trong phn loi hc v nghin cu a dng vi sinh vt. Nu nh
cc phng php truyn thng ch tp trung trn mt s i tng vi sinh vt
th phng php sinh hc phn t c th p dng trn mi i tng vi sinh
vt.
Ni chung cc phng php sinh hc phn t tp trung vo cc k thut
ch yu l:
+ Phn tch acid nucleic.
+ Phn tch protein.
+ Phn tch lipopolysaccharid.
+ Ha phn loi hc.
Trong phn ny chng ti tp trung gii thiu mt s k thut phn loi
lin quan n acid nucleic. Cc phn sau chng ti ln lt gii thiu cc
phng php lin quan n polysaccharid, lipoprotein v ha phn loi.
2.1. Phn tch acid nucleic:
K thut phn tch acid nucleic lin quan ch yu n phn tch kch
thc, cu trc acid nucleic, mi tng quan v trnh t acid nucleic thng qua
gii trnh t ADN v lai ADN.
a. Phn tch ADN plasmid:
Phng php phn tch y bao gm tch plasmid, so snh cc loi
plasmid v kch thc sau tinh sch plasmid v x l bng enzym ct hn
ch, sau in di trn gel agarose v so snh s a hnh ca cc mnh ct
phn bit cc chng vi sinh vt vi nhau.

Plasmid vng

Streptomyces v Borrelia
Plasmid l nhn t di truyn ngoi nhn v sao chp c lp vi nhim
sc th. Cu trc plasmid l si i ADN khp kn theo vng. Tuy nhin, c
nhiu trng hp ngoi l l si kp ADN mch thng (i vi vi khun Borrelia
v Streptomyces). Plasmid c tm thy hu ht cc vi khun v mt s t
cc vi sinh vt nhn thc bc thp nh nm men.
+ Tch plasmid:
Thng thng lng plasmid c trong t bo chim < 5% tng s acid nucleic
ca t bo. Nh vy, ta phi thc hin php tch plasmid ra khi ADN ca
nhim sc th. Ni chung c nhiu phng php tch plasmid t vi khun
nhng nguyn tc chung l ph t bo vi khun dng enzym, siu m hay
trong dung dch kim c cht ty ra l SDS hoc TritonX-100, sau l vic
loi ADN ca nhim sc th v protein. Thng thng dng phng php kt
ta vi axetat. Lc ny phn ln cc thnh phn ADN nhim sc th v protein
ca t bo b kt ta b loi bng li tm v plasmid nm trong dch trn ta
c tch khi kt ta vi ethanol hay isopropanol. y l phng php c hiu
qu tch plasmid, trn thc t hiu qu tch cc plasmid c kch thc nh
cao hn nhiu so vi cc plasmid c kch thc ln (>100 Kb). Vi cc plasmid
c kch thc ln cn cc thao tc nh nhng trnh t gy do cc nguyn
nhn c hc. Vi cch tch plasmid nh trn th sn phm thu c c ln cc
on ADN c kch thc khong 500 bp v nhim ARN. trnh nhim ARN th
cn x l vi RNase (ribonuclease A). Tuy nhin, c th tch theo cc phng
php nh:
- Tch bng Caeseium chloride.
- Tch bng KIT QIAGENE.
- Tch bng k thut khc.

+ in di plasmid trn gel agarose:

Sau khi thu c plasmid th phi tin hnh kim tra trc khi phn tch
kt qu in di trn gel agarose bit ph plasmid v kch thc ca chng.
Khi tin hnh in di th nng gel khong 0.7- 1% vi mt trong cc m
sau: TAE (40 mM Tris acetat 1mM EDTA, pH 8.0), TBE (89 mM Tris borat 89
mM boric acid, 2 mM EDTA, pH 8.0) v TPE (80 mM Tris- phosphats 2 mM
EDTA, pH 8.0). Sau khi in di, gel c nhum vi ethidium bromide v pht
hin di tia UV (310 nm). Nng ethidium bromide khong 5 mg/l v thi
gian nhum l 10 pht. Sau c ra mt ln nhanh bng nc loi ion
trc khi c nhn di UV v chp nh lm ti liu.
Mt s vn cn lu khi phn tch plasmid: trn gel thng thng
plasmid c thy 3 dng: dng siu xon, dng t gy v dng mch
thng khi b ct bi endonuclease. Dng di chuyn tc cao hn l siu xon
(Hnh 1.1).

Hnh 1.1. Di chuyn ca plasmid mch thng (L) v siu xon (OC) khi in di
i khi vic tch plasmid cng phc tp c bit trong nhng trng hp
plasmid c kch thc nh c ln cc mnh ADN ca nhim sc th. Trong mi
trng hp u c nhim cc RNA v chng di ng nhanh nht tr cc trng
hp plasmid nh vy khng c nhm ln gia plasmid siu xon v dng
chnh plasmid t gy v plasmid khc. Mt ch khc l trnh nhm ln khi
so snh kch thc gia cc plasmid siu xon v dng dui xon hay mch
thng. Thc t l ch c th so snh cc plasmid siu son vi nhau v kch
thc.
Khi tin hnh phn bit gia cc chng vi khun th ng nhin l ch
c th thc hin so snh gia cc chng cng c plasmid vi nhau. Cng nn
ch l khng phi cc chng u ging nhau v c tnh min dch nu c
chung kt qu phn tch plasmid nh nhau. Php phn tch s c hiu qu
trong cc mu c nhiu loi plasmid, tuy nhin cng phi tnh n vic cc
plasmid cng c th b mt trong qu trnh bo qun.
+ K thut du vn tay (finger printing) phn tch
plasmid vi enzym ct hn ch:
Cho n nay, ngi ta tm thy hn 400 enzym ct hn ch. Mc ch
ca k thut ny b sung cho k thut so snh ph kch thc plasmid trn.
Tc l ngi ta c th phn bit c s khc nhau ca cc plasmid c cng
kch thc. Nh vy, khi x l plasmid vi mt hay kt hp mt s enzym ct
hn ch th kt qu s thu c l cc on ADN c ct c kch thc khc
nhau. Kt qu ny c lp li cao, do d s dng khi so snh cc mu
khc nhau.
Hin nay, c nhiu enzym ct hn ch c sn xut v tiu th trn th
trng, cc enzym ny c im nhn bit thay i t 4-6 cp nucleotid. Thng
thng x l enzym trong 1 gi sau mu c pht hin trn gel agarose
hoc polyacrylamid Tu thuc vo kch thc ca cc mnh ct m s dng
agarose c nng khc nhau:
Bng 1.1. Nng agarose v kch thc mu ADN tng ng.

Nng agarose (%) Kch thc ADN (kb)
0.3 1-7
0.5 0.7-45
0.8 0.4-20
1.0 0.3-10
1.2 0.2-8
1.5 0.2-6
2.0 0.1-5
Theo kinh nghim ca nhiu tc gi (Grimont-1991) ph cc bng ADN
c ngha cho so snh khng nn di 10 bng v nn c c bng kch thc
nh v kch thc ln. Tuy nhin, cc bng ln khng nn vt qu 10 do bng
c kch thc ln nh vy kh di chuyn trn gel. Trong cc trng hp so
snh th nn dng thang chun ADN so snh kch thc v php phn tch
du vn tay cho cc ln phn tch khc nhau. Nh trnh by trn, nu nh
phn tch cc chng vi sinh vt da vo kch thc plasmid c ngha i vi
cc i tng c nhiu plasmid th phng php x l enzym ct hn ch li c
ngha ln trong cc trng hp nghin cu dch t hc trn cc chng c
cng ph plasmid hay plasmid c kch thc ging nhau. Ngi ta ng dng
k thut ny trong nghin cu chng E.coli (0157: H7) gy bnh t thc phm
(Hamburger). Cc plasmid t cc chng khc nhau th c ph khc nhau v cc
c im ct bi enzym ct hn ch. Kt qu to ra cc mnh ADN c trng
cho c th lm c s cho php so snh. Tuy nhin kh c th so snh kt qu
thu c t cc phng th nghim khc nhau, s d nh vy l do khng dng
cng mt loi enzym ct hn ch. Mt l do na cng cn c cp n l
s xut hin cc t bin ngu nhin ti v tr enzym ct, kt qu ny to ra s
khc bit v ph thu c t cc mnh ct.
b. Phn tch ADN nhim sc th:
Phng php phn tch y bao gm vic tch ADN ca nhim sc th
v ct bng enzym ct hn ch phn bit gia cc vi sinh vt vi nhau. Mt
ch khi tch ADN nhim sc th phi thao tc nh nhng trnh t gy do
nguyn nhn c hc. Ni chung cc mnh ct nn c kch thc nh hn hoc
bng 50 kb. Vic tch cc mnh ct c thc hin da vo k thut in di
trong trng xung in (PFGE = Pulsed Field Gel Electrophoresis ).style="text-align:
justify; margin-top: 6.0pt"> Nh vy k thut du vn tay khng ch p
dng trong trng hp trn t bo mang plasmid m k thut ny cn c s
dng vi nhim sc th cho mi trng hp.rng hp.ng hp.ng hp.
Lin quan n vn ny phi k n k thut BRENDA: phn tch kt
qu x l enzym ct hn ch vi vi sinh vt. y cch chn loi enzym ct
hn ch v cng quan trng, theo cch chn ny c th to ra qu nhiu mnh
ct nn khng th phn bit c cc bng ring r trong khi i vi cc
trng hp khc li to ra qu t mnh ct c kch thc ln kh tch theo cc
k thut in di hin c. Cc vi sinh vt khc nhau c t l GC khc nhau (dao
ng t 25-75%) cc mnh ct thu c sau khi x l enzym ct hn ch rt
khc nhau. Theo Nei M. v Li W. H. (1979) th s mnh ct c th thu c
tnh trn l thuyt l:
a = (g/2)
r1
x {1-(g/2)}
r2

Trong : g - t l GC ca ADN
r1 - s cp GC
r2 - s cp AT trong v tr ct ca enzym gii hn s
dng
a - s v tr ct khi s dng enzym ct hn ch.
Mt khc, ngi ta cng cn c vo kt qu nghin cu cc v tr ct ca
b gene vi sinh vt lm c s cho cch chn enzym ct. Thng thng cho mi
php phn tch ngi ta ghi c s cc bng ct bi enzym v tnh ton kch
thc cc mnh to thnh lm c s cho cc php phn tch v sau. Tuy nhin,
mt trong nhng nguyn nhn hn ch cho php phn tch trn l cc phng
php tch ADN thng thng u dn n thay i kch thc do t gy ADN
nhim sc th, mt khc s c mt ca plasmid ln cng to ra s khc bit
gi trong kt qu phn tch, khc phc nhc im trn ngi ta phi thc
hin php phn tch ADN nhim sc th nguyn vn pht hin cc ngun
ADN tp nhim ln vo kt qu phn tch.
+ K thut in di trong trng xung in (in di
trong trng in thay i, PFGE)
- Nguyn tc: Trc tin cc mu a vo phn tch phi c x l
trc bng loi enzym ct hn ch m c rt t im ct. Kt qu phi to ra
c cc mnh c kch thc ln (50 kb 12 Mb), vi kch thc ny khng
th in di theo phng php thng thng m ch c th tch ra bng k
thut PFGE (t PFGE (t PFGE (ut PFGE (Pulsed-Field Gel Electrophoresis ). K
thut PFGE ln u tin c Schwartz va Cantor (1984) gii thiu. Tuy nhin
sau c nhiu tc gi m t li k thut ny, tuy c sai khc t nhiu
nhng c s l thuyt ca cc phng php ny l nh nhau: Mc ch ca k
thut ny l tng kh nng di ng ca cc mnh ADN c kch thc ln trong
in trng, do cc thnh phn gel v m hu nh khng thay i theo
cc phng php in di thng thng. Tuy nhin theo phng php thng
thng th s in di lin quan n s di ng ca cc mnh ADN c kch thc
khc nhau trn gel agarose trong mt trng in khng i. Kt qu y l
phn t ln kh di ng hn phn t nh qua mt xch agarose, to ra s tch
bit trong trng in di. Trong trng hp PFGE lc lm thay i kh nng di
ng li l trng in tch thay i lin tc dn n cc mnh ADN c kch
thc khc nhau thay i hng di ng. Nh vy kt qu l cc mnh c kch
thc khc nhau s c kh nng di ng khc nhau. Kch thc cng ln th
mc di ng cng chm. S di ng khc nhau ca ADN ch yu ph thuc
vo kch thc ch khng phi do gel agarose nh phng php in di
thng thng, hnh 1.2.

Hnh 1.2. S dng k thut PFGE phn tch nhim sc th
sau khi x l vi Sma I vi 7 chng Haemophilus nluenzae.
Tip theo cc m t ca Schwartz va Cantor, Smith v Codemine (1990)
cp n s di chuyn ca cc mnh ADN khc nhau l hm s tuyn tnh
vi kch thc ca chng. Trn c s c th iu chnh cc xung in v
in trng. Tuy nhin cc nhn t khc cng c mi tng tc ln nhau v
nh hng n kh nng di ng ca ADN nh: nhit , in th, nng
agarose cng nh nng ion trong m.
- Chun b ADN cho PFGE: Chun b ADN t nhim sc th khng ging
nh cc phng php chun b ADN plasmid. Mt vn thng xy ra l s
t gy ngu nhin ADN dn n sai khc trong kt qu phn tch. hn ch
iu ny ngi ta phi thc hin k thut tch ADN NST khi t bo mu
agarose c nhit nng chy thp LMT (Schwartz va Cantor 1984 v
Smith, Klco 1988). Theo phng php ny hn dch t bo (dch nui cy hoc
dch huyn ph) c trn vi LMT agarose ti 37
o
C. Hn dch u tin c x
l vi enzym v cht ty ra tch ADN NST khi thnh, mng t bo, RNA
v protein. Sau l x l vi EDTA, bt hot nuclease t bo vi proteinase K
v N-lauroylsarcosine loi cc thnh phn khc ca t bo. Kt qu l phn
LMT agarose c cha ADN NST c dng lm mu chy gel 0.5-1.0% (nn
lm tan 65
o
C) v a ln mu chy gel PFGE, ti liu ca Smith (1988) m
t chi tit cch chun b mu t cc t bo c v khng c thnh t bo: vi
khun, nm men, nm si, t bo thc vt v ng vt. Ni chung vi cc
trng hp c thnh t bo th cn n enzym ph thnh t bo. Lng ADN
NST thng dao ng trong khong 0.5-20 g ADN l thch hp. Nu qu
nhiu ADN th khng th phn tch c, cn qu t th cng khng th pht
hin c cc bng ADN trong kt qu phn tch. Mu ADN dng cho k thut
PFGE c th c gi 1 nm trong lnh c cha 0.5M EDTA.
- Sau khi thu c ADN NST trong mu LMT agarose th tin hnh x l
vi enzym gii hn loi c t im ct. Tuy nhin mu u tin phi c x l
vi PMSF bt hot protein K sau PMSF li phi loi b sau mt s ln ra
vi cht ty ra v EDTA. Sau ch cn phn nh mu trong agarose c x
l vi m v enzym ct hn ch qua m trong tube v sau ton b mu
ny c s dng tch trong trng xung in. Bng 1.2 di y lit k
cc enzym ct hn ch c dng cho phn tch PFGE.
Bng 1.2. Mt s enzym ct hn ch c dng cho k thut PFGE.

Enzym V tr ct
AatII
GACGT/C
ApaI
GGGCC/C
ClaI
AT/CGAT
MluI
A/CGCGT
NarI
GG/CGCC
NheI
G/CTAGC
NotI
GC/GGCCGC
NruI
TCG/CGA
PvuI
CGAT/CG
SacII
CCGC/GG
SalI
C/TCGAC
SfiI
GGCC(N)4/NGGCC
SmaI
CCC/GGG
XhoI
C/TCGAG

- ng dng ca k thut phn tch PFGE:

K thut PFGE c s dng cho nghin cu trn nhiu i tng khc
nhau (xem bng 1.3).
Bng 1.3. Minh ho cc chng vi sinh vt c phn tch da trn kt qu
PFGE thu c t cc on nhim sc th.
Vi sinh vt nghin cu Ti liu tham kho
1. Acinetobacter baumannii
2. Brucella spp
3. Campylobacter hyointestinalis
4. Campylobacter jejuni
5. CADNida arabicans
6. CADNida prapsilosis
7. Coxiella burnettii
8. Enterobacter cloacae
9. Enterococcus faecium
10. Escherichia coli
11. Listeria monocytogenees
12. Leptospira spp
13. Mycobacterium tuberculosis
14. Neisseria meningitidis
15. Psedomonas aeruginosa
16. Shigella spp
17. Staphylococcus aureus
18. Streptococci ssp
Gouby et al (1992)
Allardet, Servent et al (19980
Salama et al (1992)
Yan et al (1991)
Vasquez et al (1991)
Carruba et al (1991)
Heizen et al (1990)
Haertl ADN BADNlow (1993)
MirADNa et al (1991)
Bohm ADN Karch (1992)
Brosch et al (1991)
Herrmann et al (1992)
Zhang et al (1992)
Bygraves ADN Maiden (1992)
Boukadida et al (1987)
Sodati ADN Piffaretti (1991)
Prevost et al (1992)
Single ADN Martin (1992)

Mi mt i tng c c trng ring v kt qu phn tch PFGE v c
dng cho nghin cu so snh vi nhau v s tng ng. i vi nhiu i
tng c nhim sc th th khng nht thit phi thc hin i vi mi nhim
sc th m ch cn trn mt s nhim sc th m thi. V d trong trng hp
ca C.albicans Monod (1990) ch cn thc hin php phn tch vi mt trong 4
t hp ca mt s nhim sc th l . Cho d theo cc cch chun b nhim
sc th khc nhau th kt qu ca php phn tch PFGE u ging nhau. Php
phn tch PFGE c ng dng cho cc nghin cu mc loi vi loi.
Mt s im cn lu khi s dng k thut PFGE:
+ K thut ny i hi phi c trang thit b chuyn dng, k thut cng
nh kinh nghim v vic tch nhim sc th i khi gp kh khn trn mt s
i tng vi sinh vt.
+ K thut PFGE khng nhy pht hin nhng sai khc nh gia
cc mu, c bit khi thc hin vi mt enzym th kt qu c th ging nhau
nhng khng chc chn l hai mu ging nhau hon ton. iu c ngha l
k thut ny nn dng xc nh s khc nhau cn vic xc nh s ging
nhau th khng tin cy. Tuy nhin ngay c khi xc nh s khc nhau i khi
cc mu c plasmid nh hay thc khun th cng cn xt n v chnh ngun
ADN ny cng to ra nhng sai khc gi.
+ S khc bit gia cc mu c th pht hin c khi dng cc enzym
ct hn ch khc nhau.
Tm tt:
Phng php phn tch plasmid l phng php c dng ph bin
trong cc phng th nghim chun on vi sinh vt. Vic kt hp gia phn tch
plasmid vi s dng enzym ct hn ch s to c s phn tch chnh xc vi
cc thng tin ng tin cy i vi cc mu phn tch. Tuy nhin, khi phn tch
cc mu da trn plasmid th cn lu l cc plasmid c th b mt qua cc
th h phn chia, dn n sai lch cc kt qu nghin cu. Khi thc hin php
phn tch PFGE vi cc plasmid ln s khc phc c hn ch ca cc php
phn tch hin ti vi plasmid nh nh trn. Phng php PFGE hin ang c
dng rng ri ti cc phng th nghim vi sinh vt, p dng trn cc i tng
d nui cy c th cho cc kt qu tt hn nhng hn ch v gi thnh thit b
cng nh yu cu cao v k thut v kinh nghim. Mt khc khi s dng cc
enzym ct hn ch him c th s kh pht hin c mc sai khc. Nh
vy s kt hp gia cc php phn tch plasmid, PFGE v lai ADN s cho kt
qu tin cy hn.
2.2. Lai ADN
Nguyn tc c tin hnh nh sau: ADN tng s c tch ra v x l
vi enzym ct hn ch (c trng hp khng cn x l vi enzym ct hn ch)
sau in di trn gel agarose v chuyn ln mng lai. Mu d (probe) c
chun b v lai vi mng ADN trn. Kt qu php lai cho bit s khc bit
gia cc mu ADN khc nhau.
Php lai c tin hnh y da vo cu trc si i ADN t hai si
n vi cc cu lin kt hydrogene theo nguyn tc b sung. Bt u l t
gy cc lin kt hydrogene do ho cht (kim) hay bin tnh nhit, lc ny hai
si n ADN tch nhau c gi l trng thi bin tnh ADN (denature). Nu
ti thi im ny ngi ta cho vo mt ADN nh du bin tnh (mu d) sau
to iu kin hi bin xut hin (renature), lc s cho php cc si n
ca mu d bt cp vi cc si n ca mu ADN ban u (target ADN). Mc
lai s ph thuc vo tnh c hiu (s tng ng) gia mu d v mu gc
cng nh iu kin cho php lai thc hin (nhit , nng mui, pH, t ln
mu d, kch thc mu d). Nh vy, vic chn iu kin chnh xc cho php
lai c ngha rt quan trng cho tnh c hiu ca kt qu php lai. Sau khi
lai, bc tip theo l ra bng m thch hp loi mu d d v cui cng l
php o bc x nh du (tu theo phng php nh du phng x hay ho
cht pht x hunh quang) nh gi mc tng ng ca php lai.
Ni tm li mt s nhn t tham gia vo kt qu ca php lai l: mu d
ADN, phng php nh du ADN, mu ADN lai v iu kin tin hnh v
phng php nh gi kt qu php lai.
+ Chn mu d:
Vic chn mu d c ngha quan trng cho php lai. Nhn chung cc
nh phn loi hc vi sinh vt thng khng trc tip thit k mu d. V mt
ny chng ti a ra mt s ni dung ch yu v tiu ch chn mu d theo
Stahl v Amann (1991) nh sau: Mu d s lai vi ADN ch (target) v khng
lai vi cc ngun ADN khc c mt trong mu lai. Khi phn tch cc mu vi sinh
vt vi nhau th mu d nn l on nucleotid c trng cho cc mu phn tch
phn bit vi cc sinh vt khc. Tuy vic chn mu d cng mang tnh kinh
nghim, mu d i khi khng cn phi l ton b gene m ch l mt on
gene m ho cho mt c tnh c trng cho i tng nghin cu (c th l
yu t khng nguyn b mt, c t hay mt gene chc nng no trn
plasmid). Vi cch chn mu d c kch thc nh (14-40 bp), c thun li l
cc mu d c tng hp nhn to v php lai thc hin nhanh (di 30
pht) trong khi vi cc mu d ln thi gian ko di hn (khong 16 gi).
Hn ch ln nht i vi cc mu d nh l kh khn khi nh du v dn n
gim tnh c hiu ca php lai.
Mt cch thng c s dng l thit k cc mu d t cc chui ARN
thng tin 16S. Cch chn c th cn c vo on ADN c mt trong cc i
din mc di loi, trong loi hay thuc chi nghin cu.
+ Cc phng php nh du:
Cc k thut nh du ADN c xem l lnh vc pht trin mnh nht
trong sinh hc phn t. Ni chung k thut nh du c chia thnh k thut
nh du trc tip v gin tip. Trong phng php trc tip th phn nh
du pht hin c gn vo acid nucleic. i vi phng php gin tip th
c mt phn chc nng c gn vo acid nucleic v phn ny li c pht
hin gin tip da vo protein bm c hiu c pht hin bng k thut
min dch. Sau y l chi tit cc phng php nh du.
nh du trc tip:
- Bng di y a ra cc loi c cht dng cho phng php nh du
trc tip da vo vic nhn ADN c nh du ln s tng nhy so vi
phng php nh du ch c thc hin vi cc mi n l. i vi cc
phng php nh du trc tip th cc nhm to tn hiu c gn trc tip
bng lin kt ho tr vi nucleic ca mu d.
- nh du trc tip bao gm 2 bc chnh: to tn hiu da vo lin kt
ho tr ca nhm tn hiu vi mu d v thc hin php lai gia mu nghin
cu v mu d.
Bng 1.4. Mt s c cht dng cho nh du trc tip.

Ngun to tn hiu Ti liu
32
P
35
S
Alkaline phophatase
Ethidium
Fluorescein
Horseradish peroxidase
Microperoxidase
Nitrobenzofuran
Tetramethylorhodamine
Southern (1975)
Collins& Hunsaker (1985)
Renz&Kurz (1984)
Albarella & ADNerson(1986)
Amman & ctv (1988)
Urdea ctv(1988)
Heller& Shneider(1983)
Draper (1984)
Amman&ctv(1990)


Hnh 1.3. Minh ho phng php nh du trc tip (A) v gin tip (B)

nh du gin tip:

nh du gin tip c thc hin theo 3 bc: thc hin php lai gia
mu d v mu ADN ch, phn ng gia nhm chc nng v protein bm c
hiu vi nhm chc tn hiu thng bo (reporter) v cui cng l to ra tn hiu
t nhm chc tn hiu.

Bng 1.5. Lit k mt s h thng nh du gin tip.


Nhm chc nng bm protein
Nhm tn hiu
(reporter)
Ti liu dn
Biotin-dUTP/steptavidin
Digoxigenein-dUTP/antidigoxigenein
Sulphone/antisulphone
Dinitrophenyl/antinitrophenyl
Poly(dA)/poly(dT)
Acetyllaminofluorene/anti-
acetylaminofluorene

Alkaline phosphatase
Alkaline phosphatase
Europium chelate
Piruvate kinase
Horseradish peroxidase
Alkaline phosphatase

Leary et al (1982)
Kessler et al (1990)
Syvanen et al
(1986)
Leller et al (1990)
Morrisey & Collins
(1989)
Tchen et al (1984)

Mc d c nhiu h thng nh du v pht hin tn hiu nhng kinh
nghim cho thy h thng biotin v digoxidenin cho nhy cao hn c. Hai
h thng ny c th pht hin ti mc di picrogam acid nucleic. Trong
trng hp vi biotin th i khi c mc nhiu nn cao do biotin c mt
trong cc sinh phm, cn i vi digoxigenein th c th khng gp kh khn
ny.
+ nh du phng x v ghi phng x:
Phng php nh du phng x l phng php c dng ph bin
trong cc phng th nghim lai ADN vi cc thnh phn nh du l
32
P v
35
S.
Kt qu ca php lai gia mu d v ADN ch c pht hin trn X-phim
hoc nhp nhy lng. u im ni bt ca phng php nh du phng x l
nhy c th t ti di mc picrogam ADN ch. Hn ch ca phng php
ny l khng t c s thch hp cn thit cho cc th nghim chn on lin
quan ti xc nh mu vi sinh vt, v d thi gian bn hu mu d ngn, nguy
him cho ngi s dng v cn yu cu an ton nghim ngt cho phng th
nghim v c nhn s dng cng nh vic qun l cht thi.
Xut pht t thc t trn m cng ngy ngi ta cng quan tm n cc
phng php nh du phi phng x. Mc tiu l t c mt h thng nh
du c nhy tng ng vi phng php dng cht phng x. Bng di
y lit k cc yu cu l tng cho phng php nh du mu d:
1, D gn vi acid nucleic theo phng php n gin v c lp li
cao.
2, Bn trong iu kin lai v bo qun.
3, Khng b nh hng v lm nh hng n phn ng lai.
4, C th thc hin vi iu kin phn ng lai trong dch th (liquid
phase) hay c cht mang (solid phase).
5, Phng php pht hin nhy v n gin.
6, D b loi b sau phn ng lai.
7, D phn bit gia mu lai v mu cha lai trong cng iu kin.
8, C th ng dng vi h thng t ng ho.
Tuy nhin, nu theo cc yu cu l tng trn th cha c h thng nh
du phi phng x no tho mn.
Hin nay, c nhiu h thng nh du phi phng x t nhy cao v
l l do cc phng php ny ang c ng dng rng ri c bit l
phng php da trn cc enzym nh Alkaline phosphatase, Horseradisk
peroxidase. Tuy nhin phng php s dng cc cht phng x hin ti vn
ang c dng cho mt s phng th nghim c bit.
Chin lc nh du vi cht phi phng x.
C 3 phng php nh du phi phng x:
1, Dng c cht ho pht x v sinh pht x, trong trng hp ny th
c c cht ho v sinh pht quang u to ra cc bc x nh sng v sau l
phng php pht hin bc x ny.
* Vi ngun c cht ho pht quang c loi nh AMPPD (Bronstein,
Edward & Voyta, 1989) c th c dng trc tip nh l c cht cho enzym
Alkaline phosphatase trong php lai ADN t nhy cao trong thi gian ngn
(Bronstein et al., 1990).
* Phng php pht x s dng enzym Alkaline phosphatase trn c s
gii phng D-luciferin t c cht, v d D-luciferin-O-phosphate (Miska v
Geiger., 1987). Hai phng php ny tng i nhy v ang c s dng
rng ri.
2, Phng php pht hin da vo thay i mu:
Phng php o mu c th thc hin trn mi trng dch th hay cht
mang v kh nhy so vi phng php pht quang. Ngi ta to ra cc c
cht sinh mu khi c mt enzym (chng hn nh Alkaline phosphatase). Li
th ca phng php ny l vic pht hin n gin do kt qu l s thay i
mu d pht hin v ng dng trong cc phng th nghim vi sinh vt.
Mt phng php c th lm tng nhy ca phn ng mu bng cch
thm mt enzym kch hot phn ng mu vo h thng. Mt v d cho iu
ny l vai tr loi nhm phosphat ca phn t NADP thnh NAD do Alkaline
phosphatase trong h thng pht hin mu d nucleic. Trong h thng ny th
NAD c vai tr kch hot chu trnh oxy ho m c s tham gia ca alcohol
dehydrogenease v diaphorase. Trong mi mt chu trnh th NAD s b kh
thnh NADPH + H
+
. Phn ng oxy ho ny li km theo phn ng oxy ho m
NADPH + H
+
li b oxy ho thnh NAD, mi phn ng xy ra gn lin vi vic
kh -indonitro-tetrazolium mu tm thnh formazan. Sn phm cui cng
formazan c nh lng bng php so mu (Self. 1985).
3, C cht hunh quang v pht x hunh quang theo
thi gian.
S dng c cht pht x hunh quang l phng php kh nhy v c
th pht hin c ti mt phn t cht pht x (fluorescein). Ni chung vi
cc mu sinh phm thng c mc nhiu tn hiu nn cao. Thc t ny c
th c ci thin vi c cht fluorophore bn b kch hot bi sng nh sng.
S pht x sau c ghi li khi cc bc x nn b gim. i vi phng
php dng Alkaline phosphatase th vic pht hin nhn t gn lanthanide theo
thi gian i km vi hot tnh enzym ny gi l phng php pht quang
lanthanide khuych i bi enzym (EALL: Evangelista, Pollack & Templeton,
1991). Trong trng hp ny, c cht khng c kh hnh thnh phc h gn
vi lathanide pht x. Di tc dng ca Alkaline phosphatase th c cht v
lanthalide b chuyn thnh phc h hot ng. Phng php ny kh nhy
nhng hn ch ln nht ca n l cn thit b kch hot v o bc x hunh
quang.
+ Qu trnh lai:
Ni chung qu trnh lai (Hybridization ) c tin hnh theo mt trong 3
cch sau nh danh hay xc nh vi sinh vt: Lai vi vi sinh vt (in situ), lai
trong dch th v lai vi cht mang (solid support).
a. K thut lai vi vi sinh vt (in situ), hnh 1.4.

Hnh 1.4. Minh ho k thut lai in situ vi k thut FISH (fluorescence in
situ hybridization) pht hin vi khun Helicobacter pylori.
K thut ny c thc hin nh v chui acid nucleic trong vi sinh
vt sau khi c c nh trong cc tiu bn hin vi. Tuy nhin, do hu ht
cc vi sinh vt c kh nng thm (hay cho php) cc on ngn oligonucleotid
nh du i vo t bo sau khi c nh mu v vy khi s dng mu d nh
du vi cht nhum hunh quang c bit c th nhn thy c trc tip vi
sinh vt di knh hin vi hunh quang. iu ng ch l phn ng lai phi
tin hnh trong thit b c hn kn nhm hn ch vic bay hi ca dch lai.
Vic bay hi dch lai ny dn n vic bm khng c hiu ca cht nhum
hunh quang vi t bo. Nhit lai ph thuc vo oligonucleotid mu d. Sau
phn ng lai th mu phi c xem ngay hoc c bo qun trong ti trong
thi gian 6 thng. Nu mu d c hiu cho RNA th nhy tng ln rt ln v
c ti hng ngn bn copy ca RNA trong mi t bo (Stahl v Amman, 1991).
Lai trong mi trng dch th:
Phn ng lai trong mi trng dch th xy ra nhanh hn nhiu so vi
mi trng c cht mang pha rn (soild). Do c phn t ADN ch v mu d
u c th di ng t do trong mi trng do phn ng t kt qu cao
nht. Ni chung vi php lai thc hin trong dch th th thi gian kt thc
nhanh hn t 5-10 ln so vi trong iu kin l cht mang (Bryan, et al.,
1986). Tuy nhin, cng c th b sung thm cht mang lm tng phn ng lai.
Cn thm bc cui cng l tch phc hp mu d-si ADN ch. Hn ch
y l mu trong dch cho nn c th to ln cc nn km c hiu, do vy cn
cc gii php lm hn ch mc nhiu ca nn cho thch hp.

Hnh 1.5. Minh ho k thut lai trong mi trng dch th.
Trn thc t hu ht cc KIT thng phm dng cho vi sinh vt u tin
hnh trong mi trng dch th. Vi sinh vt trong mu u tin c phn gii
trong iu kin thch hp ADN ch lai vi mu d. Sau l bc lai v
tch phc h mu d v ADN ch da vo cc thng s ca php lai theo kiu
kp dp c th. Cc php lai theo kiu kp dp (hnh 1.9) cn c hai on ADN
c trnh t khc nhau; mt on bm gi ADN ch gn vo cht mang v
mt on l mu d. iu quan trng l hai on ADN ny phi c trnh t
khc nhau cho d chng u gn vi hai vng khc nhau trn on ADN ch
theo nguyn tc b sung. Mu d nh du c b sung vo trong dung dch
c cc on ADN ch nghin cu. Nh vy theo hnh v phc hp ADN mu d
nh du pht hin gn vi ADN ch v phc hp mu d- ADN ch gn
vo cht mang c hnh thnh. Phc hp ny c tch ra khi dung dch
v khi c mt c cht thch hp pht hin c mu d nh du. Khi dng
h thng pht hin da vo cc ho cht hunh quang hay to mu c th
dng thit b pht hin kt qu mt cch t ng.
+ K thut lai da vo mng:
Tc gi u tin gii thiu k thut c nh ADN trn mng l Nygaard
v Hall (1963, 1964). Sau chnh Southern (1975) l ngi m t vic tch
ADN khi gel sau khi in di v chuyn chng ln mng nitrocellulose. Cc
on ADN ch c pht hin bng k thut lai v y l bc tin quan trng
ca sinh hc phn t. T nm 1977 n nay c nhiu ci tin v php lai
cng nh bc chuyn ADN ln mng ca cc tc gi khc nhau nh: Meinkoth
v Wahl (1984). Ngi ta cng s dng mt s mng nynol, mng
nitrocellulose hot ho hay c bn cho cc php lai. i vi cng vic nh
loi vi sinh vt vi k thut c nh trn mng cho php lai ADN th cn chm
mu (l ADN sch, hay t bo vi sinh vt hoc sinh phm c vi sinh vt nghin
cu) ln mng thch hp. Mu c ly gii v ADN b bin tnh, sau ADN
c c nh trn mng khi a vo t xy ti 80
o
C trong 2 gi hoc a vo
x l vi tia UV trong trng hp s dng mng lynon. Mu d nh du sau
c a vo dung dch. Bc tin lai c thc hin hn ch cc mu
bm vo nhau khng c hiu. Sau l bc lai, mng sau khi lai c ra
loi cc mu d cn d. Bc ra tip theo nh gi mc bm c
hiu ca php lai. Sau cc mu d nh du lai vi v tr ADN mu trn
mng c pht hin bng cc h thng pht hin tng thch. Ton b qu
trnh lai trn mng c Meinkoth v Wahl (1984) m t chi tit.
Khi pht hin typ vi sinh vt, chng ta phi s dng k thut Southern.
Trong phng php ny ADN ca nhim sc th c tinh sch v c x l
vi enzym ct hn ch thch hp, sau mu li c in di trn gel agarose
v to ra du vn (finger print) ca nhim sc th. Sau khi in di gel c t
di mng nitrocellulose hay mng nylon nm gia mt s lp giy. Lp giy
lc pha trn c t trn cng phn gel v mng kp gia giy lc nh trong
hnh 1.6.

Hnh 1.6. M t phng php chuyn ADN ln mng cho k thut Southern
Blot.
Kt qu l m t pha di c thm mt cch t nhin ln trn. iu
gip cho vic chuyn cc mnh ADN t gel ln mng v bm cht vo mng
vi kch thc v phin bn ng nh trn gel sau khi in di (m t chi tit
theo Sambrook 1989).
Phng php truyn thng ny cng c ci tin khi dng mng nylon
chuyn ADN trong iu kin bin tnh (Read v Mann 1985). Thi gian
chuyn c th vi gi hoc qua m. Vic c nh ADN trn mng c thc
hin sau bng cch x l nhit hay tia UV nh trnh by trn. Tuy
nhin, nhiu phng php ci tin c thc hin nhanh nh chuyn bng in
di (Bittner, Kupfere, Morris, 1980) hay s dng bm chn khng (Medveczky,
1987; Olszewsk, 1988).
Trong nhiu trng hp dng in chuyn ADN t gel agarose ln
mng th trc tin gel phi c x l bin tnh v c lm trung ho tr li.
Gel c t gia hai bn in c cc bn giy thm (hnh 1.7).

Hnh 1.7. Minh ho bc chuyn ADN ln mng.
Sau ngun in c ni v lc ADN s c chuyn ln mng.
Thi gian chuyn s ph thuc vo kch thc on ADN nhng thng dao
ng trong khong 1-3 gi. Phng php c th thc hin theo chiu thng
ng hay chiu nm ngang. Hin nay c mt s thit b bn trn th trng
c dng cho k thut ny. Vi thit b nm thng ng, th ton b c
ngm trong m v c th tng cng dng in (ch v c th lm tng
nhit ), phng php ny cn dng nhiu m. Ngc li theo phng php
nm ngang hay phng php semi-dry, y gel v mng c kp gia cc
bn giy lc tm t bng m thch hp. Trng hp ny cn rt t m v
cng dng in l 1mA/cm
2
l thch hp.
Trong trng hp chuyn ADN ln mng bng p lc do chn khng,
vic thit lp h thng c trnh by m gel c t trn mng v s
dng lc ht chn khng a ADN ln mng. Dng lc ht chn khng t
c hiu qu chuyn ADN ln mng cao hn phng php thm t nhin.
Hiu qu ti a cho chuyn gel vi trng hp geneom sau x l enzym ct
hn ch c th t c sau 1 gi.
+ Phn tch RELPs vi k thut lai ADN.
Gii thiu phng php:
Nh trnh by, s phc tp trong k thut RELP to ra nhng kh
khn cho vic xc nh cc tc nhn gy bnh trong cc nghin cu v dch t
hc. iu ny cn kh khn v phc tp hn trong khi so snh cc kt qu thu
c trn cc bn gel khc nhau hoc ti cc phng th nghim khc nhau.
Nguyn nhn l do s lng cc bng ADN ln ti mc khng th xc nh kch
thc ca chng hay cc bng thu c khng lp li cc kt qu nghin cu.
Mc d vy, theo phng php ny cc ph ADN phc tp sau khi x l
vi enzym ct hn ch s c lm bin tnh v chuyn ln mng
nitroxenluloza hay nylon. Mng s c lai vi mu d thch hp, kt qu ph
php lai s r v khng qu phc tp do n ch hin th cc mnh ADN bt cp
vi mu d. Vi mt s lng khng ln cc mnh hin th thu c sau php
lai s cho php xc nh c chnh xc hn v kch thc. iu ny lm c s
cho so snh gia cc gel vi nhau cng nh kt qu thu c t cc phng th
nghim khc nhau.
Khi s dng phng php ny cn ch mt s mu d sau:
1, Mu d c th l on RNA ribosom t mt loi sinh vt no : v d
rRNA ca E.coli c th c cc cng ty thng phm cung cp (Boeringer
Mannheim), y l mu d kh thun li v c nh du phng x hay vi
cc c cht hunh quang. u im chnh ca mu d ny ch phn RNA l
on kh bo th do mu d c th dng lai vi cc sn phm x l enzym
ct hn ch ca nhiu loi vi khun khc nhau. C mt s loi khi lai vi mu
d ch cho mt kt qu ging nhau trong khi loi khc khi lai cc chng
vi mu d th li thu c kt qu khc nhau. y l c s cho phng php
ribotyping.
2, Mu d c th l on ADN ngu nhin c chc nng khng xc nh
(Tompkins et al., 1986). Mu d ny thng c dng cho cc loi c cha
chnh cc on ADN ny. S dng cc mu d ny i khi rt c ngha trong
vic phn bit cc mu sau khi tin hnh phng php ribotyping (Saunders et
al., 1990).
3. Mt cch khc na cng c dng l s dng mu d l mt on
ADN tch dng t mt gene bit. V d dng on gene m ho cho c t
ngoi bo (exotoxinA) s dng nh typ Pseudomonas aeruginosa (Ogle et
al., 1987). Mu d dng gene m ho cho c t Cholera c dng xc
nh cc typ cho cc chng thuc h phy khun sinh c t (Yam, Li Lung &
Ng, 1989). Vic s dng bt k loi mu d no cng to ra ph c trng ca
php lai c ngha cho so snh gia cc chng vi nhau.
Hn ch chnh ca k thut ny ch kt qu thu nhn ch khu tr phn
gene bt cp vi mu d m khng c trng cho c h gene.
Bng 1.6. Kt qu thu c khi thc hin k thut RFLP vi dng cc mu d
khng phi l ribosom.

i tng Tc gi nghin cu v ti liu dn
Aeromonas spp Altwegg an Luthyhottenstein (1991)
Borrelia burgorferi Wallich et al (1992)
Candida albicans Schmid et al (1992)
Chlamydia trachomatis Scieux et al (1992)
Corynebacterium diphtheriae Groman et al (1993)
Cryptococcus noeformans Spitzer (1992)
Escherichia coli Bohm ADN Karch (1992)
Hemophilus influenzae Forbes et al (1992)
Histoplasma capsulatum Leathet al (1992)
Lactobacillus helveticus Delostreyesgavilan et al (1992)
Mycobacterium tuberculosis Mazurek et al (1991)
Pseudomonas aeruginosa Tompkins(1991)
Salmonella spp Sodati ADN Piffaretti (1991)
Staphylococcus aureus Goh et al (1992)

- K thut ribotyping:
Nh trnh by trn mi mu d u cho cc kt qu c sc thuyt
phc tuy nhin k thut dng mu d l on RNA ca ribosom a ra mt
cch tip cn mi trong nghin cu dch t phn t vi cc vi khun c s khc
bit ln trong khi cc mu d khc ch gii hn vi mt loi hay ch c
ngha cho cc chng trong cng mt loi. K thut ny ln u tin c
Grimont m t nm 1986 v nhanh chng tr thnh phng php hu hiu
hin nay cho nghin cu dch t hc vi sinh vt mc phn t.
Tnh hp l cho vic s dng k thut ny l ch gene m ho cho RNA
ribosom c bo th cao. Cng c th pht hin thy nhng thay i cht t
trong qu trnh tin ho i vi cc chui ADN trong cc vi khun nghin cu.
Gene RNA ribosom c t chc thnh cc operon m cc gene ring r m
cho cc RNA kch thc 5S, 16S v 23S chng c cch nhau bng cc on
ADN spacer khng m cho gene no. Nu dng mu d hn hp gia 16S v
23S th kt qu php lai s hin th cc mnh tng ng vi phn ca gene ny
trong khi dng mu d l on ca cc gene c tch dng c th a n
kt qu hin th c phn gene tng ng v chui spacer. Nh vy s khc
nhau ca kt qu ph thuc vo loi mu d s dng.
- Yu cu k thut:
thu c kt qu ti u cn c cc yu cu k thut c th cho tng
bc thc hin.
u tin l phi to ra c ph du vn tay thu c sau khi x l mu
vi enzym ct hn ch. Ph vn tay ti u khi cc mnh ct phi c
tch r v phn b ng u v kch thc trn gel. S lng cc mnh
ADN thu c ph thuc vo mu ADN v loi enzym ct hn ch c
s dng. Thng thng phi chn mt s mu x l th trc vi tng
loi enzym (hay ng thi x l vi mt s enzym). C th thy r l cc
enzym c 5 nucleotid ti v tr ct s to ra sn phm nhiu mnh hn
cc enzym c 6 nucleotid ti v tr nhn bit.
Thc t cng cho thy khi dng enzym ct hn ch (mt hay ng thi
vi loi) thu c ph cc on ct hp l cho vic phn tch
ribotyping th cc kt qu ny cng rt khc nhau. Nh vy, iu quan
trng nn tin hnh trc cc php phn tch ny, phi chn c mt s
chng c trng v th vi mt s enzym ct hn ch chn gii php
cho nghin cu dch t hc vi k thut ny. Trong mt s trng hp
kt qu phn tch khi ch tin hnh vi 1 hay 2 enzym ct hn ch c th
khng a ra c kt qu chnh xc.
Nh vy, khi c c kt qu hp l sau khi x l cc mu ADN vi
enzym ct hn ch th tin hnh cc bc chuyn cc on ADN trn gel
ln mng theo cc phng php c m t trn. Phng php
chuyn bng chn khng l hp l hn c v kt qu cho vic chuyn cc
bng c kch thc gn nhau li c tch ra sc nt trn mng. Khi thc
hin php lai nn ch nu nh dng ngun ARN ribosom ca mt
chng no nh du lm mu d th cn phi thay i iu kin lai
nh nhit php lai c thc hin tt vi cc on ADN t cc
chng vi sinh vt khc nhau.
C mt s phng php nh du mu d l ADN ribosom. Phng php
nh du phng x (Grimont, 1986), y l phng php c u th l ch
cn vi bc n gin cho php lai v ra mu nhng li mang nhiu
hn ch nh: thi gian bn hu ngn, yu cu an ton cho phng th
nghim ring bit, nguy him cho ngi dng v gp kh khn vi vic
x l cht thi phng x. Mi y c mt s phng php nh du mu
d phi phng x c s dng kh thnh cng. Pitcher (1987) m t
phng php tng hp mu d gn vi bilatin pht hin ARN ribosom
ca Providencia stuartii. Cht kch hot quang ho c Koblavi v
Grimont m t (1990). ARN ribosom cng c nh du bng
acetylaminofluorence (AAF) do Grimont (1989) m t. Cng ty
Eurogeneetik (B) cung cp phc hp AAF-rARN trn th trng.
Gn y Gustafero v Persing (1992) a ra mt phng php mi
m y phc hp horseradisk-peroxysase-polyethyleneimine vi rARN
v kch hot bng quang ho. Cc mu d c nh du bng cc cht
phi phng x ny c th cho kt qu nhanh tng ng vi trng hp
dng cc cht phng x. Nh vy kt qu tin hnh php lai cc mu ca
cc chng khc nhau trong cng gel chuyn ln c th so snh vi nhau
khi dng k thut ny. Trong trng hp xc nh chnh xc kt qu cc
mnh lai vi mu d c th tin hnh so snh cc kt qu thu c t
cc phng th nghim khc nhau.
+ ng dng k thut ribotyping:
Xc nh typ vi khun bng k thut ribotyping c mt s u im so vi
mt s k thut nh typ khc ch:
Thc t l cc gene ca ribosom kh bn do chng nm trn nhim sc
th. Hu ht cc vi sinh vt u cha nhiu phin bn ca operon
ribosom do khi lai vi mu d thch hp th kt qu l to ra mt s
mnh lai c kch thc khc nhau. Hin nay, ngun rARN ca E.coli nh
du l mu d kh ph bin c thng mi ho.

Hnh 1.8. Kt qu ribotyping vi Helicobacter pylori.
Do tnh a dng v phc tp ca k thut nh typ theo ribosom do
trn thc t khi nhn kt qu bng mt thng kh c th pht hin c s
khc nhau hay ging nhau gia cc chng trong cng mt loi khi tin hnh
nghin cu dch t hc trn din rng. Owen v cng s (1992) nghin cu
kh nng tr gip ca computer so snh kt qu thu c khi nghin cu
cc chng Helicobacter pylori. Tng t nh vy Bialkowska-Hobranska v
cng s (1990) dng thit b laze o mt cc mnh ADN lai cng vi s tr
gip ca computer phn tch cc kt qu thu c t cc chng cu khun
nha bo gram m. Phng php ny c th a ra s liu chung lm c s cho
xc nh s ging nhau gia cc loi khc nhau. Cc phn tch thu c t cc
s liu ca cc cc th khc nhau c th ch ra c cc c th mi lm c s
cho nh danh cng nh theo di.
Mi nghin cu cho thy mt iu quan trng v c ngha nht l cch
chn enzym ct hn ch v loi mu d dng cho php lai (Saunder v cng s
1991).
Tm tt:
Nhn chung phng php lai ADN chng t u th ca n nh l mt k
thut quan trng cho nh typ v nghin cu dch t hc nhiu loi vi sinh vt
khc nhau. V nguyn tc phng php ny cng c u im v nhc im
ca n.
u im:
- S dng cho nhiu i tng vi sinh vt.
- C cc mu d vn nng c bn rng ri trn th trng. Kt
qu lai c lp li cao v kh n gin cho vic phn tch kt qu.
- C th s dng s tr gip ca computer lu gi v phn tch
kt qu th nghim.
Hn ch:
- Phng php s dng kh phc tp v tn thi gian.
- Thng tin kt qu ch phn nh cho on gene lai vi mu d s
dng. Hin nay, c th s dng cc on ADN tng hp lm mu d v
iu ny thc t lm ci thin nhiu mt ca k thut lai nh: khi c
ADN tng hp th khng phi thc hin cc k thut tch v tinh sch
cc mnh ADN tch dng c th ln ADN plasmid. Mt khc khi lai vi
mu d tng hp th phn ng tin hnh nhanh vi c hiu cao
hn.
Cho d k thut ny c s dng tt cho nhiu i tng vi sinh vt
khc nhau nhng nm 1992 Heimberger v cng s tin hnh phn
tch ADN c x l vi enzym ct hn ch trong trng xung in
(PFGE), kt qu ny rt c ngha cho php phn tch su hn v phn
bit cc chng vi sinh vt lin quan n s bng pht dch i vi cc
chng vi sinh vt khc.
Ribotyping v PFGE c chung nguyn tc da vo s phn b ca cc v
tr enzym ct hn ch trn ADN ca ribosom. Tuy nhin, s khc bit
ch ribotyping phn nh s phn b ca v tr cc enzym ct hn ch
nm trong cc gene m ho cho rRNA hay nm trong vng nhim sc
th c bo th cao, cn i vi PFGE phn nh s phn b ca cc
enzym ct hn ch phn b trn ton b gene nghin cu. Nghin cu
ca Prevost (1992) cho thy l k thut PFGE c th hiu qu hn k
thut ribotyping i vi php phn tch cc chng Staphylococus aureus
khng methicillin.

2.3. Nhn gene v k thut gii trnh t ADN.
K thut lai ADN c s dng rng ri cho cc nghin cu xc nh
typ ca nhiu i tng vi sinh vt. Ngun ADN ch i khi ch c s lng t
tr khi cc tin hnh nui cy vi sinh vt. Trong mt s trng hp vic nui
cy khng th thc hin c vi nhiu loi vi sinh vt hay virt hoc trong cc
trng hp khc cn thi gian di nui cy vi sinh vt c ADN cho cc k
thut lai hay nh typ. Kh khn ny c khc phc bng cch lm tng
ngun ADN ch mt cch nhanh chng bng k thut nhn gene PCR.
a. K thut nhn gene PCR (Polymerase Chain Reaction)
Vic nhn ADN cho php lm tng s lng gp hng triu hoc nhiu
hn na i vi on ADN hay ARN nghin cu. C nhiu phng php khc
nhau phn tch ngun ADN khuych i theo cch ny. Phng php n
gin nht l s dng in di xc nh kch thc sn phm ca phn ng
PCR. C th xc nh nhy v tnh c hiu cao hn nu sn phm PCR
c lai vi mu d c hiu c nh du. Phng php a li thng tin
nhiu nht l xc nh c trnh t ADN v ARN ca sn phm PCR. Vic xc
nh c sai khc ca chui ADN s cho php xc nh v nh typ chnh xc
nht cc i tng vi sinh vt nghin cu. Kt qu xc nh trnh t ADN cn
cho php xc nh mi lin h tin ho v dch t hc ca cc chng vi sinh
vt.
- i nt v phn ng chui PCR:
Phn ng chui PCR nhm khuych i ADN ln u tin c Saki
(1985) gii thiu v y c coi l mt trong nhng tin b khoa hc quan
trng nht v sinh hc trong thp k 80. Phn ng PCR s dng enzym chu
nhit to ra nhiu phin bn theo hm s m. C th v d, ch cn 1 si ADN
sau vi gi c th nhn ln 10
11
phin bn ADN tng ng vi 100 ng ADN.
Sau khi thc hin phn ng PCR th cn tin hnh mt s k thut xc nh
sn phm, n gin nht l in di v xc nh kch thc ca sn phm PCR.
Trong cc nghin cu chn on th kt qu ny rt c ngha, n chng t s
c mt ca ADN ch trong mu nghin cu. C th nhn c tnh c hiu v
nhy cao hn khi s dng k thut RFLP vi sn phm PCR hoc lai vi mu
d c hiu. Tuy nhin, thng tin y nht vn l kt qu xc nh trnh t
ADN ca sn phm PCR.
Ngay t khi c gii thiu th PCR c xem l phng php a li
kt qu kh nhy trong vic xc nh v pht hin vi sinh vt. Tnh u vit ca
n th hin ch c th ch cn mt ht virus hay mt t bo vi khun no
cng cho phn ng xy ra v khuych i ti mc c th pht hin c
trong thi gian ngn. K thut PCR cn c dng nhn ADN t khun RNA
sau khi c chuyn thnh cDNA sau phn ng phin m ngc (RT).
Phng php ny nhanh chng c ch v tr ln ph bin cho cc nghin
cu pht hin vi khun hay virus nng thp trong cc mu mi trng v
bnh phm. V d vic pht hin mu mu nhim HIV c ngha l pht hin
phn nh ADN ca virus trong h gene ngi c kch thc gp 100 000 ln.
Mt khc khi dng k thut min dch vi khng th th phi cn ti 8 ngy mi
c p ng min dch trong mu, trong khi vi k thut PCR th ton b quy
trnh pht hin ch mt vi gi (i vi ngun ADN hay RNA). Li th ca k
thut PCR c tn dng cho vic chun b ADN lm mu d vi s lng ln
cho cc php lai ADN trnh by trn. Cc mu d c th c nh du
bng phng x hay phi phng x khi tin hnh phn ng khuych i. Mu d
nh du vi k thut PCR c u th hn cc phng php nh du truyn
thng bng k thut tng hp cc mch t gy (nick translation) hay nh
du vi mi ngu nhin (rADN random primer labeling). Cc u th ny c th
k n, c th nh l cn lng nh si ADN khun, c th tin hnh nh du
vi cc mu d c kch thc ngn, chun b mu d khng cn tinh sch ADN
khun. Bng 1.7. di y lit k mt s v d ng dng k thut PCR cho
pht hin nhiu i tng vi sinh vt khc nhau. Rt nhiu cc kt qu nghin
cu nh vy c trnh by trong cc tp ch chuyn ngnh nh: nh nh: nh
nh: nh nh: Journal of Clinical Microbiology, Applied Environmental
Microbiology.
Bng 1.7. Mt s v d v ng dng k thut PCR trong xc nh vi sinh vt.


Vi sinh vt Ti liu
Acanthamoeba sp
Bordetella pertussis
Borrelia burgdorferi
Brucella sp
Candida albicans
Carnobacterium spp
Chlamydia trachomatis
Clostridium difficile
Coxiella burneti
Cryptosporidium parvum
Cytomegalovirus
Dengue virus
Epstein-barr virus
Erwinia amylovora
Escherichia coli
Frankia spp
Gaeumannomyces spp
Helicobacter pylori
Vokin et al (1992)
Houard et al (1989)
Marconi ADN Garon (1992)
Herman an Derider (1992)
Miyakawa et al (1992)
Brook et al (1992)
Hayes et al (1992)
Gumerlock et al (1991)
Stein an Raoult (1992)
Laxer et al (1991)
Gozlan et al (1991)
Henchal et al (1991)
Samoszuk (1991)
Bereswill etal (1992)
Jackson (1992)
Simonet et al (1990)
Henson (1992)
Claton et al (1992)
Hepatitis C virus
Human immunodeficiency virus
(HIV)
Influenza virus
Leptospira spp
Litsteria monocytogenees
Luteovirus
Mycobacterium leprae
Mycobacterium tuberculosis
Neisseria meningitis
Papillomavirus
Parvovirus
Plsmodium falciparum
Pneumocystis carini
Polio virus
Pseudomonas solanacearum
Rickettsia spp
Rotavirus
Salmonella spp
Staphylococcus spp
Toxoplasma gondii
Treponema pallidum
Trichomonas vaginalis
Trypanosoma cruzi
Vibrio vulnificus
Yersinia

Okamoto et al (1992)
Dawood et al (1992)
Claas et al (1992)
Merien et al (1992)
Niederhauser et al (1992)
Robertson et al (1991)
Hackel et al (1990)
Altamirano et al (1992)
Maiden et al(1992)
Charlotte et al (1993)
McOmish et al (1993)
Barker et al (1992)
Olsson et al (1993)
Yang et al (1991)
Seal et al (1992a)
Gage et al (1992)
Taniguchi et al (1992)
Rahn et al (1992)
Murakami et al (1991)
Vanevan et al (1991)
Grimprel et al (1991)
Riley et al (1992)
Breniere et al (1992)
Hill et al (1991)
Nakajima et al (1992)

Sau y l nhng nt chung nht ca k thut PCR:
Nguyn tc c bn ca PCR:
+ Khi qut chung:
PCR l phng php dng enzym khuych i chn lc mt on ADN
theo lut s m. Phn ng nhn gene c thc hin vi enzym ADN chu
nhit, hai mi tng hp v 4 loi deoxyribonucleic (dATP, dGTP, dCTP v
dTTP). Mi mt chu k phn ng nhn gene gm 3 bc (xem hnh v minh
ho): Bc 1 l bin tnh ADN, c tc dng tch hai si n t si khun xon
kp. Bc 2 l bt cp hai mi vo hai si n ca khun. Bc 3 l ko di
chui theo mi, chiu 5-3 vi qu trnh trng hp gn cc dNTP dc theo si
khun to thnh phin bn mi theo nguyn tc b sung. Tnh c hiu ca
phn ng c quy nh bi mi v s sao chp trc tip theo trnh t si
khun gia hai mi. Nh vy, kt qu to ra hng triu phin bn si khun
trong vi gi.
Mi cho phn ng l cc on ADN c kch thc 20-30 nucleotid c
thit k theo trnh t ca on gene cn khuych i da theo d liu c sn.
Mi cp mi phi hon thnh chc nng ca mnh trong ton b phn ng, tc
l chng phi bm c hiu vo ng v tr ring cho tng mi. S d nh vy
l do nu chng khng bm vo v tr c hiu th khng th khuych i c
on gene ch. Vi cc gene c bo th cao nh rADN th khi cp mi c
thit k th chng c th nhn c gene t nhiu i tng. Ngc li, trong
nhiu trng hp dng mi khng c hiu th c th nhn c cc sn phm
PCR khng c hiu. Tuy nhin, c hai kt qu trn u c dng cho nh
typ vi sinh vt.
Th nghim phn ng PCR ln u tin dng mnh Klenow ca enzym
ADN polymerase I t E.coli. Tuy nhin, enzym ny b bin tnh ti 94
o
C v nh
vy cn phi b sung enzym mi sau mi chu k phn ng. n nay qu trnh
ny c ci thin do dng enzym Taq ADN polymerase (Taq = Thermus aquaticus
) chu nhit. Enzym ny c tch ra t vi khun sng sui nc nng. Tc
xc tc cho phn ng ca enzym ny rt nhanh, c th t khong 8000
bp/pht ti 755
o
C. Hot tnh enzym gim mt na khi x l ti 92.5
o
C trong
230 pht hoc 40 pht ti 95
o
C. Nh vy khi dng enzym ny th khng cn b
sung enzym mi sau mi chu k phn ng.

+ Cc thng s k thut:

Bt k mt phn ng PCR no cng bt u bng vic tch ADN lm si
khun. Ni chung, theo l thuyt ch cn mt si khun cng cho phn ng
to ra sn phm nhng trong thc t cn khong 10-100 ng/phn ng. i khi
chun b ADN theo phng php n gin khng cn tinh sch cng ph hp
cho phn ng PCR. Tuy nhin, iu quan trng l trnh s c mt ca EDTA
(acide thylne-diamine-ttraactique) hoc m phosphat v kt qu s lm
gim Mg
++
(do EDTA hoc to thnh mui Mg
3
(PO4)
2
khi nhit cao). Phn
ng PCR c th nhn c on gene di 10 Kb nhng trong thc t thng
dng nhn cc on c kch thc ngn hn (<2 Kb). C th tham kho cc
thng s thnh phn phn ng PCR trong bng di y.

Bng 1.8. Thnh phn phn ng PCR.

