Tham Khảo Thực Hành VI Sinh PDF

Trng i hc Cng nghip
thc phm TP.HCM
2010
THC HNH PHN TCH

VI SINH THC PHM
ThS. L Thy Linh

0
Lu hnh ni b
Trng i hc Cng nghip thc phm TP.HCM
NI DUNG Trang
Bi 1: nh lng Coliforms v E. coli 2
1.1. Tm tt kin thc c bn 2

1.2. Quy trnh nh lng Coliforms v E. coli bng phng php MPN 3
1.3. Cch tin hnh th nghim 4
1.4. Cch tnh kt qu 10
Bi 2. nh lng Staphylococcus aureus 10

2.2. Quy trnh nh lng Staphylococcus aureus bng phng php MPN 10
2.3. Cch tin hnh v cch tnh kt qu 11
Bi 3. nh tnh Salmonella 14

3.2. Qui trnh nh tnh Salmonella trong thc phm 14
Bi 4. nh lng Bacillus cereus 20

4.2. Quy trnh nh lng Bacillus cereus bng phng php m khun lc 20
Bi 5. nh lng tng vi khun hiu kh v tng nm men-nm mc 24

5.2. Quy trnh phn tch 26
Ti liu tham kho
1. Trn Linh Thc, Phng php phn tch vi sinh vt trong nc, thc phm v m
phm, NXB Gio dc, 2006
2. http://www.microbelibrary.org
3. http://www.chromagar.com
4. http://www.rapidmicrobiology.com
5. http://www.marietta.edu
Bin son: L Thy Linh Page | 1

Homepage: http://lethuylinh.weebly.com
Bi 1: nh lng Coliforms v E. coli
1.1 Tm tt kin thc c bn Hnh 1: pht hin Coliforms v E.

1.1.1 nh ngha coli trong thc phm hay nc bng
mi trng CHROMagar ECC
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 v
sinh hi 370C trong 24 48 gi. 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 loi vi sinh vt khc. Nhm Coliforms gm 4 ging l:
E. coli, Citrobacter, Klebsiella, 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 l IMViC.
Coliforms chu nhit l nhng Coliforms c kh nng ln men lactose sinh hi
trong khong 24h khi 440C trong mi trng canh EC.
Coliforms phn (Faecal Coliforms) l Coliforms chu nhit c kh nng sinh
indole khi khong 24h 440C trong canh Trypton. E. coli l Coliforms phn cho kt
qu th nghim IMViC l ++-- (Indol +, Methyl Red +, Voges-Proskauer -, Citrate -)
1.1.2 Phng php Most Probable Number (MPN)
y l phng php nh lng da trn kt qu nh tnh ca mt lot th nghim
c lp li mt s pha long khc nhau. Thng thng, vic nh lng ny c
thc hin lp li 3 ln 3 pha long bc 10 lin tip, tng cng 3 x 3 = 9 ng nghim.
Quy trnh thc hin nh sau:
Pha long mu phn tch 3 nng pha
long bc 10 lin tip.
nhit v thi gian thch hp
Kt qu biu kin chng minh s tng

trng ca vi sinh vt cn kim nh
Ghi nhn s lng ng nghim dng tnh

tng pha long. Tra bng Mac Crady
Mt vi sinh vt MPN/100 ml hay MPN/1g mu

Ch :
a s cc bng MPN cung cp s liu dng s MPN trong 100ml dung dch
c s dng phn tch cc liu lng l 10ml, 1ml, 0.1 ml. Trn thc t phn tch,
ngi ta thng dng 1ml cho mu pha long 10-1, 10-2, 10-3. Lng mu vi cc
pha long ny tng ng vi 1/100 lng mu s dng theo bng MPN/100ml tnh
theo lng mu l 10ml, 1ml, 0.1 ml nu trn. Vy s liu ca bng MPN/100ml tnh
theo lng mu l 10ml, 1ml, 0.1 ml tng ng vi MPN/ml mu cha pha long khi
1ml ca cc dung dch c pha long 10-1, 10-2, 10-3 c dng phn tch.
Trng hp mt vi sinh vt qu cao, cn phi s dng lot nng pha long

tip theo cn phi nhn tr s tra bng MPN nu trn vi h s pha long. V d: s dng
1ml cho mi pha long 10-2, 10-3, 10-4 tc l ch s dng 1/10 lng mu mi nng
so vi trng hp dng 1ml mu cc pha long 10-1, 10-2, 10-3. Do vy, tr s tra
bng MPN cn nhn thm h s pha long l 10.
1.2 Quy trnh nh lng Coliforms v E. coli bng phng php MPN
Chun b dch ng nht hoc pha long mu c

pha long 10-1, 10-2, 10-3
Chuyn 1ml dung dch 10-1, 10-2, 10-3 vo ng 10ml canh

LSB, mi nng c 3 ng lp li, 370C, 48 gi
Ghi nhn cc ng LSB (+) (sinh hi) mi nng pha long
Cy vo ng canh BGBL, Cy vo ng canh EC,

370C 10C, 48 gi 44.50C 0.20C, 24 gi
S ng (+)(sinh hi ) S ng (+) mi pha long

mi pha long
Cy ln thch EMB, 370C,
24 gi
Coliforms
Xem
trang
sau

Tip
theo
Chn khun lc (trn, dt hnh a v c

nh kim xanh), cy vo Trypton, MR-VP,
SC Citrate, 44.50C 0.20C, 24 gi
Th nghim IMViC
m s ng canh EC (+) v
IMViC ++--, tra bng MPN
E. coli
1.3 Cch tin hnh th nghim
Mu phn tch: nc ma nguyn cht (dng 10ml cho mt t)

