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Phn Tch Vi Sinh Thc

BPhm
CNG THNG

Nhm 6

TRNG I HC CNG NGHIP THC PHM


THNH PH H CH MINH

PHN TCH VI SINH


THC PHM
ti: Quy trnh nh tnh Salmonella
Danh sch nhm
1.
2.
3.
4.
5.

Hunh Phc Duy


Nguyn Thnh Ngc Phc
V Th Mai Trang
Nguyn Ngc Khnh
Trn Th Thu H

2005130382
2005130267
2005130370
2005130263
2005130387

GVHD : Nguyn Th Kim Oanh


Thc hin nhm 6
Lp Th 3, Bui chiu, Tit 7-8
ngy: Nguyn
24 thngTh
04 nm
Trang 0Thnh Ph H Ch Minh,
GVHD
Kim 2016
Oanh

Phn Tch Vi Sinh Thc Phm

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GVHD : Nguyn Th Kim Oanh

Nhm 6

Phn Tch Vi Sinh Thc Phm

Nhm 6

DANH SCH NHM V BN PHN CNG VIC NHM 6


H v tn

Mssv

Hunh Phc Duy

2005130382

Nguyn Thnh Ngc Phc


V Th Mai Trang
Nguyn Ngc Khnh
Trn Th Thu H

2005130207
2005130370
2005130263
2005130387

Cng vic
Quy trnh nh tnh Salmonella v hon
thin bi word
c im Salmonella
Tng hp v vit bo co
Cc phng php truyn thng
Cc phng php hin i

MC LC
DANH SCH NHM V BN PHN CNG VIC NHM 4........................1
MC LC.............................................................................................................1
DANH MC HNH NH.....................................................................................1

GVHD : Nguyn Th Kim Oanh

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Phn Tch Vi Sinh Thc Phm

Nhm 6

DANH MC VIT TT.......................................................................................1


LI M U.......................................................................................................1
CHNG 1

TNG QUAN V QU TRNH OXY HA..............................1

1.1 Mt s nh ngha.....................................................................................1
1.2 C ch qu trnh oxy ha..........................................................................1
1.3 Nguyn nhn qu trnh oxy ha................................................................1
1.3.1 Do ch n ung ca bn.......................................................................1
1.3.2 Do nhim mi trng............................................................................1
1.3.3 Do li sng...............................................................................................1
1.3.4 Do Bc x in t.....................................................................................1
1.3.5 Bnh mn tnh...........................................................................................1
1.4 Hu qu ca cc qu trnh oxy ha...........................................................1
CHNG 2 TC DNG CA THC PHM CHC NNG I VI
VN OXY HA.............................................................................................1
2.1 Tc dng chng lo ha v ko di tui th.............................................1
2.1.1 Khi nim.................................................................................................1
2.1.2 Biu hin ca lo ha...............................................................................1
2.1.3 Tc dng...................................................................................................1
2.2 Tc dng to sc khe sung mn..............................................................1
2.2.1 Khi nim.................................................................................................1
2.2.2 Cc yu t to nn sc khe sung mn.....................................................1
2.2.3 Tc dng...................................................................................................1
2.3 Tc dng tng sc khng, gim nguy c bnh tt................................1
2.3.1 Khi nim.................................................................................................1
2.3.2 Phn loi sc khng.............................................................................1
2.3.3 Tc dng lm tng sc khng, gim nguy c bnh tt........................1
2.4 Tc dng h tr lm p cho con ngi...................................................1

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GVHD : Nguyn Th Kim Oanh

Phn Tch Vi Sinh Thc Phm

Nhm 6

CHNG 3 MT S THC PHM CHC NNG C TC DNG


CHNG OXI HA...............................................................................................1
3.1 Enjuvenate Unicity tr ha t bo.............................................................1
3.1.1 Cng dng ca Enjuvenate Unicity tr ha t bo...................................1
3.1.2 Thnh phn Enjuventane..........................................................................1
3.1.3 Cc hp cht c trong Enjuventane..........................................................1
3.2 Fine Pure Collagen thuc chng nm, xa m np nhn, chng li qu
trnh lo ha.......................................................................................................1
3.2.1 Tc dng ni bt ca tng thnh phn trong Fine Pure Collagen............1
3.2.2 Thnh phn...............................................................................................1
3.3 Ageloc Vitality Nuskin Chng lo ha.....................................................1
3.3.1 Tc dng...................................................................................................1
3.3.2 Thnh phn ageloc vitality nuskin chng lo ha....................................1
3.4 Natures Bounty Hair,Skin &Nails...........................................................1
3.4.1 Cng dng................................................................................................1
3.4.2 Thnh phn...............................................................................................1
3.4.3 Tc dng ni bt ca cc thnh phn........................................................1
3.5 AGEL EXO THC PHM GEL CHNG OXI HA.........................1
3.5.1 Cng dng................................................................................................1
3.5.2 Thnh phn...............................................................................................1
TI LIU KHAM KHO....................................................................................1

DANH MC HNH NH
Hnh 1.1. Cc tc nhn gy ra qu trnh oxy ha..................................................1
Hnh 3.1.Thc phm chc nng Enjuvenate Unicity............................................1
Hnh 3.2. Thc phm chc nng Fine Pure Collagen...........................................1
Hnh 3.3. Thc phm chc nng Ageloc Vitality Nuskin.....................................1

GVHD : Nguyn Th Kim Oanh

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Phn Tch Vi Sinh Thc Phm

Nhm 6

Hnh 3.4. Thc phm chc nng Natures Bounty Hair........................................1


Hnh 3.5. Bng lit k cc thnh phn trong Natures Bounty Hair......................1
Hnh 3.6. Thc phm chc nng Agel EXO.........................................................1

DANH MC VIT TT
TPCN: Thc phm chc nng

LI M U
Nhng nm gn y vn v sinh an ton thc phm lun l tm im ca cc
phng tin truyn thng i chng. Ngy cng c nhiu v ng c thc, nht l vn

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GVHD : Nguyn Th Kim Oanh

Phn Tch Vi Sinh Thc Phm

Nhm 6

ng c thc phm do tnh trng mt v sinh an ton thc phm ca cc c s ch


bin c xu hng ngy cng gia tng. Cc bo co cho thy phn ln chng l do vi
sinh vt gy nn.
C rt nhiu vi sinh vt gy ng c thc phm, v d nh Clostridium
butolinum, Escherichia Coli, Listeria monocytogenes... Trong , Salmonella l loi vi
sinh vt gy ng c rt nguy him. Salmonella thuc h Enterobactriaceae, gy ra
bnh thng hn, nhim trng huyt v nhiu bnh nghim trng khc.
Hin nay c khong 4% ngi thng mang mm bnh. Tt c cc tiu chun
v sinh thc phm iu khng cho php c Salmonella trong thnh phm. S hin din
ca chng c kim tra gt gao bi cc c quan chc nng.
kim sot c chng, cc c quan nh nc a nhng quy trnh pht
hin Salmonella c quy nh bng cc vn bn rt c th. Nhm gii thiu mt trong
nhng quy trnh trn, chng ti thc hin ti: Quy trnh trnh nh tnh
Salmonella
Chng ti hy vng qua ti ln ny s cung cp thm cho cc bn mt s kin
thc v Salmonella, cc phng php c th dng pht hin Salnonella v qu trnh
pht hin Salmonella. Trong qu trnh thc hin ti khng trnh khi mt s sai st,
mong c v cc ng gp gip nhm hon thin bi.
Nhm 6

GVHD : Nguyn Th Kim Oanh

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Phn Tch Vi Sinh Thc Phm

Nhm 6

TNG QUAN V SALMONELLA


1.1 Lch s pht hin

Nm 1885 Slamon v Smith (M) tm c Salmonella t ln mc bnh dch t


v gi tn l Baclus cholerasus, hin nay gi l Salmonella. Nhng sau
Schweinittz v Dorset 1903 chng minh bnh dch t l do mt loi vi rt gy nn
v xc nh S.choleraesuis l vi khun gy bnh ph thng hn.
Nm 1888 A.Gartner phn lp c mm bnh t tht b v lch ngi bnh,
ng gi vi khun ny l Bacillus enteritidis v ngy nay vi khun ny c gi l
S.entertds. Vi khun ny cng c gi bng nhiu tn khc nh: Bacterium
enteritidis, Bacillus gartner...
Nm 1889 Klein phn lp c S.gallinarum v Rettger cng phn lp c

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GVHD : Nguyn Th Kim Oanh

Phn Tch Vi Sinh Thc Phm

Nhm 6

S.pullorum nm 1909. Trc y ngi ta cho rng y l hai loi vi khun gy ra hai
bnh khc nhau ln gi chung l bnh ph thng hn g Typhus avium) v cn
bnh c tn chung l S.gallinarum - pullorum.
Nm l896 C.Archard v Rbensauded phn lp c vi khun Sparatyphi
equi v Sparatyphi bacilus. Ngy nay vi khun ny c gi tn l Sparatyphi B v
n nm 1898, Sparatyphi A tm c do N.Guyn v H Keyser.

1.2 Phn loi


V phn loi khoa hc, Salmonella c xp vo:

Gii:

Bacteria

Ngnh: Proteobacteria
Lp: Gamma
B:

proteobacteria
Enterobacteriales

H:
Enterobacteriaceae
Hin nay theo phn
loi ca h thng Vin
Chi:
Salmonella
Pasteur th gii, Trung
tm Kim sot v ngn
nga dch bnh ca M Loi: Salmonella bongori
CDC (Control and Prevent
Salmonella
enterica
Disease Center), ging
Salmonella c chia lm
hai loi: S. enterica v S. bongori. Trong S. bongori gm tt c cc kiu huyt thanh
(serotype) cua loi ph s V, v S. enterica c chia lm 6 loi ph nh s: I, II. IIIa,
IIIb, IV v VI. Cc loi v loi ph ny c th phn bit c bng phan ng sinh ha.
Trong mi loi ph c nhiu kiu huyt thanh.
Cho n nay xc nh c trn 2463 kiu huyt thanh thuc ging Salmonella.
Theo h thng ca Kauffman White, cc kiu huyt thanh ny c chia da trn
khng nguyn thn O, khng nguyn tim mao H v khng nguyn b mt Vi. Phn
ln cc kiu huyt thanh c t tn theo cng thc khng nguyn, mt s khc c
t tn ring nh Enteritidis (S. enteritidis), Typhi (S. typhi), Paraphi ( S. paraphi),
Typhimurim ( S.typhimurium).

