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TRNG I HC AN GIANG

KHOA NNG NGHIP TI NGHUYN THIN NHIN

BI BO CO

NH LNG COLIFORMS TRONG NC


UNG NG CHAI AQUA MOON

GIO VIN HNG DN

Ths. BNG HNG LAM

THNH VIN NHM 2:


NGUYN YN NHI
PHM HUNH PHI LIL
H TH THIN NHU
NG TH TUYT PHNG
PHAN TH THY DNG
NGUYN TH HNG THNG

AN GIANG, 2013

Mc lc
Ni dung
Trang
Mc lc ................................................................................................................ i
Danh sch bng .................................................................................................. ii
Danh sch hnh .................................................................................................. iii
Chng 1 t vn .......................................................................................... 1
Chng 2 Ni dung ............................................................................................ 2
2.1. nh ngha .................................................................................................................... 2
2.2. nh lng Coliforms, Coliforms chu nhit, Coliforms phn bng phng
php MPN ............................................................................................................................. 3
2.2.1. Khi nim ................................................................................................. 3
2.2.2. Nguyn tc ............................................................................................... 3
2.2.3. Mi trng v ha cht ............................................................................ 4
2.2.4. Quy trnh phn tch................................................................................... 4
2.2.5. Cch c kt qu ...................................................................................... 5
2.3. nh lng Coliforms, Coliforms phn bng phng php m khun lc 7
2.3.1. Khi nim ................................................................................................. 7
2.3.2. Nguyn tc ............................................................................................... 7
2.3.3. Mi trng v ha cht ............................................................................ 7
2.3.4. Quy trnh phn tch................................................................................... 7
2.3.5. Cch tnh kt qu ...................................................................................... 9
Chng 3 Kt lun v kin ngh ..................................................................... 11
3.1. Kt lun ....................................................................................................................... 11
3.2. Kin ngh..................................................................................................................... 11
Ti liu tham kho ............................................................................................ 12
Ph chng....................................................................................................... 13

Danh sch bng


Bng 1: Biu hin sinh ha cc ging ca Coliforms ........................................ 2

ii

Danh sch hnh


Hnh 1: Quy trnh nh hng Coliforms, Coliforms chu nhit, Coliforms
phn bng phng php MPN ........................................................................... 6
Hnh 2: Quy trnh nh lng Coliforms, Coliforms phn bng phng php
m khun lc .................................................................................................... 9
Hnh 3: Hnh dng Coliforms ........................................................................... 13
Hnh 4: Khun lc Coliforms trn mi trng m - ENDO .............................. 13
Hnh 5: Mi trng lng Lauryl Sulphate Broth LSB (bn tri): mu vng;
Mi trng canh BGBL (bn phi): mu xanh ................................................ 13
Hnh 6: Coliforms sinh hi trong mi trng canh BGBL .............................. 14
Hnh 7: Qui trnh kim tra s c mt ca Coliforms bng phng php MPN14

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Chng 1
t vn
Ngy nay, khi cuc sng con ngi ngy cng c nng cao th iu m h
quan tm l v sinh n ung. Hng nm c hng trm ca ng c thc phm
phi nhp vin, gy hu qu nghim trng n sc khe cng ng. iu ny
khng ch ph bin Vit Nam m ngay c cc nc khc trn th gii cng
vy. Mt trong nhng nguyn nhn gy ra tnh trng ny l s hin din qu
mc cho php cc vi sinh vt gy hi trong thc phm.
Kim sot cht lng v sinh v an ton thc phm lun l mi quan tm ca
mi x hi, mi thi i, c bit l hin nay, khi cuc sng ca con ngi
ngy cng cao, cht lng mi trng sinh quyn ngy cng thp, ngha l
him ha t cc tc nhn l, ha v nht l sinh hc t mi trng vo thc
phm ang tr nn ngy mt ln. Trong xu th hi nhp hin nay, cc x
nghip ch bin thc phm nu mun sn phm ca mnh sc cnh tranh
khng ch trn th trng ni a m c trn cc th trng nhp khu ln vi
yu cu v sinh ngy cng mt khc khe. Do vy buc cc nh sn xut phi
c nhng qui trnh kim tra v sinh tht khc khe t c cc tiu chun
quc t, mt trong nhng ch tiu m h quan tm n l s lng
Coliforms trong cc sn phm ca mnh.
Coliforms l sinh vt ph bin trong ng tiu ha ca con ngi v ng
vt. hu ht chng tn ti mt cch t nhin khng gy hi cho con ngi. Tuy
nhin, trong mt s trng hp, nht l khi sinh l c th thay i, stress th
mt s dng Coliforms mang gen gy c c th gy bnh trn ngi v mt
s loi ng vt. Coliforms l mt ch tiu thng dng c dng nh gi
mc an ton trong v sinh thc phm. S hin din mt s lng nht nh
Coliforms trong thc phm l nh gi thc phm khng an ton cho ngi
s dng.

