Bai Giang LT Phan Tich Vi Sinh DH-CD

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TRNG I HC CNG NGHIP THC PHM TP.HCM

KHOA CNG NGH THC PHM

PHN TCH VI SINH THC PHM
(Dnh cho h CNKN)

Bin son : Nguyn Thy Hng

Tp.HCM 09/ 2011

2
MC LC

Chng 1: i tng vi sinh vt v cc ch tiu trong thc phm.3
Chng 2: K thut c bn trong phn tch vi sinh vt..17
Chng 3: Quy trnh phn tch vi sinh vt theo phng php truyn thng...39
Chng 4: Quy trnh phn tch vi sinh vt theo phng php hin i...85
Chng 5:nh gi v sinh cng nghip.95

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Chng I
I TNG VI SINH VT V CC CH TIU TRONG THC PHM

1.1GII THIU
Ng c thc phm trong nhng nm gn y c ghi nhn kh thng xuyn,
tr thnh mi quan tm ca ton x hi. C nhiu nguyn nhn khc nhau c th gy
ra cc v ng c thc phm nhng phn ln cc trng hp l c ngun gc t vi
sinh vt, do s hin din ca vi sinh vt gy bnh hay s hin din ca c t tit ra
bi cc vi sinh vt ny trong nc ung, thc phm. Ngy nay, an ton, nht l v
phng din vi sinh vt, tr thnh mt trong nhng yu cu khng th thiu i vi
cht lng thc phm.
Vit Nam l mt nc nng nghip c tim lc ln v sn xut nng sn, thy
hi sn, thc phm. Ngoi th trng tiu th ni a cho hn 80 triu dn, thc phm
v thy sn ca nc ta cng xut khu c ra th trng th gii (Chu u, Bc
M, Nht Bn), mang li nhiu ngoi t cho t nc, gii quyt vic lm cho mt
s lng ln ngi lao ng c nng thn v thnh th. Do nhn thc ngy cng
c nng cao ca ngi tiu dng trong nc v an ton v sinh thc phm v s
tng cng v qun l ca Nh nc, kim tra, gim st ca cc c quan chc nng,
yu cu nghim ngt v ch tiu vi sinh ca th trng th gii, vic phn tch vi sinh
vt gy bnh, thc hin cc bin php nhm m bo sn xut, ch bin thc phm t
tiu chun an ton v thc hin cc bin php m bo sn xut, ch bin thc phm
t tiu chun an ton v sinh thc phm ngy cng c cc n v sn xut, ch bin
thc phm ni a quan tm thc hin. Tuy nhin khng phi v th m ng c thc
phm, s v ng c thc phm, mc ng c thc phm khi con ngi tiu th
thc phm c thuyn gim.
Nc v thc phm nui sng con ngi nhng cng c th gy ng c hoc
nhim bnh cho con ngi do c th cha cc c t vi sinh vt, c t ho hc hoc
cha cc vi sinh vt gy bnh. C rt nhiu v ng c hay nhim bnh gy ra bi vi
sinh vt hin din trong nc v thc phm. Ng c thc phm thng c hiu l
cc triu chng gy ra bi c t ca vi sinh vt hin din trong thc phm v nhim
bnh bi vi sinh vt trong thc phm l trng hp nhim bnh do s dng thc n
cha cc vi sinh vt gy bnh. Tuy nhin hin nay, khi nim ng c thc phm cn
bao gm trng hp thc n c cha cc vi sinh vt gy bnh hin din mc rt
thp trong nguyn liu hay b nhim vo trong qu trnh ch bin v trong qu trnh ch
bin hay bo qun, cc vi sinh vt ny v c t ca chng c th tng nhanh n mc
gy ng c.
Ng c thc phm thng xy ra ng thi nhiu ngi, to ra nhng triu
chng chung sau khi tiu th thc phm. Tuy nhin, mc tc ng s khc nhau
ph thuc kh nng p ng vi c t v th trng khc nhau ca tng ngi. Cc
triu chng thng gp ca ng c thc phm l tiu chy, chng mt, nn ma, au
nhc ngi, st, au u. Triu chng ny thay i cc v ng c khc nhau tu
thuc vo tc nhn vi sinh vt v c gy ra bi c t do vi sinh vtk to ra v tit
vo thc phm (trng hp ny c gi l ngoi c t) hay bi c t nm trong t
bo vi sinh vt (ni c t).
i vi nc v thc phm, cc vi sinh vt c quan tm cn kim sot l cc
vi sinh vt gy ng c v gy bnh. Cc vi sinh vt gy ng c hay gy bnh
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ngi khi b o thi khi c th bng ng phn s lm nhim ngun nc b
nhim phn ny. Nc tr thnh mi trng phn tn, lan truyn mm bnh cho ngi
khi s dng m khng c tinh sch ng quy cch. Nc l mi trng sng, mi
trng nui trng ca cc loi thu sn nn khi nc b nhim th vi sinh vt gy
bnh c th nhim vo cc loi thu hi sn, lm nhim ngun nguyn liu ca cc
thc phm thu hi sn ch bin. l mt trong nhng con ng nhim vi sinh vt
gy bnh vo thc phm. Mt khc, trong qu trnh sn xut, ch bin thc phm, vi
sinh vt gy bnh cng c th nhim vo thc phm thng qua tip xc vi b mt
thit bi, cng nhn. Mc d c th gy ng c, s lng t bo vi sinh vt hin din
hoc c t ca chng tit ra trong thc phm khi c s dng phi ln. Tuy mt
vi sinh vt ban u trong nc, trong nguyn liu hoc trong thc phm c th rt
thp nhng nhng iu kin nht nh thch hp cho s tng trng v to c t ca
vi sinh vt trong qu trnh ch bin hoc bo qun thc phm, mt vi sinh vt c
nhn ln nhanh n mc gy bnh hay hay sn xut lng c t gy hi. Do
vy, mt cho php ca vi sinh vt gy bnh trong nc, thc phm l rt thp, trong
a s cc trng hp l bng khng.
1.2. TNH HNH CC V NG C THC PHM TRONG C NC V
TP. H CH MINH TRONG THI GIAN VA QUA
Theo thng k ca t chc y t th gii, mi nm Vit Nam c 8 triu ngi
(chim xp x 1/10 tng dn s) b ng c thc phm hoc ng c do lin quan
n thc phm.
Hng nm Vit Nam c khong hn 3 triu trng hp nhim c t thc phm, gy
thit hi hn 200 triu USD. y l con s c T chc Y t Th gii (WHO) a ra
trong mt hi tho v an ton thc phm - dantri.com.vn - 18:02 15-04-2011.
Theo s liu thng k ti Cc An ton V sinh thc phm, t 1997 ti 2004
c 2.237 v ng c thc phm vi 43.655 ngi mc v 429 ngi t vong. Nguyn
nhn gy ng c thc phm ch yu l do vi sinh vt (42,2%), sau l ho cht
(24,9%) v c t t nhin trong thc phm (25,2%), cn li khng xc nh c
nguyn nhn. 4 l do cha th xc nh c nguyn nhn cc v ng c l: Ly
mu chm, khng ly c mu, khng c cch no iu tra c hoc cn phi xt
nghim.