GVHD : Nguyn Th Kim Oanh

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Phn Tch Vi Sinh Thc Phm

Nhm 6

Loi ph S. enterica hu nh chim a s (99%), n c tm thy hu ht cc


ng vt mu nng. Loi ph ny chim 59% (1454/2463) trong tng s kiu huyt
thanh ca Salmonella c kh nng xm nhp v gy nhim cao cho ngi v ng vt
mu nng, trong khi cc loi ph khc hu nh ng vt mu lnh v mi trng.
Nh vy c ti 41% (1009/2463) s kiu huyt thanh khng c phn lpp t bnh
phm ca ngi, y l mt t l rt c ngha cc c quan chc nng xem xt v
sa i cc tiu chun hin hnh trong thc phm to iu kin thun li cho cc nh
sn xut tiu th thc phm.

1.3 Cc loi in hnh


Salmonella typhi: Gy bnh thng hn
Hnh 1.1. Vi khun Salmonella typhi
Salmonella paratyphi: Gy bnh ph thng hn
Salmonella cholera_suis: Gy bnh nhim trng mu

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GVHD : Nguyn Th Kim Oanh

Phn Tch Vi Sinh Thc Phm

Nhm 6

Hnh 1.2 Vi khun Salomonella cholera _suis


Salmonella entertidis: Gy ri lon tiu ha
Hnh 1.3 Vi khun Salmonella entertidis
Mt s chng Salmonella khc: s. thompson, s. derbv, s. newport, s. kissangani,
s. sen/lenberg, s. meleagnidis, s. anatum, s. aberdeeh, s. panama, s. montevideo, s.
dubin, s. london, s. anatum, s. minnesota, s. ganarum...
Mt s bnh thng gp ng vt do Salmonella gy ra: ph thng hn b
v ln, bnh sy thai cu v nga, bnh bch l g, bnh thng hn chut, bnh
vim phi b...

1.4 c im ca Salmonella
1.4.1 c im hnh thi
Salmonella l trc khun gram (-), hnh que, kch thc trung bnh t 2 3 x
0.5 1m, hiu kh v k kh ty nghi, c tin mao, c th di ng (tr S.gallinarum
v S.pullorum), khng to bo t, sinh acid t glucose v mantinol nhng khng len
mn saccharose, khng sinh indol v khng phn gii ur. Hu ht chng u dinh H 2S
(tr S.typhi), chng pht trin tt nhit 6C - 42C, thch hp nht 35C - 37C,
pH t 6 - 9 v thch hp nht pH = 7,2. nhit t 18C - 40C c th sng n
15 ngy.
Salmonella l vi khun k kh ty nghi, pht trin c trn cc mi trng
nui cy thng thng. Trn mi trng thch hp, vi khun s pht trin sau 24 gi.

GVHD : Nguyn Th Kim Oanh

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Phn Tch Vi Sinh Thc Phm

Nhm 6

C th mc trn nhng mi trng c cht c ch chn lc nh DCA (deoxycholate


citrate agar) v XLD (xylose lysine deoxycholate), trong mi trng XLD t cht
c ch hn nn thng c dng phn lp Salmonella. Khn lc c trng ca
Salmonella trn mi trng ny l trn, li, trong sut, c tm en, i khi tm en ln
bao trm khn lc,mi trng xung quanh chuyn sang mu .
1.4.2 c im ha sinh
Salmonella khng ln men lactose, ln men ng glucose hu ht chng u
sinh H2S, c th sinh hi. Thng khng ln men sucrose, salicin v inositol, s dng
c citrate mn trng Simmons.
Tuy nhin khng phi loi Salmonella no cng c nhng tnh cht trn, cc
ngoi l c xc nh l s.typhi ln men ng glucose khng sinh hi, khng s
dng citrate trong mi trng Simmon, hu ht cc chng s.paratyphi v S.Cholerasuis
khng sinh H2S, khong 5% cc chng Salmonella sinh c t sinh bacteriocin chng
li E.Coli, Shigella v ngay c 1 s chng Salmonella khc.
1.4.3 Cu trc
Salmonella c ba loi khng nguyn l nhng cht khi xut hin trong c th th
to ra kch thch p ng min dch v kt hp c hiu vi nhng sn phm ca s
kch thch gm: khng nguyn thn O, khng nguyn lng H v khng nguyn v
K. Vi khun thng hn (S.typhi c khng nguyn V (Virulence) l yu t chng thc
bo gip cho vi khun thng hn pht trin bn trong t bo bch cu.

Hnh 1.4 Cc khng nguyn trn b mt Salmonella

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GVHD : Nguyn Th Kim Oanh

Phn Tch Vi Sinh Thc Phm


1

Nhm 6

Khng nguyn vch t bo (khng nguyn thn O)

Thnh phn c bn l vch t bo c cu trc phc tp gm 2 lp. Trong cng


l mt lp peptidoglycan mng, cch mt lp khng gian chu cht v ti lp mng
ngoi (outer membrane) l phc hp lipidpolysaccharide gm lipoprotein v
lipopolysaccharide.
Bao bn ngoi lp peptidoglycan l lp phospholipid A v B (quyt nh c t
ca Ni c t), sau l hai lp polysaccharide khng mang tnh c hiu. Khng
nguyn ca ni c t c bn cht ha hc l lypopolysaccharide (LPS). Tnh c hiu
ca khng nguyn O v LPS l mt, nhng tnh min dch th khc nhau: khng
nguyn O ngoi LPS cn bao gm c lp peptidoglycan nn tnh sinh min dch ca
n mnh hn LPS. Mng ngoi c cu trc gn ging t bo cht nhng phospholipid
hu nh ch gp lp trong, cn lp ngoi l lipopolysaccharide dy khong 8-10
nm gm 3 thnh phn: Lipid A, Polysaccharide li, Khng nguyn O. Mng ngoi cn
c thm cc protein:
Protein c cht: protein vi khun cn gi l protein l xuyn mng vi chc nng
cho php mt s loi phn t i qua chng nh dipeptide, disaccharide, cc ion v
c.
Protein mng ngoi: chc nng vn chuyn mt s phn t ring bit v a qua
mng ngoi.
Lipoprotein: ng vai tr lin kt lp peptidoglycan bn trong vi lp mng ngoi.
2

Khng nguyn v (khng nguyn K khng nguyn Vi)

Bn cht ha hc ca v vi khun l polypeptid hoc polysaccharide. V ca vi


khun gy min dch khng mnh nhng khi gn vi t bo vi khun v vn gy c
min dch. Khng nguyn v c dng phn loi cc chng Salmonella.
Khng nguyn K: Khng phc lp, c mt khng nguyn v l khng nguyn
Vi v cng c 2 Ivpe huyt thanh s.typhi v s.paratyphi. Khng nguyn Vi - angtigen
c Felix v cc cng s pht hin nm 1935. Khng nguyn Vi gy hin tng
ngng kt chm v xut hin cc ht v, khng nguyn Vi l khng nguynn v bao
bc bn ngoi khng nguyn o, khnh nguyn Vi khng tham gia vo qu trnh gy
bnh.
3

Khng nguyn lng (khng nguyn H)


c tng hp t cc acid amin dng D (dng t gp trong t nhin). Do

GVHD : Nguyn Th Kim Oanh

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Phn Tch Vi Sinh Thc Phm

Nhm 6

vic x l khng nguyn ca cc t bo min dch khng thun li v p ng khng


th khng mnh. Khi cc si lng b kt hp bi cc khng th c hiu, lng s b bt
ng, vi khun khng th di chuyn c. Khng nguyn lng c dng phn loi
mt s chng Salmonella.
Khng nguyn H: Ch c cc Salmonella c lng. Hu ht Salmonella u c
lng ch tr S.galHarum, s.pulorum gy bnh cho gia cm. Khng nguycn H l mt
loi khng nguyn c bn cht l protit, km bn hn khng nguyn O. Khng nguyn
H rt d b ph hu nhit cao hoc x l bng cn, axit yu.
Khng nguyn H chia lm 2 phase:
Phase 1: C tnh cht c hiu gm c 28 loi khng nguyn lng c biu th
bng cha s La tinh thng: a, b, c...
Phase 2: Khng c tnh cht c hiu, loi ny c th ngng kt vi cc loi khc
i khi thnh phn ny c th gp E.coli. Pha 2 gm c 6 loi c biu th bng
cha s Rp 1-6 hay cha s La Tinh e, n, X...
Bng 1.1 Bng phn bit cc khng nguyn O, K, H

Khng nguyn

Tnh chu nhit

n nhit 2h30 100C

K
H

Tc ng
ca alcohol Formol 50%
50%
B ngn tr ngng
Khng
kt

Bin nhit 1506 100C Nhy cm


Bin nhit 2h 100C

Nhy cm

Khng
Khng

1.4.4 iu kin sinh trng


Salmonella pht trin c trn cc mi trng nui cy thng thng. Trn
mi trng thch hp, vi khun s pht trin sau 24 gi. C th mc trn nhng mi
trng c cht c ch chn lc nh DCA (deoxycholate citrate agar) v XLD (xylose
lysine deoxycholate), trong mi trng XLD t cht c ch hn nn thng c
dng phn lp Salmonella. Khun lc c trng ca Salmonella trn mi trng
XLD l trn, li, trong sut, c tm en, i khi tm en ln bao trm khn lc, mi
trng xung quanh chuyn sang mu .
Salmonella km khng vi iu kin bn ngoi, b ph hy bi qu trnh
tuyt trng bng phng php Pasteur, Tia bc x v un si nu k. Tuy nhin,