Thnh phn S lng
ADN khun 10-100 ng
dNTP (mi loi) 200 mM
Mi xui 0.1 1.0 mM
Mi ngc 0.1 1.0 mM
Taq polymerase 1 U
KCL 50 mM
Tris HCL (pH 8.4) ti 25
o
C 10 mM
MgCl
2
2.85 mM
Gelatin 100 mg/l
Nonidet-40 0.05%

S chu k cho phn ng to sn phm c hiu t 20-30 chu k. iu
ch l cc mi cho phn ng phi c nhit bt cp vi ADN ch gn nhau,
c trnh t khng b sung nhau trnh bt cp vi nhau. Trnh t u 3 ca
cc mi cng phi khc vi trnh t vng trung tm ca sn phm trnh to
thnh hin tng kp tc. Ni chung, ngy nay ngi ta c th ti u ho vic
chn mi nh s tr gip ca cc chng trnh my tnh.
i khi cng xut hin hin tng nhn sn phm PCR khng c hiu,
iu ny thng hay xut hin khi dng cc cp mi suy bin t chui acid
amin. Tuy nhin trong hu ht cc trng hp ny th vic thay i iu kin
phn ng c th hn ch c cc sn phm khng mong mun. Cc thnh
phn nh nhit , nng Mg
++
v enzym c nh hng ln n tnh c
hiu. S c mt ca nonidet-40 c tc dng tng hiu sut ca phn ng.
Vic ng dng k thut RFLP vi sn phm PCR c th l cch nhanh
chng tm s khc bit i vi cc gene khc nhau. Vi cch ny yu cu
sn phm PCR phi sch v ng nht. Theo cch ny trnh t c th c
thc hin nh sau: Sau khi in di kim tra sch ca sn phm PCR, tin
hnh kt ta vi ethanol. Kt ta c ho vi m thch hp v sau c
x l vi enzym ct hn ch thch hp. Kim tra kt qu thng thng trn gel
agarose, trong mt s trng hp c th trn gel polyacylamid thu c
phn gii cao hn. Ch rng thng thng hot tnh enzym gim 5% sau mi
chu k nhit. Nh vy c th c lng 50% hiu qu khuych i mt i
trong ton b qu trnh phn ng.
+ Mt s kh khn vi phn ng PCR:

PCR l k thut c ngha ln trong nghin cu sinh hc phn t nhng cng ny sinh
mt s vn cn ch c bit khi pht hin mt s bnh virus hay vi khun. Vn y
l acid nucleic ca c t bo cht v t bo sng u cho kt qu dng tnh trong phn ng
PCR. Phn ng nhy nn mi s nhim ADN u c th a n sn phm dng tnh gi
trong kt qu. hn ch thc t ny cn tin hnh vi cc mu i chng cn thit bit
c s nhim tp t cc thnh phn phn ng hay t dng c th nghim.

Mt vn na cng cn cp l sai s trong phn ng nhn gene vi enzym taq
ADN polymerase. Sai st thm nucleotid xy ra vi mi mt n v sao chp khong 9000
nucleotid v t bin dch khung xy ra vi s lng tng ng khong 40 000 nucleotid.
+ Mt s k thut PCR khc:
Mt s k thut pht trin trn c s ca k thut PCR c ngha quan
trng cho nh danh v nh typ vi sinh vt l nested PCR (PCR lng), revert
transtriptase PCR (sao chp ngc) v asymmetric PCR (PCR mt chiu). PCR
lng l phn ng PCR bao bm hai phn ng ng thi, phn ng u vng
ngoi gene v sn phm ca n li lm khun cho phn ng sau cho on nm
trong gene. Nh vy phn ng sau cn 1 hay 2 mi nhn phn gene nm
trong sn phm PCR t hai mi pha ngoi to ra. Vai tr k thut ny lm
tng nhy v c hiu ca phn ng PCR.
Phn ng PCR phin m ngc c dng nhn ADN t ARN
ca virus. Vic kt hp phn ng ny vi PCR lng c tc dng pht hin ARN
ti thi im nht nh ca mu.
K thut PCR mt chiu c ng dng trong phng php gii trnh t
ADN trc tip t sn phm PCR.phm PCR.
+ S dng k thut PCR cho xc nh typ vi sinh vt:
Nh trnh by trn, dng cp mi c hiu c th xc nh c
gene c trng cho loi hay thm ch mt chng vi sinh vt no . Cc chin
lc ng dng li th ca k thut PCR dng trong xc nh typ vi sinh vt
trong nghin cu dch t hc c chia lm 4 nhm sau:
- Phn tch RFLPs vi sn phm PCR khi dng cp mi c
hiu.
Trong trng hp ny dng kt hp k thut PCR v x l sn phm PCR vi enzym ct
hn ch v quan st kt qu sau khi in di sn phm trn gel agarose hoc polyacrylamid Mt
dn chng cho k thut ny l kt qu nhn gene synthase citrat trong Ricketsiae sau sn
phm PCR c x l vi enzym ct hn ch phn bit gia cc chng vi nhau (Regnery v
Ctv, 1991). Cc v d sau (bng 1.8) cho thy hiu qu ca phng php ny.
Bng 1.9. Mt s v d cho k thut RFLPs (Restriction Fragment Length
Polymorphism) phn tch cc gene c hiu c nhn ln bng k thut PCR.
Chng vi sinh vt nghin cu Tc gi nghin cu v ti liu dn
Chlamydia spp. Kaltenboek et al (1992)
Clostridium spp. Gurtler et al. (1991)
Helicobacter pylori Foxall et al. (1992)
Hepatilis C virus Okamoto et al.(1992)
Mycobacterium tuberculosis Ross &Dwyer(1993)
Nitrobacter spp. Navarro et al.(1992)
Rickettsia spp. Regnery et al. (1991)
Streptococcus spp. McClellADN et al. (1992)

+ Dng k thut PCR cho ribotyping:
K thut PCR ribotyping l ng dng cc cp mi c hiu nhn
cc vng gene ca ARN (thuc ribosom hay ARN vn chuyn). Tuy nhin, trong
thc t k thut ny yu cu kinh nghim, k thut v tn thi gian thc
hin php lai. khc phc hn ch ny mt cch tip cn c trnh by
y l phn tch a hnh (du vn tay) ca vng gene ny hay vng gene xen
k ca rARN hoc tARN.
Hu ht cc chng vi khun u cha 2-22 phin bn rARN trong mi
t bo (Gottlie v Rundner, 1985) trong khi vng xen k gia 16S v 23S
cha mt s cc on ADN lp li (Bacot v Reeves, 1991). Trong khi on
gene m ho cho tARN kh bo th th vng xen k ca tARN li thay i t 2-
35bp i vi Bacillus subtilis (Vod, 1985) v t 2-208bp trn i tng E.coli
(Jinks-Roberson v Nomura, 1987). Nh vy khi s dng cp mi nhn ton b
vng gene tARN c th to ra c s khc bit gia cc chng vi sinh vt. Kt
qu ng dng k thut ny trn mt s i tng c trnh by trong bng
1.9.

Bng 1.10.. Mt s v d s dng k thut PCR cho ribotyping.
Vi sinh vt Tc gi v ti liu dn
Vi khun lactic Klijin etal.(1991)
Listeria Czajka et al.(1993)
Mycoplasma Van Kuppeveld et al.(1992)
Pseudomonas cepacia Kostman et al.(1992)
Pseudomonas solanacearum Seal et al.(1992b)
Staphylococcus spp. Welsh &McClellADN(1992)
Streptococcus spp. McClellADN(1992)

Khi s phin bn rARN tng c ngha l lm tng nhy ca phng
php ng dng. Engstrand v cng tc vin (1920) cng b c th s dng
mu d l on nucleotid b sung vi on 16S ca rARN nh l mt trong s
mi cho php nhn phin m ngc ARN v php phn tch c thc hin nh
k thut PCR nhn sn phm cDNA c dng cho pht hin Helicobacter spp.
nhy ca phng php pht hin ny tng 50 ln so vi phng php
truyn thng.
+ K thut rep-PCR:
Mt phng php trc tip cho kt qu finger printing khng cn s
dng enzym ct hn ch l rep-PCR da trn nguyn tc nhn cc on gene
lp li. Thut ng rep-PCR l ch phng php chung s dng cp mi c hiu
nhn cc chui lp li ph bin trong cc vi sinh vt thuc c th nhn s.
Cc chui lp li c bo th cao trong cc i tng thuc
Enterobacteriaceae v mt s i tng khc (Versalovic v Lupsky, 1991).
Dng cc on mu d bo th c th lai vi c hai on ADN lp li: on c
kch thc 126bp (Enterobacterial repetitive intergeneic concensus-ERIC) v
on 38bp (repetitive extrageneic palindromic-REP) t b gene ca
Enterobacteriaceae vi khun gram m v mt s chng c quan h xa khc. C
th trc tip thit k cp mi cho cc on gene bo th thu c kt qu
finger printing sau khi nhn gene dng b gene ca cc vi sinh vt c mang
cc on gene bo th ny. Sau khi in di c th pht hin c trn agarose
cc on gene c kch thc khc nhau t cc ngun vi sinh vt khc nhau. K
thut ny c ng dng em li kt qu tt khi nghin cu mi quan h
trn cc i tng Bacillus subtilis (Versalovic v cng s, 1992), Citrobacter
diversus (Woods v cng s, 1991), Rhizobium meliloti (Brujin, 1992). Thc t
phng php to ra kt qu finger printing a dng t hu ht cc i din ca
Enterobacteriaceae (Versalovic v cng s, 1991) v c ng dng trn tt
cc vi khun c mang cc on gene bo th lp li trn.
Phng php tng t cng cho php pht hin s khc nhau t cc
ngun ADN trn cc i tng nhn thc thuc chi Naegleria nh m t ca
Belkim v Quint (1992). Phng php ny dng cho pht hin a hnh cc
on gene chung cho c vi sinh vt nhn s v nhn chun.
+ K thut RAPD (Random amplified of
polymorphic DNA):
Hn ch ca cc phng php nghin cu trc y l phi dng cp mi
c hiu. Theo cch ny phi c cc thng tin lin quan n on gene nht
nh v i tng nghin cu. Kh khn ny c loi b trong trng hp
dng cc mi ngu nhin hay tu tin, nh vy phng php phn tch khng
cn n thng tin v geneome ca i tng nghin cuC s ca phng
php ny c Welsh v McClell ADN (1990) m t khi dng mi ngu
nhin nhn gene v to ra ph finger printing c trng cho tng h gene
(geneome). Mi ngu nhin c th ch gm 5 bp nhn gene c th to ra
kt qu finger printing kh a dng. C th xem kt qu minh ho trong bng
v hnh sau.
Bng 1.11. V d minh ho cho k thut RAPD.

i tng Tc gi v ti liu dn
Agaricus bisporus Khush&ctv(1992)
Aspergillus fumigatus Aufauvre-Brown (1992)
Bacillus thuringiensis Brousseau&ctv (1993)
Brucella spp. Fekete&ctv(1992)
Campylobacter spp. Mazurier&ctv(1992)
CADNida spp. Lehmann 7 ctv (1992)
Casuarina spp. Sellstedt& ctv(1992)
Clostridium difficile McMillan& Muldrow(1992)
Frankia spp Sellstedt& ctv(1992)
Fusarium solani Crowhurst & ctv(1991)
Helicobacter pylori Akopyanz & ctv(1992)
Histoplasma capsulatum Woods& ctv (1993)
Lactococcus lactic Cancilla & ctv (1992)
Leptosphaeria maculans Goodwin&Annis(1991)
Listeria monocytogenees Czajka & ctv (1993)
Porphyromonas gingivalis Menard &ctv (1992)
Rhizobium spp. Harrison &ctv (1992)
Staphylococcos spp. Weish &McClellADN (1990)
Streptococcus spp. Weish &McClellADN (1990)
Streptococcus uberis Jayarao &ctv (1992)


Hnh 1.9. Minh ho kt qu phn tch s dng k thut RADP.
b. Gii trnh t ADN
Phng php chnh xc v a li nhiu thng tin nht cho nh danh v
nh typ vi sinh vt l xc nh chnh xc trnh t chui ADN ca mt vng quy
nh trn nhim sc th. Phng php gii trnh t ADN pht trin nhanh
chng, kt qu so snh s tng ng ca trnh t gene nghin cu tr thnh
phng php phn loi chun theo sinh hc phn t trong nghin cu h thng
hc v cy ph h di truyn gia cc i tng nghin cu. Cc gene bo th
c gii trnh t lm c s xc nh v tr ca sinh vt nghin cu, trong
khi cc trnh t khng tng ng (khc bit) li lm c s cho s bit ha
cng nh xc nh mi quan h gia cc c th gn vi nhau.
+ Nguyn tc:
Phng php gii trnh t ADN theo nguyn tc to ra cc mnh ADN
c nh du ti u 5 hoc 3. Cc mnh c cc base nh u c tch ra
trn gel polyacrylamid Cc mnh c phn bit v v tr trn gel vi s sai
khc ch vi mt nucleotid. Vic nh du v c kt qu theo cc k thut v
phng php khc nhau. Vic to ra cc mnh ADN sai khc nhau v 1
nucleotid c trnh by theo 2 phng php: phng php ha hc (Maxam &
Gilbert, 1997) v phng php enzym kt thc phn ng chui (Sanger,
1997). Ngy nay phng php enzym c dng ph bin hn c.
+ Phng php enzym kt thc phn ng chui ca
Sanger:
Phng php kt thc phn ng chui c thc hin da trn vic nhn
ADN t si khun khi c on ADN mi vi xc tc ca Klenow hay T7-ADN
polymerase v 4 loi deoxyribonucleotid (dNTPs). Nh vy, c 4 phn ng
c tin hnh ng thi v trong mi phn ng th c mt loi dNTP b thay
i bi mt dn xut lm dng phn ng (ddNTP). Kt qu l phn ng
chui b dng c hiu cho mi mt base xc nh xy ra mt cch ngu nhin
trong mi phn ng. Trnh t ca ADN c xc nh trn gel. u tin phng
php ny c xy dng cho xc nh trnh t si ADN n to ra bng cch
tch dng ADN vo vect thc khun th M13 (Sanger v cng s, 1978). Sau
phng php xc nh trnh t si i ADN c Chen, William (1986)
m t khi tch dng ADN vo plasmid. Ngy nay ngi ta la chn phng
php Sanger gii trnh t ADN cho nghin cu phn loi v xc nh typ vi
sinh vt thay cho phng php ha hc.
Mt s li th ca phng php ny ch: Phng php ny n gin v
khng tn nhiu thi gian nh phng php ha hc. Khi nghin cu phn loi
v xc nh typ th cc gene nghin cu c bo th nht nh v vy ta c
th dng chung mi cho nhiu i tng khc nhau. Mt trong hn ch ca
phng php ny ch cc on ADN cn c tch dng trc khi tin hnh
gii trnh t ADN. Tuy nhin cho n nay ngi ta ci tin phng php ny
c th gii trnh t trc tip sn phm PCR.
+ Gii thiu k thut gii trnh t ADN theo Sanger:
C hai phng php: Phng php gii trnh t truyn thng c kt qu
trn bn gel (nh du bng phng x l ch yu) v phng php gii trnh
t v c kt qu t ng (nh du bng ha cht hunh quang).
- Phng php truyn thng:
Cc bc chnh cho gii trnh t ADN nh sau:
Chun b ADN khun
Bt cp ADN khun v mi
Tin hnh phn ng gii trnh t ADN
Tch sn phm trn gel polyacrylamid bin tnh vi urea.
c kt qu.
Tuy nhin cn ch mt s im sau:
1. Chun b ADN khun.
2. iu kin bt cp mi.
3. Thc hin phn ng gii trnh t ADN.
4. Tch sn phm trn acrylamid gel.
5. c kt qu.
Mt s im cn c chi tit thm cho mi bc nh
sau:
1, Chun b ADN khun:
Si khun cn c hiu l vic gii trnh t c thc hin vi si n
(vect M13) hay si i ADN. Vic c trnh t theo si n thng cho kt qu
l kch thc on gene c di v chnh xc cao. Ngy nay ngi ta thng
c si i v vic chun b mu d dng hn, ngoi ra khi s dng hai phn
ng vi hai mi tng thch th qu trnh c li chnh xc hn. l u th
ca cch c si i ADN. Do vy, rt cn c kt qu t c hai si ADN. Hin
nay phng php c trnh t ADN trc tip t sn phm PCR ang ngy cng
tr ln ph bin.
2, Chun b si ADN mi cho bc bt cp vo si
khun:
Ngi ta c th dng cc on ADN mi tiu chun c hiu cho trnh t
ADN ca vect gn st vi si khun hay primer c hiu ca si khun m n
s bt cp vo. Tin ch ca vic dng mi chun ch mi iu kin c
la chn cho si khun v mi c dng lp i lp li nhiu ln cho cc si
khun khc nhau. Hn ch ca loi mi ny ch ch c c mt on ngn
ca gene (300 500 bps) trong trng hp phi c cc gene c kch thc
ln cn phi tin hnh subclone c tip. iu ny c th thc hin bng
cch to ra cc on ADN ngu nhin t ton b gene sau li gn vo vect
c cho ht trnh t ca gene.
Phng php tip theo l s dng mi c thit k theo nguyn tc b
sung vi trnh t ca gene. Theo cch ny, ln lt cc on c c m
khng cn thao tc subclone. Tuy nhin theo cch on mi c thit k
c gene nh vy th iu kin cho tng phn ng gii trnh t ADN vi cc
mi khc nhau s khng c ti u. Tuy vy, hin nay vic tng hp mi
tr nn d dng v ph bin vi gi thnh thp, hn th na vi s tr gip
ca tin hc th bc thit k mi c ti u ho. V vy phng php ny
ngy cng tr ln thng dng hn. Sau khi mi c thit k th vic tip
theo l chn cch nh du vo mi hay vo on ADN s c tng hp. u
th ca vic nh du ngay ti u 5 ca mi ch cc bng ADN thng c
cng mt cng tn hiu. Thng thng vi phng php c trnh t th
cng th P
32
thng c dng. u th ca n l mang gc photphat cao nng
bta mnh, do ch cn trong thi gian ngn l thu c tn hiu. Hn ch
ca phng php ny l do mang nng lng cao nn kh phn bit gia cc
bng khc nhau so vi dng S
35
. Hin nay gii quyt vn ny ngi ta
dng P
33
tuy gi thnh cao nhng cho kt qu tt hn.
3, Thc hin phn ng gii trnh t ADN:
Enzym thng dng thng dng l sequenase (l mt dng ca T7 DNA
polymerase), enzym ny c u th hn hn so vi Klenow c dng trc
y. Phn ng c thc hin nhanh hn v thu c kt qu ti u nu c
mt ion Mn
++
, ion ny gip cho qu trnh sao chp c chnh xc cao. Trc
y dNTPs nh du vi P
32
c dng nh du sn phm. Tuy nhin ngy
nay nhiu ngi chuyn sang dng S
35
thay th cho P
32
, kt qu l cho
phn gii cao hn.
Mi y phng php gii trnh t da trn k thut PCR c gi l chu
k gii trnh t trc tip ADN ang thay th phng php c dng enzym
sequenase. V nguyn tc th cc phng php ny l ging nhau. u th ca
phng php ny l: lng ADN i hi t v khng yu cu phi c mc
tinh sch cao. Ngoi ra, c mt u im na l phn ng xy ra nhit
cao, do khc phc c nhng hn ch ca cc phng php khc khi
gii trnh t i vi cc si khun c cu trc bc 2 (Fan & cng s, tp ch
Biotechniques 21: 1132-1137).
4, Tch sn phm trn gel urea-acrylamid:
Cho n nay c nhiu tin b trong k thut in di gel k t nm
1970 khi m ngi ta pht hin ra phng php ny. Tin b quan trng nht
l gel gradient to ra dn cch thch hp c c thm t 50-100 base.
u tin, gradient c to ra gia acrylamid v gradient mui khi gel. C
th c cch khc na l ngi ta thay i dy ca gel bng spacer. Phng
php d nht l b sung nng mui NaOAc vo b m di khi chy gel,
kt qu s to ra mt gradient cn thit m khng my kh khn.
C nhiu cch ci tin khc nhau to ra gel c phn gii cao, v d
to ra gradient i vi acrylamid mch thng hay b sung mt s cht to ra
gradient (Long Ranger). Mt khc vic gel cng c n gin ha bc s
dng bng dn. Mt s ngi dng cc gel siu mng t phn gii cao.
Nhiu thit b c pht minh nhm thu nhn cc bng c tch ring khi
chng ra khi gel, v gn y nhng thit b ny c thng mi ha.
Nhum bc l phng php tng i hiu qu xc nh cc trnh t
ADN, cch ny hn ch c nhiu bc dng trong cc k thut da vo nh
bc x m khng cn s dng cht phng x.
5, c trnh t gene:
Hin nay k thut c phim t ng thay th cng vic c trnh t
ADN bun t trc y. C hng lot cc thit b t loi c th ghi li trnh t
da vo vic chn tng loi base n nhng thit b c th thc hin c ton
b qu trnh ny mt cch t ng.
Hy vng rng vi nhng bn lun ngn gn ny s gip bn lm quen
dn vi nhiu loi k thut khc nhau. Ngy nay cc thit b gii trnh t t
ng cng tr ln thng dng v vic s dng loi thit b v ha cht khc
nhau rt a dng. Vic chn la thit b, ha cht v phng php thch hp
gii trnh t ADN nhanh, chnh xc v kinh t l iu cn c quan tm n.

* Mt s phng php c th thng c dng cho k thut
gii trnh t ADN.
Phng php 1: Tinh sch Plasmid cho gii trnh t ADN.
Hin nay c nhiu k thut tinh sch plasmid khc nhau, phng php
c trnh by di y l phng php thu c plasmid c tinh sch cao,
c th dng cho c gii trnh t ADN th cng v t ng ha. Qu trnh ny l
ci tin phng php s dng dch kim lm tan t bo, sau s dng sc k
qua ct trao i ion. Cng nh cc sn phm c lu hnh trn th trng ca
cc cng ty, hn ch ln nht ca phng php ny l gi thnh ca nha trao
i ion tng i t. Thc t c rt nhiu cng vic phi gii trnh t mt cch
th cng v cc cn b nghin cu tm ra nhiu phng php tinh sch
khc nhau thu nhn ADN t sch cn thit, thm ch c th dng cho
gii trnh t ADN t ng. Tuy vy, nhiu ngi vn thch s dng ct trao i
ion, kt qu c th c c ti 600 bps hoc nhiu hn na.
Qu trnh tinh sch nh sau:
+ Nui cy vi khun mang plasmid trn mi trng chn
lc vi khng sinh tng ng (khong 3 ml).
1, Phn 3 ml mi trng LB cha 100 mg/ml apicilin trong ng
nghim v trng (12X100 nm c nt).
2, V trng u que cy trn la v lm ngui trn mt a thch.
3, Ly sinh khi t mt khun lc v a vo ng nghim cha mi
trng nui cy trn.
4, Nui cy vi khun 37
o
C qua m c lc va phi.
+ Chun b ADN dng ct Qiagene:
Cc ct Qiagene c chun b d dng v nhanh tinh sch ADN
khng cn dng gradient CsCl. ADN c chun b theo phng php ny t
kt qu rt tt cho vic bin np v gii trnh t gene.
C hai ch quan trng khi dng ct Quiagene l: (1) Khng s dng qu nhiu mu
ln ct, iu ny rt quan trng v nu dng th tch qu nhiu kt qu l s c nhiu protein
nh hng n kh nng bm ct ca ADN. Vi cch ny c th nhn c lng ADN c cht
lng cao hn t th tch t hn. Nu nh lng ADN thu c t t ln th u tin th cn
thc hin ng theo ch dn ln th th 2 vi th tch ln hn. Khi c mt lot th tch mu
c Quiagene khuyn co ln ct t c hiu sut tinh sch cao nht th nn dng lng
dch ln ct c th tch nh nht. (2) m bo ct phi c ra hai ln trc khi gii phng
ADN ra khi ct, iu ny cng rt c ngha v vic ra nh vy s loi tr c protein v
cc thnh phn tp nhim khc.
+ Cc bc dng ct trao i Qiagene (ti liu hng
dn s dng Quiagene):
1. Ly tm khong 1,3 ml dch vi khun mang plasmid. Lm tan vi
0.3 ml m P1 c RNase, chuyn sang tube mi.
2. Thm vo 0.3 ml m P2 v trn u, gi nhit phng 5
pht (khng nn lu hn).
3. Thm vo 0.3 ml m P3 trn u ngay nhng phi nh nhng,
ly tm 4
o
C trong 30 pht vi tc 15000 v/pht. Thu ly phn
trn ta.
4. Ly tm li nh bc 3 mt ln na loi cc phn cn nu c.
5. Cn bng m cho ct Qiagene-tip 20 vi 1ml QBT v cho
m chy ht t do.
6. a phn dch thu c bc 4 ln ct v t chy qua ct.
7. Ra ct 2 ln, mi ln 1 ml m QC.
8. Thu ADN vi 0.8 ml m QF.
9. Kt ta ADN vi 0.7 th tch isopropanol (trc a v nhit
phng). Ly tm 15000 v/pht ti 4
o
C.
10. Ra ADN thu c vi ethanol 70%, kh 5 pht v ho tan
li trong th tch m thch hp (25ml). Ch trong trng hp
mu dng cho gii trnh t ADN t ng hunh quang th ho tan
mu trong nc.
Ni chung s dng ct Qiagene thu c trn 90% ADN v thng c
dng cho tinh sch Plasmid v cosmit (kch thc di 2 Kbs n ln hn 50
Kbs). S lng plasmid thu nhn c t t bo E.coli ph thuc vo h t bo
ch, loi plasmid t thp trung bnh n s lng plamid cao v iu kin nui
cy (c th tham kho ti liu lin quan t cc ch dn ca Qiagene quyt
nh s lng t bo dng x l cho mi loi kch thc ct).
Ch cch lm sch v ti s dng ct:
1, Thm ethanol y vo ct, ly tm.
2, B ethanol v ly tm li mt ln na.
3, Lp li bc 1 v 2 vi nc ct.
4, Gi ct trng thi kh cho n ln s dng v sau.
(Ct c th c dng cho nhiu ln tuy nhin khi mun tinh sch ADN
dng cho tch dng th nn dng ct mi).

Cc loi dch m dng cho ct Qiagene:
m P1 50 mM Tris -HCl, 10 mM EDTA, pH 8.0, Rnase A
pha ngay trc khi dng trong m P1 nng 100 g/ml
Bo qun
4
o
C
m P2 200 mM NaOH,1%SDS Nhit
phng
m P3 2,55M KAc, pH 4,8 Nhit
phng
m QBT 750mM NaCl,50mM MOPS,15% ethanol, pH 7,0, 0,15%
Triton X-100
Nhit
phng
m QC 1M NaCl, 50mM MOPS, 15% ethanol, pH 7,0 Nhit
phng
m QF 1,25M NaCl, 50mM MOPS, 15% ethanol, pH 8,2 Nhit
phng

Phng php 2. Chun b gel cho gii trnh t ADN
Gii thiu chung:
Phng php ny gii thiu cch gel, ln mu v chy gel gii trnh t
ADN. C nhiu cch gel, c th tham kho cc cch khc nhau chn cch
tin ch nht.
iu quan trng nht l cc bn knh phi c ra sch tuyt i.
1, Mang gng tay v loi b ht bt gng tay i. Ra bn knh mt ln
vi nc nng. Sau dng bn chi ra vi 0.5N NaOH. Lau bn knh
vi dung dch Alconox hay cht ty ra c chc nng tng ng. Sau
ra sch li bng nc.
2, Ra cc bn knh vi nc trao i ion sau dng ln gi cho kh,
trnh bi bm vo knh. Lau vi ethanol v dng giy Kimwipes hay
Scott Utinity lau cho sch khng to vt. Kim tra li bi bm vo
knh lau cho sch, sau knh c lau bng lp dch Rain-X hay
Sigmacott, Gel Slick ca hng AT Biochem. Mc ch dng cht ny l
gip cho vic gel d dng hn v gel khng b dnh vo cc bn knh.
3, Ra li bn m cch gel (spacer) vi nc t vo dc theo hai cnh
i xng ca bn knh.
4, Ghp hai bn knh vi nhau. t cn thn hai bn knh bng nhau sao
cho hai bn spacer st tn cui bn gene (to mt phng cng vi hai
bn knh). Nu khng m bo mt phng th d b chy gel trong qu
trnh gel. Kp 2 cnh ca gel ti 3 v tr.
5, Chun b gel acrylamid 8% Long Ranger (Hoefer Rig).
Trn u hn dch:
16 ml Acrylamid Long Ranger (LR) 50%.
5 ml 10X TBE
70 ml 10M Urea (46 gam)
a n th tch 100 ml vi nc
Cho vo 800 l 10% Ammonium persulphat.
50 l TEMED.
Qu trnh polyme ha c thc hin trong 15 -20 pht.
Chy gel LR trong di nhit 50-55
o
C, nhit cao hn 55
o
C s ph
cc lin kt acrylamid LR gel l dng ci bin cho gel acrylamid c mt s
u im sau: Th nht l LR gel vi nng cao hn nhng vic chy
gel th cng nh gel c nng thp thng thng, v d LR gel 5%
tng ng 4% gel acrylamid vi bisacrylamid Th 2 l LR gel c kh
nng phn gii cao hn v chy vi tc nhanh hn 30-40% gel thng
thng, gel LR kh nhanh hn v khng b dnh.
Mt s ch khi thao tc::
a. Trn gel nh nhng (trnh trn mnh).
b. Ht dch acrylamid bng pitpet hay c th dng cc ong.
c. t bn knh nghing 15
o
v gel bng cc ong hay pipet. Kim
sot tc gel bng gc nghing. Trnh dng gel khi ngt
qung v iu ny s dn n to bt. Nu phi dng li ht dch
mi vo syringer th lc phi h dn bn knh xung v tr nm
ngang trc khi dng gel. Sau ly dch gel mi vo syringer v
tip tc gel trc khi gel ln khi v tr nm ngang. C thao tc
nh vy n khi y bn gel.
Nu xut hin bt khi gel th phi dng li ngay v nghing tm
knh cho bt kh i ln, c th g nh tay vo bn knh cho bt
kh i ln ht. Phi loi ht bt kh ra khi gel, sau mi tip tc
gel.
6, t gel nm ngang v a cnh bng ca lc rng c mp vo gel,
to mt gc hi nghing nhm trnh to bt kh. Chc chn rng t
cnh bng ca lc vo pha trn ca gel v phn rng c mp s nm
dc theo nh ca bn knh di (lc s nm ngc vi v tr trong hnh
1.10). B sung thm gel v y thm lc vo ng v tr v c nh lc
li bng kp trnh x dch.
7, Bao bc gel vi giy nilon SaranWrap trnh gel tip xc vi khng
kh. Ngoi ra c th thm giy lc vi m TBE y ln. iu ny gi
cho gel lun m v trnh gel tip xc vi oxy (oxy lm cn tr qu trnh
polyme ha).
8, Qu trnh polyme xy ra t 30 pht n 1 gi. xc nh polyme
ha c th ht mt t gel vo pipt pastuer v cho n khi qu trnh
polyme ha xy ra. Hoc c th ly gel cho vo ng falcon v y cht
np li, trong nhiu trng hp th gel ch nn polyme ha trong 15-20
pht hoc nhanh hn. Nu qu trnh polyme ha khng xy ra th li phi
tin hnh ra knh v li gel. Ch nhiu kh nng qu trnh polyme
ha khng xy ra do Ammonium persulphat khng cn tt khi dng lu,
nn chun b dch ny dng trong tun. iu cn ch na l gel phi
c bc k vi SaranWrap qua m.
9, Chun b gel trc khi chy:
a. Ly SaranWrap ra khi gel.
b. t gel vo thit b in di (bn knh di t p vo thit b), chc
chn l ct b lp bng dnh y gel vic in di xy ra.
c. 0,5X TBE vo b m di ca thit b, lng m cn phi
cho bn gel nm trong m.
d. m vo b trn ca thit b, m bo gel nm di lp m. Kim
tra xem m c b chy ra hay khng.
e. Chy th 20-30 pht ti hiu in th 2000V (phi cn thn v rt
nguy him), sau nhit ca bn gel nn t ti 50
o
C.
10, X l nhit vi cc mu ADN in di ti 85
o
C trong 5 ht v trn u,
trn cho n khi ln mu.
11, Tt ngun in vo thit b in di (nh ngt ngun in vo thit b
trong bt k trng hp no khi thao tc vi thit b).
12, Trong khi x l nhit vi cc mu th ra b mt vi m trong b
in di, dng pipet hay syringer 60 ml. Mc ch ca thao tc ny l loi
lp urea trn b mt gel.
13, nh du v tr nh ca mt gel trn bn knh trc ca gel.
14, t lc rng c mp ln mt gel, cc rng c t trn mt gel v
ngp su vo mt gel khong 1 mm. Chc chn l vng khng gian gia
cc rng c gii hn gia cc rng v gel. Nh vy phn lc rng c
mp tr thnh cc cnh bn ca ging, b mt gel l y ca ging tra
mu (hnh 1.10).

Hnh 1.10. V tr t lc sau khi gel.

Phi m bo chc chn lm sch b mt gel trc khi t lc rng
c mp ln trn gel. Nu khng thc hin vic ny s khng c c
phn kt qu pha trn ca gel. Nu quyn thc hin bc ny c th ra
sch tng ging tra mu nhng vic ny s kh khn hn. Nu ln nhiu
mu th trc cn phi phi lm sch ging v urea s khuych tn v
tch t ti b mt gel ti thi im ln mu.
15, Vic x l nhit 85
o
C i vi cc mu in di nhm lm bin tnh
ADN, ch ln khong 2-4 ml mu cho mi ging (ln mu theo th t
ACGT), c th dng bt vit ln knh nhm trnh nhm ln.
16, Qu trnh in di vi hiu in th 2000V cho n khi mu BPB
(Bromophenolblue) di chuyn n tn cng bng gel (1-2 gi). Nu chy
gel c gradient mui s cho php c c nhiu base hn trn mt gel
do vic lm ngn li khong cch gia cc bng ADN phn cui bn
gel. Nn dng in di t 45 pht 1 gi. Tt ngun in, b sung 1/2
th tch 3M NaOAc vo b m y, trn u. Li tip tc cng vic chy
gel cho n khi BPB di chuyn n cui bn gel. Ch s c mt ca
mui nng cao s dn n nhit tng, do cn phi kim sot
nhit ca qu trnh chy gel nhm trnh v knh v hng gel.
Chun b gel cho pht hin bng nh phng x.
17, Ly bn gel ra khi thit b, ch b m y cha cht phng x,
cc nucleotid d s i ra khi bn gel.
18, b b m trn.
19, t bn gel trn bn th nghim sao cho tm knh ngn ln trn. Ph
ln mt lp SaranWrap, dng lnh t ln trn khong 2 pht cho
gel co li, dng thanh nha tch cc lp knh ra, gi nguyn khng cho
tm knh trn tip xc li vi gel v nh vy gel s dnh v b v. Lc ny
gel s ch dnh vo tm knh di khng c silicon c ph khi gel.
20, t tm giy Whatman ln trn gel v vut u nh nhng, lc
gel s dnh vo giy v c th ly ra d dng (Ch trong trng hp s
dng cht phng x l S
35
th nn c nh mu theo cch di y).
21, Sau khi ly gel ra hi tm knh y th bc gel vi SaranWrap, ch
trnh khng kh i vo gia lp SaranWrap v gel, iu ny s cn tr
nh phng x trn phim.
22, C th a vo thit b sy gel.
23, Sy gel trong khong 45 pht ti 80
o
C.
24, t phim X-ray theo ng ch dn vo gel trong hp cassette (c phim ch c mt mt
th mi thao tc phi ng c nh phng x). Thi gian nhn c bc x l 12-14 gi i
vi P
32
v 24-48 gi i vi S
35
.
25, c kt qu.
C nh gel: Mc ch ca bc ny l loi bt urea khi gel v s c mt ca urea s hn
ch s phn cch gia cc ADN, cc bc c thc hin nh sau:
+ t bn knh c gel vo khay chm trong hn dch 15% ethanol v 10% acetic
acid.
+ C nh trong 10-15 pht urea khuych tn khi gel.
+ Dng giy lc thm ht dch c nh khi gel.
+ Tip tc lm theo cc bc nh quy trnh hin nh phng x.
Ha cht v cch chun b:
Phng php: Gii trnh t ADN si kp s dng KIT bin tnh nhanh vi
sequenase.
Phng php ny thch ng vi vic gii trnh t cc plasmid si i.
Bt u bng vic gy bin tnh trong kim (NaOH) hoc glycerol, sau kt
ta ADN v bt cp vi ADN mi dng gii trnh t. Phng php gii trnh
t ADN vi si i cn lng ADN mi cao gp 10-20 ln so vi si n. Vic
gii trnh t gm hai phn ng: phn ng nh du v phn ng dng ti im
cui. Trong phn ng nh du cc si ADN b sung c gn kt vi cht
nh du phng x trong vic ko di chui. Sau phn ng ko di chui
ADN b dng mt cch ngu nhin do nng thp ca nucleotid trong dch
phn ng (0.08 mM). Bc tip theo l kt thc chi vi dideoxynucleotid khi
c nng cao ca deoxyribonucleotid (33,3 mM). Cc trnh t tip theo ca
si khun s c xc nh vi nng cao ca deoxynucleotid trong khi phn
ng nh du xy ra trc vic b sung dideoxynucleotid vo hn hp phn
ng. Quy trnh sau y c rt gn, c nhiu phn tin ch chi tit hn v
thay i trong cc ti liu hng dn.
iu quan trng c kt qu tt trong vic gii trnh t ADN l cht
lng cao ca ADN plasmid. Trong cc khu chun b vic xut hin cc phn
t t gy s dn n kt qu khng r, kh xc nh cc bng. Cc plasmid
si n phi c lm bin tnh cho m xon trc khi gii trnh t. Bin tnh
cc plasmid xon do lin kt ha tr khng phi lc no cng t c bng x
l nhit ti nhit bt cp mi hay trong m phn ng, bi l nhit m
xon thng l 105ng l 105
o
C. C hai phng php kh nhanh v tin li: Mt
phng php xut pht t thc t l glycerol hoc ethylen glycerol c tc dng
lm gim nhit m xon, cn phng php th 2 l m xon bng mi
trng kim. C hai phng php u thc hin n gin cho hu ht cc loi
ADN khun.
i vi hai phng php bin tnh ADN, nn dng lng ADN khun nng
0.5 pmol (05- 0.3 mg) v lng primer 2-5 pmpol. Nu s dng th tch
ADN nh hn th phi pha n nng thch hp bng nc. Kt qu tt nht
thu c l theo t l phn t mi : si khun l 4 : 1. T l ny nn c duy
tr khi tin hnh vi lng ADN khun khc nhau. Trong mi trng hp khi m
s lng thnh phn no qu t th u dn n bng khng r cui bn gel.
Lng ADN khun cn theo phng php ny t hn so vi s dng sequenase
KIT 2.0 do khng b mt ADN trong bc kt ta vi ethanol. Tc nhn gy
bin tnh ADN khun trong KIT ny l m 40:50 glycerol v ethylen glycerol.
Vic b sung m ny vo dung dch ADN theo t l ch dn; 40% glycerol s
lm gim nhit bin tnh ADN xung khong 20
o
C. Ch khi dng m
glycerol s gy nn vic di chuyn khng u trn gel in di ti vng c
khong cch 300-400 k t on ADN mi. Nn s dng m chy gel hn ch
tc dng ca glycerol (thay taurin cho m chy gel TBE) s khc phc c
iu ny.
ADN plasmid s bin tnh ti bt k nhit no pH kim (pH 13). iu
quan trng i vi phng php ny l vic trung ha bc bin tnh kim
bng HCL phi c thc hin mt cch chnh xc. Nh vy nn dng mt loi
pipet cho vic b sung kim v trung ha bng acid HCL sau . Ni chung
dung dch NaOH v HCL c chun b c nng 1M, trong trng hp dng
ha cht ring phi m bo nng ch dao ng trong khong 0,95- 1.05M.
Bc gy bin tnh kim v kt ta vi ethanol cng c th c thc hin vi
cc ha cht trong KIT nhng i khi cng khng t c kt qu mong mun
v cn phi ci tin.
Trong trng hp gii trnh t vi ADN si n, khng phi thc hin bc
bin tnh ADN, hn dch c chun b n gin gm: ADN plasmid, 05 pmol
primer, 2 ml m phn ng v b sung nc cho 15 ml.
Phn ng tt s c c trnh t di khong 400 bps.
Cch tin hnh c th:
1. Bin tnh ADN (theo hai cch trnh by trn).
A. Bin tnh vi glycerol:
ADN: 0.5 0.3 mg (th tch ti a 7 ml).
m bin tnh plasmid: 5 ml.
Primer: 1 ml.
Nc ct dn n: 13 ml.
Trn u, trong 5 pht 90 100
o
C, lm lnh nhanh trn .
B sung thm m phn ng: 2 ml t c tng th tch l 15
ml.
B. Bin tnh vi kim (khng cn kt ta vi ethanol).
Thnh phn phn ng gm:
ADN: 0.5 - 3mg (th tch ti a l 8ml)
NaOH 1M: 2 ml.
Primer: 1 ml.
Nc ct dn n: 11 ml.
Trn u, gi trong 10 pht ti 37
o
C, lm lnh nhanh
trn .
iu quan trng trong phng php ny l bc trung ha phi
c thc hin chnh xc vi HCL.
Sau b sung HCL 1M : 2 ml.
m phn ng plasmid: 2 ml.
Tng th tch l: 15 ml.
2. Bt cp mi vo si khun:
Gi hn dch ADN v mi 37
o
C trong 10 pht, sau gi trn
lnh (mu ch nn dng trong vng 4 gi).
3. Chun b tube cha hn dch kt thc chui:
Trong khi thc hin bc bt cp si khun v si mi, tin thnh ht
2,5 ml hn dch kt thc chui cho tng loi (ddA, ddC, ddG, ddT) cho
vo tng tube ring bit. y np, trn dng cho cc bc 5
v 7.
4. Pha long dch nh du:
Pha long dch nh du 5 ln vi nc v gi trn . Khng nn pha
long dch nh du khi mun c on xa mi.
Dch nh du: 2 ml
Nc: 8 ml
Tng s: 10 ml.
5. Lm m cc mu chun b trong bc 3:
Bn mu c chun b trong bc 3 ln lt l ACGT c 37
o
C
trong 1 pht.
6. Phn ng nh du (k t bc ny th mi u cn v pipt s thc
hin vi ha cht phng x, do cn phi c cc bin php bo v v
eo gng tay).
B sung ln lt cc thnh phn sau vo 15 ml hn dch bt cp
c chun b trong bc 1.
DTT 0.1M : 1 ml
Dch nh du (bc 4): 2 ml
Ha cht phng x: S
35
(P
33
, P
32
): 0.5 ml
Enzym T7 sequenase cho plasmid: 2 ml
Tng th tch : 20.5 ml
(Th tch ny s c chia ra 4 phn trong bc 7)
Trn u (trnh bt), hn dch ti nhit phng 2 -5 pht (i vi
plasmid lm bin tnh trong glycerol th 5-10 pht ti nhit phng
hoc 5 pht ti 37
o
C). Thng b sung hn dch T7 sequenase cho
plasmid cui cng, sau khi cc thnh phn khc c b sung. Khng
bao gi b sung hn dch T7 sequenase cho plasmid vo hn dch nh
du, DTT hay hn dch khc khng phi l m.
Ch : Nu trong trng hp cht phng x S
35
qu lu th c th
tng lng ln 2 ml cho mi phn ng, sau gim lng nc tng
ng.
7. Phn ng kt thc chui:
a. Ly 4,5 ml hn dch thu c t bc 6 cho vo thnh trn ca 4 tube
kt thc chui c lm m bc 5 (A, C, G, T). y np li v li
tm nhanh trn u, tip tc trong 5 pht ti 37
o
C (c th ko di
ti 30 pht). Nu thy cn thit th c th li tm nhanh thu ly hn
dch trc khi tin hnh phn ng nh du.
b. Ti thi im cui ca 5 pht, b sung 4 ml hn dch dng phn ng
vo thnh trn ca cc tube v ng np nhanh. Nh vy c th dng
phn ng ti thi gian thch hp bng cch li tm dch dng phn ng
i xung hn dch phn ng.
c. Ngay lp tc li tm dch dng phn ng i xung hn dch phn
ng, sau bc ny phn ng nh du hon tt v mu c th c
gi 4
o
C s dng trong ngy hm sau.
d. X l nhit 75
o
C cho cc mu khong 2-3 pht ngay trc khi tra
mu ln gel, vi cc mu cha glycerol th vic ln mu d dng vi u
cn vng.
e. Ln khong 2-3 ml mu cho mi ging trn gel gii trnh t. Nn a
mt lng ng mu trn cc ging. Cch tt nht ln mu khng b
bt l ht 3 ml mu nhng khi tra ch dng 2 ml l .
Ha cht:

Hn dch kt thc chui:

Tube A C G T
dATP 80M 80M 80M 80M
dCTP 80M 80M 80M 80M
7-deaseGTP 80M 80M 80M 80M
dTTP 80M 80M 80M 80M
ddATP 8M
ddCTP 8M
ddGTP 8M
ddTTP 8M
NaCl 50 mM 50 mM 50 mM 50 mM

Dch dng phn ng:
95% Formamide
20mM EDTA (Na2), pH 8.0
0.05% Bromopheno Blue (BPB).
0.5% Xylen CyanolFF (XC).
Ch , nu bn gp trc trc vi vic gii trnh t ADN th cc plasmid
nn c ti tinh sch vi Genee-Clean hoc sc k trao i ion.




Hnh 1.11. Tra mu trn thit b chy gel.
Cc phn di y cp n mt s vn ny sinh v cch gii quyt
khi tin hnh gii trnh t ADN.
- Mun c c trnh t ADN di hn:
Yu cu u tin l phi c c on gene n mc c th trn gel.
Thng thng th kch thc c cho mi phn ng trn gel l 250-300
nucleotid. Vi thi gian chy gel lu khong 8 gi th c th c c
kch thc gene di hn 400 nucleotid. Nu s dng gel gradient s
khng ph hp cho mc ch ny v khong cch gia cc bng ADN s
hp ti mc khng th c c. Nng gel acrylamid dao ng 4-6%
l thch hp cho kt qu tt. C mt gii php na l c th s dng gel
vi gradient mui nhng vn gi nguyn thi gian chy gel (xem phng
php 2, bc 16).
- S dn cc bng:
Nguyn nhn chung khng th hon tt vic gii trnh t ADN l cc
bng khng r do cu trc bc hai trong sn phm ca phn ng ko di
chui, iu ny thng c m t l s dn p cc bng do cc bng b
thu hp mt cch bt thng trn nh fim ca kt qu gii trnh t ADN.
Cc khong cch hon ton nh nhau khi m khong 3 lin kt G-C b
sung km vi 2-5 base c th dn n kt qu khng th c c on
gene chim mt khong nh trn bn gel. Khong trng bt thng l do
nh hng ca chui vi khong cch ngn ca cu trc bc 2 s c cng
tc di chuyn nh l si thng c chiu di ngn, ti v tr bn fim
ngoi vng dn bng th khong cch cc bng s rng khc thng do
s km bn ca cu trc bc 2 lm cho chiu di ca chui tng ln v
s di ng ca chui tr nn nh bnh thng.
Vic gii trnh t qua vng gene dn nn c th c khc phc bng
cch thc hin vic c trnh t trn c hai si. S bt thng v vic di
chuyn ny sinh cc chui c chiu di khng cho vic bt cp. iu
ny c ngha vng gene c cc bng khng r rng s c khc phc
bng cch s dng dITP thay cho dGTP trong phn ng gii trnh t ADN.
L do l base I ch c 2 lin kt vi C thay v 3 lin kt trong trng hp
vi G, kt qu l ADN s d dui hn. K thut ny thng c dng,
do dITP c trong KIT sequenase.
Mt cch khc c th lm km bn vng base dn nn do cn cp i
l thc hin chy gel trong iu kin bin tnh (v d s dng formamide
trong nc dng gel). Trong trng hp ny khi thay th 25%
formamide cho nc s t c kt qu tt nht. Gel c formamide
thng c dng khi vng dn nn kh gn mi, nn x l formamide
qua gel trao i ion ngay trc khi chun b gel.
Trong cc trng hp m cc cu trc cp i phc tp hn khi m vng
dn nn li nm ngay ti v tr tng ng trn si i din. Khi cch
gii quyt s tr nn kh khn v vi cc k thut hin c thng a li
cc kt qu khc nhau.
- Cng cc bng thiu s ng nht:
Mt vn khc trong phn ng gii trnh t ADN l cng cc bng
khng ng nht. iu ny thng xut hin trong phng php gii trnh t
vi thit b t ng da vo phng php pht hin bc x hunh quang. S
thiu ng nht l do s c mt ca sequenase cn Mg
++
gn dideoxy kt thc
chui khc nhau ti cc v tr khc nhau (vn ny c bn lun k trong ti
liu USB sequenase).

b. K thut gii trnh t ADN trn thit b gii trnh t
ADN t ng:



Hnh 1.12. Thit b gii trnh t ADN t ngg
(3100-Avant Genetic Analyzer)

Trong phn ny chng ti cp phn ng gii trnh t ADN trn 2
loi thit b ch yu hin ang c dng ph bin l: Beckman Coulter v ABI
3100-Avant
Chun b mu gii trnh t ADN:
1. Thnh phn phn ng (cho 1 phn ng 20 ml):
- Terminator Ready Reaction Mix*: 8 l
- Template: 80-100 ng
( vi plasmid l: 300ng)
- Primer : 3.2 pmol
- Nc ct v trnh: 20 l
Chun b Teminator Ready Reaction Mix cho 4 phn ng:
15 l Bigdye Sequencing Buffer 3.1 (5X) + 15 l nc = 30 l (2.5X)
20 l m 2.5X + 20 l Ready Reaction Premix= 40 l Terminator Ready
Reaction Mix (dng 8 l) cho mt phn ng.
2. Tin hnh phn ng:
Phn ng c thc hin trong 200 ml tube hay trong microplate.
Cycling c thc hin trong my PCR theo chng trnh sau:
Denature 96
o
C: 1 pht
96
o
C: 10 giy
50
o
C: 5 giy
60
o
C: 4 pht
Cycling: 25 cycles.
3. Kt ta sn phm PCR:
- Cho vo 5l EDTA 125 mM + 60l E-OH 100%E-OH 100%
- Trn u, 15 pht nhit phng. Ly tm 15 000 v/pht
- Ra li bng 60l E-OH 70% v li ly tm (4
o
C)
- Lm kh mu nhit phng (Speed Vac).
- Khi mu kh, thm 10l Hi-Di formamide, trn u sau 96
o
C
trong 2 pht.
- trn trc khi cho vo seq.
4. a mu vo my c trnh t.

Phng th nghim "o" :
http://web.mit.edu/7.02/virtual_lab/PBC/PBC5virtuallab.html
Formazan gm MTT (3-(4, 5-dimethyl-2-thiazolyl)-2, 5-dihphenyl-2H-tetrazolium bromide) v
nc ha tan XTT (23-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide).
Bi 13 Gii thiu mt s k thut bo qun vi sinh vt
1. Phiu thng tin v chng vi sinh vt bo qun:

i hc Quc gia H Ni
Trung tm Cng ngh Sinh hc
Bo tng Ging chun vi sinh vt (VTCC)
Thng tin chung v
chng vi sinh vt bo qun

1. Nm si Nm men X khun Vi khun
2. Tn khoa hc:
Ging (Genus) Loi (Species) Di loi (Subspecies)
Tn khc nu c (synnonym):
3. Ngun phn lp:
Ni phn lp:
4. Thi gian bt u bo qun ti VTCC:
5. Ngi phn lp:
6. Ngi cung cp: Ni cung cp:
7. K hiu chng VTCC:
8. K hiu l l lch chng t cc bo tng khc:
VTCC < < < < <
9. Chng chun (Type) Chng t nhin (wild) t bin (c th)
10. Hnh thc sinh sn:
11. Gy bnh cho: Ngi ng vt Thc vt Khng
12. Du chun di truyn (nu c):
13. Hnh thi t bo, khun lc:
14. Kh nng ng dng:
15. Ti liu lin quan:
16. Cc phng php bo qun:
ng kh Lnh su Nit lng Cy truyn
17. Mi trng nui cy thch hp:
18. Nhit nui cy thch hp:
19. Ghi ch:
2. Chc nng ca b su tp vi sinh vt:
Bo qun vi sinh vt c tm quan trng c bit, lm nn tng cho cc nghin cu c bn v
ng dng lin quan n nhiu lnh vc: sinh hc, y hc, nng nghip v mi trng.
Nhim v quan trng nht ca B su tp chng vi sinh vt l thu thp, lm giu cc chng
vi sinh vt hu ch v bo qun chng theo phng php thch hp. Vic thu thp cc chng vi sinh
vt c th bng nhiu cch nh phn lp, tuyn chn t mi trng, trao i trong nc v quc t.
Cc chng vi sinh vt phi c nh hng theo tng mc tiu c th ca tng B su tp, v d
cc chng vi sinh vt chun, cc chng c hot tnh sinh hc v cc chng lm c s cho tra cu khi
nghin cu tnh a dng ca vi sinh vt.
Bo qun cc chng vi sinh vt l cng vic khng d dng, xut pht t mc ch ca bo
qun khng nhng l duy tr kh nng sng ca vi sinh vt, thun chng, trnh tp nhim m cn
m bo tnh n nh di truyn v cc c tnh sinh hc trong sut qu trnh bo qun. Thc t
khng c mt phng php bo qun no l vn nng dng chung cho cc nhm vi sinh vt m mi
nhm vi sinh vt ch thch hp vi mt vi phng php bo qun nht nh.
Cc chng vi sinh vt bo qun s c cung cp cho ngi s dng do nhim v quan
trng ca b su tp vi sinh vt l thu thp v cung cp cc thng tin quan trng ca chng vi sinh
vt bo qun cho ngi s dng nh: mi trng nui cy, nhit , nhu cu dinh dng, tnh an
ton sinh hc, tn phn loi v.v..Nh vy yu cu i vi cn b ph trch B su tp vi sinh vt
phi c kin thc vng v vi sinh vt, di truyn hc, sinh ho hc, sinh l vi sinh vt v bnh hc vi
sinh vt kim sot c cc c tnh quan trng ca cc vi sinh vt bo qun.
3. Mt s im lu i vi mt B su tp vi sinh vt:
3.1. Duy tr kh nng sng ca vi sinh vt bo qun:
Trong khi thc hin cc phng php bo qun v trong qu trnh bo qun cc t bo vi sinh
vt s b cht do phi p dng phng php bo qun thch hp nhm hn ch thp nht kh
nng cht ca t bo.
3.2. Quan tm n s lng t bo khi tin hnh bo qun:
Trong qu trnh bo qun th s lng t bo vi sinh vt gim dn theo thi gian do vy cn
tnh ton s lng vi sinh vt ti thi im bo qun thch hp duy tr s lng vi sinh vt sng
trong thi gian bo qun di.
3.3. Duy tr c tnh di truyn n nh ca chng vi sinh vt
bo qun:
Ni chung vi cc chng vi sinh vt bo qun, c bit vi cc chng vi sinh vt chun, cc
chng vi sinh vt c ng dng trong cng nghip th yu cu duy tr c tnh sinh hc, tnh trng di
truyn l rt quan trng. Cc phng php bo qun khng thch hp c th dn n t bin hoc
mt plasmid. V vy cn phi chn cc phng php bo qun thch hp cho cc chng ny.
3.4. Tnh thun chng ca vi sinh vt bo qun:
Chng vi sinh vt t khi bo qun n khi s dng phi m bo thun chng ng theo tn v
cc c im sinh hc c trng. y cng l yu cu tin quyt i vi cng vic ca mt Bo
tng vi sinh vt, do vy m cc thao tc v phng php tin hnh phi c thc hin sao cho hn
ch ti mc ti thiu i vi cc chng bo qun nhm trnh tp nhim.
3.5. Kinh ph cn cho B su tp vi sinh vt:
Kinh ph bao gm kinh ph v lng cho cn b, thit b, vt t ho cht, nh xng v in
nc tiu hao. Cc kinh ph ny tu thuc vo quy m ca B su tp ging vi sinh vt v phng
php bo qun, phm vi dch v thc hin i vi khch hng.
Tn Bo tng vi
sinh vt (vit tt)
Nc
S lng chng vi sinh vt
ATCC M 73507
DSMZ c 14460
NBRC Nht 18300
Bng 1. Quy m ca mt s b su tp ging vi sinh vt.

STT Tn Bo tng vi sinh vt, Nc Gi thnh (USD)
1 ATCC, M 80
2 CBS, H Lan 60
3 VKM, Nga 45
4 Thi Lan 40
Bng 2. Gi thnh cho bo qun mi chng vi sinh vt hng nm

3.6. Bo qun cc chng c gi tr:
i vi cc chng vi sinh vt c gi tr th tu theo yu cu m cn thc hin nhiu phng
php khc nhau cng nh bo qun ti cc ni khc nhau hn ch kh nng mt cc c tnh qu
cng nh mt chng do nhng ri ro ngu nhin (chy n, ng t, chin tranh v.v..).
3.7. Cung cp chng ging cho khch hng:
i vi cc chng cn cung cp nhiu cho khch hng (hoc cc chng cn cho nghin cu
thng xuyn) th cn phi bo qun vi s lng ln vi phng php thch hp cho vic vn
chuyn n khch hng.
3.8. Cung cp cc thng tin lin quan n chng bo qun:
Cht lng ca B su tp ging lin quan n cc s liu, thng tin v tng chng vi sinh vt
bo qun. Do , nhu cu nghin cu thu nhn cc thng tin cn thit cung cp cho khch hng
l rt quan trng.
3.9. Kim tra cht lng chng vi sinh vt bo qun:
Cc chng ca B su tp vi sinh vt cn phi tun theo cc qui nh nghim ngt kim tra
nh k theo tng thi gian bo qun nh t l sng st, mc tp nhim, thay i c tnh di truyn
nh du chun di truyn, s tn ti ca plasmid, c im phn loi, kh nng sinh cc cht c hot
tnh sinh hcMt trong cc cch nh gi kh nng sng st ca chng vi sinh vt bo qun l
da theo phng trnh sau:
t = 8Log(So/Log So-Log Sac)
Trong : t - Thi gian ca mu bo qun trong ampoule.
So - S lng t bo sng st ngay sau khi bo qun.
Sac - S lng t bo sng st ngay sau khi tin hnh th nghim.
3.10. C s d liu ca B su tp vi sinh vt:
Ngy nay, khi tin hc l lnh vc xm nhp v lm thay i su sc n mi lnh vc i sng
th vic qun l B su tp vi sinh vt khng cn l mt ngoi l. Ngi ta ng dng cc phn
mm my tnh ph hp qun l cc chng vi sinh vt bo qun, cc thng tin lin quan cng nh
chng trnh kim tra cht lng nh k. S dng tin hc l cng vic c tin ch ln, n gip cho
ngi qun l nm bt c ton b thng tin cng nh l lch ca cc chng vi sinh vt v d dng
tra cu, lm bo co khoa hc theo cc tiu ch ring. Ni chung vi cc b su tp c s lng vi
sinh vt khng ln th c th s dng phn mm: Microsoft Access, Access Visual Basic.
4. Gii thiu chung v mt s phng php bo qun vi sinh
vt:
Trong phn ny chng ti m t cc phng php c dng chung cho cc i tng vi sinh
vt chnh (vi khun, nm si, nm men, x khun, vi to).
4.1. Phng php cy truyn vi sinh vt:


Hnh 1. Bo qun bng phng php cy truyn trn mi trng thch
y l phng php bo qun n gin, cc chng vi sinh vt c cy trn mi trng thch
hp (dch th hay trn thch) trong ng nghim hay bnh tam gic v trong iu kin thch hp
cho vi sinh vt pht trin. Sau cc chng vi sinh vt ny c chuyn n ni bo qun c nhit
thch hp. Qu trnh ny c lp li trong mt thi hn nht nh, m bo chng vi sinh vt
lun c chuyn n mi trng mi trc khi gi v cht. Thc t c nhiu chng vi sinh vt
thch hp vi phng php bo qun ny nh: Staphylococi, Coliform... c th sng c vi nm
theo cch ny. Cho d phng php ny l phng php kh ph bin c dng trong cc c s
nghin cu v s dng cc chng vi sinh vt c bit l cc chng ang dng cho nghin cu. Tuy
nhin, phng php ny cng bc l nhiu nhc im sau:
- D b tp nhim v d dn n mt chng ging gc.
- Mt hay nhm ln nhn hiu giu cc chng trong qu trnh bo qun.
- Phi nghin cu v theo di thi gian cy truyn thch hp i vi cc chng bo
qun.
- Tn nhiu cng sc cy truyn.
- Ging gc c th mt do sai st khi dng mi trng cy truyn khng thch hp.
- Chng vi sinh vt cy truyn d b thay i cc c im sinh hc do t bin xut
hin sau mi ln cy truyn.
* Phng php lm mt nc trong mi trng bo qun:
Phng php ny thng dng cho cc chng nm si v nm men. Theo phng php ny
cc chng vi sinh vt c th c bo qun vi cc cht mang ph bin nh sau:
a. Trn t, ct v silicagel. Cc nghin cu cho thy l bo t nm c th
sng 4-5 nm khi b lm kh trong t m khng b thay i cc c tnh sinh hc.
Ngy nay silicagel l cht mang c dng ph bin v c hiu qu i vi bo
qun nm men, nm si.
b. Bo qun trn giy: Cc chng nm men v nm si c lm kh trn
giy v sau c bc bng giy bc v ng trong hp kn. u th ca phng
php ny l bo qun c lng mu ln.
c. Bo qun trn gelatin: thc hin phng php ny, ngi ta to dch
huyn ph chng vi sinh vt trong mi trng c gelatin. Sau cc git mu
c lm kh trong a petri. Phng php ny c th bo qun c vi khun
trong vi nm.
Nhn chung, khng c phng php no l vn nng cho bo qun cc nhm vi sinh vt
khc nhau. Thc ra l rt kh khi nh gi mt cch y xt theo mi yu cu c t ra
trn. Chnh v vy m cc phng php bo qun phi c kim nghim thc t vi tng loi vi
sinh vt, t kt qu c th chn ra phng php thch hp hoc ng thi s dng cc phng
php khc nhau.
4.2. Phng php ng kh vi sinh vt v phng php ng
kh trc tip:
a. Phng php ng kh:

Hnh 2. ng kh vi sinh vt.
ng kh l qu trnh m nc c ly ra khi mu khi cc mu ang trng thi lnh
su. y vi sinh vt c huyn ph trong mi trng thch hp v c lm lnh trong mi
trng chn khng. Thit b ng kh s ht nc v cui cng mu c lm kh n mc nht
nh. Mu c hn kn cho mi trng cha mu l chn khng. y l phng php ph bin
c hiu qu cao cho bo qun cc i tng vi sinh vt khc nhau nh nm si, nm men, vi khun
v mt s virut. Tuy nhin, phng php ny t c ng dng i vi to, ng vt nguyn sinh v
t bo ng vt.
b. Phng php ng kh dch th trc tip (L-drying):
Ngoi phng php ng kh nh m t trn, cn c phng php ng kh trc tip.
Khc bit vi phng php trn ch dch huyn ph vi sinh vt c lm kh nhanh ch
chn khng thch hp m mu khng cn lm lnh t trc. Phng php ny c bit c ngha
i vi nhm vi khun khng c kh nng sng trong nhit thp ca giai on tin ng. Cc
thng s quan trng cn c quan tm khi thc hin phng php ny l:
- Tui ca vi sinh vt bo qun.
- Thnh phn dch huyn ph t bo vi sinh vt.
- Tc ng kh.
- Nhit ng kh thp nht.
- Khong thi gian lm kh mu v m cui cng ca mu.
Phng php ny nhanh v thun li cho cc t bo qun s lng ln mu. Thng thng
theo phng php ny vi sinh vt c bo qun t 10-20 nm. Ni chung c hai phng php ny
c nhiu u im so vi cc phng php trc nh thi gian bo qun lu, tit kim c cng
sc v sai st nhn mc v tp nhim. Tuy nhin, nhc im ln nht ca phng php ny l gi
thnh thit b. n nh ca cc chng vi sinh vt bo qun theo cc t ng kh l khc nhau.
Hn th na cc chng trc khi em ra s dng phi c hot ho trn mi trng thch hp mt
s ln phc hi cc c tnh sinh hc.
4.3. Phng php bo qun lnh su:
i vi phng php bo qun lnh su th vi sinh vt c bo qun trong mi trng dch
th v nc cn cho hot ng sng ca vi sinh vt b bt hot nhit lnh su (-196

C -> -80

C).