1.3.1 Pha mi trng v kh trng dng c
Bc 1: pha mi trng
Tn mi trng Thnh phn Tng th tch Ghi ch

SPW NaCl : 42.5g 500 ml nc Phn phi cho mi t 27ml
(Saline Pepton Pepton: 5g ct SPW (9ml/ng nghim)
Water) trc khi kh trng. (bui 1)
LSB Tryptose: 20g 1000 ml nc Phn phi 90ml LSB cho
(Lauryl Sulphate Lactose: 5g ct mi t (10ml/ng nghim)
Broth) Sodium chloride: 5g pH 6.8 0.2 trc khi kh trng. (bui 1)
KH2PO4: 2.75g
K2HPO4: 2.75g
Lauryl sulphate: 0.1g
BGBL Peptone: 10g 1000 ml nc Phn phi 90ml BGBL cho

(Brilliant Green Lactose: 10g ct mi t (10ml/ ng nghim c

Bile Lactose Mt b: 20g pH 7.4 cha ng Durham) trc khi
Broth) Brilliant green: 0.0133g kh trng. (bui 1)
EC Trypticase or tryptose: 1000 ml nc Phn phi 90ml BGBL cho
(E.coli medium) 20g ct mi t (10ml/ ng nghim c
Mui mt No.3: 1.5g pH 6.9 cha ng Durham) trc khi
Lactose: 5g kh trng. (bui 1)
KH2PO4: 1.5g
K2HPO4: 4g
NaCl: 5g
EMB Pancreatic digest of 1000 ml nc Phn phi 100ml EMB cho
(Eosin Methylene gelatin: 10g ct. mi t (20ml/petri) (bui 2)
Blue Agar) Lactose: 10g pH 7.1 0.2
K2HPO4: 2g
Eosin Y: 0.4g
Methylene blue: 65mg
Agar: 15g
Tryptone water Tryptone hoc 1000ml nc Mi t ch dng 20ml, phn
trypticase:10g ct phi 10ml/ng nghim.
pH 7.2 0.2 (bui 3)
MR-VP Both Mi trng 1: 1000ml nc Mi t ch dng 30ml, phn
(Glucose Buffered peptone-water ct. phi 10ml/ng nghim.
Phosphate) powder: 7g pH 6.9 0.2 Kim tra li mi trng cung
Glucose: 5g cp no pha mi trng.
K2HPO4: 5g (bui 3)
Mi trng 2:
Casein Pancreatic
Digest: 3.5g
Peptic digest of animal
tissue:3.5g
Dextrose: 5g
Potassium phosphate: 5g

pH 6.9 0.2
Mi trng 3:
Peptone: 5g
Glucose: 5g
Phosphate buffer: 5g
pH 7.5 0.2
SCA Sodium citrate: 2g 1000ml nc Mi t ch dng 50ml, phn
(Simmons Citrate NaCl: 5g ct. un nng phi 10ml/ng nghim.
Agar) K2HPO4: 1g nh v thnh (bui 3)
NH4H2PO4: 1g thong lc.
MgSO4: 0.2g un si 1-2
Bromothymol blue: pht cho n
0.08g khi ha tan
Agar: 15g ht. pH 6.8
0.2
Bc 2: kh trng dng c v mi trng pha.
Bao gi dng c v phn phi mi trng vo dng c, gn nt v bao gi. Hp

tit trng 1210C trong 15 pht.
1.3.2 Cch tin hnh:

Bc 3: pha long mu
1ml 1ml 1ml 1ml 1ml 1ml
ng 9ml
mu
10-1 10-2 10-3 10-4 10-5 10-6 pha long
Hnh 2: quy trnh pha long mu
Pha long mu thnh 3 nng pha long 10-1, 10-2, 10-3.

Bc 4: Cy mu vo canh LSB
Chuyn 1ml dung dch 10-1, 10-2, 10-3 vo ng 10ml canh LSB, mi nng c 3
ng lp li, 370C, 48 gi.
Hnh 3: S cy mu t cc pha long
10-1 10-2 10-3
Bc 5: nh lng Coliform v E. coli
nh lng Coliform nh lng E. coli

Ghi nhn s ng c sinh hi (+) Ghi nhn s ng c sinh hi (+)
Dng que cy vng cy chuyn dch mu Dng que cy vng cy chuyn dch mu
t cc ng LSB (+) sang cc ng c cha t cc ng LSB (+) sang mi trng canh
canh BGBL v 370C 10C, 48 gi EC, 44.50C 0.20C, 24 gi
Ghi nhn s ng c sinh hi (+) ng vi Ghi nhn s ng c sinh hi (+) ng vi
mi pha long mi pha long
Tnh kt qu (xem 1.4) Dng que cy vng ria dch mu t cc ng
(+) trn canh EC sang mi trng thch a
EMB. 370C, 24 gi.
Chn khun lc ng knh ln hn 1mm
(trn, dt hnh a v c nh kim xanh)
(xem hnh 4), cy vo Trypton, MR-VP, SC
Citrate, 44.50C 0.20C, 24 gi

Hnh 4: E.coli nui trn mi trng thch EMB

cho khun lc mu kim xanh (theo Naowarat
Cheeptham, Thompson Rivers University,
Kamloops, BC, Canada)
Bc 6: th nghim IMViC
Hnh 5: Kt qu IMViC ca Escherichia coli sau 24gi 37C. (Anne Hanson, University of Maine, Orono)
ng A: dng tnh th nghim indole trn mi trng tryptone. Xut hin lp mu trn b mt mi
trng sau khi nh thuc th Kovcs.
ng B: dng tnh th nghim methyl red xut hin mu ca methyl red.
ng C: m tnh th nghim Voges-Proskauer biu hin qua s khng thay i mu sau khi cho thuc
th Barritts A v Barritts B.
ng D: m tnh th nghim citrate biu hin qua s thay i mu sc v khng c khun lc trong ng
nghim.
a. Th nghim Indol (I):