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GVHD : Nguyn Th Kim Oanh

Phn Tch Vi Sinh Thc Phm

Nhm 6

Salmonella c th sng st trong 1 thi gian di cc thc phm khi v p lnh. Do


, khi lm tan thc phm ng lnh vi khun ny d pht trin tr li.
Chng pht trin tt nht 6C - 42C thch hp nht 35C - 37C , pH t 6-9
v thch hp nht pH= 7,2. nhit t 18C - 40C vi khun c th sng n 15
ngy.
Kh nng chu nhit ca vi khun ny rt km: 50C trong 1 gi, 70C
trong 15 pht v 100C trong 5 pht . Chng b tiu dit bi phenol 5%, Chloramin
1% v chlorur thy phn 0,2% trong 5 pht. Vi nng mui 8 -19% th s pht trin
ca vi khun ngng li.
Vi khun ny khng gy mi v kh chu cho thc phm, chng hin din trong
tt c cc loi thc phm nh: trng, tht, thy hi sn, rau.
Salmonella c kh nng len men glucose v manitol, sinh acid nhng khng ln
men saccharose v lactose, khng phn gii urea, khng c kh nng tch nhm amine
t tryptophane. Salmonella khng sinh indol, khng lm lng gelatin.
Bng 1.2 Cc tnh sinh ha c bn ca Salmonella
Tnh cht
Kh Nitrate
Ln men Glucid

Kt qu
(+)
(+)

Ln men Glucose sinh kh

(+)

S dng Citrate

(+)

Sinh H2S

(+)

S dng Lactose
S dng Saccharose
H enzyme:
+ Catalase
+ Beta -galactosidase
+ Urease
+ Decarboxylase Lysine
(LDC)
Ornithine (ODC)
Arginine dihydrolase
(ADH)
Desaminase Phenylalanine

(-)
(-)

Ghi ch
Km theo s tng hp acid
Tr mt s chng thuc loi s.
ty phi v s. gallinarnm
Tr s. typhi v s. paratyphi A
Tr mt s chng thuc loi s.
paratyphi A v s. choleraesuis
Tr s. anizonae

(+)
(-)
(-)

Tr mt s loi ph

(+)

T r s. paratyphi A

(+/-)

GVHD : Nguyn Th Kim Oanh

(-)
(-)

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Phn Tch Vi Sinh Thc Phm


Tryptophane
Tetrathionate reductase
Cc tnh cht khc
Mannitol
Indole
Acetylmethylcarbinol
(VP)

Nhm 6
(-)
(+)
(+)
(-)
(-)

Ghi ch: (+): dng tnh; (-): m tnh; (+/-): c th dng tnh hay m tnh ty
chng.
1.4.5 Yu t c lc
Vi khun Salmonella c th tit ra 2 loi c t: Ngoi c t v ni c t.
Ni c t ca Salmonella rt mnh gm 2 loi: Gy xung huyt v mn lot, c
t rut gy c thn kinh, hn m, co git.
Ngoi c t ch pht hin khi ly vi khun c c tnh cao cho vo ti colodion ri
t vo bng chut lang nui, sau 4 ngy ly ra, ri li cy truyn nh vy t 5
n 10 ln, sau cng em lc, nc lc c kh nng gy bnh cho ng vt th
nghim. Ngoi c t ch hnh thnh trong iu kin invivo v nui cy k kh.
Ngoi c t tc ng vo thn kinh v rut.
4

Ni c t - Endotoxin

Mng ngoi t bo vi khun gram m ni chung v vi khun Salmonella ni


ring, c cu to bi thnh phn c bn l lipopolysaccharide (LPS). LPS c cu to
phn t ln, gm 3 vng ring bit vi c tnh v chc nng ring bit: Vng a
nc, vng li v vng lipit A.
Vng a nc bao gm mt chui polysaccharide cha cc n v cu trc
khng nguyn O. Vng li c bn cht l acid heterooligosaccharide, trung tm ni
khng nguyn o vi vng lipit A. Vng lipit A m nhn chc nng ni c t ca vi
khun. Cu trc ni c t gn ging cu trc ca khng nguyn O. Cu trc ni c
t bin i s dn n s thay i c lc ca Salmonella.
Ni c t thng l lipopolysaccharide (LPS) c phng ra t vch t bo vi
khun khi b dung gii. Trc khi th hin c tnh ca mnh, LPS cn phi lin kt
vi cc yu t lin kt t bo hoc cc receptor b mt cc t bo nh: Te bo lm ba
cu B, lm ba cu T, t bo i thc bo, tiu thc bo, t bo gan, lch.Rt nhiu cc
c quan trong c th chu s tc ng ca ni c t LPS: Gan, thn, c, h tim mch,

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GVHD : Nguyn Th Kim Oanh

Phn Tch Vi Sinh Thc Phm

Nhm 6

h tiu ho, h thng min dch; vi cc biu hin bnh l: Tc mch mu, gim
trng lc c thiu oxy m bo, toan huyt, ri lon tiu ho, mt tnh thm n...
Ni c t tc ng trc tip ln h thng min dch ca c th vt ch, kch
thch hnh thnh khng th.
LPS tc ng ln cc t bo tiu cu, gy st ni c t, theo c ch :
Gii phng cc cht hot ng mnh nh: Histamin.
Ngng kt cc tiu cu ng mch.
ng vn, tc mch qun.
LPS tc ng ln qu trnh trao i gluxit: LPS lm tng cng hot lc ca cc
men phn gii glucoz, cc men phn gii glycogen, lm gim hot lc cc men tham
gia qu trnh tng hp glycogen
5

c t ng rut

V c ch min dch v di truyn cc Enterotoxin ca Salmonella c quan h


gn gi vi Choleratoxin, nn c gi l Choleratoxin like enterotoxin (CT). Cn v
c tnh sinh hc Enterotoxin ca Salmonella khng ch vi ging CT m cn ging
vi Enterotoxin ca E.Coli.
c t ng rut ca vi khun Salmonella c hai thnh phn chnh: c t
thm xut nhanh Rapid permeability facto (RPF) v c t thm xut chm Delayed
permeability facto (DPF).
RPF gip Salmonella xm nhp vo t bo biu m ca rut, n thc hin kh
nng thm xut sau 1-2 gi v ko di 48 gi v lm trng cc t bo CHO (Chinese
Hamster Ovary cell). c t thm xut nhanh c cu trc, thnh phn ging vi c t
chu nhit ca E.Coli, c gi l c t chu nhit ca Salmonella. ST c kh nng
chu c nhit 100c trong 4 gi, bn vng nhit thp, c th bo qun
nhit -20c. Cu trc phn t gm mt chui polysaccharide v mt chui
polypeptide.
RPF kch thch co bp nhu ng rut, lm tng s thm thu thnh mch, ph
hu t chc t bo biu m rut, gip vi khun Salmonella xm nhp vo t bo v
pht trin tng nhanh v s lng. Vi khun tch cc tng cng sn sinh c t lm
ri lon cn bng trao i mui, nc v cht in gii. Qu trnh bnh l ng rut
v hi chng tiu chy cng thm phc tp v nghim trng.

GVHD : Nguyn Th Kim Oanh

Trang 16

Phn Tch Vi Sinh Thc Phm

Nhm 6

DPF ca Salmonella c cu trc, thnh phn ging c t khng chu nhit ca


vi khun E.Coli, nn c gi l c t khng chu nhit ca Salmonella. N thc hin
chc nng phn ng chm t 18-24 gi. LT b ph hu 70c trong vng 30 pht v
56c trong vng 4 gi. LT c cu trc gm 3 chui polypeptid.
DPF lm thay i qu trnh trao i nc v cht in gii, dn n tng cng
bi xut nc v cht in gii t m bo vo lng rut, cn tr s hp thu, gy thoi
ho lp t bo villi ca thnh rut, gy tiu chy.
6

c t t bo

Khi c th ngi v ng vt b tiu chy thi km theo hin tng mt nc v


mt cht in gii l hin tng hng lot cc t bo biu m rut b ph hu hoc b
tn thng cc mc khc nhau. S ph hu hay tn thng l do c t t bo
ca Salmonella gy nn, theo c ch chung l: c ch tng hp protein ca t bo
Eukaryotic v lm trng t bo CHO.
t nht 3 dng c t ca t bo:
Dng th nht: Khng bn vng vi nhit v mn cm vi trypsin. Dng ny c
pht hin rt nhiu serovar Salmonella', c trng lng phn t trong khong t
56 n 78 kDa; khng b trung ho bi khng th khng c t Shigella toxin hoc
Shigella - like. c t dng ny tc ng theo c ch l c ch tng hp protein
ca t bo Hela v lm teo t bo.
Dng th hai: C ngun gc t protein mng ngoi t bo vi khun c cu trc v
chc nng gn ging cc dng c t t bo do Shigella v cc chng (ETEC) sn
sinh ra. Dng c t ny cng ph bin hu ht cc serovar Salmonella gy bnh.
Dng th ba: C trng lng phn t khong 62 kDa; c lin h vi c to
Hemolysin. Hemolysin lin h vi cc c t t bo c s khc bit vi cc
Hemolysin khc v trng lng phn t v phng thc tc ng ln t bo theo c
ch dung gii cc khng bo ni bo.
1.4.6 C ch gy bnh
Tt c cc kiu huyt thanh Salmonella u mang cm gen inv (invasion) gip
cho qu trnh xm nhim vo trong thnh rut ca ngi v ng vt, m u ca tin
trnh gy bnh. Cm gen ny nm trong h thng gen SPI - 1 {Salmonella
pathogenicity island) c mt trong tt c cc Salmonella, t nhm tin ho thp nht l
s.bongori n nhm tin ho cao nht l s.enterica.