Hnh 3. Bo qun lnh su
Vi phng php ny, t bo c th b v trong qu trnh lm lnh v lm tan mu. Mt
nguyn nhn dn n lm v t bo l vic tch lu cc cht in gii trong mu bo qun v hnh
thnh cc tinh th nc trong t bo. khc phc nhc im ny ngi ta b sung cc cht
lm hn ch tc lnh su v lm tan nhanh nh glycerol, DMSO (dimethyl sulfoxide). Vic bo
qun theo phng php lnh su ny c thc hin cc thang nhit khc nhau nh -20

C, -
30

C, -40

C, -70

C, -140

C v -196

C. Ni chung mc nhit cao hn -30

C cho hiu qu thp
do t bo chu nng mui cao sinh ra t cc cht in gii. Phng php bo qun ny c hiu
qu vi nhiu nhm vi sinh vt khc nhau nh nm si, nm men, vi khun, x khun v virut.
c bit vi phng php bo qun lnh su trong nit lng l phng php vn nng hn c.
Phng php ny thch hp vi nhiu i tng vi sinh vt khc nhau nh vi khun, nm si, nm
men, virut, to v c cc dng t bo ng vt. Tuy nhin, phng php ny cng bc l mt s
nhc im nh u t kinh ph cho thit b v in, nit lng hoc ri ro nh chy n... c bit
phng php ny khng thch hp vi cc chng vi sinh vt thng xuyn dng n. Ni chung
phng php ny thng c dng vi cc chng vi sinh vt c nhng c tnh qu m khng
thch hp vi phng php ng kh.

Hnh 4. Bo qun trong nit lng.
5. Mt s phng php ph bin s dng trong bo qun cc
nhm vi sinh vt c th:
Trong phn ny chng ti gii thiu c th mt s phng php thng dng nht.
5.1. Cc phng php dng cho bo qun vi khun v x
khun:
Bo qun vi khun:
a. Phng php ng kh (Freeze drying/
lyophilization):
y l phng php ph bin c s dng ti mi bo tng vi sinh vt trn th gii.
Phng php ng kh hn ch c s thay i cc c tnh ca vi sinh vt v bo qun c
trong mt thi gian di.
Phng php ng kh c trnh by y l phng php c dng ti NCTC (National
Colleciton Type Culture, Anh).
1, Nui vi khun cho ng kh:
Vi khun c cy trn mi trng ng thch nghing thch hp. Tuy nhin, cng c th
chun b dch vi khun trn mi trng dch th nhng phi lm m c t bo bng li tm. Tui
ca t bo cng rt quan trng do thng chn t bo nm trong pha sinh trng log.
2, Chun b m mu:
C th dng mt trong hai loi m sau:
+ m huyt thanh- inositol;
Meso-inositol: 5 gam
Huyt thanh nga (s 3): 100 ml
Thanh trng bng phng php lc vi khun, sau phn 5ml vo cc bnh v trng.
+ m inositol:
Mi trng dinh dng bt s 2: 2.5 gam
Meso-inositol: 5 gam
Nc ct: 100 ml.
Phn 5 ml vo cc bnh v kh trng ti nhit 121
O
C.
3, Chun b dch t bo:
Mt t bo c ngha quan trng i vi cht lng bo qun. Thng thng c th thu
sinh khi t mi ng thch nghing ch dng cho 1-2 ml m pha mu to dch huyn ph t bo.
Mt vi khun thng thng cn t 10
10
(i vi vi sinh vt khng c bo t). Mt trn c th
gim vi cc vi sinh vt c bo t. Hai m pha mu trn c th dng vi mi vi khun nhng trong
trng hp enterobacteria th khng dng m huyt thanh v s gy ra thay i c tnh min dch
ca vi khun.
4, Chun b ng ng kh:
ng ng kh c ra sch sau ngm qua m vi dch acid long (HCl 2%) v li c
ra sch, lm kh v chun b nt bng, ng c thanh trng kh hoc kh trng theo phng
php thng thng.
5, Chun b mu trc khi ng kh:
Lng dch huyn ph vi khun c a cn thn bng pipet pasteur vo ng ng kh
khong 0.1- 0.2 ml. Ch mi thao tc phi cn thn trnh dnh mu vo thnh hay pha trn ca
ng ng kh.
6, Bc tin ng kh:
Mc tiu ca bc ny l lm bay hi ht nc c th b lnh ng to . Trc khi tin hnh
ng kh mu c chun b qua bc tin ng (nh trnh by trn) sau bc tin ng kh
tin hnh khi mu c lp vo h thng ng kh v qu trnh lm mt nc trong iu kin lnh
c tin hnh khong 3 gi.
7, To ch tht trn ng ng kh:
Sau bc tin ng kh, yu cu phi to vt tht. Vic to vt tht c thc hin vi h
thng n hn. Tuy nhin, hin nay cc c s bo qun vi sinh vt s dng h thng hn v to vt
tht trn thit b t ng.
8, Hu ng kh:
Trong bc ny mu li tip tc c lm kh cho n m cui cng t khong 1%. Thi
gian cho bc ny c th thay i t 1-2 gi.
9, Hn ng ng kh:
Vic hn ng trc tip trn thit b ng kh (manifold) cn c kinh nghim v cn thn.
Trc ht phi tin hnh kho van v sau mi thc hin thao tc hn ng ng kh.
10, Kim tra chn khng ca ng ng kh:
Ngi ta s dng thit b l n pht hin mc chn khng. Vi cc mu t chn
khng cn thit th xut hin mu xanh tm nht. i vi cc mu khng t tiu chun th khng c
tn hiu hoc mu tm m.
11, Kim tra kh nng sng ca mu:
m s lng t bo l phng php ch yu nh gi cht lng bo qun. Vic m
c thc hin trc khi v ngay sau khi ng kh. Cc bc kim tra tip theo c th c thc
hin sau 1 hoc 5 nm nh gi cht lng ca mu ng kh. Ni chung kh nng sng ca vi
sinh vt c bo t cao hn vi sinh vt khng c bo t v loi gram dng cao hn vi sinh vt gram
m. Tuy nhin, vi vi sinh vt k kh th yu cu phi c tin hnh theo ng qui trnh, trnh vi
sinh vt tip xc vi oxy.
12, Bo qun mu:
Mu thng thng c bo qun ti 4
O
C hay nhit phng.
b. Phng php gi trong lnh su:
1, Chun b t bo cho lnh su:
T bo c nui cy trn mi trng v nhit thch hp nht ti gia hoc u pha log.
2, Pha dch t bo vi glycerol hoc DMSO thanh trng trc t nng cui cng
10% v mt t bo 10
6
.
3, Dch huyn ph t bo c a vo ng lnh su v ng np (trong trng hp hn ng
thu tinh, cn trong dch xanh metylene 0.05% pht hin cc ng b r r).
Ton b mu nhit phng trong 30 pht cho cn bng p sut thm thu trong v
ngoi t bo. Trong trng hp c nit lng th mu c chuyn sang bnh nit lng.
Mu a vo lnh su cng cn theo tc nht nh. Thng thng tc lnh dao ng 1-3
O
C/pht t ti nhit -30
O
C ban u. Sau mu c lm lnh tip vi tc cao hn 10-15
O
C/pht t ti nhit lnh cn thit (-100
O
C n -80
O
C). Hin nay, c cc thit b lm lnh
su c chng trnh ho vi tc mong mun. Tuy nhin, cng c th n gin hn l mu ban
u c t trong kh sau li a vo t lnh su v t thi gian vi gi t nhit -
65
O
C.
4, Hot ho mu: mu trong lnh (lnh su hay nit lng) c hot ho bng cch lm tan
nhanh ti mc c th (thng thng a ngay vo t m 37
O
C trong 40-60 giy). Nh vy, theo
cch trn c th t c kh nng sng 95% t bo sau 10 nm bo qun.
c. Mt s phng php bo qun khc: Bo qun trn gelatin v silicagel.
Bo qun x khun:
X khun mang c c tnh ca vi khun v nm si (c si v bo t) do cn c nhng
phng php bo qun thch hp.
Thng thng x khun cng c bo qun theo cc cch thng thng nh cy truyn,
ng kh v lnh su nh vi vi khun trn. Tuy nhin, c phng php kinh t m vn m bo
cht lng chng bo qun m chng ti trnh by y, l phng php bo qun trong t.
Cc bc tin hnh bo qun trong t:
1, Chun b t: ly t vn lm kh ti 150
O
C trong 6 gi lm cht cc vi sinh vt c
bo t v trng giun nu c. t sau khi thanh trng c nghin nh qua li c kch thc 30
mesh. t sau khi nghin c a vo ng nghim chun b nt bng v thanh trng trong 60
pht. Trc khi tin hnh bo qun phi kim tra nhim s khun trong t bng cch cy vng que
cy t t ng nghim t vo mi trng giu dinh dng cho vi khun v gi 24
O
C, 28
O
C v
37
O
C, kim tra sau 2-4 ngy.
2, Chun b dch huyn ph x khun. Nc ct v trng c dng to dch huyn ph x
khun (hay bo t x khun) t ng thch nghing. Ly 1 ml dch huyn ph cho vo mi ng
nghim v kh ti nhit phng (24
O
C) trong thi gian 1 thng. p nh vo thnh ng cho cc
ht tch u ra v bo qun mu ti 4
O
C.
3, Hot ho mu n gin bng cch ly vng que cy mu a ln mi trng (thch hoc
dch th) v nhit thch hp.
5.2. Bo qun vi to:
Vi to thng thng c bo qun theo 3 phng php: Cy truyn, lnh su v ng kh.
Cc phng php c trnh by chi tit trong phn vi khun. Mt s im cn ch khi bo
qun vi to l ch: Nn dng t bo gi pha cn bng, phng php lnh su cho kt qu tt hn
phng php ng kh.


5.3. Cc phng php dng cho bo qun nm si:
a. Phng php cy truyn:
Phng php ny thng c ng dng vi cc si nm c bo t. Cc chng nm si c
nui trn mi trng thch thch hp v cho si nm pht trin n mc cc i, sau l
qu trnh hnh thnh bo t, cc ng ging ny s c bo qun theo cc cch khc nhau:
- Cc ng thch c trong t lnh (4-7
O
C).
- Bo qun trong thch di lp du: Cc ng thch c bo qun di lp du
parafin c t trng tng i l 0.83- 0.89 c kh trng nhit 121
O
C trong 15
pht. Lp du cch b mt thch khong 1 cm. Nhit bo qun l 15-20
O
C.
- Bo qun trong nc, nm si c nui trn mi trng thch a. Sau khi si pht
trin cc i th ct thnh tng ming nh kch thc khong 6 mm
2
v cho vo l nc
thanh trng. Bo qun ti nhit phng.
b. Bo qun nm si c bo t trong silicagel:
Chun b:
1, L ng silicagel (10-15ml) c np chu nhit (st hay nha) thanh trng nhit 170
O
C trong 3 gi.
2, Silicagel: Loi trong sut, khng c mu, kch thc v s lng ht silicagel a vo
khng qu 1/3 th tch ca l, thanh trng nhit 170

C trong 3 gi.
3, Mi trng sa tch b (Skimmilk) 20% kh trng t (115
O
C/20 pht).
4, ng thch nghing (c th a petri) cha bo t nm si, tu theo tng chng, mi
trng nui cy m thi gian thu bo t thch hp khc nhau (10-15 ngy).
Cch tin hnh:
1, Dng pipet pasteur ly khong 3-4 ml sa thanh trng cho vo ng nghim cha bo t
nm si, dng que cy to dch huyn ph bo t c mt cao nht (i vi mt s chng c s
lng bo t thp th c th to dch bo t t mt vi ng thch bo t khc nhau).
2, Dng pipet pasteur ly dch bo t cho vo l ng silicagel. Ch silicagel ht nc v
sinh nhit do vic a dch bo t vo phi c thc hin trong chu nc . Lng dch bo
t a vo khng vt qu 1/3 th tch ht silicagel trong l. Sau dng tay lc u m bo cc
ht silicagel u dnh dch bo t nm si. Dn nhn, ghi cc thng tin cn thit nh s m chng,
ngy bo qun, mi trng v nhit thch hp...
3, L silicagel c y np cn thn nhng khng vn cht (ni 1,5 vng) sau gi trong
bnh ht m ( m 35-40%), 25
O
C trong 1 tun. Vn cht nt v qun giy parafil gi trong 4
O
C.
Kim tra sng st sau khi bo qun:
Mu silicagel 4

C, dng que cy v trng ly ra 3 ht silicagel trn mt thch mi trng

mm thc (Maltagar), lc u cho ht silicagel tip xc ln mt mi trng. nhit thch
hp, kim tra tra khun lc nm theo thi gian thch hp.
c. Bo qun nm si c bo t theo phng php ng kh (freez-drying):
Chun b:
1, ng ng kh ra sch (sonic cleaner) trong 15 pht sau kh trng 170
O
C trong 3 gi.
2, Mi trng sa tch b (Skimmilk) 20% kh trng t (115
O
C/20 pht).
3, ng thch nghing (c th a petri) cha bo t nm si, tu theo tng chng mi trng
nui cy m thi gian thu bo t thch hp khc nhau (10-15 ngy).
Cch tin hnh:
1, Dng pipet pasteur ly khong 3-4 ml sa thanh trng cho vo ng nghim cha bo t
nm si, dng que cy to dch huyn ph bo t c mt cao nht (i vi mt s chng c s
lng bo t thp th c th to dch bo t t mt vi ng thch bo t khc nhau).
2, Dng pipet pasteur ly dch bo t cho vo ng ng kh (0,2 - 0,3 ml), thng thng
chun b 13 ng cho mi chng. Dn nhn, ghi cc thng tin cn thit nh m s chng, ngy bo
qun, mi trng v nhit thch hp. Sau y bng nt bng hay giy bc.
3, Gi -80

C trong 3 gi trc khi tin hnh ng kh, ng thi bt my Dura-Stop, khi

nhit t -40
O
C, a mu vo t ng kh ca Dura-Stop, bt my Dura-Dry, khi mc chn
khng t 45 minitor, nhit -55
O
C, tng nhit Dura-Stop ln -5
O
C, qua m.
4, Tng nhit Dura-Stop ln 25

C, khi t nhit cn thit, tt my ht chn khng nhng

khng tt Dura-Dry.
5, Tt Dura-Stop, ln lt ni tube mu vo h thng manifold, bt my Dura-Dry, tin hnh
hn ng khi chn khng t 45-48 minitor, nhit -55
O
C.
Ch : Trong trng hp ch dng Dura-Dry c th c thc hin nh sau:
- Tin hnh cc bc 1,2,3,4 nh trn.
- Mu c tin ng kh -80

C trong 3 gi. Bt my Dura-Dry, khi t nhit -

60
o
C n -55
o
C, chn khng 45 minitor, gn ng mu vi h thng manifold tin
hnh hn ng khi chn khng t 45-48 minitor, nhit -55
o
C.
Kim tra sng st sau khi ng kh:
1, M ng ng kh bng cch t nng u hn trn n cn v lm lnh t ngt bng cn
t.
2, Dng pipet pasteur ly khong 1ml nc ct v trng t ng nghim cha 9,8 ml nc ct
v trng lm tan u mu ng kh. Ht ton b dch huyn ph sang ng nghim cha 9,8 ml
nc ct. Trn u v nh vo 3 v tr khc nhau (mi v tr 3 git) trn mi trng thch a thch
hp v nghing cho dch chy u dc theo mt hng nh hnh v:




3, Sau y np a, bao quanh bng parafil v nhit thch hp.
4, Cch nh gi:
Rt tt: 100 khun lc.
Kh: 50 100 khun lc.
Trung bnh: 10-50 khun lc.
Km: < 10 khun lc.
Khng mc.
Bo qun c thc hin vi cc mu t kh tr ln. Tuy nhin, c mt s nm si m khun
lc mc trn lan th c th nh gi nh sau: Nu cc khun lc mc u khp trn b mt a l tt.
M ng kh l thnh cng nu nh s khun lc mc chim t nht l na b mt mi trng trong
a petri. Cc mu c s lng khun lc mc di mc trn phi lm tng mt bo t trc khi
ng kh nh thay i mi trng sinh bo t, hoc chun b dch huyn ph bo t t 3-4 ng
nghim bo t nm si.
d. Bo qun nm si bng phng php ng kh trc tip:
Phng php ng kh trc tip thc hin tng t nh phng php ng kh trnh by
trn v c thc hin nh sau:
Chun b:
1, ng ng kh ra sch (sonic cleaner) trong 15 pht sau kh trng 170
o
C trong 3 gi.
2, Mi trng L-drying chun c thnh phn nh sau:
m phosphat: 0.1M : 100 ml.
Monosodium glutamat: 3 g
Adonitol: 1.5 g.
(Kh trng 115
o
C/20 pht).
3, ng thch nghing (c th a petri) cha bo t nm si, tu theo tng chng mi trng
nui cy m thi gian thu bo t thch hp khc nhau (10-15 ngy).
Cch tin hnh:
1, Dng pipet pasteur ly khong 3-4 ml mi trng thanh trng trn cho vo ng nghim
cha bo t nm si, dng que cy to dch huyn ph bo t nm c mt cao nht (i vi mt
s chng c s lng bo t thp th c th to dch bo t t mt vi ng thch bo t khc nhau).
2, Dng pipet pasteur ly dch bo t cho vo ng ng kh (0,2 ml), thng thng chun b
13 ng cho mi chng, dn nhn, ghi cc thng tin cn thit nh s m chng, ngy bo qun, mi
trng v nhit thch hp. Sau y bng nt bng hay giy bc.
3, ng thi bt my Dura-Dry, khi mc chn khng t 45 minitor, nhit -75
o
C, ln
lt ni ampoule mu vo h thng manifold (ch kho van chn khng khi ni ampoule mu vi
tube manifold sau li m ra, m bo chn khng cn thit khi lm ng kh phi ln lt
a mu vo khi tn hiu n ch th cho php). Khi chn khng t 45-48 minitor, nhit -
55
o
C tin hnh qu trnh lm kh trong 3 gi.
4, Hn kn ampoule mu:
Khi my c Dura-Dry ang chy m bo chn khng cn thit, ln lt kho van cho
tng ampoule mu v tin hnh hn vi cc mu ny ti v tr ng b tht. Dng ngn la gas t
nng phn ng tht cho u cc pha v xoay u cho thu tinh chy bt kn ng. Sau dng ngn
la gas ct ri phn trn (ni vi manifold) v phn di cha mu. Ht t li kho cc van ca
cc ampoule mu tip theo v hn ln lt nh trn.
Kim tra cht lng ngay sau khi bo qun:
- kim tra sng st ca mu nm si bo qun, c th thc hin theo cch
c m t phn ng kh.
- Kim tra thun chng: cc mu c cy trn mi trng thch hp v quan st
hnh thi v cc c im sinh hc c trng cn thit nh gi kh nng tp nhim
v c tnh sinh hc.
- Th nghim nh gi nhanh thi gian bo qun: cc mu sau ng kh trc tip c
gi 37
o
C trong 2 tun. sng st ca mu bo qun gim trong 2 tun 37
o
C tng
ng vi bo qun 10 nm 4
o
C.
e. Bo qun nm si bng phng php lnh su -80
o
C v nit lng (-196
o
C):
Chun b:
1, Chun b dung dch glycerol 10% (3 ml trong ng nghim) kh trng 121
o
C trong 15
pht. ng nha nt xoy (2 ml), ra sch trong sonic cleaner, kh trng 170
o
C trong 3
gi.
2, Chun b mu nm si trn mi trng thch a c lng bo t ln.
Cch tin hnh:
1, Dng pipet pasteur ht khong 0,3 ml glycerol thanh trng vo ng nha ng mu. Sau
dng dao v trng ct ly 1 ming nh mi trng cha c si v bo t nm si cho vo ng
nha. Lng glycerol ph ln ming thch mu, y nt v bao bng parafil sau dn nhn.
2, Mu trc tin phi 5
o
C (to iu kin cho glycerol i vo t bo), sau c lm
lnh su t 25
o
C xung -55
o
C vi tc -1
o
C/pht. Trong trng hp khng c thit b lm lnh th
c th -20
o
C trong 3 gi, sau chuyn sang -80
o
C hay nit lng (-196
o
C).
Ch : Vic bo qun theo phng php lnh su thch hp vi hu ht cc nm si sinh bo
t v t bo c vch ngn. Nhng phng php ny t ra khng thch hp vi cc nm si khng
sinh bo t v khng c vch ngn. Trong trng hp ny cn nui cy nm si trn mi trng
thch hp to bo t. Nu khng khc phc c th phi tin hnh nui trn mi trng dch th
thu ly sinh khi v gi trong glycerol 10% nh trn.
nh gi kh nng sng ca mu bo qun:
1, Lm tan mu (mu ly t t lnh su -80
o
C hoc nit lng -196
o
C) trong b n nhit 37
o
C
trong 2 pht.
2, Dng que cy ly cc ming mi trng thch t vo 3 v tr khc nhau trn mi trng
thch a thch hp. nhit thch hp v xem kh nng mc sau thi gian thch hp (2-4
ngy). Ch khi ly mu cn ly c si nm a vo mi trng thch a.
Mt s iu ch i vi bo qun nm si:
- nh hng ca nh sng vi qu trnh hnh thnh bo t: Phn ln trng hp th s
thnh cng ca phng php bo qun ph thuc vo s lng bo t. nh sng l mt
tc nhn quan trng i vi qu trnh hnh thnh bo t ca nm si, nh sng c bc
sng ngn c tc dng kch thch qu trnh hnh thnh bo t, nh sng gn vng cc tm
(3100 4000 nm) c tc dng thay i mu sc v kch thc khun lc.
- Thnh phn mi trng: Ni chung cc mi trng c thnh phn dinh dng ngho
th thng cho lng bo t ln.
- Trnh nhim tp cc con mt (mite), cc chng nm si lu gi thng b mt ph
hoi. u tin chng n chng ging v lm hng, sau gy tp nhim do mang bo t
v vi khun trn c th t ng ny sang ng khc. V l do trn m cn c bin php hn
ch mt, thng thng cc chng ging phi c bo qun ni sch, nhit 4-8
o
C.

5.4. Phng php bo qun nm men:
Tng t nh nm si, c nhiu phng php bo qun nm men. Chng ti ch cp n
cc phng php thng dng nht ang c s dng ti cc b su tp nm men ln trn th gii.
a. Phng php cy truyn:
Cy truyn l phng php n gin, nhanh v r tin cng nh thng dng nht, tuy nhin
nhng hn ch ca phng php ny l d tp nhim cng nh nhng thay i cc c im sinh
hc, tnh trng di truyn s l bt li khi bo qun theo phng php ny.
Cy truyn trn mi trng dch th:
- Cch tin hnh rt n gin l chun b 10 ml mi trng nui cy nm men (thng
thng l mi trng YM Yeast extract Maltose) trong l McCartney kh trng
nhit 121
o
C trong 15 pht. Thng lm 2 l cho mi chng bo qun.
- Mt vng que cy chng nm men bo qun c a vo mi trng bng thao tc
v trng v gi 25
o
C trong 72 gi. Phi kim tra s pht trin ca chng bo qun.
- Sau cc mu c gi 4
o
C.
- Thng thng theo cch ny thi gian bo qun cc chng thng thay i t 2- 6
thng.
Cy truyn trn mi trng thch:
Cy truyn trn mi trng thch tng t nh mi trng dch th nhng y mi trng
c b sung thch (1.6%). Ging sau khi cy c gi trong 72 gi nhit thch hp t
pht trin cn thit, sau c 4-8
o
C. Theo phng php ny ging c thi gian sng lu hn
phng php cy truyn dch th, thng thng ging c bo qun t 3-6 thng.
Thi gian bo qun c th c ko di t 2-3 nm nu nh cc ng ging c bo qun
trong du parafil thanh trng v c ln trn mi trng thch sao cho to mt lp dy
khong 1cm.
b. Phng php bo qun trong lnh su v ng kh (xem phn cc phng php dng cho
bo qun vi khun v x khun).

Bi 14 Dinh dng ca vi sinh vt

13.1. YU CU DINH DNG CA VI SINH VT
13.1.1. Thnh phn ho hc ca t bo vi sinh vt
C s vt cht cu to nn t bo vi sinh vt l cc nguyn t ho hc. Cn c vo mc yu
cu ca vi sinh vt i vi cc nguyn t ny m ngi ta chia ra thnh cc nguyn t a lng v
cc nguyn t vi lng. Cc nguyn t ch yu bao gm: C, H, O, N, P, S, K, Mg, Ca v Fe. Trong
s ny c 6 loi ch yu (chim n 97% trng lng kh ca t bo vi sinh vt), l C, H, O, N,
P v S. Cc nguyn t vi lng thng l Zn, Mn, Na, Cl, Mo, Se, Co, Cu, W, Br v B. T l cc
nguyn t ho hc tham gia cu to t bo vi sinh vt l khng ging nhau cc nhm vi sinh vt
khc nhau. V d nm men, nm si v vi khun c lng cha trung bnh ca 6 nguyn t ch yu
l khng ging nhau (bng 13.1):
Bng 13.1: Lng cha trung bnh cc loi nguyn t ch yu trong t bo mt s nhm vi sinh vt
(% trng lng kh)
Nguyn t Vi khun Nm men Nm si
C ~50 ~50 ~48
H
O
N
P
S
~8
~20
~15
~3
~1
~7
~31
~12
-
-
~7
~40
~5
-
-
Theo cc ti liu ca Tempest (1969), Pirt (1975) v Herbert (1976) th thnh phn trung bnh
ca cc nguyn t to nn t bo vi sinh vt ni chung l nh sau:
Bng 13.2: Thnh phn cc nguyn t cu to nn sinh khi t bo

Nguyn t % trng lng kh
*


Cc ngun dinh dng in hnh c s dng cho
sinh trng VSV trong mi trng
Trung bnh Bin
C
O
N
H
P
S
K
Mg
Ca
Cl
Fe
Na
Nhng
nguyn t
50
21
12
8
3
1
1
0.5
1
0.5
0.5
1
0.5
45-58
18-31
5-17
6-8
1.2-10
0.3-1.3
0.2-5
0.1-1.1
0.02-2.0
0.01-5.0
CO
2
, hp cht hu c
H
2
0, 0
2
, cc hp cht hu c
NH
3
, NO
3
-, cc hp cht hu c cha N
Nc, cc hp cht hu c.
Phosphate v cc hp cht cha P.
SO4
-2
, H
2
S, v cc hp cht cha S.
K
+
(c th thay th bng Rb
+
)
Mg
2+

Ca
2+

Cl-
Fe
3+
, Fe
2+
v phc cht ca Fe
Na
+

Ly t cc ion v c khc

khc,Mo, Ni,
Co, Mn, Zn,
..
*Cc t bo bao gm 70% trng lng l nc v 30% l cc nguyn liu kh khc. Mc trung bnh
ny c tnh theo sinh trng ca vi khun Gr(-) trong iu kin d tha cht dinh dng nui
cy theo m.
Vi khun lu hunh (sulfur bacteria), vi khun st (iron bacteria) v vi khun i dng (marine
bacteria) c lng cha cc nguyn t S, Fe, Na, Cl nhiu hn so vi cc nhm vi khun khc. To
Silic (diatom) c cha lng SiO
2
kh cao trong thnh t bo. Thnh phn cc nguyn t ho hc
cn thay i trong mt phm vi nht nh tu thuc vo tui nui cy v iu kin nui cy. Khi
nui cy trn cc mi trng c ngun N phong ph th lng cha N trong t bo s cao hn so
vi khi nui cy trn cc mi trng ngho ngun N.
Cc nguyn t ho hc ch yu tn ti trong t bo vi sinh vt di dng cht hu c, cht v c
v nc. Cht hu c thng bao gm protein, carbon hydrat, lipid, acid nucleic, vitamin v cc sn
phm phn gii ca chng cng nh cc cht trao i cht. phn tch cc thnh phn hu c
trong t bo thng s dng hai phng php: mt l, dng phng php ho hc trc tip chit
rt tng thnh phn hu c trong t bo, sau tin hnh phn tch nh tnh v nh lng. Hai l,
ph thnh t bo, thu nhn cc thnh phn kt cu hin vi ri phn tch thnh phn ho hc ca tng
kt cu . Cht v c thng ng ring r di dng mui v c hoc kt hp vi cht hu c.
Khi phn tch thnh phn v c trong t bo ngi ta thng phn tch tro sau khi nung t bo
nhit 550
0
C, cht v c thu c di dng cc oxit v c c gi l thnh phn tro. Dng
phng php phn tch v c c th nh tnh hay nh lng tng nguyn t v c.
Bng 13.3:Thnh phn ha hc ca t bo vi khun (theo F.C.Neidhardt et al.,1996)
Phn t kh (1) / t bo % khi lng S phn t S loi phn t
- Nc
- Cc i phn t
+Protein
+Polysaccharide
+Lipid
+ADN
+ARN
- Cc n phn t
+Aminoacid v tin th
+ng v tin th
+Nucleotid v tin th
- Cc ion v c
Tng cng
-
96
55
5
9,1
3,1
20,5
3,0
0,5
2
0,5
1
100
24 609 802
2 350 000
4 300
22 000 000
2,1
255 500



1
khong 2500
khong 1850
2 (2)
4 (3)
1
khong 660
khong 350
khong 100
khong 50
khong 200
khong 18



Ch thch:
(1) -Khi lng kh ca t bo vi khun Escherichia coli ang sinh trng l khong 2.8 x 10
-13
g.
(2) - Gi thit Peptidoglycan v Glycogen l 2 thnh phn ch yu.
(3) - T bo cha vi loi phospholipid, do tnh a dng ca thnh phn acid bo gia cc chi vi
khun khc nhau v do nh hng ca iu kin sinh trng m c nhiu hnh thc tn ti ca mi
loi phospholipid.
Nc l thnh phn khng th thiu duy tr hot ng sng bnh thng ca t bo. Nc
thng chim n 70-90% trng lng t bo. chnh lch gia trng lng ti v trng lng
kh chnh l lng nc trong t bo, thng biu th bng t l % tnh theo cng thc sau y:
(Trng lng ti - Trng lng kh) / Trng lng ti x 100%.
n v trng lng t bo trong dch nui cy thng c biu th bng n v g/l hay mg/ml.
Phng php nung kh t bo nhit 550
0
C thng lm phn gii mt s hp cht ca t bo v
vy khi tnh trng lng kh ca t bo nn dng phng php sy kh 105
0
C hay lm kh
nhit khng cao trong chn khng, hoc lm kh nhanh nh tia hng ngoi...
13.1.2. Cc cht dinh dng v chc nng sinh l
Vi sinh vt ch yu thu nhn c cht dinh dng t mi trng bn ngoi. Cn c vo chc
nng sinh l khc nhau trong t bo m ngi ta thng chia cc cht dinh dng thnh 5 nhm
ln:
1) Ngun carbon (source of carbon)
L ngun vt cht cung cp C trong qu trnh sinh trng ca vi sinh vt. Trong t bo ngun C
tri qua mt lot qu trnh bin ho ho hc phc tp s bin thnh vt cht ca bn thn t bo v
cc sn phm trao i cht. C c th chim n khong mt na trng lng kh ca t bo. ng
thi hu ht cc ngun C trong cc qu trnh phn ng sinh ho cn sinh ra trong t bo ngun nng
lng cn thit cho hot ng sng ca vi sinh vt. Mt s vi sinh vt dng CO
2
lm ngun C duy
nht hay ch yu sinh trng, khi ngun C khng phi l ngun sinh nng lng.
Vi sinh vt s dng mt cch chn lc cc ngun C. ng ni chung l ngun C v ngun
nng lng tt cho vi sinh vt. Nhng tu tng loi ng m vi sinh vt c nhng kh nng s
dng khc nhau. V d trong mi trng cha glucose v galactose th vi khun Escherichia coli s
dng trc glucose (gi l ngun C tc hiu) cn galactose c s dng sau (gi l ngun C tr
hiu). Hin nay trong cc c s ln men cng nghip ngi ta s dng ngun C ch yu l glucose,
saccharose, r ng (ph phm ca nh my ng) tinh bt (bt ng, bt khoai sn...), cm go,
cc ngun cellulose t nhin hay dch thu phn cellulose.
Nng lc ng ho cc ngun C cc vi sinh vt khc nhau l khng ging nhau. C loi c
kh nng s dng rng ri nhiu ngun C khc nhau, nhng c loi kh nng ny rt chn lc.
Chng hn vi khun Pseudomonas c th ng ho c ti trn 90 loi hp cht C, nhng cc vi
khun thuc nhm dinh dng methyl (methylotrophs) th ch ng ho c cc hp cht 1C nh
methanol, methane...
Ngun C ch yu c vi sinh vt s dng gm c ng, acid hu c, ru, lipid,
hydrocarbon, CO
2
, carbonat... (Bng 13.4)
Bng 13.4: Ngun C c vi sinh vt s dng
Ngun C Cc dng hp cht
ng glucose, fructose, maltose, saccharose, tinh bt, galactose, lactose,
mannite, cellobiose, cellulose, hemicellulose, chitin...
Acid hu c acid lactic, acid citric, acid fumaric, acid bo bc cao, acid bo bc thp,
aminoacid...
Ru ethanol
Lipid lipid, phospholipid
Hydrocarbon kh thin nhin, du th, du paraffin
Carbonate NaHCO
3
, CaCO
3
, phn
Cc ngun C
khc
Hp cht nhm thm, cyanide, protein, pepton, acid nucleic...

Hnh 13.1: Sn lng sinh trng ti u khi vi sinh vt d dng
s dng cc ngun C khc nhau
Ngun carbon thng c s dng trong cng nghip ln men l r ng (molasses). S khc
nhau gia r ng ma v r ng c ci c thy r trong bng 13.5
Bng 13.5: Thnh phn ha hc ca r ng c ci v r ng ma
Thnh phn T l R ng c ci R ng ma
ng tng s % 48-52 48-56
Cht hu c kh ng % 2-17 9-12
Protein (N x 6,25) % 6-10 2-4
K % 2-7 1,5-5,0
Ca % 0,1-0,5 0,4-0,8
Mg % khong 0,09 khong 0,06
P % 0,02-0,07 0,6-2,0
Biotin mg/kg 0,02-0,15 1,0-3,0
Acid pantoteic mg/kg 50-110 15-55
Inositol mg/kg 5000-8000 2500-6000
Tiamin mg/kg khong 1,3 khong 1,8
T l cc nguyn t trong cc hp cht cao phn t vi sinh vt c th thy r trong bng sau
y:


Bng 13.6: T l cc nguyn t trong cc cao phn t t bo vi sinh vt

Thnh phn % trng lng kh %C %H %O %N %S %P
Trung bnh Bin dao ng
Protein 55 15
c
-75 53 7 23 16 1 -
RNA
d
21 5
c
30
e
36 4 34 17 - 10
DNA
d
3 1
c
5
f
36 4 34 17 - 10
peptidoglycan 3 0
g
20
h
47 6 40 7 - -
Phospholipit 9 0
i
-15 67 7 19 2 - 5
Lipopolysaccharide 3 0
h
-4
j
55 10 30 2 - 3
Lipit trung tnh - 0-45
k
77 12 11 - - -
Acid Teichoic - 0
l
-5
d
28 5 52 - - 15
Glycogen 3 0-50
k
28 6 49 - - -
PHB - 0-80
k
45 7 37 - - -
PHA (C8)
m
- 0-60
k
56 9 23 - - -
Polyphosphat
d
- 0-20
n
68 - 61 - - 39
Cyanophycin
o
- 0-10 - 15 25 27 - -
a. Theo Herbert (1976). Cc thng s c thu nhn t cc vi sinh vt khc nhau, khng in
hnh cho mt nhm no.
b. E. coli (trong pha sinh trng log). Theo Neidhardt et al. (1990).
c. Cc t bo c ngun d tr C.
d. Bao gm cc cao phn t nh ARN, ADN, polyphosphate hoc mt s thnh phn ca thnh
t bo.
e. Ti mc c t l sinh trng cao.
f. Cc t bo sinh trng chm.
g. Cc loi k sinh khng c thnh t bo.
h. Vi khun Gram(+).
i. Cc chng thay th ngun phospholipid bng cc cht tng t cha P t do, trong iu kin
hn ch ngun P
j. Vi khun Gram(-)
k. Cc t bo trong iu kin hn ch ngun N.
l. Hn ch ngun P.
m. PHA (polyhydroxyaldehyde) cha 3-hydroxyoctanoic acid.
n. Mt s nm men v vi khun.
o. Mt s vi khun lam c ngun d tr N cyanophycin [(asp-arg)].
n

*PHB= Poly- - hydroxy butyrate
2) Ngun N (source of nitrogen)
Ngun N l ngun cung cp N cho vi sinh vt tng hp nn cc hp cht cha N trong t
bo. Thng khng l ngun nng lng, ch mt s t vi sinh vt t dng (thuc nhm ammon
ho-ammonification, nhm nitrate ho- nitrification) dng mui ammone, mui nitrate lm ngun
nng lng. Trong iu kin thiu ngun C mt s vi sinh vt k kh trong iu kin khng c oxy
c th s dng mt s aminoacid lm ngun nng lng. Ngun N thng c vi sinh vt s dng
l protein v cc sn phm phn hu ca protein ( peptone, peptide, aminoacid...), mui ammone,
nitrate, N phn t (N
2
), purine, pyrimidine, urea, amine, amide, cyanide...(bng 13.7)
Bng 13.7: Ngun N c vi sinh vt s dng
Ngun N
Cc dng hp cht
Protein v cc sn
phm phn gii ca
protein
peptone, peptide, aminoacid... (mt s vi sinh vt tit men
proteinase phn gii protein thnh cc hp cht phn t nh hn ri
mi hp thu c vo t bo)
Ammone v mui
ammone
NH
3
, (NH
4
)
2
SO
4,
... (d c hp thu)
Nitrate KNO
3
(d c hp thu)
N phn t N
2
(vi vi sinh vt c nh N)
Cc ngun N khc purine, pyrimidine, urea, amine, amide, cyanide (ch mt s nhm vi
sinh vt mi c th ng ho c)
Ngun N thng c s dng nui cy vi sinh vt gm c pepton, bt c, bt nhng tm,
bt u tng, bt kh lc, cao ng, cao tht, cao nm men... Vi sinh vt s dng chn lc i vi
ngun N. Chng hn x khun sn sinh terramycin s dng cao ng vi tc nhanh hn so vi s
dng kh u tng hay kh lc, bi v ngun N trong cao ng l cc sn phm phn gii d hp
thu ca protein. Cao ng c coi l ngun N tc hiu, cn kh du c coi l ngun N tr hiu.
Loi N tc hiu l c li cho s sinh trng ca vi sinh vt, cn loi tr hiu li c li cho s hnh
thnh cc sn phm trao i cht. Khi sn xut terramycin chng hn, ngi ta phi hp s dng
cao ng v kh du theo mt t l nht nh phi hp gia giai on sinh trng to sinh khi v
giai on sinh tng hp cc sn phm trao i cht, nhm mc tiu l nng cao sn lng
terramycin.
Nng lc hp thu mui ammone v nitrate vi sinh vt l kh mnh. Ion NH
4
+
sau khi c t
bo hp thu c th c trc tip s dng, do cc ngun mui ammone c coi l ngun N tc
hiu. Cn nitrate sau khi c hp th cn kh thnh NH
4
+
ri mi c vi sinh vt s dng. a s
cc vi khun hoi sinh (saprophyte), vi khun ng rut, vi sinh vt gy bnh ngi, ng vt,
thc vt...u c th dng mui ammone, mui nitrate lm ngun N. Chng hn cc vi khun
Escherichia coli, Enterobacter aerogenes, Bacillus subtilis, Pseudomonas aeruginosa...u c th
s dng ngun (NH
4
)
2
SO
4
v NH
4
NO
3
lm ngun N; x khun c th s dng KNO
3
lm ngun N;
nm si c th s dng KNO
3
lm ngun N. Lc dng cc mui nh (NH
4
)
2
SO
4
lm ngun N
nui cy vi sinh vt cn ch l sau khi vi sinh vt hp thu NH
4
+
th s lm h thp pH ca mi
trng. Ngi ta gi l nhng mui c tnh sinh l acid. Ngc li khi dng cc mui nitrate
(nh KNO
3
) sau khi vi sinh vt hp thu NO
3
-
th s lm nng cao pH ca mi trng. Ngi ta gi
l cc mui c tnh sinh l kim. lm cho pH trong cc mi trng nui cy vi sinh vt t b
bin ng ngi ta b sung thm cc cht c tnh m (buffer substance).
3) Ngun mui v c (source of inorganic salt)
Cc mui v c l ngun cht dinh dng khng th thiu i vi s sinh trng ca vi sinh
vt. Chng c cc chc nng sinh l ch yu l: tham gia vo thnh phn ca cc trung tm hot
tnh cc enzyme ca vi sinh vt, duy tr tnh n nh ca kt cu c i phn t v t bo, iu tit
v duy tr cn bng p sut thm thu ca t bo, khng ch in th oxy ho kh ca t bo v l
ngun vt cht sinh nng lng i vi mt s loi vi sinh vt (bng 13.8).
Bng 13.8: Mui v c v chc nng sinh l ca chng
Nguyn t Hp cht s
dng
Chc nng sinh l

P

KH
2
PO
4
,
K
2
HPO
4

L thnh phn ca acid nucleic, nucleoprotein,
phospholipid, coenzyme, ATP... Lm nn h thng m
gip iu chnh pH mi trng.

S

(NH
4
)
2
SO
4
,
MgSO
4

L thnh phn ca cc aminoacid cha S, mt s vitamin;
glutathione c tc dng iu chnh in th oxy ho kh
trong t bo.


Mg


MgSO
4

L thnh phn trung tm hot tnh ca enzyme phosphoryl
ho hexose, dehydrogenase ca acid isocitric, polymerase
ca acid nucleic, thnh phn ca chlorophyll v bacterio-
chlorophyll.

Ca

CaCl
2
,
Ca(NO
3
)
2

To tnh n nh ca mt s cofactor, enzyme duy tr, cn
cho s dng trng thi cm th ca t bo.

Na

NaCl
Thnh phn ca h thng chuyn vn ca t bo, duy tr p
sut thm thu, duy tr tnh n nh ca mt s enzyme.

K

KH
2
PO
4
,
KH
2
PO
4

L cofactor ca mt s enzyme, duy tr p sut thm thu
ca t bo, l nhn t n nh ca ribosome mt s vi
khun a mn.


Fe


FeS0
4

Thnh phn ca sc t vi khun v mt s enzyme, l vt
cht ngun nng lng ca mt s vi khun st, cn thit
tng hp chlorophyll v c t vi khun bch hu.
Trong qu trnh sinh trng vi sinh vt cn cn ti mt s nguyn t vi lng. Nhng nguyn t
ny cng c vai tr quan trng mc du ch cn vi s lng rt nh, khong 10
-8
-10
-6
mol/ L mi
trng nui cy. Nguyn t vi lng tham gia vo thnh phn enzyme v lm hot ho enzyme.
(Bng 13.9)
Bng 13.9: Tc dng sinh l ca nguyn t vi lng
Nguyn t Tc dng sinh l
Zn C mt trong alcohol dehydrogenase, lactodehydrogenase, phosphatase
kim, ARNpolymerase, ADNpolymerase...
Mn C mt trong peroxyd dismutase, carboxylase ciitric synthetase
Mo C mt trong reductase nitrate, nitrogenase, dehydrogenase formic.
Se C mt trong reductase glycin, reductase formic.
Co C mt trong mutase glutamic.
Cu C mt trong cytochrome oxydase.
W C mt trong dehydrogenase formic.
Br C mt trong urease, cn cho s sinh trng ca vi khun hydrogen.
Nu thiu nguyn t vi lng trong qu trnh sinh trng th hot tnh sinh l ca vi sinh vt b
gim st, thm ch ngng sinh trng. Do nhu cu dinh dng ca vi sinh vt l khng ging nhau
cho nn khi nim v nguyn t vi lng chi c ngha tng i. Vi sinh vt thng tip nhn
nguyn t vi lng t cc cht dinh dng hu c thin nhin, cc ho cht v c, nc my hay
ngay t trong cc dng c nui cy bng thu tinh. Ch trong nhng trng hp c bit mi cn b
sung nguyn t vi lng vo mi trng nui cy vi sinh vt.
V nhiu nguyn t vi lng l kim loi nng cho nn nu d tha s gy hi cho vi sinh vt.
Khi cn b sung thm nguyn t vi lng vo mi trng cn lu khng ch chnh xc liu lng.
4) Nhn t sinh trng
Nhn t sinh trng (growth factor) l nhng hp cht hu c m c nhng vi sinh vt cn thit
sinh trng tuy vi s lng rt nh v khng t tng hp so vi nhu cu.
Cc vi sinh vt khc nhau c nhng yu cu khng ging nhau v chng loi v liu lng ca
cc nhn t sinh trng. Sau y l mt s v d (bng 13.10).
Bng 13.10: Cc nhn t sinh trng cn thit di vi mt s loi vi sinh vt
Vi sinh vt Cht sinh trng Nhu cu / ml
Acetobacter suboxydans APAB, Acid nicotinic 0-10 ng
Clostridium acetobutylicum
Streptococcus pneumonia
Leuconostoc mesenteroides
Staphylococcus aureus
Corynebacterium diphtheria
Clostridium tetani
Lactobacillus arabinosus


Streptococcus faecalis


Lactobacillus delbruckii

Lactobacillus casei
APAB
choline
pyridoxal
thiamin
b-alanin
uracil
acid nicotinic
acid pantothenic
methionine
acid folic
arginine
tyrosine
thymonucleoside
biotin
ephedrin
3 mg
0,15 ng
6 mg
0,025 mg
0,5ng
1,5 mg
0~4 mg
0,1 mg
0,02 mg
1,0 mg
0,02 mg
50 mg
8 mg
0-2 mg
1 ng
Ch thch: 1 mg= 10
-6
g; 1ng= 10
-9
g
Vi sinh vt t dng v mt s vi sinh vt d dng (nh Escherichia coli) thm ch c th sinh
trng m khng cn bt k nhn t sinh trng no. Mt khc, cng mt loi vi sinh vt nhng
nhu cu i vi nhn t sinh trng cng thay i tu theo iu kin mi trng. V d Mucor
rouxii khi sinh trng trong iu kin k kh th cn thiamin (B1) v biotin (H), nhng trong iu
kin hiu kh th li t tng hp c cc vitamin ny. C trng hp cha gii thch c bn cht
ca nhu cu v nhn t sinh trng mt s loi vi sinh vt. Thng thng b sung vo mi trng
cc cht hu c nh cao nm men, cao tht, dch un ng thc vt (nhng, gi ) l c th p
ng c nhu cu v nhn t sinh trng.
Cn c vo s khc nhau v cu trc ho hc v chc nng sinh l ca cc nhn t sinh trng
ngi ta chia nhn t sinh trng thnh cc nhm vitamin, aminoacid, purine v pyrimidine.
Vitamin l nhn t sinh trng c tm thy bn cht ho hc sm nht. Hin nay ngi ta pht
hin c nhiu loi vitamin c tc dng l nhn t sinh trng. Mt s vi sinh vt c th t tng
hp c vitamin, nhng nhiu loi khc li cn c cung cp vitamin trong mi trng dinh
dng th mi sinh trng c. Vitamin ch yu l coenzyme hay cofactor ca cc enzyme tham
gia vo qu trnh trao i cht. Mt s vi sinh vt khng t tng hp c nhng aminoacid no ,
cn b sung vo mi trng cc aminoacid hay b sung peptide chui ngn. Chng hn vi khun
Leuconostoc mesenteroides cn ti 17 loi aminoacid mi sinh trng c. Mt s vi khun cn
cung cp D-alanin tng hp thnh t bo. Purine v pyrimidine ch yu c dng lm
coenzyme hay cofactor ca cc enzyme cn thit cho qu trnh tng hp nucleoside, nucleotide v
acid nucleic.
Bng 13.11: Chc nng ca mt s vitamin thng thng i vi vi sinh vt
Vitamin Chc nng V d v cc vi sinh vt cn cung cp

Biotin (H)
-Carboxyl ha (c nh CO
2
)
-Trao i cht mt carbon
Leuconostoc mesenteroides (B)
Saccharomyces cerevisiae (F)
Ochromonas malhamensis (A)
Acanthammoeba castellanii (P)

Vitamin B
12

-Sp xp li phn t
-Nhm mang methyl trong trao i cht
mt carbon
Lactobacillus spp. (B)
Euglena gracilis (A)
To silic v nhiu vi to khc (A)
Acanthammoeba castellanii (P)
Acid folic -Trao i cht mt carbon Enterococcus faecalis (B)
Tetrahymena pyriformis (P)
Acid lipoic -Chuyn nhm acyl Lactobacillus casei (B)
Tetrahymena spp. (P)
Acid
pantotenic
-Tin th ca CoA (oxy ha pyruvat,
trao i axit bo)
Proteus morganii (B)
Hanseniaspora spp. (F)
Paramecium spp. (P)
Pyridoxin
(B6)
-Trao i acid amin Lactobacillus spp. (B)
Tetrahymena pyriformis (P)

Niacin
-Tin th ca NAD, NADP Brucella abortus (B)
Haemophilus influenza (B)
Blastocladia pringsheimii (F)
Crithidia fasciculata (P)

Riboflavin
(B
2
)
-Tin th ca FAD, FMN Caulobacter vibrioides (B)
Dictyostelium spp. (F)
Tetrahymena pyriformis (P)
Bacillus anthracis (B)

Thiamin (B1)
-Chuyn nhm aldehyd (kh carboxyl
pyruvat, oxy ha acid -keto)
Phycomyces blakesleeanus (F)
Ochromonas malhamensis (A)
Colpidium campylum (P)
Ch thch: B-Vi khun; F-Vi nm; A-Vi to; P-ng vt nguyn sinh
5) Nc
Nc l thnh phn khng th thiu vi sinh vt c th sinh trng. Chc nng sinh l ca
nc trong t bo l:
- Ho tan v chuyn vn cc cht, h tr cho vic hp thu cht dinh dng, gii phng cc sn
phm trao i cht.
- Tham gia vo hng lot cc phn ng ha hc trong t bo.
- Duy tr cu hnh thin nhin n nh ca cc i phn t nh protein, acid nucleic...
- L th dn nhit tt, hp thu tt nhit lng sinh ra trong qu trnh trao i cht v khuch tn
kp thi ra bn ngoi duy tr s n nh ca nhit bn trong t bo.
- Duy tr hnh thi bnh thng ca t bo.
- Thng qua qu trnh thu phn hay kh nc khng ch kt cu ca t bo (enzyme, vi ng,
tin mao...) v s tho lp virt.
Tnh hu hiu ca nc i vi s sinh trng ca vi sinh vt thng c biu th bng
hot ng (hot ) ca nc (water activity, a
w
). l t l gia p lc hi nc ca dung dch
trong nhng iu kin nhit v p lc nht nh vi p lc ca hi nc thun khit trong cng
nhng iu kin nh vy:
a
w
= p
w
/ p
w
0

y P
w
l p lc hi nc ca dung dch, cn a
w
0
l p lc ca hi nc thun khit. P
w
0
ca
nc thun khit l 1.0. Dung dch cng cha nhiu dung cht (cht ho tan) th a
w
cng nh. Vi
sinh vt thng sinh trng trong iu kin c a
w
trong khong 0,6-0,99. i vi mt s loi vi sinh
vt khi a
w
qu thp th tc sinh trng v tng sinh khi gim. Cc vi sinh vt khc nhau c a
w

thch hp khng ging nhau (bng 13.12)
Bng 13.12: a
w
thch hp nht cho sinh trng mt s nhm vi sinh vt

Vi sinh vt a
w

Vi khun ni chung
Nm men
Nm si
Vi khun a mn
Vi nm a mn
Nm men a p sut thm thu cao
0,91
0,88
0,80
0,76
0,65
0,60
Nhn chung a
w
thch hp nht cho s sinh trng ca vi khun cao hn ca nm men v nm
si. Vi sinh vt a mn c a
w
thch hp nht cho s sinh trng l kh thp.
Phn nc c th tham gia vo cc qu trnh trao i cht ca vi sinh vt c gi l nc t do.
Phn ln nc tn ti trong t bo vi sinh vt l nc t do. Phn nc lin kt vi cc hp cht
hu c cao phn t trong t bo c gi l nc lin kt. Nc lin kt mt i kh nng ho tan v
lu ng.
13.1.4. Khi nim v s sinh trng trong iu kin hn ch cc cht dinh dng
mi trng nui cy lc trong phng th nghim, khi tt c cc cht dinh dng c cung
cp cho s sinh trng ca vi sinh vt c thit k ti u th s d tha xy ra vo lc u v
cc t bo sinh trng theo logarit vi tc sinh trng l ln nht. Tuy nhin, trong mi h thng
mi trng v k thut nui cy, s sinh trng ca vi sinh vt khng th tip din mi m khng b
gii hn trong mt khong thi gian di. Mt tnh ton n gin chng minh nhn nh ny l:
sau 2 ngy sinh trng theo logarit, mt t bo vi sinh vt c 20 pht li nhn i mt ln s to ra
xp x 2 x 10
43
t bo. Gi s khi lng trung bnh ca mi t bo l 10
-12
g th ton sinh khi t
bo trn s c khi lng gp gn 400 ln khi lng ca qu t. V vy, trong mi mt th tch
nui cy, s sinh trng lun lun sm b gii hn do s cn kit ca mt hoc vi cht dinh dng.
Thut ng cc cht dinh dng hn ch c s dng vi rt nhiu ngha, v thng vn b
nhm ln. Cc cht dinh dng hn ch c kh nng nh hng n s sinh trng trong cc mi
trng nui cy vi sinh vt theo hai cch ring bit: ha hc v v ng hc. S hn ch ha hc
c nh ngha l khi lng ln nht sinh khi c th c to ra trong iu kin gii hn cc
cht dinh dng. Nguyn l Liebig bt ngun t cc nghin cu v s mu m trong nng nghip
ca Justus von Liebig vo nm 1840. Trong nghin cu ny ng tm ra rng hm lng ca mt
cht dinh dng no s quyt nh n nng sut ma mng, min l tt c cc cht dinh dng
khc c mt mt cch d tha (phng trnh 1). Gii hn ng hc xut hin khi nng cc
cht dinh dng l thp (trong phm vi t miligram ti microgram trong mi lit), s hn ch cc
cht dinh dng s iu khin tc sinh trng ring ca t bo (). iu khin ng hc v tc
sinh trng thng ko theo cc ng lc bo ha v phng trnh Monod (phng trnh 2)
c s dng m t mi quan h gia nng ca cc cht dinh dng i vi tc sinh
trng ring ca t bo ().
X = X
0
+ ( S
0
- S) x Y
X/S
(1)
=
max
x x s / (K
S
+ S) (2)
Trong S
0
l nng ban u v s l nng cui cng ca cc cht dinh dng b hn ch S;
X(X
0
) l nng sinh khi (ban u); l sn lng sinh khi thu c i vi cht dinh dng S,

max
l tc sinh trng ring ln nht, v K
S
l hng s i lc c cht Monod.
iu ny th hin r trong hnh 13.2 i vi s sinh trng trong h thng nui cy kn. Cc t
bo ban u sinh trng khng gii hn cho n khi s tiu th cc cht dinh dng hn ch b ht
dn, dn n tc sinh trng suy gim dn, sau tc sinh trng ngng hn. l lc t
n nng cui cng ca sinh khi. Trong nui cy lin tc, ngi b sung mi trng mt cch
lin tc v mt lng mi trng d tha c loi b. Tc b sung thm vo ca cc cht dinh
dng b hn ch s iu khin ng thi c v nng sinh khi trong mi trng nui cy (Pirt,
1975; Kovarova v Egli, 1998).

