[Type text]
[Type text]
[Type text]
Trng i Hc K Thut Cng Ngh TP.HCM Khoa Cng Ngh Thc Phm
ti:
-Tp.HCM thng 11/2011Mc lcChng 1 : Tng
quan..............................................................................................4[Type
text] Page 1
[Type text]
[Type text]
[Type text]
I. S lc v vi khun
E.coli.....................................................................................4
1. c im sinh
hc..................................................................................................4
1.1 Hnh
thi................................................................................................................4
1.2 Tnh cht nui
cy.................................................................................................4
1.3 Tnh cht ha
sinh..................................................................................................5
1.4 Khng
nguyn........................................................................................................6
1.5 Phn
loi................................................................................................................6
2. Kh nng gy
bnh................................................................................................7
3. Chn on vi sinh
vt............................................................................................12
3.1 Chn on trc
tip................................................................................................12
3.2 Chn on gin
tip...............................................................................................12
4. Nguyn tc iu tr v phng
bnh.......................................................................13
4.1 iu
tr...................................................................................................................13
4.2 Phng
bnh............................................................................................................13
Chng II : Cc phng php pht hin
E.coli......................................................14 I.
Phng php truyn
thng....................................................................................14
1. nh lng E.coli bng phng php
MPN........................................................14 1.1
Nguyn
tc.............................................................................................................14
1.2 Quy trnh phn
tch................................................................................................14
1.3 Cch c kt
qu....................................................................................................15
2. nh lng E.coli bng phng php m khun
lc........................................15 2.1 Nguyn
tc.............................................................................................................15
2.2 Mi trng v ha
cht..........................................................................................15
2.3 Quy hoch phn
tch..............................................................................................16
2.4 Cch tnh kt
qu...................................................................................................16[Type
text] Page 2
[Type text]
[Type text]
[Type text]
3. Phng php lm giu vi
khun...........................................................................17
3.1 Nguyn
l..............................................................................................................17
3.2 Thit b v thy
tinh..........................................................................................17
3.3 Mi trng v thuc
th........................................................................................17
3.4 Cch
lm................................................................................................................17
II. Phng php hin
i..............................................................................................22
1. Phng php
PCR.................................................................................................22
1.1 Nguyn
tc.............................................................................................................22
1.2 Mi trng tng
sinh.............................................................................................22
1.3 Mi trng phn
lp..............................................................................................22
1.4 Trch ly
DNA.........................................................................................................22
1.5 Qui trnh
multiplex-PCR........................................................................................24
1.6 u v nhc im ca phng php
PCR.............................................................24
2. Phng php
ELISA.............................................................................................25
2.1 Nguyn
tc.............................................................................................................25
2.2 Cc phng php
ELISA.......................................................................................26
2.3 Pht hin E.coli O157:H7 bng phng php
Elisa...............................................29 2.4 ng dng
ca khng th n dng trong xc nh E.coli
O157:H7.......................30 2.5 u im ca phng php
E.lisa...........................................................................30
3. Pht hin E.coli bng phc hp khng th + ht nano silica pht
quang..................30 Kt
Lun.....................................................................................................................34
Ph
Lc......................................................................................................................35
Ti liu tham
kho.....................................................................................................47
[Type text]
Page 3
[Type text]
[Type text]
[Type text]
Chng I: TNG QUAN V VI KHUN ESCHERICHIA COLI
I. S LC V VI KHUN ESCHERICHIA COLI (E.COLI)
Escherichia coli do Theodore Escherich (1857-1911), mt nh vi
khun hc ngi o, pht hin ln u tin nm 1885. Chi Escherichia thuc h vi
khun ng rut. Trong cc loi thuc chi ny, E.coli c chn lm in hnh v c
vai tr quan trng nht trong y hc. E.coli l mt trong nhng thnh vin
chnh ca h vi khun bnh thng rut nhng cng l cn nguyn ca nhiu bnh nhim
trng, trong c nhng bnh n l cn nguyn ng u.
E.coli c s dng lm m hnh nghin cu v sinh hc phn t trong lnh vc vi
sinh hc ni ring v sinh hc ni chung.[Type text]
Hnh 1.1: Hnh chp vi khun E.coli di knh hin vi vi kch thc 2 m
Page 4
[Type text]
[Type text]
[Type text]
E.coli K12 l vi khun c s dng lm m hnh nghin cu nhiu nht. nhiu
thnh tu v di truyn hc, ha sinh hc c thu trn c s nghin cu vi khun
ny. Ngy nay E.coli cng c s dng nhiu trong cng ngh sinh hc. Mc d
E.coli l loi vi khun c nghin cu su nht, cho n nay n vn tip tc c
quan tm nghin cu, vi rt nhiu pht hin mi tm phn t, c bit c ch bnh
sinh ca cc type gy bnh (pathotype).1. c im sinh hc
1.1 Hnh thi E.coli l trc khun Gram m. Kch thc trung bnh t 2 n 3m
x 0,5m; trong nhng iu kin khng thch hp (v d nh trong mi trng c khng
sinh) vi khun c th rt di nh si ch. Rt t chng E.coli c v, nhng hu ht
c lng v c kh nng di ng. 1.2. Tnh cht nui cy E.coli pht trin d dng
trn cc mi trng nui cy thng thng. Mt s c th pht trin trn mi trng tng
hp rt ngho cht dinh dng. Hiu k kh ty . C th pht trin nhit t 5-400C.
Nhit thch hp xung quanh 370C. Trong iu kin thch hp E.coli pht trin
rt nhanh, thi gian th h ch khong 20 n 30 pht. Cy vo mi trng lng (nh
canh thang) sau 3 n 4 gi lm c nh mi trng, sau 24 gi lm c u; sau hai
ngy trn mt mi trng c vng mng. Nhng ngy sau, di y ng c th thy lng
cn. E.coli khng mc trn canh thang Selenit. Trn mi trng thch thng,
sau khong 8 n 10 gi, dung knh lp c th quan st c khun lc. Sau 24 gi
khun lc c kch thc khong 1,5mm. Hnh thi khun lc in hnh dng S, nhng c
th gp dng R, hoc M. Trn mi trng phn lp, ty theo cht ch th mu,
E.coli c khun lc mu vng (nh trn thch lactose) hoc mu (nh trn thch
MacConkey). Khng mc c trn mi trng SS. Mt s loi E.coli c tnh cht nui
cy ring c gi tr trong sng lc nhanh, nh EAEC to thnh vng c trng khi
nui cy trn canh thang Muller-Hinton. 1.3. Tnh cht ha sinh E.coli c
kh nng ln men nhiu loi ng v c sinh hi. Hu ht E.coli u ln men
lactose v sinh hi, tr E.coli tr (inactive) (trong c EIEC) khng hoc
ln men rt chm. Mt s chi khc trong h vi khun ng rut cng c kh nng
ln
[Type text]
Page 5
[Type text]
[Type text]
[Type text]
men nhanh lactose (nh Klebsiella, Enterobacter, Serratia v
Citrobacter) c gp vo mt nhm vi khun c tn chung l coliform. E.coli c
kh nng sinh indole. Khng sinh H2S. Khng s dng c ngun carbon ca
citrate trong mi trng Simmons. C decarboxylase, v vy c kh nng kh
carboxyl ca lysine, ornithin, arginin v acid glutamic.