Chng 2
Ni dung
2.1. nh ngha
Coliforms l nhng trc khun gram m, khng sinh bo t, hiu kh hoc k
kh ty , c kh nng ln men lactose sinh acid l sinh hi 370C trong 24
48 gi. Trong thc t phn tch, coliforms cn c nh ngha l cc vi khun
c kh nng ln men sinh hi trong khong 48 gi khi c 370C trong
mi trng canh Lauryl Sulphate v canh Brilliant Green Lactose Bile Salt.
Nhm Coliforms hin din rng ri trong t nhin, trong rut ngi, ng vt.
Coliforms c xem l nhm vi sinh vt ch th: s lng hin din ca chng
trong thc phm, nc, hay cc loi mu mi trng c dng ch th kh
nng hin din ca cc vi sinh vt gy bnh khc. Nhiu nghin cu cho thy
rng khi s Coliforms ca thc phm cao th kh nng hin din ca cc vi
sinh vt gy bnh khc cng cao. Tuy nhin, mi lin h gia cc vi sinh vt
gy bnh v vi sinh vt ch th ny vn cn nhiu tranh ci. Nhm Coliforms
gm 4 ging l: Escherichia vi mt loi duy nht l E. coli, Citrobacter,
Klebsiella v Enterobacter. Tnh cht sinh ha c trng ca nhm ny c
th hin qua cc th nghim Indol (I), Methyl Red (MR), Voges-Proskauer
(VP) v Citrate (iC) thng c gi tm tt chung l IMViC.
Bng 1: Biu hin sinh ha cc ging ca Coliforms
Voges
Phn ng
Indol
Methyl Red
Citrat
Proskauer
Escherichia
+(-)
+
Citrobacter
-(+)
+
+
Klebsiella
-(+)
+
+
Enterobacter
-(+)
+
+
Ghi ch: + phn ng dng tnh, - phn ng m tnh, +(-): a s phn ng
dng tnh v (+): a s phn ng m tnh.
Coliforms chu nhit l nhng Coliforms c kh nng ln men lactose sinh hi
trong khong 24 gi khi c 440C trong mi trng canh EC. Coliforms
phn (Faecal Coliforms hay E. coli gi nh) l Coliforms chu nhit c kh
nng sinh indole khi c khong 24 gi 44,50C trong canh Trypton.
Coliforms phn l mt thnh phn ca h vi sinh ng rut ngi v cc
ng vt mu nng khc v c s dng ch th mc v sinh trong qu
trnh ch bin, bo qun, vn chuyn thc phm, nc ung cng nh ch
th s nhim phn trong mu mi trng.

Coliforms pht trin tt trn nhiu loi mi trng, nhiu loi thc phm. C
nhng nghin cu cho thy chng c th pht trin nhit thp n -2oC v
cao n 50oC. trong thc phm chng pht trin yu v rt chm 5oC tuy
cng c ti liu ghi nhn s pht trin ca chng 3-6oC.
Ngng pH Coliforms c th pht trin l 4,4 9.
Chng pht trin tt trn mi trng thch thng, cho nhng khun lc thy
sau 12-16 gi 37oC, pht trin tt rt nhiu loi thc phm trong iu kin
thch hp.
Coliforms gm 2 nhm:
- Coliforms c ngun gc t phn pht trin nhanh, khong 16 gi, trong
mi trng dinh dng 44oC, khng mc 4oC trong 30 ngy. L loi vi
khun a nhit, nhit thch hp nht l 41oC.
- Coliforms khng c ngun gc t phn, chng c ngun gc thy sinh
hay t t, mc nhanh 4oC trong 3-4 ngy v 10oC trong 1 ngy. Khng mc
41oC, 44oC c ch hon ton s pht trin ca tt c cc Coliforms khng
c ngun gc t phn
2.2. nh lng Coliforms, Coliforms chu nhit, Coliforms phn bng
phng php MPN
2.2.1. Khi nim
MPN l phng php dng nh gi mt vi sinh vt theo s c xc sut
ln nht ca lng vi sinh vt c trong mt th tch mu, vi chnh xc
tng i cao. Phng php ny cn gi l phng php pha long ti hn
hay phng php chun , chng da trn k thut nui cy vi sinh vt cn
nh lng trong mt mi trng lng thch hp vi mt s ln lp li nht
nh. Pha long mt s ln mu c cha vi sinh vt, sau kim tra xem ti
pha long no cn pht hin s c mt ca vi sinh vt cn kim tra.
2.2.2. Nguyn tc
S lng Coliforms, Coliforms chu nhit, Coliforms phn trong mu nc,
cha mt thp ca nhm vi khun ny 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 nhau 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 3-5 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 cho phn ng dng tnh mi nng pha long v da

