VI Sinh Vat Hoc

Vi sinh vat hoc Nguyen Lan Dung
VI SINH VT HC
GS Nguyn Ln Dng ch bin Vi hng ni u Lc s ngnh Vi sinh vt Nhng c im chung ca Vi Sinh Vt Cu trc t bo Vi khun Cc nhm Vi khun ch yu X khun C khun Phng php thc nghim dng nh tn cc loi vi khun Virus Vi nm (Microfungi) Nm si (Filamentous Fungi) Nm men Vi to 1 (Microalgae) Vi to 2 Nm m Blazei - sn xut v ng dng Phn loi vi sinh bng Sinh Hc Phn T Gii thiu mt s k thut bo qun vi sinh vt http://vietsciences.free.fr v http://vietsciences.or
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. 2
Vi sinh vat hoc Nguyen Lan Dung 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). 3
Vi sinh vat hoc Nguyen Lan Dung 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). 4
Vi sinh vat hoc Nguyen Lan Dung 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)
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), 7
Vi sinh vat hoc Nguyen Lan Dung 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 8
Vi sinh vat hoc Nguyen Lan Dung 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
10
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 Nhn c mng nhn v
Bacteria Khng Khng 11
Archaea C
Eukarya
Vi sinh vat hoc Nguyen Lan Dung hch nhn Phc hp bo quan c Khng mng Hu ht c peptidoglycan Thnh t bo cha acid muramic Cha lin kt este, cc acid Mng lipid bo mch thng Ti kh C Thymine c trong phn ln tARN ARN vn chuyn tARN m u cha Nformylmethionine mARN a cistron Intron trong mARN Ghp ni, gn m v gn ui polyA vo mARN Kch thc Yu t ko di EF2 Mn cm vi cloramphenicol v kanamycin Mn cm vi anisomycin C Khng Khng
Khng Nhiu loi khc nhau, khng cha acid muramic Cha lin kt ete, cc chui aliphatic phn nhnh C Khng c thymine trong nhnh T hoc TyC ca tARN tARN m u cha methionine C Khng Khng
C Khng cha acid muramic Cha lin kt este, cc acid bo mch thng Khng C thymine tARN m u cha methionine
Khng C C
Ribosom 70S 70S Khng phn ng vi c t C phn ng bch hu Mn cm Khng Khng Mn cm ARN polymerase ph thuc ADN S lng enzym Mt Cu trc 4 tiu n v Mn cm vi rifampicin Mn cm Promoter typ Polymerase II Khng Tng t ATPase Sinh methane C nh N2 Quang hp vi dip lc Ho dng v c Khng Khng C C C Mt s 8-12 tiu n v Khng C Trao i cht C C C Khng C C Khng Khng C Khng Ba 12-14 tiu n v Khng C 80S (ribosom t bo cht) C phn ng Khng Mn cm
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 12
Vi sinh vat hoc Nguyen Lan Dung 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 1cm3 th chng c din tch b mt rng ti ...6 m2 !
13
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
14
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 15
Vi sinh vat hoc Nguyen Lan Dung 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 1300C, lnh n 0-50C, 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).
16
Vi sinh vat hoc Nguyen Lan Dung 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.
17
Vi sinh vat hoc Nguyen Lan Dung Vt tch vi khun lam cch Vt tch Gloeodiniopsis cch y 1,5 Vt tch Palaeolyngbya cch y 950 y 3,5 t nm t nm triu nm
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 :
Thnh phn Peptidoglycan Acid teicoic (Teichoic acid) Lipid Protein
Gram dng Gram m T l % i vi khi lng kh ca thnh t bo 30-95 5-20 Cao 0 Hu nh khng c 20 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) 18
Vi sinh vat hoc Nguyen Lan Dung -Acid N-Acetylmuramic (N-Acetylmuramic acid, NAM) -Tetrapeptid cha c D- v L- acid amin
19
Thnh t bo vi khun Gram dng
20
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
21
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 22
Vi sinh vat hoc Nguyen Lan Dung 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) 23
Vi sinh vat hoc Nguyen Lan Dung 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 3.109, cha 4,6.106 cp base nit. Th nhn l b phn cha ng thng tin di truyn ca vi khun.
24
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...)
25
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
26
Vi sinh vat hoc Nguyen Lan Dung 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 10-20 v c ng m 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
27
Tin mao VK Gram dng
Tin mao VK Gram m
28
Tin mao vi khun G +
29
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 : 30
Vi sinh vat hoc Nguyen Lan Dung -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.
31
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
32
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 22,5nm), 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. 33
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. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. -Aquificae -Thermotogae -Thermodesulfobacteria -Deinococcus-Thermus -Chrysiogenetes -Chloroflexi -Nitrospirae -Defferribacteres -Cyanobacteria -Proteobacteria -Firmicutes -Actinobacteria -Planctomycetes -Chlamydiae/Nhm Verrucomicrobia -Spirochaetes -Fibrobacteres /Nhm Acidobacteria -Bacteroidetes/Nhm Chlorobia -Fusobacteria -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. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. -Nhm Oxy ho Hydrogen -Nhm Chu nhit -Nhm Vi khun khng lu hunh mu lc -Nhm Deinococcus -Nhm Vi khun lam -Nhm Proteobacteria -Nhm Chlamydia -Nhm Planctomyces -Nhm Spirochaetes (Xon th) -Nhm Vi khun lu hunh mu lc -Nhm Cytophaga --Nhm Vi khun Gram dng
34
35
Vi sinh vat hoc Nguyen Lan Dung 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 H2O 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: aNhm I (c tc gi gi l b Chroococcal):
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
36
Chamaesiphon
Chroococcus
Glooeothece
Gleocapsa
37
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
38
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 3467%. Cc chi tiu biu l:
-Lyngbya
-Osscillatoria
-Prochlorothrix
-Spirulina
-Pseudanabaena
39
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 40
-Aphanizomenon
-Nostoc
-Scytonema
-Calothrix
Anabaena
Anabaena trong Bo hoa du
41
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 42
-Geitlerinema
Fischerella
Geitlerinema
Theo NCBT (2005) th Vi khun lam bao gm nhng b sau y:
43
-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
44
Vi sinh vat hoc Nguyen Lan Dung 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
45
Vi sinh vat hoc Nguyen Lan Dung 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
46
Vi sinh vat hoc Nguyen Lan Dung 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
47
Vi sinh vat hoc Nguyen Lan Dung 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
AB-
Catalase dng tnh, khng di ng hay di ng nh tin mao cc Thng di ng nh tin mao cc, khng k sinh ng vt c xng sng 48
Vi sinh vat hoc Nguyen Lan Dung 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 49
Vi sinh vat hoc Nguyen Lan Dung 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
50
Pasteurella
Pasteurella
Enterobacter
Morganella
Salmonella
Salmonella
Yersinia
Citrobacter
Serratia
Serratia
Klebsiella
Klebsiella 51
Escherichia
4-Trc khun Gram m , khng ln men , hiu kh hoc k kh khng bt buc
A- Sinh trng c 60C 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 60C B-C th oxy ho etanol thnh axit axetic C-C th oxy ho etanol ti CO2 v H2O D-C th oxy ho acid DL-lactic ti CO2 v H2O 4.5- Chi Acetobacter DD-Khng oxy ho acid DL-lactic ti CO2 v H2O 4.6- Chi Acidomonas CC-Khng th oxy ho ethanol ti CO2 v H2O D-C th oxy ho acid DL-lactic ti CO2 v H2O 4.7- Chi Frateuria DD-Khng oxy ho acid DL-lactic ti CO2 v H2O 52
Vi sinh vat hoc Nguyen Lan Dung 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 53
Vi sinh vat hoc Nguyen Lan Dung 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 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 54 ngun carbon duy nht
Vi sinh vat hoc Nguyen Lan Dung 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 55
Vi sinh vat hoc Nguyen Lan Dung 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.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 520C 4.35- Chi Hydrogenophaga LL- Sinh trng 520C 4.36- Loi Ps. hydrogenothermophila KK- Khun lc khng mu vng 4.36- Chi Pseudomonas (P. saccharophila, P.facillis, P.hydrogenovora) 56 (P.stanieri, P.perfectomarina,
Vi sinh vat hoc Nguyen Lan Dung 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 57
Vi sinh vat hoc Nguyen Lan Dung 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
58
Thermus
Gluconobacter
Azotobacter
Methylobacterium
Bordetella
Rhizobium 59
v nt sn trn r
Sinorhizobium
v nt sn trn r
Methylococcus
Bradyrhizobium
v nt sn trn r
Agrobacterium
v nt sn
Pseudomonas
60
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 61
Vi sinh vat hoc Nguyen Lan Dung 5.4- Chi Veillonella
Megasphaera
Acidaminococcus
Veillonella
6-Trc khun, phy khun, xon khun Gram m, k kh :
I-
Sinh trng nhit 550 C A-Khng ln men v ng ho carbohydrat , s dng peptid, aminoacid, cao nm men... B- T bo c bao, khng kh Fe3+. 6.1- Chi Thermosipho BB- T bo khng c bao C-S dng cc loi acid hu c v H2 kh Fe3+ 6.2- Chi Deferribacter CC-Khng kh Fe3+ 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, H2/CO2 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, H2/CO2 6.5- Chi Thermotoga 62
Vi sinh vat hoc Nguyen Lan Dung 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 B12 6.6- Chi Acetothermus DD-Thnh t bo khng in hnh Gram m, cn H2S 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 H2/CO2 v hn hp acid F-Sn phm ln men gm acid acetc, ethanol, H2/CO2 6.10- Chi Thermohydrogenum FF-Sn phm ln men gm acid acetic v ethanol, hoc acid acetic v H2/CO2 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 550 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 H2 /CO2 6.13- Chi Syntrophobacter 63
Vi sinh vat hoc Nguyen Lan Dung CC-Phy khun, sn phm phn gii acid bo l acid acetic, acid propionic, acid isobutiric, acid isopentanoic v H2//CO2 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 H2/CO2 B-Acid acetic l sn phm ln men duy nht C- Di ng nh chu mao 6.21- Chi Acetoanaerobium 64
Vi sinh vat hoc Nguyen Lan Dung 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 ESn 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 H2/CO2 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 65
Vi sinh vat hoc Nguyen Lan Dung EE- T bo hnh cong, sinh kh 6.31- Chi Lachnospira DD- Sn phm ln men l acid lactic, acid acetic v acid succinic ET 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 H2/CO2 C-Khng di ng, a mn, cn NaCl 1% D- Ch sn sinh acid propionic v CO2 6.35- Chi Propionigenium DD- Ngoi acid propionic v CO2 cn c cc acid khc E- Sn sinh acid propionic, CO2 ,acid acetic v ethanol 6.36- Chi Pelobacter EE- Sn sinh acid propionic, CO2 , 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 66
Vi sinh vat hoc Nguyen Lan Dung 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 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 67 acetic
Vi sinh vat hoc Nguyen Lan Dung 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 CO2 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 CO2 6.53- Chi Oxalobacter CC-Sn sinh acid formic v cc acid khc nhng khng sinh CO2 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 68
Vi sinh vat hoc Nguyen Lan Dung 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 CO2 6.56- Chi Malonomonas EE- Sn sinh acid succinic, acid acetic, khng sinh CO2 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 FT 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
69
Thermosipho
Syntrophobacter nui cy chung vi Methanobrevibacte
Wolinella
Porphyromonas gingivalis
vt cy trn thch mu
Leptotrichia
Bacteroides 70
Bacteroides
Fusobacterium
Fusobacterium
Mitsuokella
7- Cu khun Gram dng hiu kh v k kh khng bt buc A- Hiu kh B-Di ng 71
Vi sinh vat hoc Nguyen Lan Dung 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 72
Vi sinh vat hoc Nguyen Lan Dung 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 10C G- Sinh kh t glucose 7.13- Chi Leuconostoc GG- Khng sinh kh t glucose H- Sinh trng 45C 7.14- Chi Enterococcus HH- Khng sinh trng 45C I-Di ng, sng trong nc 7.15- Chi Vagococcus II- Khng di ng 7.16- Chi Lactococcus FF- Khng sinh trng 10C G-Lng cha G + C ca ADN>35mol % 7.17- Chi Streptococcus GG-Lng cha G + C ca ADN< 35mol % 7.18- Chi Gemella 73
Micrococcus
Deinococcus
Deinococcus
Melissococcus
Melissococcus
Khun lc Stomatococcus
Staphylococcus
Staphylococcus 74
Pediococcus
Aerococcus
Leuconostoc
Leuconostoc
Enterococcus
Lactococcus
Lactococcus
75
Vi sinh vat hoc Nguyen Lan Dung 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 (H2), MK-7 (H2) 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
76
Vi sinh vat hoc Nguyen Lan Dung 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 EHnh 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
77
Vi sinh vat hoc Nguyen Lan Dung 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
78
Vi sinh vat hoc Nguyen Lan Dung EE- Khng di ng 8.24- Chi Rothia DD Peptidoglycan khng cha lysin EPhn 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 EThng 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)
79
Vi sinh vat hoc Nguyen Lan Dung 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
80
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 81
Vi sinh vat hoc Nguyen Lan Dung 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 CO2 F- Thch hp sinh trng 30C 9.6- Chi Acetobacterium FF-Thch hp sinh trng 38C 9.7- Chi Acetitomaculum EE- Ln men hydrat carbon sinh acid lactic, khng tng hp ra acid acetic t H2 v CO2 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 82 tng hp ra acid acetic t H2 v
Vi sinh vat hoc Nguyen Lan Dung 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 9.11AA- Gram (-) A- T bo hnh que, a nhit, khng di ng 9.12BB- 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 Chi Acetogenium Chi Coribacterium
Thermoanaerobacter di knh hin vi in t
Bifidobacterium
Eubacterium
83
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
84
Peptostreptococcus
Peptostreptococcus
Ruminococcus
X khun
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 85
Vi sinh vat hoc Nguyen Lan Dung 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. 86
Vi sinh vat hoc Nguyen Lan Dung 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.
87
Khun lc x khun
Khun ty x khun v bo t
Chui bo t trn cung sinh bo t dng xon
88
Bo t x khun
S hnh thnh hai loi khun ty sau khi bo t x khun ny mm
89
Si bo t v chui bo t trn
Mt s dng bo t x khun (SV t ch thch tng hnh) 90
Mt s dng nang bo v nang bo t x khun
91
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:
92
93
94
95
96
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 (%) 97
Vi sinh vat hoc Nguyen Lan Dung 1 2 3
9 5167 6572 6369 6465 68 73 6270 6472 6069 6673 7172 7273 7173 7173 6972 6973 71 69 70 5763 73 6668 68
H Corynebacteriaceae v mt s h lin quan Corynebacterium Turicella Gordonia Williamsia Skermania Dietzia Mycobacterium Nocardia Rhodococcus Tsukamurella IV IV IV IV IV IV IV IV IV IV A A A A A A A A A A A1 A1 A1 A1 A1 A1 + + + + + + + + + 1b 1b 1a 1b 1b 1b 1b 8(H2), 9(H2) 10,11 9(H2) 9(H2) 8(H4) 8(H2) 9(H2) 8(H4) 8(H2), 9(H2) 9 PII PII PII PII PII PII PII PII PI
H Micromonosporaceae Micromonospora Actinoplanes Cattelatospora Catenuloplanes Couchioplanes II II II VI VI D D D D D D D A3 A3 A1 A1 A1 A1 A1 3b 2d 2d 2b 3b 3b 9(H4.6), 10(H4,6) 9(H4,6), 10(H4,6) 9(H4,6), 10(H8,6) 10(H4), 11(H4) 9(H4) 9(H4,6,8) 9(H4,2) 10(H4) 9(H4) PII PII PII PIII PII PII PII PII PII
Dactylosporangium II Pilimelia Spirilliplanes Verrucosispora II II II
H Propionibacteriaceae v Nocardia Propionibacterium I Friedmaniella Luteococcus Microlunatus I I I A3 A3 A3 A3 1a 2d 9(H4) 9(H4) 9(H4) 9(H4) 98 PI PI PII
Vi sinh vat hoc Nguyen Lan Dung Propioniferax Tessaracoccus Nocardioides Aeromicrobium Hongia Kribbella Marmoricola I I I I I I I A3 A3 A3 A3 A3 A3 A3 1a 3a 3a 2d 9(H4), 7(H4) 8(H4) 9(H4) 9(H4) 9(H4) 9(H4) 8(H4) PI PI PI PII PI PIII PI PIII/ PII PIV PIII PII PII PII PII PIII PII 5963 74 6772 6972 71 6870 72 6879 71 6468 6669 66 6769 6974 7077 6973 6971 73 7071 7076
H Pseudonocardiaceae Pseudonocardia Actinobispora Actinopolyspora Amycolatopsis III IV IV III A A A A A A A A C A1 A1 A1 A1 A1 A1 3c 3f 2a 2c 2c 1 8(H4) 7(H2), 9(H2) 9(H4), 10(H4) 9(H2,4,6) 9(H4) 9(H2,4) 9(H4) 9(H4) 9(H4)
Kibdelosporangium III Prauserella IV
Saccharomonospora III Saccharopolyspora IV Thermocrispum III
H Actinosynnemataceae Actinosynnema Actinokineospora Kutzneria Saccharothrix III III III III C C C C A1 A1 A1 9(H4,6) 9(H4) 9(H4) 9(H4), 10(H4) PII PII PII PII
H Streptosporangiaceae Streptosporangium III Acrocarpospora Herbidospora Microbispora III III III B B B B A1 A1 A1 3c 3c 9(H0,2,4) 9(H2,4) 10(H4,6) 9(H0,2,4) 99 6971 68PIV/ PII 69 PIV 71 PIV 67PIV
Vi sinh vat hoc Nguyen Lan Dung Microtetraspora Nonomuraea Planobispora Planomonospora Planotetrespora III III III III III B B B B B A1 A1 A1 A1 3c 3c 3c 3c 3c 9(H0,2,4) 9(H0,2,4) 9(H0,2,4) 9(H0,2,4) PIV PIV PIV PIV PIV 74 66 6469 7071 72
H Nocardiopsaceae Nocardiopsis Thermobifida III III C C B B B A1 A1 A1 A1 3d 3c 3e 3e 3a 3e 10(H2,4,6) 10(H6,8) 9(H6,8) 9(H4,6) 9(H6,8) 9(H4,6) PIII PII PI PII PI PI 6469
H Thermomonosporaceae Thermomonospora III Actinocollaria III Actinomadura Spirillospora III III 73 6670 6971 7173 6978 6673 6671 7376 68 70 70 6479 71 5355
H Glycomycetaceae Glycomyces II 2c 10(H4), 11(H4) PI
H Streptomycetaceae Streptomyces Kitasatospora I I/III A3 A3 2c 9(H6,8) 9(H6,8) PII PII