Betagalactosidase dng tnh. Th nghim VP (Voges Proskauer) sau 24 gi
m tnh, sau 48 gi c th dng tnh. Bng 1.1: Tnh cht ha sinh ca mt s loi
thuc chi Escherichia Cc loi Th nghim CNPG Indole methyl
VogesProskauer Citrate (Simmons) Lysine decarboxylase Arginine
dihydrolase Ornithine decarboxylase Di ng D-Glucose acid E.coli
(bnh thng) + + + T T T + + E.coli (inactive ) T T + T T + E.blatta
e + T + + + E.fergus onii T + + T + + + + E.herma nnii + + + + + +
+ T T + + + + E.vulneri E.alberti s i + + T T + + + T + + + T ? + +
+ + + + -
D-Glucose sinh + + + hi Lactose + T Sucrose T T D-Mannitol + + +
Adonitol + Cellobiose + D-Sorbitol + T D-Arabitol L-Ramnose T T + +
Sinh sc t vng ONPG: Ortho-nitrophenyl-beta-Dgalactopyranoside +:
>90% dng tnh -: < 10% dng tnh.[Type text]
Page 6
[Type text]
[Type text]
[Type text]
T: 10-90% dng tnh 1.4. Khng nguyn Khng nguyn O: ngi ta bit ti gn
160 yu t khng nguyn O ca E.coli. Khng nguyn K: Khong 100 yu t khng
nguyn K c xc nh v c chia thnh ba loi: A, B v L, trong A di dng v
quan st c bng knh hin vi quang hc thng thng, B v L di dng mng rt
mng ch c th quan st c nh knh hin vi in t. Khng nguyn H: hn 50 yu t
khng nguyn H c xc nh. 1.5 Phn loi Da vo cu trc khng nguyn, E.coli c
chia thnh cc type huyt thanh. Vi s t hp ca cc yu t khng nguyn O, K
v H s c rt nhiu type huyt thanh khc nhau. Mi type huyt thanh c k
hiu bng khng nguyn O v K, v d O86B7 (yu t khng nguyn O s 86, yu t
khng nguyn K s 7 loi B). Da vo v tr gy bnh cc E.coli c kh nng gy
bnh ngi c chia thnh 2 nhm: th nht l nhm gy bnh ng rut
(IPEC-intestinal pathogenic E.coli) hay E.coli gy tin chy
(DEC-Dierrheagenic E.coli), th hai l nhm gy bnh ngoi ng rut
(ExPEC-extraintestinal pathogenic E.coli).-
Cc loi E.coli gy bnh ng rut (IPEC) c bit gm:
EPEC (Enteropathogenic E.coli): E.coli gy bnh ng rut ETEC
(Enterotoxigenic E.coli): E.coli sinh c t rut EIEC (Enteroinvasive
E.coli): E.coli xm nhp rut EAEC (Enteroaggregative E.coli): E.coli
ngng tp rut DAEC (Diffusely adherent E.coli): E.coli bm dnh phn tn
EHEC (Enterohaemorrhagic E.coli): E.coli gy xut huyt rut-
Hai loi E.coli gy bnh ngoi ng rut quan trng nht l:
MAEC (Meningitidis-associated E.coli): E.coli gy vim mng no UPEC
(Uropathogenic E.coli): E.coli gy nhim khun ng tit niu 2. Kh nng gy
bnh
[Type text]
Page 7
[Type text]
[Type text]
[Type text]
E.coli l thnh vin thuc nhm vi h bnh thng ca ng tiu ha, chim t l
cao nht trong s cc vi khun hiu kh (khong 80%). Tuy nhin, E.coli cng
l 1 vi khun gy bnh quan trng, n ng u trong cc vi khun gy tiu chy,
vim ng tit niu, vim ng mt; ng hng u trong cc cn nguyn gy nhim khun
huyt. E.coli l cn nguyn thng gp trong vim mng no, vim phi tr mi
sinh. E.coli cn gp trong nhim trng ngoi khoa, nhim trng trong bng.
Theo bo co ca chng trnh quc gia gim st tnh khng thuc ca cc vi khun
gy bnh thng gp (1988-1994) th E.coli ng th hai (sau S.aureus) v t l
phn lp c (tnh chung tt c cc loi bnh phm) nc ta. Nhng type huyt
thanh c kh nng gy bnh thng gp trn lm sng l: O111B4, O86B7, O126B16,
O55B5, O119B4, O127B8, O26B6, O25B15, O128B12. C ch gy bnh ca
E.coli khc nhau ty loi: ETEC-E.coli sinh c t rut Hai yu t ng lc
quyt nh kh nng gy bnh ca ETEC l: kh nng bm dnh vo nim mac rut v sn
xut c t. Kh nng bm dnh v c tr trn t bo biu m rut non l iu kin u tin
c th gy bnh. Kh nng ny c vai tr ca khng nguyn CFA (clonisation
factor antigen) c m ha bi gen trn plasmid. Quyt nh kh nng gy tiu
chy ca ETEC l c t rut. C hai loi c t rut: loi khng chu nhit LT
(heat labile toxin) v loi chu nhit ST (heat stable toxin). Mt chng
E.coli c th sinh mt trong hai hoc c hai c t . c t rut LT l ngoi c
t, gm hai loi LT I c m ha bi gen trn plasmid v LT II c m ha bi gen
trn nhim sc th. LT I v LT II c cu trc v c ch tc ng ging nhau v ging
vi c t rut ca vi khun t. Cu to ca LT gm 1 tiu phn A (active) vi phn
t lng 25.000 Dalton v nm tin phn B (binding) vi phn t lng 11.500
Dalton. LT bm vo th th GM1 ca t bo biu m rut non nh tiu phn B. Tiu
phn A c y vo t bo biu m hot ha enzyme adenylate cyclase lm tng AMP
vng (cAMP-cyclic adenosine monophosphate), dn n lm gim hp thu Na+,
tng tit Cl-. Hu qu ca qu trnh ny l p lc thm thu trong lng rut tng,
nc c ko t t bo ra lng rut gy tiu chy. c t chu nhit ST c trng lng
phn t xp x 5000 Dalton. ST tc ng ln rut bng s hot ha enzyme
guanylate cyclase lm tng GMP vng (cGMPcyclic guanosine
monophosphate). GMP vng gy ri lon chuyn ha nc v in gii ging nh AMP
vng. ST cn lm mt hoc lm teo mt phn nhung mao ca t bo biu m rut. Ngi
ta ni n ST-I v ST-II. ST-I gm ST-Ia v ST-Ib. Hu ht cc chng sn xut
ra ST-Ib phn lp c t ngi, cn ST-Ia th hu ht t ng[Type text] Page
8
[Type text]
[Type text]
[Type text]
vt hoc cc thc n c ngun gc ng vt. Cha gp cc chng sinh ST-II gy
tiu chy ngi. Tiu chy do ETEC thng khi pht t ngt, phn ton nc khng c
nhy, mu. EIEC-Ecoli xm nhp rut EIEC gy bnh ch yu do kh nng xm nhp
vo nim mc i trng. Kh nng xm nhp c m ha bi gen trn plasmid 140MDa.
Cc gen trn plasmid ny m ha cho cc khng nguyn xm nhp (IpaA n IpaD,
Ipa-Invasion plasmid antigen). EIEC cho kt qu (+) tnh trong th
nghim kh nng gy vim kt gic mc chut lang (th nghim Sereny). EIEC cn
c kh nng sn xut c t rut ging mt s Shigela. Gen m ha cho c t ny c tn
l sen (Shigella enterotoxin). C ch gy bnh ging vi khun l. V vy
trong y vn hin nay vit cn nguyn gy bnh l trc khun (bacillary
dysentery) gm Shigella v EIEC. EIEC xm nhp vo trong t bo biu m i
trng, lm tiu cc ti thc bo v nhn ln trong bo tng, ph hy t bo ri mi
xm ln sang cc t bo khc. Tn thng nht chnh l vim lot hoi t nim mc i
trng. Triu chng lm sng in hnh l i ngoi phn t, c ln nhy mu.
EAEC-E.coli bm dnh kt tp rut EAEC thng gy tiu chy ko di hoc mn tnh,
nht l tr em. N cng l mt trong nhng cn nguyn quan trng ca nhim khun
ng tiu ha khch du lch. C ch bnh sinh trong tiu chy do EAEC vn cha c
sng t hon ton. Nhng yu t ng lc chnh ca EAEC c ni n gm cc dim bm dnh
kt tp AAF (aggregative adhesion fimbriae), Yu t iu ha bm dnh kt tp
aggR, protein Pet v c t EAST-1 (enteroaggregative heat-stable
toxin-1). Dim bm dnh kt tp c cho l yu t quyt nh c lc. Ba loi AAF c
ni n l AAF/I, AAF/II v AFF/III, trong loi I v II c cu trc b, loi
III c dng si ring bit. Cc AFF to nn kiu bm dnh hnh chng gch trn t
bo Hep-2. Yu t aggR c vai tr iu ha s biu hin ca cc AFF. Protein Pet
c tit qua mng ngoi vi khun, gy tch t dch v gy c cho biu m tiu ha.