Thng tin t B Y t cho bit, trong nm 2010 ton quc xy ra 132 v ng
c thc phm vi 4.676 ngi mc, 3.281 ngi nhp vin v c 41 trng hp t
vong. Ring trong qu IV nm 2010, c nc xy ra 18 v ng c lm 4 ngi t
vong do c t c nc ti cc tnh Ph Yn, Bn Tre, Bnh Thun, trong c 3 v
ng c ln t 30 ngi tr ln. S ngi b ng c l 323 ngi vi 242 ngi
nhp vin. So vi cng k nm 2009, s ngi mc gim 189 ngi, s ngi i vin
gim 186 ngi. Tuy nhin s ngi t vong li tng ln 2 ngi. S v ng c thc
phm c quy m ln (trn 30 ngi) gim 3 v, s ngi mc gim 215 ngi, s
ngi i vin gim 174 ngi.
Theo TS. Lm Quc Hng - Trng phng Qun l ng c thc phm, Cc
ATVSTP, trong nm 2010, hu ht cc v ng c thc phm c chn on, xc
nh nhanh nguyn nhn v x l kp thi, thng tin ghi nhn nhanh chng, chnh xc
hn. Cng tc qun l ng c thc phm c nhiu chuyn bin tch cc. Mc d
vy, TS. Hng cho hay, ng c thc phm ti gia nh chim gn 60% tng s v
5
ng c thc phm trn c nc, c bit l ng c c nc, iu cho thy, cc v
ng c thc phm din ra ti khu vc h gia nh cha c xu hng gim. Do ,
ngi dn cn phi nng cao thc trong vic thc hin ATVSTP ngay chnh ti bp
n ca gia nh mnh.
Nu nh nm 2000, ng c ch yu do vi sinh vt (chim 70%) th ti nm 2010,
ng c vi sinh vt thp i (<50%), ng c ch yu do ha cht, chim ti trn 60%.
Ring trong qu IV nm 2010, c nc xy ra 18 v ng c lm 4 ngi t vong do
c t c nc ti cc tnh Ph Yn, Bn Tre, Bnh Thun, trong c 3 v ng c
ln t 30 ngi tr ln. Xu th ng c cho ha cht cng ngy cng tng ln, ng c
do vi sinh vt c xu hng kim sot tt hn nhng khng c ngha l nguy c gim
i.
So vi cng k nm 2009, s ngi mc gim 189 ngi, s ngi i vin gim 186
ngi. Tuy nhin s ngi t vong li tng ln 2 ngi. S v ng c thc phm c
quy m ln (trn 30 ngi) gim 3 v, s ngi mc gim 215 ngi, s ngi i vin
gim 174 ngi.

Nm 2010, s v ng c do ha cht l ch yu, chim trn 60%.

TS. Lm Quc Hng - Trng phng Qun l ng c thc phm, Cc ATVSTP -
nhn xt: Cc v ng c thc phm c chn on, xc nh nhanh nguyn nhn v
x l kp thi, thng tin ghi nhn nhanh chng, chnh xc hn. Cng tc qun l ng
c thc phm c nhiu chuyn bin tch cc. c bit, thi gian va qua, khng
c v ng c no xy ra trong sut thi gian din ra i l 1.000 nm Thng Long
H Ni.

