TH SINH HA CA E.

COLI

I. Dng c v thit b
- T 37oC
- T cy v trng
- Chng E.coli (nghi ng) gi trn mi trng NA 2-4oC (*)
- Que cy vng
- n cn
II. Cc phn ng sinh ha
1. Kh nng to thnh indol
1.1 Nguyn tc:

Mt s vi khun c cha enzym tryptophanase, enzym ny c kh nng phn hu

tryptophan to ra cc sn phm l indol, acid pyruvic v hp cht amoni. xc nh kh
nng ny ngi ta nui cy vi khun vo mi trng c tryptophan (thng l mi trng
Ur - Indol), nu vi khun c enzym tryptophanase th trong mi trng s xut hin indol
v n c pht hin khi nh thuc th Kovac. Vic pht hin indol c thc hin bng
phn ng ca phn t ny vi cc para dimethybezaldehide (p-DMABA). Nhn pyrrol ca
Indol cha nhm CH2 s kt hp vi nhn benzene ca p-DMABA to nn mt phc cht
dng quinone mu (Phn ng dng tnh).(1)

1.2 Mi trng- thuc th

- Canh dinh dng tryptophan (Merck KgaA)
- Thuc th Kowacs (Merck KgaA)
1.3 Quy trnh:
- Dng que cy vng, bng thao tc v trng, cy vi khun kim nh (*) vo mi
trng canh dinh dng tryptophan
- 37oC
- c kt qu sau 24 gi nui cy.
1.4 nh gi kt qu:
 Nh 5 git thuc th vo mi trng nui cy E.coli (kim nh) 37oC/ 24 gi.
yn vi pht, quan st mu ca vng xut hin trn b mt mi trng. Ch khi nh thuc
th khng c lc ng mi trng.

Nu vng xut hin trn b mt mi trng c mu : Phn ng Indol dng tnh.

Nu vng xut hin trn b mt mi trng khng c mu : Phn ng Indol m
tnh. (2)
2. Phn ng MR (Methyl red)
2.1 Nguyn tc:

Phn bit vi sinh vt da trn s khc bit v kh nng to ra v duy tr cc sn phm

bin dng c tnh acid bn trong mi trng trong qu trnh ln men glucose. Ch th
methy 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 5,0-5,8. Mu vng khi pH trn 6,0. (1)

Phn ng: 2 glucose + H2O Acid lactic + acid acetid + ethanol + CO2 + H2O (3)

2.2 Mi trng v thuc th:

- Mi trng canh dinh dng c cha 1% ng glucose (mi trng MR-PV) hay cn
gi m mi trng Clark-Lubs
- Thuc th Methyl red
2.3 Thc hin:
- Dng que cy vng, bng thao tc v trng, cy vi khun kim nh (*) vo canh
thang MR-VP
- 37oC
- c kt qu sau 24 gi nui cy.
2.4 c kt qu
- Nh 2-3 git methyl red vo ng canh trng c nui cy 37oC sau 24 gi, c
kt qu ngay.
- Mi trng c mu , phn ng MR dng tnh
- Mi trng c mu vng, phn ng MR m tnh (1)
3. Phn ng VP (Voges-proskauer)
3.1 Nguyn tc. Mt s vi sinh vt c kh nng oxy ha ng glucose to thnh acid
pyruvic, nhng li khng chuyn ha acid pyruvic thnh cc acid hu c m thnh cc
hp cht acetyl methyl carbinol (acetoin). Acetoin trong mi trng kim cao s b oxh
thnh diacetyl. Diacetyl sinh ra kt hp vi guanidin trong pepton to phc diacetyl
guanidin c mu vi thuc th apha naphtol. (2)

Phn ng: Acetoin+ Naphthol Diacetyl ((KOH 40%, O2)

Diacetyl + Guanidine nucleus mu hng nht.

3.2 Mi trng v thuc th
Mi trng: Mi trng MR-VP (himedia)
Thuc th: KOH 40%
-naphtol 5% trong cn
3.3 Thc hin:

Dng que cy vng, bng thao tc v trng, cy vi khun kim nh (*) vo canh
thang MR-VP

Nh 6 git thuc th A (5% naphtol) v 2 git thuc th B( KOH 40%), Lc k. i

5-10 pht

3.4 c kt qu:

Phn ng VP dng tnh c mu trn mt mi trng

Phn ng VP m tnh khng c mu trn mt mi trng (1)

4. Phn ng Citrate:
4.1 Nguyn tc:

Xc nh vi sinh vt c kh nng s dng citrat l ngun hydratcarbon duy nht. Sn

phm chuyn ha lm kim ha mi trng (mi trng sodiumcitrat Na3C6H5O7 vi sinh
vt s dng citrat v lm tha gc Na+) v thay i mu ca ch th mu. VSV tit enzyme
citrase thy phn citrate thnh acid oxaloacetic v acidacetic.Acid oxaloacetic thy phn
thnh acid pyruvic v CO2. CO2 kt hp vi cc thnh phn ca mi trng to hp cht
alkalin, lm cht ch th pH (bromothymol blue) chuyn t xanh l sang xanh bin.

4.2 Mi trng: Simmons citrate agar (Biolife)

4.3 Thc hin:

Cy vi khun (*) vo mi trng Citrat Simmons, 35 37oC/24 gi

4.4. c kt qu:

Phn ng dng tnh: mu xanh nc bin

Phn ng m tnh: mi trng khng i mu

Ti liu tham kho

1. Trn Minh Hng, 2002. Phng php phn tch vi sinh vt trong nc, thc phm
v m phm. Nh xut bn gio dc.
2. Nguyn Ngc Hi, 2012. Thc hnh nghin cu vi sinh vt. Nh xut bn lao ng.
3. http://s1.downloadmienphi.net/file/downloadfile2/200/1400665.pdf
4. http://himedialabs.com/TD/M070.pdf
5. http://www.masciabrunelli.it/public/cartellina-allegati-schede-
certificazioni/schede-tecniche-inglese/TS-4020452.pdf