EAST-1 c kh nng ph hy t bo biu m. Ngoi cc yu t dc lc nu trn EAEC cn
tit ra 1 protein c kh nng lm tan mu v lm mt thng bng vn chuyn ion
qua mng. Cc yu t c lc nu trn ca EAEC phn ln c m ha bi cc gen nm trn
plasmid c phn t lng 60 MDa. Mt s yu t c lc c m ha bi cc gen trn
nhim sc th ang c nghin cu. Khi nui cy trn canh thang Muller-Hinton,
EAEC to thnh vng c trng. Tnh cht ny c dng sng lc nhanh cc chng
E.coli thuc loi EAEC.[Type text] Page 9
[Type text]
[Type text]
[Type text]
Bng 1.2: Tm tt cc c im lin quan n kh nng gy bnh ca cc E.coli gy
bnh ng rut Loi E.coli Gen ng lc hoc yu t c lc Plasmid 50-70 MDa
(EAF): M ha BFP (bundle-forming pilus), Per (plasmid-encoded
regulator) v Ler (LEE-encoded regulator. PAI nhim sc th (LEE) TTSS
gm: intimin (eaeA), cc protein tit; Tir, EspA, EspB, EspD, EspF,
EspG v MAP (mitochondriaassociated protein) EAST-1 CDT (Cytolethal
distending toxin) Cc CFA m ha bi plasmid: CFA-I (fimbriae cng),
CFA-III (to b), CFA-II v CFA-IV (fimbriae mm) v pili di lin quan vi
type IV c im lm sng Bm dnh ti ch nh Cc tn BFP thng A/E Tn thng m
A/E Tiu chy cp C ch gy bnh
EPEC
ETEC
Bm vo nim mc rut non (CFA I-IV) v sn xut cc c t rut LTI, LTII,
STa, STb
ETEC
EIEC
EAEC
c t chu nhit LT I v LT II (m ha bi plasmid) Hot ha adenylate
cyclase- tng cAMP gim hp thu Na+/tng tit Cl- - tiu chy c t vng bn
vi nhit STa v STb Hot ha guanylate (m ha bi plasmid v transposon)
cyclase- tng cGMP tit chloride v/hoc gim hp thu NaCl tiu chy Cc gen
trn plasmid 140 MDa m Vi khun bm v xm ha cc khng nguyn xm nhp nhp
vo biu m i (invasion plasmid antigen) IPaA trng, nhn ln gy IpaD vim
lot hoi t nim mc i trng im bm dnh kt tp AAF AAF to nn kiu bm
(aggregative adhesion fimbriae) dnh hnh chng gch aggR iu ha s biu
Yu t iu ha bm dnh kt tp aggr hin ca AAF Protein pet gy tch t dch v
gy c t cho biu m tiu ha c t EAST-1 (enteroaggregative EAST-1 ph hy
t heat-stable toxin-1) bo biu m Protein lm tan mu v mt thng Protein
lm tan mu
Tiu chy cp, phn thng ton nc khng c nhy mu
Hi chng l trc khun Tiu chy ko di
[Type text]
Page 10
[Type text]
[Type text]
[Type text]
bng vn chuyn ion qua mng
EHEC/ VTEC
DAEC
v lm mt thng bng vn chuyn ion qua mng Plasmid 60 MDa m ha
heamolysin, c t Shiga: LCT, EspP - Hy hoi cc vi nhung mao hp thu c
t bo biu m rut. PAI nhim sc th - c ch qu trnh tng hp protein ca t
bo biu m i trng, dn n lm cht t bo, c t Shiga (Stx1, Stx2, Stx2v) m
- gy hi chng HUS ha bi cc gen trn nhim sc th Cc yu t bm dnh Afa/Dr
Afa/Dr gy bm dnh (AIDA) phn tn EAST-1 EAST-1 ph hy t bo biu m Cc
geb set (enterotoxin) C th c TTSS vi esc
Tiu chy ra mu (do xut huyt i trng)
Tiu chy phn thng khng c mu
EHEC cn c gi l E.coli sinh c t Shiga. Yu t ng lc chnh ca EHEC l
c t Stx (Shiga toxin) hay c t gy c i vi t bo Vero VT
(veroccytotoxin) v vy loi ny thuc nhm c tn l VTEC Verocytotoxin
E.coli. Hin nay, c ba loi Stx do EHEC sinh ra c xc nh l Stx1, Stx2
v Stx2v. Cc c t ny c m ha bi cc gen prophage thch hp trn nhim sc
th. c t Shiga hy hoi chn lc cc vi nhung mao ht thu ca t bo biu m
rut. N cng xm nhp vo t bo biu m i trng, c ch qu trnh tng hp protein
dn n lm cht t bo. Hu qu l vim i trng xut huyt, gy tiu chy phn nh
mu. Nhng trng hp hoi t nng c th gy thng rut. Stx cn c th gy hi chng
tng ure huyt tan mu (HUS-haemorrhagic uremic syndrome). Stx vo mu n
thn gy tn thng t bo biu m tiu cu thn, lm hp v tc mao mch tiu cu
thn. Hu qu ca qu trnh ny l mc lc ca thn suy gim, bnh nhn b suy thn
cp. EPEC E.coli gy bnh ng rut EPEC c bt u nghin cu rt sm, t nhng nm
1940. EPEC bm dnh vo nim mc rut gy tn thng c trng l s ph hy vi
nhung mao rim bn chi ca nim mc rut. Loi vi khun ny c vai tr rt quan
trng trong vim d dy rut gy tiu chy tr em. DAEC E.coli bm dnh phn
tn[Type text] Page 11
[Type text]
[Type text]
[Type text]
DAEC l type gy bnh c m t tng i gn y. N c xc nh l mt type gy bnh
ring v khng c cc gen c lc c trng ca cc type khc c m t trc. Khc vi
E.coli gy bnh ng rut (IPEC), cc E.coli gy bnh ngoi ng rut (ExPEC) l
nhng vi khun gy bnh c hi khi lc ch. Chng c th l thnh vin ca h vi
khun bnh thng rut nhng khi vo mu, vo dch ty no, vo ng tit niu th tr
nn gy bnh, nht l nhng ngi c c ch khng b suy gim. Tuy nhin xc nh c
nhng gen c lc ca ExPEC thng gp trong cc nhim trng ngoi ng rut. MAEC
c khng nguyn v c lin quan v mt ha hc v min dch vi polysaccharide
nhm B ca Neisseria meningitides. Cho n nay, hiu bit v s nhy cm ca
tr s sinh vi loi E.coli ny cha y . Nhiu tc gi cho rng c s lin quan
ch yu n c a ca tr. Theo kt qu ca nhiu nghin cu, ti 80% cc trng hp
vim mng no tr s sinh do E.coli. UPEC l nguyn nhn ch yu ca nhim trng
ng tit niu. yu t c lc quan trng ca UPEC l P-pili. Nh Pili ny,
E.coli c th gn c hiu vo khng nguyn P, l mt trong cc khng nguyn nhm
mu. Ngoi ra n con c mt s yu t c lc khc cng tham gia vo c ch gy bnh.
v\cc nhm huyt thanh hay gp l O1, O2, O4, O6, O7 v O75. Cc chng c
khng nguyn K1, K2, K3, K5, K12,K13 l hay gp nht. nhng vi khun ny
thng c cc gen m ha cho cc yu t c lc nh yu t bm dnh, v, cc c t.3.
Chn on vi sinh vt
3.1. Chn on trc tip Bnh phm khc nhau ty bnh: l phn vi nhim khun
ng tiu ha, nc tiu vi nhim khun ng tit niu, mu nu l nhim khun mu Qui
trnh chun on trc tip E.coli c bn gng vi qui trnh chun on cc vi khun
ng rut. C th lm tiu bn soi trc tip i vi mt s loi bnh phm nh cn ly
tm nc tiu hoc nc no ty. i vi vim mng no m c th pht hin khng nguyn c
hiu ca vi khun bng phn ng ngng kt latex. D lm, cho kt qu rt nhanh v
tin cy rt cao. Nguyn l ca phn ng ny nh sau: cc ht latex c gn khng
th khng E.coli gy bnh c trn dch no ty, nu trong bnh phm c khng
nguyn c hiu ca E.coli th phn ng s dng tnh. l phn ng ngng kt th
ng.