Mc d vy, ng Hng cng khuyn co ngi dn trc tnh trng ng c thc
phm ti gia nh chim gn 60% tng s v ng c thc phm trn c nc, c bit
l ng c c nc. iu cho thy: Cc v ng c thc phm din ra ti khu vc h
gia nh c xu hng tng, thc ngi dn cha cao trong vic thc hin ATVSTP.
Trong khi , theo kt qu iu tra ca Cc ATVSTP, nhn thc ca ngi dn v
vn ATVSTP u c nng cao hn t 2005-2008. Theo , nhn thc ca ngi
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sn xut sn phm thc phm tng t 47,8% ln 55,7%; nhn thc ca ngi kinh
doanh thc phm tng t 38,6% ln 49,4%; nhn thc ca ngi s dng thc phm
tng t 38,3% ln 48,65%.
Nh , s v ng c thc phm trong c nc vn cao (8 triu ngi/nm) nhng
bt u c chiu hng gim. S ngi mc ng c thc phm nm 2005 gim
gn mt na (43,34%) so vi nm 1994, s ca t vong cng gim 28,17%.
Cc c s m bo ATVSTP tng mnh. Nm 2006, c nc ch c 1.106 c s t
yu cu. Nhng ch sau 2 nm, con s ny ln ti 17.592 c s.
Theo bo co ca B Trng B Y t Nguyn Quc Triu, trong nm 2010, t l
ngi kinh doanh thc phm c hiu bit ng n v VSATTP tng t 65%
(2008) ln 73% (2010); ngi tiu dng c hiu bit ng tng t 65,5% (2009) ln
84,5% (2010); ngi sn xut v ch bin thc phm c hiu bit ng tng t 44,4%
(2009) ln 79,4% (2010). Trng Ban ch o, Ph Th tng Nguyn Thin
Nhn nh gi s chuyn bin trong nhn thc ca ngi dn l mt trong nhng
im sng ca cng tc VSATTP.
Nm 2011 : trong qu I/2011, s v ng c thc phm gim 50% v s ca t
vong gim ti 75% so vi cng k nm ngoi (2010).
Trong 3 thng u nm 2011, ton quc xy ra 16 v ng c thc phm vi 442
ngi mc v 5 ngi t vong, trong c 1 v ng c tp th 30 ngi. So vi
cng k nm ngoi, s v ng c gim 10 v (gim 50%), s ngi mc gim 321
ngi, s t vong gim 11 trng hp (gim 75%). Theo thng k t Cc V sinh an
ton thc phm trong 6 thng u nm 2011 ton quc xy ra 53 v ng c vi
1.776 nn nhn, trong c 9 trng hp t vong. Nguyn nhn gy ng c do vi
sinh vt (17 v), do ha cht (10 v), do c t t nhin (17 v)...
1.3. I TNG VI SINH VTTC NHN CHNH GY NG C THC PHM
Cc qu trnh h hng ca thc phm thng do vi sinh vt gy nn. Trong
rt nhiu trng hp cc vi sinh vt ny cn to ra cc c t gy nh hng n sc
khe con ngi. Chnh v vy vic tm hiu v cc vi sinh vt gy bnh t tm ra
cc bin php khng ch chng l vic lm cn thit.
C 2 dng gy bnh c ngun gc t vi sinh vt:
- Vi sinh vt tit ra c t v s hin din ca cc c t ny trong thc phm l
nguyn nhn gy bnh. V d: Clostridium botulinum tit ra c t gy nn bnh
botulism (chng ng c tht); Staphylococcus aureus gy nn bnh t cu khun
- Vi sinh vt hin din trong thc phm, t xm nhp vo c th con ngi.
S hin din ca chng hoc ca cc sn phm hnh thnh t qu trnh phn gii cc
cht trong c th l nguyn nhn gy bnh. V d: Salmonella spp., Vibrio
parahaemolyticus, Escherichia coli,
Tuy nhin c nhiu vi sinh vt mang c tnh ca c 2 dng gy bnh ny. V d
Clostridium perfringens v Bacillus cereus.

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1.3.1.Tng vi khun hiu kh:
Vi sinh vt hiu kh l nhng vi sinh vt ch sinh trng trong iu kin c oxy.Tng
s vi sinh vt hiu kh c m bng cch a v trong iu kin hiu kh 30
o
C
trong khong 3 ngy.
Ch s ny c mt s tn gi khc nhau nh:
- S vi sinh vt hiu kh (Aerobic Plate Count _ APC)
- Tng s m trn a (Total Plate Count _ TPC)
- Tng s vi sinh vt sng (Total Viable Count _ TVC)
- S m a chun (Standard Plate Count _ SPC)

1.3.2.Tng nm men, nm mc:
Nm mc l vi nm c kh nng to si nm, sinh sn bng bo t hay khun ty
Nm men l nhng t bo n tnh sinh sn ch yu dng v tnh theo kiu ny
chi.
Aspergillus
Aspergillus flavus v A. parasiticus l hai loi nm mc quan trng trong cc
loi nm mc c kh nng to c t mycotoxin gy ng c thc phm. c t
mycotoxin c th c to ra bi nhiu chng ca hai loi ny c gi chung l
aflatoxin c kh nng gy ung th. Cc nm mc ny c th pht trin nhit t 7-
40
0
C v ti u 24-28
0
C, do vy c kh nng tng trng tt trn cc loi ng cc,
nng sn (go, la, u phng, bp). T cc nguyn liu ny, nm mc v c t c
th nhim vo thc phm gy ng c cho ngi.

Hnh 9. Asperillus flavus (Chp qua knh
hin vi in t)

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1.3.3. Salmonella
Nu phn loi theo mc gy bnh ca Salmonella ta c cc loi sau:
- Loi ch gy bnh cho ngi (vd: S. typhi gy st thng hn ti ngi)
- Loi ch gy bnh cho ng vt (vd: S. typhimurium gy st thng hn ti
chut)
- Loi gy bnh cho c ngi v ng vt (chim a s)
Salmonella l trc khun gram (-), khng to bo t, c tin mao (tr S.
gallinarum), c kch thc t bo vo khong 0,5 3 m. Ging nh nhiu loi vi
khun khc Salmonella c kh nng pht trin trong khong nhit tng i rng t
6 n 45.6
0
C, tuy nhin nhit pht trin ti u l 37
0
C. Salmonella pht trin trong
khong pH t 4.1 9.0. Trong cc mn salad c pH khong 5.5 5.7 khng nhn
dng c s thay i mi v khi Salmonella pht trin. m ti u cho s pht trin
ca Salmonella l 0.93 0.95.

Hnh 1. Salmonella typhy
(Chup qua knh hien vi ien
t)