H Frankiaceae v mt s h lin quan Frankia Geodermatophilus III III A1 A3 1 2b 9(H4,6,8) 9(H4) 8(H4) 9(H6,8) 9(H6,8) PI PII PII PI
Microsphaera III Cryptosporangium II Sporichthya I
Cc h khc Kineosporia Thermobispora I/III III A3 9(H4) 9(H0,2) 7,9 100 PIII PIV
Thermoactinomyces III
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 I II III IV V VI VII VIII
Acid diamino Cc acid amin khc LL-A2pm meso- A2pm meso- A2pm meso- A2pm Lys, Orn Lys Lys, A2bu Gly* Gly**
ng
Ara, Gal*** Asp Asp Asp
Tt c cc loi thnh t bo u cha alanine, acid glutamic, glucosamine v acid muramic. A2pm: Acid diamino pimelic c th gm cc ng phn quang hc LL- A2pm, DL- A2pm (mesoA2pm), DD- A2pm. Ngoi ra cn c OH- A2pm khi c thm nhm OH gn vo v tr C th 4. Lys: Lysine Orn: Ornitin A2bu: 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
101
Vi sinh vat hoc Nguyen Lan Dung 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 A B C D Cc loi ng ch yu Arabinose, Galactose (khng cha Xylose) Madurose (khng cha Arabinose hay Xylose) Khng c ng c trng Arabinose, Xylose
Peptidoglycan: Theo Schleifer v Kandler (1972), peptidoglycan x khun c chia thnh cc nhm nh sau: V tr ca K mi lin Cu ni gia hai mch tetrapeptid hiu kt 3-4 1 Trc tip
K hiu A
K Acid amin hiu v tr th 3 L-Lys L-Orn meso- A2pm L-Lys L-Lys L-Orn meso- A2pm L-Lys L-Orn meso- A2pm L-A2bu L-Lys L-Hsr L-Glu L-Ala L-Orn
2 3
2-4
Tiu n v peptid L-acid amin cha mt nhm carboxyl (gly) hoc chui peptid ngn ca acid amin D-acid amin cha hai nhm carboxyl (D-Asp hay D-Glu) L-acid amin (Glyn-L-Lys) D-acid amin (D-Orn hay D-A2bu) 102
Vi sinh vat hoc Nguyen Lan Dung L-Hsr L-A2bu
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- A2pm. Thnh phn acid bo: 10Methyl- Cyclopropane 18 ++ +++ (+) (+) +++ ++ -
K hiu 1 a b c a b c d a b c d
Khng IsoIsoBo bo ha 14/16/18 15/17 ha +++ +++ +++ ++ (+) + + +++ + + + +++ +++ +++ + + (v) + ++ + + + +++ ++ +++ +++ +++ +++ ++ +++ + +++ + +++ (+) +++ + ++
Anteiso15/17 (+) + +++ +++ (+) ++ + +++
10Methyl-17 (+) ++ +++ (+)
(v): t hn 5%
Thnh phn menaquinone: X khun ch cha menaquinone, khng cha ubiquinone. K hiu 8(H2) hay MK-8(H2) ch menaquinone cha mt trong s tm n v isoprene b hydro ha. Thnh phn phospholipid: K Thnh phn phospholipid c trng hiu PI Phosphatidylglycerol(v) PII Phosphatidylethanolamine 103
Vi sinh vat hoc Nguyen Lan Dung Phosphatidylcholine, phosphatidylethanolamine(v), phosphatidylmethylPIII ethanolamine(v), phosphatidylglycerol(v), khng cha phospholipid cha glucosamine Phospholipid cha glucosamine, phosphatidylethanolamine(v), PIV 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 104
Vi sinh vat hoc Nguyen Lan Dung Cc chi: Dermacoccus, Demetria, Kytococcus H Dermatophilaceae Chi: Dermatophilus H Intrasporangiaceae Cc chi: 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 105 Intrasporangium, Janibacter, Terrabacter, Terracoccus,
Vi sinh vat hoc Nguyen Lan Dung 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, 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 106 Luteococcus, Microlunatus,
Vi sinh vat hoc Nguyen Lan Dung 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 107
Vi sinh vat hoc Nguyen Lan Dung Cc chi: Bifidobacterium, Gardnerella Di lp Acidimicrobidae B Acidimicrobiales H Acidimicrobiaceae Chi: Acidimicrobium Di lp Coriobacteridae B Coriobacteriales H Coriabacteriaceae Cc chi: Coriobacter, Atopobium, 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 oC, 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. Collinsella, Cryptobacterium,
108
Vi sinh vat hoc Nguyen Lan Dung 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(H2) v MK-9(H2). 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(H4). 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:
109
Vi sinh vat hoc Nguyen Lan Dung 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 28-37oC, khng mc c 45oC. 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(H4). Hiu kh. Ha d dng hu c. Nhit thch hp cho sinh trng l 35-50oC, 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 110
Vi sinh vat hoc Nguyen Lan Dung 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 20-30oC. 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 oC. T l mol GC trong ADN l 71-73%. 111
Vi sinh vat hoc Nguyen Lan Dung 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(H4), MK10(H4), MK-10(H6) v MK-12(H6). 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 20-40oC. 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 oC, 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(H6), MK-9(H8). 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 oC. 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(H4) v MK-9(H6). T l mol GC trong ADN l 65-69%. Loi chun: Actinomadura madurae
112
Vi sinh vat hoc Nguyen Lan Dung 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(H4). 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 oC, 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 90oC 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(H4) v MK-9(H6). Gram dng, khng acid fast, sinh 113
Vi sinh vat hoc Nguyen Lan Dung 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(H2,4,6) hoc MK-9(H4,6). 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
114
Mt s loi Streptomyces
115
Mt s chi x khun: A-Microtetraspora; B- Planomonospora; C- Actinosynnema; D- Actinobispora; ESaccharothrix; F- Crosiella
116
Chi Actinomadura
117
Cc chi: A- Micromonospora; B- Dactylosporangium; C- Verrucosispora D- Pilimelia; E- Catenuplanes
118
Chi Actinoplanes
Loi Actinomyces viscosus
119
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:
120
Vi sinh vat hoc Nguyen Lan Dung Mi trng HV (Humic acid-vitamin agar) Thnh phn: Acid humic CaCO3 FeSO4.7H2O KCl MgSO4.7H2O Na2HPO4 Hn hp vitamin nhm B* Cycloheximide Thch Nc ct pH 1g 0,02 g 0,01 g 1,71 g 0,05 g 0,5 g 5 ml 500 mg 18 g 1L 7,2
*Hn hp vitamin nhm B Thiamine-HCl Riboflavin Niacin Pyridoxin-HCl Inositol Ca-Pantothenate Acid p-Aminobenzoic Biotin Nc ct 10 mg 10 mg 10 mg 10 mg 10 mg 10 mg 10 mg 10 mg 10 ml
Mi trng YS (Yeast extract-Soluble starch agar) Cao nm men Tinh bt tan Thch Nc ct pH 2g 10 g 18 g 1L 7,3
Thng s dng mi trng YS pha long 5 ln phn lp x khun v pha long 2 ln nghin cu. 121
Vi sinh vat hoc Nguyen Lan Dung Mi trng Maltose-Bennette Cao nm men Cao tht NZ amine Maltose monohydrat Thch Nc ct pH 1g 1g 2g 10 g 18 g 1L 7,3
Mi trng ISP-2
Cao nm men Cao malt Glucose Thch Nc ct pH
4g 10 g 4g 18 g 1L 7,2
Mi trng dch th YG (Yeast extract-Glucose) Yeast extract Glucose 1% 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 28oC trong 1,5 gi 122
Vi sinh vat hoc Nguyen Lan Dung 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 28oC 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 40oC trong 20 pht Pha long bng nc ct v trng Tri trn mi trng HV Nui cy 28oC 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 100oC trong 30 pht Rc mt lng rt nh mu ln mi trng HV Nui cy 28oC trong 7-14 ngy
Mt s phng php ha phn loi x khun: Chun b thnh t bo 123
Vi sinh vat hoc Nguyen Lan Dung 1. T bo t (1-2 ml) + 5ml nc ct v trng + ht thy tinh (loi ln v nh, ng knh 0.110.12mm). 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 4oC. 2. 3. 4. 5. 6. 7. 8. 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. Thm 3ml SDS 4% (SDS c tc dng loi lipid v protein). Nu dng cng vic y, gi mu nhit phng. Gi 100oC trong 40 pht, nh vn cht np. Ly tm vi tc 17000 rpm trong 30 pht 30oC (nu gi nhit thp, SDS s b kt tinh). Loi b dch trn. Ra vi nc t nht 3 ln vi iu kin ging trn (17000rpm, 30 pht) Nu cn thit, x l bng RNase v DNase. ng kh qua m.
Phn tch thnh phn acid amin trong thnh t bo 1. 2. 3. 4. 2-3mg thnh t bo + 200l HCl 4N, vn cht np, gi 100oC qua m Lm kh bng ht chn khng (khong 1 ngy 1 m) Thm 200 l nc ct, trn u, chuyn sang ng eppendorf, ly tm vi tc 12000 rpm trong 5 pht. Lc mu. Chy sc k bn mng TLC: Dung mi: Methanol Nc ct 6N HCl Pyridine 80ml 17.5ml 4ml 10ml
Cellulose TLC Chun (1l): DAP, Ala, Gly, Glu, Lys Mu: 3-5l
124
Vi sinh vat hoc Nguyen Lan Dung Nhn bit bng phun ninhydrin, gi 100oC 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% CH3CN, dung dch B: 60% CH3CN Tc dng chy: 1ml/pht
Chng trnh Thi gian (pht) 0 15 15.1 25 25.1 30 Nng dung dch B(%) 0 70 100 100 0 Dng
Phn tch thnh phn menaquinone 1. 2. 3. 100-500mg t bo kh + 10-15 ml Chloroform: methanol (2:1), trn bng khuy t chm trong 2-3 gi. Chuyn sang ng falcon, ly tm 3000 rpm trong 5-10 pht, ly dch trn, lm kh bng c quay Ha tan bng 1,5 ml acetone, chuyn sang ng eppendorf, lm kh bng ht chn khng 125
Vi sinh vat hoc Nguyen Lan Dung 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. Co vch c nhn bit bng UV, cho vo ng eppendorf, thm 1,5ml acetone, trn u. Ly tm vi tc 3000 rpm trong 5 pht, ly dch trn, lm kh bng Nit lng. Thm 50-100 l ethanol, lc v dng lm mu chy HPLC v Mass spectrometer (MS) iu kin chy HPLC
6. 7. 8. 9.
- Dung mi: Methanol: Propanol (2:1) Tc dng chy: 0.2ml/min Nhit ct: 40oC 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. 2. 3. 4. 5. 30-50mg t bo kh + 1ml H2SO4 1N, lc nh, vn cht nt, gi 100oC trong 2 gi. ngui, chuyn sang cc thy tinh, chnh pH 5.2-5.5 bng dung dch Ba(OH)4 bo ha. 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 Ha tan bng 300 l DW, chuyn sang ng eppendorf, ly tm vi tc 3000rpm trong 5 pht, ly dch trn, lc mu. Chy sc k bn mng TLC: TLC cellulose 3 l mu 1 l chun 126
Vi sinh vat hoc Nguyen Lan Dung 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 100oC 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 127
Vi sinh vat hoc Nguyen Lan Dung Nhit : 65oC Phn ng nhn bit: Dung dch C: 5g arginine + 15g acid boric trong 500ml DW Tc dng chy: 0,5ml/min Nhit : 150oC Bc sng: Ex=320nm, Em=430nm Chng trnh Thi gian (pht) 0 50 100 65 Nng Sol B 0 100 57.1 dng 0 Dung dch C: 0.2ml/min 57
Phn tch thnh phn acid bo 1. 5mg t bo kh + dung dch kim (R1) 1ml, vortex 5- 10 sec, gi 100oC trong 5min, vortex 5-10 sec, gi tip 100oC trong 25min, ngui xung nhit phng, c th t trc tip di vi nc sau khi h xung 80oC Thm dung dch methyl ha (R2): 2ml, gi 80oC trong 10min, ngui xung nhit phng. Qu trnh ny thy xut hin khi trng Thm dung dch tch (R3) 3ml, o ln u ui nh nhng trong10min, loi b lp di (lp nc) 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) 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.
2. 3. 4. 5.
Ch : Cc bc cn thc hin lin tc, trc khi tin hnh cn chun b heatblock 100 and 80oC.
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 128
Vi sinh vat hoc Nguyen Lan Dung R5: 100ml MQ + 40g NaCl
6. Mu c phn tch bng sc k kh (h thng MIDI)
Phn tch thnh phn phospholipid 1. 2. 3. 4. 5. 6. 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. 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. Lm kh bng Nit lng. Nu ngng cng vic, gi -20oC. Ha tan bng 100 l ethanol. Chm mu ln 4 bn HP-TLC (high performance) 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 p-anisaldehyde H2SO4 Ethanol 90ml 1ml 5ml 5ml
Phun thuc th, gi 110oC trong 15 pht Cc phospholipid cha ng nh GlcNU cha NPG v PIMs, glycolipit s hin ln di dng cc chm mu vng xanh.
129
Vi sinh vat hoc Nguyen Lan Dung Bn 2: Nhn bit cc amino lipit Phun ninhydrin ln bn TLC. Gi 120oC 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 H2SO4 100mg MoO3 4,011g
Ha tan bng cch un 90oC n khi dung dch tr nn trong SolII Iot Mo 50mg 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: 1,7g 20ml 80ml 10g 25ml
Sol A: Basic bismuth nitrate [Bi(OH)2NO3] Acetic acid Water Sol B: KI Nc
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: 130
Vi sinh vat hoc Nguyen Lan Dung 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. 2. 3. 4. 25l ADN (2-25g), gi 60oC trong 1 gi. Gi 100oC trong 2 pht. Lm lnh lp tc trong chu , spin down. Mu ADN 25l + m acetate (40mM, pH 5,3) cha 2mM ZnSO4 25l + dung dch nuclease P1 (20U/ml) 25l. Spin down. Gi 37oC trong 1 gi. Glycine buffer (0,1 M, pH 10,4) 25 l + alkaline phosphatase 2l (0.35U/l). Spin down. Gi 37oC trong 1-2 gi. S dng lm mu chy HPLC iu kin HPLC: Ct Cosmosil5 C18 Nhit 35oC 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 28oC 131
Vi sinh vat hoc Nguyen Lan Dung Ly 1,5 ml dch nui cy, ly tm vi tc 16000g trong 5 pht 4oC, 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 37oC 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 C trong 30 pht
o
Thm 400 l phenol: chloroform: isoamylalcohol (25:24:1), trn u, ly tm vi tc d 16000g trong 10 pht 4oC, 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 T1, gi 37oC trong 20 pht Thm 50 l proteinase K, gi 37oC trong 1 gi Thm 400 l phenol: chloroform: isoamylalcohol (25:24:1), trn u, ly tm vi tc d 16000g trong 10 pht 4oC, 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. 132
Vi sinh vat hoc Nguyen Lan Dung 2. Miyadoh S. (ed.). 2001. Identification manual of actinomycetes. Business Center for Academic Societies Japan. 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 (9th edition). Lippincott Williams & Wilkins 3.
C khun (Archaea)
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.
133
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 Sinh vt nhn tht c im Vi khun (Bacteria) C khun(Archaea) (Eukarya) Pseudo-peptidoglycan, protein, cellulose, carbonat, Thnh t bo Peptidoglycan polysaccharid, glycoprotein silicat, chitin Mng t bo Este-lipid Ete-lipid Este-lipid Ch c mt loi C nhiu loi C ba loi ARN polymeraza (trn khun ADN) 4 n v 2 7 12 n v 7 12 n v Ribosom 70 S 70 S 80 S Phn ng ca ribosom khng Mn cm Mn cm vi c t bch hu
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 134
Vi sinh vat hoc Nguyen Lan Dung 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 (C20cacbuahydro tng hp t isopren) v biphytanyl (C40). 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 2 1 1 v mt nhn t 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 Hnh 2. Lipid trong mng t bo ca c thng tin (mARN) sinh vt nhn tht gm 10-12 chui khun (ete-lipid) khc vi ca vi khun v sinh polypeptid c kch thc tng t nh ARN-polymeraza ca vt nhn tht (este-lipid) 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 135
Vi sinh vat hoc Nguyen Lan Dung 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. 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 109 Da, trong khi ADN ca Thermoplasma acidophilum l 0,8 x 109 Da, hay ca Methanobacterium l 1,1 x 109 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 Sulfolobus + Vi khun Sinh vt nhn tht Escheri- Saccharomyces chia coli cerevisae + + + + + +
Methanobacterium Cycloheximid c ch bc khi u Virginiamycin, c ch bc ko + pulvomycin di Neomycin, Dng tng hp sm + puromycin c ch enzyme Rifamycin ARN polymeraza Erythromycin, Tng tn s mc li streptomycin, v mt s hiu ng chloramfenicol 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 CO2 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 (LjungdahlWood). 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 O2, S0 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. 136
Vi sinh vat hoc Nguyen Lan Dung 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 CO2 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 CO2 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 CO2 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 di p sut cao nh cc ming ni la di C 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 Cn trng ng vt n bo To Nm Vi khun thng C khun 38 C 50 50 56 60 90 113
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 137
Vi sinh vat hoc Nguyen Lan Dung 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.
138
Vi sinh vat hoc Nguyen Lan Dung 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) 139
Vi sinh vat hoc Nguyen Lan Dung 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)
140
Vi sinh vat hoc Nguyen Lan Dung 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 CO2 hoc nhm methyl trong cc hp cht C1 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 C6H12O6 + 6 O2 6 CO2 + 6 H2O (G0 = 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 F420 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 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 4H2 + CO2 CH4 + 2H2O 4 Format CH4 + 3CO2 + 2H2O 4 Isopropanol + CO2 CH4 + 4 Aceton + 2H2O 2 Etanol + CO2 CH4 + 2 Acetat Metanol + H2 CH4 + H2O 4 Metanol 3CH4 + CO2 + 2H2O 4 Methylamin + 2H2O 3CH4 + CO2 + 4NH4+ 2 Dimethylamin + 2H2O 3CH4 + CO2 + 2 NH4+ 4 Trimethylamin + 6H2O 9 CH4 + 3CO2 + 4 NH4+ 2 Dimethylsulfid + 2H2O 3CH4 + CO2 + H2S Acetat CH4 + CO2 Nng lng c gii phng 0 (kJ/ methane) G 135,6 130,1 36,5 116,3 112,5 104,9 75,0 73,2 74,3 73,8 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 CO2 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 141
Vi sinh vat hoc Nguyen Lan Dung 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 CO2, 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. Hnh 9 : Cng sinh gia Methanobrevibacter (t bo trc khun) v Synthrophobacter (t bo hnh oval). 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 Methanobacteriales -nt-nt-ntMethanococcales Methanomicrobiales -nt-nt-nt-nt-nt-nt-nt-nt-nt-ntH Methanobacteriaceae (T) -nt-ntMethanothermaceae Methanococcaeae (T) Methanosarcinaceae -nt-nt-nt-nt-nt-ntMethanomicrobiacaea (T) -nt-nt-nt142 Chi Methanobacterium (T) Methanobrevibacter Methanosphaera Methanothermus (T) Methanococcus (T) Halomethanococcus Methanococcoides Methanohalobium Methanohalophilus Methanolobus Methanosarcina (T) Methanosaeta (Methanothrix) Methanoculleus Methanogenium Methanolacinia Methanomicrobium (T)