vo bng MPN suy ra s lng nhm vi sinh vt tng ng hin din trong
1ml mu nc ban u.
2.2.3. 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)
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.
- Canh Tryptone
- Mi trng rn Simmon Citrate Agar (thch Simmon Citrate) mi trng
c cha sodium citrate l ngun carbon duy nht. Khi vi khun s dng
sodium citrate s kim ha mi trng v mi trng thch s c mu xanh.
- Dung dch nc mui pepton SPW (Saline Pepton Water)
- Thuc th Kovac,s
- Canh MR- VP
- Thuc th Methyl Red
Thuc th -napthol
2.2.4. Quy trnh phn tch
Thng tin v sn phm
Nc ung ng chai AQUA MOON sn xut ti c s An Hnh (a ch:
22D2, H Huy Tp, khm ng Thnh 6, phng M Phc, Long Xuyn, An
Giang).
2.2.4.1. nh lng Coliforms
Chun b mu nc ng chai AQUA MOON, pha long mu, c mu c
pha long 10-1.
Tun t cy 1ml dung dch mu pha long 10-1 vo 3 ng nghim ging
nhau, mi ng cha 10ml canh LSB. Thc hin tng t vi dung 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. Nu nghi ng s lng Coliforms trong mu
qu cao, phi s dng cc mu c bc pha long cao hn. cc ng nghim
37oC trong 48 gi. Ghi nhn s ng c sinh hi. Dng que cy vng cy
chuyn dch mu t cc ng LSB (+) sang cc ng c cha canh BGBL v
37oC trong 48 gi. Ghi nhn s ng cho kt qu (+) (c sinh hi) ng vi mi
pha long.

2.2.4.2. nh lng Coliforms chu nhit


Dng que cy vng chuyn mt vng dch mu t cc ng canh LSB (+) sang
mi trng canh EC, 44,5 0,2oC trong 24 gi. m s lng cc ng cho
kt qu (+) (sinh hi) mi pha long.
2.2.4.3. nh lng coliforms phn
Dng que cy vng ria dung dch mu t cc ng (+) trn mi trng canh EC
sang mi trng thch a EMB. cc a ny 37oC trong 24 gi. Cc
khun lc trn, dt hnh a v c nh kim tm l khun lc ca Coliforms phn
hay E.coli gi nh. Chn khun lc ng knh ln hn 1mm v cy truyn
vo canh Trypton, 44,5 0,2oC trong 24 gi. Nh thuc th Kovacs vo
cc ng nghim. ng nghim c s xut hin ca mu trong mi trng
trong vi pht l ng (+) . Thc hin tng t cho tt c cc ng (+) trn mi
trng EC. Ghi nhn s lng cc ng cho kt qu (+) trn mi trng
Trypton tng ng vi mi pha long.
2.2.5. Cch c kt qu
tt c cc trng hp nu trn, t s lng cc ng nghim c kt qu
dng tnh mi pha long ca mu, dng Bng MPN thch hp 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.

Chun b mu nc, pha long mu c pha


long 10-1, 10-2, 10-3
Chuyn 1ml dd 10-1, 10-2, 10-3 vo ng 10ml canh
LSB, mi nng 3 ng lp li
37oC, 48 gi