Trang 17

GVHD : Nguyn Th Kim Oanh

Phn Tch Vi Sinh Thc Phm


7

Nhm 6

C ch gy bnh thng hn

Bnh thng hn do s.typhi v s.paratyphi A, B, c gy ra. Cc yu t c lc


chnh ca vi khun thng hn l kh nng bm v xm nhp vo t bo ch, kh nng
nhn ln trong i thc bo v ni c t. Khng nguyn Vi c mt s.typh v
Spararatyphi c l yu t c lc quan trng, nhng chng vi khun gy bnh thng
hn khng c khng nguyn Vi thi s lng cn thit gy bnh cao hn rt nhiu so
vi nhng chng c khng nguyn ny.
Vi khun xm nhp vo c th qua ng tiu ha do thc n hay nc ung b
nhim bn, s lng cn thit gy bnh vo khong 105 n 107
u tin, vi khun thng hn phi vt qua mi trng axit ca d dy, mc
d chng c kh nng khng vi axit nh c gen art (acid response tolerance),
nhng ngi bnh thng, vi khun thng hn khng th tn ti lu, nui cy dch
d dy m tnh sau 30 pht. Sau khi vt qua c ro cn d dy, vi khun di
chuyn xung rut non ri nhn ln , nhng trong tun u s c 1 t vi khun o
thi theo phn, cy phn dng tnh trong 5 ngy khng c ngha l bnh thng hn
s xy ra. T rut non, vi khun thng hn i vo hch mc treo rut nh t bo M,
mt i thc bo ca mng Peyer. Sau theo ng bch huyt v mu gy nhim
trng ton thn. Sau khong mt tun, nhim khun huyt th pht xut hin. Vi khun
theo gan qua ng mt li tip tc xm nhp vo mt non, tip tc nhn ln cc
mng Peyer. T mu, vi khun ti lch v cc c quan khc. Trong mt non, vi khun
cht v gii phng ra ni c t. Ni c t kch thch dy thn knh giao cm mt
gy ra hoi t chy mu v c th gy thng mt, v tr gy tn thng thng nm
cc mng Peyer. y l bin chng hay gp c bnh nhn n sm khi cha bnh
phc, nht l n cc thc n cng. Ni c t theo mu ln h thn kinh v kch thch
trung tm thn kinh thc vt no tht ba. Giai on ton pht bnh, bnh nhn st
cao, biu thn nhit tng ln theo thi gian. Thn nhit tng nhng nhit khng
tng gy ra hin tng mch v nhit phn ly.
Thi k cha c iu tr bng khng sinh, khon mt tun bnh, din bin in
hnh ca bnh thng hn tri qua 4 giai on, mi giai on khong mt tun, Tun
th nht thn nhit tng cao, tun th hai th au bng, gan v lch to dng thi c s
xut hin ca cc m hng trn da, tun th 3 c th xut hin thm cc bin chng
nh xut huyt, thng rut, tun th 4 s xut hin thm cc bin chng nguy him
dn n t vong, nu khng bnh nhn s bnh phc.
Bnh nhn thng au u, bun nn hoc nn, chng bng, tiu chy, khon
50% bnh nhn s thy gan v l lch di sn. Nhng trng hp nng thng c
du hiu ly b, hn m, try tim mch, khng cha tr kp thi c th dn n t vong.

GVHD : Nguyn Th Kim Oanh

Trang 18

Phn Tch Vi Sinh Thc Phm

Nhm 6

Nhng bin chng khc nh vim phi cp,, vim mng no, vim gan, vim ty
xng cng c th gp.
Nhng bnh nhn qua khi c khong 5 - 10% vn tip tc thi vi khun qua
phn trong qu trnh hi phc v 1- 4% tr thnh ngi mang vi khun lu di do vi
khun vn tn ti trong ti mt. Tnh trng ny c th ko di n nhiu nm v h tr
thnh ngun mang bnh rt nguy him.
8

C ch gy nhim khun v nhim c thc n

Bnh thng xy ra do n phi thc n b nhim Salmonella, nhng cng c th


ly truyn trc tip. Cn nguyn thng do vi khun S.enteritidis v s.typhimurum.
Vi khun xm nhp qua rut non nh cc t bo M ca mng Peyer. Trn b
gen ca Salmonella c cc tc nhn gy c ch cc cht khng khun c trong
lysosome, bin i t bo ch m bo cho s tn ti ca vi khun, lm cn tr hot
ng ni bo nh gim lng NADH oxidase rt cn thit cho vic sn xut cc hp
cht khng khun.
S hy hoi ca i thc bo v cc t bo biu m ln cn, s cht ca vi
khun gii phng ni c t gy nn tn thng cho c th vt ch v gy ra cc
triu chng. Thi gian bnh trung bnh t 10-48 gi. Sau thi gian bnh, ngi
nhim thng c biu hin nh st, nn, au bng v tiu chy. Mc st khc nhau
ty vo th trng tng ngi, tiu chy phn thng khng c mu. ngi ln
thng ch dn n tnh trng ri lon ng tiu ha, nhng tr s sinh thng gy
ra nhim trng rt nng, c th dn n tnh trng nhim khun huyt, vim mng no
v vim xng. nhng ngi khe mnh, nhim trng do nhim c thc n thng
t khi sau 2- 5 ngy ri t khi.
1.4.7 Ngun gc ly nhim
Cc sn phm tht ni chung, nht l tht gia cm v tht ln. Tt c cc thc n
ti sng c ngun gc ng vt u c th l ngun vi khun Salmonella. Vi khun
ny sng t do trong rut ng vt v c trn lng. Gia cm c nhiu Salmonella nht,
tip theo l cc ng vt nui trong nh v ng vt hoang (vt, ra, ch, ch, chim
mng bin, loi gm nhm, rn). Vi khun ny c th c trong thnh phn dn xut cc
cht t ng vt nh gelatin hoc nc bt ng vt, bi cn trng, loi gm nhm,
chim hoc cc sn phm tht nhim khun gy nhim vo thc phm. Ngoi ra c th
b nhim t ngi khe mnh c mang vi khun ny. Thc phm c ngun gc thc
vt t c nguy c nhim khun. V l do b c ch bi pH < 4 v c mt vi khun lactic

Trang 19

GVHD : Nguyn Th Kim Oanh

Phn Tch Vi Sinh Thc Phm

Nhm 6

nn cc sn phm ln men t b nhim. Trng v cc sn phm trng v d nh bt


nho, nc st mayonnaise, protit ng t tch t sa, gia cm l ngun mang nhiu
Salmonella, nn trng ca n cng b nhim v vi khun ny c th xuyn qua v trng
v sinh sn trong lng trng.
Cc sn phm sa nh sa khng thanh trng, phomt t sa ti, kem cht
bo sa, v cc sn phm t sa ni chung c ch bin t cc nng tri, cc thit b
u c th gy nhim vo nguyn liu, to mi trng thun li choSalmonella, v t
gy nhim c cho sn phm sa. Neu tin hnh axit ha chm thi vi khun d
dng sinh sn trong phomt nhng n b ph hy vi pH < 4,5. Nhng sn phm c
sa phi c gim st cht ch bi chng khng c thanh trng na, v vy nu c
Salmonella trong sa bt thi chng vn c th sinh sn c bi chng c kh nng
tn ti iu kin kh hn v ly nhim sang cc sn phm khc.

CHNG 2

PHNG PHP PHT HIN SALMONELLA

2.1 Phng php truyn thng


2.1.1 Nguyn tc
Salmonella c th c pht hin (phn tch nh tnh) bng mt qui trnh bao
gm 4 bc: bc tng sinh, tng sinh chn lc, phn lp v khng nh. Salmonella

thng c mt trong mu vi s lng nh, b tn thng v cng hin din chung vi


mt s lng ln vi sinh vt thuc h Enterobacteriaceae c tnh cnh tranh mnh, c
ch s tng trng ca Salmonella.
2.1.2 Mt s mi trng ha cht
Buffered pepton water (BPW)
Rappaport Vassiliadis Soya Pepton (RVS)
Malachite Green Magnesium Chloride
Tetrethionate Muller- Kauffmanm ( MKTTn)
Xylose Lysine Deoxycholate (XLD)
Hektoen Entric Agar (HE)

GVHD : Nguyn Th Kim Oanh

Trang 20

Phn Tch Vi Sinh Thc Phm

Nhm 6

Bismuth Sulphite Agar (BS)


Brillian Green Phenol Red Lactose Sucrose (BPLS)
Triple Sugar Iron (TSI)
Urea
Lysine Decarboxylase
Ornithine Decarboxylase
Manitol
Sucrose
Sorbitol
Tryptone
Thuc th Kovacs
Khng huyt thanh Salmonella a gi
2.1.3 Cc bc thc hin
9

Bc tng sinh:

Ty theo c tnh thnh phn ha hc ca mu m cn chn qui trnh tng sinh


ph hp. Thng thng t l gia mu v mi trng l 1:9 nhng c th thay i.
i vi cc loi mu thng thng, tin hnh cn 25g mu vo ti PE v
trng,b sung 225ml dung dch BPW v ng nht mu trong 15 hoc 30 giy.
37oC trong 18-24 gi. i vi mt s loi mu c cha cc cht gy c hoc c ch
s pht trin ca Salmonella cn tin hnh quy trnh c bit.
i vi tht gia cm ti sng: t gia cm trong ti PE ln, cho thm 1 lt
BPW, lc bng my lc 30 giy mi trng thm vo ton b mu.
i vi mu sa kh: cn 25g mu vo ti PE v trng , cho thm 225ml BPW,
yn nhit phng khong 60 pht. Sau a lc cho tan hon ton .
i vi cc mu gia v hay cc loi thc phm cha nhiu gia v: ng nht
mu vi t l 1/100 trong BPW( thay v 1/10 nh bnh thng) trc khi tng sinh.