[Type text]
Page 12
[Type text]
[Type text]
[Type text]
Phng php chun on ch yu nht l nui cy phn lp. Bnh phm phn c nui cy
trn mi trng phn lp c cht c ch chn lc nh DCL, Endo. Nc tiu gia dng c
tin hnh cy m trn mi trng c, trc y thng s dng thch thng, hin nay c
mi trng uriselect va c gi tr cy m, va c kh nng nh danh. Tin hnh cy
mu khi nghi c nhim khun mu. Sau khi phn lp c vi khun thun nht th xc
nh tnh cht sinh vt ha hc v nh tn bng phn ng ngng kt trn phin knh vi
cc khng huyt thanh mu. xc nh cc E.coli sinh c t ngi ta c th s dng
nhiu phng php khc nhau nh phng php quai rut, phng php th nghim trn
t bo nui, phng php ng ngng kt, ELISA K thut khuch i gen PCR cng c p
dng trong chn on E.coli. nc ta hin nay ch yu mi s dng PCR trong nh
danh khi vi khun c phn lp thun nht. Cc k thut PCR xc nh E.coli trc
tip t bnh phm ang c nghin cu pht trin. 3.2. Chn on gin tip Trn thc
t phng php huyt thanh hc khng c s dng chn on cc nhim khun do
E.coli.
4. Nguyn tc iu tr v phng bnh
4.1 iu tr E.coli thuc vo cc vi khun c t l khng thuc cao, nht l
cc chng phn lp c t nc tiu, v vy cn phi lm khng sinh chn khng sinh
thch hp. Ngoi vic s dng khng sinh, mt s vic khc rt c gi tr trong iu
tr nh bi ph nc, in gii trong trng hp a chy (vic ny nhiu khi c vai
tr quyt nh cu sng bnh nhn), gii quyt cc cn tr trn ng tit niu, rt ng
thng sm nu c th c. 4.2. Phng bnh Hin nay cha c phng php no phng bnh
c hiu. phng nhim khun ng tiu ha do E.coli, thc hin cc bin php phng
bnh chung khng c hiu ging nh i vi cc vi khun ng rut khc.[Type text]
Page 13
[Type text]
[Type text]
[Type text]
Thc hin cc bin php ngn nga nhim trng bnh vin v E.coli l mt trong
cc vi khun gy bnh c hi quan trng. phng nhim khun ng tit niu do
E.coli: thc hin v sinh vng hu mn v b phn sinh dc ngoi, thc hin
nghim tc nguyn tc v trng khi phi tin hnh thm d hoc t thng ng tit
niu. iu tr loi tr cc yu t nguy c khc.
Chng II: CC PHNG PHP PHT HIN E.COLI
I. Phng php truyn thng 1. nh lng E.coli bng phng php MPN 1.1
Nguyn tc S lng E.coli trong mu nc, thc phm cha mt thp c th c xc nh
bng phng php MPN ( Most Probable Number). Phng php ny da vo nguyn
tc mu c pha long thnh mt dy thp phn (hai nng k tip khc nhau 10 ln);
3 hoc 5 mu c pha long thp phn lin tip c trong ng nghim cha mi trng
thch hp c ng by kh Durham. Mi nng pha long c t 3 n 5 ng lp li. Theo
di s sinh hi v i mu nh tnh s hin din trong tng ng th nghim; y l cc
ng dng tnh. Ghi nhn s ng nghim
[Type text]
Page 14
[Type text]
[Type text]
[Type text]
cho phn ng dng tnh mi nng pha long v c vo bng MPN suy ra s lng
nhm vi sinh vt tng ng hin din trong 1g (hoc 1ml) mu ban u. Mi trng
v ha cht :-
Mi trng lng Lauryl Sulphate Broth LSB (canh Lauryl Sulphate) Mi
trng lng Brilliant Green Lactose Bile Salt (canh BGBL) Mi trng lng
E.coli (E.coli medium, canh EC)
-
Cc mi trng lng trn c chun b trong cc ng nghim cha ng Durham p
ngc. Sau khi kh trng, ch s dng cc ng nghim khng c bt kh bn trong ng
Durham. Mi trng thch a EMP Mi trng rn Simmon Citrate Agar (thch
Simmon Citrate) Canh MR-PV Thuc th Methyl Read Thuc th
-napthol.
I.2 Quy trnh phn tch Tun t cy 1ml dung dch mu pha long 10 -1vo 3
ng nghim ging nhau, mi ng cha 10ml canh LSB. Thc hin tng t vi dch
mu pha long 10-2 v 10-3. y l trng hp xc nh MPN bng h 3 dy nng v 3
ng nghim lp li (h 3 x 3 hay 9 ng nghim). nu nghi ng s lng vi sinh
vt trong mu qu cao, phi s dng cc mu c bc pha long cao hn. cc ng
nghim 37 0C trong 48 gi. Ghi nhn ng c sinh hi. Dng que cy vng cy
chuyn dch mu t cc ng LSB (+) sang cc ng c cha canh BGBL v 37 0C 10C
trong 48 gi. Ghi nhn s ng cho kt qu (+) (c sinh hi) ng vi mi pha
long. Dng que cy vng chuyn mt vng dch mu t cc ng canh LSB (+) sang
mi trng canh EC, 44,5 0,20C trong 24 gi. m s lng cc ng cho kt qu
(+) (sinh hi) mi pha long. Dng que cy vng ria dch mu t cc ng (+)
trn mi trng canh EC sang mi trng thch a MBN. cc a ny 370C trong 24
gi tm khun lc E.coli gi nh (trn, dt hnh a v c nh kim tm). Chn khun
lc c ng knh ln hn 1mm v cy vo cc mi trng MRVP, Simmon Citrate Agar
thc hin cc th nghim IMViC. Khun lc E.coli gi nh cho kt qu th nghim
IMViC tun t l + + - - chnh l E.coli. ng nghim cho kt qu trong mi
trng EC v khun lc E.coli[Type text] Page 15
[Type text]
[Type text]
[Type text]
gi nh trn mi trng EMB cho kt qu th nghim IMViC nh trn l ng nghim
c E.coli (+). Thc hin tng t cho tt c cc ng nghim cho kt qu (+)
trong mi trng EC v to c khun lc E.coli gi nh trn mi trng EMB. Ghi
nhn s lng cc ng nghim c E.coli (+) mi pha long ca mu. 1.3 Cch c kt
qu tt c cc trng hp nu trn, t s lng cc ng nghim c E.coli (+) mi pha
long ca mu, dng bng MPN thch hp (bng 3 x 3 tc 9 ng nghim) tnh ra mt
vi sinh vt trong mu v biu din di dng tr s MPN/g hay MPN/ml mu ban u
cha pha long.2. nh lng E.coli bng phng php m khun lc
2.1
Nguyn tc
Mu c ng nht ha c cy mt lng nht nh ln mi trng thch chn lc thch hp
cha lactose, 440C trong 24 gi, m cc khun lc c hnh dng c trng ca
Coliforms. Khng nh khun lc m l E.coli bng cc th nghim IMViC. 2.2 Mi
trng v ha cht
- Mi trng canh Tryptone Soya Agar (TSA) v mi trng Violet Red
Bile
Agar (VRB) c chun b v trng trong cc chai thy tinh, un chy v lm
ngui nhit 450C trong b iu nhit trc khi s dng.- Mi trng canh
Escherichia coli broth (EC Broth) c phn phi 10ml trong
mi ng nghim cha ng Durham p ngc, hp kh trng ngui v kim tra khng
c s hin din ca bt kh trong ng Durham. Cc ng EC ny c bo qun 2 80C. -
Mi trng canh Lactose Tryptone Lautyl Sulphate Broth (LST Broth) c
chun b tng t mi trng canh EC.- Mi trng canh Tryptone Broth c phn
phi 5ml vo mi ng nghim, hp
kh trng.- Mi trng canh MR-VP Broth c phn phi 5ml vo mi ng nghim,
hp
kh trng, ngui v bo qun 2-80C cho n khi s dng. - Mi trng thch
Simmon Citrate c chun b di dng cc ng thch nghing c mu xanh lc. -
Thuc th Kovacs.