Vi sinh vat hoc Nguyen Lan Dung -nt-nt-nt-nt-ntMethanocorpusculaceae T = h/ chi chun; -nt- nh trn
Methanoplanus Methanospirillum Methanocorpusculum (T)
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 H2 + CO2, mt s loi s dng.
143
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. 144
Vi sinh vat hoc Nguyen Lan Dung Soi knh: dng vt knh du 100 . Kt qu: Vi khun Gram (+) bt mu tm, Gram () bt mu .
Hnh 1.1. 1.2. Nhum tin mao Vt liu, ho cht:
Cc bc tin hnh nhum Gram v v d minh ho kt qu.
Dung dch A: Acid tannic FeCl3 Formalin NaOH 1% Nc ct
5g 1,5 g 2 ml 1 ml 100 ml
Dung dch B: 2 g AgNO3 ho tan trong 100 ml nc ct, ly 10 ml d ring. Nh dung dch NH4OH m c vo 90 ml cn li, thy hnh thnh ta rt c, tip tc nh NH4OH vo cho n khi tan ht ta. Ly 10ml AgNO3 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. 145
Vi sinh vat hoc Nguyen Lan Dung Cc bc tin hnh: Kt qu: T bo vi khun bt mu nu thm, tin mao bt mu nu. 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.
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: Kt qu: 146 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.
Vi sinh vat hoc Nguyen Lan Dung 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: 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 147 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
Vi sinh vat hoc Nguyen Lan Dung 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: Kt qu: Bo t bt mu , t bo bt mu xanh 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.
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: 148
Vi sinh vat hoc Nguyen Lan Dung Mc tu Metanol Dung dch safranin 0,5%
Cc bc tin hnh: kh. 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%. 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. 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
Cc bc tin hnh: Kt qu: 149 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.
Vi sinh vat hoc Nguyen Lan Dung 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 450 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 CuSO4 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. 150
Vi sinh vat hoc Nguyen Lan Dung Ra vt bi bng dung dch sulfat ng 20%, thm kh. Kt qu: T bo c mu sm, v nhy mu nht xung quanh. Soi knh: dng vt knh du 100.
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: Kt qu: Thnh t bo bt mu tm, t bo cht mu tm nht. 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
1.7. Nhum ht d nhim Vt liu, ho cht: Dung dch A: Xanh Toluidin (Toluidine blue) Lc malachite Acid acetic (glacial) Ethanol 95% Nc ct Dung dch B: Iod (I) Iodid kali (IK) Nc ct Cc bc tin hnh: 151 3g 300 ml 0,15 g 0,2 g 1 ml 2 ml 100 ml 2g
Vi sinh vat hoc Nguyen Lan Dung Lm vt bi, c nh vt bi. 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) Etanol 70% 0,3 g 100 ml. 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.
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: 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. 152 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
Vi sinh vat hoc Nguyen Lan Dung Khi dng pha long 10 ln. Cc bc tin hnh: Kt qu: Tinh th bt mu . Bo t tch ri c vng mu 1.10. Nhum vi khun khng acid Lm vt bi, c nh vt bi Nhum trong 1 pht, ra nc, hong kh Soi knh: dng vt knh du
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 Trn u 2 dung dch vi nhau. Dung dch B: Etanol 95% HCl c Dung dch C: Dung dch Xanh metylen bo ho trong etanol ( 2%) Dung dch KOH 0,01% trong nc Cc bc tin hnh: Kt qu: 153 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 30 ml 100 ml 100 ml 3 ml 100 ml
Vi sinh vat hoc Nguyen Lan Dung 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 0C 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 0C 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)
154
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 0C. T 37 0C 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 0C trong 30 pht sau t vo t m 35-37 0C, nui trong 48 gi.
155
Vi sinh vat hoc Nguyen Lan Dung Nu vi khun pht trin c l c tnh bn nhit. Dng chng vi khun Enterococcus faecalis lm i chng dng tnh.
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
156
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. 157
Vi sinh vat hoc Nguyen Lan Dung 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.
158
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. 159
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 160
Vi sinh vat hoc Nguyen Lan Dung 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 Trong hnh ny, khi g 2 VP1 hnh ng protein l VP1, VP2, VP3 v VP4 gic, s thy c bn trong VP4 mu (VP4 khng thy trn hnh). VP1 c vng hnh ng gic , i xng bc 5 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 161
Vi sinh vat hoc Nguyen Lan Dung 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 162
Vi sinh vat hoc Nguyen Lan Dung 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 163
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 164
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 165
Vi sinh vat hoc Nguyen Lan Dung 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). 166
Vi sinh vat hoc Nguyen Lan Dung * 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 167
V d
Vi sinh vat hoc Nguyen Lan Dung Virus parvo Chui n, dng thng ADN n Chui n, khp vng Herpes, adeno, coliphage T, phage l. Chui kp, dng thng Coliphage T5 Phage jX174, M13, fd
ADN kp
Chui kp, dng thng, trn mt mch c Vaccinia, Smallpox nhng ch t cu ni phosphodieste. Chui kp vi hai u khp kn Chui kp khp vng kn Chui n, dng dng thng Chui n, m, dng thng 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) Orthomyxo (cm) Reo (rota), mt s virus gy u thc vt, NPV cn trng, phage j6 v nhiu virus nm (mycovirus). Polioma (SV40), papiloma, phage PM2, virus m hoa l
ARN n
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
ARN kp
Chui kp, dng thng, phn on
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, Qb) c kch thc 1x106 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 1,6x108 Da c th m ha cho 100 protein. Genom ADN * Cc virus ADN c kch thc rt nh (nh jx 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. 168
Vi sinh vat hoc Nguyen Lan Dung * 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 l, 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. 169
Vi sinh vat hoc Nguyen Lan Dung 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 170
Vi sinh vat hoc Nguyen Lan Dung 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. 171
Vi sinh vat hoc Nguyen Lan Dung 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. 172
Vi sinh vat hoc Nguyen Lan Dung 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 Myoviridae Vt ch Vi khun Hnh thi Phc tp Cc virus i din T4
Lp I. Virus c genom ADN kp
173
Vi sinh vat hoc Nguyen Lan Dung Siphoviridae Rodoviridae Papovaviridae Adenoviridae Herpesviridae Poxviridae Baculoviridae Vi khun Vi khun ng vt ng vt Phc tp Phc tp Khi, khng v ngoi Khi, khng v ngoi l 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
ng vt Khi, c v ngoi ng vt Cn trng Phc tp Xon, c v ngoi Khi, c v ngoi Khi, khng v ngoi
Hepadnaviridae ng vt Caulimoviridae Thc vt Microviridae Parvoviridae Geminiviridae Reoviridae Vi khun ng vt Thc vt ng vt
Lp II. Virus c genom ADN n Khi, khng v ngoi jX174 Khi, khng v ngoi Virut parvo, virut lin quan Adeno Khi, hai ht gn thnh i Sc ng Khi, khng c v ngoi Rota, reo 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. Di, hoi t vng rau dip. Quai b, si, cm. Hanta, Phelebovirus (St thung lng Rift). Lassa, Junin, Machupo. Ebola, Marburg AIDS, HTLV-I, HTLV-II
Lp III. Virus c genom ARN kp 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.
Lp V. Virus c genom ARN n, m. Rhabdoviridae Paramyxoviridae Bunyaviridae Arenaviridae Filoviridae Retroviridae ng, thc vt. ng vt ng vt ng vt ng vt ng vt Xon, c v ngoi Xon, c v ngoi Xon, c v ngoi Xon, c v ngoi Xon, c v ngoi Khi, c v ngoi
Lp VI. Virus c genom ARN dng, nhn ln cn ADN kp trung gian.
174
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).
175
Vi sinh vat hoc Nguyen Lan Dung 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. 176
Vi sinh vat hoc Nguyen Lan Dung 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) 177
Vi sinh vat hoc Nguyen Lan Dung 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 (AAVdefective adeno-associated virus), cn gi l virus km adeno. C. Virus thc vt 178
Vi sinh vat hoc Nguyen Lan Dung 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 1,2x106 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. 179
Vi sinh vat hoc Nguyen Lan Dung 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 2,4x106 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 4x106 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 6065 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 9-11x106 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 180
Vi sinh vat hoc Nguyen Lan Dung 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 3-3,5x106 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 2x106 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 2x106 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)
181
Vi sinh vat hoc Nguyen Lan Dung a. Genom: Gm hai phn t dng thng, 5,9-3,5 Kb, khi lng phn t 2,4x106 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 4,6x106 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 100430 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 5-7x106 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 4,2x106 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 182
Vi sinh vat hoc Nguyen Lan Dung 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 4,5x106dalton. 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 3x106 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. 183
Vi sinh vat hoc Nguyen Lan Dung 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 2-3,5x106dalton. 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
184
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 u ma (smallpox Da viriola) H Poxviridae Molluscipox H Da Poxviridae Herpes simplex-1 Mi, da H Herpesviridae Herpes simplex-2 C quan sinh dc H Herpesviridae Varicella-Zoster ng h hp, H Herpesviridae mt, mu, da
Tn bnh u ma
ng ly truyn Qua sol kh, vy mn cha virus
U mm ly Herpes mi, mn rp. Herpes sinh dc, mn rp Thy u, Zona 185
Ly ngi sang ngi qua tip xc Qua nim mc, vt xc da ng tnh dc. Ngi sang ngi
Vi sinh vat hoc Nguyen Lan Dung Herpes 6 (HHV6) H Da, hch lympho Herpesviridae Papiloma Biu m da, nim ngi (HPV) H mc Papovaviridae Parvo B19 H Da (m, tay, i, Parvoviridae mng, mnh) Coxsackie A H Ming, hng, da Picornaviridae Rubella H Togaviridae
Ban o (roseola)
Qua sol kh, nc bt
Mn cm
Tip xc trc tip, ng sinh dc
Ban truyn nhim, bnh Qua sol kh cha virus th 5 Vim hng,mn nc (taychn-ming) vim kt mc, ng phn, ming xut huyt, vim mng no v khun. ng h hp
Da, ng h hp, Rubella hch lympho, mu ng h hp, h bch huyt, tun Si (measles) H hon, no, niu, Si ( Rubeola) Paramyxoviridae thn kinh trung ng.
ng h hp (sol kh).
2. Virus gy bnh h thn kinh. Tn virus Polyoma JC H Popovaviridae Polio H Picornaviridae Flavi H Flaviviridae C quan nhim Cc t bo thn, mu, no, h thn kinh trung ng. T bo rut, hng, no h thn kinh trung ng tu sng Tn bnh Bnh no bch cu nhiu tin trin (PML) Bi lit tr em ng ly truyn Qua ng h hp ng tiu ha, phn, ming Truyn qua bn chn khp.
Toga H Togaviridae Di (Rabies) H Rhabdoviridae Bunya H Bunyaviridae HIV H Retroviridae
Vim no St. Louis, Ty Mu i khi no, gan, da sng Nile, Xun h Nga, v mch mu vim no Nht Bn, st Dengue, st vng. Vim no nga min ty Mu i khi no, gan, da (WEE), vim no nga min v mch mu ng (EEE), vim no nga Venezuela C vn, nron, tu sng, Di no tuyn nc bt. Vim no california, st Mu, no, thn, gan, thung lng Rift mch mu T bo TH, i thc bo, AIDS t bo c trn, t bo tua 186
Truyn qua bn chn khp Qua nc bt ng vt di qua vt cn da. Truyn qua bn chn khp. Qua ng tnh dc, tim chch ma ty, tip vi dch c th ngi b
Vi sinh vat hoc Nguyen Lan Dung Priron No Bnh xp no ( JD), Kuru nhim HIV 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
T bo biu m ca hng, U lympho Burkit, Epstein Barr (EVB) tuyn nc bt mang tai, bnh bch cu n Nc bt H Herpesviridae mu v lympho B nhn. Qua dch tuyt ca c th nh nc bt, nc tiu c t cung, Cytomegalo (CMV) Gan, hch lympho D tt bm sinh truyn qua ng tnh dc, sinh H Herpesviridae n, truyn mu, ghp tng, tim chch. Vim c tim, vim mng ngoi tim, Coxsackie B H C tim, da vim no v trng, Qua ng phn ming Picornaviridae chng au nhi ngc. Ung th bch cu HTLV-1, HTLV-2 t bo T ngi, Qua ng tnh dc, truyn T bo lympho T H Retroviridae ung th bch cu t mu, tim chch bo lng. Nhiu loi t bo, c bit Marburg, Ebola H St xut huyt do Ly trc tip qua dch c th, l t bo gan v i thc Filoviridae virus trc ht l mu. bo Hi chng bnh Hanta H Ht phi virion t bi phn v Mu, phi phi do virus Hanta Bunyaviridae nc tiu ca chut. km sc tim
4. Virus gy bnh ng h hp
Tn virus Adeno H Adenoviridae Rhino H Picornaviridae
C quan nhim ng h hp ng h hp trn
Tn bnh Cm lnh thng Cm lnh (s mi)
ng ly truyn Qua sol kh ng h hp Qua sol kh ng h hp, vt b nhim hoc tip xc trc tip vi ngi bnh.
Corona ( Hcov) ng h hp H Coronaviridae
Hi chng vim ng h hp cp nng (SARS), cm Qua sol kh ng h hp. lnh thng 187
Vi sinh vat hoc Nguyen Lan Dung Quai b (mumps) Cc t bo ng H Quai b h hp trn Paramyxoviridae Hp bo h hp ng h hp, Bnh hp bo h hp (RSV) phi Cm H Phi, ng h orthomyxoviridae Cm hp
Dch tit ng h hp (qua ho, ht hi), nc bt. Qua bi nn, chn tay b nhim, nht l vn tr Qua git tit ng h hp
5. Virus gy bnh ng tiu ho Tn virus Vim gan B (HBV) H H Hepadnaviridae Vim gan A (HAV) H Picoraviridae Vim gan C (HCV) H Flaviridae Vim gan D (HDV) Cha phn loi Vim gan E (HEV) H Hepeviridae Rota H Reoviridae C bo (CMV) (Cytomegalo) H Herpesviridae C quan nhim T bo gan T bo gan T bo gan T bo gan T bo gan Tn bnh ng ly truyn
Vim gan huyt thanh Qua ng tnh dc, qua dch c (vim gan B), ung th gan th b nhim, tim chch ma ty. Vim gan nhim trng (vim gan A) Vim gan mn tnh Vim gan Delta Vim gan E Qua ng tiu ha. Qua ng tnh dc, truyn mu, ghp tng, tim chch. Qua ng tnh dc, bm tim b nhim Qua ng phn-ming Qua ng phn-ming Qua dch tit ca c th (nc bt, dch nhn, sa, nc tiu, phn, dch t cung, qua quan h tnh dc, truyn mu.
ng h hp v Tiu chy, vim d dy ng tiu ha rut tr em Hch lympho, gan D tt bm sinh
6. Virus gy bnh ng niu v sinh dc Tn virus C quan nhim Tn bnh Herpes simplex-2 Herpes sinh dc, mn C quan sinh dc H Herpesviridae rp. Papilloma (HPV) Biu m da, nim H Mn cm mc. Papovaviridae Vim gan B Vim gan huyt thanh (HBV) H Gan ung th Hepadnaviridae T bo T, i thc Hi chng suy gim HIV H bo, t bo c min dch mc phi Retroviridae trn, t bo tua. (AIDS). 188 ng ly truyn Qua quan h tnh dc Qua tip xc trc tip, qua dng c b nhim, qua quan h tnh dc. Qua tip xc trc tip vi dch c th b nhim. Qua quan h tnh dc, bm tim b nhim. Qua quan h tnh dc, tim chch ma ty, truyn mu, tip xc vi dch c th b nhim, ghp tng.
5. Hnh nh mt s virus thng thng:
Kch thc Virus (1000nm= 1m)
H Orthomyxoviridae
189
Virus cm (Influenzavirus)
Virus cm gia cm H5N1 (HPAV- Highly pathogenic avian influenza)
190
H Paramyxoviridae
Virus quai b (Mumps virus)
191
192
Vi sinh vat hoc Nguyen Lan Dung Virus si (Measles virus)
H Togaviridae Virus si c (Rubella virus)
193
Virus hp bo h hp (RSV- Respiratory syncytial virus)
H Coranoviridae
194
Vi sinh vat hoc Nguyen Lan Dung 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)
195
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)
196
H Togaviridae Virus vim gan E ( HEV- Hepatitis E virus)
Virus st xut huyt Dengue (Dengue fever virus)
197
Virus HIV (Human immunodeficiency virus)
Virus bi lit (Poliomyelitis virus) 198
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)
199
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.
200
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.
201
Vi sinh vat hoc Nguyen Lan Dung 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 Labyrinthomorpha
Lp Labyrinthulea Thraustochytriacea Hyphochytriomycetes
Lp ph hoc nhm
Pseudofungi Chytridiomycota Zygomycota Ascomycota Trichomycetes Archiascomycetes Saccharomycetes 202 Oomycetes Chytridiomycetes Zygomycetes
Vi sinh vat hoc Nguyen Lan Dung Ascomycetes discomycetes Major licheneized orders plectomycetes pyrenomycetes loculoascomycetes powdery mildews Laboulbeniomycetes conidial ascomycetes (Hyphomycetes, Coelomycetes) Urediniomycetes Basidiomycota Urediniomycetidae Ustilaginomycetes Tremellomycetidae Dacrymycetidae Hymenomycetes Auriculariomycetidae Hymenomycetidae Platyglomycetidae
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, fruitbody) 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 203
Vi sinh vat hoc Nguyen Lan Dung 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.
I1.
CU TO CHUNG CA SI NM 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: Thnh phn thnh t bo: Glucan Cellulose Chitine Chitosan Mannan Protein Lipid 1 16 58 10 2 54 36 0 10 <1 5 3 3 0 0 9 33 2 6 8 4 43 0 19 2 11 5 5 29 0 1 0 31 13 9 6 6 0 10 <3 7 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: 204
Vi sinh vat hoc Nguyen Lan Dung Acrasiales Cellulose, Glycogen Oomycetes Cellulose, Glucan Hyphochytriomycetes Zygomycetes Cellulose. Chitine
Chitin, Chitosan Basidiomycota (dng si),