Trang 21

GVHD : Nguyn Th Kim Oanh

Phn Tch Vi Sinh Thc Phm

Nhm 6

Bin php ny nhm gim nng cc cht c ch s sinh trng ca Salmonella


trong bc tng sinh.
i vi cc loi mu nh casein , phomat, b v cc sn phm tng t khc:
thc hin ng nht mu trong mi trng tng sinh c lm m n 40 oC.
i vi cc sn phm cha coca: ng nht mu trong mi trng Skim Milk
Broth c lm m n 40 oC.
i vi da, cc sn phm t da v cc mu c hm lng cht bo cao: ng
nht mu trong mi trng BPW, sau cho thm 2-3 git Triton X-100 trc khi
tng sinh.
10

Bc tng sinh chn lc:

Cc mi trng tng sinh chn lc dng d pht hin Salmonella trong mu thc
phm l Rappaport Vassiliadis (RV), Selenite Cystein Broth,Tetrathionate Mueler
Kaumanm Broth (TT)...nhng nghin cu gn y cho thy mi trng RV c th thay th
cho cc mi trng khc. Hin nay, ngi ta khuyn khch l nn dng t nht 2 loi mi
trng tng sinh chn lc pht hin tt c Serotype Salmonella nu c hin din trong mu.

11

Bc phn lp

Nhm tch v nhn dng Salmonella khi cc qun th vi sinh vt khc trong
mu. Nhiu loi mi trng rn khc nhau c s dng phn lp Salmonella. Hin
nay, vcc tiu chun v sinh an ton thc phm khuyn khch s dng t nht 2 loi
mi trng phn lp khc nhau tng cng kh nng pht hin tt c cc dng
Salmonella, c bit l mi trng XLD c s dng nhiu nht.
12

Bc khng nh

Nhm xc nh li cc khun lc c trng ca Salmonella xut hin trn mi


trng phn lp. Bc ny c thc hin trn cc th nghim sinh ha v cc th
nghim huyt thanh c trng cho Salomonella. Cc th nghim sinh ha c khuyn
khch s dng l KIA/TSI, Indol, LDC (lysine decarboxylase), ODC (omithine
decarboxylase), urea, Manitol, sorbitol, cc th nghim huyt thanh O hay H.
13

Bo co kt qu

Qui trnh kim nghim ny ch cho php phn tch nh tnh Salmonella, gip kt
lun c hay khng c Salmonella hin din trong 25 g mu, khng cho php phn bit
c cc dng Salmonella hin din trong mu. khng nh c tn chng
Salmonella phn lp c cn phi tin hnh th nghim ngng kt vi cc loi khng

GVHD : Nguyn Th Kim Oanh

Trang 22

Phn Tch Vi Sinh Thc Phm

Nhm 6

huyt thanh n gi khc nhau hay cn gi cc chng Salmonella phn lp c n


cc phng th nghim chuyn nh danh vi sinh vt.
Cn lu Salmonella l nhm cha cc dng gy bnh nguy him. Do vy cn
tun th trit cc bin php an ton khi lm vic vi cc chng Salmonella dng lm
i chng (+) cng nh khi thao tc trn cc chng phn lp c. Cc mi trng
hay mu vt sau khi nui cy cn phi hp kh trng cn thn trc khi ra.

2.2 Phng php hin i


Vi phng php truyn thng s mt thi gian di t 3 n 5 ngy, do cc
chuyn gia ca cc hng pht trin cc phng php nhanh rt ngn thi gian phn
tch, chnh xc cao v cc bc n gin hn. Vi b dng c sng lc v kim tra
nhanh c sn s dng vi cc cng ngh khc nhau nh cng ngh mi trng
tin tin, lai DNA, PCR hoc Real time PCR. Tt c cc cng ngh u tri qua qu
trnh tng sinh v s dng k thut chuyn bit ca tng hng pht hin Salmonella
v thi gian c kt qu khng qu 48 gi.
2.2.1 Phng php nh tnh Salmonella s dng kit th nhanh 3MTM PetrifilmTM
Salmonella Express System
14

Nguyn tc.

Phng php ny s dng cc a cy cha mi trng dinh dng kh c chn


lc, sau s dng 1 a sinh ha b sung khng nh chnh xc s c mt ca
Salmonella. Dung dch mu s c tng sinh Salomonella trong mi trng 3M
Salmonella Enrichment Base plus 3M Salmonella Enrichment Supplement medium
sau cy chuyn sang a m 3M TM PetrifilmTM Plate 41,5oC trong 242h, cc
khun lc c mu nu hoc vng trn a m 3M TM PetrifilmTM Plate v chuyn
mu xanh sau khi t a khng nh sinh ha 3M TM PetrifilmTM Salmonella Epress
Confirmation Disk ln chnh l nhng khun lc Salmonella.
15

Mi trng thuc th
Mi trng R-V R10 (RAPPAPORT-VASSILIADIS R10 BROTH)
B test nhanh 3MTM PetrifilmTM Salmonella:

Dung dch 3M Salmonella Enrichment Base


Cht b sung 3M Salmonella Enrichment Supplement

Trang 23

GVHD : Nguyn Th Kim Oanh

Phn Tch Vi Sinh Thc Phm

Nhm 6

a m 3MTM PetrifilmTM Plate


a khng nh sinh ha 3MTM PetrifilmTM Salmonella Epress Confirmation Disk.

Hnh 2.1 B test nhanh 3MTM PetrifilmTM Salmonella


16

Thit b ph tr
Thit b trn mu
T m
Thit b m khun lc
Ni hp tit trng

17

Quy trnh
a.

Cc bc thc hin

Bc 1: Tng sinh Salomonella: trn X(g) mu (X<25g) vi Y(ml) dung


dch tng sinh 3M Salmonella Enrichment Base plus 3M Salmonella Enrichment
Supplement medium vi t l X/Y=1/9 , 41,5oC trong 18h.
i vi nn mu nhim vi khun cao (>10 4CFU/g): chuyn 0,1ml dch
tng sinh vo 10ml mi trng R-V R10 (RAPPAPORT-VASSILIADIS R10
BROTH), 41,5oC trong 8h, s dng dch mu sau bc ny lm mu th.
Bc 2: Cy 0,01 ml dch mu sau tng sinh ln a Petrifilm,
41,5 C trong 242h. nh du nhng khun lc nghi ng (c mu nu hoc vng
sng).
o

Bc 3: t thm a khng nh sinh ha vo, tip tc 41,5 oC trong


4-5h v c kt qu ngay sau khi .
b.

Bo co kt qu th nghim.

Mu dng tnh: Khun lc c nh du chuyn mu xanh

GVHD : Nguyn Th Kim Oanh

Trang 24

Phn Tch Vi Sinh Thc Phm

Nhm 6

Mu m tnh: Khng c s chuyn mu ca khun lc c khoanh hay khng


c khun lc nghi ng c nh du.

Hnh 2.2 Mu dng tnh vi Salmonella


2.2.2 Phng php nh tnh Salmonella s dng kit th nhanh IRIS Salmonella
18

Nguyn tc.

y l phng php pht hin nhanh Salmonella trong thc phm, sa, trng,
thc n chn nui v mu mi trng trong vng 37 gi da vo s pht trin v hnh
thnh khun lc c trng trong mi trng chn lc ca Salmonella. Mu s c
tng sinh Salmonella trong mi trng Salmonella Enrichment, sau c cy
chuyn sang mi trng trng IRIS Salmomella Agar 371oC vi thi gian 243
h. Cc khun lc c mu hng sau chnh l khun lc Salmonella.
19

Mi trng thuc th
B kit th nhanh IRIS SalmonellaR:

Salmonella Enrichment
IRIS Salmonella Supplement
IRIS Salmomella Agar
20

Thit b ph tr
Thit b trn mu
T m
Thit b m khun lc
Ni hp tit trng

Trang 25

GVHD : Nguyn Th Kim Oanh

Phn Tch Vi Sinh Thc Phm


21

Nhm 6

Quy trnh
a

Cc bc thc hin

Bc 1: Tng sinh Salmonella: cn X(g) mu (X<25g), b sung Y(ml) mi


trng Salmonella Enrichment vi t l X/Y=1/9 (g/ml) v thm Z(ml) cht b sung
chn lc IRIS Salmonella Supplement vi t l Z/X=1/10(ml/g). nhit
41,51oC vi thi gian 182 gi.
Bc 2: Cy 0,01ml dung dch mu sau tng sinh ln mi trng IRIS
Salmomella Agar. nhit 371oC vi thi gian 243 gi.
c kt qu ngay sau khi kt thc .
c.

Bo co kt qu th nghim.

Mu xut hin khun lc mu hng l mu c cha Salmonella

Hnh 2.3 Mu m-dng tnh vi Salmonella


2.2.3 Phng php pht hin Salmonella s dng b kit xt nghim MCC test
(M)
22

Nguyn tc.

Phng php ny da trn s thay i mu ca cht ch th mu c trng


c gn trn cc que th ca b kit xt nghim MCC test (M) khi c mt ca
cc enzym c trng sinh ra bi Salmonella.
23

Mi trng thuc th
Nc dng pha long mu
B kit th E.coli - MCC test:

GVHD : Nguyn Th Kim Oanh

Trang 26

Phn Tch Vi Sinh Thc Phm

Nhm 6

Dung dch Buffer


12 que th Salmonella (Salmonella test trip)/ 12 que th E.coli (E.coli test trip)

Hnh 2.4 B kit th E.coli/Salmonella MCC Test


24

Quy trnh
a

Cc bc thc hin

Bc 1: Ra mu hoc pha long mu cn phn tch bng nc v trng.