Salmonella xm nhp vo c th bng hai con ng: t phn ngi hoc ng
vt; t ngi bnh. Trong phi k n tc ng ca ng vt lng v, trng ca
chng v nht l phn ca chng lm cho vic lan truyn Salmonella d dng hn.
Ngoi ra chut, mo, rui cng l nhng tc nhn gin tip dn n vic Salmonella
lan rng hn khi chng truyn phn vo cc thc phm khng c bo qun k.
Trong qu trnh git m cng cn phi phng s nhim Salmonella nu khng thc
hin ng quy trnh an ton thc phm.
Cc triu chng do Salmonella gy ra thng l tiu chy, i ma, bun nn.
Thi gian bnh k t khi tiu th thc phm b nhim cho n khi cc triu chng
c biu hin l 12-36 gi. Triu chng ng c thng ko di t 2-7 ngy.
1.3.4. Coliforms
y l nhng vi sinh vt hnh que, G
-
, khng to bo t, c kh nng ln men
latose v sinh kh trong qu trnh ln men ny. Pht trin trong khong nhit (t 2
n 50
oC
), pH t 4,0 n 8.5, c th sng hiu kh hoc k kh ty . Sau 12 16 gi
trn mi trng thch to ra nhng khun lc c th nhn thy c. Coliform i vo
thc phm t nc hoc nguyn liu thc phm c nhim phn. Cc loi tiu biu cho
Coliform: Escherichia coli v Enterobacter aerogenes. Tuy nhin, ngoi ra cn c
khong 20 loi mang nhng tnh cht c trng cho Coliform, trong bao gm c cc
Enterobacteriaceae khc v cc loi thuc Aeromonas.
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Nhm Coliform phn l nhng Coliform c kh nng pht trin trong mi trng
c nhit t 44.5 45
0
C (cc Coliform khc c t
0
opt
=37
0
C). y cng l c im
nhn bit v phn bit Colifom phn. Tuy nhin vic xc nh ny ch l gi tr dng
tham kho cho vic xc nh iu kin sng ca Coliform.
Di y l mt vi tnh cht c trng cho vic lm h hng thc phm ca
Coliform:
- Chng c kh nng phn gii nhiu loi c cht khc nhau: carbohydrate, cc
cht hu c sinh nng lng, cc hp cht cha nit n gin,..
- C kh nng tng hp hu ht cc vitamin cn thit.
- C kh nng pht trin tt trong mi trng c nhit t 10 46
0
C.
- C kh nng to ra acid v sinh gas t ng
- Sinh ra mi v h hng, kh chu
Escherichia coli:
c pht hin l vi sinh vt gy bnh t nm 1700. E. coli thuc h
Enterobacteriaceae. Cc loi E. coli hin din din rng ri trong mi trng b
nhim phn hay cht thi hu c, pht trin v tn ti rt lu trong mi trng. Cc.
Thng sng trong rut gi ca ngi v ng vt, theo phn i ra ngoi, nhim vo
thc phm t nguyn liu hay thng qua ngun nc trong qu trnh sn xut ch bin.
C kh nng ln men nhiu loi ng, sinh hi, kh nitrat thnh nitrit. Gy nhy
nht, h hng thc phm.
Kh nng gy bnh ca E. coli rt a dng: gy nhim khun ng tiu; vi c
th yu gy nhim khun mu; gy vim mng no tr s sinh; gy tiu chy.

Hnh 2. Escherichia coli (Chp
qua knh hin vi in t)

1.3.5. Staphylococcus
Hin nay tm thy 31 loi. Staphylococcus l cu khun, G
+
, cc t bo thng
lin kt thnh hnh chm nho. Khng di ng, khng to bo t. Nhit ti u l
37
oC
. Chu c kh hn, hi nng ( 50
oC
vn sng c trong 30 pht). C kh nng
sng c trong nng NaCl l 9-10%. pHopt= 6-7 ; aw
opt
= 0,83- 0,90.
Staphylococus c kh nng pht trin trn nhiu loi mi trng khc nhau nht l trn
cc mi trng cha cht hu c. Tuy nhin phi c nit trong mi trng. Ngun
10
nit s dng l acid amin. Trong t nhin Staphylococcus c tm thy trn da, mi,
tc hay lng ca cc ng vt mu nng. Staphylococcus sinh mt s c t ng
rut enterotoxin bn nhit v khng b phn hu 100
0
C trong 30 pht. Khi n phi
thc phm c cha c t ny, sau 4-6 gi ngi ng c c cc triu chng tiu chy,
nn ma ko di 6-8 gi. Ngoi ra Staphylococcus cn to mn nht, lm ng huyt
tng, t gy nn cc bnh nh vim phi, vim mng no, vim c tim, vim thn,
vim ty xng ngi. Cc loi thc phm c cha nhiu mui nh jambon, kem
tng hp, nc sp v cc loi thu sn, thc phm ng hp thng hay nhim vi
sinh vt ny. Con ng ly nhim ch yu thng qua tip xc t nh bp, qu trnh
ch bin

Hnh 3. Staphylococus aureus (Chp
qua knh hin vi in t)

Staphylococcus aureus
c trng nht ca loi nay l gy vim rut dn n bnh chy mu rut.
Mang tnh cht c trng ca loi Staphylococcus, kt li vi nhau thnh dng
chm nho hoc thnh tng chui ngn. C mt vi loi Staphylococcus aureus c kh
nng chu mc rt tt (10 20% NaCl) v cng chu c tc ng ca nitrit. Chnh v
vy phi rt kim tra s hin din ca Staphylococcus aureus i vi cc sn phm tht
nh: xc xch, dm bng, lp xng. Staphylococcus aureus cng pht trin tt trong
mi trng c nng ng cao (50 60% ng). Chng ln men v phn gii
ng nhng li khng to mi v kh chu cho sn phm.
Ngun gy nhim Staphylococci vo c th ngi thng t nhng ngi b
vim mi gy nn vim xoang, t cc ung nht, hoc cc vt thng b nhim trng, t
da ngi tip xc vi ngi bnh. Staphylococci cn gy nn chng vim v b dn
n lm nhim sa v cc sn phm t sa. Ring khng kh khng phi l ngun gy
nhim c trng tr phi trong mi trng c qu nhiu Staphylococci.
Cc sn phm thc phm thng c Staphylococci : tht v cc sn phm t tht,
c v cc sn phm t c, sa v cc sn phm t sa, salad, pudding, cream. Nu
khng c tr lnh ng mc th Staphylococci s pht trin mnh lit v gy bnh.
1.3.6. Vibrio
C khong 28 loi. Trong c 4 loi thng gp nhiu trong hi sn:
- Vibrio vulnificus
- V. cholerae
- V. parahaemolyticus
- V. alginolyticus
11
Vibrio l phy khun. Phn ln thuc gram (-). Di ng nhanh nh n mao
u. Khng sinh nha bo, phn ng oxydase dng tnh. Thuc loi hiu kh ty tin.
Vibrio l loi vi khun a mn, trong mi trng sng yu cu c khong 1 3%
NaCl. C th pht trin trong mi trng c nng mui n 7.0%. Nhit pht
trin ti u l 35 37
0
C, tuy nhin vn pht trin c khong nhit t 10
44
0
C. Khong pH m Vibrio c th pht trin t 5.0 11.0. Vibrio cholerae to ra c
t t cholerae-toxin c c tnh mnh, ch cn 5g gy nhim qua ng ming c th
gy tiu chy cho ngi trng thnh. Ngoi c t ny, loi ny cn tit c t
hemolysin c c tnh tng t tetrodotoxin ca cc nc.
Thng c mt hi sn, cc sn phm hi sn, nc bin. Ngun thc phm c
nguy c nhim v lan truyn dch t l nc ung, nc tri cy v cc sn phm sa,
cc loi thu hi sn ti sng. Tuy nhin b tiu dit di tc ng ca nhit . Ni
mt cch khc, khi nu chn thc phm th khng phi lo s s tc ng ca Vibrio.