Chytridiomycetes, Ascomycota (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 Thnh phn mng NSC Protein Lipid Polysaccharide Acid nucleic 1 52,0 43,0 9,0 0,3 2 38,5 40,4 5,2 1,1 3 46,5 41,5 3,2 7,5 4 45,5 31,0 25,0 0,5 5 25,5 40,0 30,0 -
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. 205
Vi sinh vat hoc Nguyen Lan Dung 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 58nm( 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 206
Vi sinh vat hoc Nguyen Lan Dung 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
207
Cu trc ca si nm
Si nm c vch ngn
Si nm khng vch ngn
H si nm 208
S pht trin ca h si nm
Khun lc nm
209
Mt v d v cu trc ca Ribosome Eukaryote
Th Golgi v Mng li ni cht
210
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 211
Vi sinh vat hoc Nguyen Lan Dung 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)
212
Vi sinh vat hoc Nguyen Lan Dung 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. 213
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):
214
Vi sinh vat hoc Nguyen Lan Dung 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.
215
216
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
217
Nm Rhizopus
218
Nm Absidia
219
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:
220
Vi sinh vat hoc Nguyen Lan Dung 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: 221
Vi sinh vat hoc Nguyen Lan Dung 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 222
Vi sinh vat hoc Nguyen Lan Dung 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. JuYoung 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. 223
Vi sinh vat hoc Nguyen Lan Dung 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 250C, 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). 200C 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). 224
Vi sinh vat hoc Nguyen Lan Dung 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 450 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: 225
Vi sinh vat hoc Nguyen Lan Dung 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 250C, 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. 200C 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.
226
Vi sinh vat hoc Nguyen Lan Dung 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 250C 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 227
Vi sinh vat hoc Nguyen Lan Dung - 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, 228
Vi sinh vat hoc Nguyen Lan Dung bo t trn hoc bo t sinh ra trong hoc trn cc bo t c bit 2a. Bo t sinh ra trong ti bo t
NM TI (ASCOMYCETES) 3
2b. Bo t hoc bo t trn khng sinh ra trong ti bo t 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. 229
Vi sinh vat hoc Nguyen Lan Dung - 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.
230
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 231
Vi sinh vat hoc Nguyen Lan Dung - Ny mm t trong
a-b. Dng tn. a- To chui. b- n c
232
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)
233
Vi sinh vat hoc Nguyen Lan Dung * 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 234
Vi sinh vat hoc Nguyen Lan Dung 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. 235
Vi sinh vat hoc Nguyen Lan Dung 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. 236
Vi sinh vat hoc Nguyen Lan Dung d. Bo t xon c - Trc bo t un cong hn 1800. 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. 237
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
238
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 239
Vi sinh vat hoc Nguyen Lan Dung 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
240
Vi sinh vat hoc Nguyen Lan Dung 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.
241
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
242
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.
243
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
244
Vi sinh vat hoc Nguyen Lan Dung 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.
245
Vi sinh vat hoc Nguyen Lan Dung 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).
246
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. 247
Vi sinh vat hoc Nguyen Lan Dung 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 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 248 nhau nhiu hn 1 mt
Vi sinh vat hoc Nguyen Lan Dung - 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. 249
Vi sinh vat hoc Nguyen Lan Dung - 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. 250
Vi sinh vat hoc Nguyen Lan Dung - 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. 251
Vi sinh vat hoc Nguyen Lan Dung + 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
252
Vi sinh vat hoc Nguyen Lan Dung + 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 253
Vi sinh vat hoc Nguyen Lan Dung - 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. 254
Vi sinh vat hoc Nguyen Lan Dung 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. 255
Vi sinh vat hoc Nguyen Lan Dung 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: Podosporium, Didymobotrium. Dratomyces* (74,76), Graphium*, Stibella, Tubercularia, Phaeoisaria,
256
Hnh 1: 1. Geotrichum candidum, 2. Arthrographis sp., 3. Geomyces pannorum, 4. Oidiodendron flavum, 5. Wallemia sebi, 6. Scytalidium lignicola, 7. Sympodiella acicola.
257
Hnh 2: 8. Mammaria echinobotryoides, 9. Chrysosporium sp., 10. Nigrospora oryzae, 11. Humicola grisea, 12. Arthrinium phaeospermum, 13. Gilmaniella humicola
258
Hnh 3: 14. Basipetospora rubra, 15. Aureobasidium pullulans, 16. Sepedonium chysospermum, 17. Cladosporium sp., 18. Mycogone perniciosa, 19. Hyalodendron.,
259
Hnh 4: 20. Dimorphospora foliicola, 21. Oedosephalum glomerulosum, 22. Torula herbarum, 23. Chrimelosporium sp., 24. Botrytis cinerea
260
Hnh 5: 25. Idriella lunata, 26. Tritirachium oryzae, 27. Verticicladium trifidum, 28. Sporothrix sp., 29. Rhinocladiella sp., 30. Beauveria bassiana.
261
Hnh 6: 31. Selenosporella curvispora., 32. Calcarisporium arbuscula, 33. Subulispora procurvata, 34. Costantinella micheneri, 35. Hansfordia pulvinata.
262
Hnh 7: 36. Dactylaria sp., 37. Beltraniella odinae, 38. Beltrania rhombica, 39. Beltraniopsis esenbeckiae.
263
Hnh 8: 40. Acremonium butyli, 41. Gliomastix murorum var. felina. 42. Monocillium indicum, 43. Chalara sp., 44. Phialophora sp., 45. Gonytrichum macrocladum
264
Hnh 9: 46. Codinaea sp., 47. Chloridium chlamydosporis, 48. Torulomycrs lagena, 49. Eladia saccula, 50. Stachylidium bicolor, 51. Phialocephala bactrospora.
265
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: 266
Vi sinh vat hoc Nguyen Lan Dung 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.
267
N= nhn; M= ty th; Va= khng bo; ER= mng li ni cht; Ves= bo nang
268
Bo t bn
Phn ct t bo
Ny chi
Bo t ti
Bo t mng dy
Bo t t
269
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. 270
Vi sinh vat hoc Nguyen Lan Dung - Sinh trng ti cc nhit khc nhau: 25, 30, 35, 37, 420C. - 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 600C 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 6o 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 5,7o Baume. C ti liu li s dng nng 5-80 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 25-300C 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 271
Vi sinh vat hoc Nguyen Lan Dung 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 Iodua Kali Nc ct 2g 4g 100ml
(Nghin nh I v KI trong ci s ri sau dng nc ho tan dn). - Dung dch xanh methylen (methylene blue) Xanh methylen Nc ct 1g 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) Cn 900 Dung dch phenol 3% 0,1g 10ml 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 272
Vi sinh vat hoc Nguyen Lan Dung 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 Cn 90% 0,05g 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 Cn 70% 0,3g 100ml
(Trn u, 24 gi ri mi s dng. Dng trong vng mt thng). - Thuc nhum safranin: Dch safranin 2,5% (trong cn 95%) Nc ct 10ml 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 FeNH4(SO4).12H2O 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 FeNH4(SO4).12H2O 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 Axetit acetic glacial Dung dch fomol long + Dung dch ngm FeNH4(SO4)2.12H2O FeNH4(SO4)2.12H2O 3g 273 75 phn 5 phn 20 phn
Vi sinh vat hoc Nguyen Lan Dung Nc + Dung dch thuc nhum hematoxilin Hematoxilin Cn Nc
100ml
1g 10ml 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) Nc 1g 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) Cao nm men (yeast extract) Glucoza Pepton Nc 3g 3g 10g 5g 1000ml
274
Vi sinh vat hoc Nguyen Lan Dung Nui cy trong bnh nn 25-280C 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 Glucoza Thch Nc - Mi trng khoai ty - glucoza: Nc chit khoai ty 10% Glucoza Thch 1000ml 20g 20g 10g 20g 20g 1000ml
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 600C 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 275
Vi sinh vat hoc Nguyen Lan Dung 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 600C, 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 0C 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 0C 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 0C 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 276
Vi sinh vat hoc Nguyen Lan Dung 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. 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 250C 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 (1200C trong 30 pht). Cy nm men ti (t thch nghing mi nui cy 48 gi tui). Gi 250C 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: Glucoza: KH2PO4: CaCl2: MgSO4: FeSO4: (NH4)2SO4: 100 ml 5g 0,2g 0,05g 0,05g 0,001g 0,5g 277 Nghin cu hnh dng bo t v s lng ca bo t ti
Vi sinh vat hoc Nguyen Lan Dung 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% KH2PO4. c. Mi trng Gorodkowa (1908) Nc tht: Pepton: NaCl: Glucoza: Thch: Nc: 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: Axetat Na khng ngm nc: Thch: Nc: 20 g 1000 ml 0,4 g 1,4 g 1g 1g 0,5 g 0,25 g 2g 100ml
g. Mi trng dch tinh bt khoai ty 0,5% (Almeida v Lacaza) Cy nm men ln mi trng trn ri nui cy 370C, sau quan st bo t ti theo tng thi im thch hp. h. Mi trng Kleyn: Natri glutamat (hay asparagin, pepton, glixin): Glucoza: 0,25g 0,062g 278
Vi sinh vat hoc Nguyen Lan Dung NaCl: Natri axeta: KH2PO4: K2HPO4: Biotin (c th khng cn): Dch hn hp I: Thch: Nc:
0,062g 0,5g 0,012 g 0,02g 2 1 ml 2g 100 ml
Cch pha dch hn hp I: MgSO4 - 0,4g; CuSO4 - 0,002g; FeSO4 - 0,2g; MnSO4 - 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 10-150 Baling (xem phn Quan st hnh thi t bo nm men v o kch thc). Nui cy 250C 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 12-150 Baling sau t m 25-300C. 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 250C trong 14 n 30 ngy. Ly ra quan st khun lc ln. Cn ch miu t: - Kch thc khun lc (v hnh). 279
Vi sinh vat hoc Nguyen Lan Dung - 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 1200C. 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 107 t bo/ml, 250C sau mt tun. - Quan st vic to CO2 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 CO2 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 25-300C 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:
280
Vi sinh vat hoc Nguyen Lan Dung - 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, Lrhamnoza, D-glucosamin, N-acetyl-D-glucosamin, methanol, ethanol, glycerol, erythritol, ribitol, galactitol, Dmannitol, D-glucitol, a- metyl-D-glucosid, salicin, glucono-d-lacton, D-gluconat, 2-ketogluconat, 5ketogluconat, 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 25x106/ml (hay mt A640 = 1,0). - Sau ly ra 100 l 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 15-400) 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 450C 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 0C sau 30 pht cho ng thch. C th sau p ngc 37 0C 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 281
Vi sinh vat hoc Nguyen Lan Dung 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 NH3 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 I2 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 : (NH4)2SO4: KH2PO4: MgSO4.7H2O: Glucoza: Thch: Nc ct: pH : 1g 1g 0,5 g 10 g 25 g 1000 ml 4,5
Kh trng mi trng nhit 1100C (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 250C 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: (NH4)2SO4: KH2PO4: MgSO4.7H2O: CaCl2.2H2O: NaCl: 5g 1g 0,5 g 0,1 g 0,1 g 282
Vi sinh vat hoc Nguyen Lan Dung Cao nm men: Glucoza: Nc:
1g 30 g 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 25-300C 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: Biotin: Acid folic: Myo - inositol: Acid nicotinic: Pantotenat (Ca): Pyridoxin (HCl): Riboflavin: Tiamin HCl: 200 mg 20 mg 2 mg 10 mg 400 mg 2 mg 400 mg 200 mg 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 250C trong 4 tun. Ch trnh cho mi trng b kh bng cch bc bng giy nn (wax-paper). 283
Vi sinh vat hoc Nguyen Lan Dung 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 : NaCl: KH2PO4 : glucoza : thch: nc: 1g 5g 2g 5g 20g 1000ml
un tan mi trng, thm 6ml dung dch phenol (phenol red) c nng 0,2% trong cn. Kh trng mi trng ni hp (1150C/15 pht). i ngui n 500C 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 260C 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 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.
0
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: D-Glucoza: 9,80 1,00 284
Vi sinh vat hoc Nguyen Lan Dung NaCl: MgSO4.7H2O: Cao nm men: Thch: Kh trng:
1,20 0,70 2,50 20 1200C/15 pht
2. Mi trng thch Gorodkowa (Dodder v Kreger - van Rij, 1952) (g/l) D-glucoza: Pepton: NaCl: Thch: Kh trng: 1,0 10,0 5,0 20,0 1200C/15 pht
3. Mi trng cao ng (Lodder v Kreger - van Rij, 1952) Ho tan 12,5g cao ng maize extract) vo 300ml nc 600C 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 1200C/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 200C. Bnh A c lm tan thch v trn u vi bnh B v phn vo cc ng nghim kh trng 1200C 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 1200C 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: (NH4)2SO4: L-Asparagin: 3,5 g 1,5g 285
Vi sinh vat hoc Nguyen Lan Dung Ngun carbon D-glucoza: Aminoacid L-Histidin: DL-Methionin: DL-Triptophan: 10 mg 20 mg 20 mg Cht sinh trng Acid P-aminobenzoic: Biotin: Acid folic: Myo-inositol: Acid nicotinic: Pantotenat (Ca): Pyridoxin HCl: Riboflavin: Tiamin HCl: 200 mg 20 mg 2 mg 10 mg 400 mg 2 mg 400 mg 200mg 400 mg Vi lng H3BO3: CuSO4.5H2O: KI: FeCl3.6H2O: MnSO4.4H2O: Na2MoO4.H2O: ZnSO4.7H2O: 500 mg 40 mg 100 mg 200 mg 400 mg 200 mg 400 mg Mui khong KH2PO4: 850 mg 286 10 g
Vi sinh vat hoc Nguyen Lan Dung K2HPO4: MgSO4.7H2O: NaCl: CaCl2.6H2O:
150 mg 500 mg 100 mg 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 (NH4)2SO4), 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 (NH2)2SO4, 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.
287
Vi sinh vat hoc Nguyen Lan Dung 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 ng ho nitrat : Yu v chm : - (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) T bo ny chi a cc, ch hnh thnh khun ty gi
Sinh sn sinh dng: Sinh sn hu tnh: c im sinh l:
Cc ti (asci) cha 1-2 bo t ti hnh cu, x x. 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. 288
Vi sinh vat hoc Nguyen Lan Dung 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 :: -/+ : rt yu ::-
4.
Babjevia (1 loi) T bo ny chi a cc, khun ty tht him khi to thnh, khun ty gi s ng
Sinh sn sinh dng:
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 : -/+ :289
Vi sinh vat hoc Nguyen Lan Dung 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) T bo ny chi a cc, i khi c khun ty gi. Khng c khun ty tht.
Sinh sn sinh dng: Sinh sn hu tnh: c im sinh l:
Ti cha 1-4 bo t ti hnh chu Ln men ng ho nitrat :+ :-
Mng trn mi trng dch th : C cht ging tinh bt ng ha inositol Hot ho Ureaza :::290
Vi sinh vat hoc Nguyen Lan Dung Ho lng gelatin Phn ng DBB
::-
8.
Coccidiascus (1 loi) T bo hnh cu hay ovan, khng to khun ty tht v khun ty gi.
Sinh sn sinh dng:
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 - 400C. Ln men ng ho nitrat :: yu.
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 : 291
Vi sinh vat hoc Nguyen Lan Dung 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: c im sinh l: Ti khng tip hp v tr nn d chy, c 1-4 bo t ti dng m hoc dng cu. 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: 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 ::Ti hnh kim hay
:+ :+ :::-
13. Dipodascus (13 loi) Sinh sn sinh dng: Sinh sn hu tnh: c im sinh l: Hnh thnh bo t t, c khun ty tht. Ti hnh kim, tr, elip hay cu. Mi ti cha 4-128 bo t ti trong sut, hnh elip. Ln men ng ho nitrat : -/yu :-
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 :-
293
Vi sinh vat hoc Nguyen Lan Dung 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) 294
Vi sinh vat hoc Nguyen Lan Dung Sinh sn sinh dng: Hnh thnh bo t t, c khun ty tht. Sinh sn hu tnh: c im sinh l: Ti bo t dng gn cu, trong cha 1 bo t hnh elip. Ln men ng ho nitrat : -/yu :-
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 :Phn ng DBB ::::Ha lng gelatin
19. Issatchenkia (4 loi) Sinh sn sinh dng: Sinh sn hu tnh: c im sinh l: Cc t bo ny chi a cc, c khun ty gi, khng c khun ty tht. Mi ti cha 1-4 bo t ti hnh trn, b mt c th x x Ln men ng ho nitrat :+ :295