Bc 2: Ly 32ml ca phn nc ra mu/ huyn ph pha long ng vo
trong ti nha.
M chai nc ra buffer, thm 3 git ca buffer, thc hin thao tc bp dung
dch trong ti nha 5 giy, yn trong 3-4 pht.
Bc 3: M ti MCC test v ly test trip (ty thuc vo mc ich xc nh
E.coli hay Salmonella s s dng E.coli test trip hay Salmonella test trip)
M bao nha v nhng test trip trong dung dch 30 giy
Ly test trip ra, t ln b mt sch v i trong 20 pht cho kim tra
Salmonella v 45-50 pht cho kim tra E.coli ri c kt qu.
d.

Bo co kt qu th nghim.

Test trip khng c mu l m tnh, chuyn mu l dng tnh vi vi khun:


Test Salmonella dng tnh: chuyn mu xanh.

Trang 27

GVHD : Nguyn Th Kim Oanh

Phn Tch Vi Sinh Thc Phm

Nhm 6

Test E.coli dng tnh: chuyn mu .

Hnh 2.5 Kt qu dng tinh E.coli v Salmonella


2.2.4 nh tnh Salmonella bng phng php PCR
25

Nguyn tc

PCR l phng php in vitro tng hp mt trnh t xc nh ADN nh


enzyme. Phn ng s dng 2 on mi oligonucleotide gn c hiu vi chui b tr
v ko di chui theo trnh t ADN ch cn khuch i nh enzyme polymerase.
PCR l mt qu trnh lp li ca 3 phn ng lin tc: bin tnh ADN si kp
thnh ADN si n; gn mi c hiu vo chui ADN si n ti u 5- 3; v ko
di chui nh hot tnh enzyme t u 3 theo hng 3 5 tng hp chui ADN
b tr mi.
26

Quy trnh
a

Bc 1:

Chun b mu: Ti nilong v trng cha 25g mu v 225 ml dung dch mi


trng buffer peptone. Sau khi ng ha mu 37oC trong 5h, chuyn 1ml mu
nui cy qua bnh tam gic cha 9ml mi trng tetra thionate broth base v nui
420C trong 4h. Cho 1ml mu va nui sang bnh tam gic c cha 9ml mi trng
Rapport-Vasiliadis R10 Broth nui 420C trong 15h. Mu sau nui cy c trch ly
DNA kim tra s hin din ca Salmomella
Thit k cc on mi PCR t vi khun Salmonella: cc on mi c thit k
cho Salmonella da trn trnh t gen invA. Trnh t nucleotid ca gen invA sn c
t GenBank.

GVHD : Nguyn Th Kim Oanh

Trang 28

Phn Tch Vi Sinh Thc Phm


e.

Nhm 6

Bc 2: Ly trch DNA bng phng php phenol-chroroform

Cho vo tup nha 2 ml dung dch nui vi khun, ly tm vi tc 13000


vng/pht trong 10 pht. b phn dung dch ni trn mt, ho tan phn ta vi 1ml
TE (10 mM Tris + 1 mM EDTA, pH 8), ly tm 13000 vng/pht trong 5 pht. b
dung dch ni trn mt, ho tan phn ta vi 350l TE, cho tip 30 l lysozyme (50
mg/ml) lc u nh, t trn nc khong 30 pht. Cho vo 40l SDS 10%, lc u
cho n khi dung dch ng nht. Thm 40l proteinase K (10mg/ml), lc u v
nhit phng t 27-30oC trong 2-3 gi. Tip tc cho vo 800 l phenol (pH 7), lc
cho n khi c mu trng c, ly tm 13.000 vng/pht trong10 pht. Ht phn dung
dch pha trn cho vo tup nha 1.5ml c sn 150 l TE. Thm vo mt lng tng
ng (khong 700 l) Phenol-Chloroform-isoamyl alcohol (25:24:1) lc cho n khi
dung dch c mu trng c, ly tm 13.000 vng/pht trong 10 pht. Sau khi ly tm,
chuyn phn trong pha trn sang tup nha 1.5 ml mi c cha sn 75 l Sodium
acetate (3M, pH 7.2), lc u bng tay. Thm vo 1 ml ethanol 96 %, lc u, t trong
nc 10 pht. Ly tm 13000 vng/pht trong 15 pht, b dung dch, gi li phn
ta. Thm ethanol 70 %, ly tm 13000 vng/pht trong 5 pht, b dung dch, lm
kh phn ta v ho tan DNA vi 30 l nc ct v trng, tr -20oC.
f.

Bc 3: Phn ng PCR

Phn ng PCR vi mt cp mi: Phn ng PCR c thc hin vi cc thnh


phn nh sau: 50-100ng DNA, 2.5 l buffer 10X (Tris 100mM, KCl 500mM, pH 9.0,
1% (v/v) Triton X-100), 3 l MgCl2 25mM, 4 l dNTP (0.2 mM mi loi), 0.4 M
ca mi loi mi (mi ngc v mi xui), 0.25 l DNA polymerase (5U/ l), 0.25 l
BSA(0.1%). Thm nc ct v trng cho th tch 25 l. Phn ng khuch i c
tin hnh 95 oC trong 5 pht, sau lp li 35 chu k vi cc bc nh sau: bin
tnh 95 oC trong 30 giy, bt cp mi vo khun 60 oC trong 30 giy, ko di 72
oC trong 1 pht. Cui cng phn ng c duy tr 72 oC trong 10 pht.
g.

Bc 4: Phn tch sn phm PCR

Sn phm PCR sau khi khuch i c phn tch bng in di trn gel 1.5%
agarose trong dung dch m TBE 1X v chp bng my chp hnh gel Biorad UV
2000. Thang chun 100bp ca cng ty Fermentas c s dng c lng kch
thc on sn phm PCR

Trang 29

GVHD : Nguyn Th Kim Oanh

Phn Tch Vi Sinh Thc Phm

Nhm 6

Hnh 2.6 Nguyn l ca PCR v RT-PCR

CHNG 3
QUY TRNH NH TNH SALMONELLA TRONG
SA V CC SN PHM SA
3.1 Phm vi p dng
Quy trnh ny tham chiu theo TCVN 6402:2007 dng pht hin Salmonella
trong sa v cc sn phm t sa.

3.2 Nguyn tc
Phng php ny ch dng nh tnh v kt lun pht hin hay khng pht
hin Salmonella trong mu sa v cc sn phm sa ly.
Quy trnh pht hin Salmonella bt buc phi tri qua nm giai on v s dng

GVHD : Nguyn Th Kim Oanh

Trang 30

Phn Tch Vi Sinh Thc Phm

Nhm 6

chng i chng trong ton b qu trnh phn tch.


Tin tng sinh: mu c tin tng sinh trong mi trng tng sinh khng chn
lc (BPW) m 37oC trong khong t 16h n 20h.
Tng sinh: sau giai on tin tng sinh, dch mu c chuyn qua mi trng
tng sinh chn lc RVS 41,5C trong 24 h v tip theo 24 h na, v mi trng tng
sinh chn lc TT 37 C trong 24 h v tip theo 24 h na.
Phn lp: Cy ria dch mu t mi trng tng sinh chn lc sang hai mi
trng chn lc. Mt l mi trng thch lc sng/ phenol t 37 C v kim tra
sau 20 h n 24 h, nu cn, sau 40 h n 48 h kim tra li s c mt ca cc khun lc.
Hai l trong mi trng XLD 37 C trong 24 h, nu cn, sau 40 h n 48 h kim
tra li s c mt ca cc khun lc.
Phc hi: Cy chuyn cc khun lc nghi ng Salmonella trn cc mi trng
phn lp sang mi trng dinh dng.
Cy truyn cc khun lc c coi l Salmonella v khng nh qua cc th
nghim sinh ha v huyt thanh hc.

3.3 Mi trng v ha cht


3.3.1 Vai tr ca cc mi trng-ha cht
Bng 3.1 Mi trng ha cht v mc ch s dng
Mi trng v ha cht
Dung dch Buffered Peptone Water (BPW)
Canh Rappaport-Vassiliadis Soya Peptone (RVS)
Selenit xystin / Tetrathionat (TT)
Thc lc sng/ phenol- Brilliant-green

Mc ch
Tng sinh s b

Phenol-red Lactose Sucrose Agar (BPLS)

Phn lp

Xylose Lysine Desoxycholate (XLD)


Tryptone Soy Agar (TSA)
Lysine Decacboxylase broth (LDC)
Voges-Proskauer (VP)
Ure Broth
Trypton Water
Kligler Iron Agar (KIA) / TSI

Trang 31

Tng sinh chn lc

Phc hi
Th nghim sinh ha khng
nh Salmonella

GVHD : Nguyn Th Kim Oanh

Phn Tch Vi Sinh Thc Phm

Nhm 6

Thuc th pht hin -galactosidaza


Dung dch creatine
Khng huyt thanh
Dung dch a-napthon
KOH 40%
Thuc th Kovacs
HCl v NaOH 10%

Thuc th cc phn ng sinh ha


Chnh pH

3.3.2 Thnh phn ca cc mi trng


27
Dung dch Buffered Peptone Water (BPW)
Pepton
Natri clorua (NaCl)
Dinatri hydrooctophosphat ngm 12 phn t nc (Na2HPO4.12H2O)
Kali dihydrooctophosphat (KH2PO4)
Nc
28

Canh Rappaport-Vassiliadis Soya Peptone (RVS)


a

Dung dch A

Dch thy phn u tng bng enzym


Natri clorua (NaCl)
Kali dihydro phosphat (KH2PO4)
Dikali hydro phosphat (K2HPO4)
Nc
h.

Dung dch B

Magi clorua ngm 6 phn t nc (MgCl2.6H2O)


Nc
i.

Dung dch C

Malachit xanh oxalat


Nc

GVHD : Nguyn Th Kim Oanh

Trang 32

Phn Tch Vi Sinh Thc Phm


29

Nhm 6

Selenit xystin / Tetrathionat (TT)


a

Mi trng c bn

Trypton
Lactoza
Dinatri hydro phosphat ngm 12 phn t nc (Na2HPO4.12H2O)
Natri hydro selenit
Nc
j.