Nguyn tc: Tryptophan l mt acid amin c th b oxi ha bi mt s vi sinh vt
c h enzyme tryptophanase to nn cc sn phm cha gc indol. Vic pht hin indol
c thc hin bng phn ng ca phn t ny vi cc thuc th cha p-
Dimethylaminbenzaldehyde (p-DMABA) to nn mt phc cht dng quinone c mu
.
Phng php tin hnh: Mi trng s dng cho th nghim ny l nc
tryptone. Sinh khi chng thun c cy vo mi trng lng tryptone, nhit
370C trong 24-48 gi. 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 kt qu: (+) xut hin ca lp mu trn b mt

mi trng. (-) lp mu vng ca thuc th. i khi c s
xut hin ca mu cam do skatol, l tin cht ca methyl ha
ca indol, to ra.
Hnh 6: th nghim Indol
b. Th nghim Methyl Red (MR):
Nguyn tc: ch th methyl red gip phn bit nng H+ hin din trong mi
trng sau khi vi sinh vt ln men glucose. Ch th ny thay i mu nh sau: pH<4.4 ;
pH 5.0-5.8 mu cam; pH>6.0 mu vng.
Phng php tin hnh: Mi trng s dng cho th nghim ny l mi trng

lng Glucose Phosphate (MR-VP Broth). Dng que vng cy mt lng nh khun lc
trn mi trng MR-VP, 370C trong khong 2-5 ngy. Thm vo 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 kt qu: (+) mi trng c mu sau khi b sung

thuc th. (-) khi c mu vng. Trng hp mu cam, cn tip
tc ng mi trng c ging trong 4 ngy v thc hin li
Hnh 7: th nghim MR
th nghim.
c. Th nghim Voges-Proskauer (VP) (xem mc 3.3, bi 3)

d. Th nghim Citrate (iC)
Nguyn tc: s bin dng citrate bi vi sinh vt s to ra CO2 lm km ha mi

trng. Mt khc mi VSV 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. S gia tng gi tr pH ny c ch th bng s i mu ca ch th pH trong mi
trng.


lng SCA. Dng que vng cy ria mt lng nh khun lc ln
mi trng SCA rn trong ng nghim thch nghing, 350C
trong khong 24-48 gi.
c kt qu: (+) 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.
1.4 Cch tnh kt qu Hnh 8: th nghim Citrate
T s lng cc ng nghim c E. coli (+) mi pha long ca mu, dng bng

MPN lot 3 ng nghim 3 nng pha long lin tip tnh ra mt vi sinh vt
trong mu v biu din di dng tr s MPN/ml mu ban u cha pha long.
Bi 2. nh lng Staphylococcus aureus
2.1. Tm tt kin thc c bn

Staphylococcus aureus l vi khun hiu kh hay k kh ty ,
hnh cu, gram dng, c th nghim coagulase, phn ng
DNAse, phosphatease (+) c kh nng ln men v sinh acid t
mannitol, trehalose, sucrose. Tt c cc dng S. aureus u mn
cm vi novobiocine, c kh nng tng trng trong mi trng
cha n 15% NaCl. Mt s dng c kh nng tng trng lm
Hnh 9: S. aureus trn BPA tan mu trn mi trng thch mu. S. aureus c th nhim vo
thc phm qua con ng tip xc vi ngi thao tc trong qu trnh ch bin thc phm.
S hin din vi mt cao ca S. aureus trong thc phm ch th iu kin v sinh v
kim sot nhit km ca qu trnh ch bin.
2.2 Quy trnh nh lng Staphylococcus aureus bng phng php MPN
Phng php ny c dng nh lng S. aureus trong mu c mt S. aureus thp
nhng mt vi sinh vt cnh tranh cao.


lng SCA. Dng que vng cy ria mt lng nh khun lc ln
mi trng SCA rn trong ng nghim thch nghing, 350C
trong khong 24-48 gi.
c kt qu: (+) 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.
1.4 Cch tnh kt qu Hnh 8: th nghim Citrate
T s lng cc ng nghim c E. coli (+) mi pha long ca mu, dng bng

MPN lot 3 ng nghim 3 nng pha long lin tip tnh ra mt vi sinh vt
trong mu v biu din di dng tr s MPN/ml mu ban u cha pha long.
Bi 2. nh lng Staphylococcus aureus
2.1. Tm tt kin thc c bn

Staphylococcus aureus l vi khun hiu kh hay k kh ty ,
hnh cu, gram dng, c th nghim coagulase, phn ng
DNAse, phosphatease (+) c kh nng ln men v sinh acid t
mannitol, trehalose, sucrose. Tt c cc dng S. aureus u mn
cm vi novobiocine, c kh nng tng trng trong mi trng
cha n 15% NaCl. Mt s dng c kh nng tng trng lm
Hnh 9: S. aureus trn BPA tan mu trn mi trng thch mu. S. aureus c th nhim vo
thc phm qua con ng tip xc vi ngi thao tc trong qu trnh ch bin thc phm.
S hin din vi mt cao ca S. aureus trong thc phm ch th iu kin v sinh v
kim sot nhit km ca qu trnh ch bin.
2.2 Quy trnh nh lng Staphylococcus aureus bng phng php MPN
Phng php ny c dng nh lng S. aureus trong mu c mt S. aureus thp
nhng mt vi sinh vt cnh tranh cao.

ng nht mu v pha long mu thnh cc dy nng

thp phn 10-1, 10-2, 10-3
Chuyn 1ml dung dch 10-1, 10-2, 10-3 vo ng 10ml canh

MSB, mi nng c 3 ng lp li, 370C, 48 gi
Chn ng (+) c mi pha long
Ria ln a thch BPA, 370C, 48 gi
Chn khun lc c trng (trn li, en

bng, c qung trong v qung sng
xung quanh (i khi ch xut hin 1
trong 2 qung))
Cy vo TSA, 370C 10C , 24 gi
Th nghim ngng kt coagulase
Ghi nhn s coagulase (+) mi pha