[Type text]
Page 16
[Type text] 2.3 Quy trnh phn tch
[Type text]
[Type text]
Mu c ng nht ha bng cch i vi mu rn xay nhuyn mu trong iu kin v
trng ri cn chnh xc 10g (hoc 25g), i vi mu lng ht 10ml (hoc 25ml), b
sung vo trong bnh tam gic cha 90ml (hay 225ml) nc SPW c hp kh trng.
Lc u trong mt thi gian nht nh (t nht l 2 pht i vi mu rn). Sau khi c
lm ng nht bng cch trn, dung dch mu thu c c pha long l 10-1 so vi
ban u. Dch mu ng nht c tip tc pha long theo dy thp phn bng cch
chuyn 1ml dch mu vo ng nghim cha 9ml dung dch pha long. Dung dch ny
c pha long 10-2, sau tip tc chuyn 1ml dch mu ny vo ng nghim th hai
cha 9ml dung dch pha long ta s thu c dch mu c pha long 10-3. Tip tc
thc hin c cc pha long thp phn tip theo sao cho cha 60% v sau 3 ngay
vn phc hp vn gi nguyn hoat tinh. Gii han phat hin cua ky thut nay i
vi vi khun ich la 102 CFU/ml, thi gian phat hin nhanh hn cac phng
phap thng thng nhiu ln.
1. 2. nh 1: TEM E. coli O157:H7 c bao bc bi phc hp khng th + ht
silica nh 2: SEM E. coli O157:H7 c bao bc bi phc hp khng th + ht
silica Hnh nh SEM (knh hin vi qut) va kinh hin vi ng tiu cua t bo
E.coli O157:H7 c nh du bi phc hp khng th + ht nano silica chng minh
rng phc hp ny c th bao bc u hon ton v nh nhau ln b mt t bo vi khun.
Qua c th khng nh cc ht nano silica hp sinh vi khang th vn gi nguyn
tnh cht pht quang hiu qu v cc phn t khng th khi gn vi ht nano
silica vn gi c hot tnh v c kh nng nhn bit v lin kt vi vi khun
ch
Anh huynh quang: (a) phc hp ht nano silica khng th; (b) VK gn vi
phc hp khng th - Silica; (c) VK gn vi phc hp khng th - QDs
[Type text]
Page 34
[Type text]
[Type text]
[Type text]
Ph hunh quang ca phc hp ht silica khng th gn kt vi vi khun E.
coli O157:H7. y l c s xy dng ng chun: cng ph - s lng E.coli O157:H7
pht hin nhanh, nhy s lng vi khun ny mt thp
nh hunh quang phc hp Silica E. coli O157:H7: chp bng knh hin vi
ng tiu Nikon C1plus Ti-E. Vin mu bao bc xung quanh t bo vi khun l
phc hp ht silica + khng th c hiu
[Type text]
Page 35
[Type text]
[Type text]
[Type text]
KT LUN Hin nay, ng c thc phm tr thnh mi quan tm ca ton x hi. C
nhiu nguyn nhn gy ra cc v ng c thc phm nhng phn ln cc trng hp c
ngun gc t vi sinh vt. Ng c thc phm thng xy ra do nguyn liu dng ch
bin hay thc phm b nhim vi khun v c t ca vi khun. Mt trong nhng loi
ngc c thc phm gy ra bi vi sinh vt thng gp l ng c thc phm do vi khun
E.coli. Trong thc t cuc sng, theo di kho st m bo v sinh an ton thc
phm hn ch s nhim v tc hi ca vi khun trong qu trnh thu hoch cc sn
phm nng nghip, chn nui, nh bt hi sn n qu trnh bo qun ch bin v nu
nng ti tay ngi tiu dng thng rt phc tp v gp nhiu kh khn. Do cc phng
php pht hin vi khun E.coli c vai tr quan trng trong nhim v m bo v
sinh an ton thc phm nhm lm gim t l ng c do vi khun gy nn.
[Type text]
Page 36
[Type text]
[Type text]
[Type text]
PH LC I. TH NGHIM SINH HA 1. Th nghim sinh indol a. Nguyn tc
Trytophan l mt acid amin c th b oxi ha bi mt s vi sinh vt c h
enzyme trytopanse to nn cc sn phm cha gc indol. Phn ng trung gian
chnh ca phn ng oxi ha tryptophan l indolpyruvic acid IPA. Phn t ny
sau b bin i theo hng loi nhm amin (deamination) thnh indol hay theo
hng loi nhom1carboxyl (decarboxylation) thnh skatol. Trytophanase
xc tc phn ng loi nhm amin to thnh indol. Vic pht hin indol c thc
hin bng phn ng ca phn t ny vi cc thuc th cha
p-Dimethylaminobenzaldehyde (p-DMABA). Nhn pyrrol ca indol cha nhm
CH2 s kt hp vi nhn benzene ca p-DMABA to nn mt phc cht dng quione
mu . Th nghim ny c dng phn bit E.coli (+) vi Klebsiella (-),
Proteus mirabilis (-) vi cc loi Proteus khc (+), Bacillus alvei (+)
vi cc Bacillus khc (thng l -). Heamophilus heamoglobinophilus (+) v
H.influenzae (-) vi cc loi Haemophilus khc (thng l -), Pasteurella
multocida (+) v P.pneumotropica (+) vi P.haemolytica (-) v P.urea
(-). Chng dng lm i chng (+) cho th nghim ny l Proteus rettgeri, (-)
l Serratia marcescens. b. Phng php tin hnh Mi trng s dng cho th
nghim ny c th l nc tryptone, hoc cc mi trng kt hp nh MIU (Motility
Indol Urea), SIM (Sulfide Indol Motility) khi cn kim tra cng lc
nhiu c tnh sinh ha cn thit. hai loi thuc th c th s dng cho th nghim
ny l thuc th Kovacs v thuc th Erlich. Sinh khi chng thun c cy vo ng
mi trng lng thuc mt trong cc loi nu trn, nhit 370C trong 24-48 gi.
Trc khi b sung thuc th c th b sung 1ml ether hoc xylene vo ng
nghim, lc u chit tch indol ln lp dung mi hu c. Nh 5 git thuc th vo
ng dch nui cy, yn vi pht, theo di s to mu trong lp dung mi hu c. c.
c kt qu
[Type text]
Page 37
[Type text]
[Type text]
[Type text]
Th nghim l (+) khi c s xut hin ca lp mu trn b mt mi trng; l (-)
khi c lp mu vng ca thuc th trn b mt mi trng. i khi c s xut hin ca
mu cam do skatol, l tin cht methyl ha ca indol, to ra.