L-Xystin

Dung dch natri hydroxit, c(NaOH) = 1 mol/l


Nc v trng
30

Thc lc sng/ phenol- Brilliant-green Phenol-red Lactose Sucrose Agar


(BPLS)
a

Mi trng c bn

Cao tht b
Pepton
Cao men
Dinatri hydrophosphat (Na2HPO4)
Natr dihydrophosphat (NaH2PO4)
Thch
Nc
k.

Dung dch ng/ phenol

Lactoza
Sucroza
phenol
Nc

Trang 33

GVHD : Nguyn Th Kim Oanh

Phn Tch Vi Sinh Thc Phm


l.

Nhm 6

Dung dch lc sng

Lc sng
Nc
31
Xylose Lysine Desoxycholate (XLD)
Bt cao nm men
Natri clorua (NaCI)
Xyloza
Lactoza
Sucroza
L-Lyzin hydroclorua
Natri thiosulfat
St (III) amoni xitrat
Phenol
Natri deoxycholat
Thch
Nc
32
Tryptone Soy Agar (TSA)
Trypticase peptone
Phytone peptone
NaCI
Agar
Nc ct
33
Lysine Decacboxylase broth (LDC)
L-Lystin monohydroclorua

GVHD : Nguyn Th Kim Oanh

Trang 34

Phn Tch Vi Sinh Thc Phm

Nhm 6

Cao men
Glucoza
Bromocresol ta
Nc
34
Voges-Proskauer (VP)
Pepton
Glucoza
Dinatri hydro phosphat (K2HPO4)
Nc
35

Ure Broth
a

Mi trng c bn

Pepton
Glucoza
Natri clorua
Kali dihydrooctophosphat (KH2PO4)
phenol
Thch
Nc
m. Dung dch ur

Ur
Nc

36
Trypton Water
Tryptone hoc trypicase
Nc ct

Trang 35

GVHD : Nguyn Th Kim Oanh

Phn Tch Vi Sinh Thc Phm

Nhm 6

37
Kligler Iron Agar (KIA) / TSI
Cao tht
Cao men
Pepton
Natri clorua
Lactoza
Sucroza
Glucoza
St (II) xitrat
Natri thiosunfat
phenol
Thch
Nc
Thuc th pht hin -galactosidaza

38
a
n.

Toluen
Dung dch m

Natri dihydro phosphat (NaH2PO4)


Dung dch natri hydroxit 10 mol/l
Nc
o.

Dung dch ONPG

o-Nitrophenyl -D-galactopyranosid (ONPG)


Nc

GVHD : Nguyn Th Kim Oanh

Trang 36

Phn Tch Vi Sinh Thc Phm

Nhm 6

3.4 Quy trnh phn tch

Trang 37

GVHD : Nguyn Th Kim Oanh

Phn Tch Vi Sinh Thc Phm

Nhm 6

(371)oC/(243)h
3.5 Cc bc

m tnh

3.5.1 Chun b

tin hnh
mu

chun
b dung dch ban u, cho 25
g mu th vo 225 ml mi
trng tin tng sinh, l t l ca mu
th vi mi trng tin tng
sinh c quy nh trong phng php
ny. Nu phn mu th khc vi 25 g, th s dng mt lng mi trng tin tng sinh
cn thit to ra pha long 1/10 (khi lng/th tch).
39

Sa nguyn liu, sa x l nhit v cc sn phm sa dng lng.

Dng pipet ly 25 ml mu th cho vo bnh tam gic cha 225 ml mi trng


tin tng sinh v lc u.
40

Sn phm sa kh
Chun b mt bnh c np y cha 225 ml mi trng tin tng sinh .

Cn 25 g mu th mt cch v trng v ln b mt cht lng ng trong


bnh. y np bnh, khng lc. yn nhit phng trong 60 pht 10 pht trc
khi em nui m. Khng cn chnh pH. Nu sau 1 gi m m sa bt vn cha tan,
th dung tay lc bnh trn lng cha trong bnh hoc khuy bng dao trn v
trng.
41

Lactoza

Cn 25 g mu th mt cch v trng cho vo 1 bnh c np y cha 225 ml


mi trng tin tng sinh v lc cho n tan.
42

Casein, caseinat v phomat

Cn 25 g mu th mt cch v trng cho vo cc ng v trng ca my trn


tc cao hoc loi nhu ng. Thm 225 ml mi trng tin tng sinh nhit 45
C. Trn cho n khi mu th phn tn u (t 1 pht n 3 pht). Gi nhit khng
c qu 45 C.
Bo co kt qu
Khng pht hin Salmonella trong 25g/25ml
GVHD
: Nguyn Th Kim Oanh

Trang 38

Phn Tch Vi Sinh Thc Phm


43

Nhm 6

Lc mu th c lm tan chy. Dng pipet chuyn 25 ml phn mu th


c trc n khon 45 C, cho vo bnh tam gic cha 225 ml mi trng tin
tng sinh v lc u.
44

Sn phm sa ng lnh (k c kem thc phm)

Dng pipet chuyn 25 ml mu th c lm tan chy v trc n


khong 37 C, cho vo bnh cha 225 ml mi trng tin tng sinh v lc u.
45

Sa ln men, sa chua, bnh kem v mn trng ming

Cn 25 g mu th mt cch v trng cho vo bnh c np y cha cc vin bi


thy tinh v 225 ml mi trng tin tng sinh v lc cho tan.
n gin vic kim tra, khi mu kim tra gm nhiu phn mu th 25 g
c ly ra t l hng sa hoc sn phm sa qui nh cn kim tra, v khi chng
minh c vic to thnh mu chung (gp chung cc phn mu th) khng lm nh
hng n kt qu ca sa hoc sn phm sa th c th gp li cc thnh phn mu
th. V d, nu kim tra 10 mu th, mi mu 25 g th kt hp 10 mu vi nhau to
thnh mu th chung l 250 g v em ha tan hoc khuch tn trong 2,25 lt mi
trng tin tng sinh.
Hoc cch khc, cc phn 0,1 ml mi trng tin tng sinh (mi trng RVS)
v 10 ml mi trng tin tng sinh (selenit/xystin) t 10 phn ring r c th kt hp
li em tng sinh tng ng trong 0,1 lt v 1 lt mi trng chn lc.Kim tra pH ca
huyn ph v chnh n 6,8 0,1 nu cn, tr khi c qui nh khc
3.5.2 Tin tng sinh (Tng sinh s b)
Mu c tin tng sinh trong mi trng tng sinh khng chn c (BPW)
37 C 30C trong khong 16h-20h.
0

3.5.3 Tng sinh chn lc


Cho 0,1 ml dch cy thu c vo ng cy c cha 10 ml mi trng RVS.
Chuyn 10 ml dch cy thu c vo bnh tam gic c cha 100 ml mi trng
selenit/xystin .
mi trng RVS cy trong ni cch thy hoc trong t m t 41,5 C
trong 18 gi n 24 gi. mi trng selenit/xystin cy trong t m t 37 C

Trang 39

GVHD : Nguyn Th Kim Oanh

Kh

Phn Tch Vi Sinh Thc Phm

Nhm 6

trong 18 gi n 24 gi.

Hnh 3.1 Salmonella trn mi trng selenit/xystin


Bc 3: Phn lp
Hnh 3.2 Salmonella trn mi trng XLD v BPLS
Dng que cy vng cy phn lp t mi canh thang tng sinh chn lc RVS v
selenit/xystin ln mi a thch cha mi trng chn lc XLD v BPLS. Sau khi cy,
lt ngc cc a sao cho y hng ln trn v 370C trong khong 20h 24h
Sau khi 20h 24h kim tra cc a v s c mt ca cc khun lc in hnh
v cc khun lc khng in hnh m c th nghi ng l Salmonella. Trn mi trng
XLD khun lc Salmonella in hnh c mu hng trong sut, c hoc khng c tm
mu en. Trn thch lc sng/ phenol, cc khun lc Salmonella in hnh c mu
hng vi vng mi trng bao quanh ti. . nh du v tr cc khun lc ny trn
y a.
Sau khi m tip mi trng RVS v mi trng selenit/xystin trong 18 gi
n 24 gi, lp li vic cy v qui trnh m.Sau mi ln m (9.4.3 v 9.4.4)kim tra
cc a v s c mt cc khun lc Salmonella in hnh. Nu cc khun lc mc yu
v khng c mt cc khun lc Salmonella in hnh th m li cc a thm 18 gi
n 24 gi trong t m t 37 C v kim tra li cc a ny v s c mt cc khun
lc Salmonella in hnh.
3.5.4 Phc hi trn mi trng dinh dng NA / TSA
nh du 5 khun lc in hnh hoc nghi ng t mi a trn mi trng phn
lp XLD v BPLS. Nu trn mt a c t hn 5 khun lc in hnh hoc khun lc
nghi ng, th ly tt c cc khun lc in hnh hoc nghi ng cy ria ln NA / TSA.

GVHD : Nguyn Th Kim Oanh

Trang 40

Phn Tch Vi Sinh Thc Phm

Nhm 6

370C 10C trong khong 24h 3h


Hnh 3.3 Salmonella trn mi trng TSA
Trng hp 1: t mi a th mt khun lc c trng, nu cho cc kt qu th
nghim sinh ha ph hp th kt lun pht hin Salmonella trong mu
Trng hp 2: nu khun lc u tin cho kt qu th nghim sinh ha khng ph
hp th tin hnh th 4 khun lc cn li c nh du, mt trong bn khun
lc ny cho kt qu th nghim sinh ha ph hp th kt lun pht hin Salmonella
trong mu v ngc li th kt lun khng pht hin Salmonella trong mu
3.5.5 Khng nh sinh ha v khng huyt thanh
T cc khun lc chn cy ria ln NA / TSA,dng que cy cy vo cc mi
trng sau:
46

Thch TSI

Nguyn tc: xc nh kh nng s dng ngun carbohydrate c th kt hp vi


mi trng tng trng cn bn, c hoc khng c sinh hi v hydrogen sulfide (H2S)
Cch ly mu: cy ria trn b mt nghing ca thch v cy m su xung y
370C 10C trong khong 24h 3h
Din gii nhng thay i trong mi trng sau:
Cy m su
Mu vng glucose dng tnh (s dng glucose).
Mu hoc khng i mu glucose m tnh (khng s dng glucose).
Mu en sinh hydro sunfua.
Bt hoc vt rn sinh kh t glucose.