long
Tra bng MPN
Mt S.aureus
(MPN/g hoc MPN/ml)
2.3 Cch tin hnh v tnh kt qu

Mu phn tch: tht heo sng, khi lng 50g cho 1 lp

Bc 1: pha mi trng
SPW NaCl : 42.5g 500 ml nc Phn phi cho mi t 27ml
(Saline Pepton Pepton: 5g ct SPW (9ml/ng nghim)
Water) trc khi kh trng. (bui 2)
MSB Cao tht: 1g 1000 ml nc Phn phi 90ml MSB cho
(Mannitol Salt Polypeptone: 10g ct mi t (10ml/ng nghim)
Broth)
NaCl: 75g pH 7.4 0.2 trc khi kh trng. (bui 2)
Mannitol: 10g
Phenol red: 0.025g
BPA Mi trng c bn 1000 ml nc Phn phi 100ml BPA cho
(Baird Parker Tryptone:10g ct cho mi mi t (20ml/petri) (bui 2)
Agar) Cao tht: 5g trng c bn
Cao nm men: 1g pH 7.0 0.2
Sodium pyruvate: 10g
Glycine: 12g
Lithium chloride.6H2O:
5g
Agar: 20g
Mi trng hon chnh
5ml Bacto EY tellurite
enrichment (45-500C)
vo 95ml mi trng c
bn c lm nng
chy. Mi trng hon
chnh phi c.
TSA Trypticase peptone: 15g 1000 ml nc Phn phi 50ml BGBL cho
(Tryptone Soya Phytone peptone: 5g ct mi t (25ml/petri) (bui 3)
Agar) NaCl: 5g pH 7.3 0.2
Agar: 15g


Bc 3: ng nht mu v pha long mu
Cn 10g mu trong ti PE v trng, thm 90ml dung dich SPW, ng nht mu
bng my dp mu khong 30 giy. Pha long mu (xem bi 1, mc 1.3.2)
Bc 4: Cy mu vo canh MSB
Chuyn 1ml dung dch 10-1, 10-2, 10-3 vo ng 10ml canh MSB, mi nng c 3
ng lp li, 370C, 48 gi. (xem bi 1, mc 1.3.2). Chn cc ng (+) c vi sinh vt pht
trin lm c mi trng tin hnh phn lp khun lc n trn BPA
Bc 5: phn lp khun lc n trn BPA
Dng k thut cy ria ln a thch BPA, 370C, 48 gi.

Chn khun lc c trng :trn li, en bng, c qung trong v
qung sng xung quanh; i khi ch xut hin 1 trong 2 qung (xem
hnh 10). Hnh 10: hnh dng S. aureus trn BPA
Chn khun lc c trng ca S. aureus trn BPA th nghim sinh ha

coagulase
Bc 6: th nghim sinh ha coagulase
Nguyn tc: cc loi thuc ging Staphylococcus c kh nng tit ra mi trng

enzyme coagulase c tc dng lm kt t cc thnh phn ca huyt tng, to thnh cc
khi ng lm ng huyt tng.
Phng php tin hnh: Huyt tng ngi hay th ng kh thng phm c
dng cho th nghim ny. Cho vo ng nghim 0.5ml huyt tng ngi hay th khng
pha long, b sung 0.5ml dch nui cy chng thun hoc mt lng ln sinh khi khun
lc. Xoay nh ng trn u VSV. ng th nghim 370C trong
4 gi. Quan st, ghi nhn s ngng kt mi 30 pht. Tip tc n
24 gi nu khng thy xut hin khi kt t.
c kt qu: (+) c s kt t; (-) khng c s kt t, hn hp
vn ng nht
Hnh 11: th nghim coagulase. ng trn (+); ng di (-)

Bi 3. nh tnh Salmonella
3.1 Tm tt kin thc c bn

Hnh 12: Salmonella trn thch XLD
Salmonella l trc trng gram m, hiu kh v k kh ty
, c kh nng di ng khng to bo t, ln men glucose v
mannitol sinh acid nhng khng ln men saccharose v lactose,
khng sinh Indole, khng phn gii ure, khng c kh nng
tch nhm amine t tryptophane, hu ht cc chng u sinh
H2S.
Salmonella c th phn tch nh tnh bng m quy trnh
gm 4 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 cc loi vi khun khc thuc h Enterobacteriaceae
c tnh cnh tranh mnh v c ch s tng trng ca Salmonella.
3.2 Quy trnh nh tnh Salmonella trong thc phm
ng nht 25g mu trong 225ml mi trng tng sinh

BPW, 370C, 18-24 gi
Cy 0.1ml dch tng sinh sang mi trng tng sinh chn

lc RV, 420C, 18-24 gi
Phn lp khun lc n trn t nht 2 mi trng chn

lc c bit (XLD, HE, BS, SS), 370C, 24 gi
Chn cc khun lc c trng cho Salmonella, cy sang

BHI hay TSA, qua m
Th nghim sinh ha cho kt qu:
- Trn KIA/TSI: /vng, c/khng H2S, sinh hi/khng

- Urea (-); Indol (-); VP (-); LDC (+); ODC (+); Manitol
(+); Sorbitol (+)
Salmonella dng tnh/

m tnh trong 25g mu

Mu phn tch: c hoc rut c, khi lng 25g cho 1 lp
Bc 1: pha mi trng
BPW Pepton: 10g 1000 ml nc Phn phi cho mi t 20ml
(Buffered Pepton NaCl : 5g ct BPW trc khi kh trng.
Water) Na2HPO4: 3.5g pH 7.2 0.2 (bui 2)
KH2PO4: 1.5g
RV Mi trng c bn 1110 ml Phn phi 30ml RV cho mi
(Rappaport- Tryptone:5g pH 5.5 0.2 t (10ml/ng nghim) trc
Vassiliadis Soya
Pepton) NaCl: 8g khi kh trng. (bui 2)
KH2PO4: 1.6g
Nc ct: 1000 ml
Dung dch MgCl2
MgCl2.6H2O: 400g
Nc ct: 1000 ml
Dung dch Malachite
green oxalate
Malachite green oxalate:
0.4g
Nc ct: 100 ml
Mi trng hon chnh
Mi trng c bn:
1000ml
Dung dch MgCl2:
100ml
Dung dch Malachite
green oxalate: 10ml
XLD Cao nm men: 3g 1000 ml nc Phn phi 50ml XLD cho
(Xylose Lysine L-lysine: 5g ct mi t (25ml/petri) (bui 3)
Desoxycholate) Xylose: 3.75g pH 7.4 0.2
Lactose: 7.5g un si mi
Sucrose: 7.5g trng.
Sodium deoxycholate: Khng hp.