2. Th nghim KIA, TSI a. Nguyn tc Mi trng KIA (Ligler Iron agar)
v mi trng TSI (Triple Sugar iron Agar) c s dng kt hp th nghim ng
thi kh nng s dng cc ngun carbon khc nhau (glucose, lactose) v kh
nng sinh H2S ca chng vi sinh vt V ngun carbon, mi trng KIA cha hai
loi ng l 1% lactose v 0,1% glucose. Tng t, mi trng TSI c thnh phn
nh mi trng KIA nhng c them 1% sucrose (saccharose). Kh nng s dng c
hai ngun ng l glucose v lactose hay ch s dng glucose thu ly nng lng
cn cho tng trng l ty thuc vo di truyn ca chng vi sinh vt v c th c
theo di trn ng thch cha mi trng KIA. Khi chng cy ln mi trng ny, c
ba trng hp c th xy ra i vi s tng trng ca chng vi sinh vt th nghim:
ch s dng glucose, s dng cc glucose v lactose, khng s dng c hai loi
ng ny. Kh nng ny ca vi sinh vt c th c xc nh thong qua s i mu ca ch
th pH trong mi trng trn b mt v bn trong mi trng trong ng thch
nghing.- Nu vi sinh vt ch ln men glucose, sau 18-24 gi nui cy mi
trng b mt
ca ng thch nghing tr nn c pH kim v phn su trong ng c pH acid. iu
ny c th c gii thch l lng glucose trong phn b mt ca mi trng c vi
sinh vt oxi ha hon ton thnh H2O v CO2 thu nng lng. p ng nhu cu nng
lng cho tng trng, vi sinh vt tin hnh d ha pepton trong mi trng qua
gii phng NH3 lm phn b mt ca mi trng mi c pH kim. Ngc li, phn su
trong mi trng mi c iu kin oxi khng y , glucose c ln men k kh sinh
ra cc acid hu c lm gim pH ca mi trng. nu trong mi trng c cht ch th
l phenol th trn mt thch nghing s c mu v phn su c mu vng. - Nu vi
sinh vt c th s dng c glucose v lactose, sau 18-24 gi nui ton b mi
trng u tr nn c pH acid v s bin dng ng thi c glucose v lactose b mt
mi trng gip vi sinh vt nng lng tng trng m khng cn phi s dng n
pepton. - Nu vi sinh vt khng s dng c c hai ngun carbon ny, th
pepton c s dng bin dng thu nng lng v vt cht cn cho s tng trng ca vi
sinh vt.[Type text] Page 38
[Type text]
[Type text]
[Type text]
Tuy nhin, do pepton ch c bin dng trong iu kin hiu kh nn hin tng
kim ha mi trng ch din ra trn phn ca b mt mi trng v ch phn ny tr nn
c mu . Trong khi , phn mi trng su trong ng nghim s khng c hin tc i
mu. Trong cc trng hp nu trn, nu s ln men c to ra cc sn phm kh th kh
s tch t bn trong thnh bt kh hay s lm v thch. Trng hp s dng mi trng
TSI, cc hin tng lien quan n s dng ngun carbon cng xy ra tng t. Th
nghim kh nng sinh H2S c th thc hin ng thi trn hai mi trng ny do
thnh phn mi trng c cha sodium thiosulphate. Vi sinh vt kh sulfate c
th kh hp cht ny gii phng H2S to ra s c pht hin da vo phn ng to kt
ta mu en FeS gia H2S v ion Fe2+ ca ch th ferric ammonium citrate
hin din trong mi trng.
b. Phng php tin hnh Mi trng KIA hay TSI c pha ch, hp kh trng v
chuyn vo cc ng thch nghing sao cho nh thch nghing cch np ng nghim
khong 2,5cm v phn su c chiu cao khong 2,5cm. dung que cy thng cy
sinh khi t khun lc ca chng thun vo phn su ca ng thch nghing nhng
trnh chm y ng, sau ria trn b mt thch nghing. Sau khi cy ging, cc ng
thch nghing c 370C t 18-24 gi. Ghi nhn mu, s sinh kh phn su, mu mt
thch nghing, s to thnh mu nu bi FeS. c. c kt qu - Th nghim kh nng s
dng ng: xem mc nguyn tc.- Th nghim sinh H2S: th nghim l (+) nu xut
hin ta mu nu en bn
trong mi trng, l (-) nu khng xut hin ta nu en. 3. Th nghim MR
(Methyl Red) a. Nguyn tc Th nghim MR nhm phn bin vi sinh vt da trn
s khc bit v kh nng to ra v duy th cc sn phm bin dng c tnh acid bn
trong mi trng trong qu trnh ln men glucose. Ch th methyl red gip
phn bit nng ion H + hin din trong mi trng sau khi vi sinh vt ln men
glucose. Ch th ny c mu thay i khc nhau ty dy pH hay nng ion H+: khi
ph thp hn 4,4 mu cam trong vng pH[Type text] Page 39
[Type text]
[Type text]
[Type text]
5,0 5,8, mu vng khi pH trn 6,0. Hm lng ion H + ny ph thuc vo t l
CO2 v H2v con ng chuyn ha ng ca tng loi vi sinh vt. i vi cc vi sinh
vt ng rut, thi gian nui cy th nghim phn ng MR thng c xc nh trpong
khong 18-24 gi. Tuy nhin cc vi sinh vt ny u to acid trong mi trng
ngay khi chng bt u tng trng. khi ho di thi gian nui cy, cc vi sinh
vt c phn ng MR (+) c c tnh l tch ly acid trong mi trng ngy cng nhiu
hn v pH trong mi trng ngy cng gim; ngc li, trng hp cc vi sinh vt
cho phn ng MR(-) s tip tc chuyn ha cc sn phm c tnh acid c to ra
thnh cc sn phm trung tnh lm cho pH trong mi trng chuyn dn v pha
trung tnh. Th nghim MR ph thuc rt nhiu vo thi gian nui cy v thng c
tin hnh trong thi gian khong 3-5 ngy 370C. Th nghim ny gip phn bit
E.coli (+) vi Klepsiella (-); Enterbacter aerogenes (-) vi
Enterbacter cloacae (+); Yersinia spp. (+) vi cc loi Bacillus Gram
m khc khng thuc ng rut (-); xc nh Listeria monocytogenes (+). Chng
dng lm i chng (+) cho th nghim ny l Proteus rettgeri, (-) l
Serratia marcescens.
b. Phng php tin hnh Th nghim c tin hnh trong cc ng mi trng lng
Glucose Phosphate (MR-VP broth). Dung que cy vng cy mt lng nh sinh
khi t khun lc chng thun trn mi trng KIA, 370C trong khong 2-5 ngy.
Them vi git thuc th methyl red (0,02% trong hn hp cn nc c t l 3:2,
bo qun 40C) vo trong ng nghim dch nui cy, c kt qu ngay. c. c kt qu
Th nghim MR l (+) khi mi trng c mu sau khi b sung thuc th; l (-)
khi c mu vng. Trng hp mu cam, cn tip tc ng mi trng c ging trong 4
ngy v thc hin li th nghim. 4. Th nghim VP (Voges-Prokauer) a. Nguyn
tc vi sinh vt, bin dng nng lng bng phng thc ln men t glucose qua
con ng ng phn s to ra cht trung gian ch yu l pyruvic acid. khi phc
d tr NAD+ trong t bo phc v cho con ng ng phn, cc loi vi sinh vt,
sau pyruvic acid tip tc c chuyn ha theo cc con ng sinh ha khc nhau
to ra cc sn phm ln men cui cng khc nhau. H Enterbacteriaceae c c
tnh[Type text] Page 40
[Type text]
[Type text]
[Type text]
chung l ln men sinh hn hp acid nh acid formic, acid acetic, acid
succinic, ethanol, H2 v CO2. h ny c th c chia thnh hai nhm l nhm
khng sinh 2,3butanediol (v d nh E.coli) v nhm sinh 2,3-butanediol
(v d nh Klebsiella, Enterobacter). Phn t 2,3-butanediol c th chuyn
ha qua li thnh aceton: trong iu kin c oxi v mi trng c tnh kim,
2,3-butanediol b oxi ha thnh aceton nh xc tc ca enzyme
2,3-bute\anediol dehydrogenase; ngc li, acetoin c th b kh thnh
2,3-butanediol do hot tnh ca enzyme diacetyl reductase. Ngoi ra,
acetoin cn b oxi ha thnh diacetyl. Nh vy, khi c oxi v pH kim,
2,3-butanediol b chuyn ha thnh acetion, n lt mnh acetion b oxi ha
thnh diacetyl, cht ny tham gia vo phn ng to mu trong th nghim VP.