Ghi nhn cc ng LSB (+) mi nng pha long

Cy vo ng canh EC

Cy vo ng canh BGBL

44,5 0,2oC
24 gi

37oC 1oC
48 gi

S ng (+) mi pha long

S ng (+) mi pha long

Cy ln thch EMB, 37oC, 24 gi

Chn khun lc in hnh cy


vo canh Tryptone
44,5 0,2oC
24 gi

Th nghim Indol

m s ng canh EC (+) v
indol (+), tra bng MPN

Coliforms

Coliforms chu nhit

Coliforms phn

Hnh 1: Quy trnh nh hng Coliforms, Coliforms chu nhit,


Coliforms phn bng phng php MPN

2.3. nh lng Coliforms, Coliforms phn bng phng php m khun


lc
2.3.1. Khi nim
Phng php m khun lc l phng php cho php xc nh s lng t
bo vi sinh vt cn sng hin din trong mu. T bo sng l t bo c kh
nng phn chia to thnh khun lc trn mi trng chn lc.
2.3.2. Nguyn tc
Mu nc em v c pha long v cy mt lng nht nh ln mi trng
thch chn lc thch hp cha lactose. m s khun lc ln men lactose v
sinh acid sau khi 37oC trong 24 - 48 gi. Ngoi lactose, mi trng chn
lc cho Coliforms cn cha mui mt c ch vi khun gram dng v cht ch
th pH nh neutral red, crystal violet. Trn mi trng ny khun lc
Coliforms c mu n mu m, ng knh > 0,5 mm, xung quanh
khun lc c vng ta ca mui mt. Vic khng nh c thc hin bng
cch nui cy trn mi trng canh chn lc nh BGBL. nh lng
Coliforms phn, thc hin tng t nhng thay i nhit l 44oC. Mt
Coliforms hay Coliforms phn c tnh da trn s lng khun lc in hnh
m c, t l khng nh v pha long mu trc khi cy vo a.
2.3.3. Mi trng v ha cht
Mi trng Troptone Soya Agar (TSA) c chun b trong cc binhg
hay chai thy tinh, hp kh trng v bo qun 4-8oC. Trc khi s dng,
mi trng c un chy v lm ngui 45oC trong b iu nhit.
Violet Red Bile Agar (VRB) c chun b v trng trong cc chai
thy tinh, un chy v lm ngui 45oC trong b iu nhit trc khi s dng.
C th s dng mi trng Desoxycholate Agar thay cho VRB.
Mi trng canh Brilliant Green Bile Lactose Broth (BGBL) c
phn phi 10ml vo mi ng nghim v trng cha mt ng Durham p
ngc. Hp kh trng. Sau khi hp kim tra cc ng m bo khng c bt
kh trong ng Durham.
Mi trng canh EC Broth c phn phi 5ml vo mi ng nghim,
hp kh trng.
Mi trng canh Trypton Broth c phn phi 5ml vo mi ng
nghim, hp kh trng.
Thuc th Kovacs hay Indol.
2.3.4. Quy trnh phn tch
Chun b mu nc ng chai AQUA MOON, pha long mu, c mu c
pha long 10-1.
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Mu c pha long tng t nh phn nh lng tng s vi khun hiu kh


sao cho cha <100 t bo Coliforms trong 1ml dung dch pha long. Chuyn
1ml dch pha long mu chn vo a petri. B sung vo mi a cy
mu khong 5ml mi trng TSA c un chy v n nh trong b iu
nhit 45oC. Lc trn a petri xui v ngc chiu kim ng h, mi chiu
3-5 ln trn iu dch mu vi mi trng. nhit phng trong 1-2
gi hi phc cc t bo b tn thng. B sung vo mi a 10-15ml mi
trng thch VRB nhit 45oC ln trn mi trng TSA. Ch cho mi
trng trong a ng c, lt ngc a v 37oC trong 24-48 gi. Thc
hin tng t trn mu 2 nng pha long lin tip sao cho mi a s xut
hin t 10-100 khun lc v lp li t nht 2 a ng vi mi nng pha
long.
- Th nghim khng nh Coliforms
Trong trng hp mu c cha cc ngun carbon khc khng phi lactose,
trnh cc trng hp vi sinh vt s dng cc ngun carbon trong mu ln
men v to khun lc c hnh dng tng t Coliforms cn thc hin thm
bc khng nh nh sau: Chn t nht 5 khun lc nghi ng, dng que cy
vng cy truyn sang cc ng nghim cha mi trng BGBL (trng hp
khng nh Coliforms tng s) hoc mi trng EC ( trng hp khng nh
Coliforms phn). cc ng BGBL 37 v cc ng EC 44oC trong 24 - 48
gi. Kt qu khng nh l (+) khi vi khun tng trng lm c mi trng
v sinh hi trong ng Durham. Tnh t l khng nh l t s gia s khun lc
cho kt qu (+) vi s khun lc c dng trong th nghim khng nh.
Ngoi ra, trng hp th nghim khng nh Coliforms phn, cc khun lc
cho kt qu (+) trn EC cn c thc hin th nghim indol 44oC. Th
nghim khng nh Coliforms phn ch c xem l (+) khi va l (+) trn
mi trng EC va l (+) trn th nghim indol.