Hnh 4. Vibrio cholerae (Chp qua
knh hin vi in t)

1.3.7. Shigella
Shigella thuc h Enterobacteriaceae, l vi khun gy bnh l trc trng. Ch
gy bnh cho ngi v linh trng. Sinh c t enterotoxin. L trc khun gram(-),
khng di ng, khng sinh bo t, k kh ty tin. Pht trin trong khong nhit t
10 40
oC
, t
0
opt
= 37
0
C, pH = 6 8, nng mui 5 6%. Nhy cm vi nhit.

Hnh 5. Shigella sonnei (Chp qua
knh hin vi in t)

12

Bao gm 4 loi
S. dysenteriae
S. boydii
S. plexnery
S. sonnei

Shigella to c t gy nn cc triu chng l (bnh l trc trng), kch thch
ln thnh rut, ln h thn kinh trung ng, gy tiu chy, c ch hp thu
ng v acid amin rut non. Nu tc ng ln h thn kinh th c th gy t
vong. Khi b nhim Shigella, chng s tn cng lp biu m nim mc rut
gi, gy hoi t, lm lot, xut huyt. Khi rut gi b tn thng gy au bng
d di, tiu chy nhiu ln, phn nhy nht, c mu. Liu lng gy ng c
thc phm do Shigella rt thp, c th mc 10 t bo/g sn phm. Khng
c php hin din trong 25g thc phm

Thng nhim vo c, qu, rau, tht, cc loi salad t nc hoc phn ngi.
Thc phm b nhim Shigella t nguyn liu hay thng qua tip xc b mt trong qu
trnh sn xut, ch bin thc phm.
1.3.8. Yersinia
Hin nay tm thy 11 loi. L trc khun gram(-). Trong mt s trng hp c
kh nng chuyn ng. Thuc loi k kh ty tin, khng to bo t, khng sinh nha
bo. Khong nhit pht trin ti u t 25 32
oC
. B tiu dit 60
oC
trong vng 1
3 pht. i din tiu biu l Yersinia enterocolitica. Yersinia enterocolitica pht trin
trn nhiu loi thc phm khc nhau: b, heo, dch trng, pho mt mm, sa, c, tm,
cua, g, u ph, Y. enterocolitica thng gy bnh ng rut, gy vim d dy ti
ngi. Sau 24 36 gi sau khi s dng thc phm c mm bnh th triu chng ca
bnh bt u xut hin nh au bng, st, tiu chy, cc vt lot d dy hnh thnh.
Nu lu s dn n chy mu d dy.

Hnh 6. Yersinia pestis (Chp qua
knh hin vi in t)

13
1.3.9. Clostridium
Hin nay pht hin ra hai chng gy ng c thc phm:
- Clostridium botulinum: to c t gy hi n h thn kinh
- Clostridium perfringens: gy au bng, tiu chy v gii phng nhiu kh. Khi
vi khun hnh thnh bo t to ra c t rut, gy ng c cho ngi.
Clostridium botulinum l trc khun gram (+), khng di ng, ym kh, to bo
t. Bo t rt chu nhit. Nhit pht trin ti u l : 43 47
oC
, pH t 5 9, b c ch
bi NaCl 5%, hoc NaNO
3
2,5%
C.botulinum l vi khun sinh c t. T bo C.botulinum b tiu nhit 80
0
C
n 90
0
C. Trong a s trng hp C.botulinum sinh gas v to mi kh chu. T c
im ny ta c th loi b thc phm nhim C.botulinum mt cch d dng. Tuy nhin
cn phng mt s trng hp ngoi l, lc ny ta khng nhn bit c bng cm
quan v s nhim vi khun C.botulinum ca thc phm, do ngi s dng s b
nhim c t gy hi chng botulism (ng c tht).
S ng c tht xy ra khng thng xuyn, nhng n thng gy t vong. Theo
s pht trin ca khoa hc, t l t vong do botulism ngy cng gim. V d vo
nhng nm 1970 1973 chim khong 23%, vo nm 1899 1994 t l ny chim
trn 60% ti M. Theo nh thng k ca Trung tm kim sot dch bnh ti M, th
cc sn phm c lm ti nh d b nhim C.botulinum hn l cc sn phm sn xut
trong cng nghip.