Hnh 13.2: ng hc ca s gii hn sinh trng ca vi sinh vt trong nui cy ng do gii hn
nng ca cht dinh dng (c cht) S. S
0
l nng c cht ban u, s l nng thc ca c
cht, X l nng sinh khi; X
0
: nng sinh khi ban u; Y: sn lng sinh khi thu c i
vi c cht S.
Trong thc nghim, ngi ta c th nui cy cc t bo trong cc iu kin c bit r, nh
cc cht dinh dng hn ch s c xc nh. i vi vic nui cy cc vi sinh vt d dng
nghin cu v to ra cc sn phm sinh khi, mi trng c thit k ph bin vi ngun carbon
v nng lng gii hn, tt c cc cht dinh dng khc c cung cp d tha. Tuy nhin, trong
qu trnh cng ngh sinh hc, s gii hn bi cc cht dinh dng ch khng phi ngun carbon
gi chc nng iu khin cc trng thi sinh l v qu trnh trao i cht ca vi sinh vt. S hn ch
cc cht dinh dng no thng kch thch hoc tng cng s to thnh rt nhiu cc sn phm
trao i cht v cc enzyme ca vi sinh vt. V d, nng sut s c tng ln trong qu trnh ln
men to cht khng sinh do sinh trng trong mi trng hn ch photphat, s sn xut acid citric
trong mi trng c s hn ch Fe-, Mn-, hoc Zn. Cn s sinh tng hp ca NAD l c thc
hin trong iu kin hn ch Zn-Mn. Vic tch ly cc nguyn liu d tr ni bo PHB hoc PHA
(cht do sinh hc-bioplastic) s b gii hn bi ngun cung cp hp cht giu nitrogen.
R rng l s sinh trng ca vi sinh vt c iu khin thng xuyn khng phi ch bi mt
cht dinh dng m bi s kt hp ca hai hay nhiu cht dinh dng ng thi (Kovarova v Egli,
1998).
13.1.5. Thit k v phn tch mi trng sinh trng ti thiu
sinh trng v tng hp cc nguyn liu t bo cho bn thn mnh, vi sinh vt phi thu nhn
cc thnh phn cu trc (hay cc tin cht ca chng) v nng lng cn thit t mi trng sng.
Do , nui cy vi sinh vt trong phng th nghim th cc cht dinh dng phi c cung cp
y vo mi trng v cc cht dinh dng phi dng m cc vi sinh vt ny c th s dng
c.
Do c s a dng sinh l ca th gii vi sinh vt m c v s cc mi trng vi thnh phn
dinh dng khc nhau c a ra, vi mc ch hoc l lm giu mt cch chn lc hoc l
nui cy mt nhm v sinh vt c th no (LaPage v cs, 1970; Balows v cs 1992; Atlas,
1997). Tt c cc mi trng ny u cha cc thnh phn vi cc chc nng dinh dng r rng,
c bit l cn nhc v chc nng cu trc hoc sinh nng lng. Tuy nhin, hu ht cc nghin cu
v cht dinh dng c tin hnh nh tnh ch khng phi nh lng v cc cht dinh dng
khc nhau c thm vo nhiu hn hay t hn mt cch ty . Ngoi ra, rt nhiu cc mi trng
nui cy c cha cc thnh phn khng c bit r rng bi v s dng cc nguyn liu hu c
nh ng, khoai ty,
Trong cng nhng iu kin nh: nhit hoc pH, tc sinh trng ring ln nht ca vi
sinh vt b nh hng bi s a dng ca cc cht dinh dng trong mi trng. iu ny c
minh ha mt cch c th i vi s sinh trng ca Salmonella typhimurium (th nghim bi
Schaechter v cs, 1958). H s dng 22 mi trng c thnh phn khc nhau v nhn thy cc
tc sinh trng khc nhau cc mi trng trong cc iu kin d tha cc cht dinh dng.
Kt qu cho thy cht lng cc tin cht a vo mi trng khong cho php iu chnh tc
sinh trng mt cch r rng nht.
A. Thit k mi trng v kim tra cc cht dinh dng gii hn
1. Thit k mi trng sinh trng
Trong thit k mi trng sinh trng, quyt nh u tin c a ra l chn la nng
cao nht cho php to ra sinh khi (X
max
), v xc nh cc cht dinh dng gii hn (theo nguyn l
Liebig). in hnh, mi trng sinh trng cho cc vi sinh vt d dng c thit k vi ngun
nng lng - carbon ring bit s gii hn lng sinh khi c to ra, nhng ngc li tt c cc
cht dinh dng khc (c thm vo di dng cc hp cht n) c cung cp d tha. Da
vo gi tr X
max
, c th tnh ton c nng ti thiu ca cc nguyn t khc nhau cn thit
trong mi trng nui cy. m bo s d tha ca tt c cht dinh dng khng gii hn trong
mi trng th nng ca chng c nhn vi nhn t d (F
E
). Bng cch ny, nng ca cht
dinh dng i hi trong mi trng tng trng (E
req
) gp x ln theo l thuyt i vi ngun
carbon.
E
req =
X
max
/ Y
X/E
x F
E
(3)
Y
X/E

(the individual average elemental growth yield) l sn lng tng trng trung bnh da
trn tng nguyn t.
Mt v d cho vic thit k mi trng khi gii hn ngun carbon, cho php to sn lng sinh
khi kh t 10g/l sinh (bng 13.13). Cn ch rng, trong mi trng ny cc thnh phn c
la chn sao cho c th thay i nng ca mi nguyn t (v d c th thay th MgCl
2
v
NaHSO
4
bng MgSO
4
). Hn na, mi trng ny ch c tnh cht m yu (weakly buffered), do
cn thit phi khng ch pH trong sut qu trnh sinh trng.
Cch thc ny c s dng cho vic thit k mi trng nui cy cc vi sinh vt hiu kh vi
mt sinh khi thp v trung bnh. Phc tp hn l thit k ca mi trng cho nui cy vi sinh
vt k kh, trong rt nhiu thnh phn ca mi trng d dng kt ta ti th oxy ha kh cn
thit, hoc mt t bo cao trong c cha cc cht ha tan hoc vn c tnh ca mt s mi
trng.


Bng 13.13: Thit k mi trng ti thiu b gii hn bi ngun C cho php sn lng sinh khi
kh t 10g/l
a,b

Thnh phn
mi trng
Ngun Nng sut sinh
trng
(g sinh khi
kh/g nguyn
t)
Cc nhn t
d tha vi
ngun carbon
tng ng
Khi lng
cc nguyn t
(g/l)
Khi lng cc
thnh phn cu
to (g/l)
Glucose C, nng
lng
1 1 10 25.0
NH
4
Cl N 8 3 3.75 14.33
NaH
2
PO
4
P 33 5 1.52 5.88
KCl K 100 5 0.5 0.95
NaH
2
SO4 Na 100 5 0.5 1.87
MgCl
2
Mg 200 5 0.25 0.98
CaCl
2
Ca 100 10 1.0 2.77
FeCl
2
Fe 200 10 0.5 1.13
MnCl
2
Mn 10
4
20 0.02 0.046
ZnCl
2
Zn 10
4
20 0.02 0.042
CuCl
2
Cu 10
5
20 0.002 0.0042
CoCl
2
Co 10
5
20 0.002 0.0044
a. Da vo sn lng tng trng ca cc nguyn t trong sinh khi kh.
b. Theo Pirt (1975), Egli v Fiechter (1981). Sn lng tng trng ca C v cc nguyn t vt
Zn, Cu, Mo, Mn
Nhn t Y
X/E
c phn tch t sinh khi kh khi nui cy trong iu kin khng gii hn tng
trng ca h thng ng. i vi carbon, oxy, v hydro, Y
X/E
khng th c tnh ton chnh xc
trc tip t cc thnh phn c bn ca t bo do nhng thnh phn ny khng ch to nn sinh khi,
m cn c cc chc nng trao i cht khc.Ngoi ra, trong bng khng ni n mt s lng ln
cc cht nhn in t cn thit phi c m bo cho qu trnh sinh trng.
Tnh cht ha hc ca cc thnh phn trong mi trng sinh trng phi c tnh n khi
chn F
E
. V d, phn ln cc nguyn t vi lng d dng kt ta trong mi trng sinh trng pH
trung tnh hoc kim v do gim bt kh nng hp th sinh hc (kh khn xc nh). Do ,
chng c thm vo nhiu gp 10 ti 20 ln (Bridson v Brecker, 1970).
Trong cng ngh sinh hc, qu trnh nui cy theo m (batch) v nui cy theo m c b sung
(fed-batch) c nghin cu t lu v rt c li khi thit k cc mi trng cha tt c nguyn t
vi lng chnh xc lng cn thit, sao cho tt c cc nguyn t phi c tiu th ht ti cui k
tng trng. Tuy nhin, rt kh khn c th t c iu ny do tnh a dng ca cc nguyn t v
s ph thuc ca chng vo iu kin nui cy.Tt nhin, trong cng ngh sinh hc, vic ti u ha
mi trng l rt quan trng tnh ton s tiu th cht dinh dng v hn ch ti a s hao ph
nguyn liu, ha cht.
Bng 13.14: Cc nhn t tng trng sn lng ca cc cht cho v nhn in t
Cc cht cho in t
H
2
Y
X/H2 =
12g/mol
S
2
O
3
Y
X/S2O3 =
4g/mol
Fe
2+
Y
X/Fe2+ =
0.35g/mol
NH
4
+
- NO
3
-
Y
X/NH4 =
1.3-2.6/mol
NO
2
_
- NO
3
Y
X/NO2 =
0.9-1.8g/mol
Cht nhn in t
O
2
Y
X/O2 =
10
a
-42
b
g/mol
NO
3
-
- N
2
Y
X/NO3 =
27g/mol
c

NO
2
-
- N
2
Y
X/NO2 =
17g/mol
c

N
2
O
-
- N
2
Y
X/N2O =
9g/mol
c

a. i vi cc c cht kh l methane hoc n-alkanes.
b. i vi cc cht oxy ha l glucose.
c. i vi Paracoccus denitrificans vi ngun carbon l glutamate
13.2. CC LOI HNH DINH DNG CA VI SINH VT
Vi sinh vt c tnh a dng rt cao cho nn cc loi hnh dinh dng (nutritional types) l kh
phc tp. Cn c vo ngun C, ngun nng lng, ngun in t, c th chia thnh cc loi sau y
(bng 13.15)
Bng 13.15: Cc loi hnh dinh dng ca vi sinh vt (I)

-Ngun C (Carbon sources)
+T dng (autotroph) +D dng
(heterotroph)

CO
2
l ngun C duy nht hay ch yu
Ngun C l cht hu c
-Ngun nng lng (Energy sources)
+Dinh dng quang nng
(phototroph)
+Dinh dng ho nng
(chemotroph)

Ngun nng lng l nh sng

Ngun nng lng l nng lng ha
hc gii phng ra t s oxy ho hp
Ngun in t (Electron sources)
+ Dinh dng v c
(lithotroph)
+ Dinh dng hu c
(organotroph)

Dng cc phn t v c dng kh cung
cp in t
Dng cc phn t hu c cung cp
in t


C th m hnh ha chc nng sinh l ca cc cht dinh dng i vi s sinh trng ca vi
sinh vt qua hnh 13.3 sau y:

Hnh 13.3: M hnh s lc v chc nng sinh l ca cc cht dinh dng i vi
s sinh trng ca vi sinh vt.

C th em phn ln vi sinh vt phn thnh bn nhm chnh (bng 13.16)
Bng 13.16: Cc loi hnh dinh dng ca vi sinh vt (II)
Loi hnh dinh dng Ngun nng lng;
Hydrogen; in t;
Carbon
i din
-T dng quang nng v c
(photolithoautotrophy)
Quang nng; H
2
, H
2
S,
S hoc H
2
O; CO
2

Vi khun lu hunh, mu ta,mu lc;
Vi khun lam.
-D dng quang nng
hu c (photoorganohetero-
trophy)
Quang nng; Cht hu
c
Vi khun phi lu humh mu ta, mu
lc.
-T dng ho nng
v c (chemolithoauto-
trophy)
Ho nng (v c); H
2
,
H
2
S, Fe
2+
, NH
3
, hoc
NO
2
-
, CO
2

Vi khun oxy ho S, vi khun
hydrogen, vi khun nitrt ho, vi
khun oxy ho st.
-D dng ho nng
hu c (chemoorganohetero-
trophy)
Ho nng (hu c);
Cht hu c
ng vt nguyn sinh, nm, phn ln
cc vi khun khng quang hp (bao
gm c cc vi khun gy bnh).


Loi T dng quang nng v c cn c gi l Photolithotrophic autotrophy; loi D dng
quang nng hu c cn c gi l Photoorganotrophic heterotrophy; loi T dng ha nng v
c cn c gi l Chemolithotrophic autotrophy; loi D dng ha nng hu c cn c gi l
Chemoorganotrophic heterotrophy.
Chng ta s xem xt k hn cc qu trnh trao i cht ca tng nhm vi sinh vt ny trong
chng Trao i cht.
Cc vi sinh vt thuc loi hnh T dng quang nng v c v D dng quang nng v c c
th li dng nh sng sinh trng. Chng c vai tr quan trng trong qu trnh din bin ca mi
trng sinh thi trong giai on c xa ca Tri t. Vi sinh vt T dng ho nng v c phn b
rng ri trong t v trong nc, chng tham gia tch cc vo cc vng tun hon vt cht trn Tri
t. Vi sinh vt D dng ho nng hu c dng cht hu c va lm ngun carbon va lm ngun
nng lng. Hu ht cc loi vi khun, nm, ng vt nguyn sinh bit u thuc loi hnh D
dng ho nng hu c. Tt c cc vi sinh vt gy bnh bit u thuc loi ny. Trong loi hnh
d dng ho nng hu c li chia thnh hai nhm: Nhm Hoi sinh (metatrophy) dng cht hu c
cht (xc ng thc vt) lm ngun carbon. Nhm K sinh (paratrophy) k sinh trn c th thc
vt, ngi v ng vt hp thu cht dinh dng. Chng khng th sng c khi tch ri khi
vt ch. Tuy nhin gia hai nhm ny cn c nhng loi hnh trung gian l Hoi sinh khng bt
buc (facultive metatrophy) v K sinh khng bt buc (facultive paratrophy).
Mt s chng vi sinh vt pht sinh t bin (t bin t nhin hay t bin nhn to) mt i
nng lc tng hp mt (hoc mt s) cht cn thit cho sinh trng (thng l nhn t sinh trng
nh aminoacid, vitamin), chng ch sinh trng c khi b sung vo mi trng cc cht ny.
Ngi ta gi chng l loi hnh Khuyt dng (auxotroph). Cc chng hoang di tng ng c
gi l loi hnh Nguyn dng (prototroph). Ngi ta thng s dng cc chng vi sinh vt khuyt
dng trong nghin cu Di truyn hc vi sinh vt.
Khng c ranh gii tuyt i gia cc loi hnh dinh dng ca vi sinh vt. Vi sinh vt d dng
khng phi tuyt i khng s dng c CO
2
m ch l khng th dng CO
2
lm ngun carbon
duy nht hay ch yu sinh trng. Trong iu kin tn ti cht hu c, chng vn c th ng
ha CO
2
to ra t bo cht. Tng t nh vy, vi sinh vt t dng khng phi l khng c th
s dng cht hu c sinh trng. Ngoi ra, mt s vi sinh vt c th thay i loi hnh dinh
dng khi sinh trng trong nhng iu kin khc nhau. V d vi khun phi lu hunh mu ta
(purple nonsulfur bacteria) khi khng c cht hu c c th ng ha CO
2
v thuc loi vi sinh vt
t dng; nhng khi c cht hu c tn ti th chng li c th s dng cht hu c sinh trng
v lc chng l cc vi sinh vt d dng. Hn na, vi khun phi lu hunh mu ta trong iu
kin k kh v c chiu sng c th sinh trng nh nng lng ca nh sng v thuc loi dinh
dng quang nng; nhng trong iu kin hiu kh v khng chiu sng th chng li sinh trng
nh nng lng sinh ra t qu trnh oxy ha cht hu c v thuc loi dinh dng ha nng. Tnh
bin i loi hnh dinh dng vi sinh vt r rng l c li cho vic nng cao nng lc thch ng
ca chng i vi s bin i ca iu kin mi trng.
13.3. MI TRNG NUI CY (Culture medium)
Mi trng nui cy l cc c cht dinh dng c pha ch nhn to nhm p ng cho yu
cu sinh trng, pht trin v sn sinh cc sn phm trao i cht ca vi sinh vt. Mi trng dinh
dng dng trong nghin cu vi sinh vt v trong qu trnh sn xut cc sn phm ca vi sinh vt.
Mi trng dinh dng l yu t quan trng trong cng nghip ln men, cng nghip sinh tng hp
nh vi sinh vt.
13.3.1. Nguyn tc pha ch mi trng nui cy
1) Chn cc cht dinh dng thch hp: Ni chung mi trng dinh dng cn p ng cc nhu
cu ca vi sinh vt v ngun C, ngun N, ngun mui khong, ngun nhn t sinh trng v nc.
V loi hnh dinh dng ca vi sinh vt l phc tp, cc vi sinh vt khc nhau c nhng yu cu
khng ging nhau v cc cht dinh dng cho nn c rt nhiu cng thc pha ch mi trng nui
cy. Sch Danh lc mi trng nui cy (A Compilation of Culture Media) xut bn t nm 1930
cng ghi ti trn 2500 loi mi trng nui cy khc nhau.
Mi trng nui cy vi sinh vt t dng hon ton pha ch t cc hp cht v c. V d
nui cy vi khun Thiobacillus thiooxidans gm c cc thnh phn nh sau (g/l): (NH
4
)
2
SO
4
-0.4;
MgSO
4
.7H
2
O - 0,5; FeSO
4
- 0,01; KH
2
PO
4
- 4; CaCl
2
- 0,25; S- 10; pH: 7,0, kh trng 121

C
trong 20 pht. Cc vi khun ny s dng CO
2
trong khng kh (hay ha tan trong nc) cung cp
ngun carbon. Vi cc vi sinh vt t dng quang nng ngoi vic cung cp cc cht dinh dng
cn tit cn cn chiu sng cung cp nng lng cho chng. i vi vi sinh vt d dng cn
cung cp cht hu c v nhu cu dinh dng ca cc nhm khc nhau l rt khc nhau. nui cy
vi khun Escherichia coli c th dng mi trng kh n gin sau y (g/l): Glucose - 5;
NH
4
H
2
PO
4
- 1; MgSO
4
.7H
2
O - 0,2; K
2
HPO
4
- 1; NaCl - 5; pH: 7,0-7,2, kh trng 112

C trong 30
pht. Nhng cngc nhng vi khun d dng yu cu nhng mi trng nui cy rt phc tp. V
d vi khun Lactobacillus bifidus cn mi trng sau y (trong 1 lt mi trng m c gp i) :
K
2
HPO
4
- 5g; Na-Acetat - 50g; NZ Case Peptone- 10g; Lactose- 70g; Alanin, Cystin, Tryptophan-
mi loi 0,4g; Asparagin- 0,2g; Xanthin, Adenin, Guanin, Uracin - mi loi 0,02g; Dung dch Mui
B- 10ml; Pyridoxin-HCl - 2,4mg ; Tiamin-HCl - 0,4mg; Riboflavin- 0,4mg; Acid nicotinic- 1,2mg;
Ca-Pentotenat - 0,8mg ; Biotin- 8,0 mcg (microgram); Acid folic- 20 mcg; Acid p-aminobenzoic-
20 mcg; Tween 80 - 1g. iu chnh pH n 6,8. Thm bng thao tc v trng 100ml dung dch Acid
ascorbic 1% lc qua nn lc vi khun. iu chnh n pH 6,5. Li thm bng thao tc v trng
Sa ngi loi kem sao cho nng t c l 2%. Dung dch Mui B c thnh phn nh sau
(g/l): MgSO
4
.7H
2
O-10; FeSO
4
.7H
2
O-0,5; NaCl-0,5; MnSO
4
.2H
2
O- 0,337.
Thng thng thay cho cc nhn t sinh trng ngi ta thng dng Peptone (thay cho
tng aminoacid) v cao nm men (thay cho cc nhn t sinh trng). Mi trng thng dng
nui cy cc vi khun d dng l Mi trng Cao tht-Pepton vi thnh phn nh sau (g/l): Cao
tht (Beef extract) - 5; Peptone- 10; NaCl- 5; pH: 7,0-7,2; kh trng 121

C trong 20 pht. Mi
trng nui cy vi khun Brevibacterium spp. c thnh phn nh sau (g/l): Cao nm men (Yeast
extract)-10; Glucose- 20; CaCO
3
- 20.
Ngi ta chia mi trng nui cy thnh nhiu loi khc nhau.
Cn c vo thnh phn mi trng ta c: mi trng thin nhin, mi trng tng
hp.
Mi trng thin nhin (complex medium): y l loi mi trng cha cc cht hu c thin
nhin khng bit r thnh phn ha hc hoc thnh phn ha hc khng n nh, v vy cn c
gi l mi trng khng xc nh v ha hc (chemically undefined medium). Cc mi trng Cao
tht-Pepton, mi trng Mch nha, mi trng LB (Luria-Bertani) l cc v d ca loi mi trng
ny. Thnh phn ca mi trng LB l nh sau (g/l): Peptone - 10; Cao nm men - 5; NaCl -10; pH:
7,0; kh trng 121
0
C trong 21 pht. Cao tht l nc chit tht c c c li. Cao tht cha cc
cht m hu c, ng, vitamin, mui khong- tt c u d tan trong nc. Peptone l dng thy
phn bng protease hay bng acid i vi tht, casein, gelatin sau lm kh li thnh dng bt.
Peptone cha phong ph cc cht m hu c, cng c mt s vitamin v ng. Cao nm men l
dch t phn (autolysate) t bo nm men c c c li. Cao nm men cha phong ph vitamin
nhm B, cng c cha cc cht m hu c v ng.
Ngoi cc loi ni trn mi trng thin nhin cn c ch to t cc nguyn liu khc nh
nc chit khoai ty, nc chit gi u, nc chit t, nc chit rm r, nc chit lng v bt
ng, cm go, sa, huyt thanh, nc p c rt, nc da. Vi sinh vt a phn (coprophilous
microorganisms) c th dng nc phn lm cht dinh dng. Gi thnh ca mi trng thin
nhin thng thp, cho nn khng ch c s dng trong phng th nghim m cn c th c s
dng trong cc x nghip ln men cng nghip.
Bng 13.17: Thnh phn mt s loi peptone v dch thy phn protein
Ch phm(CP) Phn ng
Biure
Cc thnh phn (% trong protein)
(1) (2) (3)
Peptone Pharmacon + 63.5 9.7 27.8
Peptone Vitte + 44.1 26.7 29.2
Peptone Canbaum + 40.2 27.4 32.4
Peptone Gee + 44.5 23.2 32.3
Peptone Roche + 25.3 11.7 63.0
CP thy phn nh pepsin + 42.1 25.2 32.7
CP thy phn nh trypsin + 2.0 14.9 82.1
Dch thy phn gluten - 0 0 100
Dch thy phn gelatin - 0 0 100
Ch thch:
(1)- Cc polypeptid cao phn t c kt ta bng tannin khi c mt 2% H
2
SO
4
.
(2)- Cc polypeptid c kt ta bng tannin khi mi trng c phn ng trung tnh.
(3)- Cc aminoacid t do v cc peptid khng b t ta bi tannin.
Mi trng tng hp (synthetic medium): y l loi mi trng c thnh phn ha hc c
bit r cho nn cn c gi l mi trng xc nh v ha hc (chemically defined medium). V
d mi trng Gause thch hp cho X khun vi thnh phn nh sau (g/l): Tinh bt tan - 20;
KNO
3
- 1; NaCl - 0,5; K
2
HPO
4
.3H
2
O

- 0,5; K
2
HPO
4
.3H
2
O

- 0,5; FeSO
4
.7H
2
O- 0,01, pH: 7,2-7,4;
kh trng 121
0
C trong 21 pht. Mi trng tng hp c gi thnh cao v trn loi mi trng ny
vi sinh vt pht trin tng i chm, ni chung thch hp s dng trong phm vi phng th nghim.
C nhng vi khun i hi cc mi trng tng hp kh n gin, chng hn nh vi khun
Escherichia coli vi mi trng sau y: K
2
HPO
4
-7,0g; KH
2
PO
4
-2,0g; (NH
4
)
2
SO
4
-1,0g; MgSO
4
-
0,1g; CaCl
2
-0,02g; Glucose-4-10g; Nguyn t vi lng (Fe,Co,Mn,Zn,Cu,Ni,Mo)-mi loi 2-10g;
Nc ct- 1000ml.



Escherichia coli
C nhng vi sinh vt i hi cc mi trng tng hp rt phc tp (v t tin). Sau y l v d
v mi trng tng hp dng nui cy vi to Euglena: acid glutamic-6g; acid aspartic-4g;
Glycine-5g; Sacchaose-30g; Acid malic-1,04g; Acid boric-1,14mg; Thiamine HCl-12mg; KH
2
PO
4
-
0,6g; MgSO
4
-0,8g; CaCO
3
-0,16g; (NH
4
)
2
CO
3
- 0,72g; FeCl
3
-60mg; ZnSO
4
- 40mg; MnSO
4
-6mg;
CuSO
4
- 0,62mg; CoSO
4
- 5mg ; (NH
4
)
2
MoO
4
- 1,34mg; Nc 1000ml.
Mt v d khc v mi trng tng hp nui cy vi khun Leuconostoc mesenteroides:
K
2
HPO
4
- 0,6g; KH
2
PO
4
- 0,6g; NH
4
Cl- 3g; MgSO
4
- 0,1g; Glucose- 25g; Na acetate- 20g; Cc
amino acid- mi loi 100-200g (gm c alanine, arginine, asparagine, aspartate, cysteine,
glutamate, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine,
proline, serine, threonine, tryptophane, tyrosine, valine); Purinevaf Pyrimidine mi loi 10mg
(gm c adenine, guanine, uracil,xanthine); Vitamin- mi loi 0,01-1mg (gm c biotin, folate,
nicotinic acid, pyridoxal, pyridoxamine, pyridoxine, ribolavine, thiamine, pantothenate, para-
aminobenzoic acid). Nguyn t vi lng - mi loi 2-10g; Nc ct- 1000ml.

Cn c vo trng thi ca mi trng ngi ta chia ra thnh mi trng c, mi trng
bn c v mi trng dch th.
Mi trng c (solid medium): y l loi mi trng c lm ng c li nh c b sung
thm thch (agar-agar), gelatin hay silica gel. Mi trng c phi m bo c cc yu cu sau
y:
- Khng b vi sinh vt nui cy s dng.

Rau cu ch vng
-Gi c trng thi c trong nhit nui cy vi sinh vt. D ha tan khi un nng (thng
c iu chnh bng lng cha cht lm c trong mi trng)
-Nhit lm c mi trng khng qu thp.
-Cht lm c mi trng khng c hi i vi vi sinh vt
-Cht lm c khng b ph hy khi kh trng mi trng
-Gi c trng thi trong sut ca mi trng.
-Gi thnh khng qu cao, pha ch mi trng d dng.
Thch l sn phm ch to t Rau cu ch vng (Gracilaria verucosa) hay cc to bin khc
thuc chi Gracilaria hay Gelidium. Thch c cha khong 70% agarose v khong 30%
agaropectin. lm tan thch cn un mi trng n 100
0
C v lm gi mi trng thch
trng thi lng cn gi nhit khong 50-60
0
C (trong ni cch thy). lm cho mi trng
c li cn h nhit xung 40-45
0
C. Ty cht lng ca thch m khi lm mi trng ngi ta
cho vo vi t l 15-20g/l. Khi cn nui cy vi sinh vt trn cc mi trng thch c pH t 6 tr
xung th cn iu chnh mi trng ti pH trung tnh trc khi kh trng, sau mi iu chnh
li n pH thch hp (nu khng thch c b thy phn trong iu kin pH thp v nhit cao).


Hnh 13.4: Robert Koch (1843-1910)

Hnh 13.5: Fannie Eilshemius (1850-1934) v
Walther Hesse (1846-1911)


Ngi u tin ngh n s dng mi trng c trong nghin cu vi sinh vt l Robert Koch
khi tnh c thy cc khun lc ca vi khun trn c khoai ty v ng dng cc lt khoai ty
lm mi trng phn lp vi khun vo nm 1881. Ngi u tin dng gelatin ch to mi
trng c cng vo nm ny l mt tr l ca Koch, ng Frederick Loeffler. Vic dng thch
lm cht ng c l do c Minora Tarazaemon pht hin; khi nu thc n vi to bin v khi
ngui c thy thc n ng c li (1882). Ngi u tin dng thch thay th gelatin trong mi
trng nui cy l v ca Walther Hess (mt tr l khc ca Koch) - b Fannie Eilshemius Hess.
Sau y l vi c im ch yu ca Thch v Gelatin:
c im Thch Gelatin
Nng thng dng (%) 1,5-2,0 5-12
Nhit ha tan (
0
C) 96 25
Nhit ng (
0
C) 40 20
pH Hi acid Acid
Cht khong (%) 16 14-15
CaO (%) 1,15 0
MgO (%) 0,77 0
N (%) 0,4 18,3
Vi sinh vt c th s
dng lm cht dinh dng
Tuyt i a s khng s dng Nhiu vi sinh vt c th s dng

Mi trng bn c (semisolid medium):
Mi trng bn c l mi trng ch cha 0,2-0,7% thch v thng c s dng quan st
kh nng di ng ca vi sinh vt, quan st hiu gi thc khun th (phage)...
Mi trng dch th (liquid medium):
Mi trng dch th hay mi trng lng l cc mi trng khng b sung cc chy lm ng
c mi trng. thng kh phi dng ti my lc hay cc ni ln men c h thng thi kh v
trng (v khun) v h thng khuy o lm tan u bt kh. Mi trng dch th ngoi vic s
dng trong nghin cu ti phng th nghim cn c s dng rng ri trong sn xut ln ti cc
nh my ln men cng nghip.
- Cn c vo mc ch s dng ngi ta chia mi trng nui cy thnh nhiu loi khc nhau
Mi trng c s (minimum medium): Cc vi sinh vt tuy c yu cu dinh dng khng ging
nhau nhng ni chung v c bn th cc cht dinh dng l ging nhau. Mi trng c s l mi
trng c cha cc cht dinh dng c bn cn thit cho s sinh trng, pht trin ca a s vi sinh
vt. Mi trng c s thng dng l Mi trng cao tht - peptone. Mi trng c s c dng
lm thnh phn c bn cho nhng mi trng c bit, ty theo yu cu ca tng nhm vi sinh vt
m b sung thm cc cht dinh dng cn thit.
Mi trng lm giu hay cn gi l mi trng gia ph (enrichment medium): Trn mi trng
c s cho thm mt s cht dinh dng c bit thch hp vi vic nui cy mt s nhm vi sinh
vt. Cc cht b sung thm c th l mu, huyt thanh, cao nm men, m ng vt hay thc vt. V
d nui cy vi khun Bordetella pertussis ngi ta dng mi trng c s Difco 0048 nhng b
sung bng thao tc v trng mu th ( lc qua nn lc) sau khi kh trng mi trng 15 pht
121
0
C, sao cho nng cui l 15%.

Bordetella pertussis
Mi trng gim bit (differential medium)
Mi trng gim bit dng trong vic gim nh cc loi vi sinh vt khc nhau xc nh v tr
phn loi ca chng. Cc mi trng gim bit v phng php s dng c trnh by trong
Tp I (Th gii vi sinh vt). Chng hn khi xc nh kh nng sinh protease th b sung casein hay
gelatin, kh nng sinh amylase th thm tinh bt tan, kh nng sinh lipase th thm du n v ch th
mu, kh nng sinh H
2
S th thm Pb acetat, ..Ngi ta thng dng mi trng EMB (Eosin
Methylene Blue) gim bit vi khun ng rut. Mi trng ny c thnh phn nh sau:
Peptone-10g; Lactose-5g; Saccharose-5g K
2
HPO
4
- 2g; EosinY-0,4g; Methylene Blue-0,065g; Nc
ct-1000ml; pH=7,2. Mi trng ny c ch vi khun Gram (+) v mt s vi khun Gram (-). T
mi trng ny kim tra thm mt vi th nghim vi cc khun lc xut hin c th phn lp c
nhiu loi vi khun ng rut Gram (-) theo s sau y:
a- Ln men lactic, sinh acid.
b- Sinh acid mnh,khun lc chiu sng thy c mu ta, phn quang c mu lc nh kim
...Escherichia coli
bb-Sinh acid yu, khun lc c mu nu g ...Enterobacter,Serratia, Klebsiella, Hafnia
aa- Khng ln men lactic, khng sinh acid, khun lc trong v mu... Proteus, Salmonella,
Shigella.
Mi trng chn lc (Selective medium)
Dng mi trng chn lc phn lp tng nhm vi sinh vt ring bit t mt qun th vi sinh
vt trong t nhin. Da vo yu cu dinh dng c bit ca tng nhm vi sinh vt hoc tnh mn
cm khc nhau i vi ha cht, vi cht khng sinh m a thm vo mi trng nhng cht
tng thch, nhm c ch s sinh trng ca cc nhm vi sinh vt khc v gip cho phn lp c
nhm vi sinh vt cn nghin cu. C nhng mi trng chn lc c thit k da trn nhu cu
dinh ng c bit ca tng nhm vi sinh vt nht nh. V d dng cellulose hay du parafin lm
ngun carbon duy nht khi phn lp nhm vi sinh vt phn hy celluose hay phn hy parafin, dng
protein lm ngun nitrogen duy nht phn lp vi sinh vt sn sinh proteinase, dng mi trng
khng cha nitrogen phn lp vi sinh vt c nh nitrogen. V d mi trng v m Ashby
dng phn lp vi khun Azotobacter c thnh phn nh sau: Mannit-1%; KH
2
PO
4
-0,025%,
MgSO
4
.7H
2
O-0,02%; NaCl-0,02%; CaSO
4
.2H
2
O-0,01%; CaCO
3
-0,5%.
Cng c loi mi trng chn lc thm 10% phenol s lm c ch s sinh trng ca vi khun
v vi nm nhng li c th phn lp c x khun. Nu thm vo mi trng Bi sulphat th c th
c ch c cc vi khun Gram (+)v phn ln cc vi khun Gram (-), nhng li phn lp c vi
khun thng hn (Salmonella typhi). Thm vo mi trng Brilliant green hay Crystal violet th
c ch c vi khun Gram (+) nhng li phn lp c vi khun Gram (-). Trm vo mi trng
Streptomycin th c th c ch c nhiu loi vi khun nhng li phn lp c vi nm. Thm vo
mi trng Na propionat c th c ch c nm si nhng li phn lp c nm men. Trong K
thut di truyn (Genetic engineering) ngi ta thng xuyn s dng cc mi trng chn lc cha
cc khng sinh xc nh tch ra cc chng mang gen ti t hp.
Trong thc t c nhng mi trng va l mi trng chn lc, va l mi trng gim bit. V
d phn lp t cu khun vng (Staphylococcus aureus) ngi ta thm vo mi trng 7,5%
NaCl, Mannit v ch th mu acid-kim. Vi khun ny va chu c nng NaCl cao , va
chuyn ha mannit thnh acid.

Staphylococcus aureus
Sau y l mt s cht c b sung vo mi trng (MT) chn lc khi cn thit phn lp
mt s nhm vi sinh vt nht nh: Potassium tellurite (MT Mueller tellurite) phn lp
Corynebacterium diphtheriae; Tellurite v Crystal violet (MT Mitis-salivarius) phn lp
Streptococcus; Na azide (MTAzide glucose) phn lp Streptococcus; Phenylethanol (MT
Phenylethanol) phn lp Staphylococcus v Streptococcus; Nc p c chua (MT nc p c
chua) phn lp vi khun lactic t nc bt; Desoxycholate, citrate (MT Desoxycholate citrate)
phn lp vi khun ng rut Gram(-); Mt(bile),citrate, brilliant green (MT SS) phn lp
Salmonella v Shigella; Malachite green dye (MT Lowenstein-Jensen) phn lp Mycobacterium;
Chloramphenicol (MT Emmon) phn lp nm; Rose Bengal v Streptomycin (MT Martin)
phn lp nm...

Corynebacterium diphtheriae
Ngoi cc loi mi trng k trn cn c cc loi mi trng c bit khc. l Mi trng
phn tch (assay medium) dng nh lng vitamin, cht khng sinh... l Mi trng kh
(reduced medium) dng nui cy cc vi sinh vt k kh. l Mi trng nui cy m (Tissue-
culture medium) chuyn phc v cho vic nui cy t bo v m ng, thc vt, hoc dng nui
cy trn t bo cc nhm vi sinh vt chuyn k sinh nh virt, Chlamydia, Rickettsia, Spirochete.
Mt s virt v Rickettsia khng pht trin c trn cc mi trng nhn to m phi nui cy trn
phi g, trn t bo thn kh, trn c th ng vt thc nghim.
Di y l mt vi gi quan trng khi chun b mi trng nui cy.
Rt nhiu ng d b phn gii trong qu trnh kh trng pH kim (c bit vi s c mt
ca photsphate v peptone), lm cho mu mi trng chuyn thnh mu nu v cc sn phm to
thnh c th c ch s sinh trng ca vi sinh vt. trnh tnh trng , ngi ta kh trng mi
trng pH acid nh hoc kh trng ring bit i vi ng.
Tt c cc kim loi vi lng d dng to nn mui photphat khng tan v kt ta trong mi
trng nui cy. iu ny c th c trnh bng cch b sung thm cc nhn t c i lc vi kim
loi (metal-chelating agents) nh l EDTA (Ethylene Diamine Tetraacetic Acid) hay NTA
(Nitrilotriacetic acid) hay i khi l cc axit cacboxylic nh citrate hay tartrate. Vic thm cc nhn
t ny c hiu qu hai mt. Mt mt, n ngn chn s kt ta ca cc kim loi vi lng, mt khc
n hot ng ging nh mt b cha cc kim loi , bng cch ny c th lm gim tnh c nh
gim nng t do ca chng (ti mc m cc vi sinh vt c th s dng c).
mi trng pH >7, cc kim loi kim th Ca v Mg (di dng vi lng) d dng kt ta vi
s hin din ca photphate (hay s c mt ca ion carbonate khi s dng mi trng m l
bicarbonat, hay c sn trong nc cng) to nn hm lng mui khng tan cao. Nhng kt ta ny
i khi kh thy bng mt thng, c bit trong cc bnh nui cy lc do th tch nh ca mi
trng. trnh iu ny, mi trng c th c kh trng pH hi axit (pH c iu chnh
sau), hay mui photphat c kh trng ring r vi mi trng v kt hp sau khi lm ngui.
Cn ch rng a s cc mi trng c in s dng trc nhng nm 60 ca th k trc
thng khng bao gm cc nguyn t vi lng. S thm vo thng l khng cn thit bi v cc
nguyn t vi lng c cha sn trong cc mui khng tinh sch c s dng chun b cho
mi trng. Mi trng hin nay c chun b vi cc mui tinh sch cao nn khng ng ngc
nhin l s tht bi trong vic to ra nhiu sinh khi sn phm nu khng c b sung cc nguyn
t vi lng vo trong mi trng. Mt v d in hnh l mi trng c in M9 c s dng rt
rng ri cho s sinh trng ca E.coli trong cc nghin cu di truyn. Mi trng ny khng cung
cp thun li cc nguyn t vi lng cho s pht trin ca E.coli trong mt vi th h, sau chng
sinh trung chm li v cui cng l ngng li.

4. S HP THU CC CHT DINH DNG VI SINH VT
tn ti, sinh trng v pht trin, t bo vi sinh vt phi thng xuyn trao i vt cht v
nng lng vi mi trng bn ngoi. Mt mt chng tip nhn cc cht dinh dng t mi trng,
mt khc chng thi ra mi trng mt s sn phm trao i cht. T bo vi sinh vt s dng cc
cht dinh dng bt u t vic hp thu chng. C ch ca s hp thu ny c tnh chuyn ha, ni
cch khc chng ch hp thu cc cht cn thit, vic hp thu cc cht khng s dng c l bt li
i vi t bo. Vi sinh vt thng sng trong cc mi trng ngho cht dinh dng, do chng
phi c nng lc vn chuyn cht dinh dng t mi trng c nng thp vo mi trng c
nng cao bn trong t bo, tc l ngc li vi gradient nng . Nh th l gia trong v ngoi
t bo c mt hng ro thm thu, l mng sinh cht c tnh thm thu chn lc. Chng cho php
cc cht dinh dng xm nhp vo t bo v cn tr cc cht khc. Do tnh a dng v phc tp ca
cc cht dinh dng nn vi sinh vt c nhiu phng thc khc nhau vn chuyn cc cht dinh
dng. Quan trng nht l cch Khuch tn xc tin (Facilitated diffusion), cch Vn chuyn ch
ng (Active transport) v cch Chuyn v nhm (Group translocation). cc vi sinh vt c nhn
tht khng thy c cch Chuyn v nhm nhng c cch s dng qu trnh Nhp bo
(Endocytosis).Cu to ca mng sinh cht c biu th qua hnh 13.6 sau y:

Hnh 13.6: Cu trc ca mng sinh cht (Theo sch ca Prescott, Harley v Klein).

13.4.1. S khuch tn xc tin (Facilitated Diffusion)
Mt s t cc cht, nh glycerol, c th i qua mng t bo cht theo phng thc Khuych tn
b ng (Passive diffusion). Khuych tn b ng cn c gi tt l Khuych tn, l vic cc
cht dinh ng chuyn t ch c nng cao n ch c nng thp. Khuych tn b ng
mun lm cho t bo hp th c hiu qu mt s cht dinh dng cn c nng cht ny bn
ngoi t bo cao hn bn trong. Tc hp thu ty theo lc t bo tng lng hp thu cht ny m
gim xung. Tr phi loi cht dinh dng ny sau khi xm nhp t bo lp tc c s dng v
khng lm nng cao nng cht trong t bo. Ch c nc (H
2
O), O
2
v CO
2
, l nhng phn t
rt nh mi thng c vn chuyn qua mng bng phng thc khuch tn b ng. Cc phn t
tng i ln hn, cc ion v cc cht c tnh cc (polar substances) kh c th i qua mng sinh
cht bng phng thc khuch tn b ng.

Hnh 13.7: Khuch tn b ng (ng thng) v khuch tn xc tin (ng cong)
(Theo sch ca Prescott, Harley v Klein).
Protein mang (carrier protein) cn gi l enzim permease l mt loi protein gn trn mng. Vi
s h tr ca permease c th nng cao rt nhiu tc khuch tn qua mng c tnh thm thu
chn lc. Phng thc vn chuyn qua mng vi s h tr ca permease c gi l s khuch tn
xc tin (facilitated diffusion). Tc ca qu trnh khuch tn xc tin tng ln khi s chnh lch
nng cht dinh dng gia trong v ngoi t bo tng ln. Khi nng cc cht dinh dng
tng i thp th khun kh tng ln cao hn so vi phng thc khuch tn b ng. Lc gradient
nng t ti mt tr s nht nh th dn n hiu ng bo ha. S tham gia ca Permease lm
dn n hiu ng bo ha (hnh 13.7)
ng ch l, lc permease b bo ha, s khuch tn xc tin khng tng ln do s tng mc
chnh lch cht dinh dng trong v ngoi t bo. Quan h gia tc khuch tn xc tin v
gradient nng cht dinh dng tong t nh mi quan h gia enzyme v c cht, v khc hn
vi ng biu din thng phn nh s khuch tn b ng. Ngoi ra s ging nhau gia permease
v enzyme cn ch c tnh chuyn nht i vi cht vn chuyn, mi loi permease ch c th vn
chuyn mt cch chn lc i vi mt s cht tng thch. D c s tham gia ca permease nhng
khuch tn xc tin vn ng l phng thc vn chuyn khuch tn. Vic vn chuyn vn phi da
vo s chnh lch nng cht dinh dng gia trong v ngoi mng. Khi mt i s chnh lch
nng s vn chuyn s dng li. Qu trnh ny khng cn ti nng lng trao i cht
(metabolic energy) ca t bo. Gradient nng c th duy tr khi t bo chuyn bin cht dinh
dng c vn chuyn thnh mt hp cht khc hoc chuyn cht dinh dng ti mt v tr
khc ca mng ( sinh vt c nhn tht). Tht th v khi thy mt s permease ny lin quan n
protein ch cht ca thu knh mt ng vt c v, l cc protein thuc h MIP. Trong vi
khun 2 loi knh MIP phn b rng ri nht l aquaporins vn chuyn nc v glycerol facilitators
(cc nhn t xc tin glycerol) vn chuyn glycerol.
Mc du c rt nhiu nghin cu i vi c ch khuch tn xc tin nhng qu trnh ny vn
cha c hiu bit mt cch y . Hnh nh phc hp permease xuyn ngang qua mng t bo.
Sau khi cht dinh dng c kt gn bn ngoi mng, cu hnh ca permease pht sinh bin ha
phng thch c cht dinh dng vo bn trong mng. Permease sau li hi phc li cu
hnh ban u v sn sng n nhn phn t dinh dng khc bn ngoi mng. Kt qu ca qu
trnh ny l mt phn t khng tan trong lipid c th i vo t bo p li gradient nng ca n.
Nn nh rng, c ch ny c th o ngc bi gradient nng , nu nng mt s vt cht trong
t bo cao hn bn ngoi th cng c th thng qua phng thc ny m chuyn vn ra ngoi t bo.
V thng qua hot ng trao i cht m t bo tiu hao rt nhanh cc cht dinh dng a vo t
bo nn khng c chuyn cht dinh dng b a ngc ra ngoi (hnh 13.8).
cc sinh vt nhn nguyn thy qu trnh khuch tn xc tin khng phi l phng thc vn
chuyn ch yu v nng cht dinh dng bn ngoi t bo thng rt thp cho nn khng th
thc hin c qu trnh khuch tn xc tin hp th cht dinh dng. Glycerol c vn chuyn
bi qu trnh khuch tn xc tin E.coli, Salmonella typhimurum, Pseudomonas, Bacillus v nhiu
vi khun khc. S khuch tn xc tin thng gp t bo sinh vt nhn thc, chng dng phng
thc vn chuyn ny chuyn vn cc loi ng v amino acid vo t bo.

Hnh 13.8. Mt kiu Khuch tn xc tin (Theo sch ca Prescott, Harley v Klein)
13.4.2. S vn chuyn ch ng (Active Transport)
Mc du s khuch tn xc tin gip chuyn vn c hiu qu cht dinh dng vo bn trong t
bo khi nng cht ha tan bn ngoi cao hn bn trong t bo, nhng khng th vn chuyn
c cht dinh dng khi nng cht ha tan trong t bo cao hn bn ngoi. Vi sinh vt thng
sng trong cc mi trng c nng cht dinh dng rt thp, c th sinh trng v pht trin
chng phi c th vn chuyn v hp thu c t mi trng cc cht dinh dng c nng thp.
Khi khuch tn xc tin khng cn l phng thc vn chuyn hu hiu na m phi c nhng
phng thc vn chuyn khc, trong quan trng nht l phng thc vn chuyn ch ng
(active transpore) v phng thc chuyn v nhm (group translocation); c hai phng thc ny
u cn ti nng lng.
S vn chuyn ch ng l loi phng thc vn chuyn cc phn t cht ha tan ti ni c
nng cao hn, tc l ngc li vi gradient nng v cn phi tiu hao nng lng. V s vn
chuyn ch ng cn ti cc protein mang (permease) nn tng t vi s khuch tn xc tin trong
mt s phng din. Permease c tnh chuyn nht cao i vi cc phn t c vn chuyn. Cc
phn t cht ha tan c tnh cht tng t c th ln kt vi permease trong c hai trng hp -
khuch tn xc tin v vn chuyn ch ng. Trong trng hp nng cc cht dinh dng kh
cao s vn chuyn ch ng cng c hiu ng bo ha (hnh 13.9). Tuy nhin, s khc nhau ln
nht gia hai loi ny l vn chuyn ch ng c th vn chuyn ngc nng nhng cn tiu hao
nng lng trao i cht. Cc cht c ch trao i cht c th lm tr ngi vic sn sinh nng lng
do lm c ch s vn chuyn ch ng, nhng khng lm nh hng n qu trnh khuch tn
xc tin (ngay c trong thi gian ngn).
Vi khun, c khun v cc vi sinh vt nhn tht c cc h thng vn chuyn protein kt hp
(Binding protein transport systems) hoc protein vn chuyn hnh hp kt hp vi ATP (ATP-
binding cassette transporters) hay cn gi l protein vn chuyn ABC (ABC transporter). Loi
protein vn chuyn ny thng c to thnh mt phc th nh s kt hp gia hai vng xuyn
mng a nc (hydrophobic membrane - spanning domain) trn b mt t bo cht v hai vng gn
vi nucleotide (hnh 13.9).

Hnh 13.9: Cng nng ca protein vn chuyn hnh hp c kh nng kt hp vi ATP (Theo sch
ca Prescott, Harley v Klein)
(1)=Protein mang cht ha tan c gn vi c cht vn chuyn v hng n phc cht protein
vn chuyn ABC
(2)=Protein mang cht ha tan gn vo protein vn chuyn v phng thch c cht, chuyn qua
mng nh nng lng ca s thy phn ATP
Vng xuyn mng hnh thnh mt l nh trong mng v vng kt hp nucleotide s gn vi
ATP ri thy phn ATP hp th cht ha tan. Protein vn chuyn ABC tn dng protein lin kt
c cht chuyn bit nm trn khe chu cht ca vi khun Gram m hoc bm trn mng lipid ti mt
ngoi ca mng sinh cht vi khun Gram dng. Cc protein lin kt ny (cng tham gia vo qu
trnh ha hng ng-chemotaxis) s gn vi phn t c vn chuyn, ri tng tc vi protein
vn chuyn mng chuyn phn t ha tan vo trong t bo. Vi khun E.coli dng c ch ny
vn chuyn nhiu loi ng (arabinose, maltose, galactose, ribose) v aminoacid (glutamate,
histidine, leucine).
Cc cht a vo vi khun Gram (+) phi i qua mng ngoi trc khi pht huy tc dng ca
protein vn chuyn ABC v cc h thng vn chuyn ch ng khc. Cc phn t ng c th s
dng mt protein l ph bin nh OmpF. Cc phn t ln hn phi dng ti cc protein l mng
chuyn bit. Trong mt s trng hp, v d vic hp thu st v vitamin B
12
phi dng ti cc
protein vn chuyn v protein tip nhn mng ngoi c i lc cao chuyn bit.
ng ch l protein vn chuyn ABC sinh vt nhn tht nhiu khi c tm quan trng ln
trong y hc. Mt s t bo ung th s dng cc protein vn chuyn ny bm thuc ra. Vic x
ha nang l kt qu ca mt t bin lm bt hot mt protein vn chuyn ABC i vi chui
chuyn ion chloride trong phi.
Vi khun cng dng gradient proton pht sinh ra khi chuyn vn in t thc y s vn
chuyn ch ng. Cc protein vn chuyn mng chu trch nhimi vi qu trnh ny thiu ht cc
protein lin kt chu cht chuyn bit kt hp vi cc cht dinh dng. Lactose permease vi
khun E.coli l mt v d in hnh. Permease ny l mt protein n c phn t lng khong 30
000. N vn chuyn phn t lactose khi c mt proton xm nhp t bo (nng proton cao bn
ngoi t bo l do hot ng ca chui chuyn vn in t). S vn chuyn lin kt ca hai c cht
theo cng mt hng c gi l vn chuyn ng hng (symport). Trong qu trnh ny nng
lng tch t trong gradient proton c huy ng vn chuyn vt cht. Mc du c ch ca
phng thc vn chuyn ny cn cha c hiu bit y nhng ni chung c cho rng
proton v permease sau khi kt hp s ci bin hnh dng v i lc hp th cht dinh dng. Vi
khun E.coli cng dng s vn chuyn ng hng vi proton vn chuyn aminoacid v cc acid
hu c nh succinate v malate.
Mt gradient proton cng c th thng qua vic hnh thnh mt gradient ion natri gin tip
tc ng ln s vn chuyn ch ng (hnh 13.10).
Cc cht vn chuyn c chuyn xuyn mng theo phng hng tng phn nh vy c
gi l vn chuyn ngc hng (antiport). Gradient natri sinh ra trong ngoi t bo do h thng vn
chuyn proton ngc hng ny s c th dn n vic hp thu ng v acid amin vo t bo. Mt
ion natri c th lin kt vi mt protein mang v gy ra s bin i hnh dng. Protein mang s kt
hp mt thit vi ng hay acid amin v nh hng chng chuyn vo bn trong t bo. Do nng
natri trong t bo thp, ion natri c th tch ri ra khi protein mang v cht dinh dng c
vn chuyn cng c tch ra theo. Cng vi vic ion natri di ng vo t bo vi khun E.coli th
protein mang cng s chuyn ng melibiose v acid amin glutamate vo t bo ny.

Hnh 13.10: Tc dng ca gradient proton v natri trong vn chuyn ch ng (Theo sch ca
Prescott, Harley v Klein).
1- Proton bm ra ngoi mng sinh cht khi vn chuyn in t
2-Gradient proton thng qua c ch vn chuyn ngc hng (antiport mechanism) y ion
natri ra ngoi
3- Ion natri lin kt vi phc hp protein mang (carrier protein complex)
4-im kt hp vi cht ha tan (dung cht) bin i hnh dng v gn vi dung cht (ng hoc
aminoacid)
5-Cu hnh ca protein mang (carrier) thay i, ion natri c chuyn vo trong t bo v sau
dung cht cng ri khi protein mang
S vn chuyn ng hng (symport) hay vn chuyn hip ng (cotransport) natri cng l mt
qu trnh quan trng cc t bo nhn tht (eucaryotic) khi hp thu ng v acid amin. Nhng
gradient ion natri thng sinh ra do vic thy phn ATP ch khng phi do lc chuyn ng ca
proton.
Vi sinh vt thng thng qua nhiu h thng vn chuyn hp thu mt cht dinh dng. V d
nh vi khun E.coli, t nht cng c ti 5 h thng vn chuyn hp thu ng galactose, 3 h
thng vn chuyn hp thu glutamate v leucin, 2 h thng vn chuyn hp thu ion kali. Khi c
nhiu h thng vn chuyn nh vy i vi cng mt c cht, cc h thng c s khc nhau v
ngun nng lng tiu hao, v i lc i vi cht dinh dng v v phng thc iu tit h thng
vn chuyn. Tnh a dng v cc phng thc vn chuyn nh vy gip cho vi sinh vt cng c
u th cnh tranh mnh m trong iu kin mi trng d bin i.
13.4.3. S chuyn v nhm (Group Translocation)
Trong vic vn chuyn ch ng, cc phn t ha tan vn chuyn qua mng m khng cn ci
bin. Nhiu vi sinh vt nhn s cn c th thng qua vic chuyn v nhm hp thu cht dinh
dng. Trong qu trnh ny vt cht c vn chuyn s c pht sinh bin ha ha hc. Nhm ny
c th xp vo loi vn chuyn ph thuc nng lng v cn s dng nng lng trao i cht. H
thng chuyn v nhm quen bit nht l Phosphoenolpyruvate: h thng phosphotransferase ng
(PTS). Nhiu loi ng thng qua phng thc vn chuyn ny chuyn vo t bo vi sinh vt
nhn s khi b phosphoryl ha v s dng phosphoenolpyruvate (PEP) lm th cho phosphate:
PEP + ng (ngoi bo) Pyruvate + ng + P (ni bo)
PET rt phc tp, vi khun E.coli v Salmonella typhimurium PTS to thnh bi 2 enzym (EI
v EII) v 1 protein n nh nhit c phn t lng thp (HPr). HPr v Enzym EI l tn ti trong t
bo cht. Enzym EII c nhiu bin ha trong cu trc, thng do hp thnh bi 3 tiu n v
(subunits) hay vng kt cu (domains), trong EIIA (trc y gi l EIII) l protein t bo cht
c th ha tan, EIIB cng l mt protein a nc (hydrophilic), EIIC l protein k nc nm trn
mng t bo, hai loi sau thng kt hp li vi nhau. Trong qu trnh vn chuyn, di tc dng
ca EI v HPr mt phosphate cao nng t PEP chuyn n enzym EII vi s gip ca Enzym I
v HPr. Sau EII a 1 phn t ng qua mng vo t bo v c phosphoryl ha. EII ch vn
chuyn chuyn ha tng loi ng v trong cc h thng PTS khc nhau c cc EII khc nhau, cn
EI v HPr l ging nhau trong mi h thng PTS.

Hnh 13.11: Chuyn v nhm (Theo sch Microbiology ca Prescott, Harley v Klein)
H thng PTS phn b rng ri cc vi sinh vt nhn s. Cc chi vi khun Escherichia,
Salmonella, Staphylococcus v cc vi khun k kh khng bt buc (facultatively anaerobic) khc
u c h thng PTS. Mt s vi khun k kh bt buc, nh thuc chi Clostridium, cng c h
thng PTS. Mt s vi khun thuc chi Bacillus c c hai h thng ng phn (Glycolyse) v PTS.
Nhng cc vi khun hiu kh hu nh khng c h thng PTS. Nhiu carbohydrate c vn
chuyn bi h thng ny. Vi khun E.coli hp thu glucose, fructose, mannitol, saccharose, N-
acetylglucosamine, cellobiose v nhiu carbohydrate nng phng thc chuyn v nhm. Ngoi vai
tr dng vn chuyn, protein PTS cn l c quan cm th ha hc - cm th kh trong qu trnh
ha hng ng.
13.4.4. S hp th St (Iron Uptake)


Hnh 13.12. Siderophore
Hu nh tt c vi sinh vt u cn s dng st (Fe) cu to nn cc Cytochrome v nhiu
enzym. St rt kh hp th v ion st (Fe
3+
) v cc dn xut ca chng rt kh ha tan, trong mi
trng thng c rt t cc hp cht st d ha tan c th vcn chuyn vo t bo. Vic hp thu
st ca vi sinh vt l ht sc kh khn. Nhiu vi khun v nm phi khc phc kh khn ny bng
cch thng qua th mang st (siderophore). l nhng phn t c phn t lng thp li lin kt
vi st v chuyn vn c vo t bo, thng l cc mui hydroxamates hoc phenolates-
catecholates. Ferrichrome l mt loi hydroxamate c sinh ra bi nhiu nm; enterobactin l loi
catecholate c sinh ra bi E.coli... Trong hnh bn ta thy 3 loi th mang st di cc dng khc
nhau.
Khi mi trng c cha vi hm lng thp cc st c th s dng vi sinh vt s tit ra cc th
mang st (siderophones) kt hp vi st v chuyn n mng t bo. Lc s kt hp tip vi
protein th th (receptor-protein) chuyn st vo trong t bo, hoc ton b phc th St-
siderophone c chuyn vo trong nh mt protein vn chuyn ABC (ATP-binding cassette
transporter). vi khun E.coli th th siderophone nm mng ngoi (outer membrane), sau khi Fe
3+

c chuyn n khe chu cht (periplasmic space) th vi s h tr ca protein vn chuyn st s
c chuyn qua mng sinh cht (plasma membrane), sau Fe
3+
s c kh thnh Fe
2+
. V st rt
cn cho qu trnh sinh trng, pht trin ca vi sinh vt cho nn vi sinh vt cn s dng nhiu
phng thc hp thu khc nhau p ng nhu cu ny.
Bi 15 Sinh trng v pht trin ca vi sinh vt
Sinh trng l biu th s tng trng cc thnh phn ca t bo. i vi cc vi sinh vt c hnh
thc sinh sn bng ny chi hay phn i th sinh trng dn ti s gia tng s lng t bo. T bo
tng trng n mt mc nht nh th s phn ct thnh hai t bo th h con c kch thc hu
nh bng nhau. i vi cc vi sinh vt a nhn th s phn cch nhn khng ng hnh vi s phn
ct t bo - s sinh trng lm tng kch thc t bo m khng lm tng s lng t bo. V vi sinh
vt rt nh b cho nn l i tng rt khng thun tin nghin cu v sinh trng v pht trin.
Chnh v vy m khi nghin cu v sinh trng, ngi ta thng xt n s bin i v s lng
ca c qun th vi sinh vt.
14.1. NG CONG SINH TRNG
S sinh trng qun th vi sinh vt c nghin cu bng cch phn tch ng cong sinh trng
trong mt mi trng nui cy vi sinh vt theo phng php nui cy theo m (batch culture) hoc
trong mt h thng kn. C ngha l vi sinh vt c nui cy trong mt thit b kn, trong qu trnh
nui cy khng thay i mi trng v thi gian nui cy cng ko di th nng cht dinh dng
cng gim st, cc cht ph thi ca trao i cht cng tng ln. Nu ly thi gian nui cy l trc
honh v ly s logarit ca s lng t bo sng lm trc tung s c th v c ng cong sinh
trng ca cc vi sinh vt sinh sn bng cch phn i. ng cong ny c 4 giai on (phases)
khc nhau.

Hnh 14.1: ng cong sinh trng trong h thng kn
(Theo sch ca Prescott, Harley v Klein)
14.1.1. Giai on Tim pht (Lag phase)
Khi cy vi sinh vt vo mt mi trng mi s lng thng khng tng ln ngay, l giai on
Tim pht hay pha Lag. Trong giai on ny t bo cha phn ct nhng th tch v khi lng tng
ln r rt do c s tng cc thnh phn mi ca t bo. Nguyn nhn l do t bo trng thi gi,
thiu ht ATP, cc cofactor cn thit v ribosome. Thnh phn mi trng mi khng ging mi
trng c cho nn t bo cn mt thi gian nht nh tng hp cc enzyme mi nhm s dng
c cc cht dinh dng mi. Cc t bo cng c th b thng tn v cn mt thi gian hi
phc. Bt k v nguyn nhn g th kt qu vn l t bo phi t trang b li cc thnh phn ca
mnh, ti to ADN v bt u tng khi lng. Giai on tim pht di hay ngn lin quan n bn
thn tng loi vi sinh vt v tnh cht ca mi trng. Nu tnh cht ha hc ca mi trng mi sai
khc nhiu vi mi trng c th giai on tim pht s ko di. Ngc li, nu cy t giai on
logarit vo mt mi trng c thnh phn tng t th giai on tim pht s rt ngn li. Nu cy
vi sinh vt t giai on tim pht hay t giai on t vong th giai on tim pht s ko di.
14.1.2. Giai on logarit (Log Phase) hay Pha Ch s (Exponential Phase)
Trong giai on ny vi sinh vt sinh trng v phn ct vi nhp ti a so vi bn tnh di truyn
ca chng nu gp mi trng v iu kin nui cy thch hp. Nhp sinh trng ca chng l
khng thay i trong sut giai on ny, cc t bo phn i mt cch u n. Do cc t bo sinh
ra ch khc nhau rt t cho nn ng cong sinh trng l mt ng trn nhn ch khng gp khc
(hnh 14.1). Qun th t bo trong giai on ny c trng thi ha hc v sinh l hc c bn l nh
nhau cho nn vic nui cy giai on ny thng c s dng nghin cu sinh ha hc v
sinh l hc vi sinh vt.
Sinh trng logarit l sinh trng ng u, tc l cc thnh phn t bo c tng hp vi tc
tng i n nh. Nu cn bng dinh dng hay cc iu kin mi trng thay i s dn n s
sinh trng khng ng u. S sinh trng khi nhp tng hp cc thnh phn ca t bo tng
i bin ha s bin i theo cho n khi t ti mt s cn bng mi. Phn ng ny rt d quan st
thy khi lm thc nghim chuyn t bo t mt mi trng ngho dinh dng sang mt mi trng
giu hn. T bo trc ht phi to nn cc ribosome mi c th nng cao nng lc tng hp
protein, sau l s tng cng tng hp protein v ADN. Cui cng tt yu dn n tc pht
trin nhanh chng.
Lc chuyn qun th t bo t mt mi trng giu dinh dng ti mt mi trng ngho th cng
c kt qu v s sinh trng khng ng u nh vy. Vi sinh vt trc c th thu c t mi
trng nhiu thnh phn ca t bo nhng khi chuyn sang mi trng ngho chng cn c thi
gian to ra cc enzyme cn thit sinh tng hp cc thnh phn khng c sn trong mi trng.
Sau t bo mi c th phn ct, ADN mi c th ti to, nhng vic tng hp protein v ARN l
chm cho nn t bo nh li v t chc li s trao i cht ca chng cho n khi chng c th sinh
trng tip. Sau s sinh trng cn bng s c hi phc v tr v li giai on logarit.
Cc th nghim trn y cho thy s sinh trng ca vi sinh vt c kim sot mt cch chnh xc,
phi hp v phn ng nhanh chng vi nhng s bin i ca mi trng.
Khi s sinh trng ca vi sinh vt b hn ch bi nng thp ca cc cht dinh dng cn thit th
sn lng t bo cui cng s tng ln cng vi s tng ln ca cc cht dinh dng b hn ch
(hnh 14.2a). y chnh l c s s dng vi sinh vt trong vic nh lng vitamin v cc nhn t
sinh trng khc. Tc sinh trng cng tng ln cng vi s tng nng cc cht dinh dng
(hnh 14.2b). Hnh dng ca ng cong hu nh phn nh tc hp thu cht dinh dng nh s
chuyn vn protein ca vi sinh vt. Lc nng cht dinh dng cao th h thng vn chuyn s
bo ha v tc sinh trng khng tng ln cng vi s tng ln ca nng cht dinh dng.