2.5g Khng gi
Ferric ammonium qu 1 ngy
citrate: 0.8g
Sodium thiosulfate: 6.8g
NaCl: 5g
Agar:15g
Phenol red: 0.08g
HE Proteose Peptone: 12.0g 1000 ml nc Phn phi 50ml HE cho mi
(Hektoen Entric Yeast Extract: 3.0g ct t (25ml/petri) (bui 3)
Agar) Bile Salts No. 3: 9.0g pH 7.5 0.2
Lactose: 12.0g un si mi
Saccharose: 12.0g trng.
Salicin: 2.0g Khng hp.
Sodium Chloride: 5.0g
Sodium Thiosulfate:
5.0g
Ferric Ammonium
Citrate : 1.5g
Agar: 14.0g
Bromthymol Blue:
65.0mg
Acid Fuchsin : 0.1g
TSA Trypticase peptone: 15g 1000 ml nc Phn phi 50ml TSA cho
(Tryptone Soya Phytone peptone: 5g ct mi t (25ml/petri) (bui 4)
Agar) NaCl: 5g pH 7.3 0.2
Agar: 15g
KIA Polypeptone peptone: 1000 ml nc Phn phi 20ml KIA cho
(Kliger Iron 20g ct mi t (10ml/ng nghim)
Agar) Lactose: 20g pH 7.4 0.2 (bui 5)
Dextrose: 1g
NaCl: 5g

Ferric ammonium
citrate: 0.5g
Sodium thiosulfate: 0.5g
Agar: 15g
Phenol red: 0.025g
K2HPO4: 5g (bui 5)
Mi trng 2:
Casein Pancreatic
Digest: 3.5g
tissue:3.5g
Dextrose: 5g
pH 6.9 0.2
Mi trng 3:
Peptone: 5g
Glucose: 5g
pH 7.5 0.2
RSU Dipotassium Phosphate: 1000ml nc Mi t ch dng 30ml, phn
(Rustigian-Stuart 9.5g ct. phi 3ml/ng nghim.
Urea Broth) Yeast Extract: 0.1g pH 6.8 0.2 (bui 5)
Urea: 20g
Monopotassium
Phosphate: 9.1g
Phenol Red: 0.01g

Bao gi dng c v phn phi mi trng vo dng c, gn nt v bao gi. Hp tit trng
1210C trong 15 pht.
Bc 4: Tng sinh
i vi cc loi mu thng thng tin hnh cn 25g mu trong ti PE v trng,

b sung 225ml dung dch BPW v ng nht mu bng Stomacher trong 15 hoc 30 giy.
370C, 18-24 gi. i vi mt s loi thc phm (mu gia cm ti sng, sa kh,
cc gia v hay cc loi thc phm cha nhiu gia v, casein, pho mt, b, sn phm cha
cocoa, cc sn phm ca da) c cha cc cht c th gy c hoc c ch s tng trng
ca Salmonella cn thc hin quy trnh tng sinh c bit (xem TLTK).
Bc 5: Tng sinh chn lc
Lc trn u dch tng sinh, v chuyn 0.1ml sang 10ml mi trng tng sinh
RV c m 420C. 420C, 18-24 gi. Khi cn thit c th ko di thi gian
thm 24 gi.
Bc 6: Phn lp v nhn din
Dng que cy vng thc hin k thut cy phn lp khun lc n vi ging t

dch tng sinh chn lc ln a mi trng chn lc phn bit c trng cho Salmonella
nh XLD v HE. Biu hin ca Salmonella trn tng mi trng nh sau:
+ Mi trng XLD: khun lc c mu hng trong sut, c

hay khng c tm en. Mt s dng Salmonella c th c tm en
bng rt ln c th chim gn ht khun lc.
Hnh 13: khun lc Salmonella trn XLD

Hnh 14: khun lc Salmonella trn HE
+ Mi trng HE: khun lc Salmonella c mu thay i t

xanh dng n mu xanh lc, c hay khng c tm en. Mt s
dng Salmonella c th c tm en bng rt ln c th chim gn
ht khun lc.
Bc 7: Khng nh
T mi mi trng chn lc phn bit chn v cy chuyn t nht 5 khun lc nghi

ng sang mi trng khng chn lc TSA. 370C, 18-24 gi. Cc khun lc xut hin
trn mi trng ny c s dng cho cc th nghim sinh ha nh sau:
+ Th nghim sinh H2S trn mi trng KIA (Kligler Iron Agar): Mi trng KIA
c s dng th nghim kh nng s dng cc ngun carbon khc nhau (glucose,
lactose) v kh nng sinh H2S.
V ngun carbon, mi trng KIA cha 2 loi ng l 1% lactose v 0.1%

glucose. Khi cy chng vi sinh vt ln mi trng ny c 3 trng hp xy
ra i vi s tng trng ca VSV: ch s dng glucose, s dng glucose v
lactose, khng s dng c hai ng ny. Salmonella ch ln men c
ng glucose v th phn nghing ca mi trng c mu , phn su c
mu vng. Thi gian t 18-24 gi.
V sinh hi: a s cc dng Salmonella u c kh nng sinh H2S nn c

xut hin cc vt mu en trong mi trng ny. C th thy hin tng
sinh hi qua hin tng lm v thch mi trng hoc mi trng b y
ln trn to mt khong khng bn di y ng nghim.
Hnh 15: th nghim KIA. ng 1: mi trng KIA khng c
VSV. ng 2: xut hin mu vng chng t VSV ln men
lactose tt (ln men glucose cng tt). ng 3: c sinh kh,
chng t ln men c sinh kh. ng 4: mu trn phn thch
nghing, mu vng di y chng t VSV ch ln men
glucose. ng 5: mu en xut hin chng t c qu trnh kh
lu hunh v sinh H2S.
+ Th nghim urea (-): c thc hin trn mi trng