Hnh 7. Clostridium botulinum v bo t hnh vin n (Chp qua KHV in t)
Ty theo tng loi C. botulinum m triu chng bnh xut hin sau khi s dng
thc phm cha c t xut hin sau cc khong thi gian khc nhau (t 12 36 gi).
Khi b ng c cc triu chng u tin xut hin l: nn ma, tiu chy, mt mi,
chng mt v au u. Sau l cc triu chng to bn, nhn mt thnh hai. Ri cc
hin tng kh nut, kh pht m xut hin. Ngi bnh cm nhn c s kh
ming, kh th, li b sng v xut hin mng. C th ln cn st hoc khng. Tip
theo l c b t cng, ri n h thng h hp b t cng v cui cng l tim ngng
p. Kt qu l ngi b b t vong do t lit h thng h hp. S t vong ny thng
din ra sau 3 6 ngy s dng thc phm nhim c t.
Phng thc duy nht cha tr l s dng cc cht khng c t. Tuy nhin n
ch hiu nghim nu s dng khi pht hin ra cc triu chng u tin ca bnh. Cn
14
khi cc triu chng c trng xut hin th mun ri. Chnh v vy vic xc nh
bnh l rt kh khn. Km theo vic s dng cht khng c t l phi gi cho ngi
bnh trng thi yn tnh, s dng phng php th nhn to, to s cn bng cc
cht lng trong c th,..
ngn chn s nhim C.botulinum vo c th cn ch thc thi cc vic sau:
- Nn x l nhit cc hp trc khi s dng.
- Loi b cc hp c triu chng phng, mo m.
- Khng nm cc thc phm c mi kh chu.
- Khng s dng cc thc phm c nu chn nhng khng c bo qun
tt v khng c hm nng.
- Cn un nng li thc phm nghi ng trong khong 15 pht.
- i vi cc sn phm xng khi th cn ch mt s im sau:
- Sn phm c gi sch, tay trc khi cm sn phm c ra sch.
- Trrong qu trnh xng khi cn gi t nht 82
0
C trong 30 pht.
- Lm lnh nhanh sau khi ng gi v bo qun lnh.
- Tt c cc bao b s dng phi l loi c b Y t cho php v chu lnh tt.

1.3.10. Bacillus cereus
B. cereus l trc khun G(+), sinh bo t, k kh ty , tng trng c trong
khong nhit t 5-50
0
C, ti u 35-40
0
C. Vi khun ny hin din trong t, bi,
cc loi thc phm (sa, tht, rau qu, hn hp gia v, sn phm kh).
Vi khun c th tit ra hai loi c t chnh l diarrhoeal toxin gy tiu chy v
emetic toxin gy nn ma. Ng c thc phm gy ra bi B. cereus khi thc phm
c chun b m khng c gi lnh vi gi trc khi s dng. Triu chng ng
c ph bin l au bng, tiu chy, khng st, bt u 14-16 gi sau khi n thc
phm b nhim v ko di trong 12-24 gi.

Hnh 8. Bacillus cereus (Chp qua
knh hin vi in t)

1.3.11. Feacal streptococcus (Streptococcus phn)
Streptococcus phn c s dng nh l ch th cht lng v sinh ca thc
phm. Streptococcus phn l cc lin cu khun c ngun gc t phn, hnh cu hay
hnh oval ko di, Gram dng, thng t tp thnh hnh i hay hnh chui, khng di
15
ng, khng sinh bo t, mt s dng c to v nhy. Hu ht cc loi ny sng hiu
kh ty nhng pht trin tt trong iu kin k kh, tit bacteriocin trong qu trnh
tng trng.
Cc loi ny c th pht trin c trong mi trng cha 6,4% mui NaCl, pH
9,6 45
0
C.

1.4. CH TIU VI SINH VT TRONG NC V TRONG THC PHM
1.4.1. Ch tiu vi sinh vt trong nc
* Nc dng cho mc ch sinh hat v sn xut
Cc loi nc dng cho mc ch ny c gi chung l nc mt, tiu chun
quc gia Vit Nam (TCVN 5942 1995) quy nh hai mc nh sau:
- Loi A dng l m ngun cp nc sinh hat nhng phi qua qu trnh x l,
gii hn ti a s coliform cho php l 5.000 MPN/100ml
- Loi B dng cho cc mc ch khc, gii hn ti a s coliform cho php l
10.000 MPN/100ml
* Nc ung
Nc ung bao gm nc ng chai, nc gii kht (khng cn, c cn). Tiu
chun (TCVN 6096 1995, TCVN 5042 1994) quy nh v vi sinh vt ca cc dng
nc ny nh sau:
- Nc ung ng chai
Ch tiu Mc ti a cho php
1. Coliform (MPN/100ml)
2. Coliform phn (MPN/100ml)
3. E. coli (CFU/100ml)
4. Clostridium kh sulphat (CFU/100ml)
5. Streptococci phn (CFU/100ml)
0
0
0
0
0
- Nc gii kht khng cn
Ch tiu
Mc ti a cho php
Khng ng chai ng chai
1. Tng vi khun hiu kh (CFU/100ml)
3. Clostridium perfringens
4. Leuconostoc (CFU/100ml)
5. Nm men nm mc (CFU/100ml)
5x10
4

0
0
0
10
3

10
2

0
0
0
0
16
Ch tiu
Mc ti a cho php
6. Staphylococus aureus 0 0
- Nc gii kht c cn
Ch tiu
Mc ti a cho php
1. Tng vi khun hiu kh (CFU/100ml)
3. Clostridium perfringens
4. Vi khun gy c (quan st bgng mt)
5. Nm men nm mc (CFU/100ml)
6. Staphylococus aureus/vi khun gy bnh
ng rut
10
3
0
0
0
10
2

0
10
2

0
0
0
0
0

1.4.2. Ch tiu vi sinh vt trong thc phm
Cc ch tiu vi sinh vt trong thc phm c quy nh bi tiu chun ca B Y
t thng 4/1998 bao gm cc ch tiu:
- Tng vi khun hiu kh
- Coliform
- E. coli
- Staphyloccocus aureus
- Salmonella
- Vibrio parahaemolyticus
- Bacillus cereus
- Streptococcus faecalis
- Pseudomonas aeruginosa
- Clostridium botulinum
- Clostridium perfringens
- Tng s nm men, nm mc