Chun b mu nc, pha long mu c pha


long 10-1, 10-2, 10-3
Cy 1ml dd mu vo a petri, b sung 5ml mi
trng TSA, lc u, yn 1 2 gi
Rt vo mi a 10 - 15ml mi trng thch VRB,
ng, 37oC trong 24 48 gi
m cc khun lc mu n m, c vng ta
mui mt, ng knh 0,5 mm; chn 5 khun lc

Cy vo ng canh BGBL,
37oC, 24 48 gi

Cy vo ng canh EC,
44oC, 24 48 gi

m s ng canh (+) (sinh hi);


tnh t l khng nh Coliforms

Chn ng canh (+) (sinh hi)

Mt Coliforms

Th nghim Indol

m s ng canh EC (+) v indol


(+), tnh t l khng nh Coliforms
phn

Mt Coliforms phn
Hnh 2: Quy trnh nh lng Coliforms, Coliforms phn
bng phng php m khun lc
2.3.5. Cch tnh kt qu
Da vo s khun lc m c v t l khng nh, tnh mt ca Coliforms
v Coliforms phn theo cng thc sau:
A (CFU/g hay CFU/ml) =
xR

N: tng s khun lc m c
ni: s i c s khun lc c chn ti mi pha long
V: dung tch mu (ml) cy vo mi a
fi: pha long c s khun lc c chn ti cc a m
R: t l khng nh

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Chng 3
Kt lun v kin ngh
3.1. Kt lun
Coliforms l sinh vt ph bin trong ng tiu ha ca con ngi v ng
vt. khi gp iu kin thun li chng s gy hi cho con ngi nht l khi
sinh l c th thay i, stress
Coliforms l mt ch tiu thng dng c dng nh gi mc an ton
trong v sinh thc phm. S hin din mt s lng nht nh Coliforms trong
thc phm l nh gi thc phm khng an ton cho ngi s dng.
Coliforms c xem l nhm vi sinh vt ch th. S lng hin din ca chng
trong thc phm, trong nc c dng ch th cho kh nng hin din ca
cc vi sinh vt gy bnh khc. S lng Coliforms cao th kh nng hin din
ca vi sinh vt gy bnh khc cao. So vi hu ht cc vi khun gy bnh
ng rut, Coliforms c kh nng sng lu hn. Nu trong mt th tch nc
nht nh (100 mL) khng pht hin thy vi khun ch th ng rut ny th
c th dng lm nc ung hoc nc sinh hot.
3.2. Kin ngh
Do Coliforms hin din khp mi ni nn phi kim tra trn nhiu sn phm
khc nhau.
S dng thm nhiu phng php khc kim tra nh lng Coliforms,
Coliforms phn, Coliforms chu nhit.
Nn n ung hp v sinh, chn thc phm an ton trnh cc bnh ly nhim
ng rut.

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Ti liu tham kho


Trn Linh Phc. 2012. Phng php phn tch vi sinh vt trong nc, thc
phm v m phm. H Ni: Nh Xut Bn Gio Dc Vit Nam.
L Vn Vit Mn v Li Mai Hng. 2006. Th nghim vi sinh vt hc thc
phm. TP. HCM: Nh Xut Bn i Hc Quc Gia Tp. H Ch Minh
Nguyn c Lng. Phan Th Huyn v Nguyn nh Tuyt. 2006. Th
nghim vi sinh vt hc. TP. HCM: Nh Xut Bn i Hc Quc Gia TP.
HCM
on Ngc Tun. 2006. Kho st tnh hnh nhim E. Coli v Coliforms trong
nc ung, nc c gas, nc c cn trn a bn qun Th c. Lun
vn k s chuyn ngnh Cng Ngh Sinh Hc. B mn Cng Ngh Sinh
Hc. Trng i hc Nng Lm Thnh Ph H Ch Minh.
Trang web:
http://www.tuanvietnam.net/mot-so-vi-khuan-chu-yeu-trong-thuc-pham

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Ph chng

Hnh 3: Hnh dng Coliforms

Hnh 4: Khun lc Coliforms trn mi trng m - ENDO

Hnh 5: Mi trng lng Lauryl Sulphate Broth LSB (bn tri): mu


vng; Mi trng canh BGBL (bn phi): mu xanh

13

Hnh 6: Coliforms sinh hi trong mi trng canh BGBL

Hnh 7: Qui trnh kim tra s c mt ca Coliforms bng phng php


MPN

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