urea lng RSU (Rustigian Stuarts Urea Broth), cha ch th
phenol, chuyn mu t vng sang (vng chuyn mu l
pH 6.8-8.4). Dng que cy vng cy lng sinh khi ln t
khun lc ca chng thun vo ng cha 3ml mi trng v
trng, lc nh ng trn u vi khun v 370C trong 48
gi. Salmonella khng phn gii urea nn khng lm thay i
pH mi trng; sau khi nui cy mi trng vn gi nguyn Hnh 16: kt qu th nghim Urea
mu vng cam.
+ Th nghim VP (-): mi trng c s dng l mi

trng lng Clark-Lubs (mi trng MR-VP), pH 6.9. Dng que
cy vng cy vo cc ng mi trng MR-VP mt t sinh khi t
khun lc ca chng thun 18-24 gi trn mi trng KIA.
Sau thi gian , b sung thuc th trc tip vo ng mi trng.
Thuc th Barritt gm dung dch A l 5% -naphthol trong cn
Hnh 17: kt qu th
Bin son:
nghimLVPThy Linh Page | 19
tuyt i, dung dch B l 40% NaOH hay KOH. Trc tin nh 6 git dung dch A, sau
nh 2 git dung dch B. c kt qu sau 20 pht hoc chm nht 4 gi. Salmonella
cho phn ng m tnh vi th nghim VP c hin tng khng i mu trn b mt mi
trng. Th nghim VP (+) khi c mu trn b mt mi trng.
Bi 4. nh lng Bacillus cereus

B. cereus l nhng trc khun, gram dng, hiu kh v k kh ty , di ng, to
ni bo t, ln men glucose sinh hi, phn ng VP (+), c kh nng s dng nitrate. Loi
ny tng trng c trong khong nhit t 50C 500C, ti u 350C 400C; pH dao
ng t 4.5-9.3, d to bo t v bo t ny mm rt d dng. Trn mi trng chn lc
loi ny to khun lc rt ln, mc lan, ra nhn.
4.2 Quy trnh nh lng Bacillus cereus bng phng php m khun lc
Cn 25g mu, ng nht trong 225ml BPW, ng

nht bng Stomacher, pha long 10-1
Pha long n cc pha long 10-2, 10-3
Tri 0.1ml mi pha long ln mi trng thch

MYP, 300C, 24 gi
Chn 5 khun lc c mu hng eosin, lecithinase

dng tnh (ta lecithinase bao quanh khun lc)
cy sang thch nghing MYP, 300C, 24 gi
Nhum Gram v th nghim sinh ha nh: ln

men glucose, th nghim VP.
Kt lun B. cereus

tuyt i, dung dch B l 40% NaOH hay KOH. Trc tin nh 6 git dung dch A, sau
nh 2 git dung dch B. c kt qu sau 20 pht hoc chm nht 4 gi. Salmonella
cho phn ng m tnh vi th nghim VP c hin tng khng i mu trn b mt mi
trng. Th nghim VP (+) khi c mu trn b mt mi trng.
Bi 4. nh lng Bacillus cereus

B. cereus l nhng trc khun, gram dng, hiu kh v k kh ty , di ng, to
ni bo t, ln men glucose sinh hi, phn ng VP (+), c kh nng s dng nitrate. Loi
ny tng trng c trong khong nhit t 50C 500C, ti u 350C 400C; pH dao
ng t 4.5-9.3, d to bo t v bo t ny mm rt d dng. Trn mi trng chn lc
loi ny to khun lc rt ln, mc lan, ra nhn.
4.2 Quy trnh nh lng Bacillus cereus bng phng php m khun lc
Cn 25g mu, ng nht trong 225ml BPW, ng

nht bng Stomacher, pha long 10-1
Pha long n cc pha long 10-2, 10-3
Tri 0.1ml mi pha long ln mi trng thch

MYP, 300C, 24 gi
Chn 5 khun lc c mu hng eosin, lecithinase

dng tnh (ta lecithinase bao quanh khun lc)
cy sang thch nghing MYP, 300C, 24 gi
Nhum Gram v th nghim sinh ha nh: ln

men glucose, th nghim VP.
Kt lun B. cereus

Mu phn tch: tm kh, khi lng 50g cho 1 lp

Bc 1: pha mi trng
BPW Pepton: 10g 1000 ml nc Phn phi cho mi t 50ml
(Buffered Pepton NaCl : 5g ct BPW trc khi kh trng.
Water) Na2HPO4: 3.5g pH 7.2 0.2 (bui 4)
KH2PO4: 1.5g
MYP Mi trng c bn Mi trng Phn phi 100ml MYP cho
(Mannitol-Egg Cao tht: 1g c bn em mi t (12.5ml/petri). (bui
Yolk-Polymycin)
Pepton: 10g kh trng, 4)
Mannitol: 10g ngui 500C.
NaCl: 10g Sau trn
Phenol red (1% trong cc thnh
ethanol 90%): 2.5ml phn cn li
Agar: 15g
Nc ct: 900 ml
Dung dch Polymixin
0.1%
Ha tan 500 000 n v
polymixin B sulfate
trong 50ml nc ct.
Lc v trng v ct
trong ti 40C cho n
khi dng.
Egg yolk emulsion 50%
(sn phm thng mi)
Mi trng cui cng
Mi trng c bn:
225ml ( 500C)
Dung dch Polymixin B:
2.5ml
Egg yolk emulsion (lng
trng): 12.5ml
PRG Proteose peptone No. 3: 1000 ml nc Phn phi 30ml PRG cho
(Phenol Red 10 g ct mi t (3ml/ng nghim)
Glucose Broth) NaCl: 5 g pH 7.4 0.2 (bui 4)
Beef extract (optional):
1g
Dextrose: 5 g
Phenol red (7.2 ml of
0.25% solution): 0.018 g
K2HPO4: 5g (bui 4)
Mi trng 2:
Casein Pancreatic
Digest: 3.5g
tissue:3.5g
Dextrose: 5g
pH 6.9 0.2
Mi trng 3:
Peptone: 5g
Glucose: 5g
pH 7.5 0.2