Hnh 14.2: Nng cht dinh dng v sinh trng
(a )- nh hng ca s hn ch cht dinh dng i vi sn lng chung ca vi sinh vt. Lc nng cao th sn
lng chung s t ti n nh.
(b)- nh hng ca s hn ch cht dinh dng ti tc sinh trng.
14.1.3. Giai on n nh (Stationary Phase) hay Pha Cn bng
Qua giai on Logarit s sinh trng ca qun th cui cng s dng li, ng cong sinh trng i
ngang (hnh 14.1). Nng vi khun trong giai on n nh thng vo khong 10
9
/ml. Vi cc vi
sinh vt khc thng khng t c n nng ny. Vi ng vt nguyn sinh v vi to thng
ch t n nng 10
6
/ml. ng nhin, s lng t bo cui cng quyt nh bi nh hng
chung ca iu kin dinh ng, chng loi vi sinh vt v cc nhn t khc. Trong giai on ny s
lng t bo sng l khng thay i, c th do s lng t bo mi sinh ra cn bng vi s lng t
bo cht i, hoc l t bo ngng phn ct m vn gi nguyn hot tnh trao i cht.
C nhiu nguyn nhn lm cho qun th vi sinh vt chuyn sang giai on n nh. Trong
nguyn nhn ch yu l s hn ch ca cht dinh dng. Nu mt cht dinh dng thit yu b thiu
ht nghim trng th s sinh trng s chm li. Vi sinh vt hiu kh thng b hn ch bi nng
oxygen. Oxygen thng ha tan t trong nc, O
2
trong ni b mi trng rt nhanh chng b tiu
th ht, ch c cc vi sinh vt sinh trng b mt mi trng mi c nng O
2
sinh
trng. V vy khi nui cy vi sinh vt phi s dng ti my lc hay cc bin php thng kh khc.
Qun th vi sinh vt cng c th b nh ch sinh trng khi gp s tch ly ca cc sn phm trao
i cht c hi. Mt s vi sinh vt k kh (nh Streptococcus) c th ln men ng lm sn sinh
mt lng ln acid lactic hay cc acid hu c khc, lm acid ha mi trng v c ch s sinh
trng ca vi sinh vt. ng thi s tiu hao ht ng cng lm cho t bo i vo giai on n
nh. Sau na l, mt s chng c cho thy khi s lng vi sinh vt t n mt gii hn nht nh
th s sinh trng c th b nh ch. S sinh trng ca vi sinh vt chuyn sang giai on n nh
c th do kt qu chung ca rt nhiu nhn t khc nhau
Nh chng ta. thy vi khun khi nui cy theo m s chuyn sang giai on n nh khi thiu
thc n. Trong t nhin, do nhiu mi trng c nng ch dinh dng rt thp nn vi sinh vt
thng chuyn sang giai on n nh. i vi vi khun vic chuyn sang giai on n nh c th
l mt loi thch ng tt. Nhiu loi vi khun khng c s bin ha r rt v hnh thi (nh hnh
thnh bo t ni sinh-endospore) nhng chng c th thu nh kch thc li, thng do cht
nguyn sinh co li v nhn gi (nucleoid) m c li. Mt bin i quan trng hn l, khi thiu
thc n vi khun s sinh ra mt loi protein i (starvation proteins) lm cho t bo khng nhiu
hn vi cc thng tn bng nhiu con ng khc nhau. Chng lm tng cc lin kt
peptidoglycan v s bn vng ca thnh t bo. Chng hn Dps (DNA-binding protein from starved
cells), mt loi protein kt hp vi ADN ly t cc t bo i, c th bo v cho ADN. Phn t
Chaperones cn tr s bin tnh ca protein v hi phc li c cc protein b tn thng. V
nhng vic v nhiu c ch khc m cc t bo i c th kh b cht i v khng c vi
tnh trng b i, vi s bin ha ca nhit , s tn thng v xy ha v s thm thu, cng nh
tng sc khng vi cc ha cht c hi (nh chlorine chng hn). Nhng ci bin ny rt c hiu
qu v lm cho mt s vi khun c th sng li sau vi nm b i. R rng vic hiu r nhng vn
ny s c tm quan trng thc tin to ln i vi y hc v vi sinh vt hc cng nghip. Chng
cn c th chng minh vi khun thng hn (Salmonella typhimurium) v nhiu vi khun gy bnh
khc c th c kh nng gy bnh mnh hn khi b i.
14.1.4. Giai on t vong (Death Phase)
Vic tiu hao cht dinh dng v vic tch ly cc cht thi c hi s lm tn tht n mi trng
sng ca vi sinh vt, lm cho s lng t bo sng gim xung. l c im ca giai on t
vong. Ging nh giai on logarit, s t vong ca qun th vi sinh vt cng c tnh logarit (t l t
bo cht trong mi gi l khng i). Tng s t bo sng v t bo cht khng thay i v cc t
bo cht cha b phn hy. Mun xc nh s lng t bo sng phi pha long ra ri cy ln thch
a v a vo iu kin thch hp xc nh s khun lc xut hin. Mc du phn ln vi sinh vt
t vong theo phng thc logarit nhng sau khi s lng t bo t nhin gim xung th tc
cht ca t bo chm li. l do mt s c th sng li nh c tnh khng c bit mnh. V
iu ny v nhng nguyn nhn khc lm cho ng cong ca giai on t vong c th kh phc
tp.
14.1.5. Tnh ton v qu trnh sinh trng
Khng t cc nh vi sinh vt hc tnh ton v tc sinh trng ca vi sinh vt trong giai on
logarit. Tnh ton nhp sinh trng s lm c s cho cc nghin cu v sinh l hc, sinh thi hc
vi sinh vt, v cn gii quyt mt s vn ng dng trong sn xut cng nghip.
Trong giai on logarit mi c th vi sinh vt tin hnh phn ct trong mt thi gian hng nh. S
lng t bo tng theo phng thc 2
n
. Thi gian gia hai ln phn chia lin tip hay thi gian cn
cho s tng i s t bo c gi l thi gian th h (generation time hay doubling time). V d
a mt t bo vo mi trng nui cy, c 20 pht phn ct mt ln th sau 20 pht c 2 t bo,
sau 40 pht c 4 t bo v tip tc nh vy (bng 14.1)
Bng 14.1: Mt v d v sinh trng theo logarit
Thi gian
*
S ln phn ct 2
n
S lng (N
0
x 2
n
) lg
10
N
t

0 0 2
0
=1 1 0,000
20 1 2
1
=2 2 0,301
40 2 2
2
=4 4 0,602
60 3 2
3
=8 8 0,903
80 4 2
4
=16 16 1,204
100 5 2
5
=32 32 1,505
120 6 2
6
=64 64 1,806
*Thi gian th h l 20 pht, gi thit l nui cy t 1 t bo

S lng logarit t bo l 2
n
, n l s th h. C th biu th cc s liu trong bng 14.1 bng cng
thc sau y:


Trong : N
0
l s lng t bo ban u; N
t
l s lng t bo thi gian t; n l s th h.
T cng thc trn c th bin i nh sau v s th h n c tnh bng logarit thp phn:


Khi nui cy phn m (batch culture) tc sinh trng trong giai on logarit c th biu th bng
hng s tc sinh trng bnh qun k (mean growth rate constant k). l s th h sinh ra trong
n v thi gian, thng biu th bng s th h trong 1 gi:

Thi gian cn thit tng gp i tng s t bo l thi gian th h bnh qun (mean generation
time) hay thi gian tn gp i bnh qun (mean doubling time) v c biu th bng g. Nu t=g
th N
t
= 2N
0
. Thay vo cng thc trn ta c:

Thi gian th h bnh qun l o s ca hng s tc sinh trng bnh qun:

Thi gian th h bnh qun g c th cn c trc tip vo th bn logarit (semilogarithmic plot) v
hng s tc sinh trng tnh ra (hnh 14.4). V d ,s lng vi khun ti gi th 10 l t 10
3

tng ln n 10
9
th :
(th h/h)
gi/th h hay 30 pht/th h


Hnh 14.3: Sinh trng th h ca vi sinh vt (biu th 6 th
h)

(Theo sch ca Prescott,Harley v Klein).
Hnh 14.4; Xc nh thi gian th h.

Thi gian th h c th xc nh bng ng cong sinh
trng ca vi sinh vt. Ly thi gian l trc honh v ly
s lng t bo lm trc tung. Thi gian tng gp i s
lng ca qun th (thi gian th h) c th c trc tip
trn th
Thi gian th h thay i ty theo chng loi vi sinh vt, iu kin nui cy. Mt s vi khun thi
gian th h khng qu 10 pht (0,17h) trong khi mt s vi sinh vt nhn thc (eucaryotic) li di
ti vi ngy (Bng 14.2). Thi gian th h trong t nhin thng l di hn so vi khi nui cy.
Bng 14.2: Thi gian th h ca mt s loi vi sinh vt
Vi sinh vt Nhit (
0
C) Thi gian th h (gi)
Vi khun v Vi khun lam
Beneckea natriegens 37 0,16
Escherichia coli 40 0,35
Bacillus subtilis 40 0,43
Staphylococcus aureus 37 0,47
Pseudomonas aeruginossa 37 0,58
Clostridium botulinum 37 0,58
Rhodospirillum rubrum 25 4,6-5,3
Anabaena cylindrica 25 10,6
Mycobacterium tuberculosis 37 Khong 12
Treponema pallidum 37 33
To
Scenedesmus quadricauda 25 5,9
Chlorella pyrenoidosa 25 7,75
Asterionella formosa 20 9,6
Euglena gracilis 25 10,9
Ceratium tripos 20 82,8
ng vt nguyn sinh
Tetrahymena geleii 24 2,2-4,2
Leishmania donovani 26 10-12
Paramecium caudatum 26 10,4
Acanthamoeba castellanii 30 11-12
Giardia lamblia 37 18
Nm
Saccharomyces cerevisiae 30 2
Monilinia fructicola 25 30
14.2. XC NH S SINH TRNG CA VI SINH VT
C nhiu cch thng qua vic xc nh s bin i s lng v cht lng vi sinh vt hiu c
s sinh trng ca vi sinh vt, bit c tc sinh trng v thi gian th h. Di y s gii
thiu cc phng php thng dng nht cng cc u, khuyt im ca cc phng php ny.
Khng c phng php no l tt nht, la chn phng php no cn ph thuc vo tng trng
hp c th.
14.2.1. Xc nh s lng t bo
Phng php n gin nht xc nh s lng t bo l m trc tip di knh hin vi. Dng
cc phng m m va nhanh chng, d dng, li r tin nht, li c th quan st thy kch c
v hnh dng t bo. Thng dng phng m Petroff-Hausser m t bo ng vt nguyn sinh.
Dng phng m hng cu c th m c cc t bo nhn nguyn thy cng nh t bo nhn
tht. Vi t bo nhn nguyn thy cn nhum mu hoc l dng knh hin vi tng phn pha hay
knh hin vi hunh quang (phase-constrast or fluoresence microscope) d quan st hn. Phng
m c cu trc c mt su nht nh li c chia ra thnh cc nh (hnh 14.5). Khi m s
lng ta a dch pha long vo phng m, y l knh (lamelle/ cover glass) ln trn, sau tin
hnh m s lng di knh hin vi. Khuyt im ca phng php ny l khng xc nh c
vi cc mu c s lng vi khun qu nh, chnh xc cng khng cao v khng phn bit c
gia t bo sng v t bo cht.
Hnh 14.5: Phng m Petroff-Hauser:
(a)- Mt nhn nghing ca phng m- Phng m cha dch huyn ph vi khun
l khong khng gian bn di l knh; (b)- Gia phin knh c phng m vi
cc nh; (c) phng i khong x 400-500 tin hnh m s lng vi khun
trong cc nh. Ly s lng bnh qun tnh ra mt vi khun trong mu
vt. Trong phm vi 1mm
2
c 225 nh , do s lng vi khun trn 1mm
2
l (s
vi khun/mm) x 25; v phng m c chiu dy l 0,02mm do nng vi khun
trong phng m l: (s vi khun)/m
2
x 25 (tng s nh) x 50= s vi khun/mm
3
.
V 1 cm
3
=1 mm
3
x 10
3
cho nn gi th s lng vi khun bnh qun trong mi
nh l 28 th trong 1 cm
3
c nng vi khun l 28 x 25 x 50 x10
3
= 3,5 x 10
7
vi
khun. Nhn vi pha long ban u (nu c) s bit c nng vi khun
trong mu kim tra.




Vi ng vt nguyn sinh, vi to v nm men c th dng my m in t nh loi my Coulter
Counter xc nh s lng. Nguyn l l hai bn mi l nh c in cc v ni in. Khi t bo
trong dch huyn ph i qua l nh th c mi t bo i qua th in tr li tng ln (hoc tnh dn
in gim xung) v sinh ra mt tn hiu in, my m s t ng ghi s. Kt qu xc nh ca
loi my ny kh chnh xc, c th ng dng rng ri xc nh s lng hng cu v bch cu,
nhng phng php ny khng thch hp xc nh s lng vi khun v d b can thip bi cc ht
nh v cc vt cht dng si trong mu vt.

C hai phng php ni trn u khng phn bit c t bo sng v t bo cht. xc nh s
lng t bo sng ngi ta thng dng phng php cy dch pha long ln b mt mi trng
thch a. Sau khi nui cy mi vi khun s to thnh 1 khun lc. V d pha long 1 x 10
-6

m c 150 khun lc th c ngha l mt vi khun trong mu l 1,5 x 10
8
.
Dng dng c m khun lc cng thm thun tin. Phng php ny cho bit s lng cc t bo
sng ca vi sinh vt. Phng php ny n gin, nhy cm v thch hp ng dng rng ri xc
nh s lng vi sinh vt sng khi phn tch cc mu thc phm, nc, t...Tuy nhin kt qu cng
chu nh hng ca mt s nhn t. Nu vi khun dnh thnh khi khng tch ri nhau ra th kt
qu thu c l thp hn thc t., v mi khun lc khng pht trin t mt t bo ring r. V vy
kt qu thu c t phng php ny c coi l s n v hnh thnh khun lc (CFU-colony
forming unit). CFU khng hon ton ph hp vi s t bo sng trong mu vt. Trong qu trnh s
dng phng php ny nn s dng pha long no cho s khun lc xut hin trn a ch nm
trong phm vi khong 30-300 m thi. ng nhin mi trng dinh dng khng th p ng
chung cho mi loi vi sinh vt, do kt qu thu c bao gi cng thp hn thc t. Khi trn
thch vi dch pha long th thch ngui khng lm cht vi khun hay lm thng tn vi
mt s loi mn cm vi nhit . Vic cy cy dch pha long trn b mt ri dn u bng que
gt thy tinh thng cho kt qu cao hn v s lng vi sinh vt so vi phng php trn vi mi
trng thch cha ng.

Hnh 14.6: Tch khun lc v phng php kim tra s lng vi sinh vt thng qua m khun lc mc trn mi trng
thch a.
(a) (b)- Cch ria cy tch khun lc ring r (khng dng m s lng) (c)(d)- Cch pha long ri trn vi mi
trng thch cha ng
(e)(f)- Cch dn dch pha long bng que gt trn mt thch (cho s lng khun lc nhiu hn).Theo sch ca
K.P.Talaro,2005.
xc nh s lng vi sinh vt cn c th nui cy giy lc lc dch pha long mu vt.
Phng php ny gi l phng php mng lc (membrane filter). Dng mt thit b lc c bit
t va mt giy lc hnh trn c cc l nh hn kch thc vi khun v cc vi sinh vt khc. Sau
khi lc t giy lc ln mi trng thch thch hp hoc thm t mng lc bng dch mi trng
thch hp ri nui cy 24 gi. m s khun lc mc trn giy lc tnh ra mt vi khun
sng c mt trong mu vt (hnh 14.7)



Hnh 14.7: Phng php lc mng xc nh s lng vi sinh vt

Phng php ny thch hp s dng phn tch vi sinh vt trong nc. C th dng cc mi
trng khc nhau thch hp vi cc nhm vi sinh vt khc nhau (hnh 14.8)

Hnh 14.8: Cc loi khun lc mc trn mng lc.
Theo sch ca Prescott,Harley v Klein (2005)
(a)- Tng s vi khun mc trn mi trng tiu chun, Dng ch th mu nhum khun lc cho d im;
(b)- Dng mi trng thch hp kim tra nhm vi khun coliform c ngun gc t phn (khun lc bt mu xanh);
(c)- Dng mi trng thch m-Endo xc nh vi khun E.coli v cc Coliform khc- khun lc c mu lc;
(d)- Nm si v nm men mc trn mi trng Thch - Mch nha.
Phng php mng lc cn dng m trc tip vi khun. Dch mu vt c lc qua mt mng
polycarbonate mu en. Vi khun trn mng lc c nhum mu hunh quang bng thuc nhum
acridine da cam hoc DAPI (diamidino-2-phenylindole). Quan st di knh hin vi hunh quang
c th thy cc t bo vi sinh vt hin ln mu da cam hay mu lc trn mt nn en. Hin c
nhng kit thng mi cho php phn bit t bo sng v t bo cht khi kim tra.
14.2.2. Xc nh khi lng t bo
S sinh trng ca vi sinh vt khng ch biu hin s lng t bo m cn c s tng trng ca
tng khi lng t bo. Phng php trc tip nht l xc nh trng lng kh ca t bo. Trc
ht cn ly tm thu nhn sinh khi t bo. Sau ra t bo ri lm kh trong l sy ri cn trng
lng kh. Phng php ny thch hp xc nh s sinh trng ca nm. Phng php ny tn
thi gian v khng tht mn cm. i vi vi khun v trng lng tng c th l rt nh, thm ch
phi ly tm ti vi trm ml mi s lng xc nh trng lng sinh khi kh.
Phng php nhanh hn, mn cm hn l dng phng php o c nh tn x nh sng. Mc
tn x nh sng t l thun vi nng t bo. Lc nng vi khun t n 10
7
t bo/ml th
dch nui cy s vn c, nng cng tng th c cng tng theo v lm cn tr nh sng i
qua dch nui. C th o tn x nh sng bng quang ph k (spectrophotometer). mt mc
hp th nh sng thp, gia nng t bo v gi tr hp th nh sng c quan h tuyn tnh (hnh
14.9). Ch cn nng vi sinh vt t ti nng c th o c l u c th dng phng php
o c trn quang ph k xc nh s sinh trng ca vi sinh vt. Nu hm lng mt s vt
cht trong mi t bo l ging nhau th tng lng cht trong t bo c tng quan trc tip vi
tng sinh khi vi sinh vt. Chng hn, thu t bo trong mt th tch nht nh ca dch nui cy, ra
sch i ri o tng lng protein hay tng lng nitrogen, c th thy s tng qun th vi sinh vt l
ph hp vi s tng tng lng protein (hay N). Cng tng t nh vy, vic xc nh tng lng
chlorophyll c th dng o sinh khi to; o hm lng ATP c th bit c sinh khi ca cc
vi sinh vt sng.

Hnh 14.9: o s lng vi sinh vt bng phng php o c.
Thng qua vic o hp th nh sng c th xc nh c sinh khi vi sinh vt. Khi s lng t
bo tng ln s dn n vic tng c, mc tn x nh sng nhiu hn v quang ph k s o
c mc tng ln ca tr s hp th nh sng. Trn quang ph k c hai thang chia : pha
di l tr s hp th nh sng, pha trn l mc thu quang. Khi tr s hp th nh sng tng ln
th mc thu quang h xung.
14.3. NUI CY LIN TC VI SINH VT
Trong cc phn trn chng ta xem xt vic nui cy phn m (batch cultures) trong cc h thng
kn, tc l khng c chuyn b sung cht dinh dng, cng khng thi loi cc sn phm c hi sinh
ra trong qu trinh sng. Giai on logarit ch duy tr qua vi th h sau chuyn vo giai on n
nh. Nu nui cy vi sinh vt trong mt h thng h, trong qu trnh nui cy thng xuyn b
sung cht dinh dng v thi loi cc cht cn b th c th lm cho mi trng lun gi trng
thi n nh. l h thng nui cy lin tc (continuous culture system). Trong h thng ny s
sinh trng ca vi sinh vt lun gi c trng thi logarit, nng sinh khi vi sinh vt lun gi
c n nh trong mt thi gian tng i di.
Gi th ta c mt bnh nui cy trong vi khun ang sinh trng, pht trin. Ta cho chy lin tc
vo bnh mt mi trng mi c thnh phn khng thay i. Th tch bnh nui cy gi n nh.
Dng mi trng i vo b p cho dng mi trng i ra vi cng mt tc . Ta gi th tch ca
bnh l v (lit), tc dng mi trng i vo l f (lt/ gi). Tc (hay H s) pha long c gi
l D (f/v). i lng D biu th s thay i th tch sau 1 gi. Nu vi khun khng sinh trng v
pht trin th chng s b rt dn ra khi bnh nui cy theo tc :
= dX/dt = D.X
X l sinh khi t bo
Ngi ta thng dng hai loi thit b n nui cy lin tc vi sinh vt. l Chemostat v
Turbidostat.
14.3.1. Chemostat
Khi s dng Chemostat nui cy vi sinh vt ngi ta a mi trng v khun vo bnh nui cy
vi lng tng ng vi tc a mi trng cha vi khun ra khi bnh nui cy (xem hnh
14.10). Trong mi trng mt s cht dinh dng thit yu ( nh mt vi acid amin) cn khng ch
nng trong mt phm vi nht nh. V vy tc sinh trng ca vi sinh vt trong h thng
quyt nh bi tc mi trng mi c a vo h thng v nng t bo ph thuc vo nng
cc cht dinh dng c hn ch. Nhp i mi cht dinh dng biu th bi nhp pha
long D (dilution rate). Tc lu thng ca cht dinh dng (ml/h) c biu th bng f v th
tch bnh nui cy l V (ml):
D= f/V
Chng hn nu f l 30ml/h v V l 100ml th nhp pha long D l 0,30h
-1
. C s lng vi sinh vt
v thi gian th h u c lin quan n nhp pha long (hnh 14.11). Trong mt phm vi nhp
pha long tng i rng th mt vi sinh vt trong h thng l khng thay i.. Khi nhp pha
long tng ln, thi gian th h h xung (tc sinh trng tng ln), khi cht dinh dng hn
ch b tiu hao ht. Nu nhp pha long qu cao th vi sinh vt b loi ra khi bnh nui cy trc
khi kp sinh si ny n bi v lc nhp pha long cao hn tc sinh trng ca vi sinh vt.
Nng cc cht dinh dng hn ch tng ln khi nhp pha long tng cao vi c t vi sinh vt s
dng chng.

Hnh 14.10: Nui cy lin tc trong
Chemostat v Turbidostat
Hnh14.11: H thng nui cy lin tc (Chemostat)
Khi nhp pha long rt thp th nu tng nhp pha long s lm cho c mt t bo v tc
sinh trng u tng ln. l do hiu ng ca nng cht dinh dng i vi nhp sinh
trng (growth rate). Quan h ny c lc c gi l quan h Monod (Monod relationship). Trong
iu kin nhp pha long thp , ch c t i cht dinh dng c cung cp th t bo phi dng
phn ln nng lng duy tr s sng ch khng dng sinh trng, pht trin. Lc nhp pha
long tng ln, cht dinh dng tng ln, t bo c nhiu nng lng c cung cp, khng nhng
duy tr s sng m cn c th dng sinh trng, pht trin, lm tng cao mt t bo. Ni
cch khc, khi t bo c th s dng nng lng vt qu nng lng duy tr (maintenance energy)
th nhp sinh trng s bt u tng ln.

Hnh 14.12: T l pha long trong chemostat v sinh trng ca vi sinh vt
14.3.2. Turbidostat
Turbidostat l loi h thng nui cy lin tc th hai. Thng qua t bo quang in (photocell)
o hp th nh sng hay c trong bnh nui cy t ng iu chnh lu lng mi trng
dinh dng, lm cho c hay mt t bo gi mc nh d kin. Turbidostat v Chemostat
c nhiu im khc nhau. Trong h thng Turbidostat mi trng khng cha cc cht dinh dng
hn ch, nhp pha long khng c nh. Turbidostat hot ng tt nht khi nhp pha long cao
trong khi Chemostat li n nh nht v hiu qu nht khi nhp pha long tng i thp.
H thng nui cy lin tc l rt c li v cc t bo lun trng thi sinh trng thuc giai on
logarit. Hn na c th dng lm m hnh nghin cu s sinh trng ca vi sinh vt trong iu
kin nng cht dinh dng thp tng t nh mi trng t nhin. H thng nui cy lin tc
rt c ch trong vic nghin cu nhiu lnh vc khc. Chng hn, nghin cu tc dng tng h
gia cc loi vi sinh vt trong iu kin mi trng tng t nh mi trng nc ao h nc ngt.
H thng nui cy lin tc c s dng trong cc ngnh vi sinh vt hc cng nghip v thc
phm.
14.4. NH HNG CA CC NHN T MI TRNG N S SINH
TRNG CA VI SINH VT
Nh chng ta bit vi sinh vt c kh nng p ng vi s bin ha ca nng cht dinh dng,
nht l cc cht dinh dng hn ch. S sinh trng ca vi sinh vt chu nh hng rt ln i vi
cc nhn t vt l, ha hc ca mi trng sng. Hiu bit v nh hng ca cc nhn t mi
trng i vi s sinh trng ca vi sinh vt gip ch rt nhiu cho vic khng ch vi sinh vt cng
nh i vi vic nghin cu s phn b sinh thi ca vi sinh vt. ng ch l mt s vi sinh vt
c th sng c trong nhng iu kin cc oan (extreme) v kh sng (inhospitable). Cc vi sinh
vt nhn nguyn thy (Procaryotes) c th sinh tn ti mi ni c th sinh sng. Nhiu ni cc vi
sinh vt khc khng th tn ti c nhng vi sinh vt nhn nguyn thy vn c th sinh trng rt
tt. Chng hn vi khun Bacillus infernus c th sng su 1,5 dm di mt t, ni khng c
xy v c nhit cao n 60
0
C. Nhng vi sinh vt c th sinh trng c trong nhng hon cnh
h khc nh vy c gi l cc vi sinh vt a cc oan.
Trong phn ny chng ta s xem xt nh hng ca mt s nhn t ch yu ca mi trng i vi
s sinh trng ca vi sinh vt (bng 14.3)
Bng 14.3: Phn ng ca vi sinh vt vi cc nhn t mi trng
Thut ng nh ngha Vi sinh vt i din
Hot tnh ca nc v dung cht
Vi sinh vt a p (Osmotolerant) C th sinh trng trong
mt phm vi rng v hot
tnh ca nc v nng
thm thu.
Staphylococcus aureus,
Saccharomyces
Vi sinh vt a mn (Halophile) Cn sinh trng nng
NaCl cao, thng l t 0,2
mol/L tr ln.
Halobacterium,Dunaliella,
Ectothiorhodospira
pH
a acid (Acidophile) Sinh trng tt nht trong
phm vi pH 0-5,5
Sulfolobus,Picrofilus, Ferroplasma,
Acontium, Cyanidum caldarium.
a trung tnh (Neutrophile) Sinh trng tt nht trong
phm vi pH 5,5- 8,0
Escherichia, Euglena, Paramecium
a kim(Alkalophile) Sinh trng tt nht trong
phm vi pH 8,5-11,5
Bacillus alcalophilus,
Natronobacterium
Nhit
a lnh(Psychrophyle) Sinh trng tt nht 15
0
C Bacillus psychrophilus,
hay thp hn. Chlamydomonas nivalis
Chu lnh(Psychrotroph) C th sinh trng 0-7
0
C
nhng sinh trng tt nht
20-30
0
C, cn c th sinh
trng c khong 35
0
C
Listeria monocytogenes,
Pseudomonas fluorescens
a m(Mesophile) Sinh trng tt nht 25-
45
0
C.
Escherichia coli, Neisseria,
Gonorrhoeae, Trichomona vaginalis.
a nhit(Thermophile) C th sinh trng nhit
55
0
C hoc cao hn,
nhit thch hp nht
thng l gia 55 v 65
0
C
Bacillus stearothermophilus,
Thermus aquaticus, Cyanidium
caldarium, Chaetomium thermophile
a nhit cao (Hyperthermophile) Thch hp pht trin nhit
gia 80 v khong
113
0
C
Sulfolobus, Pyrodictium,
Pyrococcus.
Nng xy
Hiu kh bt buc (Obligate
aerobe)
Hon ton da vo O
2
ca
khng kh sinh trng
Micrococcus luteus, Pseudomonas,
Mycobacteriun, phn ln To, Nm
v V nguyn sinh
K kh khng bt buc
(Facultative anaerobe)
Khng cn O
2
sinh
trng nhng sinh trng
tt hn khi c mt O
2.

Escherrichia, Enterococcus,
Saccharomyces cerevisiae
K kh chu Oxy (Aetolerant
anaerobe)
Sinh trng nh nhau khi
c mt hay khng c xy
Streptococcus pyogenes
K kh bt buc (Obligate
anaerobe)
B cht khi c mt O
2
Clostridium, Bacteroides,
Methanobacterium, Trepomonas
agilis.
Vi hiu kh (Microaerophile) Cn O
2
mc thp
hn2-10% sinh trng
v b tn hi trong khng
kh (20%)
Campylobacter, Spirillum volutans,
Treponema pallidum
p sut
a p (Barophile) Sinh trng nhanh hn khi
p sut thy tnh cao
Photobacterium profindum,
Shewanella benthica,
Methanococcus jannaschii
14.4.1. Hot tnh ca nc v dung cht
V t bo vi sinh vt tch vi mi trng ca chng bng loi mng sinh cht c tnh thm thu
chn lc cho nn vi sinh vt chu nh hng ca s bin i nng thm thu trong mi trng.
Nu mt vi sinh vt c a vo dung dch c nng thm thu thp th nc s xm nhp vo
t bo, nu khng c s khng ch hu hiu th t bo s b trng ln v v ra. Nh c cc th ni
hm m c th gim thp nng thm thu ca t bo cht. Lc tnh thm thu ca mi trng
thp hn tnh thm thu ca t bo cht, cc vi sinh vt nhn nguyn thy cng c th ph v cc
knh mn cm vi p sut lm cho dung cht thm ra.
Phn ln vi khun, to v nm thng c thnh t bo vng chc, c th duy tr hnh thi v tnh
hon chnh ca t bo. Lc a t bo cc vi sinh vt c thnh t bo vng chc vo mi trng p
sut thm thu cao th nc s thot ra, mng sinh cht s tch ra khi thnh t bo v to ra tnh
trng co nguyn sinh. S mt nc ny ca t bo s tn hi ti mng t bo cht, t bo s mt kh
nng trao i cht v ngng sinh trng. iu ny lin quan n vic bo qun thc phm nh
mui mn hoc tm ng (lm mt, ngm mt ong...).
Nhiu vi sinh vt s dng dung cht hn hp (compatible solute) lm cho nng thm thu ca
nguyn sinh cht cao hn mi trng chung quanh, lm cho mng sinh cht vn gn c vi thnh
t bo. S d gi l dung cht hn hp l v dung cht c th thch hp t bo sinh trng v
pht trin ngay khi c nng cao trong t bo. Phn ln vi sinh vt nhn nguyn thy c th nng
cao nng thm thu trong t bo mi trng p sut thm thu cao l nh tng hp hoc hp
thu choline, betaine, proline, acid glutamic v cc acid amin khc. Vic nng cao nng K
+
cng
c th mt mc no gip nng cao nng thm thu trong t bo. To v nm th s dng
saccharose v cc polyol, v d nh arabitol, glycerol, mannitol,... t c mc ch nh vy.
Polyol v acid amin l nhng dung cht l tng nng cao nng thm thu trong t bo bi v
chng khng ph hy cu trc v chc nng ca enzym. Nhiu vi sinh vt nhn nguyn thy nh
Halobacterium salianarium s dng K
+
nng cao nng thm thu trong t bo, Na
+
cng c
tc dng ny nhng khng s dng c cao nh K
+
. Cc enzyme ca Halobacterium cn nng
mui cao duy tr hot tnh. ng vt nguyn sinh do khng c thnh t bo nn phi s dng cc
khng bo (vacuoles) bi xut phn nc d tha khi sng trong mi trng c nng thm
thu thp.
Tc dng tng h gia phn t nc v phn t dung cht c gi l hiu ng thm thu
(osmotic effect) cn hiu ng c cht (matric effect) l biu th cc phn t nc bi hp ph
(adsorption) trn b mt cc cht rn. Hai hiu ng ny dn n vic gim st phn nc c th
dc vi sinh vt s dng. V nng thm thu trong mi trng c nh hng su sc i vi vi
sinh vt cho nn nh lng kh nng s dng nc cc nh vi sinh vt hc thng dng khi
nim hot nc a
w
(water activity a
w
) biu th tnh hu hiu ca nc. Cng c th dng th
nng nc (water potential) tng quan vi a
w
biu th tnh hu hiu ca nc. Hot nc ca
mt dung dch l 1/100 ca m tng i ca dung dch ny (tnh theo %), cng l tng ng
vi t l gia p sut bay hi ca dung dch ny (P
soln
) v p sut bay hi ca nc tinh khit
(P
water
):

Hot nc ca mt dung dch hay mt cht rn c th xc nh bng cch a vo mt vt cha
kn v o m tng i sau khi h thng t ti trng thi cn bng (equilibrium). V d, mt
mu vt sau khi t ti trng thi cn bng trong h thng ny m khng kh t ti 95% bo ha,
th cng tc l khng kh cha 95% m khi t ti cn bng cng nhit vi mt mu nc
thun khit, hot nc ca mu vt ny l 0,95%. Hot nc t l nghch vi p sut thm
thu, nu p sut thm thu cao th tr s a
w
l thp.
Cc vi sinh vt khc nhau c nng lc thch ng khc nhau rt ln i vi mi trng c hot
nc thp (bng 14.4)
Bng 14.4: Tr s tng i v hot nc (a
w
) thp nht i vi s sinh trng ca vi sinh vt
(theo A.D. Brown, 1976)
Hot nc Mi trng Vi khun, Nm, To
1,00 Nc thun khit Phn ln VK Gram (-) khng a mn
0,95 Bnh m Phn ln trc khun Gram (-) Basidiomycetes
Fusarium
Phn ln Mucor,Rhizopus, Bacillus.
0,90 i gia sc Phn ln cu khun, Nm men c bo t ti.
0,85 Salami Staphylococcus
0,80 Thc phm mui Saccharomyces rouxii (trong mui),
Penicillium
0,75 H mui
C mui
Halobacterium, Aspergillus,
Dunaliella , Actinospora
0,70 Ng cc, ko, qu kh Aspergillus
0,60 Scla, mt ong, sa bt Saccharomyces rouxii (trong ng),
Xeromyces bisporus
0,55 ADN b ph hy
C th k thm vi v d khc v tr s a
w
: mu ngi- 0,995; qu ti- 0,97-0,98; nc bin- 0,98;
tht gia sc ti- 0,97; sir- 0,90; gim bng- 0,90; lp xng- 0,85; mt qu- 0,80; nc ng
bo ha- 0,76; bt m- 0,65...
Vi sinh vt mun gi lng nc bng cch duy tr dung cht ni bo nng cao khi sinh
trng trong mi trng c hot nc thp s gp kh khn kh ln. Nhng vi sinh vt c th
tn ti trong nhng iu kin nh vy c gi l cc vi sinh vt chu p (osmotolerant). Chng c
th sinh trng c trong mt phm vi nng thm thu hoc hot nc kh rng. V d , vi
khun Staphylococcus aureus c th nui cy trn mi trng c nng NaCl cao ti 3 mol/L.
Chng cng c th thch ng sinh trng trn da ngi. Nm men Saccharomyces rouxii c th sinh
trng trn dung dch ng c hot nc thp n 0,6. To lc Dunaliella viridis c th chu
c nng NaCl cao n 1,7mol/L hoc nng bo ha.
Mc du mt s t vi sinh vt c th thc s chu p nhng phn ln vi sinh vt ch c th sinh
trng tt hot nc khong 0,98 (tng ng vi a
w
ca nc bin) hoc cao hn na. Li
dng iu ny ngi ta s dng phng php sy kh hay dng mui, dng ng bo qun
thc phm, phng tp nhim bi vi sinh vt. Tuy nhin nhiu nm chu p vn c th lm h hng
cc thc phm sy kh hoc p mui, tm ng.
Vi sinh vt a mn (Halophile) hon ton thch ng vi mi trng cao p (hypertonic), cn nng
NaCl cao sinh trng. Phm vi nng mui cn thit sinh trng i vi nhm vi khun
a mn cc oan (extreme halophilic bacteria) l 2,8-6,2 mol/L (nng mui bo ha). Ti Bin
Cht (Dead Sea)- mt h thp nht th gii nm gia Israel v Jordan, v ti h i Dim (Great
Salt Lake) bang Utah (Hoa K) v ti cc mi trng khc c nng mui gn vi bo ha, c
th phn lp c cc C khun (archeon) thuc chi Halobacterium. Chng cng cc vi khun a
mn cc oan khc khng ging vi phn ln cc vi sinh vt chu p (osmotolerant) ch khng
phi l n gin thng qua vic nng cao nng dung cht ni bo, m ch yu l sa i cu trc
protein v mng ca mnh thch ng vi nng mui cao. Nhng vi khun a mn cc oan
ny duy tr nng kali ni bo sao cho p sut thm thu cao hn mi trng sng; nng K
+
ni bo c th ti 4-7 mol/L. Cc enzym, ribosom v protein vn chuyn ca cc vi khun ny cn
nng K
+
cao duy tr tnh n nh v hot tnh. Ngoi ra nng Na
+
cao cng gip cho s n
nh ca t bo v mng sinh cht ca vi khun Halobacterium. Nu nng Na
+
qu thp th
thnh t bo v mng sinh cht s hon ton b ph hy. Vi khun a mn cc oan thch ng thnh
cng vi iu kin mi trng mui cao, ni c th tiu dit hu ht cc sinh vt khc. Tuy nhin
chng cng bit ha (specialized), mt i tnh linh hot (flexibility) sinh thi v ch c th sinh
trng trong mt t mi trng cc oan.
14.4.2. pH
pH l s o hot tnh ion hydrogen ca mt dung dch v l s logarit m ca nng ion
hydrogen (biu th bng nng phn t):
pH= -log[H
+
]= log (1/[H
+
])
Thang pH t pH 0,0 (1,0 mol H
+
) n pH 14,0 (1,0 x 10
-14
mol H
+
). Mi n v pH i biu cho s
bin i 10 ln v nng ion hydrogen. Hnh 14.13 cho thy ni c tr m vi sinh vt c th sinh
trng l rt rng, t pH rt acid (pH 1-2) n nhng h hay t rt kim vi pH gia 2 v 10.
pH c nh hng r rt i vi s sinh trng ca vi sinh vt. Mi vi sinh vt u c mt phm vi
pH sinh trng nht nh v pH sinh trng tt nht. Vi sinh vt a acid (acidophile) c pH sinh
trng tt nht l pH 0-5,5 ; i vi vi sinh vt a trung tnh l pH 5,5-8,0 ; i vi vi sinh vt a
kim (alkalophile) l pH 8,5-11,5. Vi sinh vt a kim cc oan c mc sinh trng ti u pH 10
hay cao hn na. Ni chung, cc nhm vi sinh vt khc nhau u c phm vi sinh trng ring ca
mnh. Phn ln vi khun v ng vt nguyn sinh l a trung tnh. Phn ln nm l a hi acid (pH
4-6). Cng c nhiu trng hp ngoi l. V d, to Cyanidium caldarium v c khun Sulfolobus
acidocaldarius thng sng trong cc sui nc nng acid, chng sinh trng tt nhit cao v
pH t 1 n 3. C khun Ferroplasma acidarmanus v Picrophilus oshimae c th sinh trng
pH=0 hay rt gn vi 0.
Bng14.5: Thang pH

Mc du vi sinh vt thng c th sinh trng trong mt phm vi pH kh rng, v xa vi pH tt
nht ca chng, nhng tnh chu ng (tolerance) ca chng cng c gii hn nht nh. Khi pH
trong t bo cht c s bin ha t ngt s lm ph v mng sinh cht hoc lm c ch hot tnh
ca enzyme hay proteine chuyn mng, do lm tn thng n vi sinh vt. Vi sinh vt nhn
nguyn thy b cht khi pH ni bo gim xung thp hn 5,0-5,5. S bin i pH ca mi trng s
lm thay i trng thi in ly ca phn t cc cht dinh dng, lm h thp kh nng s dng
chng ca vi sinh vt.
Khi pH trong mi trng c s bin ha tng i ln th pH ni bo ca phn ln vi sinh vt vn
gn trung tnh. Nguyn nhn c th l do tnh thm ca H
+
qua mng sinh cht l tng i thp. Vi
sinh vt a trung tnh thng qua h thng vn chuyn s dng K
+
thay cho H
+
. Vi sinh vt a
kim cc oan nh Bacillus alcalophilus dng Na
+
ni bo thay th cho H
+
ca mi trng bn
ngoi, gi cho pH ni bo gn vi trung tnh. Ngoi ra h thng cht m ni bo (intering
buffering) cng c vai tr quan trng trong vic duy tr pH n nh.
Vi sinh vt phi c nng lc thch ng vi s bin i pH ca mi trng th mi c th sinh tn.
i vi vi khun, h thng vn chuyn ngc K
+
/H
+
v Na
+
/H
+
c th dng khc phc nhng
bin i nh v pH. Nu pH qu acid cc c ch s pht huy tc dng. Lc pH gim xung ti pH
5,5-6,0 vi khun thng hn (Salmonella typimurium) v Escherichia coli c th tng hp ra mt
lot cc protein mi v c gi l mt phn ca p ng chng chu acid. ATPase chuyn v
proton c dng sn sinh ra nhiu ATP hoc bm proton
Ra ngoi t bo. Nu pH bn ngoi gim xung cn 4,5 hay thp hn na vi khun s tng hp ra
cc phn t i km, chng hn nh cc protein gy sc acid (acid shock proteins) hay cc protein
gy sc nhit (heat shock proteins). Chng c dng phng nga s bin tnh acid ca cc
proten khc v gip sa cha li cc protedins b bin tnh.
Vi sinh vt thng sinh ra cc cht thi trao i cht c tnh acid hay kim lm thay i pH mi
trng sng. Vi sinh vt ln men s dng ngun carbonhydrat to ra cc acid hu c. Cc vi sinh
vt dinh dng ha nng v c (chemolithotrophs) nh Thiobacillus c th xy ha cc hp cht
lu hunh dng kh sinh ra acid sulfuric. Mt s vi khun khc thng qua vic phn gii cc acid
amin lm sinh ra NH
3
v lm kim ha mi trng.
Ngi ta thng b sung cc cht m (buffers) vo mi trng nui cy phng nga s c ch
qu trnh sinh trng ca vi sinh vt khi pH bin ha qu ln. Phosphat l cht m thng c s
dng, in hnh l mui H
2
PO
4
-
acid yu v mui HPO
4
2-
kim yu :
H
+
+ HPO
4
2-
H
2
PO
4
-

OH
-
+ H
2
PO
4

-
HPO
4
2-
+ HOH
Nu b sung H
+
vo h thng m n s kt hp vi HPO
4
2-
to ra acid yu. Nu b sung OH
-

vo h thng m n s kt hp vi H
2
PO
4
-
to thnh nc. Nh vy l pH mi trng khng b
bin ha qu ln. Trong cc mi trng phc tp th peptid v cc acid amin cng c nng lc m
(buffering effect) rt mnh.
14.4.3. Nhit
Cng ging nh cc sinh vt khc, nhit ca mi trng cng c nh hng rt ln i vi vi
sinh vt. Trn thc t, do vi sinh vt thng l cc sinh vt n bo cho nn chng rt mn cm vi
s bin ha ca nhit , v thng b bin ha cng vi s bin ha v nhit ca mi trng
xung quanh. Chnh v vy, nhit ca t bo vi sinh vt cng phn nh trc tip nhit ca mi
trng xung quanh. Mt nhn t quyt nh nh hng ca nhit i vi s sinh trng ca vi
sinh vt l tnh mn cm vi nhit ca cc phn ng xc tc nh enzym. Trong phm vi nhit
thp, khi nhit tng ln s lm tng tc sinh trng ca vi sinh vt, v phn ng xc tc
nh enzyme cng ging nh cc phn ng ha hc ni chung, khi nhit tng ln 10
0
C tc
phn ng s tng gp i. V cc phn ng trong t bo u tng cho nn ton b hot ng trao i
cht s tng ln khi nhit cao hn, v vi sinh vt s sinh trng nhanh hn. Lc nhit tng ln
n mt mc nht nh th nhit cng tng tc sinh trng cng gim. Khi nhit tng
qu cao vi sinh vt s cht. Khi nhit qu cao s gy ra s bin tnh ca enzym, ca cc th vn
chuyn (transport carriers) v cc protein khc. Mng sinh cht s b tn thng v hai lp lipid s
b ha tan. Do mc du nhit cng cao cc phn ng xc tc tin hnh cng nhanh nhng do
cc nguyn nhn ni trn m t bo b tn thng n mc kh hi phc v dn n vic c ch
sinh trng. Ti iu kin nhit rt thp mng sinh cht b kt ng li, enzyme cng ngng hot
ng. Ni chung, nu vt qu nhit tt nht i vi vi sinh vt, chc nng v kt cu t bo u
b nh hng. Nu nhit rt thp, tuy chc nng chu nh hng nhng thnh phn ha hc v
kt cu khng nht thit chu nh hng.
Do nh hng hai mt, va c li va c hi ca nhit i vi vi sinh vt m c th xc nh cc
loi nhit c bn (cardinal temperaturre) i vi s sinh trng ca vi sinh vt. l nhit
thp nht (minimum), nhit tt nht (optimum) v nhit cao nht (maximum) i vi s sinh
trng.

Hnh 14.13: nh hng ca nhit i vi tc sinh trng ca vi sinh vt
(Theo sch ca Prescott, Harley v Klein)
Mc du ng biu din nh hng ca nhit i vi sinh trng ca vi sinh vt l ph thuc
vo tng vi sinh vt, tng iu kin khc nhau nhng nhit tt nht thng gn vi nhit cao
nht hn l so vi nhit thp nht. Ba nhit c bn ca cng mt loi vi sinh vt khng phi l
c nh m thng ph thuc vo pH, thc n v cc nhn t khc. Chng hn, mt loi ng vt
nguyn sinh c tin mao l Crithidia fasciculate sng trong ng tiu ha ca mui c th sinh
trng trn mi trng n gin nhit 22-27
0
C nhng nhit 33-34
0
C th li khng sinh
trng c nu khng b sung vo mi trng ion kim loi, acid amin, vitamin v lipid.
Nhit c bn ca cc vi sinh vt khc nhau l khc nhau rt nhiu. Nhit tt nht c th thp
t 0
0
C n cao ti 75
0
C. Nhit thp nht sinh trng c th n -20
0
C. Nhit cao nht c
th vt qu 100
0
C. Nhn t ch yu quyt nh phm vi sinh trng ny c th l nc. Ngay
trong iu kin ti cc oan th vi sinh vt cng cn c nc trng thi dch th mi c th sinh
trng. i vi s ng vi sinh vt th phm vi nhit sinh trng thng trong khong 30
0
C.
Mt s vi sinh vt (nh Cu khun lu - Nesseria gonorrhoeae) c phm vi nhit sinh trng rt
hp. Trong khi cng c nhng vi sinh vt (nh Enterococcus facalis) li c phm vi nhit sinh
trng rt rng. Nhit sinh trng cao nht l khc nhau gia cc nhm ln vi sinh vt. Nhit
sinh trng cao nht i vi ng vt nguyn sinh (protozoa) l 50
0
C. Mt s to v nm c th
sinh trng nhit cao ti 55-60
0
C. Mt s vi sinh vt nhn nguyn thy c th sinh trng
100
0
C (nhit nc si) hay gn nh vy. Gn y ngi ta cn pht hin thy c nhng vi sinh
vt sinh trng c c nhng iu kin nhit cao hn 100
0
C. c cc thng bo cho bit
pht hin thy cc vi sinh vt nhn nguyn thy ti dch phun giu sulphid (black smoker) vt nt
di y bin - ni nhit nc cao ti 350
0
C. Nhng vi sinh vt ny sinh trng, pht trin rt
tt nhit 113
0
C v cn c th sinh trng, pht trin nhit cao hn na. p sut cao
ming ni la di y bin lm cho nc nhiu siu cao vn tn ti trng thi dch th ( p
sut 265 atm nc bin s si 460
0
C). Pht hin ny cho thy protein, mng, acid nucleic ca cc
vi sinh vt ny c tnh khng nhit rt cao. y l nhng vt liu rt tt gip cho vic nghin cu c
ch n nh ca mng v cc cao phn t sinh hc. Tng lai c th ngh n kh nng thit k cc
enzyme c th pht huy tc dng trong nhng iu kin rt cao. Nhng enzyme bn nhit t cc vi
sinh vt ny s c nhng ng dng rt quan trng trong cng nghip v trong nghin cu khoa hc.
Chng hn men Taq polymerase nhn c t c khun Thermus aquaticus c ng dng rng
ri trong phn ng chui polymerase. R rng l vi sinh vt nhn nguyn thy c th sinh trng
c nhng nhit cao hn vi sinh vt nhn tht. l v vi sinh vt nhn tht khng c th to
ra c cc mng c quan t c chc nng tng ng iu kin nhit cao hn 60
0
C. Ngoi ra
cc c quan quang hp cng hu nh khng n nh nh vy v do khng pht hin thy c s
sinh trng ca cc vi sinh vt quang hp trong mi trng nhit rt cao. Cn c vo phm vi
nhit sinh trng c th chia vi sinh vt thnh 5 nhm.
Bng 14.6: Phm vi nhit (N) i vi s sinh trng ca vi sinh vt
Vi sinh vt N thp nht N tt nht N cao nht
VSV khng quang hp
Bacillus psychrophilus -10 23-34 28-30
Micrococcus cryophilus -4 10 24
Psedomonas fluorescens 4 25-30 40
Staphylococcus aureus 6,5 30-37 46
Enterococcus faecalis 0 37 44
Escherichia coli 10 37 45
Neisseria gonorrhoeae 30 35-36 38
Thermoplasna acidophilum 45 59 62
Bacillus stearothermophilus 30 60-65 75
Thermus aquaticus 40 70-72 79
Sulfolobus acidocaldarius 60 80 85
Pyrococcus abyssi 67 96 102
Pyrodictium occultum 82 105 110
Pyrolobus fumarii 90 106 113
Vi khun quang hp v vi khun lam
Rhodospirillum rubrum - 30-35 -
Anabaena variabilis - 35 -
Osillatoria tenuis - - 45-47
Synechococcus eximius 70 79 84
To nhn tht
Chlamydomonas nivalis -36 0 4
Fragilaria sublinearis -2 5-6 8-9
Chlorella pyrenoidosa - 25-26 29
Euglena gracilis - 23 -
Skeletonema costatum 6 16-26 >28
Cyanidium caldarium 30-34 45-50 56
Nm
Candida scottii 0 4-15 15
Saccharomyces cerevisiae 1-3 28 40
Mucor pusillus 21-23 45-50 50-58
ng vt nguyn sinh
Amoeba proteus 4-6 22 35
Naegleria fowleri 20-25 35 40
Trichomonas vaginalis 25 32-39 42
Paramecium caudatum 25 28-30
Tetrahymena pyriformis 6-7 20-25 33
Cyclidium citrullus 18 43 47
Vi sinh vt a lnh (Psychrophile): l cc vi sinh vt c th sinh trng 0
0
C, sinh trng tt
nht 15
0
C hay thp hn, nhit cao nht ch l khong 20
0
C. Ti Nam cc v Bc cc d dng
phn lp cc vi sinh vt thuc nhm ny. V c ti 90% nc bin thp hn hay bng 5
0
C nn ti
c lng ln cc vi sinh vt a lnh. Chlamydomonas nivalis l mt loi to a lnh, chng sinh
bo t mu ti lm cho khi bng tuyt c mu phn hng (pink). Phn ln vi khun a lnh
thuc v cc chi Pseudomonas, Vibrio, Alcaligenes, Bacillus, Arthrobacter, Moritella,
Photobacterium, v Shewanella. C khun Methanogenum a lnh gn y c phn lp ti h
Ace Chu Nam cc.Vi sinh vt a lnh thng qua nhiu loi phng thc thch ng c vi
mi trng lnh. Chng pht huy c ch rt tt tng hp protein, enzym, cc h thng vn
chuyn. Mng t bo ca vi sinh vt a lnh c cha nhiu cc acid bo khng bo ha, c th gi
c trng thi cht bn lu (semifluid) khi gp lnh. Tuy nhin nhiu vi sinh vt a lnh nhit
cao hn 20
0
C mng t bo s b ph hi.
Nhiu vi sinh vt sinh trng tt nht nhit 20-30
0
C, nhit cao nht l cao hn 35
0
C, nhng
chng vn c th sinh trng trong iu kin 0-7
0
C.Chng thuc v nhm a lnh khng bt buc
(Psychrotrophs hay facultative psychrophiles). Nhng vi khun v nm thuc nhm ny l nguyn
nhn chnh lm h hng thc phm gi lnh.
Vi sinh vt a m (Mesophile): l cc vi sinh vt sinh trng tt nht 20-45
0
C, nhit sinh
trng thp nht l 15-20
0
C. Nhit sinh trng cao nht l khong 45
0
C hoc thp hn. Phn
ln vi sinh vt l thuc v nhm ny. Hu nh mi vi khun gy bnh cho ngi u l vi sinh vt
a m, bi v thn nhit ca ngi l 37
0
C.

Hnh 14.14 :Phm vi nhit sinh trng ca vi sinh vt
(Theo sch ca Prescott, Harley v Klein).
Vi sinh vt a nhit (Thermophile): l cc vi sinh vt sinh trng c nhit 55
0
C hay cao
hn na. Nhit sinh trng tt nht i vi chng l 33-65
0
C. Thnh phn ch yu ca nhm
ny l vi khun (ch yu l x khun), mt t to v nm (bng 14.6). Chng pht trin trong ng
phn chung , di y cc ct rm r hay c kh, trong ng dn nc nng, trong cc sui
nc nng...Vi sinh vt a nng khc vi vi sinh vt a m ch chng c h thng tng hp
enzyme v protein bn nhit (heat-stable) v c th hot ng nhit cao. Mng sinh hc ca
chng c lipid bo ha mc cao, c im si cao hn v v vy vn gi c nguyn vn nhit
cao.
C mt s t cc vi sinh vt a nhitc th sinh trng nhit 90
0
C hay cao hn. Nhit sinh
trng cao nht l 100
0
C. Ngi ta xp cc vi sinh vt c nhit sinh trng tt nht 80-113
0
C
vo nhm Vi sinh vt a siu nng (Hyperthermophiles). Chng thng khng th sinh trng
bnh thng nhit thp hn 55
0
C. Vi khun Pyrococcus abyssi v Pyrodictium occultum l v
d v nhng vi sinh vt a siu nhit c tm thy nhng y bin nng.
14.4.4. Nng oxygen
Cc vi sinh vt sinh trng trong iu kin c oxygen c gi l vi sinh vt hiu kh (aerobe),
cn cc vi sinh vt sinh trng trong iu kin khng c oxygen c hi l cc vi sinh vt k kh
(anaerobe). Hu ht cc c th a bo u phi cn sinh trng trong iu kin c oxygen, chng l
cc sinh vt hiu kh bt buc (obligate aerobes). Oxygen lcht nhn in t cui cng trong
chui vn chuyn in t khi h hp hiu kh. Ngoi ra, cc vi sinh vt nhn tht (eucaryotes) hiu
kh cn dng oxygen tng hp sterol v cc acid bo khng bo ha. Cc vi sinh vt k kh
khng bt buc (facultative anaerobes) khng cn oxygen sinh trng nhng khi c oxygen th
sinh trng tt hn. Khi c oxygen chng s dng phng thc h hp hiu kh. Cc vi sinh vt k
kh chu oxygen (aerotolerant anaerobes) nh vi khun Enterococcus faecalis c th sinh trng
nh nhau trtong iu kin c oxygen cng nh khng c oxygen. Ngc khun Bacteroides,
Fusobacterium, Clostridiun pasteurianum, Methanococcus.... s b cht khi c oxygen.

Hnh 14.15: Oxygen v s sinh trng ca vi khun
Ch thch: Cc nhm vi sinh vt xem trong bi
Mi chm biu hin khun lc ca vi khun trong hay trn b mt mi trng. SOD v catalase l biu th vi khun c
tn ti enzyme superoxide dismutase v catalase hay khng?(Theo sch ca Prescott,Harley v Klein)
Vi sinh vt k kh chu oxygen v vi sinh vt k kh bt buc khng sinh nng lng thng qua qu
trnh h hp, chng thu c nng lng thng qua qu trnh ln men hay h hp k kh (anaerobic
respiration). Sau cng, phi k n nhm vi sinh vt vi hiu kh (microaerophiles), chng khng
sinh trng c trong iu kin khng kh bnh thng (20% O
2
) v cn sinh trng trong iu
kin nng O
2
khong 2-10% Quan h gia vi sinh vt v oxygen c th xc nh bng mt th
nghim n gin nha sau: nui cy vi sinh vt trong ng nghim cha mi trng c hoc mi
trng c bit nh mi trng cha thioglycollate (l cht kh lm gim nng oxygen trong
mi trng).
Cng mt nhm vi sinh vt c th c nhiu loi quan h khc nhau vi O
2
. C 5 loi hnh u c th
thy vi sinh vt nhn nguyn thy (prpcaryotes) v ng vt nguyn sinh. Nm thng l hiu
kh, ch tr mt s loi c bit, nht l nm men, thuc loi k kh khng bt buc. To hu nh
u thuc loi hiu kh bt buc. ng ch l nng lc c th sinh trng c trong mi trnghiu
kh ln mi trng k kh lm cho vi sinh vt c tnh linh hot cao v chnh l mt loi u th
sinh thi hc.
Mc du O
2
c th lm cht cc vi sinh vt k kh bt buc, nhng trong mi trng hiu kh vn c
th phn lp c chng. l do chng thng sng chung vi loi k kh khng bt buc v b
ny tiu th ht O
2
, to nn mi trng k kh cc b gip cho vi sinh vt k kh bt buc c th
sinh trng c. V d trong khoang ming vi khun k kh bt buc Bacteroides gingivalis c th
sinh trng c trong cc khe k kh quanh rng.
S khc bit trong quan h ca vi sinh vt vi O
2
do nhiu nguyn nhn khc nhau, bao gm vic
bt hot ca protein v tc dng c hi ca O
2
trong iu kin hiu kh. Cc enzyme c th b bt
hot khi cc nhm mn cm nh sulfhydryls b oxy ha. Chng hn nh enzyme c nh m
nitrogenase l loi rt mn cm vi O
2 .
V hai in t bn ngoi ca oxygen khng thnh cp do rt d tip nhn in t v b kh.
Flavoprotein, mt s thnh phn t bo khc v s bc x u c th thc y vic kh oxy, to
thnh cc sn phm kh nh gc t do superoxide, hydrogen peroxide, gc hydroxyl.
O
2
+ e
-
O
2
-
(gc t do superoxide)
O
2
+ e
-
+ 2H
+
H
2
O
2
(hydrogen peroxide)
O
2
+ e
-
+ H
+
H
2
O + OH
-
(gc hydroxyl)
Cc sn phm kh oxy ny l cc k c hi v chng l cc cht oxy ha mnh v ph hy nhanh
chng cc thnh phn t bo. Vi sinh vt no phi c nng lc t chng li c cc sn phm kh
ny mi trnh khi b tiu dit. Bch cu trung tnh (neutrophils) v i thc bo (macrophage)
li dng cc sn phm c hi ny tiu dit cc vi sinh vt gy bnh xm nhp c th.
Nhiu vi sinh vt sinh ra cc enzyme chng li cc sn phm kh c hi ny. Vi khun hiu kh
bt buc v vi khun k kh khng bt buc thng cha cc enzyme nh superoxide dismutase
(SOD) v catalase, chng phn bit xc tc vic ph hy gc superoxide v hydrogen peroxide.
Peroxidase cng c th dng ph hy hydrogen peroxide:
2O
2
-
+ 2H
+

superoxide dismutase

O
2
+ H
2
O
2H
2
O
2
catalase

2H
2
O + O
2
H
2
O
2 +
NADH + H
+

peroxidase

2H
2
O + NAD
+

Vi sinh vt k kh chu oxygen c th thiu catalase nhng hu ht lun c superoxide dismutase.
Vi khun k kh chu oxygen Lactobacillus plantarum dng ion Mn
2+
thay th SOD phn gii
gc t do ca superoxide. Tt c cc vi sinh vt k kh bt buc u khng c hai loi enzyme ni
trn hoc c vi nng rt thp v do khng c nng lc chng chu c vi oxygen.
V vi sinh vt hiu kh cn O
2
, trong khi vi sinh vt k kh li b cht v O
2
, cho nn vic nui cy
hai nhm ny pha bng cc phng php hon ton khc nhau. Lc nui cy khi lng ln vi sinh
vt hiu kh phi nui cy trn my lc hay phi thi khng kh v khun vo bnh (hay ni) nui
cy . Cn khi nui cy vi khun k kh th phi loi b ht O
2
. C th dng cc phng php sau
y:
- S dng mi trng k kh c bit, c cha cc cht kh nh thioglycolate hay cysteine. Khi ch
to mi trng cn un ln lm tan cc thnh phn v cng ng thi loi tr ht O
2
ha tan
trong mi trng. Khi vi sinh vt k kh c th mc c ln trn b mt mi trng.
- Nui cy trong cc t nui cy k kh (anaerobic work chamber) ht chn khng v b sung
bng kh nitrogen. Thng cn cn b sung c kh CO
2
bi v nhiu vi khun k kh sinh trng tt
khi c tn ti mt lng nh kh CO
2
.
- Mt phng php rt ph bin khi nui cy mt lng nh vi sinh vt k kh l dng bnh k kh
(Gas Pak jar). Trong h thng ny li dng H
2
v cht xc tc palladium lm cho O
2
kt hp vi
H
2
to thnh nc khng c O
2
trong mi trng. Cc cht kh a vo mi trng thch cng
c th gip loi b O
2
.
- C th dng ti nha to ra cc mi trng k kh khi nui cy mt lng nh cc vi sinh vt
k kh. Trong ti nha cha CaCO
3
v cht xc tc to ra iu kin k kh giu CO
2
. Mt dung
dch c bit c a vo ti nha sau a hp lng (hp Petri) hay cc dng c nui cy khc
vo v hn kn ti nha li.
Ty tng trng hp c th m s dng cc phng php nui cy k kh khc nhau.