Vi sinh vat hoc Nguyen Lan Dung Mng trn mi trng dch th : + C cht ging tinh bt ng ha inositol Hot ho Ureaza : -/+ Phn ng DBB ::::Ha lng gelatin
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: Sinh sn hu tnh: c im sinh l: T bo ny chi a cc, khng c khun ty hoc ch c khun ty gi rt s ng. Mi ti c 4-20 bo t ti mu nu hoc h phch, nhn hoc x x, hnh elip. Ln men ng ho nitrat ::-
Mng trn mi trng dch th : +/296
Vi sinh vat hoc Nguyen Lan Dung 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: Sinh sn hu tnh: c im sinh l: T bo ny chi a cc, ch c khun ty gi. Cc ti khng tip hp, thun di, bn, mi ti c 1-2 bo t hnh van di. Ln men ng ho nitrat :+ :-
23. Metschnikowia (10 loi) Sinh sn sinh dng: gi. Sinh sn hu tnh: c im sinh l: T bo ny chi a cc, i khi c dng trng khuyt hay li lim. C to khun ty Ti bo t c dng thun di, mi ti c 1-2 bo t hnh kim. Ln men ng ho nitrat :+ :-
:::::-
24. Nadsonia (2 loi) Sinh sn sinh dng: khun ty. Sinh sn hu tnh: ti dng trn, x x. c im sinh l: T bo ny chi 2 cc. T bo to, hnh qu chanh chu u hay hnh van. Khng to Ti c pht trin t s tip hp ca t bo v chi ca n, Mi ti cha 1-2 bo t 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 ng ho nitrat : + (gluco, D-xylose) :+
:::::-
26. Pichia (91 loi) Sinh sn sinh dng: Sinh sn hu tnh: ra ng xch o. c im sinh l: T bo ny chi, c th c khun ty gi v khun ty tht. Ti c cha 1-4 (t khi 8) bo t ti nhn trn, hnh m hay sao Th, c hoc khng c 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 :: -/+ ( 1 loi) :: -/+ :-
27. Protomyces (6 loi) Sinh sn sinh dng: k sinh. Sinh sn hu tnh: nng bn ra mnh m. c im sinh l: T bo ny a phn cc, Khun ty tht c to thnh trong cc m thc vt m n Ti bo t cng to bn trong m thc vt ch, cha hng trm cc bo t ti c kh Ln men ng ho nitrat :: +/-
:+ ::: -/+( rt yu) :-
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 :+ :-
29. Saccharomycodes (1 loi) Sinh sn sinh dng: khun ty gi. Sinh sn hu tnh: c im sinh l: T bo ny chi ti hai cc. T bo dng di hay hnh qu chanh chu u. i khi c Mi ti c 1-4 bo t ti trn nhn, mi bo t c mt rnh hp. Ln men ng ho nitrat :+ :-
:::: -/+(yu) :-
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 Phn ng DBB :: +/:: + ( khng mnh) :-
31. Saturnispora (4 loi) Sinh sn sinh dng: Sinh sn hu tnh: xch o. c im sinh l: T bo ny chi a cc. C th c khun ty gi. Mi ti c 1-2, him khi l 8 bo t ti c hnh cu. B mt nhn, c rnh trn ng Ln men ng ho nitrat : + (glucose) :-
Vi sinh vat hoc Nguyen Lan Dung C cht ging tinh bt ng ha inositol Hot ho Ureaza Ha lng gelatin Phn ng DBB ng ho D-xyloza
:::: +/- ( yu) ::-
32. Schizosaccharomyces (3 loi) Sinh sn sinh dng: gii phng bo t t. Sinh sn hu tnh: c im sinh l: Cc t bo khng ny chi m phn ct. i khi c khun ty tht v thng ngt ra khi Ti c 2-8 bo t ti dng trn, van hay thn 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: Sinh sn hu tnh: c im sinh l: T bo ny chi a cc, khng c khun ty, hai loi t bo c dng lp xng. Mi ti c cha 1-4 bo t ti thnh dy, hnh t trn, elp hoc gy. Ln men : -/+ (glucose, yu) 302
Vi sinh vat hoc Nguyen Lan Dung 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 :+ 303
Vi sinh vat hoc Nguyen Lan Dung ng ho nitrat
:-
36. Wickerhamia (1 loi) Sinh sn sinh dng: khun ty. Sinh sn hu tnh: c im sinh l: T bo ny chi ti hai cc, cc t bo hnh ong hay van hoc hnh ko di. Khng c Mi ti c 1-2, him khi l 16 bo t ti, dng m bng chy. Ln men ng ho nitrat :+ :-
37. Wickerhamiella (1 loi) Sinh sn sinh dng: Sinh sn hu tnh: c im sinh l: T bo ny chi a cc, khng c khun ty. Cc ti bo t c tip hp to ra 1 bo t ti c dng di, khng i xng v x x. Ln men ng ho nitrat ::+ 304
Vi sinh vat hoc Nguyen Lan Dung 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: c im sinh l: Mi ti c 2-4 bo t ti nhn, dng cu, ovan, c ng rnh gia. Ln men ng ho nitrat : +(glucoza) : +/-
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 : + 305
Vi sinh vat hoc Nguyen Lan Dung 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 :306
Vi sinh vat hoc Nguyen Lan Dung 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 :307 :+ :::-
Vi sinh vat hoc Nguyen Lan Dung 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 :+ : +/308
Vi sinh vat hoc Nguyen Lan Dung 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 :+ :309
Vi sinh vat hoc Nguyen Lan Dung 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: men Ny chi a cc. i khi c khun ty gi s ng c im sinh l: :ng ho nitrat :Ln
Mng trn mi trng dch th : C cht ging tinh bt ng ha inositol lng gelatin : +/Nui cy dng nhy :+ : +/Ha
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 :+ ::+ :310
Mng trn mi trng dch th C cht ging tinh bt ng ha inositol
Vi sinh vat hoc Nguyen Lan Dung 53. Saitoella (1 loi) Sinh sn sinh dng: c im sinh l: Ny chi a cc. Khng c khun ty. C mu cam n mu . 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 311
Vi sinh vat hoc Nguyen Lan Dung 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 :312
Vi sinh vat hoc Nguyen Lan Dung 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 bo. c im sinh l: Ln men D-Glucuronate Myo-inositol ::+ :+ . m dng keo cha 2-4 t
C cht ging tinh bt : + ng ho nitrat Phn ng DBB Hot ho Ureaza Coenzym Xylose :+ :+ : Q10 :+ :-
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 :::313
Vi sinh vat hoc Nguyen Lan Dung 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 Xylose :+ :+ : Q8 :+ :+
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 : 314
Vi sinh vat hoc Nguyen Lan Dung ng ho nitrat Phn ng DBB Hot ho Ureaza Coenzym Xylose 62. Fibulobasidium (1 loi) :+ :+
:+
: Q10(H2) :-
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 Phn ng DBB Hot ho Ureaza Coenzym 63. Filobasidiella (1 loi) : -/+(yu) :+ :+ : Q9
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 ::+ :+ 315
Vi sinh vat hoc Nguyen Lan Dung Coenzym Xylose
: Q10 :+
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 Xylose : -/+ :+ :+ : Q9 hoc Q10 :+
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 ::+
316
Vi sinh vat hoc Nguyen Lan Dung 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 Xylose :+ :+ :+ : Q9 hoc Q10 :-
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 D-Glucuronate Myo-inositol : -/+(glucoza hoc :+ : -/+ galactoza)
C cht ging tinh bt : + ng ho nitrat Phn ng DBB Hot ho Ureaza Coenzym :+ :+ :+ : Q9 317
Vi sinh vat hoc Nguyen Lan Dung Xylose Nhit sinh trng 68. Rhodosporidium (10 loi)
:+ : di 200C
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 Xylose : -/+ :+ :+ : Q9 hoc Q10 :-
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 ::+ :+
318
Vi sinh vat hoc Nguyen Lan Dung 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 Xylose 71. Sterigmatosporidium (1 loi) : -/+ :+ :+ : Q10 :-
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 Xylose 72. Tilletiaria (1 loi) 319 ::+ :+ : Q10 :+
Vi sinh vat hoc Nguyen Lan Dung 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 Xylose 73. Tremella (11 loi) ::+ :+ : Q10 :-
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 Xylose 74. Trimorphomyces (1 loi) :: -/+ : -/+ : -/+ ::+ :+ : Q10 :+
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 320
Vi sinh vat hoc Nguyen Lan Dung 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 Xylose B. Dng sinh sn v tnh ( Anamorph) 60. Bensingtonia ( 9 loi) 321 ::+ :+ : Q10 :+
Vi sinh vat hoc Nguyen Lan Dung 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 Xylose : -/+ :+ :+ : Q9 :-
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 Xylose : -/+ :+ :+ : Q9 :+
62.
Cryptococcus ( 34 loi) 322
Vi sinh vat hoc Nguyen Lan Dung 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 Xylose : -/+ :+ :+ : Q8, Q9 hoc Q10 :+
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 Xylose ::+ :+ : Q10 :+
323
Vi sinh vat hoc Nguyen Lan Dung 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 Xylose ::+ ::+ :+ : Q9 :+
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 Xylose : +/:+ :+ : Q9 :+
66.
Kockovaella ( 2 loi) 324
Vi sinh vat hoc Nguyen Lan Dung 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 Xylose ::+ :+ :::+ :+ : Q10 :+
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 Xylose :::::+ :+ :+ : Q10 :-
325
Vi sinh vat hoc Nguyen Lan Dung 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 Xylose ::+ :+ :+ :+ :+ : Q9 :-
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 Xylose :+ ::+ :+ :+ : Q9 :-
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. 326
Vi sinh vat hoc Nguyen Lan Dung 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 Xylose
:+ :+ :::+ :+ :+ : Q10 :+
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 Xylose ::+ :+ :+ ::+ :+ : Q10 :-
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 :327
Vi sinh vat hoc Nguyen Lan Dung Myo-inositol ng ho nitrat C cht ging tinh bt Phn ng DBB Hot ho Ureaza Coenzym Xylose
::::+ :+ : Q7 :-
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 Xylose :: +/:: +/::+ :+ : Q9 hoc Q10 :-
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. 328
Vi sinh vat hoc Nguyen Lan Dung 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 Xylose :+ :+ : Q10 hoc Q10(H2) :-
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 Xylose :+ :+ : Q9 :-
76.
Sympodiomycopsis (1 loi)
Sinh trng sinh dng: Khun lc mu kem, dng b n nhy .
329
Vi sinh vat hoc Nguyen Lan Dung 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 Xylose ::+ :+ ::+ :+ : Q10 :+
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 Xylose :+ :+ : Q10 :-
78.
Trichosporon (19 loi) 330
Vi sinh vat hoc Nguyen Lan Dung 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 Xylose : +/: +/: -/+ : +/:+ :+ : Q9 hoc Q10 :+
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 Xylose :+ ::+ :+ :+ : Q9 :-
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 :331
Vi sinh vat hoc Nguyen Lan Dung D-Glucuronate Myo-inositol ng ho nitrat C cht ging tinh bt Phn ng DBB Hot ho Ureaza Coenzym Xylose
:+ :+ :: -/+(yu) :+ :+ : Q9 :+
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
332
Bo t bn v t bo sinh dng ca loi Kockovaella calophylli
Bo t bn v t bo sinh dng ca loi Kockovaella cucphuongensis
333
Vi sinh vat hoc Nguyen Lan Dung Bo t bn v t bo sinh dng ca loi Kockovaella vietnamensis
Bo t bn v t bo sinh dng ca loi Bullera hoabinhensis
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, 4th 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.
334
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) . 335
Vi sinh vat hoc Nguyen Lan Dung 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 336
Vi sinh vat hoc Nguyen Lan Dung 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).
337
Vi sinh vat hoc Nguyen Lan Dung 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 bcaroten. 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 338
Vi sinh vat hoc Nguyen Lan Dung 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:
339
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. 340
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 ):
341
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
342
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 343
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) : 344
345
Nhng nh chp chi tit hn c th thy trong cc hnh sau y :
Dysmorphococcus
Platydorina
346
Gonium
Volvox
Eremosphaera
Oedogonium
Kircheneriella
Tetraselmis
Chlorella
347
Vi sinh vat hoc Nguyen Lan Dung Hydrodiction
Pediastrum
Scenedesmus
Dunaliella
Coelastrum
Haematococcus
Botryococcus
Oocystis
348
Staurastrum
Ulothrix
Spirogyra
Stigeoclonium
Closterium
Codium
Chlorococcum
349
Asterococcus
Bryopsis
Pyrobotrys
350
Sinh sn hu tnh d phi Caulerpa
Cosmarium
Micrasterias
351
Collodiction
Pteromonas
Chlorogonium
352
Spondylomorum
Polytoma
Pandorina
Eudorina
Pleodorina 353
Lobomonas
Carteria
Sphaerellopsis
Chlorobrachys
Spermatozopsis
Platymonas
Dysmorphococcus
Coccomonas
354
Phacotus
Pedinomonas
Pyramidomonas
Golenkinia
Chodatella
Schroederia
Tetraedron
355
Ankistrodesmus
Micractinium
Characium
356
Selenastrum
Dictyosphaerium
Actinastrumm
Crucigenia
Westella 357
Coelastrum
Cosmarium
358
Elakatothrix
Sphaerocystis
Acanthosphaera
Polyedriopsis
Treubaria
Echinosphaerella
Trochiscia
359
Sorastrum
Errerella
Tetrastrum
Chaetophora
Sirogonium
Triploceras
Xanthidium
360
Vi sinh vat hoc Nguyen Lan Dung 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 ):
361
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
362
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:
363
Dinobryon
Uroglena
Synura
Uroglenopsis
Chrysostephanosphaera
Chromulina
364
Ochromonas
Mallomonas
Distephanus
Pleurasiga
Pseudopedinella
365
Pedinella
Proterospongia
Oikomonas
Dendromonas
Anthophysis
Chrysamoeba
Chrysosphaerella
Chrysidiastrum
366
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 CO2 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 367
Vi sinh vat hoc Nguyen Lan Dung 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
368
Olisthodiscus
Vacuolaria
Characiopsis
Ophiocytium
Goniochloris
Botrydiopsis
369
Chlorothecium
Gloeobotrys
Gonyostomum
Merotrichia
3) Lp To silic (Bacillariophyceae) 370
Vi sinh vat hoc Nguyen Lan Dung 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 CO2 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. 371
Vi sinh vat hoc Nguyen Lan Dung - 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 ):
372
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, 373
Vi sinh vat hoc Nguyen Lan Dung 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
374
Cyclotella
Cymbella
Epithemia
Eunotia
Diatoma
Fragilaria
Frustulia
375
Gomphoneis
Gomphonema
Navicula
Hemidiscus
Gyrosigma
Hydrosera
Melosira
Nitzschia
Planktoniella
376
Pinnularia
Pinnularia
Pleurosigma
Rhizosolenia
Skeletonema
Stauroneis
Stephanopyxis
Surirella
377
Synedra
Thalassiosira
Tabellaria
Triceratium
Ngnh To mt (Euglenophyta) 378
Vi sinh vat hoc Nguyen Lan Dung 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 CaCO3 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 CO2 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 379
Vi sinh vat hoc Nguyen Lan Dung 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
380
Menoidium
Colacium
Eutreptia
Eutreptiella
Trachelomonas
Urceolus
Astasia
381
Lepocinclis
Peranema
Hyalophacus
382
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 :
383
Porphyridium Ti liu tham kho
Rhodella
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
Nm m Blazei - sn xut v ng dng
384
Nm m Blazei c tn khoa hc l Agaricus blazei Murrill, thuc h Agaricaceae, b Acaricales, ngnh ph Basidiomycotina, ngnh Eumycota, gii Mycota. Nm m Blazei cn c cc tn gi khc l Nm m Ba T, Nm thng ln, Nm N hong, Nm ca Cha (Cognelo de Deus), Nm Himematsutake. Ngy 24-4-1944 nh khoa hc W. Blazei pht hin thy vng tho nguyn Gainesville gip bin i Ty Dng, thuc bang Florida mt chng nm l mang k hiu F. 32911. Nm ny v sau cn tm thy c min Nam California, Brazil v Peru. Ma h nm 1965 mt thng gia Brazil gc Nht tm thy vng ng c ngoi Pedade, thuc Sao Paolo (Brazil) mt loi nm n c hng v thm ngon m nhn dn a phng gi l Nm ca Cha. ng a bo t nm ny v Nht v nghin cu nui trng ra qu th. H t tn l Nm i Nguyn Nhung. Mi n nm 1967 cc nh phn loi mi thy cc nm ni trn l thuc loi Agaricus blazei Murill. Nm 1991 tin s Tin Minh Diu Trung tm nghin cu nm n , thuc Vin Khoa hc nng nghip T Xuyn (Trung Quc) a c nm ny t Nht Bn v v nui trng th nghim thnh cng. Nm 1992 nm ny c nghin cu k lng v c tnh sinh hc v c im nui cy bi S nghin cu Bo v thc vt v S nghin cu t phn, trc thuc Vin Khoa hc Phc Kin. Ngh nui trng nm m Blazei lan rng ti cc vng Phc Chu, Vu c, La Nguyn, Tin Du, Vu Kh thuc Phc Kin. Sau vic trng nm ny lan rng ra nhiu tnh Trung Quc. Sn phm nm Ba T T Xuyn v Phc Kin ch yu c bn sang Nht Bn v mt phn c p mui tiu th ni a. Nm Agaricus blazei c m hnh bn cu, v sau dt dn li. ng knh m nm thay i trong khong 512cm, mu vn nu, c cc vy mu nu.Tht nm mu trng, gia dy, ngoi mng. Phin nm mu trng, lc gi chuyn thnh mu nu en. Cung nm di khong 5-13cm, ng knh 1,0-2,5cm, mu trng hay mu vng nht. Bo t m (basidiospore) c hnh t bu dc n hnh trng vi kch thc trong khong 5,2-7,0 x 3,7- 6,31mm. m c kch thc 5,5- 7,5 x 15,2-20,0 mm, trn nh c 4 cung nh mang bo t m. Qu th gn ging vi cc nm m khc ( Agaricus bisporus, A. bitorquis) nhng c mu t nu tro nht n mu vng nu vi nhiu vy dng si. ng knh m nm khong 6-11cm. Cung di 4-14cm, ng knh 2-3cm, hnh vin tr, nm chnh gia qu th, trn di u nhau, mu sc v b mt gn ging vi m nm. Phn tht nm 385