Bc 3: Pha long mu
Cho 25g mu vo ti PE, b sung 225ml mi trng BPW ng nht c pha

long 10-1, ng nht bng Stomacher trong 1 pht. Mu tip tc pha long thnh 10-2,
10-3.
Bc 4: Pht hin bng mi trng chn lc
Tri 0.1ml mi pha long trn mi trng thch MYP,

0
30 C, 24 gi. Do B. cereus khng ln men mannitol, to lecithinase
v khng polymycin nn trn mi trng ny khun lc B. cereus c
mu hng eosin, c bao quanh bi vng c ta chng t
lecithinase c to thnh. Chn 5 khun lc (+) cy sang thch
nghing chun b cho cc phn ng khng nh. Hnh 18: khun lc B. cereus trn MYP
Bc 5: Cc th nghim khng nh
Hnh 19: nhum gram B. cereus - Nhum Gram: nh 1 git nc ct ln phin knh sch;
dng que cy vng ly 1 t sinh khi vi khun chuyn vo git
nc trn phin knh, khuy nh. C nh vt bi trn phin knh
bng cch h trn ngn la n cn. Nh vi git methyl violet
ln vt bi, gi yn 20 giy. Ra sch phm nhum bng nc.
Nh vi git dung dch KI/I2 ln vt bi, yn 1 pht. Ra
bng cn 95% n va mt mu. Ra li bng nc. Nhum bng dung dch
safranin 30 giy. Ra sch bng nc. Thm nc d bng giy lc. Quan st vi
khun bng vt knh 100X nhng trong du, Gram dng c mu xanh ta, Gram
m c mu hng.
- Th nghim ln men glucose: cy vi khun vo 3ml canh PRG, 350C, 24 gi
trong iu kin k kh. Lc ng nghim tht mnh v quan st s pht trin thng
qua c v s chuyn mu t sang vng, chng t s sinh acid t glucose
trong iu kin k kh.

Hnh 20: th nghim ln men glucose. ng

a: i chng. ng b: khng ln men
glucose. ng c: ln men yu. ng d: ln
men mnh.
- Th nghim VP (xem bi 3)
Bi 5. nh lng tng vi khun hiu kh v tng nm men-nm mc

Vi khun hiu kh l nhng vi khun tng trng v hnh thnh khun lc trong
iu kin c oxi phn t. Tng s vi khun hiu kh hin din trong mu ch th mc
v sinh ca thc phm. Ch s ny c xc nh bng phng php m khun lc mc
trn mi trng thch dinh dng t mt lng mu xc nh trn c s xem 1 khun lc
l sinh khi pht trin t 1 t bo hin din trong mu v c biu din di dng s
n v hnh thnh khun lc (colony forming unit, CFU) trong mt n v khi lng
thc phm.
Nm mc l vi nm dng si, sinh sn bng bo t hoc khun ty. Nm men l
nhng t bo n tnh pht trin theo kiu ny chi, thnh thong c th tn ti dng
khun ty gi trong cc t bo kt nhau thnh chui. n v hnh thnh khun lc ca
nm mc v nm men l mm to nn mt khun lc khi nui cy trong mi trng.
Mm c th l mt bo t, mt t bo hay mt on ca khun ty. Trong thc phm, nm
mc v nm men hin din c th tng trng lm thay i mu ca thc phm, lm pht
sinh mi hay v l, lm h hng hay thay i c cu ca thc phm.

Hnh 20: th nghim ln men glucose. ng

a: i chng. ng b: khng ln men
glucose. ng c: ln men yu. ng d: ln
men mnh.
- Th nghim VP (xem bi 3)
Bi 5. nh lng tng vi khun hiu kh v tng nm men-nm mc

Vi khun hiu kh l nhng vi khun tng trng v hnh thnh khun lc trong
iu kin c oxi phn t. Tng s vi khun hiu kh hin din trong mu ch th mc
v sinh ca thc phm. Ch s ny c xc nh bng phng php m khun lc mc
trn mi trng thch dinh dng t mt lng mu xc nh trn c s xem 1 khun lc
l sinh khi pht trin t 1 t bo hin din trong mu v c biu din di dng s
n v hnh thnh khun lc (colony forming unit, CFU) trong mt n v khi lng
thc phm.
Nm mc l vi nm dng si, sinh sn bng bo t hoc khun ty. Nm men l
nhng t bo n tnh pht trin theo kiu ny chi, thnh thong c th tn ti dng
khun ty gi trong cc t bo kt nhau thnh chui. n v hnh thnh khun lc ca
nm mc v nm men l mm to nn mt khun lc khi nui cy trong mi trng.
Mm c th l mt bo t, mt t bo hay mt on ca khun ty. Trong thc phm, nm
mc v nm men hin din c th tng trng lm thay i mu ca thc phm, lm pht
sinh mi hay v l, lm h hng hay thay i c cu ca thc phm.