Hnh 14.17: Nui cy vi sinh vt k kh trong cc bnh k kh.
(Theo sch ca Prescott,Harley v Klein).

14.4.5 p sut (Pressure)
Phn ln vi sinh vt c th sng trn lc a hay trn b mt nc, l nhng ni c p sut khng
kh l 1 atm (atmosphere) v khng chu nh hng r rt g ca p sut ny.Nhng y bin (ni
c su 1000m tr ln) li chim n 75% th tch i dng. nhng ni p p sut cao n
600-1000m, nhit lnh ti 2-3
0
C. Trong mi trng cc oan (extreme) nh vy vn c mt s
vi sinh vt thch ng tn ti. Phn ln thuc v nhm chu p (barotolerant). Tuy p sut tng
ln cao s c nh hng n chng, nhng nh hng bt li ny nh hn nhiu khi so vi cc vi
sinh vt khng chu p (nontolerant). Mt s vi khun sng trong ng tiu ha ca nhng ng
vt khng xng sng di bin su (nh amphipods v holothurians) l nhng vi khun a p
(barophilic). Chng sinh trng cng nhanh trong iu kin p sut cao. Chng c vai tr quan
trng trong vng tun hon cc cht dinh dng di y bin. Ti khe bin Mariana gn Philippine
(su khong 10 500m) ngi ta phn lp c nhng vi khun a p c th sinh trng trong
iu lin 2
0
C vi p sut khong 400-500atm. Nhng vi khun ny hin bit l thuc v cc chi
Photobacterium, Shewanella, Colwellia...Mt s thuc v C khun va a p va a nhit
(thermobarophiles), chng hn nh Pyrococcus spp., Methanococcus jannasschi...
14.4.6. Bc x (Radiation)
Th gii m chng ta ang sng y cc loi bc x in t trng (electromagnetic radiation). Cc
bc x ny hnh thnh nh sng trn mt nc v lan truyn trong khng kh. C ly gia hai nh
sng hay cui sng c gi l di sng (wavelength). Khi di sng gim i th th nng
lng bc x tng ln. Tia gamma hay tia X c nng lng cao hn bc x ca nh sng nhn thy
(nh sng kh kin) hay tia hng ngoi. Bc x in t trng cn ging nh mt dng nng lng
hp bi cc photon (quang t). Mi photon u c nng lng nht nh, nng lng cao hay thp
quyt nh bi di sng ca bc x.
nh sng mt tri l ngun bc x ch yu trn tri t, bao gm nh sng kh kin (visible light),
tia t ngoi (ultraviolet), tia hng ngoi (infraded rays) v sng radio (v tuyn in). nh sng kh
kin l loi thng thy v quan trng nht trong mi trng chung quanh chng ta: mi s sng
u ph thuc vo cc c th c kh nng quang hp da vo nng lng ca mt tri. Bc x mt
tri c 60% nm vng tia hng ngoi ch khng phi vng nh sng kh kin. Tia hng ngoi l
ngun nhit lng ch yu ca tri t. tm mt bin ch thy c rt t bc x t ngoi 290-
300nm (nanometre). Tia t ngoi c bc sng thp hn 287nm hp th bi oxygen trong khng
kh v to ra tng ozone (O
3
) cao cch mt t khong 25-50km. Tng ozone hp th cc tia t
ngoi bc sng tng i di v gii phng ra O
2
. Bi v tia t ngoi rt c hi cho sinh vt nn
vic tng ozone tiu tr bt tia t ngoi l c tc dng rt quan trng i vi s sng trn tri t.
V cc loi bc sng trong nh sng mt tri phn b ng u trong phm vi nh sng kh kin
cho nn ta thy nh sng mt tri c bn c mu trng.
Nhiu bc x in t trng l rt c hi i vi vi sinh vt, nht l cc bc x c bc sng ngn,
cao nng lng l cc bc x ion ha (ionizing radiation), chng lm nguyn t mti in t
(electron) hoc ion ha (ionize). C hai loi bc x ion ha. Mt l, tia X to ra bi con ngi, hai
l,tia gamma ( tia ) sinh ra trong qu trnh tan r cc ng v phng x (radioisotope). Bc x ion
ha mc thp s lm sn sinh cc t bin (mutations) v gin tip lm cht vi sinh vt. Bc x ion
ha cao s trc tip git cht vi sinh vt. Mc du vi sinh vt c tnh khng cao hn v cc bc
x ion ha so vi cc sinh vt khc, nhng vi liu lng cao chng s git ht vi sinh vt.
Chnh v vy c th dng bc x ion ha dit khun. Tuy vy, mt s sinh vt nhn nguyn
nthy (nh vi khun Deinococcus radiodurans v cc vi khun sinh vt sinh bo t) c th vn tn
ti c ngay c cc mc bc x ion ha kh cao.
Bc x ion ha gy cho t bo rt nhiu bin ha, c th ph v lin kt hudrogen, oxy ha lin kt
i, ph hy cu trc vng, cao phn t ha mt s phn t.Oxygen c th lm tng cc hiu ng
ny, c th l do vic sn sinh gc t do hydroxyl (OH-)... Mc du c rt nhiu thnh phn t bo
chu nh hng, nhng nguyn nhn quan trng nht gy cht l s ph hy ADN.
V bc sng ngn (10-400nm) c nng lng cao cho nn bc x t ngoi (Ultraviolet radiation)
c th tiu dit cc loi vi sinh vt. Bc x t ngoi (UV) mnh nht bc sng 260nm. Chng d
b ADN hp th, lm cho trn 1 si n ADN hnh thnh nhng song phn t (dimers) thymine,
chng lm c ch qu trnh ti to (replication) v cng nng ca ADN. Thit hi ny c th c
sa cha qua mt s con ng. Theo con ng quang hot ha mt loi enzyme quang hot ha
s dng nh sng xanh lam tch song phn t thymine. Trong hot ha ti mt on ngn ADN
c cha cc song phn t thymine c th b ct ri v i ch thnh mt on ADN bnh thng.
Thit hi ny cng c th sa cha nh cc protein recA trong qu trnh ti t hp (recombination)
hoc qu trnh SOS. Thit hi khng c th c khc phc nu nh liu lng UV qu ln, to nn
nhng tn tht qu nng.
Mc du bc x UV qu nh (thp hn 290 v 300nm) kh c th lt xung b mt tri t, bc x
UV nhng bc sng 325-400nm cng c th gy hi cho vi sinh vt. Chng phn ct tryptophan
thnh nhng quang sn phm (photoproducts) c hi. Nhng sn phm ny tc dng ng thi vi
cc bc x gn t ngoi lm ph v si ADN. C ch c th ca tc dng ny khng ging vi c
ch tc dng ca UV vi bc sng 260nm.
nh sng kh kin l ngun nng lng ch yu ca qu trnh quang hp.

(photosynthesis) v vy rt cn cho cc sinh vt. Nhng nu nh sng kh kin qu mnh s c th
gy hi hay lm cht vi sinh vt. Tham gia vo qu trnh ny c mt loi sc t gi l cht quang
mn (photosensitizers) v oxygen. Cc sc t vi sinh vt nh chlorophyll, bacteriochlorophyll,
cytochromes,v flavin c th hp th nh sng mt tri v b kch hot to ra cc cht quang mn.
Cc cht quang mn (P) khi b kch hot c th chuyn nng lng cho O
2
lm ra oxygene n -
singlet oxygen (

O
2
).
nh sng
P P (hot tnh)
P hot tnh + O
2
P + O
2
Oxygenn l cht c hot tnh rt mnh, l cht oxy ha mnh c th ph hy nhanh chng t bo,
chng cng l nhn t ch yu c cc i thc bo (phagocytes) dng dit khun.

Hnh 14.18: Phm vi bc sng ca cc bc x in t trng - Phn nh sng kh kin c trnh by pha di.
(Theo sch ca Prescott,Harley v Klein).
Nhiu vi sinh vt trong khng kh hoc sng trn b mt cc vt tip sc vi khng kh s dng sc
t carotenoid bo v chng li vi quang oxy ha (photooxidation). Carotenoid c th lm ph
hy cc oxygen n, hp thu nng lng ca oxygen n v bin thnh trng thi phi hot tnh. C
cc vi sinh vt quang hp ln vi sinh vt khng quang hp u s dng sc t vo mc ch ny.
14.5. S SINH TRNG CA VI SINH VT TRONG MI TRNG T
NHIN
14.5.1. Cc nhn t ca mi trng lm hn ch s sinh trng
Mi trng sinh sng ca vi sinh vt l phc tp v thng xuyn bin i.Vi sinh vt c trng
cho mi mi trng c th b bao bc bi s bin i ca cc cht dinh dng v cc nhn t mi
trng khc. ng l vi sinh vt sinh trng trong mt mng sinh hc (biofilm). Vi sinh vt sinh
trng trong mt vi mi trng (microenvironments) cho n khi mi trng hay cc nhn t
dinh dng t ti s sinh trng gii hn. Nguyn tc lng ti thiu ca Liebig xc nh rng:
tng sinh khi ca mt c th quyt nh bi s c mt ca cht dinh dng vi nng thp nht
theo nhu cu ca c th. Nguyn tc ny c th ph hp cho iu kin phng th nghim cng nh
trong mi trng t v nc. S tng ln ca mt nhn t dinh dng cn thit (chng hn nh
phosphate) s lm tng ln qun th vi sinh vt cho n khi mt s nhn t dinh dng khc tr
thnh nhn t gii hn. Nu mt cht dinh dng no l gii hn th tng cc nhn t dinh dng
khc khng c ch li g. Tnh hnh thc t cn phc tp hn th na. Nhiu nhn t gii hn c th
nh hng thng xuyn ln qun th vi sinh vt, chng hn nh nhit , pH, nh sng, nng
mui... nh lut chng chu ca Shelford (Shelfords law of tolerance) xc nh: vi sinh vt c mt
yu cu nht nh i vi cc nhn t mi trng, Thp hay cao hn yu cu ny th vi sinh vt
khng th tn ti v sinh trng mc du vn c y cc cht dinh dng. Chng hn mi vi sinh
vt c mt phm vi nhit sinh trng nht nh. Cng tng t nh vy i vi pH, nng
oxygen, nng mui... S sinh trng ca vi sinh vt ph thuc vo c s cung cp cht dinh
dng ln kh nng chng chu vi cc iu kin ca mi trng.
Khi vi sinh vt c iu kin dinh dng sinh trng mnh m th cng ng thi sinh ra cc
cht thi c hi v lm hn ch s sinh trng ca chng.
p ng vi mc dinh dng thp (mi trng ngho - oligotrophic environments) v c s cnh
tranh, nhiu vi sinh vt chim ot thc n v khai thc chng lm ngun cnh tranh. Vi sinh
vt thng bin i hnh thi lm tng b mt v nng lc hp thu cht dinh dng. Cc vi sinh
vt nhn nguyn thy hnh que bin thnh dng mini hay ultramini (siu nh) hoc mc ra cc
ci cung (prosthecate) p ng vi tnh trng thiu thc n. S thiu ht cht dinh dng s dn
n nhiu bin i vi sinh vt, chng hn chng c th tng bc khp li hot ng ca cc gen
lin quan n trao i cht , tr vic duy tr gen qun gia (housekeeping gene).
Nhiu nhn t c th lm ci bin mc dinh dng trong mi trng ngho. Chng hn vi sinh vt
c th tch cc cht dinh dng hn ch ra (nh l st) khin khng cn st cnh tranh na.
Khng kh cng c th cung cp cht dinh dng gip cho s sinh trng ca vi sinh vt. iu ny
c th thy c trong phng th nghim cng nh ngoi thin nhin. pht hin thy cht hu c
trong khng kh c th xc tin s sinh trng ca vi sinh vt trn mi trng pha long (dilute
media). Mi trng sinh trng c lm giu bng cht hu c trong khng kh cng c th lm
tng r rt s pht trin ca qun th vi sinh vt. Ngay trong nc ct- thng cha du vt cht
hu c, cng c th hp thu nhng hp cht 1 carbon t khng kh gip cho s sinh trng va
vi sinh vt. S tn ti cc cht dinh dng trong khng kh v tnh trng sinh trng ca vi sinh vt,
nu khng xem xt n s c th nh hng n cc thc nghim v sinh ha hc hay sinh hc phn
t, cng nh cc nghin cu v s sinh trng ca vi sinh vt trong cc mi trng dinh dng
ngho (oligotrophic).
Cc cht t nhin (natural substances) cng c th c ch trc tip ti s sinh trng v pht trin
ca vi sinh vt trong cc mi trng dinh dng thp. Cc cht bao gm phenol, tannin,
ammonia, ethylene, v cc hp cht lu hunh bay hi. y c th l mt phng thc gip vi sinh
vt trnh tn dng cc nng lng gii hn trc khi c cung cp cc cht dinh dng cn thit.
Cc ha cht ny l rt quan trng trong bnh l hc thc vt v c th gip khng ch cc bnh vi
sinh vt trong t.
14.5.2. Kim tra s lng vi sinh vt nhn nguyn thy tuy sng nhng khng
nui cy c
Khi nghin cu s sinh trng ca qun th vi sinh vt nhn nguyn thy (procariotic) trong thin
nhin bn ngoi phng th nghim cn phi xc nh s lng vi sinh vt sng. Trong lch s vi sinh
vt hc thng ngi ta nh ngha vi sinh vt sng l c th sinh trng, c th hnh thnh khun
lc (colony) hoc to ra c r rt trn mi trng dch th. John R.Postgate i hc Sussex
(nc Anh) l mt trong nhng hc gi u tin xc nh vi sinh vt chu c ch (stressed) khi sng
trong mi trng thin nhin, hoc trong nhiu mi trng phng th nghim c bit, l c bit
mn cm vi cc c ch th sinh (secondary stresses). Cc c ch ny lm cho vi sinh vt tuy sng
nhng khng c th to thnh khun lc trn cc mi trng c bnh thng vn dng nui cy
chng. xc nh c s sinh trng ca cc vi sinh vt ny Postgate a ra mt phng php
thc nghim gi l th nghim vi hot tnh Postgate (Postgate Microviability Assay). Trong th
nghim ny em vi sinh vt nui cy trn lp mt thch mng di l knh (coverslip), lm cho
chng khng qua c giai on sinh trng n bo m ch bin i hnh thi t bo, coi l
biu hin tn hiu sng (life sign)
T , nhiu nh nghin cu pht trin cc phng php hin vi mn cm v phng php cht
ng v xc nh s tn ti v ngha ca dng vi khun sng nhng khng nui cy c.
Chng hn dng khng th hunh quang v thuc nhum acridine orange dnh gi mc s
lng; hoc l dng phng php s lng kh nng ti a (MPN-most probable number)...Vic s
dng ch s gii phng hot tnh phng x ca vt cht t bo cng c dng xc nh nh
hng ca hiu ng c ch i vi vi sinh vt. Cc phng php do Postgate ra l rt quan trng.
Nhiu nghin cu cho thy c khi mt s vi khun nh Escherichia coli, Vibrio cholerae, Klebsiella
pneumoniae, Enterobacter aerogenes, Enterococcus faecalis mt nng lc sinh trng trn cc mi
trng phng th nghim theo cc k thut nui cy tiu chun, nhng chng vn gi vai tr gy
bnh truyn nhim.
Tnh trng mt qun th hn hp trong mi trng thin nhin l rt phc tp. Thng thng ch c
khong 1-10% cc t bo l c th hnh thnh khun lc. Trong tng lai c th phi tm ra cc mi
trng nui cy thch hp hn i vi cc vi sinh vt cn cha c bit n. Hin nay ngi ta
s dng k thut PCR v phn tch ARN ca tiu th ribosome nh gi tnh a dng ca qun
th cc vi sinh vt cha nui cy c.
14.5.3. Cm ng mt v cc qun th vi sinh vt
T nhiu thp k nay, cc nh vi sinh vt hc vn ngh rng qun th vi sinh vt l tp hp ca cc
c th ring bit, sinh trng v hot ng c lp vi nhau. Tuy nhin gn y ngi ta pht
hin thy nhiu vi khun c kh nng giao tip v hot ng hp tc vi nhau. Mt trong nhng
cch c bn vi sinh vt hp tc c vi nhau l cm ng mt hay cn li l s t cm
ng. l hin tng trong vi sinh vt t iu chnh mt thng qua qu trnh cm nhn hm
lng cc phn t tn hiu, i khi gi l cc cht t cm ng (autoinducer) bi v chng c th kch
thch t bo tit ra chng. Nng cc phn t tn hiu tng ln cng vi s tng ln ca s lng
vi khun trong qun th cho n khi t n ngng c trng (i vi qun th ) v ra tn hiu
cho vi khun rng mt qun th n mc ti hn hay cn gi l quorum. Vi khun lc s
bt u biu hin cc gen ph thuc mt t bo ti hn nhm iu chnh mt t bo. Cm ng
mt c pht hin c vi khun Gram m v Gram dng.
Cm ng mt c ngha quan trng vi vi sinh vt. C th ly v d v s sinh tng hp v gii
phng cc enzyme ngoi bo. Nu cc enzyme ny ch c gii phng nh mt s t vi khun,
chng s b khuch tn v khng pht huy c tc dng do b pha long. Vi cch iu khin bng
cm ng mt , vi khun s t n mt qun th ln trc khi chng gii phng enzyme v kt
qu l hm lng enzyme ln pht huy tc dng. chnh l li th ca vi sinh vt trong c
th vt ch cng nh trong cc mi trng t, nc. Nu vi sinh vt gy bnh c th t c n
nng ln ti mt im no trn c th vt ch trc khi sn sinh cc nhn t c lc v
xm nhp c vo cc m vt ch, chng s c c hi ln hn trong vic lm mt tc dng kh
nng t v ca vt ch v do c th lan ra ton b c th vt ch. iu ny gii thch mt kiu
khc ca cm ng mt . Dng nh cm ng mt rt quan trng i vi nhiu vi sinh vt
trong vic thit lp mi quan h cng sinh hay k sinh i vi vt ch.
Cm ng mt c pht hin u tin v tm hiu r nht vi khun Gram m. Cc tn hiu
thng gp nht vi khun Gram m l acyl homoserine lactones (HSLs). l cc phn t nh
c cu to bi chui acyl t 4 n 14 C lin kt vi homoserine lactone (hnh 14.19). Chui acyl
ny c th c nhm keto hay nhm v tr C th 3. Cc phn t Acyl HSLs khuch tn vo cc t
bo ch (target cell) (hnh 14.19). Khi t n hm lng ln, cc phn t Acyl HSLs s bm
vo cc protein th th c bit (R) v gy ra s thay i cu trc protein. Thng thng khi phc
h HSLs-protein c hot ha, chng c tc dng nh cht cm ng, chng bm vo cc im
ch trn ADN v kch thch s phin m ca cc gen nhy cm vi nng t bo ti hn. Cc gen
cn thit tng hp HSL cng c to ra thng xuyn, do nhiu cht t cm ng c tng
hp v gii phng.

Hnh 14.19: Cm ng mt vi khun Gram m.
(Theo sch ca Prescott,Harley v Klein).
(a) Cu trc chung ca acyl homoserine lactone, cht c bit n nh l tn hiu cm ng mt (iu chnh mt
t bo) hay cn gi l cht t cm ng.
(b) Lc minh ha cch hot ng ca cm ng mt nhu vi khun gram m. Cc protein th th hot ng vi
vai tr cht cm ng (inducer) c k hiu bng ch R. ng k gy nt biu th acyl HSL synthase khng phi lc
no cng c to ra tng ng vi cc cht t cm ng.
vi khun Gram m, rt nhiu qu trnh nhy cm vi cc tn hiu acyl HSL v cm ng mt .
Mt s v d c nghin cu k l (1) s sn sinh cht pht quang sinh hc
(bioluminescence) bi Vibrio fischeri, (2) Pseudomonas aeruginosa tng hp v gii phng cc yu
t gy bnh, (3) S chuyn cc yu t di truyn nh qu trnh tip hp Agrobacterium
tumefaciens, v (4) S sn xut khng sinh Erwinia carotovora v Pseudomonas aureofaciens.
Vi khun Gram dng cng iu ha hot ng bng cm ng mt , thng thng bng tn hiu
l cc oligopeptide. Cc v d in hnh l s tip hp Enterococcus faecalis, kch thch kh
nng ti np ADN (competence induction) Streptococcus pneumoniae, s kch thch to bo t
Bacillus subtilis, v s sn sinh rt nhiu c t v cc yu t gy bnh Staphylococcus aureus.
Cm ng mt thm ch kch thch s pht trin khun ty kh sinh v to streptomycin
Streptomyces griseus. i vi trng hp ca Streptomyces griseus th c l tn hiu l g-
butyrolaton ch khng phi l oligopeptide.
Chc nng quan trng na ng quan tm ca cm ng mt l s y mnh vic to mng
sinh hc (biofilm) bi vi khun gy bnh Pseudomonas aeruginosa, v c th n ng vai tr quan
trng trong bnh x ha u nang (crystic fibrosis). S to mng sinh hc rt c ngha i vi vi
sinh vt gy bnh v n bo v vi khun khi s tn cng ca khng sinh v cc cht ty ra. S
iu chnh mt t bo c th rt hiu qu bn trong mng sinh hc do hm lng acyl HSL t b
pha long v c th tng ln nhanh chng. Trong iu kin ny, hai loi vi khun khc nhau c th
kch thch nhau bng cch sn xut ra cc tn hiu tng t nhau, l trng hp mng sinh hc
c cha cc vi khun gy bnh P. aeruginosa v Burkhoderia cepacia.
Cm ng mt l mt v d v s hot ng a t bo trong cc c th giao tip v hp tc hot
ng nh l mt n v thng nht. Cc v d khc v hot ng phc hp nh trn l s hnh
thnh cc dng khun lc v s hnh thnh th qu Nim vi khun (Myxobacteria).

1
Bi 16 c ch vi sinh vt bng cc tc nhn vt l v ha hc
Mc du a s vi sinh vt l c ch v cn thit cho nhn loi, nhng hot ng ca vi
sinh vt cng c th gy nn nhiu tc hi cho con ngi. Chng hn nh vic gy nn
cc bnh tt cho ngi, gia sc, gia cm, vic lm h hng thc phm, nguyn vt liu...
V vy chng ta phi nm vng cc phng php tiu dit hoc c ch cc vi sinh vt
c hi, lm gim bt cc thit hi do chng gy nn. Ch yu l : (1) - Tiu dit cc vi
sinh vt gy bnh v cn tr s lan truyn ca chng. (2) - Gim bt hoc hn ch cc vi
sinh vt gy nhim ngun nc, thc phm v ph hy cc nguyn vt liu khc.
Trong mt thi k rt di, t khi cha bit n s tn ti ca vi sinh vt th t tin
chng ta bit khng t cc bin php tiu c v dit khun. Ngi C Ai Cp
bit dng la dit khun, dng cc cht tiu c x l cc vt thi ra. Ngi C
Hy Lp bit cch xng lu hunh bo qun cc vt liu kin trc. Ngi H-Br
(Hebrews) c lut thiu hy ton b qun o ca nhng ngi b bnh hi. Hin nay,
vic nm vng cc k thut tiu dit vi sinh vt vn ht sc quan trng, chng hn nh
vic s dng k thut v khun trong nghin c vi sinh vt, vic bo qun lng thc,
thc phm, vic phng chng cc bnh truyn nhim...
15.1. NH NGHA THUT NG
- Dit khun hay Kh trng (sterilization): T gc La Tinh sterilis l tuyt dc, v
sinh. C ngha l tiu dit tt c vi sinh vt, bo t, virus, viroid. dit khun c th
dng cc cht dit khun (sterilant) hoc dng cc nhn t vt l khc.
- Tiu c hay Kh c (disinfection) l tiu dit, c ch hoc loi tr cc vi sinh vt
gy bnh.. Mc tiu ch yu l tiu dit mm bnh nhng trn thc t cng l lm gim
s lng chung ca vi sinh vt. tiu c cn dng cc cht tiu c (disinfectant).
thng l cc ha cht v thng dng tiu c cc vt liu khng phi l c th ngi
v ng thc vt. Cc cht tiu c khng dit c bo t v mt s vi sinh vt, v vy
khng th dng dit khun.
-Tiu c v sinh (sanitization) c lin quan mt thit vi tiu c. Trong qu trnh
tiu c v sinh s lng vi sinh vt gim xung ti t mc an ton tr xung i vi sc
khe cng cng, tc l t n tiu chun v sinh. Cc cht tiu c v sinh (sanitizer)
thng c dng lm sch mi trng v cc vt dng khng phi c th ngi v
ng thc vt.
- Phng thi (antisepsis) l dng ha cht khng ch vi sinh vt s sinh trng
ca vi sinh vt trn cc t chc sinh vt (cc m). Gc Hy Lp , anti l i khng, sepsis
l nhim trng mu. Cht phng thi (antiseptic) nhiu ngi gi l cht st trng l cha
chnh xc, d nhm vi cht dit khun (sterilant). S dng cht phng thi phng
nhim khun, mng m nh tiu dit hay c ch vi sinh vt gy bnh, ngn nga s sinh
trng ca vi sinh vt trn cc m ca sinh vt, gim thiu tng s vi sinh vt. c tnh
ca cht phng thi thp hn cht tiu c l v cn trnh vic lm cht qu nhiu t bo
ca cc m.
2
- Cht khng vi sinh vt (antimicrobial agent) c chia thnh nhiu loi.
Cht dit khun (germicide), gc La Tinh cide l git cht, l cht c th tiu dit
cc vi sinh vt gy bnh (pathogens). Nh vy ting Vit c hai ch Cht dit khun
ch c germicide ln sterilant. Thc cht cc cht ny cng gn ging nhau, sterilant c
phm vi dit khun rng hn germicide.
Cc cht dit nm (fungicide), cht dit to (algicide), cht dit virus (viricide)
ch cc cht tiu dit tng i tng ring bit.
C nhng ha cht khng lm cht c vi sinh vt nhng c th c ch s sinh
trng ca chng. C th thng gp cc cht c ch vi khun (bacteriostatic), cht c
ch nm (fungistatic), theo gc Hy Lp th statikos l nh ch.
Tt c cc cht ni trn thng nh ngha da trn nh hng i vi cc vi sinh vt
gy hi. C loi git cht, c loi c ch, nhng trong hu ht cc trng hp u lm
gim tng s vi sinh vt ni chung (khng ch ring i vi cc vi sinh vt gy bnh).
15.2. CC PHNG PHP TIU DIT VI SINH VT
Di tc dng ca mt s nhn t gy cht qun th vi sinh vt khng cht ngay ton
b. Ging nh s sinh trng ca qun th , s cht ca qun th vi sinh vt thng xy
ra theo phng thc ch s (exponential) hay phng thc logarit (logarithmic). C ngha
l qun th vi sinh vt s gim xung tng ng vi khong cch thi gian.
Bng 15.1: Th nghim git vi sinh vt bng nhit theo l thuyt.
(Theo sch ca Prescott, Harley v Klein)
Pht S lng vi sinh vt theo
s pht
S lng vi sinh vt b cht
trong 1 pht
Log
10
ca s lng vi sinh
vt sng
1 10
6
9 x 10
5
5
2 10
5
9 x 10
4
4
3 10
4
9 x 10
3
3
4 10
3
9 x 10
2
2
5 10
2
9 x 10 1
6 10
1
9 0
7 1 0,9 -1
Ly thi gian gy cht l trc honh ta c c ng biu th l mt ng thng.
Sau khi gim a s vi sinh vt sng th tc cht ca vi sinh vt cng gim. l v
tnh khng kh cao ca cc vi sinh vt sng st.
3
nghin cu hiu lc ca nhn t gy cht phi xc nh khi no th vi sinh vt
cht. l chuyn rt kh, v kh xc nh c i vi tng t bo.Sau khi a vi
khun vo mi trng nui cy trong iu kin c th sinh trng bnh thng m thy
chng khng sinh trng c th chng t l chng cht. Vi virus nu khng cm
nhim c na vo vt ch bnh thng th cng chng t l cht.

Hnh 15.1: Phng thc cht ca vi sinh vt
(Theo sch ca Prescott, Harley v Klein). X l 121

C, trong v d D 121 l trong 1

pht.
15.3. CC IU KIN NH HNG N HIU QU CA CC
NHN T KHNG VI SINH VT
Lm cht v c ch s sinh trng ca vi sinh vt khng n gin, bi v nhn t
khng vi sinh vt (nhn t lm cht hoc c ch s sinh trng ca vi sinh vt) l chu
nh hng ca t ra l 6 yu t sau y:
1- S lng qun th vi sinh vt:
V trong mi khong cch thi gian s lng vi sinh vt cht theo mt cp s bng nhau,
cho nn thi gian lm cht mt lng ln vi sinh vt s di hn so vi mt lng nh vi
sinh vt. C th tham kho s liu bng 15.1 v hnh 15.1. Cng nguyn l nh vy i
vi cc nhn t ha hc khng vi sinh vt.
4
2- Thnh phn qun th vi sinh vt:
cc vi sinh vt khc nhau c tnh mn cm khc nhau vi mt nhn t gy cht: V vy
cng mt nhn t gy cht trong cc tnh hung khc nhau, vi cc loi vi sinh vt khc
nhau th hiu qu tc dng cng rt khc nhau. V d, bo t ca vi sinh vt c tnh
khng cao hn r rt so vi cc t bo dinh dng v cc t bo non. Mt s loi vi sinh
vt c tnh chng chu cao hn so vi cc nh hng bt li ca cc loi khc. V d vi
khun Mycobacterium tuberculosis gy bnh lao c tnh chng chu vi cc nhn t
khng vi sinh vt cao hn so vi cc vi khun khc.
3- Nng v cng ca mt nhn t khng vi sinh vt:
Thng thng (khng phi mi trng hp) nng cng cao ca mt nhn t ha hc
hay cng cng cao ca mt nhn t vt l lm cho tc vi sinh vt cht cng nhanh.
Nhng hiu sut ca cc nhn t khng ph thuc trc tip vo nng v cng .
Trong mt phm vi tng i nh th mt s tng nh v nng v cng c th lm
tng hiu ng gy cht ca nhn t khng vi sinh vt. Vt qua khong xa hn th tip
tc nng cao nng v cng khng lm tng tc gy cht vi sinh vt. C lc,
nng thp hn li c hiu qu cao hn, v d cn 70% c hiu qu dit khun cao hn
cn 95%, bi v hot tnh ca chng c nng cao khi c mt ca nc. C ti liu cho
rng vi nng cn cao phn protein bn ngoi t bo vi khun s ngng t li lm
thnh mt v bc che ch cho vi khun.
4- Thi gian tc dng:
Thi gian tc dng ca nhn t khng vi sinh vt cng di th s lng vi sinh vt cht
cng nhiu (hnh 15.1). t n mc ch dit khun th thi gian tc dng phi
cho t l sng st ch cn 10
-6
hoc thp hn na.
5- Nhit :
Tng nhit c th lm tng hiu qu hot tnh ca ha cht. Thng thng vi mt nng
thp ca cht tiu c (disinfectant) hay nhn t dit khun cn x l nhit cao
hn.
6- Mi trng bn ngoi vi sinh vt:
Vic khng ch qun th vi sinh vt khng tch ri m gn vi cc nhn t mi trng,
hoc lm tng hay lm gim tc ng gy cht. V d trong iu kin acid, nhit c
hiu qu dit khun cao hn, do i vi cc ung c tnh acid nh nc qu, nc
c chua th d dit khun theo kiu Pasteur (pasteurise) hn so vi cc thc phm c pH
cao hn nh l sa chng hn. Nhn t mi trng quan trng th hai l mt s cht hu
c c th bo v vi sinh vt khng vi tc dng ca nhit hay ca cc cht tiu c
ha hc. Mng sinh hc (biofilm) l mt v d rt r. Cc cht hu c trn b mt ca
mng sinh hc s bo v cc vi sinh vt to thnh mng sinh hc, cho nn mng sinh hc
v cc vi sinh vt trong rt kh tr kh. V vy trc khi dit khun hay tiu c mt
5
s vt phm trc ht cn ra sch. i vi ng tim v cc dng c y khoa hay nha khoa
trc khi dit khun cn phi ra sch trnh s c mt qu nhiu cht hu c gip bo
v cho mm bnh v lm tng nguy c nhim khun. Khi ch to nc ung cng cn
ch l ngun nc thnh ph vn cn cha kh nhiu cht hu c cho nn cn dng
nhiu chlorine mi sc tiu c
15.4. S DNG CC PHNG PHP VT L KHNG CH VI
SINH VT
Tng nhit v vic dng cc phng php vt l khc thng c dng dit
khun. Cc phng th nghim vi sinh vt u dng cc ni hp p sut cao (autoclave)
dit khun. Tng nhit, qua lc, chiu tia t ngoi, dng bc x in ly l 4 phng php
vt l thng c s dng.
15.4.1. Tng nhit
Ngi C Hy Lp bit dng la hay un nc si dit khun hay tiu c. Tng
nhit n nay vn l phng php thng dng nht dit khun. Ch yu c phng
php dng sc nng m v sc nng kh.
Sc nng m d dng gy cht virus, vi khun v nm (bng 15.2). Trong nc si
sau 10 pht c th lm cht cc t bo dinh dng v bo t ca cc vi sinh vt c nhn
thc. Nhng nhit si (100C) khng sc lm cht ni bo t ca vi khun. Bo t
vi khun c th tn ti vi gi trong nc si. Do cch un si ch dng un nc
ung hoc tiu c cc vt phm khng b ph hy trong nc si, khng th dng
dit khun.

Bng 15.2: iu kin c chng dit khun bng sc nng m
Vi sinh vt T bo dinh dng
Bo
t
Nm men 5 min., 50-60C
5 min.,
70-
80C
Nm si 30 min., 62C
30
min.,
80C
Vi khun 10 min., 60-70C
2-trn
800
min.,
100C
0,5-12
min,
6
121C
Virus 30 min., 60C
(Theo sch ca Prescott, Harley v Klein)
V tng nhit l bin php rt quan trng khng ch vi sinh vt cho nn cn c mt
tiu chun chnh xc i vi hiu sut dit khun bng sc nng (heat-killing efficiency).
Trc y dng im gy cht do nhit (thermal death point, TDP). l nhit thp
nht dit ht vi sinh vt trong dch huyn ph (suspention) sau 10 pht. Nhng v vi
sinh vt cht theo phng thc logarit, cho nn trn l thuyt khng c th tiu dit hon
ton vi sinh vt trong mt mu vt, tc l phi ko di thi gian tng nhit. V vy c mt
phng thc biu th chnh xc hn v c tip nhn rng ri, l Thi gian gim
thiu thp phn (decimal reduction time, D) hoc gi l Tr s D (D value). Tr s D l
thi gian cn thit dit ht 90% vi sinh vt hoc bo t trong mt mu vt mt nhit
nht nh. Trn mt th bn logarit (semilogarithmic plot) thy r s lng vi sinh
vt bin i theo thi gian tng nhit (hnh 15.2). Tr s D l thi gian cn thit s
lng vi sinh vt gim 10 ln. Tr s D lin quan n tnh khng ca vi sinh vt i
vi cc nhit khc nhau. T tr s D m tnh ra tr s Z (Z value). Tr s Z l nhit
tng ln lm gim 1/10 tr s D. Mt cch biu th khc l tr s F (F value) l
thi gian cn thit (tnh bng min.) dit ht mt qun th t bo hoc bo t mt
nhit nht nh (thng l 121C).

7
Hnh 15.2: Tnh ton tr s Z
Cn c vo tr s D cc nhit khc nhau tnh ra tr s Z. Tr s Z c th dng
tnh ton mi quan h gia nhit v thi gian sng st ca vi sinh vt. Tr s Z l
s nhit tng lm gim 10% tr s D. Trong th ny tr s Z l 10,5
0
C. Tr s
D biu th bng thang logarit. (Theo sch ca Prescott, Harley v Klein).

Tr s D v tr s Z c ng dng rng ri trng cng nghip ch bin thc phm.
Khi sn xut hp cn x l nhit sau khi a thc phm vo hp v hn hp li. Cn
x l nhit mc dit c vi khun gy ng c tht Clostridium botulinum. Vi
khun ny gy ra c t botulism rt nguy him. X l nhit di lm cho s
lng bo t ca vi khun ny nu c t 10
12
gim xung ch cn 1 bo t (10). Tr s D
i vi bo t vi khun ny 121C l 0,204 min., v vy tiu dit 10
12
bo t xung
cn 1 bo t cn 12D hay 2,5 pht. Tr s Z i vi Clostridium botulinum l 10C - tc
l tng 10C th gim c 10 ln tr s D. Nu dit khun 111C th tr s D phi tng
10 ln, tc l 2,04 pht v tr s 12D tng ln n 24,5 pht . Bng 15.3 nu ln tr s D
v tr s Z ca mt s vi khun thng gp trong thc phm.
Bng 15.3: Tr s D v tr s Z ca mt s vi khun gy bnh gp trong thc phm
Vi sinh vt C cht D(

C),pht Z (

C)
Clostridium botulinum m phosphat D
121
=0,204 10
Cl.perfringens (chng khng
nhit)
MT nui cy D
90
=3-5 6-8
Salmonella Sn phm g D
60
=0,39-0,40 4,9-5,1
Staphylococcus aureus Sn phm g
SP g ty
Dung dch NCl 0,5%
D
60
=5,17-5,37
D
60
=15,4
D
60
=2,0-2,5
5,2-5,8 6,8
5,6
(Theo sch ca Prescott,Harley v Klein)
C 3 s liu i vi t cu vng (S.aureus), cho thy tc lm cht vi khun ny
thay i ph thuc vo mi trng v hiu qu bo v ca cht hu c.
Vi sc nng m phi cn nhit cao hn 100C th mi c th dit c ni bo t
(endospores) ca vi khun, v cn c p sut cao trong iu kin bo ha hi nc. Thit
b dit khun thng dng c gi l autoclave (hnh 15.3)
8

Hnh 15.3: Hai loi autoclave nh v ln
V c bn autoclave cng tng t nh ni hm chu p lc vn thng dng trong
gia nh. Ty yu cu m c ci dng la, c ci dng in, c ci dng hi nc chuyn
vo, c ci nh, c ci va, c ci ln hoc rt ln. Autoclave do nh khoa hc
Chamberland pht minh ra vo nm 1884 v pht minh ny thc y s pht trin ca
Vi sinh vt hc. Autoclave phi c van y ht khng kh ra v trong ni ch cn c
hi nc bo ha. C th ng van ngay t u i p lc nng ln mt t ri mi m van
loi ht khng kh ra. Cng c th m van ngay t u, khi thy hi nc bay ra nhiu
mi ng van li. Thng dit khun 121C (p sut 15 pounds) trong 15 pht. C th
dit ht mi t bo vi sinh vt v bo t.
Dit khun bng sc nng m thng qua vic ph hy acid nucleic, lm bin tnh
enzym v cc protein khc, ng thi cn c th ph v mng t bo m lm cht vi sinh
vt.
Dit khun bng sc nng m phi tin hnh trit mi c hiu qu. Khi cha loi
b ht khng kh th p sut 15 pounds nhit khng th t n 121C. Cc vt cn
x l khng nn xp cht qu cn tr vic tip xc vi hi nc nng. Lc dit khun
mt bnh c th tch ln th phi gi thi gian di hn, lm cho ton b dch th phi
t ti 121C. Chng hn khi dit khun bnh 5 lt th phi x l trong 70 pht. khc
phc cc nhn t ni trn ngi ta thng xp km vi sinh vt ch th khi dit khun cc
vt phm. Khi dng ng (ampule) cha mi trng dinh dng v khun c thm
mnh giy c tm bo t vi khun Bacillus stearothermophilus hay Clostridium. sp
PA3679. Dit khun xong ph v ng trong iu kin v khun v nui cy vi ngy.
Nu sinh vt ch thi khng sinh trng th l vic dit khun thnh cng.
9
Ngi ta thng x l nhit si i vi sa v nhiu cht khc. Phng php
ny gi l phng php kh trng Pasteur (Pasteurization) k nim pht minh ny ca
ng. Vo thp k 60 ca th k 19 do ru vang b nhim khun, gy kh khn cho vic
bo qun v vn chuyn, gy kh khn cho vic sn xut ru vang Php. Pasteur
dng knh hin vi quan st thy cc trong ru b nhim c mt cc vi khun ln men
lactic v acetic. ng thy x l nhit 55-60C c th lm cht cc vi sinh vt ny v
c th bo qun tng i lu di ru vang. Nm 1886 hai nh ha hc c l V.H.
Soxhlet v F.Soxhlet s dng k thut ny bo qun sa v lm gim vic sa ly
truyn mm bnh. Nm 1889 phng php tiu c Pasteur vi sa c nhp vo Hoa
K v ngi ta dng phng php ny x l sa, bia, v nhiu loi b ung khc.
Phng php tiu c Pasteur khng t ti mc ch dit khun nhng lm cht cc
vi khun gy bnh, gim mnh cc vi khun khng gy bnh nhng lm h hng thc
phm v lm chm r rt tc bin cht ca thc phm.
C th c hai phng php kh trng sa. Phng php tng i c l x l 63 C
trong 30 pht. Cn phng php hin thng c s dng l phng php kh trng
ngn (flash Pasteurization), cn gi l phng php kh trng ngn nhit cao (high-
temperature short-term, HTST), tc l x l 72C ch trong 15 giy, sau nhanh
chng lm lnh. Trong cng nghip thc phm c lc cng cn dng phng php kh
trng siu nhit (ultrrahigh temperature, UHT), tc l x l sa v cc sn phm sa
nhit 140-150C ch trong 1-3 giy. Sa x l siu nhit khng cn bo qun lnh, c
th bo qun hai thng an ton nhit phng. Cc gi c ph kem (coffee creamer)
cung cp khch sn thng c dit khun theo phng php ny.
Nhiu vt phm c th dit khun bng sc nng kh (dry heat sterilization). a cc
vt phm ny vo t sy v gi nhit 160-170C trong 2-3 gi. Vi sinh vt b cht do
b oxy ha cc thnh phn t bo, v lm bin tnh protein. Mc du dit khun bng sc
nng kh khng c hiu qu cao nh bng sc nng m. Bo t ca vi khun Clostridium
botulinum b cht 121C sau 5 pht khi dng sc nng m nhng ch b cht nh vy
160C sau 2 gi. Dit khun bng sc nng kh c nhng u th ring v khng lm n
mn cc vt liu thy tinh v kim loi nh sc nng m, c th dng x l cc dng
bt, du v cc cht tng t. Hu ht cc phng th nghim xn l hp Petri v cc pipt
bng sc nng kh. Khng thch hp s dng phng php ny x l cc vt phm
bng cht do v cao su.

10
Hnh 15.4: T sy nhit kh
15.4.2. Nhit thp
Nhit thp c s dng c ch s sinh trng v pht trin ca vi sinh vt.
y l phng php quan trng ngnh vi sinh vt hc thc phm. nhit -20C hay
thp hn, vt phm b ng lnh, vi sinh vt b nh ch sinh trng. Mt s vi sinh vt b
cht v cc tinh th bng l ph v mng t bo,.nhng lnh su khng lm cht phn ln
cc vi sinh vt nhim trn vt phm. Trn thc t nhiu phng th nghim dng cc t
lnh su -30C hay -70C bo qun vi sinh vt. V thc phm ng lnh c th cha
nhiu vi sinh vt, cho nn khi lm tan bng phi x l ngay tiu th, trnh tn hi
v cho cc vi sinh vt gy bn pht trin.
Bo qun lnh gip lm chm s sinh trng v pht trin ca vi sinh vt, nhng
khng lm ngng hn s sinh trng. ng mng l phn ln cc vi sinh vt gy bnh
l thuc loi a m (mesophilic) v khng sinh trng c nhit 4C. Cc vt gi
lnh b h hng bi cc vi khun a lnh (psychrophilic) v chu lnh (psychrotrophic)
nht l khi c tn ti nc, cc t lnh ch dng bo qun ngn hn thc phm v cc
vt phm khc.

15.4.3. Qua lc
Phng php qua lc l phng php rt tt gim thp qun th vi sinh vt i vi
cc vt liu mn cm vi nhit v nhiu khi c th dng dit khun cc dung dch.
Qua lc ch n gin l loi vi sinh vt khi dung dch ch khng phi l dit khun. C
hai loi lc vi sinh vt. Thit b qua lc tng su (depth filter): l loi thit b cu to
bi si hay cc vt cht dng ht, to thnh mt bn lc kh dy vi nhng l rt nh.
Di sc ht chn khng dung dch s c lc qua cn vi sinh vt b gi li hay b hp
ph (adsorption) trn b mt bn lc. Nguyn liu lm ra bn lc ny thng l t
To silic (dimatomaceous earth) - l thit b lc Berkefield. Cn c th dng mt loi
s (unglazed porcelain) - l thit b lc Chamberlain. Hoc cn c th dng thch
min (asbestos) hay cc nguyn liu khc.
Gn y ngi ta dng thit b mng lc (membrane filters) thay th cho thit b
qua lc tng su. Mng lc hnh trn, dy khong 0,1mm v c ch to bi acetate
cellulose, nitrate cellulose, polycarbonate, fluoride polyvinylidene hay cc cht tng hp
khc. Cc mng lc c l vi ng knh khong 0,2m l c th dng lc b phn
ln cc t bo dinh dng ca vi sinh vt, tr virus. Dch lc thng ch t 1ml n vi
lt. Mng lc c lp c nh trn mt gi c bit (hnh 15.5)
Di p lc ca my ht chn khng dch lc c chuyn sang mt bnh v khun.
Loi thit b mng lc ny c dng trong ngnh dc, lc thuc au mt, chun b cc
mi trng nui cy, cc loi du, cht khng sinh v nhiu vt cht km chu nhit khc.
11

Hnh 15.5: Thit b mng lc
1. Bnh Erlenmeyer ng dch cn lc
2. Dch lc c y sang thit b mng lc nh my bm
3. Thit b mng lc (vi cc loi hnh cc kch c khc nhau).
Phng php dit khun nh lc cn dng lc khng kh. Hai v d thng gp l
khu trang dng trong ngoi khoa v nt bng dng cho cc ng nghim hay cc bnh
nui cy vi sinh vt. Khng kh i qua c nhng vi sinh vt th b gi li bn ngoi.
Phng cy Laminar thong kh nhng an ton sinh hc (Laminar flow biological safety
cabinet) s dng mng lc khng kh bng cc ht hiu lc cao HEPA (high-efficiency
particulate filter). N c th lc c n 99,97% cc ht c kch thc 0,3m v c
coi l mt h thng lc rt quan trng. Ngi nui cy vi sinh vt c th thao tc thoi
mi trong mt phng cy m mt phn ca nhng rt an ton nh lun c mt lung
khng kh v khun c thi t pha trong v li thot ra qua mng lc HEPA t pha
trn. Khi thao tc vi cc vi sinh vt nguy him nh vi khun lao Mycobacterium
tuberculosis, virus gy ung th, cc ADN ti t hp... nht thit cn s dng phng cy
ny. Thit b ny c dng trong cc phng th nghim, trong cng nghip, nh l cng
nghip dc phm, chun b mi trng, thao tc th nghim, nui cy m

12

Hnh 15.6: Phng cy Laminar
15.4.4. Bc x (radiation)
Bc x t ngoi (Ultraviolet radiation-UV) vi bc sng 260nm c hiu ng dit
khun rt mnh, tuy nhin khng c kh nng xuyn qua thy tinh, cc mng bn, nc
v mt s c cht khc. V vy UV ch dng dit khun trong mt s trng hp, v d
dit khun khng kh trong t cy, phng nui cy hoc bn ngoi mt s vt th. UV c
hi i vi da v mt cho nn phi tt n UV trc khi vo lm vic ni c n ny. UV
cng c th dng dit khun nc, phi l mt tng nc mng i qua n UV
sc dit mm bnh v cc vi sinh vt khc..
Bc x ion ha (ionizing radiation) hay bc x in ly c sc xuyn rt mnh v c
dng rt tt dit khun. N c th dit c t bo dinh dng ln bo t vi khun, c vi
sinh vt nhn nguyn thy (procaryotic) ln cc vi sinh vt c nhn tht (eucaryotic). Tia
gamma t ngun cobalt 60 c dng dit khun ngui i vi cht khng sinh, kch
t (hormones), ch khu vt thng, cc vt liu y hc bng cht do (plastic) nh ng
tim... Tia gamma cn c dit khun v tiu c (pasteurize) i vi tht v cc thc
phm khc. Bc x ion ha c th dit cc vi khun gy bnh nguy him nh Escherichia
coli O157:H7, Staphylococcus aureus, Campylobacter jejuni... C c quan qun l thc
phm v thuc Hoa K (FDA) ln t chc Y t th gii (WHO) u xc nh tnh an ton
ca vic chiu x ny i vi thc phm. c mt nh my chiu x thng phm
gn Tampa (bang Florida). Tic rng phng php ny cha c ng dng rng ri ti
Hoa K, nguyn nhn l do gi cn cao v nhiu ngi cn lo ngi cc nh hng bt li
ca vic chiu x ln thc phm. Gn y, Chnh ph M ph chun vic chiu x ln
tht gia cm, tht b, tht ln, tht b, tht cu non, hoa qu, rau c v cc cht iu v.
Vic chiu x trong tng lai s ngy cng c ng dng rng ri.
13
15.5. S DNG CC PHNG PHP HA HC KHNG CH
VI SINH VT
Mc du ngi ta vn thng dng cc phng php vt l tiu c nhng cc tc
nhn ha hc cng thng c dng tiu c (disinfection) v phng thi
(antisepsis). C nhiu nhn t nh hng nhiu qu tiu c v phng thi bng
phng php ha hc. Chng hn nh loi vi snh vt, nng v bn cht ca cc cht
tiu c v phng thi, thi gian x l,... Trc khi s dng cc cht tiu c hay phng
thi th b mt vt th phi c lm sch. Cn m bo s an ton khi dng ha cht
trong cc phng th nghim hay trong bnh vin. Ha cht cng c dng phng
chng s sinh gtrng ca vi sinh vt trong thc phm. C nhiu loi ha cht c dng
lm cht tiu c, mi loi u c nhng u im v nhc im ring. Trc khi chn
s dng ha cht no phi hiu r c tnh ca cht . Trong trng hp pha rt long
v c mt cht hu c th cht vn c th tc dng c hiu qu ln cc nhn t truyn
nhim (vi khun Gram dng, Gram m, vi khun khng acid, ni bo t ca vi khun,
cc loi nm v virus...), mt khc li phi khng c hi i vi c th ngi, khng lm
n mn cc vt phm ni chung. Trong thc tin, rt kh t n tiu chun va c hiu
lc va t c i vi c th. Mt s ha cht tuy hiu lc thp nhng v kh v hi nn
vn c s dng. Cht tiu c phi n nh khi bo qun, khng c mi v kh chu,
tan trong nc v trong du d xm nhp vo vi sinh vt v phi c sc cng b mt
thp xm nhp c vo cc khe trn b mt. Nu gi khng cao cng tt.
Mt vn nghim trng l vic s dng qu mc Triclosan v cc cht dit khun
(germicides) khc. Cht khng khun (antibacterial) ny hin thy c mt trong cc sn
phm nh cht kh mi (deodorant), nc sc ming, x phng, tht ct rau, chi tr
em... Triclosan hu nh ang c mt khp ni, hu qu l xut hin cc vi khun
khng Triclosan. V d trc khun m xanh Pseudomonas aeruginosa c th bi xut
cht ny ra khi t bo. Tng t nh trng hp vi khun phn ng vi vic dng qu
thuc khng sinh, vi khun cng s c s p ng nh vy khi dng qu mc cc cht
phng thi. Hin c bng chng cho thy vic s dng rng ri Triclosan lm tng
tn s xut hin cc vi khun khng thuc khng sinh. V vy vic dng qu mc cc cht
phng thi (antiseptic) c kh nng sinh ra nhng hu qu kh lng.
Bng 15.4: Nng s dng v mc hot tnh ca cc cht dit khun thng dng
Ha cht Nng s dng Mc hot tnh*
Dng kh:
Ethylene oxide 450-500mg/L Cao
Dng lng:
Glutaraldehyde dich th 2% Tng i cao
Formaldehyde + cn 8 + 70% Cao
H
2
O
2
n nh 6-30% Tng i cao
Formaldehyde dch th 6-8% Tng i cao
14
Iodophors 750-5000mg/L Tng i cao
Iodophors 75-159mg/L Tng i thp
Iodine + cn 0,5 + 70% Trung bnh
Hp cht ca Chlore 0,1-0,5% Trung bnh
Hp cht ca phenol,dch th 0,5-3% Tng i thp
Iodine, dich th 1% Trung bnh
Cn (ethyl, isopropyl) 70% Trung bnh
Hp cht Ammon bc 4 0,1-0,2% trong nc Thp
Chlorohexidine 0,75-4% Thp
Hexachlorophene 1-3% Thp
Hp cht Thy ngn 0,1-0,2% Thp
*Hot tnh cao-c th lm cht vi khun k c vi khun lao, bo t, nm, virus; Hot tnh
trung bnh- lm cht mi vi khun, tr bo t; Hot tnh thp- lm cht t bo dinh dng
ca vi khun, tr VK lao, lm cht nm, virus c lng lipid mc trung bnh. Theo
Symour S. Block, 1983.
15

Hnh 15.7: Cu trc ca mt s cht tiu c v cht phng thi thng dng
Loi Phenol
Phenol l cht phng thi v tiu c c s dng rng ri u tin. Nm 1867
Joseph Lister dng phenol lm gim nguy him ca vic nhim vi sinh vt trong
qu trtnh phu thut. Hin nay phenol v cc dn xut nh cc loi cresol, cc loi
xylenol v orthophenylphenol c dng lm cht tiu c trong cc phng th
nghim v bnh vin. Cht tiu c thng mi Lysol l mt hp cht loi phenol. Cc
cht loi phenol c tjh lm bin tnh protein v ph hy mng t bo. Chng c u im
l c th dit vi khun lao khi c mt cc cht hu c. Sau khi s dng c th duy tr tc
dng kh lu trn b mt vt th. Nhng chng c mi kh chu v c th lm kch thch
da.
Hexachlorophene l mt cht phng thi thng dng v c th lm gim s lng vi
khun trn da v duy tr c kh lu, nhng n li c th lm tn thng no cho nn
hin ch dng trong bnh vin khi c s bc pht ca T cu khun Staphylococcus.
16
Cn
Cn l mt trong nhng loi thuc tiu c v thuc phng thi thng dng. Cn c
th lm cht c vi khun v nm nhng khng lm cht c bo t. Mt s virt cha
lipid cng b cn lm cht. Ethanol v Isopopanol l hai loi cn thng dng dit
khun, nng thng dng l 70-80%, nng ny lm bin tnh protein, cn c th
lm ha tan mng lipid. dit khun nhit k v cc dng c nh cn x l bng cn
trong 10-15 pht.
Cc Halogens
Halogen l mt trong 5 nguyn t thuc nhm VIIA ca bng tun hon (Fluorin-F,
Chlorine-Cl, Bromine-Br, Iodine-I v Astatine-). trng thi t do cc phn t tn ti
di dng 2 nguyn t lin kt vi nhau. Cng c th to thnh mui vi sodium (Na)
hay cc kim loi khc. I v Cl l hai loi khng vi sinh vt quan trng. I thng c
dng lm thuc phng thi (antiseptic) ngoi da. N lm cht vi sinh vt do oxy ha cc
thnh phn t bo, iod ha (iodinating) cc protein. Vi nng cao c th lm cht bo
t, ni chung l s dng tinture diode (tinture of iodine) - tc l IK vi nng 2 % hay
cao hn iodine trong dung dch nc-ethanol. Mc du I l cht phng thi c hiu qu
nhng cng c th lm tn thng da, c th lm bin mu da, cn c th gy d ng
(allergie). Gn y ngi ta s dng iodophore - hp cht ca I vi mt cht hu c.
Iodophore tan trong nc, khng lm bn mu da, c th gii phng dn Iodine nn gim
tn hi v gim kch thch da. Loi tiu c da v dng trong phng th nghim ph bin
l loi c nhn hiu l Wescodyne, cn loi tiu c vt thng thng dng loi c nhn
hiu l Betadine.
Iodine thng c dng tiu c nc tiu dng ti thnh th v cc b bi. Cng
c dng trng cng nghip sa, cng nghip thc phm. C th dng kh chlorine,
sodium hypochlorite hoc calcium hypochlorite. Khi s dng chng bin thnh HClO ri
gii phng nguyn t oxy: S xy ra s oxy ha cc t bo dinh dng ca vi khun, nm
nhng khng c tc dng vi bo t:
Cl
2
+ H
2
O HCl + HClO
Ca(OCl)
2
+ 2H
2
O Ca (OH)
2
+ 2 HClO
HClO HCl + O
Di tc dng ca chng hu nh tt c vi sinh vt s b git cht
trong vng 30 pht. V phn ng ca cht hu c vi tc ng ca Cl
v cc dn xut ca Cl nn can thip vo tc dng dit khun ca
Cl cho nn ngi ta thng s dng qu lng Cl bo m hiu qu
dit khun. C mt kh nng l Cl phn ng vi cht hu c hnh
thnh nn nhng hp cht gy ung th trihalomethanes, cho nn
trong nc ung cn phi kim tra s tn ti ca cht ny. Ti Chu
17
u v Canada i khi ngi ta s dng thnh cng ozone thay th
cho vic chlorine ha (chlorination).
Cl l mt cht tiu c tt, khng t, li d dng s dng nn rt
hay c s dng. Vi mt lng nc ung nh c th tiu c bng
nhng vin halozone. Halozone (acid parasulfone dichloramidobenzoic)
sau khi a vo nc s t t gii phng ra chloride, sau khong na
gi c th t ti mc ch tiu c. Cht ny thng c s dng
trong trng hp thiu nc sch ung.
Dung dch Cl l cht tiu c c hiu qu trong gia nh v trong
cc phng th nghim. C th dng nng pha long 100 ln dch ty
trng gia dng (household bleach) phi hp vi cht ty khng ion ha
(non ionic detergent) sao cho nng cht ty vo khong 0,8%. Hn
hp ny va lm sch va loi b vi khun
Cc Kim loi nng
Trong nhiu nm cc ion kim loi nng nh Hg, Ag, As, Zn v Cu
thng c dng lm cht dit khun (germicides). Nhiug kim
loi nng c tc dng c ch vi sinh vt (bacteriostatic) hn l dit
khun. Hin cc cht ny c thay th bng cc cht khc c c
tnh thp hn v c hiu qu hn. Nhng cng c thng hp ngoi l,
v d dung dch 1% AgNO
3
thng c dng lm thuc nh mt
phng bnh lu mt (trong nhiu bnh vin ngi ta dng
Erythromycin thay th nitrat bc v cht khng sinh ny c hiu qu
chng c Chlamydia ln Neisseria. Bc sulfadiazine thng c dng
trong iu tr bng. CuSO
4
thng c dng dit to c hiu qu
trong ao h v cc b bi.
Kim loi nng kt hp vi protein , lm bt hot protein v cng
c th lm kt ta protein ca t bo.
Cc mui ammon bc bn (Quaternary Ammonium
Compounds)
Cc cht ty (Detergents - t gc La Tinh detergere c ngha l
loi tr) l nhng phn t hu c c dng lm cc cht gi m
(wetting agents) v nh ha (emulsifiers) v chng va c cc thn
nc (polar hydrophilic) va c nhng gc phi cc k nc (nonpolar
hydrophobic ends). Chng c th lm tan cc cht kh ha tan bi cc
phng php khc, v vy dng lm cht ty ra, git gi rt c hiu
qu, nhng c ch khc vi cc cht bo c trong x phng.
18
Mc du cc cht ty dng ion c chc nng khng vi sinh vt
nht nh nhng ch c cc cht ty ra cationic (ion dng) mi c
tc dng tiu c. Thng dng nht l cc mui ammon bc bn,
chng lm ph v mng t bo, cng c th lm bin tnh protein.
Cc cht ty cationic nh Benzalkonium chloride v
Cetylpyridinium chloride c th git cht phn ln vi khun nhng
khng git c vi khun lao Mycobacterium tuberculosis v cc ni
bo t. Chng c u im l n nh, khng c v khng gy kch
thch... nhng li b mt tc dng trong nc cng (hard water) v
nc x phng. Cc cht ty cationic thng c dng lm cht
tiu c i vi bt a, cc thit b nh v x l ngoi da. Zephiran
c cha benzalkonium chloride v Ceepryn c cha cetylpyridinium,
chloride l cc mt hng thng gp trn th trng.
Cc Aldehyde
Hai loi aldehyde thng c s dng l Formaldehyde v
Glutaraldehyde. Chng c phn ng rt mnh, c th kt hp vi acid
nucleic v protein v lm bt hot chng, cn c th lm bt hot
thng qua vic lin kt cho (crosslinking) v alkyl ha (alkylating).
Chng c th lm cht bo t, c th dng lm cht dit khun ha
hc. Formaldehyde thng c dng di dng ha tan trong nc
hay trong cn. Dung dch m 2% glutaraldehyde l mt loi cht tiu
c c hiu qu v thng c dng tiu c cc phng th
nghim v bnh vin. Glutaraldehyd trong 10 pht tiu c
nhng git cht bo t cn ti 12 gi.
Cc kh dit khun
C nhiu vt phm khng chu c nhit cao nh cc a Petri
bng cht do, cc ng tim nha, cc b phn ca my tim-phi
nhn to, cc ng dn, ng ni... cn dit khun bng kho ethylene
oxide (EtO). EtO c th kt hp vi protein, c th lm cht c vi sinh
vt ln bo t. EtO nhanh chng xuyn qua c cc bao b bng cht
do nn l mt loi cht tiu c c bit hiu qu.
Tiu c bng EtO rt ging vi tiu c trong ni cao p. Cn
khng ch nng c EtO, nhit v m. Vi EtO thun khit
thng dng nng 10-20% phi hp vi CO
2
hay
dichlorodifluorromethane. Vi cc vt dng sch cn x l 38C
trong 5-8 gi, nu 54C cn x l trong 3-4 gi. Nng EtO l l
700mg/lt.V EtO c c tnh ln cho nn sau khi tiu c cn thi kh
mnh loi tr ht EtO i.
19
Betapropiolactone (BPL) cng l loi kh dng tiu c. Trong
trng thi lng BBL dng tiu c vcxin v huyt thanh. BBL s b
ph hy thn dng v hot tnh sau vi gi ch khng kh loi tr nh
EtO. Nng lc dit khun tuy cao hn EtO nhng kh nng xuyn thu
qua vt liu li km hn so vi EtO. Hn na cht ny c th gy ung
th cho nn khng c ng dng rng ri nh EtO.
Gn y hydrogen peroxide dng bay hi cng c dng tiu
c cc phng thao tc an ton sinh hc.
15.6. NH GI HIU LC CA CC TC NHN KHNG
VI SINH VT
Ti Hoa K vic nh gi cc tc nhn khng vi sinh vt c thc
hin bi hai c quan khc nhau: C quan qun l cc cht tiu c bo
v mi trng v C quan qun l Thc phm v dc phm. Cn
nh gi cc tc nhn ny c hiu qu khng vi sinh vt hay khng, c
hiu lc t nng no. Sau tin hnh trn tng ng dng thc
tin.
Ph bin nht l Th nghim H s phenol (phenol coefficient test),
tc l so snh hiu lc ca mt s cht tiu c vi phenol. u tin
pha long vi cc mc khc nhau, sau cy vo cc pha long
ny vi khun thng hn Salmonella typhi v t cu vng
Staphylococcus aureus, 20C hay 37C. Sau 5 pht li cy sang
mi trng mi v nui cy tip 2 ngy hay lu hn. pha long cao
nht trong 10 pht c th dit ht vi khun c dng tnh ton H
s phenol. Ly bi s pha long ca cht th nghim chia cho bi s
pha long phenol t hiu qu nh nhau th thu c H s phenol. V
d bi s pha long ca phenol l 90 m bi s pha long ca cht tiu
c th nghim l 450 th H s phenol l 5. H s phenol cng cao th
biu th cht th nghim c nang lc tiu c trong cng iu kin th
nghim cng cao. H s phenol cng cao hn 1 th biu th nng lc
tiu c cng cao hn phenol (bng 15.5)

Bng 15.5: H s phenol ca mt s cht tiu c
Cht tiu c
Vi
S.typhi*
Vi
S.aureus*
Phenol 1 1
Cetylpyridinium 228 337
20
chloride
O-phenylphenol 5,6 (20C) 4,0
p-cresol 2,0-2,3 2,3
Hexachlorophene 5-15 15-40
Merthiolate 600 62,5
Mercurochrome 2,7 5,3
Lysol 1,9 3,5
Isopropyl alcohol 0,6 0,5
Etanol 0,04 0,04
Dung dich 2%I
2
trong
cn
4,1-5,2 4,1-5,2
*Nhng ch khng ch thch l x l 37

C
H s phenol l c ch s b la chn cht tiu c, nhng
trong qu trnh ng dng thc t khng th dng biu th hiu lc
cao thp ca cht tiu c. Bi v h s phenol l s liu thu c
trong nhng iu kin th nghim nht nh, vi cc vi sinh vt thun
chng, cn trong thc t vi mt qun th vi sinh vt phc tp, c tn
ti cc cht hu c, cht v c, vi cc pH, nhit khc nhau..., hiu
lc ca cht tiu c chu nh hng rt nhiu vo cc nhn t mi
trng khi s dng.
Mun nh gi thc t hn hiu lc ca cc cht tiu c c th
tin hnh cc phng php th nghim khc. Trn thc t so snh cc
cht ha hc khc nhau kim tra tc dit khun. C th dng
Th nghim pha long thc dng (use dilution test) tin hnh xc
nh. Tm nng no ca cht tiu c c th dit c 95% vi sinh
vt theo mc tin cy. Cn c th dng phng php Th nghim
thc dng (in-use test) trong cc iu kin thc t c th xc nh
nng bt u c tc dng ca tng cht tiu c.