17
Chng II
K THUT C BN TRONG PHN TCH VI SINH VT

2.1.LY MU
2.1.1.C IM CA MU THC PHM
Trong mu thc phm vi sinh vt hin din c mt thp v khng c s phn b
ng u trong mu
Trong qu trnh ch bin thc phm vi sinh vt trong nguyn liu v bn thnh phm
thng b tn thng t nhiu v gim sc sng do cc bin php vt l (nhit , nh
sng), ha hc (cht c ch, cht dit khun, nng cao ca mui, dung mi, )
c s dng nhm hn ch thp nht s hin din v tng trng ca vi sinh vt trong
qu trnh ch bin. S tn thng v sc sng yu ca cc vi sinh vt cn pht hin c
th lm sai lch kt qu cc phn ng sinh ha dng nh danh vi sinh vt.
Do vy, hu ht cc quy trnh kim nghim vi sinh trong nc v trong thc phm u
c thm cc bc nui tng sinh phc hi sc sng ca cc vi sinh vt b tn
thng v bc nui tng sinh chn lc nhm lm gia tng mt tng i ca vi
sinh vt cn pht hin, c ch s tng trng ca cc nhm vi sinh vt khng mong
mun khc.
2.1.2. PHNG PHP LY V BO QUN MU
Ly mu nguyn liu hoc sn phm thc phm xc nh cht lng bng cm quan
v phn tch trong phng th nghim (PTN) l khu u tin rt quan trng trong cng
tc phn tch, gp phn vo tnh chnh xc ca kt qu kim nghim v x l thc
phm sau ny. Trong kim nghim vi sinh, cng on ly, vn chuyn v bo qun
mu mc d c thc hin bn ngoi phng th nghim nhng li l cng on rt
quan trng trong qu trnh th nghim. Mi sai st trong cng on ny u c th dn
ti nhng sai lch nghim trng kt qu th nghim.

2.1.2.1. Nguyn tc ly mu
Vic ly mu phi m bo 02 iu kin sau:
- Mu ly phi i din cho l hng hay ni ly mu v c nhn dng r rng.
- Hm lng cc cht hay cc vi sinh vt cn xc nh khng c bin i k t
khi ly mu n khi phn tch.
Khi ly mu cn lu cc nguyn tc sau:
- Ngi ly mu c k ti liu lm cn c ly mu tng ng (ti liu lun c
th sn dng).
L hng ng nht: l l hng bao gm nhng sn phm c cng mt tn gi, cng
mt loi phm cht v cng mt khi lng, ng trong bao b cng 1 kiu, cng mt
kch thc, sn xut trong cng mt thi im nht nh theo cng mt quy trnh cng
ngh.
- Trc khi ly mu trung bnh, phi xem xt l hng c ng nht khng v
kim tra tnh trng bao b ca l hng .
- Mu thc phm phi c tnh cht i din cho c mt l hng thc phm ng
18
nht.
- T l ly mu t 0,5 1,0% ty theo s lng, nhng mi ln khng t hn
lng cn thit phn tch.
- Mu hng ly a i kim phi l mu trung bnh. Ngha l sau khi chia
thnh l hng ng nht, mu s ly u cc gc, cc pha trn di, gia l
hng v trn u..
- Mu kim phi mang tnh i din cho l thc phm. Mu kim ca mt l
hng ln hn hoc mu thc phm cha bao gi ti thiu phi t 200g, mu
bao gi ri ti thiu cn 100 g. Vi sn phm bao gi, ch nhng sn phm cn
nguyn mi c dng phn tch. i vi dng lng (nc dng trong ch
bin, nc mm..): 500ml
- Chun b cc trang b, dng c ly mu v bo qun mu ph hp.
Dng c ly mu, bo qun mu phi c tit trng
- Chun b bin bn ly mu, nhn mu.
- Trn mu cn ghi:
+ Tn mu
+ Ngi ly mu
+ Tn v a ch nh sn xut
+ S lng mu ly
+ a im, thi gian ly mu.
- Nhng mu cha s dng ngay phi c bo qun nhit -20
oC
cho n
khi phn tch. Trng hp mu khng cn bo qun ng th c th bo qun
nhit 0-4
oC
trnh s bin ng v thnh phn c tnh ca mu cng nh
vi sinh vt. Nhn chung, mu phi c xt nghim trong vng 24h. lu hn
c th b tng hoc gim vi sinh vt.
Bng 2.1. Lng mu ti thiu cn thit phn tch
STT Tn thc phm Lng mu n v
1 Tht v cc sn phm ca tht 200 500 gram
2 C, tm, cua (nguyn con) 200 500 gram
3 Trng 5 10 Qu
4 Nc mm, nc chm 500 750 ml
5 Dm 500 750 ml
6 Sa ti 500 750 ml
7 Du, m 500 750 ml
8 Ru cc loi 750 1000 ml
9 Bt ng cc, sn phm ca bt 250 500 gram
10 Bnh, mt, ko 250 500 gram
11 Gia v, mui 50 100 gram
12 Phm mu 20 50 gram
13 hp, nc gii kht 5 10 hp, chai
Ty theo l hng v yu cu kim nghim, thc phm c th ly nhiu hn mc trn.

19
2.1.2.2. K hoch ly mu
* Mt k hoch ly mu chi tit cn c cc yu t sau: n, c, m, M
- S mu c php nm trong khong gia m v M (c);
- S lng mu cn th (n);
- Gii hn vsv, m & M
+ Chp nhn (acceptable) (< m)
+ Ngng ln cn gii hn (Marginally acceptable) (> m and < M)
+ Khng chp nhn (Unacceptable) (> M);
Cc loi k hoch ly mu: K hoch hai thuc tnh v k hoch ba thuc tnh.
* K hoch hai thuc tnh
- Ch nu ra m
- Nu tt c cc mu sm hoc sc mu trong s n mu th >m th chp nhn l
hng, nu >c mu trong s n mu th >m th t chi l hng
* K hoch ba thuc tnh
- t ra c m v M
- Ch tiu m thng phn nh ngng trn ca GMP
- Ch tiu M nh du gii hn trn s nhim l nguy him v khng
chp nhn c
- Nu tt c cc mu sm hoc sc mu trong s n mu th nm trong khong >m
v sM th chp nhn l sn phm. Nu >c mu trong s n mu th nm trong khong >
m v sM hoc c mt mu >M th t chi l sn phm
* La chn k hoch ly mu
K hoch hai thuc tnh: i tng vsv quan tm khng c php c trong
thc phm. Nu cho php mt s lng nht nh vsv trong mt n v th tch th
thng s dng k hoch ba thuc tnh.
VD: S mu phi kim tra v gii hn vi khun cho tht ti