Vi sinh vat hoc Nguyen Lan Dung dy khong 11mm. Si nm c b rng khong 3-6mm. Nhit thch hp cho s pht trin ca h si nm l 10-370C, tt nht l 23-270C. Vi qu th th nhit thch hp sn sinh l 16-330C , nhng tt nht l 18-250C. Nm m Blazei thch hp trng mim Bc nc ta trong v ng Xun. Khng thch hp trng trong ma h hoc trng ti min Nam. Khi rt di 100C th si nm khng mc, di 190C si nm sinh trng chm. Vi nhit 290C tuy si nm mc rt nhanh nhng li rt yu v d lo ha. Trn 300C si nm bt u mc chm li. Ti 370C si nm b cht kh. Khi nhit qu 250C qu th vn hnh thnh nhng cung di v d xe m nm. m trong nguyn liu nui cy si nm Agaricus blazei tt nht l vo khong 58,5% ( ng vi t l 1 nguyn liu : 4 nc). m khng kh cng l yu t quan trng pht trin si nm v sn sinh qu th, m tng i thch hp nht pht trin si nm l 60-85%, sn sinh qu th l 8595%. m ca lp t ph nn l 60-65%. Cn trang b cc m k trong phng trng nm. pH thch hp cho s pht trin ca si nm l 4,5-8,5 , nhng tt nht l 6-7. Khi hnh thnh qu th tt nht mi trng nn c pH vo khong 6,5-7,5. pH ca lp t ph nn l 7. Nm m Blazei c v ngon v c gi tr dinh dng cao. Nu tnh theo trng lng ti th trong 100g nm c ti 85-87g nc. Cn nu tnh theo trng lng kh th trong 100 g nm c 40-50g prtin, 38-45g hydrat carbon, 6-8g cellulose, 5-7 g cht khong, 3-4g lipid th, ngoi ra cn c 0,3mg Thiamin (vitamin B1), 3,2mg Riboflavin ( B2), 4,92mg Axit nicotinic. Protein ca nm ny c 18 loi axit amin, trong c y 8 axt amin khng thay th (valin, leucin, isoleucin, methionin, threonin, phenylalanin, tryptophan,lysin). c bit quan trng l gi tr dc liu ca nm m Blazei. Tc dng khng khi u (chng ung th ) ca nm ny c thy r 6 loi polysacharide sau y: b-glucan Flo-a-b; a-glucan FA -1-a-a; b-galactoglucan FA-1a-b; acid ribonucleic FA-2-b-b; Proteinic glucan F III-2-b; Xyloglucan FIV-2-b. C ch tc dng v hiu qu khng khi u ca polysacharide nm m Blazei c nu ln trong rt nhiu cng trnh nghin cu c h thng ti nhiu nc. T xa xa, con ngi s dng ch lm t nm m Blazei iu tr nhiu chng bnh: cc bnh lin quan n nhng ri lon h thng min dch, tun hon, tiu ha, cng nh chng tiu ng, cc hi chng d ng hay cc ri lon v gan. Cu Tng thng M Ronald Reagan cng tng iu tr bng nm m Blazei chin thng cn bnh ung th da v qua s kin , hiu qu dc hc ca nm ny c c th gii bit n. K t khi hiu qu dc hc ca nm m Blazei c pht hin, rt nhiu nghin cu v ti ny c thc hin trn ton th gii. c bit, cc nghin cu trong sut thi gian 25 nm qua u ch ra rng nm m Blazei c tc dng rt ln trong vic phng v chng ung th. Theo cc cng trnh nghin cu ca cc nh khoa hc Nht Bn, nm m Blazei c hiu qu phng ung th t ti 99.4% v hiu qu iu tr ung th cng t ti 90 %. Cng theo cc nh khoa hc Nht Bn, nm m Blazei t ra c hiu qu cao hn 80% so vi PSK, thuc tt nht dng trong iu tr ung th. Hin nay, nm m Blazei ang c s dng rng ri trong tr liu ung th nhiu nc chu . iu ng lu l nm m Blazei l mt loi nm n ngon, giu dinh dng v khng gy ra bt k mt phn ng ph no c hi i vi c th con ngi. 386
Vi sinh vat hoc Nguyen Lan Dung S d nm m Blazei c hiu qu dc hc cao nh vy l do trong thnh phn ha hc ca si nm cng nh qu th ca nm ny c nhng hot cht c bit vi nhng tc dng min dch c hiu : - Cc phc h polysaccarit protein: bao gm ch yu l cc Beta-1,6-D-glucan, Beta-1,3-D-glucan [3], [7], [8], mt s nghin cu cho rng bao gm c cc Beta1,4aD-glucan [4]. Cc polysaccarit ny c tc dng hot ho mnh m cc i thc bo (macrophage) trong h thng min dch ca c th. Cc i thc bo rt quan trng v chng l hng ro bo v u tin ca c th. S tng cng hot ng ca cc i thc bo ng ngha vi s tng cng kh nng phng chng, ngn chn cc mm mng bnh tt ngay t giai on xm nhp u tin. S hot ho cc i thc bo cn dn n s tng hm lng cc cytokin. y l cc protein ti quan trng chu trch nhim xc tc v iu ho hng lot cc phn ng min dch ca c th. Bn cnh , cc i thc bo cn c cc tng tc vi cc t bo Lympho T, c tc dng khi ng cc phn ng min dch c hiu [5]. V th, khi hot ng ca cc i thc bo c hot ho, cc phn ng c hiu ny cng c tng cng. Ngoi ra, nhiu nghin cu cn ch ra rng cc polysaccarit k trn cng c tc dng c ch s pht trin khng bnh thng ca t bo, c bit l chng c tc dng kch thch, lm tng s lng cc t bo lymph gy git (killer cells) t nhin. Cc t bo ny c kh nng pht hin v tiu dit cc t bo pht trin khng bnh thng ca c th, cc t bo ung th. Nh vy, nhn mt cch tng th, cc polysaccarit trong thnh phn ca nm m Blazei c tc dng lm tng cng rt mnh kh nng min dch ca c th. - Cc steroid t nhin, cc dn xut Ergosterol: Cc steroid t nhin ca nm m Blazei c tc dng ngn nga ung th, c bit l ung th t cung [9]. Cc Ergosterol c bit n nh cc tin vitamin D2, mt loi tc nhn cng c kh nng chng ung th. nui cy si nm nm m Blazei trn mi trng thch nghing ( ging cp 1) nn s dng mt trong s cc mi trng sau y: MT1 MT2 MT3 MT4 MT5 Khoai ty 200g 200g 200g ng knh 20g 5g Glucose 20g 20g Bt ng 30g Phn hoai 100-150g 50g 50g MgSO4 0,5g KH2PO4 1,0g CaCl2 0,1g Fe2SO4 0,1g CaSO4 1g Thch 20g 20g 20g 20g 20g Nc 1000ml 1000ml 1000ml 1000ml 1000ml 387
Vi sinh vat hoc Nguyen Lan Dung sn xut ging cp 2 trong cc chai thy tinh ming rng c nt bng c th s dng mt trong cc mi trng sau y:
1- Rm r- 375kg; Cm go- 10kg; Phn g kh-15kg; Vi sng- 8kg; Ammon sulphat- 10kg; Super phosphat5kg; Nc- m.
2- Rm r- 350kg; Phn b kh- 75kg; V ht bng-65kg; Thch cao-%kg; Super phosphat- 5kg; Ur- 2,5kg; Nc m Ngm rm, v ht bng trong 2-3 gi. Ly ra cho ro nc, thnh ng trong 1-2 ngy. Phn b, phn g kh cn nghin nt, sng qua li st, thm nc, thnh ng trong 1-2 ngy. Sau ly ra mt tng nguyn liu th ( rm r, v ht bng...), ri n mt tng ph liu ( phn kh v cc nguyn liu khc). Xp thnh ng c mt ct ngang hnh thang, Ph bao ti m hay vi nha (nilon) ln trn. Chn hai bn bng gch cho khi bay v gi. ln men trong ngy. a mi trng vo chai dung tch 750ml , c c chai ng knh 3cm; hoc cc ti mng mng PE hay PP di 33-35cm, dy 0,04-0,05mm. Nn nguyn liu cht nhng vn m bo xp. Nu l ti nha th phi to ra c bng lun u ti qua mt on ng nha cng. Lm nt bng c cht va phi. a i hp kh trng bng hi nc si trong thi gian 7-8 gi . Khng dng ni hp p lc v nhit cao trn 1000C s lm rch ti mng mng. i ngui ly ra cy ging t ng ging cp I ( mc trn thch nghing). cy xong y li nt bng hay y bng mng mng c c thng vi l ri bao li bng giy bo ra ngoi. Gi cc bnh hoc ti cy ging trn gi t trong phng c nhit iu chnh mc 23-270C v c m khng kh 65-75. Khi si nm mc kn ti th ta dng cy vo mi trng sn xut (cp III). Cc nguyn liu dng nui trng A. blazei gm ch yu l rm r, thn cnh l ng, u, ma... v ht bng, b ru, li ng, kh du... Ngoi ra l phn gia sc, gia cm, phn m, phn ln, phn kali, MgSO4, thch cao, CaCO3, bt tit. T l C : N nn l 33 : 1. Cc nguyn liu ny cn ng ln men to nhit. Nhit c th ln ti 70 - 75oC. nhit ny cc vi sinh vt (tr bo t ca chng). Cc loi cn trng, tuyn trng cng u cht ht. Cc x khun a nhit s hot ng mnh m nhit cao ny, lm phn hu cc cht cao phn t phc tp ny thnh cc ng phn t thp. V sau si nm s dng cc hp cht phn t thp ny tng hp ra sinh khi ca chng. nguyn liu l khu u tin rt quan trng sau qu trnh phi trn nguyn liu. Dng rm kh, cha mn, cha mc, khng dng rm r ln bi bm do phi trn quc l. C 1 tn rm kh th dng 20kg vi t. Ho vi vo nc to dung dch c mu trng c nh nc vo go. Nhn chm rm r 388
Vi sinh vat hoc Nguyen Lan Dung trong 3 - 5 pht. Rm r chuyn sang mu vng nht. Vt ra ro 3 - 5 pht sau a vo ng . a rm r ny vo ng , thng c kch thc 1 x 1,5 x 1,8m. C xp rm r ln cao 30 - 50cm th ngng li rc lp ur. Li xp tip mt lp rm r khc cao 30 - 50cm. Li rc tip ur sao cho ng cao ti 1,5m. Cn phn phi ur sao cho c 1 tn rm r kh ch dng va ht 5kg ur. Khi rc lu phn di rc t m ur, cng ln trn cng nhiu ln. Nn rm trn nn b tng, bn di c lt ni lng, khng trc tip trn nn b tng v cng khng trn nn t, nn gch. xong ly ni lng ph kn c ng . Dng dy thng hoc dy ni lng buc cht xung quanh ng c hnh thang. Sau 3 ngy r ra. Ly nguyn liu pha trn xung di. Khi c dy 30 - 50cm tin hnh rc phn ln (nn dng loi ln meng chy mn - thermophotphat, ca nh my phn ln Vn in), cng lm ln lt nh trn ch c thay m bng ln, v o lp rm t trn xung di t di ln trn. Lm sao khi ht rm th cng va ht phn ln v vn theo nguyn tc khi rc phn ln th di rc t, trn rc nhiu. Dng ht 30kg phn ln nung chy cho 1 tn rm kh. Cng y ni lng v buc nh ln 1. Sau 3 ngy li r ra v lm li nh ln 2. Ln 3 thay phn ln bng bt nh, tc l CaCO3. Cng lm nhu trn, cch 30 - 50cm li rc bt nh 1 ln, di rc t trn rc nhiu. Sao cho rm lp trn o xung di, di o ln trn khi xp rm vo li. C 1 tn rm kh dng ht 25kg bt nh. Cng ph ni lng v buc cht li nh 2 ln trn. Sau 3 ngy li r ra, kim tra m, nu cn th b sung nc cho khi kh qu, o tung ng rm ln ri li nh cc ln trc nhng khng b sung thm nguyn liu g na. thm 3 ngy l . Tt c cn tng cng l 12 ngy. Khi rt mt nm nguyn liu mt gc no , nm vo bn tay v bp li, nu thy mt vi git nc r ra l tt, khi th thay ra thy nguyn liu vn kt dnh vi nhau thnh mt nm. Nu thy nc chy ra nhiu hn th cn hong phi nguyn liu 10 - 20 pht ri kim tra li m theo cch ni trn. Nu khng thy nc r ra k ngn tay cn pha 5kg vi t vo 100 lt nc sch, sau thm nc ny dn dn vo nguyn liu cho n khi no kim tra m thy va mi thi. Tip tc y ni lng thm 3 ngy na (thnh ra 15 ngy c thy). Ch khi o ng lm 4 cn gim chiu di v tng thm chiu cao cho ng . Sau khi hon thnh qu trnh rm ta cho vo cc ngn ca gi hay xp ngay trn nn nh thnh cc lung rung 1m, chiu di tu (ph thuc vo kch thc gi hoc nn nh). Chiu cao lp nguyn liu l 20 22cm. Nn dng tay cun rm r li thnh tng b pha trn c dang trn, cao 20cm, ng knh 10 - 15cm. Xp theo chiu thng nh vo lung. Trc t bao ti da lt pha bn di lung. Nu lm gi nhiu tng th tng n phi cch tng kia 50 - 60cm. Chiu cao ca gi khng nn qu 2m d chm sc. Di y tng tng u c lt bao ti da. Khi lm trn nn nh, trc khi tri bao ti da nn rc ln nn nh mt lp vi bt kh. Vo lung c 7 ngy th dng nhit k cm su khong 8 - 10cm, 5 pht ly ra xem nhit . Nu t 22 25oC l tt. Nu nhit l 25 - 28oC l do xp nguyn liu qu cht tay nn sinh nhit. Khi cn tho b bt mt vi nm nguyn liu ra v xp li nguyn liu t trn quay o xung di, di quay ln trn. Sau 1 ngy 389
Vi sinh vat hoc Nguyen Lan Dung mi cy ging. Nu 7 ngy m thy nhit t yu cu th bt u cy ging vo lung. Dng co lm bng thp vi 3 - 5 rng, cc rng di 5cm, cch nhau 3cm, cn di hay ngn tu iu kin c th. Co xi u trn mt lung theo tng m2 mt. C 1m chiu di ca lung th dng 1 ti ging c si nm mc trng ht (0,5kg). Ch co theo mt hng (hoc ngang hoc dc), khng co ngang dc ln ln. Khi co nu c phn nguyn liu mc vo rnh co th ta ly ra gom li sau dng lm nguyn liu ph trn b mt lung. Thng lm ging bng thc. Khi bp ging ti ra v rc vo nhng ng rnh co trn b mt lung. Phn ging no mc trn b mt th dng tay gt xung rnh. Sau cng ph mt lp mng nguyn liu ln trn b mt lung, dy khong 1,0 - 1,5cm. Cy ging xong dng giy bo hay giy bao xi mng ph kn ton b b mt lung. Dng bnh phun m nc lm m trn khp b mt lp giy ph. To m nh vy trong 15 ngy lin (khng to m vo ban m). n ngy th 15 b lp giy ph trn mt lung ra, ly tay nhc mt nm nh nguyn liu ra thy c si nm trng mc to lan u trn nguyn liu. Bt u n giai on ph t. Ly t rung tng canh tc, luyn tht k nh kiu lm "pho t". Ln trn ri khoanh li nh chic vng ln v ni rm mt trong 12 gi. Nu thy vng t b t ra tng on l t ln ct, khng dng c. Nu t rn chm chim l dng c. Th c t tt mi khai thc v dng. C 1 tn nguyn liu cn chun b 500kg t. Ti mng t ra phi trn sn. Dng dung dch Formol 0,05% phun vo t nhm kh trng t. Phi t 15 ngy, tri ma th y che, khi nng li m ra phi. Rc t b ra thnh tng ht, tng vin to bng khong ht ng ph ln khp b mt ca lung. dy lp t ph khong 1,0 - 1,5cm. Tuyt i khng dng t vn, t bt hoc cc cc t qu ln. Ph t xong phun m nc (phun sng bng bnh bm thuc su cha ng thuc su), c 2 gi phun m mt ln, phun lin tc trong 3 ngy u (ch phun ban ngy). Ly th mt vin t b ra quan st. Nu thy c mu nu t ngoi vo trong l t t m. Nu gia vin t c mu trng c l cha m. Khi phi phun thm 1 ngy na ri li kim tra m li theo cch ny. Sau 2 ngy to m ta bt u phun ti cho cc lung. Nu tri hanh kh mi ngy phun khng qu 4 ln, nu tri m t ch phun 1 ln vo lc chiu ti. Khi pht hin thy qu th nm bt u xut hin lm tm nh ht ht ng mu trng th phi tng thm s ln phun ti. Dng bnh phun, quay nga vi phun phun m. Phun nhiu ln trong mt t ti. Tri hanh kh phun nhiu, nhng khng qu 6 ln. Tri ma m, th khng nn phun. T lc nm xut hin n khi thu hi c qu th l khong 72 gi. Khi cn nm mc ln c 2 - 3cm k t mt t th cn thu hi. Cn chn qu th no to hi trc. Mt tay gi phn t st chn nm, mt tay t vo chn nm nh thng ln cho ra c phn r. Dng dao sc ct b phn r ca tng qu th sau mi cho nm vo r. Hi nm xong nu thy b mt lung c ch no b lm xung th phi dng t d tr b sung vo. Sau khi hi mi t phi phun nc ngay gi m. 390
Vi sinh vat hoc Nguyen Lan Dung Nu khng bn nm ti th cn ct ri sy hoc tin hnh mui nm trong cc can nha. Cc phng nui trng nm A.blazei cn sch v kn, cng ti cng tt. Khng nn m ca ra vo nhiu. Tuy nhin phi c h thng thng gi. Sau mi t nui trng nm cn dng li 5 ngy lm v sinh v kh trng phng nui trng. Dng vi bt rc ton b nh xng (5kg vi bt/100m2) sau dng Formol nng 0,05% phun. t xng khi trong thi gian l 5 gi. Sau 5 ngy mi trng tip. Mi nm nn 3 thng tri v khng trng nm kh trng nh xng. Trn y ch l mt cng thc phi trn dng rm r v phn khong, bt nh. Mi trng sn xut cn c th c theo mt trong cc cng thc khc sau y:
1. Phn gia sc kh (ln, tru, b) 55% Rm r kh 40% Kh du 2 - 3% Thch cao 1% Supe ln 0,5% Nc Khong 160%
2. Rm r 1000kg Bt tit 40kg Phn nga 100kg Ur 10kg Supe ln 40kg CaCO3 20kg t ph 500kg Nc khong 2500 lt
3. Phn nga 1000kg Phn g 100kg Thch cao 25kg Nc 200 - 800 lt
391
Vi sinh vat hoc Nguyen Lan Dung 4. Phn g (40% m) 800kg Rm r kh 1000kg Thch cao 75kg Nc 5000 lt
5. Rm r 1000kg Phn g 100kg Thch cao 10 - 20kg Nc To m 65% Da vo cc cng thc ni trn, cn c vo cc nguyn liu sn c tng a phng ngi trng nm Agaricus blazei c th t xy dng ra cc cng thc phi trn th nghim. Ti liu tham kho: 1. Antitumor effect of a peptide-glucan preparation extracted from Agaricus blazei in a double-grafted tumor system in mice. Ebina T, Fujimiya Y. Division of Immunology, Miyagi Cancer Center Research Institute, Natori, Miyagi, Japan. Biotherapy 1998;11(4):259-65 2. Selective tumoricidal effect of soluble proteoglucan extracted from the basidiomycete, Agaricus blazei Murill, mediated via natural killer cell activation and apoptosis. Fujimiya Y, Suzuki Y, Oshiman K, Kobori H, Moriguchi K, Nakashima H, Matumoto Y, Takahara S, Ebina T, Katakura R. Division of Immunology, Miyagi Cancer Center Research Institute, Natori, Japan. Cancer Immunol Immunother 1998 May;46(3):147-59 3. Anti-tumor polysaccharide from the mycelium of liquid-cultured Agaricus blazei mill. Mizuno M, Minato K, Ito H, Kawade M, Terai H, Tsuchida H. Graduate School of Science and Technology, Kobe University, Japan. Biochem Mol Biol Int 1999 Apr;47(4):707-14 4. Tumor-specific cytocidal and immunopotentiating effects of relatively low molecular weight products derived from the basidiomycete, Agaricus blazei Murill. Fujimiya Y, Suzuki Y, Katakura R, Ebina T. Division of Immunology, Miyagi Cancer Center Research Institute, Natori, Japan. Anticancer Res 1999 Jan-Feb;19(1A):113-8 5. Polysaccharides from Agaricus blazei stimulate lymphocyte T-cell subsets in mice. Mizuno M, Morimoto M, Minato K, Tsuchida H. Division of Science of Biological Resources, Graduate School of Science and Technology, Kobe University, Japan. Biosci Biotechnol Biochem 1998 Mar;62(3):434-7 6. [A stable culturing method and pharmacological effects of the Agaricus blazei]. [Article in Japanese] Higaki M, Eguchi F, Watanabe Y. Lab. of Forest Products Chemistry, Tokyo University of Agriculture, Japan. Nippon Yakurigaku Zasshi 1997 Oct;110 Suppl 1:98P-103P 7. Antitumor effects of a new polysaccharide-protein complex (ATOM) prepared from Agaricus blazei (Iwade strain 101) "Himematsutake" and its mechanisms in tumor-bearing mice. 392
Vi sinh vat hoc Nguyen Lan Dung Ito H, Shimura K, Itoh H, Kawade M. Department of Pharmacology, Mie University School of Medicine, Japan. Anticancer Res 1997 Jan-Feb;17(1A):277-84 8. Inhibitory action of a (1-->6)-beta-D-glucan-protein complex (F III-2-b) isolated from Agaricus blazei Murill ("himematsutake") on Meth A fibrosarcoma-bearing mice and its antitumor mechanism. Itoh H, Ito H, Amano H, Noda H. Laboratory of Marine Biochemistry, Faculty of Bioresources, Mie University, Japan. Jpn J Pharmacol 1994 Oct;66(2):265-71 9. [Antimutagenic and bactericidal substances in the fruit body of a Basidiomycete Agaricus blazei, Jun-17]. [Article in Japanese] Osaki Y, Kato T, Yamamoto K, Okubo J, Miyazaki T. Tokyo College of Pharmacy, Japan. Yakugaku Zasshi 1994 May;114(5):342-50 10. Illustration for China popular edible mushroom Vn Nam Xut bn x, 1999 11. C Thng Nhung ti bi k thut thuyt H Bc Khoa hc K thut Xut bn x, 2003 12. Trung Quc dc dng khun sinh sn d sn phm khai pht TrungQuc nng nghip xut bn x, 2000
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 393
Vi sinh vat hoc Nguyen Lan Dung 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: 394
Vi sinh vat hoc Nguyen Lan Dung 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
395
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.
396
Vi sinh vat hoc Nguyen Lan Dung 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.
397
Vi sinh vat hoc Nguyen Lan Dung Nng agarose (%) 0.3 0.5 0.8 1.0 1.2 1.5 2.0 Kch thc ADN (kb) 1-7 0.7-45 0.4-20 0.3-10 0.2-8 0.2-6 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. 398
Vi sinh vat hoc Nguyen Lan Dung 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.
399
Vi sinh vat hoc Nguyen Lan Dung - 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 oC. 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 oC) 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 AatII ApaI ClaI MluI NarI NheI NotI NruI PvuI SacII SalI SfiI SmaI XhoI
V tr ct GACGT/C GGGCC/C AT/CGAT A/CGCGT GG/CGCC G/CTAGC GC/GGCCGC TCG/CGA CGAT/CG CCGC/GG C/TCGAC GGCC(N)4/NGGCC CCC/GGG C/TCGAG
ng dng ca k thut phn tch PFGE: 400
Vi sinh vat hoc Nguyen Lan Dung 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 Gouby et al (1992) 2. 3. 4. 5. 6. 7. 8. 9. Brucella spp Campylobacter hyointestinalis Campylobacter jejuni CADNida arabicans CADNida prapsilosis Coxiella burnettii Enterobacter cloacae Enterococcus faecium 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)
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
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: 401
Vi sinh vat hoc Nguyen Lan Dung + 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 402
Vi sinh vat hoc Nguyen Lan Dung 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 32 P
35
Ti liu 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)
Alkaline phophatase Ethidium Fluorescein Horseradish peroxidase Microperoxidase Nitrobenzofuran Tetramethylorhodamine