5.2 Quy trnh phn tch

ng nht v pha long mu c pha
long 10-1, 10-2, 10-3, 10-4
Xc nh tng s VK hiu Xc nh tng s nm men nm

kh bng k thut hp mc bng k thut hp tri
Chn 2 nng pha long Tri 0.1ml mu ln a

thch hp, chuyn 1ml mu DRBC, nga a
vo a petri v trng (mi 250C, 5-7 ngy (mi
nng cy 2 a) nng cy 2 a)
Rt vo mi a petri 10- m khun lc nm

15ml mi trng PCA mc, nm men
c lm ngui n 450C,
lc cho mu phn tn u
vo mi trng, 300C
trong 72 gi
Tnh kt qu: tng s vi
Chn cc a c s khun sinh vt hiu kh v tng s
nm men nm mc trong
lc trong khong 25-250
mu (CFU/g hoc CFU/ml)
khun lc/a m
5.3 Cch tin hnh th nghim
Mu phn tch: nc ma nguyn cht (dng 10ml cho mt t)
1.3.1 Pha mi trng v kh trng dng c
Bc 1: pha mi trng

SPW NaCl : 42.5g 500 ml nc ct Phn phi cho mi t 27ml
(Saline Pepton Pepton: 5g SPW (9ml/ng nghim)
Water) trc khi kh trng. (bui
5)

PCA Casein enzymic 1000 ml nc Phn phi 100ml PCA cho

(Plate Count hydrolysate: 5g ct mi t (15ml/petri). (bui
Agar) Yeast extract: 2.5 g pH 7.0 0.2 5)
Dextrose: 1g
Agar: 15g
DRBC Glucose: 10g 1000 ml nc Phn phi 100ml DRBC
(Dichloran Rose Peptone: 5g ct cho mi t (15ml/petri).
Bengal KH2PO4: 1g pH 5.6 0.2. (bui 5)
Chloramphenicol MgSO4: 0.5g Lu : trn ln
Agar) Rose bengal (dung dch cc thnh phn
5%, w/v): 0.5ml tr
Dichloran (0.2%(w/v) chloramphenicol,
trong ethanol): 1ml un nng ha
Chloramphenicol: 0.1g tan ht agar, hp
Agar: 15g kh trng.
ngui n 500C,
b sung 1ml
dung dch khng
sinh 100X vo
100ml mi
trng.

Bc 3: pha long mu (xem bi 1, mc 1.3.2)
Bc 4: thc hin nh qui trnh

Cch tnh kt qu
m tt c cc khun lc xut hin trn cc a sau khi . Chn s a c s m

trong khong 25-250 tnh kt qu. Mt tng vi khun hiu kh hay tng s nm men
nm mc c tnh nh sau:
N
A (CFU/g hay CFU/ml) =
n1 x V x f1 + + ni x V x i
Trong :
A: s t bo (n v hnh thnh khun lc) vi khun (hay nm mc nm

men) trong 1g hay 1ml mu.
N: tng s khun lc m c trn cc a chn
ni: s lng a cy ti pha long th i
V: th tch dch mu (ml) cy vo trong mi a
fi: pha long tng ng.
Nu pha long cao nht, s khun lc m c trn mt a >250, v d

nng 10-5 s m ln hn 250, kt qu c ghi: >2.5 x 107 CFU/g.
Nu pha long thp nht, s khun lc m c trn mt a <25, v d

nng 10-1 s m nh hn 25, kt qu c ghi: <2.5 x 102 CFU/g.


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Trng i hc Cng nghip

thc phm TP.HCM

THC HNH PHN TCH

ThS. L Thy Linh

Bi 1: nh lng Coliforms v E. coli 2

1.1. Tm tt kin thc c bn 2

2.1. Tm tt kin thc c bn 10

3.1. Tm tt kin thc c bn 14

4.1. Tm tt kin thc c bn 20

5.1. Tm tt kin thc c bn 24

Bin son: L Thy Linh Page | 1

Bi 1: nh lng Coliforms v E. coli

1.1 Tm tt kin thc c bn Hnh 1: pht hin Coliforms v E.

nhit v thi gian thch hp

Kt qu biu kin chng minh s tng

Ghi nhn s lng ng nghim dng tnh

Mt vi sinh vt MPN/100 ml hay MPN/1g mu

Bin son: L Thy Linh Page | 2

Trng hp mt vi sinh vt qu cao, cn phi s dng lot nng pha long

Chun b dch ng nht hoc pha long mu c

Chuyn 1ml dung dch 10-1, 10-2, 10-3 vo ng 10ml canh

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

Cy vo ng canh BGBL, Cy vo ng canh EC,

S ng (+)(sinh hi ) S ng (+) mi pha long

Bin son: L Thy Linh Page | 3

Chn khun lc (trn, dt hnh a v c

1.3 Cch tin hnh th nghim

Mu phn tch: nc ma nguyn cht (dng 10ml cho mt t)

Tn mi trng Thnh phn Tng th tch Ghi ch

Bin son: L Thy Linh Page | 4

(Brilliant Green Lactose: 10g ct mi t (10ml/ ng nghim c

Bin son: L Thy Linh Page | 5

Bao gi dng c v phn phi mi trng vo dng c, gn nt v bao gi. Hp

1.3.2 Cch tin hnh:

10-1 10-2 10-3 10-4 10-5 10-6 pha long

Hnh 2: quy trnh pha long mu

Pha long mu thnh 3 nng pha long 10-1, 10-2, 10-3.

Bin son: L Thy Linh Page | 6

Hnh 3: S cy mu t cc pha long

10-1 10-2 10-3

Bc 5: nh lng Coliform v E. coli

nh lng Coliform nh lng E. coli

Bin son: L Thy Linh Page | 7

Hnh 4: E.coli nui trn mi trng thch EMB

ng B: dng tnh th nghim methyl red xut hin mu ca methyl red.

a. Th nghim Indol (I):

tch indol ln lp dung mi hu c. Nh 5 git thuc th

c kt qu: (+) xut hin ca lp mu trn b mt

Phng php tin hnh: Mi trng s dng cho th nghim ny l mi trng

c kt qu: (+) mi trng c mu sau khi b sung

c. Th nghim Voges-Proskauer (VP) (xem mc 3.3, bi 3)

Nguyn tc: s bin dng citrate bi vi sinh vt s to ra CO2 lm km ha mi

Bin son: L Thy Linh Page | 9

Phng php tin hnh: Mi trng s dng cho th nghim ny l mi trng

c kt qu: (+) khi xut hin khun lc v mi trng

1.4 Cch tnh kt qu Hnh 8: th nghim Citrate

T s lng cc ng nghim c E. coli (+) mi pha long ca mu, dng bng

Bi 2. nh lng Staphylococcus aureus