Nhu vy, th nghim VP c th gip phn bit cc loi trong Enterbacteriaceae
da trn s oxi ha acetion (acetylmethylcarbinol, AMC) t
2,3-butanediol thnh diacetyl. S oxi ha acetion thnh diacetyl c tng
cng nh xc tc ca -napthol. Diacetyl kt hp vi nhn guanidine c trong
pepton kt t thnh phc diacetyl-guanidine c mu . Trong thuc th
Koblentz v OMeara c cha creatime c tc dng b sung ngun nhn
guanidine. Th nghim VP c dung phn bit Klebsiella pneumonia (+) v
Enterbacter (+) vi E.coli (-), phn bit mt s loi ca Klebsiella, xc
nh Listeria monocytogenes (+). Chng dng lm i chng (+) cho th nghim
ny l Serratia marcescens, (-) l Proteus rettgeri. b. Phng php tin
hnh Mi trng c s dng cho th nghim VP l mi trng lng Clark-Lubs (mi
trng MR-VP), c pH 6,(. Dng que cy vng cy vo cc ng mi trng MRVP mt t
sinh khi ( trng hp dng thuc th Koblenntz nh di y th cy nhiu sinh
khi) t khun lc ca chng thun 18-24 gi trn mi trng KIA hoc TSI. yn cc
ng mi trng ny 370C trong 20-48 gi hoc n 10 ngy khi cn thit. mt s
loi nh Enterobacter hafniae c kt qu th nghim VP khng n nh 370C nhng
cho (+) 25-300C. Do vy, trng hp nghi ng nn tin hnh cc ng mi trng
song song hai iu kin nhit . Sau thi gian , b sung thuc th trc tip
vo ng mi trng. c 3 loi thuc th VP l: - Thuc th Barrit: gm dung dch
A l 5% -napthol trong cn tuyt i, dung dch B l 40% KOH hoc NaOH. -
Thuc th Koblentz: gm dung dch A l 5% -napthol trong c 95%, dung dch
B l 0,3% creatine 40% KOH hoc NaOH. - Thuc th OMeara: dung dch 0,3%
creatine 40% KOH hoc NaOH.[Type text] Page 41
[Type text]
[Type text]
[Type text]
Khi s dng cc loi thuc th c 2 thnh phn, trc tin nh 6 git dung dch
A, sau nh 2 git dung dch B. i vi thuc th 1 thanh2n phn, b sung 1ml
thuc th vo ng mi trng. lc nh ng 1 pht oxi ha acetone. c kt qu sau
20 pht hoc chm nht 4 gi. Trc khi s dng nn kim tra thuc th bng cc
chng chng nh E.coli (VP-) v Enterobacter cloacae (VP+). c. c kt qu
Th nghim VP l (+) khi c mu trn b mt mi trng, l (-) khi b mt mi trng
khng i mu. 5. Th nghim kh nng bin dng citrate a. Nguyn tc Mt s vi
sinh vt c kh nng s dng citrate lm ngun carbon duy nht thu ly nng
lng v vt cht. s bin dng citrate thng thong qua s kt hp vi acetylCoA
thnh oxaloacetate vo chu trnh tricarboxylic acid (chu trnh Krebs).
Sn phm bin dng citrate thay i ty pH ca mi tng. khi pH tng, mi trng
chuyn sang kim, lng acetate c formate to thnh s tng trong khi
lactate v CO 2 gim. pH trung tnh sn phm ch yu l COP2 v acetate. pH
acid, sn phm to ra ch yu l acetoin v lactate. Nh vy s bin dng
citrate bi vi sinh vt s to ra CO2 lm kim ha mi trng. Mt khc mi vi
sinh vt c kh nng s dng citrate lm ngun carbon duy nht u c kh nng
dng mui ammonium lm ngun m duy nht. S phn gii ca mui ammonium dng
lm ngun m trong mi trng s sinh NH3 lm kim ha mi trng. Nh vy trong
mi trng th nghim kh nng s dng citrate lm ngun carbon duy nht c cha
m dng mui ammonium, kh nng tng trng ca chng s th hin qua kh nng bin
dng ngun carbon citrate v ngun m ammonium lm tng gi tr pH ca mi
trng. S gia tng gi tr pH ny c ch th bng s i mu ca ch th pH trong mi
trng. Th nghim ny c dng phn bit nhm Klebsiella, Enterbacter (+) vi
E.coli (-) trong th nghim IMViC, hoc phn bit Edwardsiella (-) vi
Salmonella (+). Chng dng lm i chng (+) cho th nghim ny l Proteus
rettgeri, (-) l Staphylococcus epidermidis. b. Phng php tin hnh Mi
trng s dng cho th nghim kh nng bin dng citrate l mi trng rn Simmons
Citrate Agar (CSA) hay mi trng Christensens Citrate Sulfide Agar
(CCSA).[Type text] Page 42
[Type text]
[Type text]
[Type text]
Mi trng CSA cha sodium citrate hoc potassium citrate lm ngun
carbon duy nht, ch th bromothymol blue, pH 6,9. Ch th ny c mu vng
pH di 6,0 mu xanh lc pH trung tnh v mu xanh dng ph ln hn 7,6. Chng
thun trn mi trng KIA hoc mi trng thch hp khc c cy ria trn b mt mi
trng CSA rn trong ng thch nghing. ng thch c 35 0C trong 24-48 gi
hoc ko di n 4 ngy khi cn thit. Mi trng CCSA cha ngun m l cystein,
ch th mu l phenol red, pH 6,7. Mi trng cha s dng c mu thay i t mu
kem n nu. pH acid (di 6,8), mu ch th tr thnh vng v pH kim (trn 8,4)
tr thnh mu . Chng vi sinh vt c cy v trong iu kin tng t nh trng hp
mi trng CSA. c. c kt qu - Trng hp mi trng CSA, th nghim la (+) khi
xut hin khun lc v mi trng chuyn sang mu xanh dng; (-) khi khng c
khun lc v mi trng gi nguyn mu xanh lc. - Trng hp mi trng CCSA, th
nghim l (+) khi mi trng chuyn sang mu ; (-) khi mi trng khng i mu.
II. MT S MI TRNG V THUC TH THNG DNG 1. Brilliant Green Bile Lactose
Broth (canh BGBL) Peptone Lactose Mt b Brilliant green Nc ct 10 g
10 g 20 g 0,0133 g 1 lt
Ha tan peptone v lactose vo trong 500ml nc ct, mt b vo trong
khong 200ml v brilliant green trong khong 100ml nc ct. trn u 3 dung
dch t th tch cui l 1 lt; chinh pH mi trng khong 7,4. Rt vo mi ng
nghim c cha ng Durham 10ml mi trng BGBL. Hp 1100C trong 15 pht. pH
7,2 0,2 250C. 2. EC Broth ( Canh EC) Trypticase or tryptose Mui mt
No.3[Type text]
20 g 1,5 gPage 43
[Type text]
[Type text]
[Type text]
Lactose K2HPO4 KH2PO4 NaCl Nc ct
5 g 4 g 1,5 g 5 g 1 lt
Rt mi trng vo ng nghim c cha ng Durham. Hp 1210C trong 15 pht.
pH 6,9 0,2. 3. Canh thang EE Peptone Glucose Disodium hydrogen
phosphate Potassium dihydrogen phosphate Ox bile Brilliant green Nc
ct 10,0 g 5,0 g 8,0 g 2,0 g 20,0 g 0,015 g 1000,0 ml
iu chnh pH ti 7,2; chia ra bnh, c mi bnh 30ml. Tit khun 5 pht
1210C.
4. Canh thang dinh dng Beef extract Peptone Nc ct iu chnh pH ti
7,0. Tit khun 15 pht 1210C 5. Canh thang KCN Proteose peptone
Sodium chloride Potassium dihydrogen phosphate[Type text]
3,0 g 5,0 g 1000,0 ml
3,0 g 5,0 g 0,225 gPage 44
[Type text]
[Type text]
[Type text]
Nc ct
1000,0 ml
iu chnh pH ti 7,6. Dit khun 15 pht 1210C, ngui v cho vo t lnh
580C. Bng cch v khun v thn trng, cho thm 15 ml cyanid 0,5% lnh
(5-80C) lc nh nhng v phn ra ng nghim , mi ng 1 ml, ng nt bng nt bn
thm perafin v bo qun 5-60C. 6. Canh thang L-ST Tryptose, tryptone
hoc trypticase Lactose Dipotassium monohydrogen phosphate
Potasssium dihydrogen phosphate Sodium chloride Sodium lauryl
sulphate Nc ct 20,0 g 5,0 g 2,75 g 2,75 g 5,0 g 0,1 g 1000,0 ml
iu chnh pH ti 6,8. Phn ra c ng nghim, mi ng 10 ml vi ng Durham
ln ngc. Tit khun 10 pht 1210C. 7. Canh thang MacConkey Peptone
Lactose Ox gall Bromocresol purple Nc ct 20,0 g 10,0 g 5,0 g 0,01 g
1000,0 ml
iu chnh pH ti 7,3; phn vo bnh tam gic, mi bnh 225 ml. Tit khun
15 pht 1210C. 8. Canh thang Nitrate Meat extract Peptone Potassium
nitrate Nc ct[Type text]
3,0 g 5,0 g 1,0 g 1000,0 mlPage 45
[Type text]
[Type text]
[Type text]
iu chnh pH ti 7,0; phn ra ng nghim. Tit khun 15 pht 1210C. 9.