403
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.
404
Vi sinh vat hoc Nguyen Lan Dung Nhm chc nng bm protein Biotin-dUTP/steptavidin Digoxigenein-dUTP/antidigoxigenein Sulphone/antisulphone Dinitrophenyl/antinitrophenyl Poly(dA)/poly(dT) Acetyllaminofluorene/antiacetylaminofluorene Nhm tn hiu (reporter) Alkaline phosphatase Alkaline phosphatase Europium chelate Piruvate kinase Horseradish peroxidase Alkaline phosphatase Ti liu dn 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 32P v 35S. 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. 405
Vi sinh vat hoc Nguyen Lan Dung 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). 406
Vi sinh vat hoc Nguyen Lan Dung 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.
407
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 80oC 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 408
Vi sinh vat hoc Nguyen Lan Dung 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.
409
Vi sinh vat hoc Nguyen Lan Dung 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/cm2 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. 410
Vi sinh vat hoc Nguyen Lan Dung Bng 1.6. Kt qu thu c khi thc hin k thut RFLP vi dng cc mu d khng phi l ribosom.
i tng Aeromonas spp Borrelia burgorferi Candida albicans Chlamydia trachomatis Corynebacterium diphtheriae Cryptococcus noeformans Escherichia coli Hemophilus influenzae Histoplasma capsulatum Lactobacillus helveticus Mycobacterium tuberculosis Pseudomonas aeruginosa Salmonella spp Staphylococcus aureus
Tc gi nghin cu v ti liu dn Altwegg an Luthyhottenstein (1991) Wallich et al (1992) Schmid et al (1992) Scieux et al (1992) Groman et al (1993) Spitzer (1992) Bohm ADN Karch (1992) Forbes et al (1992) Leathet al (1992) Delostreyesgavilan et al (1992) Mazurek et al (1991) Tompkins(1991) Sodati ADN Piffaretti (1991) 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: 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.
thu c kt qu ti u cn c cc yu cu k thut c th cho tng bc thc hin.
411
Vi sinh vat hoc Nguyen Lan Dung 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 horseradiskperoxysase-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.
412
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 413
Vi sinh vat hoc Nguyen Lan Dung 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 1011 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 414
Vi sinh vat hoc Nguyen Lan Dung 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 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
Ti liu 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)
415
Vi sinh vat hoc Nguyen Lan Dung Frankia spp Gaeumannomyces spp Helicobacter pylori Hepatitis C virus Human immunodeficiency virus (HIV) Influenza virus Merien et al (1992) Leptospira spp Niederhauser et al (1992) Litsteria monocytogenees Robertson et al (1991) Luteovirus Hackel et al (1990) Mycobacterium leprae Altamirano et al (1992) Mycobacterium tuberculosis Maiden et al(1992) Neisseria meningitis Charlotte et al (1993) Papillomavirus McOmish et al (1993) Parvovirus Barker et al (1992) Plsmodium falciparum Olsson et al (1993) Pneumocystis carini Yang et al (1991) Polio virus Seal et al (1992a) Pseudomonas solanacearum Gage et al (1992) Rickettsia spp Taniguchi et al (1992) Rotavirus Rahn et al (1992) Salmonella spp Murakami et al (1991) Staphylococcus spp Vanevan et al (1991) Toxoplasma gondii Grimprel et al (1991) Treponema pallidum Riley et al (1992) Simonet et al (1990) Henson (1992) Claton et al (1992) Okamoto et al (1992) Dawood et al (1992) Claas et al (1992)
416
Vi sinh vat hoc Nguyen Lan Dung Trichomonas vaginalis Breniere et al (1992) Trypanosoma cruzi Hill et al (1991) Vibrio vulnificus Nakajima et al (1992) Yersinia
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 94oC 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 oC. Hot tnh enzym gim mt na khi x l ti 92.5oC trong 230 pht hoc 40 pht ti 95oC. 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 417
Vi sinh vat hoc Nguyen Lan Dung m phosphat v kt qu s lm gim Mg++ (do EDTA hoc to thnh mui Mg3(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 ADN khun dNTP (mi loi) Mi xui Mi ngc Taq polymerase KCL Tris HCL (pH 8.4) ti 25oC MgCl2 Gelatin Nonidet-40
S lng 10-100 ng 200 mM 0.1 1.0 mM 0.1 1.0 mM 1U 50 mM 10 mM 2.85 mM 100 mg/l 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 418
Vi sinh vat hoc Nguyen Lan Dung 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 Chlamydia spp. Clostridium spp. Helicobacter pylori Hepatilis C virus Mycobacterium tuberculosis Nitrobacter spp. Rickettsia spp. Tc gi nghin cu v ti liu dn Kaltenboek et al (1992) Gurtler et al. (1991) Foxall et al. (1992) Okamoto et al.(1992) Ross &Dwyer(1993) Navarro et al.(1992) Regnery et al. (1991) 419
Vi sinh vat hoc Nguyen Lan Dung 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 Vi khun lactic Listeria Mycoplasma Pseudomonas cepacia Pseudomonas solanacearum Staphylococcus spp. Streptococcus spp. Tc gi v ti liu dn Klijin etal.(1991) Czajka et al.(1993) Van Kuppeveld et al.(1992) Kostman et al.(1992) Seal et al.(1992b) Welsh &McClellADN(1992) 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 palindromicREP) 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, 420
Vi sinh vat hoc Nguyen Lan Dung 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 Agaricus bisporus Aspergillus fumigatus Bacillus thuringiensis Brucella spp. Campylobacter spp. CADNida spp. Casuarina spp. Clostridium difficile Frankia spp Fusarium solani Helicobacter pylori Histoplasma capsulatum Lactococcus lactic Leptosphaeria maculans Listeria monocytogenees Porphyromonas gingivalis Rhizobium spp. Staphylococcos spp. Streptococcus spp. Streptococcus uberis
Tc gi v ti liu dn Khush&ctv(1992) Aufauvre-Brown (1992) Brousseau&ctv (1993) Fekete&ctv(1992) Mazurier&ctv(1992) Lehmann 7 ctv (1992) Sellstedt& ctv(1992) McMillan& Muldrow(1992) Sellstedt& ctv(1992) Crowhurst & ctv(1991) Akopyanz & ctv(1992) Woods& ctv (1993) Cancilla & ctv (1992) Goodwin&Annis(1991) Czajka & ctv (1993) Menard &ctv (1992) Harrison &ctv (1992) Weish &McClellADN (1990) Weish &McClellADN (1990) Jayarao &ctv (1992)
421
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 422
Vi sinh vat hoc Nguyen Lan Dung 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. 2. 3. 4. 5. Chun b ADN khun. iu kin bt cp mi. Thc hin phn ng gii trnh t ADN. Tch sn phm trn acrylamid gel. 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 423
Vi sinh vat hoc Nguyen Lan Dung 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 P32 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 S35. Hin nay gii quyt vn ny ngi ta dng P33 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 P32 c dng nh du sn phm. Tuy nhin ngy nay nhiu ngi chuyn sang dng S35 thay th cho P32, 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 50100 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.
424
Vi sinh vat hoc Nguyen Lan Dung * 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 37oC 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. 2. 3. 4. 5. 6. 7. 8. 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. Thm vo 0.3 ml m P2 v trn u, gi nhit phng 5 pht (khng nn lu hn). Thm vo 0.3 ml m P3 trn u ngay nhng phi nh nhng, ly tm 4 oC trong 30 pht vi tc 15000 v/pht. Thu ly phn trn ta. Ly tm li nh bc 3 mt ln na loi cc phn cn nu c. Cn bng m cho ct Qiagene-tip 20 vi 1ml QBT v cho m chy ht t do. a phn dch thu c bc 4 ln ct v t chy qua ct. Ra ct 2 ln, mi ln 1 ml m QC. Thu ADN vi 0.8 ml m QF. 425
Vi sinh vat hoc Nguyen Lan Dung 9. Kt ta ADN vi 0.7 th tch isopropanol (trc a v nhit phng). Ly tm 15000 v/pht ti 4oC. 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 m P2 m P3 50 mM Tris -HCl, 10 mM EDTA, pH 8.0, Rnase A pha ngay trc khi dng trong m P1 nng 100 g/ml 200 mM NaOH,1%SDS 2,55M KAc, pH 4,8 Bo qun 4oC Nhit phng Nhit phng Nhit phng Nhit phng Nhit phng
m QBT 750mM NaCl,50mM MOPS,15% ethanol, pH 7,0, 0,15% Triton X-100 m QC m QF 1M NaCl, 50mM MOPS, 15% ethanol, pH 7,0 1,25M NaCl, 50mM MOPS, 15% ethanol, pH 8,2
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.
426
Vi sinh vat hoc Nguyen Lan Dung 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 50-55oC, nhit cao hn 55oC 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. b. c. Trn gel nh nhng (trnh trn mnh). Ht dch acrylamid bng pitpet hay c th dng cc ong. t bn knh nghing 15o 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.
427
Vi sinh vat hoc Nguyen Lan Dung 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 50oC. 10, X l nhit vi cc mu ADN in di ti 85oC 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 428
Vi sinh vat hoc Nguyen Lan Dung 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 85oC 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 S35 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 80oC. 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 P32 v 24-48 gi i vi S35. 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 429
Vi sinh vat hoc Nguyen Lan Dung 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 105oC. 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 20oC. 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: m bin tnh plasmid: Primer: Nc ct dn n: 0.5 0.3 mg (th tch ti a 7 ml). 5 ml. 1 ml. 13 ml.
Trn u, trong 5 pht 90 100 oC, 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). 430
Vi sinh vat hoc Nguyen Lan Dung Thnh phn phn ng gm: ADN: NaOH 1M: Primer: 0.5 - 3mg (th tch ti a l 8ml) 2 ml. 1 ml.
Nc ct dn n: 11 ml. Trn u, gi trong 10 pht ti 37oC, 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: Tng th tch l: 2. Bt cp mi vo si khun: Gi hn dch ADN v mi 37oC 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: Tng s: 8 ml 10 ml. 2 ml. 15 ml.
5. Lm m cc mu chun b trong bc 3: Bn mu c chun b trong bc 3 ln lt l ACGT c 37oC 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 : Dch nh du (bc 4): Ha cht phng x: S35 (P33, P32): Enzym T7 sequenase cho plasmid: Tng th tch : 1 ml 2 ml 0.5 ml 2 ml 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 oC). Thng b sung hn dch T7 431
Vi sinh vat hoc Nguyen Lan Dung 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 S35 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 37oC (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 oC s dng trong ngy hm sau. d. X l nhit 75oC 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 dATP dCTP 7-deaseGTP dTTP ddATP ddCTP ddGTP ddTTP NaCl Dch dng phn ng: 95% Formamide
A 80M 80M 80M 80M 8M
C 80M 80M 80M 80M 8M
G 80M 80M 80M 80M 8M
T 80M 80M 80M 80M
50 mM
50 mM
50 mM
8M 50 mM
20mM EDTA (Na2), pH 8.0 0.05% Bromopheno Blue (BPB). 0.5% Xylen CyanolFF (XC). 432
Vi sinh vat hoc Nguyen Lan Dung Ch , nu bn gp trc trc vi vic gii trnh t ADN th cc plasmid nn c ti tinh sch vi GeneeClean 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. 433
Vi sinh vat hoc Nguyen Lan Dung 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:
434
Hnh 1.12. Thit b gii trnh t ADN t ngg (3100-Avant Genetic Analyzer)
435
Vi sinh vat hoc Nguyen Lan Dung 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): 80-100 ng 300ng) 3.2 pmol 20 l
- Terminator Ready Reaction Mix*: 8 l - Template: ( vi plasmid l: - Primer : - Nc ct v trnh:
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 96oC: 96 C: 50oC: 60oC: Cycling:
o
1 pht 10 giy 5 giy 4 pht 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 (4oC) - Lm kh mu nhit phng (Speed Vac). - Khi mu kh, thm 10l Hi-Di formamide, trn u sau 96oC 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). 436
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 Thng tin chung v Trung tm Cng ngh Sinh hc chng vi sinh vt bo qun Bo tng Ging chun vi sinh vt (VTCC)
1. Nm si 2. Tn khoa hc: Ging (Genus)
Nm men
X khun
Vi khun
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: 7. K hiu chng VTCC: 8. K hiu l l lch chng t cc bo tng khc: VTCC < < < < < Chng t nhin (wild) t bin (c th) Ni cung cp:
9. Chng chun (Type) 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: 437
Vi sinh vat hoc Nguyen Lan Dung 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:
438
Vi sinh vat hoc Nguyen Lan Dung 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) ATCC M DSMZ c NBRC Nht S lng chng vi sinh vt 73507 14460 18300 Bng 1. Quy m ca mt s b su tp ging vi sinh vt.
Nc
STT 1 2 3 4
Tn Bo tng vi sinh vt, Nc ATCC, M CBS, H Lan VKM, Nga Thi Lan
Gi thnh (USD) 80 60 45 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: 439
Vi sinh vat hoc Nguyen Lan Dung 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:
440
Vi sinh vat hoc Nguyen Lan Dung 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. 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. 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.
b.
c.
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:
441
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 (-196C -> -80 C).
442
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, -140C 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: 443
Vi sinh vat hoc Nguyen Lan Dung 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 121OC. 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 1010 (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: 444
Vi sinh vat hoc Nguyen Lan Dung 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 4OC 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. 445
Vi sinh vat hoc Nguyen Lan Dung 2, Pha dch t bo vi glycerol hoc DMSO thanh trng trc t nng cui cng 10% v mt t bo 106. 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 OC/pht t ti nhit -30OC ban u. Sau mu c lm lnh tip vi tc cao hn 10-15 OC/pht t ti nhit lnh cn thit (-100OC n -80 OC). 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 -65OC. 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 OC 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 OC 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 OC, 28 OC v 37 OC, 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 (24OC) trong thi gian 1 thng. p nh vo thnh ng cho cc ht tch u ra v bo qun mu ti 4OC. 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.
446
Vi sinh vat hoc Nguyen Lan Dung 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 OC). 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 OC trong 15 pht. Lp du cch b mt thch khong 1 cm. Nhit bo qun l 15-20 OC. 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 mm2 v cho vo l nc thanh trng. Bo qun ti nhit phng. Bo qun nm si c bo t trong silicagel:
b.
Chun b: 1, L ng silicagel (10-15ml) c np chu nhit (st hay nha) thanh trng nhit 170 OC 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 (115OC/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%), 25OC trong 1 tun. Vn cht nt v qun giy parafil gi trong 4OC. 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): 447
Vi sinh vat hoc Nguyen Lan Dung Chun b: 1, ng ng kh ra sch (sonic cleaner) trong 15 pht sau kh trng 170OC trong 3 gi. 2, Mi trng sa tch b (Skimmilk) 20% kh trng t (115OC/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 -80C trong 3 gi trc khi tin hnh ng kh, ng thi bt my Dura-Stop, khi nhit t -40 C, a mu vo t ng kh ca Dura-Stop, bt my Dura-Dry, khi mc chn khng t 45 minitor, nhit -55OC, tng nhit Dura-Stop ln -5OC, qua m.
O
4, Tng nhit Dura-Stop ln 25C, 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 -55OC. 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 -80C trong 3 gi. Bt my Dura-Dry, khi t nhit -60oC n -55oC, chn khng 45 minitor, gn ng mu vi h thng manifold tin hnh hn ng khi chn khng t 45-48 minitor, nhit -55oC.
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:
448
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 170oC 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 115oC/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:
449
Vi sinh vat hoc Nguyen Lan Dung 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 oC, 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 -55oC 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 37oC trong 2 tun. sng st ca mu bo qun gim trong 2 tun 37oC tng ng vi bo qun 10 nm 4oC.
e. Bo qun nm si bng phng php lnh su -80 oC v nit lng (-196 oC): Chun b: 1, Chun b dung dch glycerol 10% (3 ml trong ng nghim) kh trng 121 oC trong 15 pht. ng nha nt xoy (2 ml), ra sch trong sonic cleaner, kh trng 170 oC 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 5oC (to iu kin cho glycerol i vo t bo), sau c lm lnh su t 25oC xung -55oC vi tc -1oC/pht. Trong trng hp khng c thit b lm lnh th c th -20oC trong 3 gi, sau chuyn sang -80oC hay nit lng (-196oC). 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 450
Vi sinh vat hoc Nguyen Lan Dung 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 -80oC hoc nit lng -196oC) trong b n nhit 37oC 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 4-8oC.
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 121oC 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 25oC trong 72 gi. Phi kim tra s pht trin ca chng bo qun. Sau cc mu c gi 4oC. 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, 451
Vi sinh vat hoc Nguyen Lan Dung sau c 4-8oC. 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).
452