21
Bi 17 Khi nim chung v trao i cht Vi sinh vt
16.1. NNG LNG
16.1.1. Nng lng v cng
C th nh ngha mt cch n gin nht nng lng l kh nng to nn cng hoc
gy nn nhng bin i c bit. Do , tt c cc qu trnh l, ho l kt qu ca vic s
dng hoc vn ng ca nng lng. T bo sng thc hin ba loi cng ch yu, tt c
u cn thit cho cc qu trnh sng.
Cng ho hc, bao gm vic tng hp cc phn t sinh hc phc tp t cc tin
cht n gin hn. Nng lng y c dng nng cao tnh phc tp phn
t ca t bo.
Cng vn chuyn, cn nng lng hp thu cc cht dinh dng, loi b cc
cht thi v duy tr cc cn bng ion.
Nh ta bit, nhiu phn t cht dinh dng bn ngoi mi trng phi i vo t bo
mc d nng ni bo ca cc cht ny thng cao hn ngoi bo ngha l ngc vi
gradien in ho. Vi cc cht thi v cc cht c hi cn phi c loi b khi t bo,
tnh hnh cng din ra tng t.
Cng c hc, c l l loi cng quen thuc nht trong ba loi cng. Nng lng
y cn cho vic thay i v tr vt l ca cc c th, cc t bo v cc cu trc
bn trong t bo. Hu ht nng lng sinh hc bt ngun t nh sng mt tri kh
kin chiu ln b mt tri t. Quang nng c hp thu bi cc sinh vt quang
dng trong qu trnh quang hp nh cht dip lc v cc sc t khc sau
chuyn thnh ho nng. Tri vi sinh vt quang dng, nhiu vi khun ho t
dng v c (chemolithoautotrophs) li thu c nng lng nh oxy ho cc
cht v c. Ho nng t quang hp v ho dng v c sau c th c cc
sinh vt quang t dng v c v ho t dng v c s dng chuyn CO
2

thnh cc phn t sinh hc nh Glucose (Hnh 16.1).


22
Hnh 16.1: Dng carbon v nng lng trong mt h sinh thi
(Theo: Prescott v cs, 2005)
Cc phn t phc tp do cc c th t dng tng hp (c thc vt v vi sinh vt)
c dng lm ngun carbon cho cc sinh vt ho d dng v cc sinh vt tiu th khc
vn s dng cc phn t hu c phc tp lm ngun vt cht v nng lng xy dng
nn cc cu trc t bo ca ring mnh (trn thc t cc sinh vt t dng cng s dng
cc phn t hu c phc tp). Cc sinh vt ho d dng thng s dng O
2
lm cht
nhn electron khi oxy ho Glucose v cc phn t hu c khc thnh CO
2
. Trong qu
trnh ny - c gi l h hp hiu kh - O
2
ng vai tr l cht nhn electron cui cng
v b kh thnh nc. Qu trnh trn gii phng ra nhiu nng lng. Do trong h sinh
thi nng lng c hp thu bi cc c th quang t dng v ho t dng v c. Sau
, mt phn nng lng ny c chuyn cho cc c th ho d dng khi chng s
dng cc cht dinh dng bt ngun t bn t dng (Hnh 16.1). CO
2
to thnh trong h
hp hiu kh c th li c lp vo cc phn t hu c phc tp trong quang hp v ho
t dng v c. R rng, dng carbon v nng lng trong h sinh thi c lin quan mt
thit vi nhau.
Cc t bo phi vn chuyn nng lng mt cch c hiu qu t b my sn xut
nng lng ti cc h thng thc hin cng. Ngha l, chng cn c mt ng tin chung
v nng lng tiu dng, l Adenosine 5- triPhosphate tc ATP (hnh 16.2).

Hnh 16.2. Adenosine triPhosphate v Adenosine diPhosphate.
23
(Theo Prescott, Harley v Klein, 2005)






Hnh 16.3: Chu trnh nng lng ca t bo.
Khi ATP phn gii thnh Adenosine diPhosphate (ADP) v ortoPhosphate (Pi) nng
lng gii phng ra s c dng thc hin cng hu ch. Sau , nng lng t
quang hp, h hp hiu kh, h hp k kh v ln men li c dng ti tng hp ATP
t ADP v Pi trong chu trnh nng lng ca t bo (Hnh 16.3).
ATP c to thnh t nng lng cung cp bi h hp hiu kh, h hp k kh, ln
men v quang hp. S phn gii ca ATP thnh ADP v Phosphate (P
i
) gip cho vic sn
ra cng ha hc, cng vn chuyn v cng c hc.
16.1.2. Cc nh lut v nhit ng hc
hiu c nng lng to thnh ra sao v ATP hot ng nh th no vi vai tr
l ng tin nng lng ta cn nm c mt s nguyn l c bn ca nhit ng hc.
Nhit ng hc phn tch nhng thay i v nng lng trong mt t hp vt th (v d:
mt t bo hay mt cy) c gi l mt h thng. Mi vt th khc trong t nhin c
gi l mi trng xung quanh. Nhit ng hc tp trung vo s sai khc nng lng gia
trng thi ban u v trng thi cui cng ca mt h thng m khng quan tm n tc
ca qu trnh. Chng hn, nu mt xoong nc c un n si th, v nhit ng
hc, ch iu kin nc lc ban u v khi si l quan trng, cn vic nc c un
nhanh chm ra sao v c un trn loi bp l no th khng cn ch . Trong nhit
ng hc khng th khng cp n hai nh lut quan trng sau y.
Theo nh lut th nht, nng lng khng th c to ra hoc mt i. Tng nng
lng trong t nhin l hng s mc d c th c phn b li. Chng hn, trong cc
phn ng ho hc, thng din ra s trao i nng lng (V d, nhit c thot ra
cc phn ng ngoi nhit v c hp thu trong cc phn ng ni nhit) nhng nhng s
trao i nhit ny khng tri vi nh lut trn.
24
xc nh lng nhit c s dng trong hoc thot ra t mt phn ng no
ngi ta dng hai loi n v nng lng: mt calo (cal) l lng nhit nng cn tng
nhit ca mt gam nc t 14,5 n 15,5
0
C. Lng nhit cng c th c biu hin
bng joule (joule, J) l n v ca cng. 1 cal ca nhit tng ng vi 4,1840 J ca
cng. 1000 cal hay 1 kilocalo (kcal) l lng nhit un si khong 1,9ml nc. 1
kilojoule (kj) l lng nhit un si khong 0,44 ml nc hoc gip cho mt ngi
nng 70 kg leo ln c 35 bc. Joule thng c cc nh ho hc v vt l hc s
dng, cn cc nh sinh hc li quen s dng calo khi ni v nng lng. V vy, calo
cng c s dng y khi nhng s thay i nng lng c cp.
Mc d nng lng c bo tn trong t nhin nhng nh lut th nht ca nhit
ng hc khng gii thch c nhiu qu trnh vt l v ho hc. Hy ly mt v d n
gin lm sng t iu ni trn.


Hnh 16.4: S bnh trng ca kh t xylanh cha y kh sang xylanh rng kh.
(Theo Prescott, Harley v Klein, 2005)
Gi d, ta ni mt xylanh y kh vi mt xylanh rng kh bng bng mt ng cha 1
van (Hnh 16.4). Nu ta m van kh s t xylanh y trn sang xylanh rng cho n khi
kh p cn bng 2 xylanh. Nng lng khng ch c phn b li, nhng cng c
bo tn. S bnh trng ca kh c gii thch bng nh lut th hai ca nhit ng hc
v mt trng thi vt cht c gi l entropi. C th xem entropi l i lng o tnh
hn n hoc mt trt t ca mt h thng. Tnh hn n ca mt h thng cng ln th
entropi ca h thng cng cng ln. nh lut th hai ni rng cc qu trnh vt l v ho
hc din ra theo cch sao cho tnh hn n hoc mt trt t ca c h thng v mi
trng xung quanh tng ti cc i c th. Kh bao gi cng s bnh trng sang xylanh
trng.
16.1.3. Nng lng t do v cc phn ng
Cc nh lut th nht v th hai c th kt hp trong mt phng trnh chung lin
kt nhng thay i trong nng lng c th din ra trong cc phn ng ho hc v cc
qu trnh khc.
25
G = H - T.S
G l s thay i trong nng lng t do, H l s thay i trong entalpi (enthalpi).T
l nhit Kelvin (
0
C + 273) v S l s thay i trong entropi (entropy) din ra trong
phn ng. S thay i trong entalpi l s thay i trong nhit lng. Cc phn ng trong
t bo din ra iu kin p sut v th tch khng thay i. Do s thay i trong
entalpi s tng t nh s thay i trong nng lng tng cng trong phn ng. S thay
i nng lng t do l nhit lng trong mt h thng c kh nng sinh cng nhit
v p sut khng thay i. V vy, s thay i trong entropi l i lng o t l ca s
thay i nng lng tng cng m h thng khng th s dng thc hin cng. S
thay i ca nng lng t do v ca entropi khng ph thuc vo vic h thng din ra
nh th no t lc bt u ti khi kt thc. nhit v p sut khng i mt phn ng
s xy ra ngu nhin nu nng lng t do ca h thng gim i trong phn ng, hay ni
theo cch khc, nu G l m. T phng trnh trn suy ra l mt phn ng vi s thay
i ln, dng tnh trong entropi s thng c xu hng c gi tr G m v v vy xy
ra ngu nhin. Mt s gim trong entropi s c xu hng lm cho G dng tnh hn v
phn ng t thun li.


Hnh 16.5: G
o
v cn bng. Quan h ca G
o
vi s cn bng ca cc phn ng.
(Theo Prescott, Harley v Klein, 2005)
S thay i trong nng lng t do c quan h xc nh, c th i vi hng ca cc
phn ng ho hc. Ta hy xt phn ng n gin sau y:
A + B C + D
Nu c hn hp cc phn t A v B s kt hp vi nhau to thnh cc sn phm C
v D. Cui cng C v D s tr nn m c kt hp vi nhau v to thnh A v B
vi cng tc nh khi chng c to thnh t A v B. Phn ng by gi trng thi
cn bng: tc theo hai hng l nh nhau v khng c s thay i r rt no din ra
trong nng ca cc cht phn ng v cc sn phm. Tnh hnh trn c m t l hng
s cn bng (K
eq
) lin kt nng cn bng ca cc sn phm v c cht vi nhau:
26

Nu hng s cn bng ln hn 1 cc sn phm s c nng ln hn cc cht phn
ng v phn ng c xu hng din ra n cng (Hnh 16.5).
Hng s cn bng ca mt phn ng lin quan trc tip vi s thay i trong nng
lng t do ca phn ng. Khi c xc nh cc iu kin tiu chun quy nh cht
ch v nng , p sut, pH v nhit th s thay i nng lng t do cho mt qu
trnh c gi l s thay i nng lng t do tiu chun (G
o
). Nu gi pH 7,0 (gn
vi pH ca t bo sng) s thay i nng lng t do tiu chun s c ch bi k hiu
G
o
. S thay i trong nng lng t do tiu chun c th c xem l lng nng
lng cc i m h thng c th thc hin cng hu ch cc iu kin tiu chun. Vic
s dng cc gi tr G
o
cho php ta so snh cc phn ng m khng cn quan tm ti
nhng thay i trong G, do nhng sai khc trong cc iu kin mi trng. Quan h
gia G
o
v K
eq
c th hin qua qu trnh sau:
G
o
= -2,303RTlgK
eq
.
R l hng s kh (1,9872 cal/mol hoc 8,3145 J/mol) v T l nhit tuyt i. T
phng trnh trn rt ra khi G
o
m hng s cn bng s ln hn 1, phn ng s din ra
n cng v c gi l phn ng thot nhit (Hnh 16.5). Trong mt phn ng thu nhit
G
o
l dng v hng s cn bng nh hn 1. iu c ngha l phn ng khng thun
li v t sn phm c to thnh cc iu kin tiu chun. Cn nh rng gi tr G
o

ch cho ta bit phn ng nm u khi cn bng ch khng ni ln phn ng t c
cn bng nhanh chm ra sao.
16.1.4. Vai tr ca ATP trong trao i cht
Nhiu phn ng trong t bo l thu nhit, kh din ra hon ton nu khng c s gip
t bn ngoi. Mt trong cc vai tr ca ATP l hng cc phn ng ni trn xy ra
c trit hn. ATP l mt phn t cao nng ngha l n c th b thu phn hu nh
hon ton thnh ADP v Pi vi mt G
o
khong -7,3kcal/mol.
ATP + H
2
O ADP + Pi

Vi ATP thut ng phn t cao nng khng c ngha l mt lng ln nng lng
c d tr bn trong mt lin kt c bit ca ATP m ch n gin ch ra rng vic loi
b nhnh Phosphate tn cng din ra vi s thay i nng lng t do chun l m, ln
hoc phn ng l thot nhit mnh. Ni cch khc ATP c th mnh chuyn nhm
Phosphate v d dng chuyn Phosphate cho nc. Th chuyn nhm Phosphate c
quy nh l m ca G
o
i vi vic loi b thu phn Phosphate. Mt phn t c th
chuyn nhm cao hn s chuyn Phosphate cho phn t c th thp hn.
27
Nh vy ATP thch hp kh l tng i vi vai tr l ng tin nng lng. ATP
c to thnh trong cc qu trnh hp thu v sn sinh nng lng nh quang hp, ln
men v h hp hiu kh. ng v kinh t ca t bo s phn gii ATP thi nhit lin kt
vi cc phn ng thu nhit khc nhau gip cho cc phn ng ny c hon thnh (Hnh
16.6). Ni cch khc ATP lin kt cc phn ng sinh nng lng vi cc phn ng s
dng nng lng.
16.1.5. Cc phn ng oxy ho - kh v cc cht mang electron
S thay i nng lng t do khng ch lin quan ti cn bng ca cc phn ng ho
hc thng thng m cn ti cn bng ca cc phn ng oxy ho-kh. Vic gii phng
nng lng thng bao gm cc phn ng oxy ho-kh l cc phn ng trong cc
electron c chuyn t cht cho (hoc cht kh) ti cht nhn electron (hoc cht oxy
ho). Theo quy c mt phn ng nh vy s c vit vi cht cho nm pha bn phi
ca cht nhn cng vi s (n) electron (e
-
) c chuyn:
Cht nhn + ne
-
Cht cho


Hnh 16.6. ATP nh mt tc nhn lin kt
Vic s dng ATP to thnh cc phn ng ni nng l thun li hn. ATP c
to thnh bi cc phn ng ngoi nng, sau c dng hng dn cc phn ng
ni nng.
(Theo Prescott, Harley v Klein, 2005)
Cp cht nhn v cht cho c gi l cp redox (Bng 16.1). Khi mt cht nhn
nhn cc electron n s tr thnh cht cho ca cp. Hng s cn bng i vi phn ng
c gi l th kh chun (E
o
) v l i lng o xu hng mt electron ca cht kh.
Tiu chun tham kho dng cho cc th kh l h thng hydro vi

28
(th kh pH 7,0) l -0,42V hoc -420mV.

2H
+
+ 2e
-
H
2

Trong phn ng ny mi nguyn t hydrogen cung cp mt proton (H
+
) v mt
electron (e
-
).
Th kh c ngha c th. Cc cp redox vi th kh m hn s chuyn electron cho
cc cp vi th kh dng hn v i lc ln hn i vi cc electron. Do cc electron
s c xu hng di chuyn t cc cht kh chp ca bng 16.1 n cc cht oxy ho
y v chng c th dng hn. Bng mt thng, iu ny c th c th hin dng
ca mt thp electron trong cc th kh m nht l chp (hnh 16.7).
Bng 16.1: Cc cp oxy ha - kh chn lc quan trng v sinh hc.
(Theo: Prescott v cs, 2005)

Cp oxy ha kh
Eo (Volt)
a

2H
+
+ 2e
-
H
2

Ferredoxin(Fe
3+
) + e
-
Ferredoxin (Fe
2+
)
NAD(P)
+
+ H
+
+ 2e
-
NADP(H)
S + 2H
+
+ 2e
-
H
2
S
Acetaldehyd + 2H
+
+ 2e
-
Ethanol
Pyruvate
-
+ 2H
+
+ 2e
-
Lactate
2-

FAD + 2H
+
+ 2e
-
FADH
2

Oxaloacetat
2-
+ 2H
+
+ 2e
-
Malate
2-

Fumarate
2-
+ 2H
+
+ 2e
-
Succinate
2-

Cytochrome b (Fe
3+
) + e
-
Cytochrome b (Fe
2-
)
Ubiquinone + 2H
+
+ 2e
-
Ubiquinone H
2

Cytochrome c (Fe
3+
) + e
-
Cytochrome c (Fe
2+
)
- 0,42
- 0,42
- 0,32
- 0,274
- 0,197
- 0,185
- 0,18
b

- 0,166
0,031
0,075
0,10
29
NO
3
-
+ 2H
+
+ 2e
-
NO
2
-
+ H
2
O
NO
2
-
+ 8H
+
+ 6e
-
NH
4
+
+ 2H
2
O
Fe
3+
+ e
-
Fe
2+

O
2
+ 4H
+
+ 4e
-
2H
2
O
0,254
0,421
0,44
0,771
0,815
a/ l th kh chun pH 7,0
b/ Gi tr i vi FAD/FADH
2
ng dng cho cofactor t do v n c th thay i
ng k khi lin kt vi 1 apoenzyme
c/ Gi tr i vi Fe t do khng phi Fe gn vi protein (v d cc Cytochrome).
Cc electron di chuyn t cc cht cho ti cc cht nhn xui theo gradien in th hoc
ri xung thp n cc in th dng hn. Ta hy xem trng hp ca cht mang
electron NAD
+
(nicotinamide adenine - dinucleotide). Cp NAD
+
/NADH c rt m, v v
vy c th cho electron ti nhiu cht nhn k c O
2
.

Hnh 16.7. S di chuyn ca electron v cc th kh.
30
Thp electron thng ng c cc th kh m nht nh. Cc electron chuyn dch ngu
nhin t cc cht cho cao hn trn thp (cc th hiu m hn) ti cc cht nhn thp hn
trn thp (cc th hiu dng hn). Ngha l, cht cho trn thp bao gi cng cao hn
cht nhn. Chng hn NADH s chuyn cc electron ti oxy v to thnh nc trong qu
trnh. Mt s cht cho v cht nhn in hnh c ghi bn tri v th oxy ha kh ca
chng c cho trong ngoc n. (Theo Prescott, Harley v Klein, 2005)
NAD
+
+ 2H
+
+ 2e
-
NADH + H
+
= -0,32V
O
2
+ 2H
+
+ 2e
-
H
2
O = +0,82V
V NAD
+
/NADH m hn O
2
/H
2
O cc electron s di chyn t NADH (cht kh) ti
O
2
(cht oxy ho) nh hnh 16.7.
NADH + H
+
+ O
2
H
2
O + NAD
+

Khi cc electron di chuyn t mt cht kh ti mt cht nhn vi mt th oxy ho -
kh dng hn nng lng t do s c gii phng. G
o
ca phn ng lin quan trc
tip ti mc sai khc gia th kh ca hai cp (E

o
). E

o
cng ln th nng lng t
do thot ra cng cng ln nh ch ra bi phng trnh sau: G

o
= -nFE

o
.
y n l s electron c chuyn v F l hng s Faraday (23,062 cal/mol-von hoc
96,494 J/mol-von). Vi mi thay i 0,1V trong s c s thay i 4,6 kcal tng ng
trong v K
eq
trong cc phn ng ho hc khc ngha l hng s cn bng cng ln th
cng cng ln. S khc nhau trong th kh gia NAD
+
/NADH v O
2
/H
2
O l 1,14V, mt
gi tr ln. Trong h hp hiu kh khi cc electron di chuyn t NADH ti O
2
mt lng
ln nng lng t do c dng tng hp ATP (Hnh 16.8)
NADH + H
+
+ 1/2O
2
NAD
+
+ H
2
O = 52,6 kcal.mol
-1

Khi cc electron di chuyn t cc th kh m n cc th kh dng nng lng s
c gii phng; tri li, khi cc electron di chuyn t cc in th dng hn n cc
in th m hn nng lng s cn y cc electron theo hng ngc li nh din ra
trong quang hp (Hnh 16.8), y quang nng c thu nhn v c dng y cc
electron t nc ti cht mang electron nicotinamide dinucleotide Phosphate (NADP
+
).
Nh hnh 16.1 ch dn cc sinh vt quang hp thu nhn v s dng quang nng
vn chuyn cc electron t nc (v cc cht cho electron khc nh H
2
S) n cc cht
nhn electron nh NADP
+
c cc th kh m hn. Sau cc electron ny c th di
chuyn tr li ti cc cht nhn dng hn v cung cp nng lng to thnh ATP
trong quang hp. Cc c th quang t dng s dng ATP v NADPH tng hp cc
phn t phc tp t CO
2
. Cc sinh vt ha d dng cng s dng nng lng gii phng
ra trong s vn chuyn ca cc electron nh s oxy ho cc cht dinh dng phc tp
trong h hp to thnh NADH. Sau NADH chuyn cc electron cho O
2
v nng
lng thot ra trong s vn chuyn electron c gi li dng ATP. Nng lng t nh
31
sng mt tri c s dng bi tt c cc sinh vt chnh v mi quan h ny gia dng
electron v nng lng.

Hnh 16.8: Dng nng lung trong trao i cht
Nhng v d ca mi quan h gia dng electron v nng lung trong trao i cht.
Oxy v NADP
+

c dng lm cht nhn electron ln lt t NADH v nc. (Theo
Prescott, Harley v Klein, 2005)

Hnh 16.9: Cu trc v chc nng ca NAD
+
.
(a) Cu trc ca NAD v NADP. NADP khc vi NAD ch c thm 1 Phosphate trn
mt trong cc ng ribose; (b) NAD c th nhn cc electron v 1 hydro t c cht kh
(SH
2
). Cc electron v hydro ny c mang trn vng nicotinamide. (Theo Prescott,
Harley v Klein, 2005)
32
S vn chuyn electron c ngha quan trng trong h hp hiu kh, h hp k kh,
ho dng v c v quang hp. S vn chuyn electron trong t bo cn s tham gia ca
cc cht mang nh NAD
+
v NADP
+
, c hai cht ny u c th vn chuyn electron gia
cc v tr khc nhau. Vng nicotinamide ca NAD
+
v NADP
+
(Hnh 16.9) tip nhn hai
electron ny v mt proton t mt cht cho, cn proton th hai c tch ra.

Hnh 16.10: Cu trc v chc nng ca FAD
Vitamin riboflavin bao gm vng isoalloxazine v ng ribose gn vo. FMN l
riboflavin Phosphate. Phn ca vng trc tip tham gia vo cc phn ng oxy ha kh l
phn c mu. (Theo Prescott, Harley v Klein, 2005)
Mt s cht mang electron khc c vai tr trong trao i cht ca vi sinh vt cng
c nu trong bng 16.1; cc cht ny mang electron theo cc cch khc nhau. Flavin-
adenine dinucleotide (NAD) v flavin-mononucleotide (FMN) mang 2 electron v 2
proton trn h thng vng phc tp (Hnh 16.10).
Cc protein cha FAD v FMN thng c gi l flavoprotein. Coenzyme Q (CoQ)
hoc Ubiquinone l mt quinon vn chuyn cc electron v cc H
+
trong nhiu chui vn
chuyn electron h hp (Hnh 16.11).
Cc cytochrome v mt s cht mang khc s dng cc nguyn t st vn chuyn
electron qua cc phn ng oxy ho - kh thun nghch:
Fe
3+
(st ferric) Fe
2+
(st ferrous)
Trong cytochrome cc nguyn t st ny l mt phn ca nhm hem (Hnh 16.12)
hoc ca cc vng st - porphyrin tng t khc.
33


Hnh 16.11. Cu trc v chc nng ca Coenzyme Q hoc Ubiquinone
Chiu di ca chui bn thay i ty theo c th vi n = 6 n n = 10. (Theo Prescott v
cs, 2005)
Cc chui vn chuyn electron h hp thng cha cytochrome bao gm mt protein
v mt vng st - porphyrin. Mt s protein mang electron cha st thiu nhm hem v
c gi l cc protein st khng - hem. Ferredoxin (Fd) l mt protein st khng-hem
hot ng trong vic vn chuyn electron quang hp v mt s qu trnh vn chuyn
electron khc. Mc d nguyn t st chng khng gn vi nhm hem nhng chng vn
thc hin c cc phn ng oxy ho. Cn ch rng trong s cc phn t tham gia vo
chui vn chuyn electron ni trn, mi thi im, mt s mang hai electron (nh
NAD, FAD v CoQ), s khc (nh cc cytochrome v cc protein st khng-hem) ch
mang mt electron. S khc nhau trong s lng electron c vn chuyn c ngha rt
quan trng trong hot ng ca cc chui vn chuyn electron.
34

Hnh 16.12: Cu trc ca Hem
Hem bao gm 1 vng porphyrin gn vi 1 nguyn t st. y l thnh phn khng-
protein ca nhiu Cytochrome . Nguyn t st lun phin tip nhn v gii phng 1
electron. (Theo Prescott, Harley v Klein, 2005)

16.2. ENZYME
Nh ni trn mt phn ng thot nhit l mt phn ng c G
o
m v hng s
cn bng ln hn 1 v c th din ra trit ngha l v pha bn phi ca phng trnh.
Tuy nhin, ngi ta thng c th hn hp cc cht phn ng ca mt phn ng thot
nhit m khng thy kt qu r rng mc d cc sn phm c th c to thnh. Chnh
enzyme ng vai tr trong cc phn ng ny.
16.2.1. Cu trc v phn loi cc enzyme
Enzyme l cc cht xc tc c bn cht protein, c tnh c hiu cao i vi phn ng
xc tc v vi cc phn t chu xc tc. Cht xc tc l mt cht lm tng tc ca mt
phn ng ho hc m bn thn khng b thay i. Do enzyme thc y cc phn ng
ca t bo. Cc phn t phn ng c gi l c cht v cc cht to thnh c gi l
sn phm. Nhiu enzyme l cc protein thun khit, nhng cng khng t enzyme gm
hai thnh phn: thnh phn protein (gi l apoenzyme) v phn khng - protein (gi l
cofactor); c hai cn cho hot tnh xc tc v enzyme gm c hai thnh phn trn c
gi l holoenzyme. Cofactor c gi l nhm thm (prosthetic group) nu gn cht vo
apoenzyme. Nhng thng th cofactor gn lng lo vi apoenzyme, thm ch c th
phn li khi protein enzyme sau khi cc sn phm c to thnh v mang mt trong
cc sn phm ny n mt enzyme khc. Cofactor gn lng lo ni trn c gi l
35
coenzyme. Chng hn, NAD
+
l mt coenzyme mang cc electron bn trong t bo.
Nhiu vitamin m con ngi cn ng vai tr l cc coenzyme hoc l tin cht
(precursor) ca cc coenzyme. Niacin c lp vo NAD
+
v riboflavin c lp vo
FAD. Cc ion kim loi cng c th lin kt vi cc apoenzyme v tc dng nh cc
cofactor.
Mc d t bo cha mt s lng ln v rt a dng cc enzyme nhng chng c th
c xp vo mt trong 6 nhm (Bng 16.2). Tn ca cc enzyme thng c t theo
tn c cht m chng tc dng ln v loi phn ng c xc tc. V d, Lactate
dehydrogenase (LDH) loi b hydrogen khi Lactate:
Lactate + NAD
+
Pyruvate + NADH + H
+

Lactate dehydrogenase cng c th c t tn y v chi tit hn l L-Lactate:
NAD oxydoreductase. Tn ny m t cc c cht v loi phn ng chnh xc hn.
Bng 16.2. Phn loi enzyme
(Theo Prescott, Harley v Klein, 2005)

Loi enzyme Phn ng do
enzyme xc tc
V d phn ng
Oxydoreductase Cc phn ng oxy
ha kh
Lactate dehydrogenase:
Pyruvate + NADH + H Lactate + NAD
+

Transferase Cc phn ng
chuyn nhm
gia cc phn t
Aspartate Carbamoyltransferase:
Aspartate + CarbamoylPhosphate
Carbamoylaspartate + Phosphate
Hydrolase Thy phn cc
phn t.
Glucose-6-Phosphatease:
Glucose-6-Phosphate + H
2
O Glucose + Pi
Lyase Loi b cc nhm
to thnh cc
ni i hoc b
sung cc nhm
vo ni i.
Fumarate hydratase:
L-malate Fumarate + H
2
O
Isomerase Cc phn ng xc
tc ng phn
ha.
Alanine racemase:
L-alanine D-alanine
Ligase Ni 2 phn t nh Glutamine synthetase:
36
nng lung ca
ATP (hay ca cc
nucleoside
triphosphate khc)
Glutamate + NH
3
+ ATP Glutamine + ATP +
Pi


16.2.2. C ch ca cc phn ng enzyme
Cn nh rng cc enzyme tng cng tc phn ng nhng khng lm thay i
hng s cn bng. Nu mt phn ng l thu nhit enzyme s khng chuyn dch cn bng
v nhiu sn phm hn s c to thnh. Cc enzyme ch nng cao tc m phn
ng din ra theo hng cn bng cui cng.
hiu c enzyme xc tc cc phn ng nh th no ta hy xem xt din bin ca
mt phn ng ho hc thi nhit bnh thng sau y:
A + B C + D

Khi cc phn t A v B tip cn nhau phn ng chng s to thnh mt phc hp
trng thi qu chi c c cht v sn phm (Hnh 16.13)

Hnh 16.13: Coenzyme nh cc cht mang
Chc nng ca 1 coenzyme trong vai tr mang cc cht i khp t bo. Coenzyme C
cng vi enzyme E
1
tham gia ch uyn ha A thnh sn phm B. Trong qu trnh phn
ng coenzyme C nhn X t c cht A v c th chuyn X sang c cht P trong phn ng
2. Kt qu l coenzyme li tr v dng ban u sn sng tip nhn 1X khc.
Coenzyme khng ch tham gia vo 2 phn ng m cn vn chuyn X i khp t bo.
(Theo Prescott, Harley v Klein, 2005)
Nng lng hot ho l cn nhm mang cc phn t phn ng tip xc vi nhau theo
mt cch chnh xc t c trng thi qu (hay chuyn tip). Phc hp trng
37
thi qu c th phn ly to thnh cc sn phm C v D. S khc nhau trong mc
nng lng t do gia cc cht phn ng v cc sn phm l G
o
. V vy, trong v d
nu trn cn bng s nm v pha cc sn phm v G
o
l m (ngha l cc sn phm
mc nng lng thp hn c cht). Trong hnh 16.13 r rng A v B khng th
chuyn ho thnh C v D nu chng khng c cung cp mt lng nng lng tng
ng vi nng lng hot ho. Enzyme thc y cc phn ng bng cch h thp nng
lng hot ho. Do nhiu phn t c cht hn s c nng lng y tip cn
nhau v to thnh sn phm. Mc d hng s cn bng (hoc G
o
) khng thay i nhng
cn bng s t c nhanh hn khi c mt enzyme do nng lng hot ho gim.

Hnh 16.14: Enzyme h thp nng lung hot ha
Trong tin trnh ca 1 phn ng ha hc nu trn A v B c chuyn thnh C v D. Phc hp chuyn tip
c biu th bi AB
*
v nng lung hot ha cn t c trng thi ny bi E
a
. ng biu th
tin trnh ca phn ng trong s c mt ca 1 enzyme. Cn ch , nng lung hot ha trong phn ng c
enzyme xc tc thp hn rt nhiu. (Theo Prescott, Harley v Klein, 2005)
S d enzyme c kh nng h thp nng lng hot ho ca cc phn ng v chng
mang cc c cht li gn nhau ti mt im c bit gi l v tr hot ng hoc v tr xc
tc to thnh phc hp enzyme - c cht (Hnh 16.15 v 16.16). S tng tc gia c
cht v enzyme c th din ra theo hai con ng:
1. Enzyme c hnh dng c nh, khp vi hnh dng ca c cht gip cho c cht
lin kt chnh xc v thun li cho phn ng din ra.
2. Enzyme thay i hnh dng khi gn vi c cht to iu kin cho v tr xc tc bao
quanh v khp chnh xc vi c cht.
C ch din ra theo con ng th nht c gi l m hnh kho v cha kho
(lock - and - key model). Theo con ng th hai c ch c gi l m hnh khp cm
ng (induced fit). M hnh ny c ng dng cho hexokinase v nhiu enzyme khc
(Hnh 16.16).
38

Hnh 16.15. Chc nng ca enzyme. S to thnh phc hp enzyme-c cht v
chuyn ha phc hp thnh 1 sn phm. (Theo Prescott, Harley v Klein, 2005)
Vic to thnh phc hp enzyme - c cht c th h thp nng lng hot ho theo
mt s cch. Chng hn, bng cch mang cc c cht li gn nhau ti v tr xc tc, trn
thc t, enzyme lm tng nng ca chng v thc y phn ng. Tuy nhin, mt
enzyme khng ch n gin lm m c nng cc c cht ca chng m cn lin kt
cc c cht sao cho cc c cht ny hng chnh xc vi nhau to thnh phc hp
trng thi qu . Mt s nh hng nh vy s h thp lng nng lng m cc c
cht cn t c trng thi qu . Cc hot tnh ny v cc hot tnh khc ca v tr
xc tc tng cng phn ng hng trm nghn ln bt k vi sinh vt sinh trng gia
20
0
C v khong 113
0
C. Nhng nhit ny khng cao gip cho hu ht cc phn
ng hu c trong s vng mt ca enzyme, hn na cc t bo khng th sng st
nhng nhit cao dng bi mt nh ho hc hu c trong cc qu trnh tng hp hu c
thng ngy. Enzyme gip cho s sng tn ti bng cch thc y cc phn ng c bit
nhit thp.

39
Hnh 16.16. Mt v d v s to thnh phc hp enzyme-c cht
a) M hnh y ca hexokinase nm men v c cht Glucose (mu ta). V tr hot
ng trong khe to thnh bi thy nh ca enzyme (mu lc) v thy ln (mu xm). (b)
Khi Glucose lin kt to thnh phc hp enzyme-c cht hexokinase thay i hnh
dng v bao quanh c cht. (Theo Prescott, Harley v Klein, 2005)

16.2.3. Tc ng ca mi trng ln hot tnh enzyme
Hot tnh enzyme thay i r rt vi s thay i ca cc yu t mi trng m mt
trong cc yu t quan trng nht l nng c cht. Nh ta bit, nng cc c cht
bn trong t bo thng thp. nng c cht rt thp enzyme chm to thnh sn
phm do t khi c tip xc vi phn t c cht. Nu c mt nhiu phn t c cht hn
enzyme s lin kt c cht thng xuyn hn v tc phn ng (thng c th hin
nh tc to thnh sn phm) cng ln hn nng c cht thp hn. Do tc
ca mt phn ng do enzyme xc tc tng ln theo nng c cht (Hnh 16.17).
Tuy nhin nu tip tc tng nng c cht th tc phn ng cng khng tng na
v cc phn t enzyme bo ho c cht v ang chuyn ho c cht thnh sn phm
vi tc cc i (V
max
). ng cong ca nng c cht by gi s l ng hyperbole
(Hnh 16.17). bit c nng c cht m mt enzyme cn hot ng thch hp
ngi ta thng dng hng s Michaelis (Km). y l nng c cht enzyme cn
thc hin c mt na tc cc i v c dng nh mt i lng o i lc thc s
ca mt enzyme i vi c cht. Gi tr Km cng thp c ngha l nng c cht m
enzyme xc tc phn ng cng cng thp.Hot tnh enzyme cng thay i theo s thay
i ca pH v nhit (hnh 16.18).

Hnh 16.17. ng hc Michaelis-Menten
Velocity= tc , Substrate concentration: nng c cht
40
S ph thuc ca hot tnh enzyme vo nng c cht. ng cong c cht y khp
vi phng trnh Michaelis-Menten cho trong hnh; phng trnh ny lin kt tc
phn ng (v) vi nng c cht (S) khi s dng tc cc i v hng s Michaelis
(Km). Km = nng c cht enzyme cn hot ng na tc cc i. Vmax = tc
to thnh sn phm khi enzyme c bo ha c cht v hot ng nhanh ti a.
(Theo Prescott, Harley v Klein, 2005)
Mi enzyme hot ng mnh nht mt pH thch hp nht. Khi pH chch xa khi gi
tr ti thch hot tnh ca enzyme s gim i v enzyme c th b h hi. Vi nhit
enzyme cng c gi tr ti thch cho hot tnh cc i. Nu nhit tng qu cao so vi
gi tr ti thch cu trc ca enzyme s b hu hoi v enzyme mt hot tnh. Hin tng
bin tnh (denaturation) ny ca enzyme c th l hu qu ca cc gi tr qu (tt
cng) ca pH v nhit hoc cc yu t khc. Cc gi tr ti thch ca pH v nhit
ca cc enzyme vi sinh vt thng phn nh pH v nhit ni sng ca chng. Do ,
ta d hiu, cc vi khun sinh trng tt nht nhit cao thng c cc enzyme vi
nhit ti thch cao v bn nhit ln.

Hnh 16.18: pH, nhit v hot tnh enzyme
S thay i hot tnh enzyme cng vi nhng thay i trong pH v nhit . Phm vi pH
v nhit y ch l tng trng. Cc enzyme khc nhau v v tr ca im ti thch
v hnh dng ca cc ng cong pH v nhit . (Theo Prescott, Harley v Klein, 2005)
16.2.4. S km hm enzyme
Nhiu ho cht l c i vi sinh vt v nhng cht c mnh nht chnh l nhng
cht km hm enzyme. Mt cht km hm cnh tranh trc tip cnh tranh vi c cht v
tr xc tc ca mt enzyme v ngn cn enzyme to thnh sn phm. Chng hn,
succinate dehydrogenase l enzyme xc tc s oxy ho succinate thnh fumarate trong
chu trnh Krebs. Acid malonic c cu trc tng t succinate do l cht km hm cnh
tranh ca enzyme ni trn. Sau khi lin kt vo enzyme malonat khng b oxy ho v vic
to thnh fumarate khng din ra. Cc cht km hm cnh tranh thng chi vi cc c
cht bnh thng nhng khng th b chuyn ho thnh cc sn phm.
41

Hnh 16.19: Km hm cnh tranh ca succinate-dehydrogenase
Acid malonic c cu trc tng t acid succinic do l cht km hm cnh tranh
ca enzyme ni trn. (Theo Prescott, Harley v Klein, 2005)
Cc cht km hm cnh tranh c s dng iu tr nhiu bnh do vi sinh vt.
Chng hn cc thuc sulfa nh sulfanilamit chi vi p-aminobenzoat l mt phn t dng
trong vic to thnh coenzyme acid folic. Cc thuc cnh tranh vi p-aminobenzoat i
vi v tr xc tc ca mt enzyme tham gia tng hp acid folic do ngn cn s to
thnh acid folic v km hm sinh trng ca vi khun. C th ngi khng chu tc dng
ca thuc do khng c kh nng tng hp acid folic v phi thu nhn acid ny t thc n.
16.3. TNH CHT V NGHA CA VIC IU CHNH TRAO I CHT
B my iu chnh c vai tr cc k phc tp v kh khn. Cc con ng cn phi
iu chnh v phi hp c hiu qu sao cho tt c cc thnh phn ca t bo u c mt
vi s lng thch hp, chnh xc. Hn na t bo vi sinh vt phi c kh nng p ng
rt kp thi vi nhng thay i ca mi trng bng cch s dng cc cht dinh dng
hin c v bng cch bt m cc con ng d ho mi khi c mt cc cht dinh dng
khc. V tt c cc thnh phn ho hc ca mt t bo thng khng tn ti trong mi
trng, do vi sinh vt cng phi tng hp chng v phi thay i hot tnh sinh tng
hp p ng vi nhng thay i trong vic s dng cht dinh dng. Thnh phn ho hc
ca mi trng bao quanh t bo thng xuyn thay i, do cc qu trnh iu chnh
ny cng phi nng ng v phi lin tc p ng vi cc iu kin thay i.
Vic iu chnh l cn thit cho t bo vi sinh vt duy tr c nng lng, vt cht
v cn bng trao i cht. Nu mt ngun nng lng c bit khng c mt, cc enzyme
cn cho vic s dng ngun nng lng ny l khng cn thit v vic tng hp chng
tip tc s l mt s tiu ph C, N v nng lng. Cng tng t nh vy, s l rt v ch
i vi vi sinh vt nu chng tng hp cc enzyme cn cho vic to thnh mt sn phm
cui cng no khi sn phm ny c mt vi s lng y . Nh vy, c d ho v
ng ho u c iu chnh theo cch sao cho hiu qu ca hot ng t c ti a.
C th thy r xu hng duy tr cn bng v bo tn nng lng ca vt cht trong s
p ng iu chnh vi khun E. coli... Khi sinh trng trong mt mi trng rt n
gin ch cha glucose l ngun C v ngun nng lng vi khun s tng hp cc thnh
phn m t bo cn vi s lng cn bng (chng hn acid amin tryptophan). Vic b
sung mt sn phm sinh tng hp cui cng vo mi trng s dn n km hm ngay lp
tc con ng tng hp sn phm cui cng ni trn. Vic tng hp cc enzyme ca con
42
ng cng s b ngng hoc chm li. Nu chuyn E. coli sang mi trng ch cha
lactose, vi khun s tng hp cc enzyme cn cho s chuyn ho ng ny. Tri li, khi
sinh trng trong mi trng cha c glucose v lactose E. coli s s dng glucose u
tin v y l loi ng n gip cho sinh trng ca vi khun din ra nhanh nht ch
sau khi glucose cn kit, lactose mi c s dng.
Dng carbon chuyn ho qua con ng c th c iu chnh theo ba cch ch
yu:
1. Tp trung cc cht trao i v cc enzyme trong nhng phn khc nhau ca t
bo, t nh hng n hot tnh ca con ng,
2. Cc enzyme quan trng thng c kch thch hoc b km hm trc tip nhm
thay i nhanh hot tnh ca con ng,
3. S lng cc phn t enzyme cng c th c iu ho. Cc phn t cht xc tc
c mt cng nhiu th hot tnh ca con ng cng ln. vi khun, vic iu
chnh thng chu tc dng mc phin m. Vic iu ho tng hp mRNA
chm hn vic iu chnh trc tip hot tnh enzyme nhng tit kim c nhiu
nng lng v nguyn liu v cc enzyme khng c tng hp khi khng cn
thit.
Hai c ch 1 v 2 s c trnh by y, l: khu tr trao i cht v iu ho
hot tnh enzyme.
16.4. KHU TR TRAO I CHT (Metabolic Channeling)
Mt trong cc c ch khu tr trao i cht ph bin nht l s chia khoang
(compartmentation) ngha l s phn b bit ho cc enzyme v cc cht trao i trong
cc cu trc t bo tch bit hoc cc bo quan c mng bao bc. Chng hn, s oxy ho
acid bo gp bn trong ti th nhng tng hp acid bo li din ra trong t bo cht. Chu
cht vi khun cng c th c xem l mt v d ca s chia khoang. S chia khoang
to iu kin cho vic hot ng v iu chnh ng thi nhng tch bit ca cc con
ng c th c phi hp nh s iu chnh vic vn chuyn cc cht trao i v cc
coenzyme gia cc khoang ca t bo. Gi d, c hai con ng tn ti trong cc khoang
t bo khc nhau nhng u cn NAD
+
cho hot ng. S phn b NAD
+
gia hai
khoang s quyt nh hot tnh tng i ca cc con ng cnh tranh ny v con
ng no chim d tha NAD
+
s c li th hn.
S chia khoang cng gp bn trong cc khoang nh nn t bo cht. Nn (matrix) l
vt th ng c, c cu trc gm nhiu khoang nh. sinh vt nhn tht nn cng c
chia nh bi li ni cht (endoplasmic reticulum) v b khung t bo (cytoskeleton).
Trong mt mi trng nh vy cc cht trao i v cc coenzyme khng khuch tn
nhanh v cc gradien cht trao i s c thit lp gn cc enzyme hoc cc h thng
enzyme cc b. iu ny din ra v cc enzyme mt v tr c bit chuyn ho cc cht
thnh sn phm dn n gim nng ca mt hoc nhiu cht trao i ny v tng nng
ca mt hoc nhiu cht trao i khc. Chng hn, nng sn phm s cao gn
enzyme v thp dn theo khong cch tng ln tnh t enzyme.
43
S khu tr c th to ra nhng thay i r rt trong nng cht trao i v v vy
nh hng trc tip n hot tnh enzyme. Nng c cht, ni chung, thng vo
khong 10
-3
- 10
-6
M/l, thm ch thp hn, ngha l c th trong cng phm vi nh nng
enzyme v bng hoc nh hn hng s Michaelis (Km) ca nhiu enzyme. Di cc
iu kin nh vy nng c cht ca mt enzyme c th iu ho hot tnh ca cht xc
tc v nng c cht l trong phn tng ln ca ng cong hyperbole ca s bo ho
c cht (Hnh 16.20).

Hnh 16.20: iu ha hot tnh enzyme bi nng c cht
Trong hnh l ng cong bo ha enzyme-c cht vi hng s Michaelis (Km) v tc
tng ng vi tc cc i (V
max
). Tc ban u ca phn ng (v) c dng
th i vi nng c cht. Tc cc i l tc ln nht t c vi mt s lng
enzyme c nh di nhng iu kin xc nh. Khi nng c cht bng hoc nh hn
Km hot tnh enzyme s thay i hu nh tuyn tnh vi nng c cht. Gi d, nng
c cht tng t mc A ti mc B. V nhng nng ny u trong phm vi ca
Km nn hot tnh enzyme tng ln r rt. S gim nng t B n A s h thp tc
to thnh sn phm. (Theo Prescott, Harley v Klein, 2005)
Khi nng c cht tng, c cht s c chuyn thnh sn phm nhanh hn; nng
c cht gim ng nhin dn n hot tnh enzyme thp hn. Nu 2 enzyme hai
con ng khc nhau cng s dng mt cht trao i chng c th trc tip cnh tranh
cht ny, con ng thng trong cuc cnh tranh ny, ngha l con ng vi enzyme c
gi tr Km thp nht i vi cht trao i, s hot ng gn nh hon ton thng tr. Do
s khu tr bn trong mt khoang t bo c th iu chnh v phi hp trao i cht
thng qua nhng bin i trong nng cht trao i v nng coenzyme.
16.5. IU HA HOT TNH ENZYME
Hot ng ca nhiu con ng trao i cht c th c iu ho nh vic iu
chnh hot tnh ca cc enzyme iu chnh. Mc ny m t cc enzyme ni trn v cp
vai tr ca chng trong vic iu chnh hot tnh ca con ng.
44
16.5.1. iu chnh d lp th
Cc enzyme iu chnh thng l cc enzyme d lp th (allosteric enzymes). Hot
tnh ca mt enzyme d lp th b thay i bi mt phn t nh gi l effector (effector,
cht tc ng) hoc modulator (modulator, cht iu bin). Effector lin kt thun nghch
nh lc khng - cng ho tr vo mt v tr iu chnh (regulatory site) tch bit khi v
tr xc tc (catalytic site) v gy ra s thay i trong hnh dng hoc hnh th ca enzyme
(Hnh 16.21). Hot tnh ca v tr xc tc do b thay i. Mt effector dng lm tng
hot tnh enzyme, mt effector m, tri li, lm gim hot tnh hoc km hm enzyme.
Nhng thay i nh vy trong hot tnh thng bt ngun t nhng bin i trong i lc
biu kin ca enzyme i vi c cht, tuy nhin nhng thay i trong tc cc i cng
c th din ra.


Hnh 16.21: iu chnh d lp th
Cu trc v chc nng ca 1 enzyme d lp th. Trong hnh bn effector hoc modulator
(cht iu bin) trc ht gn vo 1 v tr iu ha tch bit v lm thay i hnh th
enzyme dn n s thay i hnh dng ca v tr hot ng. V tr hot ng gi c th
lin kt c cht hiu qu hn. y effector l dng tnh v n kch thch s lin kt c
cht v hot tnh xc tc. (Theo Prescott, Harley v Klein, 2005)
Cc enzyme iu chnh thng l cc enzyme d lp th (allosteric enzymes). Hot
tnh ca mt enzyme d lp th b thay i bi mt phn t nh gi l effector (effector,
cht tc ng) hoc modulator (modulator, cht iu bin). Effector lin kt thun nghch
45
nh lc khng - cng ho tr vo mt v tr iu chnh (regulatory site) tch bit khi v
tr xc tc (catalytic site) v gy ra s thay i trong hnh dng hoc hnh th ca enzyme
(Hnh 16.21). Hot tnh ca v tr xc tc do b thay i. Mt effector dng lm tng
hot tnh enzyme, mt effector m, tri li, lm gim hot tnh hoc km hm enzyme.
Nhng thay i nh vy trong hot tnh thng bt ngun t nhng bin i trong i lc
biu kin ca enzyme i vi c cht, tuy nhin nhng thay i trong tc cc i cng
c th din ra.


Hnh 16.22: S iu chnh ACTase
Phn ng Aspartate
-
carbamoyltransferase v vai tr ca enzyme ny trong vic iu
chnh sinh tng hp pyrimidine. Sn phm cui cng CTP km hm hot tnh ca ACTase
(-) cn ATP li hot ha enzyme (+). Cacbamoyl Phosphate synthetase cng b km hm
bi cc sn phm cui cng ca con ng nh UMP. (Theo Prescott, Harley v Klein,
2005)
Cc c tnh ng hc ca enzyme khng - iu chnh chng minh rng hng s
Michaelis (Km) l nng c cht cn cho mt enzyme hot ng tc bng na tc
cc i. Hng s ny ch ng dng cho cc ng cong bo ho c cht hyperbole m
khng cho cc ng cong xch-ma thng gp vi cc enzyme d lp th (Hnh 16.23).
Nng c cht cn cho mt na tc cc i vi cc enzyme d lp th c ng
cong c cht xch-ma c gi l gi tr [S]
0,5
hoc K
0,5
.
Mt trong cc enzyme iu chnh d lp th c nghin cu k nht l Aspartate-
carbamoyltransferase (ACTase) E. coli. Enzyme xc tc s ngng t ca
cacbamoylphosphate vi aspartate to thnh cacbamoylaspartate (Hnh 16.22).
46
ACTase xc tc phn ng quyt nh tc ca con ng sinh tng hp pyrimidine
E. coli. ng cong c cht bo ho l xch-ma khi nng ca mt trong hai c cht
thay i (Hnh 16.23)


Hnh 16.23: ng hc ca Aspartate carbamoyltransferase E. coli
CTP l 1 effector m lm tng gi tr K
0,5
, cn ATP l 1 effector dng, h thp K
0,5
.
V
max
vn l hng s. (Theo Prescott, Harley v Klein, 2005)
Enzyme c trn mt v tr hot ng v s lin kt ca mt phn t c cht vo mt v
tr hot ng s kch thch s lin kt ca c cht vo cc v tr khc. Hn na, cytidine
triphosphate (CTP), mt sn phm cui cng ca sinh tng hp pyrimidine, km hm
enzyme, tri li ATP (purine) li hot ho enzyme. C hai effector thay i gi tr K
0,5

ca enzyme nhng khng thay i tc cc i ca enzyme. GTP km hm bng cch
nng cao K
0,5
hoc chuyn dch ng cong bo ho c cht ln cc gi tr cao hn. iu
ny cho php enzyme hot ng chm hn mt nng c cht c bit khi CTP c
mt. ATP hot ho bng cch chuyn ng cong ti cc gi tr nng c cht thp hn
khin cho enzyme hot ng cc i qua mt phm vi nng c cht rng ln. Do ,
khi con ng hot ng ti mc nng CTP tng qu cao hot tnh ACTase s gim
v tc to thnh sn phm cui cng b chm li. Tri li, khi nng sn phm cui
cng ATP tng ln so vi CTP, ATP s kch thch tng hp CTP thng qua tc dng ln
ACTase.
Aspartate carbamoyltransferase E. coli cung cp mt v d r rt v cc v tr iu
chnh v v tr xc tc ring r trong cc enzyme d lp th. Enzyme l mt protein ln
gm 2 di n v xc tc v 3 di n v iu chnh (Hnh 16.24a). Cc di n v
xc tc ch cha cc v tr xc tc v khng chu nh hng bi CTP v ATP. Cc di
n v iu chnh khng xc tc phn ng nhng c cc v tr iu chnh lin kt CTP v
ATP. Khi lin kt vo enzyme hon ton cc effector ny gy ra nhng thay i v hnh
th trong cc di-n v iu chnh, sau trong cc di n v xc tc v cc v tr
xc tc. Enzyme c th thay i thun nghch gia mt dng T t hot ng v mt dng
47
R hot ng hn (Hnh 16.24 b, c). Do v tr iu chnh nh hng n v tr xc tc
khong cch khong 6,0 nm.


Hnh 16.24: Cu trc v iu chnh ca Aspartate carbamoyltransferase E. coli.
(Theo Prescott, Harley v Klein, 2005)
16.5.2. Ci bin cng ho tr cc enzyme
Cc enzyme cng c th c kch thch hoc b km hm thng qua s ci bin cng
ho tr thun nghch. Thng thng iu ny din ra do vic thm v loi b mt nhm
c bit, ngha l mt dng ca enzyme c hot ng hn mt dng khc. Chng hn,
glycogen phosphorylase ca mc bnh m Neurospora crassa c gi l phosphorylase
a khi dng phosphoryl ho v phosphorylase b khi dng b loi b Phosphate (Hnh
16.25). Phosphorylase b l bt hot v cht hot ho AMP m n cn thng khng c
mt mc cao. Dng phosphoryl ho ca phosphorylase a hot ng ngay khi
khng c mt AMP. Glycogen phosphorylase c kch thch thng qua vic phosphoryl
ha phosphorylase b thnh phosphorylase a. Vic gn nhnh Phosphate lm thay i hnh
th ca enzyme chuyn n thnh dng hot ng. Phn ng phosphoryl ho v loi b
Phosphate c xc tc bi cc enzyme ring r v cc enzyme ny cng c iu
chnh.