Trang 41

GVHD : Nguyn Th Kim Oanh

Phn Tch Vi Sinh Thc Phm

Nhm 6

Cy b mt nghing ca thch
Mu vng lactose v/hoc sucrose dng tnh (s dng lactose v/hoc sucrose).
Mu hoc khng i mu lactose v sucrose m tnh (khng s dng lactose
cng nh khng s dng sucrose)
Cc khun lc Salmonella in hnh th hin tnh kim (mu ) trn b mt
nghing ca thch v khi cy m su mang tnh acid (mu vng) c sinh kh (bt kh)
v vi khong 90% trng hp sinh hydrosunfua (thch b en).
Khi Salmonella c lactoza dng tnh c phn lp, mt nghing thch TSI s
c mu vng. Do , vic khng nh s b cc Salmonella khng ch da vo kt qu
th trn thch TSI.

Hnh 3.4 Salmonella phn ng dng tnh vi TSI


47

Thch Ure

Nguyn tc: s dng pht hin kh nng phn ct ure thnh ammoniac do
hot tnh ca enzyme urease t vi sinh vt v kt qu l mi trng b kim ha do
NH3 c to ra.
Cch cy mu: cy cy ria trn b mt nghing ca thch. . 37 1oC trong
243h. Nu phn ng dng tnh, vic phn gii ur s gii phng amoniac, lm i
mu phenol sang mu hoa hng v sau di chuyn sang mu tm. Phn ng
thng xy ra sau 2 gi n 4 gi.
1

GVHD : Nguyn Th Kim Oanh

Trang 42

Phn Tch Vi Sinh Thc Phm

Nhm 6

Hnh 3.5 Th nghim Salmonella trn mi trng thch Ure


48

Lysine decarboxylase

Nguyn tc: s dng pht hin vi khun sinh cc enzyme decarboxylase, cc


enzyme ny tung tc vi cc amino acid c gc carboxyl (-COOH) cui, to thnh
amine hay diamine v carbon dioxide (CO2).
Cch cy mu: cy ngay pha di ca b mt mi trng lng, ph mt lp

paraffin hay du khong ln trn b mt mi trng. 37 1oC trong 243h.


Salmonella c phn ng lysine decarboxylase dng tnh nn sau khi nui cy, mi
trng vn gi nguyn mu tm. Phn ng m tnh khi mi trng c mu vng.
Hnh 3.6 Salmonella dng tnh trn mi trng LDC
1.
2.
3.
4.

Trang 43

Control
Escherichia coli ATCC 25922
Serratia marcescens ATCC 8100
Salmonella Typhimurium ATCC 14028

GVHD : Nguyn Th Kim Oanh

Phn Tch Vi Sinh Thc Phm


5.
49

Nhm 6

Salmonella Enteritidis ATCC 13076

Pht hin -galactosidase


Nguyn tc: xc nh kh nng ln men lactose ca vi sinh vt.

Cch cy mu: cho mt vng y cc khun lc nghi ng vo trong ng v


trng c cha 0,25ml dung dch mui sinh l. Thm mt git toluen v lc ng. t
ng ny vo trong ni cch thy 37 oC v trong vi pht( khong 5 pht ). Thm
0,25ml thuc th pht hin -galactosidase v lc u. t li ng v ni cch thy

37oC v trong 243h, kim tra ng thng xuyn. Mu vng cho thy phn
ng dng tnh. Phn ng thng xut hin sau 20 pht. Nu s dng a giy bn
sn, th theo cc ch dn ca nh sn xut.
Hnh 3.7 So snh gia Salmonella v citrobacter trong phn ng ONPG
50

Mi trng phn ng Indol

Nguyn tc: pht hin kh nng oxy ha tryptophan thnh cc dng ca indol:
indol, skatol (methyl indol ) v indol acetate.
Cch cy mu: cy khun lc nghi ng vo ng cha 5ml mi trng tryptone
water. 37 1oC trong 243h. Sau khi , thm 1ml thuc th kovacs, nu phn

ng dng tnh th trn b mt mi trng xut hin mt vng mu chng t chng


t phn ng dng tnh, xut hin mu khc l phn ng m tnh.
Hnh 3.8 Th nghim Indole

GVHD : Nguyn Th Kim Oanh

Trang 44

Phn Tch Vi Sinh Thc Phm


51

Nhm 6

Voges-Proskauer

Nguyn tc: xc nh kh nng sinh acetylmethylcarbinol(acetoin) trong qu


trnh ln men glucose ca mt s vi sinh vt.
Cch cy mu: cho mt vng y cc khun lc ngi ng vo trong ng v
trng c cha 3ml mi trng VP. 37 1oC trong 243h. sau khi , thm 2 git
dung dch creatine, 3 git dung dch -naphthol v sau thm 2 git dung dch kali
hydroxide (KOH), lc u sau mi ln thm tng loi thuc th. Khi xut hin mu
hng n mu sng trong 15 pht chng t phn ng dng tnh, cn phn ng m
tnh khi dch vi khun khng i mu.

Cn tun th nghim ngt trt t trn y. Nu, v d: cho kali hydroxit vo


trc khi dung dch 1-naphtol trong cn v pepton c mt, th kt qu c th cho dung
dch c mu hng nht m c th che khut phn ng dng tnh.
Hnh 3.8 Phn ng VP
52

Th phn ng khng huyt thanh

Vic pht hin s c mt cc khng nguyn Salmonella O-, Vi- v H- c tin


hnh bng phn ng ngng kt trn phin knh ca huyt thanh thch hp vi cc
khun lc thun khit (9.5.2) v sau khi loi tr cc chng t ngng kt (9.5.5.2).
a

Loi tr cc chng t ngng kt

Cho mt git dung dch mui ln mt phin kch ra sch cn thn. Dn u


khun lc cn th nghim trong git dung dch ny sao cho thu c mt th huyn
ph c v ng nht. Lc nh phin knh trong 30 giy n 60 giy. Quan st kt qu
trn nn en, tt nht nn dng knh lp.
Nu cc vi khun vn li thnh cc n v t nhiu c ngng kt th chng ny
c coi l t ngng kt, khng cn th phn ng huyt thanh tip theo. Nu khng c
s vn cc v vi khun ri nhau, th chng xem nh khng t ngng kt, cn phi tip

Trang 45

GVHD : Nguyn Th Kim Oanh

Phn Tch Vi Sinh Thc Phm

Nhm 6

tc th phn ng huyt thanh.


p.

Kim tra khng nguyn O,H, Vi

Nh mt git anti-O hoc H ln mt lam knh sch, dng que cy ly mt t


sinh khi vi khun t mi trng Nutrient Agar phn tn vo git huyt thanh, ch
30-60 giy, quan st trn nn en. Phn ng dng tnh khi c hin tng ngng kt
xy ra. i khi cn quan st hin tng ngng kt bng knh lp hay knh hin vi vi
phng i thp. Ngc li, nu vi khun phn b u trong huyt thanh,lm cho
git dung dch c u c xem kt qu l m tnh.
Mu c kt lun l dng tnh vi Salmonella khi c khun lc c trng
trn cc mi trng phn lp v cho nhng kt qu sinh ha sau:TSI/KIA: /vng,
c/khng c H2S v sinh hi; Ure (-); Indol(-); VP(-); ONPG(-); LDC(+).

3.6 Kt qu
Pht hin(hay khng pht hin) Salmonella trong 25g mu rn hoc 25ml mu
lng.
3.6.1 Gii thch t cc th nghim sinh ho
Bng 3.1 Gii thch cc kt qu
Php th khng nh

Phn ng m tnh
hoc dng tnh

Phn trm chng


Salmonella cho thy c
phn ng

TSI glucoza (sinh axit)

100

TSI glucoza (sinh kh)

91,9

TSI lactoza

99,2 a

TSI sucroza

99,5

TSI nidro sunfua

91,6

Phn gii ur

100

L-Lyxin decacboxyl

94,6

Phn ng -galactoxidaza

98,5

Phn ng Voges Proskauer

100

Phn ng indol

98,9

Cc Salmonella enterical sub sp, arizonae v diarizonae cho cc phn ng lactoza


dng tnh hoc m tnh nhng lun lun cho phn ng -galactosidaza dng tnh.

GVHD : Nguyn Th Kim Oanh

Trang 46

Phn Tch Vi Sinh Thc Phm

Nhm 6

Cc Salmonella nhm II c th cho cho phn ng lactoza m tnh nhng c th cho


phn ng -galactosidaza dng tnh.
3.6.2 Gii thch t cc phn ng sinh ho v huyt thanh
Bng 3.2 Gii thch t cc php th khng nh

Cc phn ng sinh ho T ngng kt

Cc phn ng huyt
thanh

Phn ng in hnh

Khng

Khng nguyn O-, Vi- Cc chng c coi


hoc H- dng tnh
l Salmonella

Phn ng in hnh

Khng

Tt c phn ng m
tnh

C th l
Salmonella

Phn ng in hnh

Khng th c

C th l
Salmonella

Phn ng khng in
hnh

Khng

Khng nguyn O-, Vihoc H- dng tnh

C th l
Salmonella

Phn ng khng in
hnh

Khng

Tt c phn ng m
tnh

Khng c coi l
Salmonella

Trang 47

Gii thch

GVHD : Nguyn Th Kim Oanh

Phn Tch Vi Sinh Thc Phm

Nhm 6

TI LIU KHAM KHO

GVHD : Nguyn Th Kim Oanh

Trang 48

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