VI Sinh Vat Hoc

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

VI Sinh Vat Hoc

Uploaded by

Copyright:

Available Formats

Vi sinh vat hoc Nguyen Lan Dung

Vi sinh vat hoc Nguyen Lan Dung

Vi sinh vat hoc Nguyen Lan Dung

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)

Vi sinh vat hoc Nguyen Lan Dung

Nhng c im chung ca Vi Sinh Vt

Carl Von Linnaeus

H thng phn loi 5 gii sinh vt

Vi sinh vat hoc Nguyen Lan Dung

H thng phn loi 6 gii sinh vt

H thng phn loi 8 gii sinh vt

Vi sinh vat hoc Nguyen Lan Dung

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

c im Nhn c mng nhn v

Bacteria Khng Khng 11

Vi sinh vat hoc Nguyen Lan Dung

Kch thc vi khun so vi u kim khu

Vi sinh vat hoc Nguyen Lan Dung

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

Lactobacillus qua KHV in t

Vi kun Escherichia coli

Nm men Saccharomyces cerevisiae

Vi sinh vat hoc Nguyen Lan Dung Nh my Vedan-Vit Nam

Thnh phn Peptidoglycan Acid teicoic (Teichoic acid) Lipid Protein

Vi sinh vat hoc Nguyen Lan Dung

Thnh t bo vi khun Gram dng

Vi sinh vat hoc Nguyen Lan Dung

Vi sinh vat hoc Nguyen Lan Dung

Sinh vin in ch thch theo hng dn ca gio vin

So snh Ribosom Vi khun v cc Sinh vt nhn tht (Eukaryotic ribosome) 23

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

Vi sinh vat hoc Nguyen Lan Dung

Th nhn trong t bo vi khun Escherichia coli.

Vi sinh vat hoc Nguyen Lan Dung

Vi khun Leuconostoc mesenteroides

Tin mao v khun mao :

Tin mao v khun mao vi khun

Vi sinh vat hoc Nguyen Lan Dung

Tin mao VK Gram dng

Tin mao VK Gram m

Vi sinh vat hoc Nguyen Lan Dung

Tin mao vi khun G +

Vi sinh vat hoc Nguyen Lan Dung

Tin mao ca vi khun c cc loi khc nhau tu tng loi : 30

Vi sinh vat hoc Nguyen Lan Dung

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

Vi sinh vat hoc Nguyen Lan Dung

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

Khun mao v Khun mao gii:

Vi sinh vat hoc Nguyen Lan Dung

Vi sinh vat hoc Nguyen Lan Dung

Vi sinh vat hoc Nguyen Lan Dung

Vi sinh vat hoc Nguyen Lan Dung

Vi sinh vat hoc Nguyen Lan Dung

c-Nhm III (c tc gi gi l b Oscillatorriales):

Vi sinh vat hoc Nguyen Lan Dung

Vi sinh vat hoc Nguyen Lan Dung

Anabaena trong Bo hoa du

Vi sinh vat hoc Nguyen Lan Dung

Vi sinh vat hoc Nguyen Lan Dung