Lauryl Sulphate Broth LSB (canh Lauryl Sulphate) Trytose Lactose
Sodium chloride K2HPO4 KH2PO4 Lauryl sulphate Nc ct 20 g 5 g 5 g
2,75 g 2,75 g 0,1 g 1 lt
Hp 1210C trong 15 pht, pH mi trng l 6,8 0,2. 10. Mi trng Indol
Tryptone Sodium chloride DL-trytophan Nc ct 10,0 g 5,0 g 1,0 g
1000,0 ml
iu chnh pH 7,0 phn ra cc ng nghim ng knh 16mm, mi ng 5 ml. Tit
khun 15 pht 1210C. 11. Mi trng ln men carbohydrate Meat extract
Peptone Sodium chlotide Ch th Nc ct 3,0 10,0 5,0 10,0 g g g g
1000,0 ml
iu chnh pH ti 7,1-7,2; phn ra cc ng nghim c ng Durham . Tit khun
15 pht 1210C.
[Type text]
Page 46
[Type text]
[Type text]
[Type text]
Glucose, lactose, sucrose m dng cui cng l 1%. ng cho vo mi trng
c bn phi dit khun trc , tr disaccharide phi tit khun bng lc hoc
1210C trong 10 pht v cho vo mi trng c bn tit khun trc. 12. Mi trng
VP Peptone Glucose Dipotassium hydrogen phosphate Nc ct 7,0 g 5,0 g
5,0 g 1000,0 ml
iu chnh pH ti 6,9 v lc. Tit khun 20 pht 1150C. 13. Simmon
Citrate Agar Sodium citrate NaCl K2HPO4 NH4H2PO4 MgSO4 Bromothylmol
blue Agar Nc ct 2 g 5 g 1 g 1 g 0,2 g 0,08 g 15 g 1 lt
un nh v thnh thong lc. un si 1 2 pht cho n khi ha tan. Phn phi
vo 1/3 ng nghim c np 1 x 100 hoc 16 x 150mm. hp 1210C trong 15 pht.
t nghing ng nghim. pH cui 6,8 0,2. 14. Thch L-EMB a.Peptone Lactose
Dipotassium hydrogen phosphate Agar Nc ct[Type text]
10,0 g 10,0 g
2,0 g 15,0 g 1000,0 mlPage 47
[Type text]
[Type text]
[Type text]
b.Eosin Y Methylen blue
0,4 g 0,0065 g
Pha dung dch a, iu chnh pH ti 7,1-7,2. Chia ra tng bnh 100ml.Tit
khun 15 pht 1210C. un chy trc khi dng, v c 100ml cho thm 2ml dung
dch eosin Y 2% v 4,3 ml dung dch methylen blue 0,15% 15. Thch
MacConkey Peptone Lactose Bile salts Sodium chloride Agar Neutral
red Crystal violet Nc ct 20,0 g 10,0 g 1,5 g 5,0 g 15,0 g 0,03 g
0,001 g 1000,0 ml
iu chnh pH ti 7,1. Tit khun 15 pht 1210C. Rt ra a petri. 16.
Thch TCBS Peptone Yeast extract Sucrose Sodium thiosulphate,
pentahydrate Trisodium citrate dihydrate Sodium cholate Ox-gall
Sodium chloride (NaCl) Ferric citrate Bromothymol blue 10,0 g 5,0 g
20,0 g 10,0 g 10,0 g 3,0 g 5,0 g 10,0 g 1,0 g 0,04 g
[Type text]
Page 48
[Type text]
[Type text]
[Type text]
Thymol blue Agar Nc ct pH
0,04 g 15,0 g 1000,0 ml 8,6 0,2
Ha tt c cc thnh phn trn trong nc ct. un nh la v khuy lin tc cho
tan thch. Ngui ti 500C v rt ra a Petri. Khng tit khun bng hp t. a
mi trng 40C dng trong 24h (ISO 1990). 17. Thch TSI Meat extract
Yeast extract Peptone Sodium chloride Lactose Sucrose Glucose Ferri
citrate Sodium thiosulphate Phenol red Agar Nc ct 3,0 g 3,0 g 20,0
g 5,0 g 10,0 g 10,0 g 1,0 g 0,3 g 0,3 g 0,024 g 12,0 g 1000,0
ml
iu chnh pH ti 7,4; phn ra cc ng nghim, mi ng 10 ml. Tit khun 10
pht 1210C, nghing phn thch y ng cao 2,5 cm.
18.
Thch ur 1,0 g 1,0 g 5,0 gPage 49
a. Peptone Glucose Soium chloride[Type text]
[Type text]
[Type text]
[Type text]
Potassium hydrogen phosphate Phenol red Agar Nc ct Tit khun
trong 2 pht 1210C b. Urea Nc ct va
2,0 g 0,012 g 15,0 g 1000,0 ml
400,0 g 1000,0 ml
Tit khun bng lc. Cho 50 ml b) vo 950 ml a) iu kin v khun, iu
chnh pH ti 6,8, phn ra cc ng nghim, mi ng 10 ml v nghing. 19.
Trypticase (Tryptic) Soy Agar (TSA) Trypticase peptone Phytone
peptone NaCl Agar Nc ct 15 g 5 g 5 g 15 g 1 lt
un nng ha tan agar, phn phi vo cc erlen. Hp kh trng 15 pht
1210C. pH cui 7,3 0,2. s dng cho cc chng Vibrio spp. a mui, cng
them NaCl vo nng cui t 2 3%. 20. Violet Red Bile Agar (VRBA) Cao nm
men Peptone hoc gelysate NaCl Mui mt Lactose Neutral red Crystal
violet Agar[Type text]
3 g 7 g 5 g 1,5 g 10 g 0,03 g 0,002 g 15 gPage 50
[Type text]
[Type text]
[Type text]
Nc ct
1 lt
Ha tan cc thnh phn trong nc, iu chnh v pH 7,4 0,2. un si trong 2
pht, khng hp kh trng. S dng lm trong mi trng a. 21. Thuc th kovacs
p- Dimethylaminobenzalhehyde (p-DMABA) Isoamyl alcohol HCl m c 10 g
150 ml 50 ml
Ha tan p-DMABA trong dung mi, b sung v khuy tng phn nh HCl cho n
khi lng. thuc th c cha trong chai mu ti trnh nh sang 40C. c th thay
th isoamyl alcohol bng amyl alcohol hoc butanol. 22. Thuc th methyl
red Methyl red Ethanol 95% Nc ct () 0,10 g 300 ml 500 ml
Ha tan methyl vo 300ml ethanol. Them nc ct vo cho th tch
500ml
[Type text]
Page 51
[Type text]
[Type text]
[Type text]
TI LIU THAM KHO 1.Vi sinh vt thc phm, tp K thut kim tra v ch tiu
dnh gi cht lng an ton thc phm, NXB Y hc. 2. , Trn Linh Thc,Cc phng
php phn tch vi sinh vt trong nc, thc phm v m phm, NXB Gio dc. 3. Vi
khun y hc, B y t, NXB Gio dc Vit nam, ch bin PGS. TS L Vn Phng. 4.
Kha lun tt nghip, ti: S dng k thut multilplex PCR pht hin cc gen c
lc ca vk Escherichia coli phn lp t phn b, phn heo tiu chy v tht b.
5. Cebula T. A., Payne W. L., and Feng P., 1995. Simultaneous
Identification of Strains of Escherichia coli Serotype O157:H7 and
Their Shiga Like toxin Type by Mismatch Amplification Mutation
Assay Multiplex PCR. J. Clin. Microbiol. 33: 248 - 250. 6. Leo
Heijnen and Gertjan Medema , Journal of water and health
04.04.2006, Quantitative detection of E. coli, E. coli O157 and
other shiga toxin producing E.coli in water samples using a culture
method combined with real-time PCR. 7. Youngsheng He, James,
E.Keen, Ralph B.Westama, E.Travis Littledike, Jimmy Kwang (1996)
Monoclonal Antibodies for Dectiction of the H7 Antigen of
Escherichia Coli, Applied and enviromental microbiology, Vol-62 No.
9p 3325-3332.
[Type text]
Page 52
[Type text]
[Type text]
[Type text]
[Type text]
Page 53