48
Hnh 16.25: S ci bin cng ha tr thun nghch ca glycogen phosphorylase.
Phosphorylase a l dng hot ng c tng hp bi phosphoryl ha v b bt hot khi
Phosphate b loi b do thy phn to thnh phosphorylase b bt hot. (Theo
Prescott, Harley v Klein, 2005)
Cc enzyme cng c th c iu chnh nh lin kt vi cc nhm khc Phosphate.
Mt trong cc enzyme c nghin cu chi tit nht l Glutamine synthetase E. coli.
y l mt enzyme ln, phc tp tn ti hai dng. Khi mt nhnh acid adenylic lin kt
vi mt trong 12 di-n v ca enzyme to thnh mt enzyme adenyl ho, glutamine
synthetase hot ng yu. Vic loi b cc nhm AMP to ra glutamine synthetase
mt adenyl hot ng hn v glutamine c tng hp. H thng glutamine synthetase
khc h thng phosphorylase hai im: 1) AMP c dng nh tc nhn ci bin; 2)
Dng ci bin ca Glutamine synthetase km hot ng. Glutamine synthetase cng c
iu chnh d lp th.
Vic s dng ci bin cng ho tr iu chnh hot tnh enzyme c mt s u im.
Cc enzyme c th chuyn ho qua li thng cng l cc enzyme d lp th. V mi
dng c th p ng khc nhau vi cc effector d lp th nn cc h thng enzyme ci
bin cng ho tr c kh nng p ng vi nhiu cht kch thch hn trong cc con ng
thay i v phc tp. Cng c th c iu chnh l cc enzyme xc tc nhng ci bin
cng ho tr, b sung vo h thng mt mc iu chnh th hai.
16.5.3. Km hm phn hi hoc km hm bi sn phm cui cng
(Feedback inhibition)
Nh ni phn trn, tc ca nhiu con ng trao i cht c iu chnh
thng qua s iu khin hot tnh ca cc enzyme iu chnh. Mi con ng c t nht
mt enzyme dn-tc (pacemaker) xc tc phn ng chm nht hoc hn ch tc
trong con ng. V cc phn ng khc din ra nhanh hn phn ng dn-tc do
nhng thay i trong hot tnh ca enzyme ny trc tip nh hng n tc ca con
ng. Thng thng, bc th nht trong mt con ng l mt phn ng dn tc
xc tc bi mt enzyme iu chnh. Sn phm cui cng ca con ng thng km hm
enzyme iu chnh ny. Qu trnh ni trn c gi l s km hm bi sn phm cui
cng. Kiu km hm ny bo m cho s to thnh cn bng ca sn phm cui cng ca
mt con ng. Nu tch lu vi nng qu cao sn phm cui cng s km hm
enzyme iu chnh v lm gim tc tng hp ca chnh sn phm ny. Khi nng
ca sn phm cui cng gim, hot tnh ca con ng li tng v nhiu sn phm hn
c to thnh. S km hm bi sn phm cui cng, nh vy, t ng phi hp vic
cung cp theo nhu cu ca sn phm ny. Aspartate carbamoyltransferase l mt v d
in hnh ca s km hm bi sn phm mt con ng sinh tng hp thng phn nhnh
to thnh trn mt sn phm cui cng. Trong tnh hnh nh vy vic tng hp cc sn
phm cui cng ca con ng phi c phi hp mt cch chnh xc. Khng th
mt sn phm cui cng ny c mt d tha trong khi mt sn phm cui cng khc li
thiu. S phn nhnh cc con ng sinh tng hp thng to nn s cn bng gia cc
sn phm cui cng qua vic s dng cc enzyme iu chnh cc im phn nhnh
(Hnh 16.26).
49
Khi c mt nng d tha mt sn phm cui cng thng km hm enzyme
im phn nhnh trn chui dn n to thnh sn phm ny, nh vy m iu chnh vic
tng hp ca chnh sn phm nhng khng nh hng n tng hp cc sn phm
khc. Hnh 16.26 cng cho thy c 2 sn phm cng km hm enzyme m u trong con
ng. S d tha ca mt sn phm lm chm dng C i vo c con ng trong khi
km hm enzyme thch hp im phn nhnh. V s phn nhnh khng hot ng cn t
C do s km hm bi sn phm cui cng ca enzyme dn tc ban u gip cho s
iu ho gia cung v cu cc con ng phn nhnh. Vic iu chnh cc con ng
phn nhnh nhiu thng c thc hin phc tp hn do s c mt ca cc izoenzyme
tc l nhng enzyme khc nhau nhng xc tc cng mt phn ng. Bc u dn tc
ban u c th do mt s izoenzyme xc tc, mi izoenzyme chu s iu ho ring r v
c lp. Trong tnh hnh nh vy, s d tha ca mt sn phm cui cng s lm gim
hot tnh ca con ng nhng khng hon ton km hm chc nng ca con ng v
mt s izoenzyme vn cn hot ng.

Hnh 16.26: Km hm phn hi
Trn hnh l s km hm phn hi trong 1 con ng phn nhnh vi 2 sn phm cui
cng. Cc enzyme im phn nhnh xc tc s chuyn ha cht trung gian E thnh F
v G c iu chnh bi km hm phn hi. Cc sn phm P v Q cng km hm phn
ng m u trong con ng. Tn hiu ch ra rng P hoc Q km hm enzyme xc tc
bc tip theo tn hiu. (Theo Prescott, Harley v Klein, 2005)
Chng 18
S DNG NNG LNG TRONG
SINH TNG HP VI SINH VT
Bin son : Nguyn nh Quyn, Nguyn Ln Dng
Nh trn d ni vi sinh vt c th thu nhn nng lng qua nhiu con ng.
Phn ln nng lng ny c dng cho sinh tng hp hoc ng ho. Trong qu
trnh sinh tng hp vi sinh vt bt u vi cc tin cht n gin nh cc phn t
v c v cc monome v kin trc nn cc phn t ngy cng phc tp hn cho ti
khi xut hin cc bo quan v cc t bo mi (Hnh 18.1). Mi t bo vi sinh vt
phi sn xut ra nhiu loi phn t khc nhau; tuy nhin, trong chng ny ch c
th gii thiu vic tng hp nhng thnh phn t bo quan trng nht.

















Cc phn t v c
Cc monome hoc
cc n v kin trc
Cp t chc
V d
T bo
Bo quan
Cc h thng siu
phn t
Cc cao phn t
Hnh 18.1: Kin trc ca cc t bo
Sinh tng hp ca cc thnh phn t bo nhn nguyn thy v nhn tht. Sinh tng hp
c t chc cc cp ngy cng phc tp hn. (Theo Prescott v cs, 2005)
V ng ho l to ra mt trt t v mi t bo c sp xp mc cao,
cc k phc tp, do sinh tng hp i hi nhiu nng lng. iu ny d nhn
thy khi ta xem xt nng lc sinh tng hp ca t bo E. coli ang sinh trng
nhanh (bng 18.1). Mc d hu ht ATP dnh cho sinh tng hp c dng cho
tng hp protein, nhng ATP cng c dng cho tng hp cc thnh phn khc
ca t bo.
Bng 18.1: Sinh tng hp E. coli (Theo: Prescott v cs, 2005)
Thnh phn
t bo
S phn t/t
bo
a

Cc phn t c
tng hp/giy
Cc phn t ATP
cn/giy cho tng hp
DNA
RNA
Polysaccarid
Lipid
Protein
1
b
15.000
39.000
15.000.000
1.700.000
0,00083
12,5
32,5
12.500
1.400
60.000
75.000
65.000
87.000
2.120.000
a/ Tnh cho 1 t bo c th tch 2,25 m
3
, trng lng 1x10
-12
g, trng lng
kh 2,5x10
-13
g v chu trnh phn bo l 20 pht.
b/ Ch : vi khun c th cha nhiu bn sao ca ADN genom.
Nng lng t do cn cho sinh tng hp trong cc t bo trng thnh c
kch thc n nh v cc phn t ca t bo lin tc b phn gii v c tng hp
li trong mt qu trnh c gi l vng quay (turnover). Cc t bo khng bao gi
chi nhau tng thi im khc nhau. Mc d vng quay ca cc thnh phn t bo
l lin tc nhng trao i cht vn c iu ho cNn thn sao cho tc sinh tng
hp ni chung, c cn bng vi tc t phn gii. N goi nng lng dng cho
quay vng cc phn t nhiu t bo khng sinh trng cng s dng nng lng
tng hp cc enzyme v cc cht khc gii phng vo mi trng.
18.1. CC NGUYN TC IU CHNH SINH TNG HP
Trao i cht trong sinh tng hp tun theo mt s nguyn tc chung, 6
trong s cc nguyn tc ny c tm tt di y:
1. Mi t bo vi sinh vt cha mt lng ln cc protein, acid nucleic v
polisaccaride. Tt c u l cc cao phn t tc l cc polime gm cc n v nh
hn lin kt vi nhau. Vic kin trc cc phn t ln, phc tp t mt vi n v
cu trc n gin hoc monome tit kim c nhiu d tr di truyn, nguyn liu
cho sinh tng hp v nng lng. Ta hy xem xt tng hp protein hiu r vn
ny. Cc protein, bt k c kch thc, hnh dng hoc chc nng nh th no,
u c to thnh ch bi 20 amino acid thng thng ni vi nhau nh lin kt
peptide. Cc protein khc nhau n gin ch l do c th t amino acid khc nhau
nhng khng phi l cc amino acid mi v khc. Gi d, nu cc protein c to
thnh khng phi bng 20 m bng 40 amino acid khc nhau, t bo s phi cn
cc enzyme sn xut ra cc amino acid nhiu gp i (hoc phi nhn c cc
acid b sung t thc n). Cc enzyme b sung i hi phi c cc gen v t bo li
phi u t thm nguyn liu v nng lng cho vic tng hp cc gen, cc
enzyme v cc amino acid b sung ny. R rng, vic s dng mt vi monome
ni vi nhau bi mt lin kt cng ho tr duy nht khin cho vic tng hp cc
cao phn t tr thnh mt qu trnh rt c hiu qu. Hu nh tt c cc cu trc t
bo u c kin trc ch yu bi khong 30 tin cht nh.
2. T bo thng tit kim cc nguyn vt liu v nng lng bng cch s
dng cc enzyme dng cho c d ho v ng ho. Chng hn, hu ht cc enzyme
ng phn u tham gia tng hp v phn gii glucose se.
3. Mc d nhiu enzyme trong cc con ng lng ho hot ng trong c
phn gii v tng hp nhng mt s bc li c xc tc bi hai enzyme khc
nhau: mt xc tc phn ng theo hng phn gii v mt theo hng tng hp
(Hnh 18.2). V vy, cc con ng d ho v ng ho khng bao gi chi nhau
mc d c nhiu enzyme chung. Vic s dng cc enzyme ring r theo hai hng
mt bc n c cho php iu chnh d ho v ng ho mt cch c lp. Cn
nh rng vic iu chnh ng ho hi khc vi iu chnh d ho. C hai con
ng u c th iu chnh c bi sn phNm cui cng cng nh bi nng
ATP, ADP, AMP v N AD
+
. Tuy nhin, trong cc con ng ng ho vic iu
chnh bi sn phNm cui cng, ni chung, c vai tr quan trng hn.
4. tng hp cc phn t mt cch hiu qu cc con ng ng ho phi
hot ng khng thun nghch theo hng sinh tng hp. T bo c th thc hin
iu ny bng cch lin kt mt s phn ng sinh tng hp vi s phn gii ATP
v cc nucleoside triphosphate khc. Khi hai qu trnh ny c lin kt nng
lng t do thot ra trong s phn gii nucleoside triphosphate s hng dn phn
ng sinh tng hp hon thnh.
5. cc vi sinh vt nhn tht cc con ng sinh tng hp thng din ra
bn trong cc khoang t bo khc vi cc con ng phn gii tng ng. Chng
hn, sinh tng hp acid bo gp trong t bo cht trong khi s oxy ho acid bo
c thc hin bn trong ti th. S phn khoang to iu kin cho cc con ng
hot ng ng thi khng ph thuc vo nhau.

Hnh 18.2: Mt con ng sinh tng hp gi thuyt.
Cc con ng lin kt G vi X, Y v Z hon ton l ng ha v chng ch c dng
tng hp cc sn phm cui cng. Con ng t A n G l lng ha, ngha l c c chc
nng d ha v ng ha. Hu ht cc phn ng c dng trong c 2 vai tr; tuy nhin, s
chuyn ha qua li ca C v D c xc tc bi 2 enzyme ring bit, E
1
(d ha) v E
2
(ng
ha). (Ngun Prescott v cs, 2005)
6. Cui cng, cc con ng ng ho v d ho thng s dng cc cofactor
khc nhau. Cc phn ng oxy ho trong phn gii, ni chung, sn ra N ADH l mt
c cht cho vn chuyn electron. Tri li, khi mt cht cho electron l cn cho sinh
tng hp th N ADPH ch khng phi N ADH thng m nhim chc nng ny.
Trao i cht ca acid bo cung cp v d th hai. Cc phn t acyl-CoA ca acid
bo b oxy ho sn ra nng lng trong khi tng hp acid bo c s tham gia
ca cc tioeste ca protein mang nhnh acyl.
S ng
ha
S lng
ha
Sau khi cc cao phn t c kin trc t cc tin cht n gin hn
chng s c tp hp thnh cc cu trc ln hn, phc tp hn nh cc h thng
siu phn t v cc bo quan (Hnh 18.1). Cc cao phn t thng cha thng tin
cn thit to thnh mt cch ngu nhin trong mt qu trnh gi l t tp hp.
Chng hn, riboxom l nhng tp hp ln gm nhiu protein v cc phn t acid
ribonucleic nhng chng c to thnh nh s tp hp ca cc thnh phn khng
cn c s tham gia ca cc yu t b sung.
18.2. C NNH QUANG HP CO
2

Mc d hu ht vi sinh vt c th c nh CO
2
t nht l trong cc phn ng
b sung tuy nhin ch cc c th t dng mi c kh nng s dng CO
2
lm
ngun carbon duy nht hoc ch yu. S kh v c nh CO
2
i hi nhiu nng
lng. Cc c th t dng thng thu nng lng nh s hp thu nh sng trong
quang hp nhng mt s nhn c nng lng t phn ng oxy ho cc cht cho
electron v c kh. S c nh CO
2
t dng c ngha quyt nh i vi s
sng trn tri t v n cung cp cht hu c cho cc c th d dng.
Vi sinh vt c th c nh CO
2
hoc chuyn phn t v c ny thnh carbon
hu c v ng ho n theo ba con ng ch yu. Hu nh tt c cc vi sinh vt
t dng u c nh CO
2
qua con ng trao i cht c bit c gi l chu
trnh Calvin (cng gi l chu trnh Calvin-Benson hoc chu trnh pentose-
phosphate kh). Mc d hot ng trong cc c th quang hp c nhn tht v hu
ht c th quang hp c nhn nguyn thu nhng chu trnh Calvin li vng mt
Archaea (C khuNn), mt s vi khuNn k kh bt buc v mt s vi khuNn hiu kh.
N hng vi khuNn ny thng s dng mt trong hai con ng khc. Mt s
archaea (Thermoproteus, Sulfolobus) v cc vi khuNn Chlorobium v
Desulfobacter s dng con ng acid tricarboxylic kh. cc vi khuNn sinh
metan, vi khuNn kh sulfate v cc vi khuNn sinh acetate (cc vi khuNn to thnh
acetate t CO
2
trong qu trnh ln men) li tn ti con ng Acetyl-CoA.
Chu trnh Calvin gp trong cht nn (stroma) ca lc lp ca cc vi sinh vt
nhn tht t dng. Vi khuNn lam, mt s vi khuNn nitrate ho v cc thiobacilli
cha cc th vi, a din gi l cacboxysom. Cacboxysom cha enzyme ribulo-
1,5-bisphosphate carboxylase, c th l v tr c nh CO
2
hoc v tr d tr
carboxylase v cc protein khc. C th chia chu trnh Calvin thnh 3 pha:
carboxyl ho, kh v ti sn. S chung ca chu trnh c gii thiu hnh
18.4.
18.2.1. Pha carboxyl ho (carboxylation phase)
S c nh CO
2
c xc tc bi enzyme ribulo-1,5-bisphosphate-
carboxylase hoc oxygenase (rubisco) (Hnh 18.3) xc tc vic gn CO
2
vo
ribulo-1,5-bisphosphate (RuBP) to thnh 2 phn t 3-phosphorus-glycerat (PGA).









Hnh 18.3: Phn ng ribulo-1,5-bisphosphate carboxylase
Enzyme xc tc b sung CO
2
vo ribulo-1,5-bisphosphate to thnh 1 cht trung gian khng
bn, sau cht ny b phn gii thnh 2 phn t 3-phosphorusglycerat. (Theo: Prescott v cs,
2005)
18.2.2. Pha kh (reduction phase)
Tip theo, PGA b kh thnh glyceraldehyde-3-phosphate. S kh c xc
tc bi 2 enzyme, thc cht l s o nghch mt phn ca con ng ng phn
mc d glyceraldehyde-3-phosphate-dehydrogenase khc vi enzyme ng phn
trong vic s dng N ADP
+
thay cho N AD
+
(hnh 18.4).
18.2.3. Pha ti sinh (regeneration phase)
Trong pha ny RuBP c ti sn v sn ra cc hidrat-carbon nh
glyceraldehyde-3-phosphate, fructose za v glucose (Hnh 18.4). Phn ny ca chu
trnh chi vi con ng pentose-phosphate v bao gm cc phn ng ca trans-
ketolase v transaldolase. Chu trnh c hon thnh khi phosphorusribulokinase
ti to RuBP. tng hp fructose -6-phosphate hoc glucose -6-phosphate t CO
2

chu trnh phi hot ng 6 ln sn ra hexose cn thit v ti to 6 phn t RuBP.
6RuBP + 6CO2 12PGA 6RuBP + Fructose -6-P
Vic c nh mt CO
2
thnh cht hu c cn 3ATP v 2N ADPH. Glucose
c to thnh t CO
2
theo phng trnh sau:
6CO
2
+ 18ATP + 12N ADPH + 12H
+
+ 12H
2
O glucose + 18ADP + 18Pi +
12N ADP
+


Hnh 18.4: Chu trnh Calvin
Trn y l s vn tt ca chu trnh ch vi cc pha carboxyl ha v kh c trnh
by chi tit. 3 ribulo-1,5-bisphosphate c carboxyl ha to thnh su 3-phosphorusglycerat
trong pha carboxyl ha. Cc cht ny c chuyn ha thnh 6 glycerat-3-phosphate ri c th
thnh dihydroxyacetone phosphate (DHAP) 5 trong s 6 triose (glyceraldehyde phosphate v

PHA KH
PHA TI TO

PHA
CARBOXYL
HA
dihydroxyacetone phosphate) c dng to li 3 ribulo-1,5-bisphosphate trong pha ti sn.
Triose cn li c dng trong sinh tng hp. Nhng con s trong ngoc n bn phi pha
di ch ra dng carbon ny. (Theo: Prescott v cs, 2005)
ATP v N ADPH c cung cp bi cc phn ng sng quang hp hay bi
s oxy ho cc phn t v c cc vi khuNn ho t dng. Sau cc ng to
thnh trong chu trnh Calvin c th c dng tng hp cc phn t cn thit
khc.
18.3. TNG HP CC NG V POLISACCHARIDE
N hiu vi sinh vt khng c kh nng quang hp v l cc c th d dng
phi tng hp ng t cc phn t hu c kh thay cho t CO
2
.


















Hnh 18.5: S ti to ng
Con ng ti to ng gp nhiu vi sinh vt. Tn ca 4 enzyme gp trong ng
phn c ng khung. Cc bc ng phn cng c biu th so snh.(Theo: Prescott v
cs, 2005)
Vic tng hp glucose t cc tin cht khng phi hidrat carbon c gi l
s ti to ng (glucose neogenesis). Mc d con ng ti to ng khng chi
con ng ng phn nhng chng c 7 enzyme chung (Hnh 18.5).
Ba bc ng phn sau y l khng thun nghch trong t bo: 1) Chuyn
ho phosphorusenolPyruvate thnh pyruvate; 2) to thnh fructose -1,6-
bisphosphate t fructose -6-phosphate v 3) phosphoryl ho glucose. Cc bc ny
phi i vng khi con ng hot ng theo hng sinh tng hp. Chng hn, s
to thnh fructose -1,6-bisphosphate bi phosphorusfructose kinase c o
nghch bi enzyme fructose -bisphosphatease, enzyme ny loi b nh thu phn
mt phosphate t fructose -bisphosphate. Thng thng t nht hai enzyme tham
gia vo vic chuyn ho pyruvate thnh phosphorusenol pyruvate (o nghch
bc pyruvate kinase).
T hnh 18.5 c th thy con ng tng hp fructose za tng t nh con
ng tng hp glucose se. Mt khi glucose v fructose za c to thnh cc
ng ph bin khc cng c sn sinh. Chng hn, mannose c hnh thnh
trc tip t fructose za qua mt s sp xp li n gin:
Fructose -6-phosphate Mannose-6-phosphate
Mt s ng c tng hp trong khi lin kt vi mt nucleoside
diphosphate. ng nucleoside diphosphate quan trng nht l uridine diphosphate
glucose (UDPG). Glucose c hot ho nh gn vi pyrophosphate ca uridine
diphosphate qua phn ng vi uridine triphosphate (Hnh 18.6).

Hnh 18.6: Uridine diphosphate glucose (Theo: Prescott v cs, 2005)
Phn UDP ca HDPG c cc enzyme nhn ra v mang glucose i khp t
bo dng tham gia vo cc phn ng ht nh ADP mang phosphate dng ATP.
UDP-galactose c tng hp t UDPG qua vic sp xp li ca mt nhm
hydroxyl. Mt enzyme khc xc tc vic tng hp UDP - acid glucuronic qua vic
oxy ho UDPG (hnh 18.7).

Hnh 18.7: Uridine diphosphate galactose v tng hp glucuronate
Trn hnh l vic tng hp UDP-galactose v UDP-acid glucuronic t UDP-glucose se.
(Theo: Prescott v cs, 2005)
Cc ng nucleoside diphosphate cng ng vai tr ch cht trong vic
tng hp cc polisaccaride nh tinh bt v glycogen. Cng li y, sinh tng hp
khng n gin ch l s o ngc trc tip ca phn gii. S phn gii glycogen
v tinh bt din ra qua s thu phn to thnh cc ng t do hay qua vic gn
thm nhnh phosphate vo cc polime ny sn ra glucose -1-phosphate. Cc
ng nucleoside diphosphate khng tham gia vo qu trnh trn. Tri li trong
vic tng hp glycogen v tinh bt vi khuNn v to adenosine diphosphate
glucose c to thnh t glucose -1-phosphate v sau chuyn glucose vo cui
chui glycogen v chui tinh bt:
ATP + Glucose -1-phosphate ADP-glucose + PPi
(Glucose se)
n
+ ADP-glucose (Glucose se)
n+1
+ ADP
Cc ng nucleoside diphosphate cng tham gia vo vic tng hp cc
phn t phc tp nh thnh t bo vi khuNn.
18.4. S NG HA PHOSPHORUS, LU HUNH (SULFUR) V NIT
(NITROGEN) V C
N goi carbon v oxy vi sinh vt cng cn mt lng phosphorus, sulfur v
nitrogen cho sinh tng hp. Mi nguyn t ni trn c ng ho hoc c c
nh thnh cc phn t hu c qua cc con ng khc nhau.
18.4.1. S ng ho phosphorus
Phosphorus gp trong cc acid nucleic, protein, phospholipid, ATP v cc
coenzyme nh N ADP. N gun phosphorus ph bin nht l cc este ca phosphate
v c v phosphate hu c. Phosphate v c c c nh qua vic to thnh ATP
thng qua mt trong ba con ng: 1) quang phosphoryl ho; 2) phosphoryl ho
oxy ho v 3) phosphoryl ho mc c cht.
ng phn cung cp mt v d ca con ng th ba. Phosphate c gn
vi glyceraldehyde-3-phosphate to thnh 1,3-bisphosphorusglycerat, sau cht
ny c dng tng hp ATP.
Glyceraldehyde-3-phosphate + Pi + N AD
+
1,3-bisphosphorusglycerat + N ADH
+ H
+

1,3-bisphosphorusglycerat + ADP 3-phosphorusglycerat + ATP
Vi sinh vt c th thu nhn cc phosphate hu c t mi trng bao quanh
dng ho tan hay dng ht. Cc este ca phosphate hu c thng b thu phn bi
cc phosphatease v tch ra phosphate v c. Vi khuNn gram m cha cc
phosphatease trong khoang chu cht nm gia thnh t bo v mng sinh cht;v
vy sau khi c gii phng phosphate c hp thu trc tip qua mng. ng vt
nguyn sinh, tri li, c th s dng trc tip cc phosphate hu c sau khi n hoc
thu phn chng trong lyzosom v tiu th phosphate.
18.4.2. S ng ho sulfur
Sulfur cn cho vic tng hp amino acid (cystein v methionine) v mt s
coenzyme (coenzyme A, tiamine-pyrophosphate v biotin) v c th thu c t
hai ngun. N hiu vi sinh vt s dng cystein v methionine dn xut t cc ngun
bn ngoi hoc t d tr amino acid ni bo. N goi ra, sulfate c th cung cp
sulfur cho sinh tng hp. N guyn t sulfur trong sulfate oxy ho hn nguyn t
sulfur trong cystein v cc phn t hu c khc, do sulfate phi b kh trc khi
c th c ng ho. Qu trnh ny c gi l s kh sulfate ng ho phn
bit vi s kh sulfate d ho din ra khi sulfate tc dng nh cht nhn electron
trong h hp k kh.
S kh sulfate ng ho i hi phi hot ho sulfate qua vic to thnh
phosphoadenosine-5-phosphosulfate (Hnh 18.8) tip theo l s kh sulfate. y
l mt qu trnh phc tp (Hnh 18.9) trong sulfate trc ht b kh thnh sulfit
(
2
3
SO

) sau thnh H
2
S. Cystein c th c tng hp t sulfua hydro qua hai
con ng.









Hnh 18.8: Phosphorusadenosin 5-phosphorussulfate (PAPS). Theo: Prescott v
cs, 2005)












Hnh 18.9: Con ng kh sulfate (Theo: Prescott v cs, 2005)
N m c th kt hp H
2
S vi serine to thnh cystein nhng nhiu vi khuNn
li gn H
2
S vi O-Acetylserine (qu trnh 1 v 2, ln lt)
(1) H
2
S + Serine Cystein + H
2
O

(2) Serine O-Acetylserine Cystein
Mt khi c to thnh cystein c th c dng tng hp cc hp cht
hu c khc c cha sulfur.
18.4.3. S ng ho nitrogen
Do l thnh phn ch yu ca cc protein, acid nucleic, coenzyme v nhiu
thnh phn khc nn nng lc ng ho nitrogen ca t bo l cc k quan trng.
Mc d kh quyn giu kh nitrogen nhng ch mt s t vi khuNn c th kh kh
ny v s dng lm ngun nitrogen. Cn hu ht vi sinh vt c kh nng ng ho
ammonia hoc nitrate.
S ng ho ammonia
N itrogen ca ammonia c th c chuyn ho thnh cht hu c tng i
d dng v trc tip v nitrogen y gp trong trng thi kh hn cc dng khc
Acet at CoA
Acet yl -
C A
H
2
S
ca nitrogen v c. Mt s vi sinh vt tng hp amino acid alanine trong mt phn
ng amine ho kh xc tc bi alanine-dehydrogenase:
Pyruvate +
4
N H

+ N ADH (N ADPH) + H
+
alanine + N AD
+
(N ADP
+
) + H
2
O









Hnh 18.10: Con ng ng ha ammonia. S ng ha ammonia nh
glutamate.dehydrogenase (GDH) v transaminease. Cc GDH ph thuc NADP hoc NAD c
th tham gia vo y. Con ng ny hot ng mnh nht nhng nng ammonia cao (
Theo Prescott v cs, 2005)
Con ng ch yu ng ho ammonia l to thnh glutamate t -
ketoglutarate (mt cht trung gian ca chu trnh TCA). N hiu vi khuNn v nm s
dng glutamate-dehydrogenase khi nng ammonia cao:
-ketoglutarate +
4
N H

+ N ADPH (N ADH) + H
+
Glutamate + N ADP
+
(N AD
+
) + H
2
O
Vic s dng N ADPH v N ADH (tc nhn kh) trong tng hp glutamate
thay i tu theo loi.
Mt khi alanine hoc glutamate c tng hp nhm -amine mi c
to thnh c th c chuyn sang cc b khung carbon khc thng qua cc phn
ng chuyn amine, t s xut hin cc amino acid khc. Cc transaminease
cha coenzyme pyridoxal phosphate c chc nng chuyn nhm amine. Vi sinh
vt c mt s transaminease, mi enzyme ny xc tc vic to thnh mt s amino
acid bng cch s dng cng mt amino acid lm cht cho nhm amine. Khi
glutamate-dehydrogenase hot ng phi hp vi cc transaminease ammonia c
th c chuyn thnh nhiu amino acid (hnh 18.10).

Hnh 18.11: Glutamine synthetase v glutamate synthase
Cc phn ng do 2 enzyme ny xc tc tham gia vo vic ng ha ammonia. Mt s
glutamate synthetase s dng NADPH l ngun electron, s khc li s dng ferredoxin kh
(Fd). (Ngun Prescott v cs, 2005)
Con ng th hai dng ng ho ammonia bao gm 2 enzyme tc dng
theo th t, l glutamine synthetase v glutamate-synthase (hnh 18.11).
Ammonia c dng tng hp glutamine t glutamate, sau nitrogen amit ca
glutamine c chuyn n -ketoglutarate to thnh mt phn t glutamate
mi. V glutamate tc dng nh mt cht cho amine trong cc phn ng ca
transaminease nn ammonia c th c dng tng hp tt c cc amino acid
thng thng khi c mt cc transaminease thch hp (hnh 18.12).
C ATP v mt ngun cc electron nh N ADPH hay ferredoxin kh u
cn. Con ng ny gp E. coli, B. megaterium v nhiu vi khuNn khc. Hai
enzyme tc dng theo th t hot ng rt hiu qu cc nng ammonia thp
khc vi con ng glutamate dehydrogenase. N h ni trn glutamine
synthetase c iu ho cht ch nh s ci bin cng ho tr thun nghch v cc
effector d lp th.
Acid glutamic
Glutamine
Phn ng glutamine synthetase
Phn ng glutamate synthase
Acid -
ketoglutaric
2 acid glutamic Glutamine

Hnh 18.12: C nh ammonia nh glutamine synthetase v glutamate synthase
Con ng ny hot ng c hiu qu nhng nng ammonia thp. (Theo: Prescott v cs,
2005)
S kh nitrate ng ho
N itrogen trong nitrate (
3
N O

) trng thi oxy ho hn nhiu so vi nitrogen

trong ammonia. N itrate trc ht phi b kh thnh ammonia trc khi nitrogen c
th c chuyn ho thnh mt dng hu c. S kh ny ca nitrate c gi l
kh nitrate ng ho. Qu trnh ny khc vi qu trnh din ra trong h hp k kh
v kh nitrate d ho. Trong s kh nitrate ng ho nitrate c chuyn thnh
cht hu c v khng tham gia vo vic sn sinh nng lng. Kh nitrate ng ho
gp ph bin vi khuNn, nm v to.
Qu trnh ni trn din ra trong t bo cht vi khuNn.
Bc th nht trong ng ho nitrate l kh nitrate thnh nitrite xc tc bi
nitrate reductase l enzyme cha FAD v molipden (Hnh 18.13), N ADPH l
ngun electron:
3
N O

+ N ADPH +H
+

2
N O

+ N ADP
+
+ H
2
O
Sau , nitrite b kh thnh ammonia thng qua mt s ln b sung 2
electron c xc tc bi nitrite reductase v c th c cc enzyme khc.
Hydroxylamine c th l mt cht trung gian. Tip theo ammonia c chuyn ho
thnh cc amino acid nh cc con ng m t.













Hnh 18.13: Kh nitrate ng ha
Con ng ny gp cc vi khun c th kh v ng ha nitrogen ca nitrate. Theo: Prescott
v cs, 2005)

18.4.4. S c nh Nitrogen (Nitrogen fixation)
S kh kh nitrogen ca kh quyn c gi l s c nh nitrogen. V nng
ca ammonia v nitrate thng thp, hn na ch mt s vi khuNn c kh nng
trn (cc t bo nhn tht hon ton khng th thc hin c c nh N
2
) nn tc
ca qu trnh ny trong nhiu hon cnh, hn ch sinh trng ca thc vt. C
nh nitrogen gp : 1) cc vi khuNn sng t do (v d: Azotobacter, Klebsiella,
Clostridium v Methanococcus); 2) cc vi khuNn cng sinh vi thc vt nh cc
cy h u (Rhizobium), cy phi lao (x khuNn Frankia) v bo du (vi khuNn lam
Anabaena azollae) v 3) cc vi khuNn lam (Nostoc v Anabaena).
S kh nitrogen thnh ammonia c xc tc bi enzyme nitrogenase. Mc
d cc cht trung gian gn vo enzyme ta cn cha r nhng c l nitrogen b kh
qua mt s ln b sung 2 electron nh Hnh 18.14 m t. S kh nitrogen phn t
thnh ammonia pht nhit mnh nhng phn ng c nng lng hot ho cao do
nitrogen phn t l mt kh tr vi mt lin kt ba gia hai nguyn t nitrogen.
V vy, s kh nitrogen l tn km v tiu th nhiu ATP. t nht 8 electron
v 16ATP, c 4ATP l cn cho mt cp electron.
N
2
+ 8H
+
+ 8e + 16ATP 2N H
3
+ H
2
+ 16ADP + 16Pi












Hnh 18.14: Kh nitrogen. Gi thuyt kh nitrogen nh nitrogenase. (Theo:
Prescott v cs, 2005)
Cc electron bt ngun t ferredoxin b kh bi mt s con ng, chng
hn qua quang hp vi khuNn lam, qua cc qu trnh h hp cc vi khuNn c
nh nitrogen hiu kh hoc qua ln men cc vi khuNn k kh. V d, Clostridium
pasteurianum (mt vi khuNn k kh) kh ferredoxin trong qu trnh oxy ho
Pyruvate, trong khi Azotobacter (mt vi khuNn hiu kh) li s dng electron t
N ADPH kh ferredoxin.
N itrogenase l mt phc h gm 2 protein ch yu: mt protein MoFe (hay
nitrogenase, MW 220.000) lin kt vi 1-2 protein Fe (hay nitrogenase reductase,
MW 64.000). Protein MoFe cha 2 nguyn t molipden v 28-32 nguyn t st;
protein Fe cha 4 nguyn t st hnh 18.15). Fe v Mo ca protein MoFe c
cha bn trong mt cofactor gi l FeMo-co v s kh N
2
din ra cofactor ny.
Trc ht protein Fe b kh bi ferredoxin sau n lin kt ATP
(hnh18.16). S lin kt ATP lm thay i hnh th ca protein Fe v h thp th
kh ca protein ny (t 100mV n 400 mV) to iu kin cho protein Fe kh
protein MoFe. ATP b thu phn khi din ra s chuyn electron ny. Cui cng,
protein MoFe kh chuyn cc electron ti nitrogen nguyn t.



Hnh 18.15: Cu trc ca protein Fe nitrogenase (Theo: Prescott v cs, 2005)
Mt s vi khuNn cha enzyme hidrogenase oxi ha H
2
thnh H
2
O v lin
kt phn ng ny vi vic to thnh ATP hay vi vic kh ferredoxin (Fd) hoc
flavodoxin (Fld) ri cc cht ny li c th chuyn electron cho protein Fe.
N itrogenase rt mn cm vi O
2
v phi c bo v sao cho khi b bt hot bi
O
2
bn trong t bo. nhiu vi khuNn lam s bo v ny c thc hin nh mt
cu trc c bit gi l d bo nang (heterocyst), c vch dy, ch cha h quang I
(dng tng hp ATP nhng khng to thnh O
2
). N itrogenase c nh N
2
bn
trong d bo nang v nhn c saccarose t cc t bo ln cn sau truyn
nitrogen c nh c cho cc t bo trn. cc vi khuNn hiu kh, c nh N
2

nh Azotobacter nitrogenase c bo v nh: (1) Lp v nhy bao quanh t bo
cn tr s khuch tn ca O
2
vo t bo; (2) Vn tc h hp cao nh O
2
b loi
b nhanh; (3) N itrogenase c kt hp vi mt protein c bit nh khng b
bt hot bi O
2
. N t r ca cc cy u thuc h Leguminosae to thnh mt
protein mu gi l leghemoglobin c kh nng lin kt O
2
t do cho qu
trnh h hp to thnh ATP nhng khng km hm hot tnh ca nitrogenase ca
vi khuNn Rhizobium.

Hnh 18.16: C ch tc dng ca nitrogenase. Qu trnh di chuyn ca 2 electron
t ferredoxin ti nitrogen c lp li 3 ln kh N
2
thnh 2 phn t ammonia. Cn bng
pha di bao gm c vic kh proton thnh H
2
. (Theo: Prescott v cs, 2005)
ng ch , y c s cng sinh di truyn gia hai c th: cy tng hp
phn protein cn vi khuNn tng hp nhm hem. Tng hp nitrogenase khng
nhng b km hm bi O
2
nhng cng bi cc hp cht nitrogen v c v hu c.
Khi thiu ngun nng lung ADP c tch ly li v c ch hot tnh ca
enzyme. iu ny lm tng ci gi ca s kh N
2
. Theo tnh ton c nh c
1 mg N Clostridium pasteurianum phi tiu th 1g C hu c trong glucose khi
vi khuNn hiu kh Azotobacter chroococcum thm ch cn ti 30g.
Trong iu kin t thiu Mo mt s vi khuNn c th tng hp 2 loi
nitrogenase khc: cha Va (vanadi) Fe hay ch cha Fe. Cc cofactor tng t
FeMo-co gp trong c 2 nitrogenase ni trn ngha l FeVa-co (trong nitrogenase
vanadi) v 1 nhm Fe-S (tng t FeMo-co v FeVa-co) nhng thiu c Mo v
Va (trong nitrogenase st)
S kh nitrogen thnh N H
3
din ra qua ba bc, mi bc cn 2 electron
(hnh 18.14 v 18.16). N h vy 6 electron s c chuyn v cn tng cng
12ATP i vi mt phn t nitrogen b kh. Tuy nhin trn thc t qu trnh
thng tiu th t nht 8 electron v 16ATP v nitrogenase cng kh cc proton
thnh H
2
. Hydro phn ng vi diamine (HN = N H) to thnh nitrogen v hydro.
Chu trnh v ch ny sn ra mt phn N
2
ngay trong iu kin thun li khin cho
vic c nh nitrogen tr nn tn km hn. Cc vi khuNn c nh nitrogen cng
sinh c th tiu th ti 20% ATP do cy ch sn ra N itrogenase c th kh mt s
phn t cha lin kt ba (nh Acetylen, xianit v azit)
HC CH + 2H
+
+ 2e H
2
C = CH
2

Tc kh Acetylen thnh etilen c dng nh gi hot tnh
nitrogenase.
Mt khi nitrogen phn t b kh thnh ammonia, ammonia c th c
chuyn thnh cc cht hu c. Rhizobium (vi khuNn c nh nitrogen cng sinh),
c l ammonia khuch tn khi t bo vi khuNn v c ng ho bi cc t bo
ca cy u bao quanh. Vic ng ho ammonia c l ch yu l tng hp
glutamine bi h thng glutamine synthetase - glutamate synthase (Hnh 18.11).
Tuy nhin, allantoin v acid allantoic (cc dn xut ca Purine) cng c tng
hp v c dng cho vic vn chuyn nitrogen ti cc phn t khc ca cy.
18.5. TNG HP CC AMINO ACID
Vi sinh vt thay i v cc ngun nitrogen c s dng nhng hu ht c
th ng ho mt vi loi nitrogen v c nh cc con ng m t. Vic tng
hp amino acid cng i hi s kin trc nn cc b khung carbon thch hp v
thng thng y l mt qu trnh phc tp bao gm nhiu bc. Do nhu cu bo
tn nitrogen, carbon v nng lng nn cc con ng tng hp acid amine, ni
chung, c iu ho cht ch bi cc c ch d lp th v c ch km hm bi sn
phNm cui cng.
E. coli, to v hu ht thc vt c kh nng tng hp tt c amino acid t cc
tin cht. Cc sinh vt khc k c ngi khng c kh nng tng hp mt s amino
acid khng thay th v phi thu c chng trong thc n. Mt s vi khuNn lactic
nh Lactobacillus hon ton khng tng hp c mt amino acid no v phi thu
nhn chng nh phn gii protein trong mi trng.Trong mc ny khng th trnh
by chi tit con ng sinh tng hp ca tng amino acid m ch gii thiu khi
qut v sinh tng hp amino acid.

Hnh 18.17: T chc ca s ng ha
Cc sn phm sinh tng hp dn xut t cc cht trung gian ca con ng lng ha.
Ch 2 phn ng c nh CO
2
b sung ch yu. (Theo: Prescott v cs, 2005)
Hnh 18.17 m t quan h ca con ng sinh tng hp amino acid vi cc
con ng lng ho. B khung ca amino acid bt ngun t Acetyl-CoA v cc
cht trung gian ca chu trnh TCA, ng phn v con ng pentose-phosphate.
cho hiu qu v kinh t nht cc tin cht dng cho sinh tng hp amino acid
c cung cp ch t mt vi con ng lng ho ch yu. Th t dn n cc
amino acid ring r phn nhnh t cc con ng trung tm ny. Alanine, aspartat
v glutamate c c tng hp nh s chuyn amine ln lt, trc tip t
Pyruvate, Oxaloacetatee v -ketoglutarate.














Hnh 18.18: Con ng phn nhnh ca tng hp amino acid. Cc con ng
dn ti methionine, threonine, izoleucine v lysine. Mc d 1 s mi tn biu th 1 bc tuy
nhin hu ht nhng s chuyn ha qua li u i hi s tham gia ca 1 s enzyme. (Theo:
Prescott v cs, 2005)
Hu ht cc con ng sinh tng hp u phc tp hn v cc cht trung
gian quen thuc thng c dng trong sinh tng hp ca cc h amino acid c
lin quan nhm mc ch tit kim hn. Chng hn, lysine, threonine, izoleucine
v methionine u c tng hp t oxaloacetatee t mt con ng ng ho
phn nhnh (Hnh 18.18). Con ng sinh tng hp cc amino acid thm
phenylalanine, tyrosine v tryptophan cng c chung nhiu cht trung gian (Hnh
18.19).














Hnh 18.19: Tng hp cc amino acid thm (phenylalanine, tyrosine,
tryptophan). Ch : hu ht cc mi tn u biu th trn 1 phn ng enzyme. (Theo: Prescott
v cs, 2005)
18.6. CC PHN NG B SUNG
Khi xem xt hnh 18.17 ta thy cc cht trung gian ca chu trnh TCA c
dng trong sinh tng hp cc pyrimidine v nhiu acid amine. Trn thc t, cc
chc nng sinh tng hp ca chu trnh ny quan trng n mc hu ht chu trnh
hot ng k kh cung cp cc tin cht sinh tng hp mc d N ADH l khng
cn thit cho vic vn chuyn electron v phosphoryl ho trong s vng mt ca
O
2
. Do chu trnh TCA c vai tr ng k trong vic cung cp carbon cho sinh
tng hp v cc cht trung gian ca chu trnh c th b cn kit nu t bo khng
c bin php duy tr chng. Tuy nhin vi sinh vt c cc phn ng hon li cc cht
trung gian ca chu trnh gip cho chu trnh TCA c th hot ng lin tc khi sinh
tng hp ang din ra mnh m. Cc phn ng thay th cc cht trung gian ca chu
trnh c gi l cc phn ng b sung (anaplerotic reactions).
Hu ht vi sinh vt c th thay th cc cht trung gian ca chu trnh TCA
bng c nh CO
2
, trong CO
2
c chuyn ho thnh carbon hu c v c
ng ho. Cn phn bit l, cc phn ng b sung khng m nhim cng chc
nng nh con ng c nh CO
2
cung cp carbon cn thit cc c th t dng.
C nh CO
2
cc c th t dng cung cp hu ht hoc ton b carbon cn cho
sinh trng. Cc phn ng b sung c nh CO
2
ch nhm thay th cc cht trung
gian v duy tr cn bng trao i cht. Thng thng CO
2
c gn vo mt phn
t cht nhn (Pyruvate hoc phosphorusenolPyruvate) to thnh cht trung gian
ca chu trnh l Oxaloacetatee (Hinh 18.17). Arthrobacter globiformis v nm men
s dng Pyruvate-carboxylase xc tc phn ng ny.
Pyruvate + CO
2
+ ATP + H
2
O
Biotin
Oxaloacetatee + ADP + Pi
Enzyme trn cn cofactor l biotin v s dng nng lng ca ATP lin
kt CO
2
vo Pyruvate. Biotin thng l cofactor ca cc enzyme xc tc phn ng
carboxyl ho. Do c chc nng quan trng nh vy nn biotin l yu t sinh trng
cn thit i vi nhiu loi vi sinh vt. Cc vi sinh vt khc nh E. coli, Salmonella
typhimurium li s dng enzyme phosphoenolpyruvate-carboxylase xc tc phn
ng di y:
Phosphoenolpyruvate + CO
2
Oxaloacetatee + Pi
Mt s vi khuNn, to, nm v ng vt nguyn sinh c th sinh trng vi
ngun carbon duy nht l acetate bng cch s dng acetate tng hp cc cht
trung gian ca chu trnh TCA trong chu trnh glioxylat (Hnh 18.20). Chu trnh
c thc hin nh hai enzyme c bit - Izocitrate liase v malat synthase - xc
tc cc phn ng sau:
Izocitrate
izoxitrat lyaza
Succinat + Glioxylat
Glioxylat + Acetyl-CoA
malat sin taza
Malat + CoA

Chu trnh glioxylat, thc ra l mt chu trnh TCA ci bin. Hai phn ng loi
carboxyl ca chu trnh TCA (bc Izocitrate dehydrogenase v -ketoglutarate
dehydrogenase) c b qua gip cho vic chuyn ho Acetyl-CoA to thnh
Oxaloacetatee m khng mt carbon ca Acetyl-CoA nh CO
2
. Theo cch ny,
acetate v bt k cc phn t no c chuyn ho thnh acetate u c th ng
gp carbon vo chu trnh v gip cho sinh trng ca vi sinh vt.


Hnh 18.20: Chu trnh glioxylat. Ch : cc enzyme ca chu trnh TCA
phn di c b qua. (Theo: Prescott v cs, 2005)
18.7. TNG HP CC PURINE, PYRIMIDIN V NUCLEOTIDE
Sinh tng hp ca purine v pyrimidine l sng cn cho mi t bo v cc
phn t ny c dng tng hp ATP, mt s cofactor, acid ribonucleic (ARN ),
acid deoxyribonucleic (ADN ) v cc thnh phn quan trng khc ca t bo. Hu
CHU TRNH GLIOXYLAT
ht vi sinh vt c th tng hp cc Purine v pyrimidine cho bn thn v cc cht
ny c vai tr quyt nh i vi chc nng ca t bo.
Purine v pyrimidine l cc baz nitrogen vng cha mt s ni i v c
cc c tnh thm r rt. Purine gm 2 vng ni vi nhau, cn pyrimidine ch c
mt vng (hnh 18.21 v 18.23). Trong vi sinh vt thng gp cc purine adenin v
guanin v cc pyrimidine uracyl, xitozin v thymine. Mt base purine hoc
pyrimidine ni vi mt ng pentose (ribose hoc deoxyribose) l mt
nucleoside. Mt nucleotide l mt nucleoside ni vi mt hoc trn mt nhm
phosphate lin kt vi ng.

Hnh 18.21: Sinh tng hp Purine.
Ch s ch dn cc ngun N v C ca b khung Purine. (Theo: Prescott v cs, 2005)
18.7.1. Sinh tng hp Purine
Con ng sinh tng hp cc purine l mt th t phc tp gm 11 bc
trong 7 phn t khc nhau gp phn vo b khung purine cui cng (Hnh
18.21). V con ng m u vi ribo-5-phosphate v b khung purine c kin
trc trn ng ny nn sn phNm purine u tin ca con ng l nucleotide acid
inosinic ch khng phi l mt base purine t do. Trong sinh tng hp ca purine
cofactor acid folic ng vai tr rt quan trng. Cc dn xut ca acid folic ng
gp carbon 2 v 8 vo b khung purine. Trn thc t, thuc sulfonamide km hm
sinh trng ca vi khuNn l do c ch tng hp acid folic. iu ny s nh hng
n sinh tng hp ca purine v cc qu trnh khc cn acid folic.
Nhm format
t acid folic
Nhm format t
acid folic
Nit amide ca
glutamine
Nit amine ca
aspactate
Glycine
Mt khi acid inosinic c to thnh, cc con ng tng i ngn s
tng hp adenosine monophosphate v guanosine monophosphate (Hnh 18.22) v
sn ra nucleoside diphosphate v triphosphate bng cch chuyn phosphate t
ATP. ADN cha deoxyribonucleotide (ribose thiu mt nhm hydroxyl trn C
2
)
thay cho ribonucleotide gp trong ARN . Cc deoxyribonucleotide xut hin t s
kh ca cc nucleoside diphosphate hoc nucleoside triphosphate qua hai con
ng khc nhau. Mt s vi sinh vt kh triphosphate nh h thng cn cofactor
vitamine B
12
. S khc, nh E. coli, li kh ribose trong nucleoside diphosphate. C
hai h thng u s dng mt protein nh cha S gi l thioredoxin lm tc nhn
kh.














Hnh 18.22. Sinh tng hp adenosine monophosoahte v Guanosine
Monophosphatee
18.7.2. Sinh tng hp pyrimidine
Sinh tng hp pyrimidine m u vi acid aspartic v cacbamoyl-phosphate
(mt phn t cao nng c tng hp t CO
2
v ammonia) (Hnh 18.23).
Aspartate-cacbamoyltransferase xc tc vic ngng t hai c cht ny to
thnh cacbamoyl-aspartat, sau cht ny c chuyn thnh sn phNm
pyrimidine u tin l acid orotic.
Sau khi b khung pyrimidine c tng hp, mt nucleotide s c to
thnh bng cch thm vo ribo-5-phosphate nh tc dng ca cht trung gian cao
nng 5-phosphorusribosyl-1-pyrophosphate. Do vic kin trc vng pyrimidine
c hon thnh trc khi ribose c thm vo tri vi vic tng hp vng
Purine bt u vi ribo-5-phosphate. Vic loi carboxyl ho ca orotidine
monophosphate sn ra uridine monophosphate v cui cng uridine triphosphate
v cytidine triphosphate.

















Hnh 18.23: Tng hp pyrimidine.
PRPP l acid 5-phosphorusribose 1- pyrophosphorusric, cht cung cp chui ribo-5-phosphate.
Phn dn xut t cacbamoylphosphate c in m. (Theo: Prescott v cs, 2005)
Pyrimidine th ba ph bin l thymine - mt thnh phn ca ADN . Ribose trong
cc nucleotide pyrimidine b kh theo cng cch nh trong cc nucleotide purine.
Sau deoxyuridine monophosphate c methyl ho vi dn xut ca acid folic
to thnh deoxythymidine monophosphate (Hnh 18.24).








Hnh 18.24: Tng hp deoxythymidine monophosphate. Ch :
deoxythymidine khc vi deoxyuridine ch c thm nhm methyl. (Theo: Prescott
v cs, 2005)
18.8. TNG HP LIPID
Vi sinh vt cha nhiu lipid c bit l mng t bo. Lipid thng cha
cc acid bo hoc dn xut ca acid bo. Acid bo l cc acid monocarboxylic vi
cc chui alkyl di thng c mt s chn carbon (chiu di trung bnh l 18
carbon). Mt s c th l cha bo ho ngha l c mt hoc trn mt ni i. Hu
ht acid bo ca vi sinh vt l chui thng nhng c mt s phn nhnh. Cc vi
khuNn gram m thng c cc acid bo cyclopropan (tc l cc acid bo cha mt
hoc trn mt cc vng cyclopropan trong chui).
Vic tng hp cc acid bo c xc tc bi phc h synthetase acid bo vi
Acetyl-CoA v malonyl-CoA nh c cht v N ADPH nh cht cho electron.
Malonyl-CoA dn xut t s carboxyl ho ca Acetyl-CoA vi s tiu th ATP.
Vic tng hp din ra sau khi acetate v malonat c chuyn t CoA n
nhm sulfihidril ca protein mang acyl (ACP, acyl carrier) l mt protein nh
mang chui acid bo ang sinh trng trong tng hp. mi thi im synthetase
li thm 2 carbon vo u carboxyl ca chui acid bo ang sinh trng trong mt
qu trnh gm hai chng (Hnh 18.25). Trc ht, malonyl-ACP phn ng vi acyl-
ACP acid bo sn ra CO
2
v mt acyl-ACP acid bo c 2 carbon di hn. Vic
mt i CO
2
hng cho phn ng hon thnh. y ATP c dng b sung
CO
2
vo Acetyl-CoA to thnh malonyl-CoA. Cng CO
2
nh vy mt i khi
malonyl-ACP chuyn cc carbon cho chui. Do CO
2
l cn thit cho tng hp
acid bo nhng khng phi lun lun c c nh. Trn thc t, mt s vi sinh vt
cn CO
2
sinh trng tt nhng chng vn c th sinh trng thun li khi
khng c CO
2
m c mt mt acid bo nh acid oleic (mt acid bo 18 carbon
khng bo ho). Trong chng th hai ca tng hp nhm -keto xut hin t phn
ng ngng t ban u b loi i trong mt qu trnh ba bc bao gm hai s kh v
mt s loi nc. Sau acid bo sn sng cho vic b sung thm 2 nguyn t
carbon na.


Hnh 18.25: Tng hp acid bo. Chu trnh c lp li cho ti khi chiu di chui
thc s t c. ACP = acyl carrier protein (protein mang acyl). (Theo: Prescott v cs,
2005)
Cc acid bo khng bo ho c tng hp theo hai con ng. Cc t bo
nhn tht v vi khuNn hiu kh nh Bacillus megaterium s dng con ng hiu
kh vi s tham gia ca c N ADPH v O
2
.

Mt ni i to thnh gia cc carbon 9 v 10 v O
2
b kh thnh nc nh
cc electron do c acid bo v N ADPH cung cp. Cc vi khuNn k kh v mt s vi
khuNn hiu kh to ra cc ni i trong qu trnh tng hp acid bo bng cch loi
nc cc acid bo hydroxy. Oxy khng cn cho vic tng hp ni i theo cch
ny. Con ng k kh hot ng mt s vi khuNn gram m quen thuc (v d: E.
coli v Salmonella typhimurium), vi khuNn gram dng (v d: Lactobacillus
plantarum v Clostridium pasteurianum) v vi khuNn lam.

















Hnh 18.26: Tng hp triacylglycerol v phospholipid.
(Theo: Prescott v cs, 2005)
R (CH
2
)
9
C SCoA +NADPH +H
+
O
2

O
R CH CH (CH
2
)
7
C SCoA +NADPH
+
+2H
2
O
O
Cc vi sinh vt nhn tht thng d tr carbon v nng lng dng
triacylglycerol, glycerol c este ho vi 3 acid bo. Glycerol xut hin t s kh
dihydroxyacetone phosphate (l cht trung gian ca ng phn) thnh glycerol-3-
phosphate, sau glycerol-3-phosphate c este ho vi 2 acid bo cho acid
phosphateidic (Hnh 18.26). Phosphate b thu phn khi acid phosphateidic to
thnh diacylglycerol v acid bo th ba c gn vo sn ra mt triacylglycerol.
Phospholipid l thnh phn ch yu ca mng t bo nhn tht v hu ht t
bo nhn nguyn thu. Tng hp phospholipid cng thng din ra theo con
ng ca acid phosphateidic. Mt cht mang c bit-cytidine diphosphate
(XDP)-ng vai tr tng t vai tr ca cc cht mang ca uridine v adenosine
diphosphate trong sinh tng hp hidrat carbon. Chng hn, vi khuNn tng hp
phosphateidinetanolamine (mt thnh phn ch yu ca mng t bo) qua vic to
thnh XDP - diacylglycerol u tin (Hnh 18.26). Sau dn xut XDP ny phn
ng vi serine to thnh phospholipid phosphateidilserine v qua vic loi
carboxyl s xut hin phosphateidinetanolamine. Theo cch ny, mt lipid mng,
phc tp c to nn t cc sn phNm ca ng phn, sinh tng hp acid bo v
sinh tng hp acid amine.
18.9. TNG HP PEPTIDOGLYCAN
Hu ht thnh t bo vi khuNn cha mt phn t ln, phc tp bao gm cc
chui polisaccaride di to thnh bi cc nhnh lun phin acid N -Acetylmuramic
(N AM) v N -Acetylglucose semin (N AG). Gn vo cc nhnh N AM l cc chui
pentapeptide. Cc chui polisaccaride c lin kt vi nhau bi cc pentapeptide
hay bi cc cu ni gian - peptide phc tp ca peptidoglycan, d nhin, cng i
hi mt qu trnh sinh tng hp phc tp, c bit cn v cc phn ng tng hp
din ra c bn trong v bn ngoi mng t bo. Tng hp peptidoglycan l mt
qu trnh nhiu bc v c nghin cu chi tit vi khuNn gram dng
Staphylococcus aureus. y c s tham gia ca hai cht mang l uridine
diphosphate (UDP) v bactoprenol (Hnh 18.27). Bactoprenol l mt alcohol 55
carbon gn vo N AM bi mt nhm pyrophosphate v vn chuyn cc thnh phn
ca peptidoglycan qua mng k nc.
CH
3
C
CH
3
CH CH
2
(CH
2
C CH CH
2
)
CH
3
CH
2
C
CH
3
CH CH
2
O P
O
O
-
P
O
O
-
O NAM

Hnh 18.27: Bactoprenol pyrophosphate. Ch : Cht ny c gn vo NAM. (Theo:
Prescott v cs, 2005)
Tng hp peptidoglycan (Hnh 18.28 v 18.29) din ra qua 8 bc:
1. Cc dn xut UDP ca acid N .Acetylmuramic v N -Acetylglucosamine
c tng hp trong t bo cht.
2. Cc amino acid ln lt c thm vo UDP-N AM to thnh chui
pentapeptide (2 D-alanine tn cng c thm vo dng dipeptide). N ng lng
ca ATP c dng sn ra cc lin kt peptide nhng khng c s tham gia
ca tRN A v riboxom.
3.












HHnh 18.28: Tng hp peptidoglycan. Ch : pentapeptide cha L-lysine
peptidoglycan ca S. aureus v acid diamineopimelic (DAP) peptidoglycan ca E. coli. Tc
dng km hm ca bacidraxin, xicloserine v vancomixin c ch r. Cc con s tng ng vi
6 trong 8 bc ni trong bi. Bc 8 c m t hnh 18. 29. (Theo: Prescott v cs, 2005)
4. N AM-pentapeptide c chuyn t UDP sang phosphate ca bactoprenol
b mt ca mng.
5. UDP-N AG b sung N AG vo N AM-pentapeptide to thnh n v lp li
ca peptidoglycan. N u mt cu ni pentaglycine l cn thit cc glycine s c
thm vo bng cch s dng cc phn t glycyl-tRN A c bit nhng khng cn
riboxom.
6. Sau khi hon thnh n v lp li ca peptidoglycan N AM-N AG c
chuyn qua mng n b mt bn ngoi nh cht mang bactoprenol
pyrophosphate. n v peptidoglycan c gn vo u ang sinh trng ca mt
chui peptidoglycan ko di chui mt n v lp li.
7. Cht mang bactoprenol tr li bn trong mng. Trong qu trnh ny mt
phosphate c tch ra cho bactoprenol phosphate gi li c th nhn mt
N AM-pentapeptide khc.
8. Cui cng, cc lin kt peptide cho gia cc chui peptidoglycan c
to thnh nh phn ng chuyn peptide (Hnh 18.29). E. coli nhm amineo t
do ca acid diamineopimelic lin kt vi D-alanine gn tn cng tch ra nhnh D-
alanine tn cng. ATP c dng to thnh lin kt peptide tn cng bn trong
mng. Khi s chuyn peptide din ra bn ngoi nng lng ca ATP l khng cn
na. Qu trnh nh vy cng c thc hin khi c s tham gia ca mt cu ni;
ch nhm phn ng vi D-alanine gn tn cng l khc.


Hnh 18.29: Chuyn peptide
Cc phn ng chuyn peptide trong vic to thnh peptidoglycan E. coli v S. aureus.
(Theo: Prescott v cs, 2005)
Tng hp peptidoglycan rt mn cm vi cc tc nhn khng khuNn. S km
hm bt k bc no trong tng hp u lm cho thnh t bo b yu i v c th
dn n ph v t bo do thNm thu. N hiu cht khng sinh nh hng n tng
hp peptidoglycan. Chng hn, penixilin km hm phn ng chuyn peptide (Hnh
18.29) v bacitraxin c ch vic loi phosphate khi bactoprenol pyrophosphate.
18.10. CC KIU TNG HP THNH T BO
sinh trng v phn chia c thun li t bo vi khuNn phi b sung
peptidoglycan mi vo thnh t bo ca mnh mt cch chnh xc v c iu ho
cNn thn trong khi vn duy tr c hnh dng v tnh nguyn vn ca thnh nu
phi tn ti iu kin p sut thNm thu cao. V peptidoglycan ca thnh l mt
mng li khng l duy nht nn vi khuNn ang sinh trng phi c kh nng phn
gii li sao cho cung cp cc u nhn dng lp vo cc n v
peptidoglycan mi. S phn gii peptidoglycan hn ch ny c xc tc bi
enzyme gi l autolyzin, trong mt s autolyzin tc dng ln cc chui
polisaccaride, s khc thu phn cc lin kt cho peptide. Cc cht km hm
autolyzin iu ho cht ch hot tnh ca cc enzyme ny.
Mc d v tr v s phn b ca hot tnh tng hp thnh t bo thay i tu
theo loi nhng c l tn ti hai kiu ph bin (Hnh 18.30) sau y.

Hnh 18.30: Cc kiu tng hp thnh
Trong hnh l cc kiu tng hp thnh t bo cc vi khun ang sinh trng v phn
chia (a) Cc streptococci v mt s cocci khc gram dng. (b) Tng hp cc vi khun hnh
que (E. coli, Salmonella, Bacillus). (Theo: Prescott v cs, 2005)

Vng vch ngn
N hiu cu khuNn Gram dng (Enterococcus faecalis v Streptococcus) ch
c mt ti mt vi vng sinh trng. Vng sinh trng chnh thng v tr to
thnh vch ngang v cc na t bo mi c tng hp gip lng nhau. Kiu tng
hp th hai gp cc trc khuNn E. coli, Salmonella v Bacillus. Tng hp
peptidoglycan mnh m din ra v tr to thnh vch ngn ngang nh trn nhng
cc v tr sinh trng cng nm ri rc dc theo phn hnh tr ca trc khuNn. Do
sinh trng c phn b trc khuNn trn lan hn cu khuNn. Vic tng hp
phi ko di cc t bo hnh que cng nh phn ct chng. C l iu ny gii
thch cho nhng s khc nhau trong kiu sinh trng ca thnh.