Mu Test N C m M
Tht ti
gia sc

Tht gia
cm
- Vi khun hiu kh a nhit trung bnh
- Samonella

- Vi khun hiu kh a nhit trung bnh
- Salmonella
5
5

5
5
3
0

3
0
10
6

0

10
6

0
10
7

10
7

2.1.2.3. Thu v vn chuyn mu
20
Thu ti nhiu thi im v v tr. i vi loi thc phm bit ch nhim b
mt th cn dng que bng v trng qut mt din tch b mt nht nh hoc ct lt
vi b dy 2-3 mm thu mu
Dng c cha bng bnh nha c np (trnh s dng dng c thy tinh d v)
Ch nhit v thi gian vn chuyn, bo qun mu (cn tu c im ca
thc phm)
i vi mu hp: Phi kim tra dng bn ngoi xem hp c b phng, bin
dng, b h cc mi ghp hay khng? Ghi li cc nhn xt trn. Lau chi bng cn
pha ngoi, a vo phng v trng kim. Ch ch kim cc hp phng khi c yu
cu.
i vi cc sn phm bao b bng thy tinh, bao nylon phi kim tra kn
ca bao b, chi cn pha ngoi, a vo phng v trng kim
2.1.2.4. Chun b mu phn tch
- i vi cc sn phm c hon ton: bnh, ko, mt Cn 10 g (hoc 25 g)
mu cho vo ci s nghin nt, sau b vo erlen 150 ml hoc 200 ml c
sn bi thy tinh v khun hay nc ct tit trng. Lc u, lng cn. Ht
phn dch cy mu, ta c pha long 10
-1
.
- i vi cc sn phm va c, va lng: hp tht cn 10-100 g mu (c
ci ln nc) cho vo ci nh v trng nghin. Cn 10 g nghin nt cho vo
erlen 250 ml c 90 ml dung dch nc mui sinh l c bi thy tinh, lc u,
lng cn. Ht phn dch cy mu, ta c pha long 10
-1
.
- i vi cc sn phm lng hon ton: nc chm, nc gii kht C th ht
trc tip mu hoc pha long tu theo mc nhim bn. Lc k. Ht 10 ml
mu cho vo erlen 250 ml c sn 90 ml nc ct hay nc mui sinh l tit
khun. Ta c pha long 10
-1
.
- i vi cc sn phm lnh ng: Mu c lau cn pha ngoi bao PE
(Polyetylen), t vo khay trng men v trng, a vo bung v trng r
ng t nhin nhit phng, hoc gii ng 2-5
oC
trong 18 h; hoc 45
oC

trong 15 pht nu cn (lin tc lc bnh cha mu nu c th tng tc gii
ng)
- C th dng Stomacher x l mu rn
- Dung dch pha long: tt hn nn dng nc peptone 0.1%, nc m phot
phat, nc mui sinh l (0.85%), trong buffer nu cn.
2.2. K THUT PHA LONG
Mu thc phm dng rn c ng nht hoc dng lng s c pha long theo
bc 10 trong dung dch pha long SPW hoc BPW. Sau khi pha long dch pha long
s c mt vi sinh vt ph hp cho k thut pht hin, nh lng hoc phn lp vi
sinh vt thnh nhng khun lc ring bit. T chng ta c th gin tip xc nh
c mt t bo hay bo t gi chung l CFU (Coloning Form Unit) c trong thc
phm cng nh phn lp to dng thun vi sinh vt.

2.3. MI TRNG NUI CY VI SINH VT
21
- Mi trng dinh dng ca vi sinh vt bao gm nhiu thnh phn dinh dng
cn thit khc nhau. Thnh phn dinh dng ny tu thuc vo nhu cu ca
tng loi vi sinh vt.
- Trong phn tch, kim nghim vi sinh vt, mi trng cn cho vic tng sinh,
phn lp, phn bit, cy chuyn, bo qun, nh danh vi sinh vt Mi trng
cn cha y cc thnh phn v ngun carbon, m, khong a lng v vi
lng cn cho s bin dng v vt cht, nng lng ca i tng vi sinh
vt quan tm. Ngoi ra, mi trng cn c mt hm lng nc thch hp, c
pH xc nh v kt cu thch hp cho s tng trng ca vi sinh vt mc
tiu.
- Mi c th sng u c cu to t nhng hp cht hu c. Cc hp cht hu
c c cu to t cc nguyn t C, H, O v N gi l cc nguyn t thit yu ca
s sng. Nhn chung cc nguyn t thit yu khng th thiu trong qu trnh
sinh trng v pht trin ca vi sinh vt. Ngoi cc nguyn t thit yu, trong
cc c th vi sinh vt cn c cc nguyn t a lng nh: S v P, cc nguyn t
vi lng nh: Fe, Cu, Mg, Mn, Zn, K, Ca, Cl, Bo, IV th trong thnh phn
dinh dng khng th cung cp n thun mt s cht no hoc mt nhm
nguyn t no m trong t nhin, VSV s dng cc ngun dng cht t c
th thc vt, ng vt hoc VSV.
- Nng ng ph hp nui cy vi khun v x khun khong 0,05
0,20%, nm mc v nm men khong 3 15%.
- Cc cht tng trng ch yu l cc vitamin v cc gc kim purin, pirimidin,
cc acid bo, cc thnh phn ca mng t bo... Nng thch hp ca cc gc
kim purin, pirimidin cho vi sinh vt vo khong 5 20 g /ml. Nng thch
hp ca cc vitamin vo khong 0,1 0,5 g/ml (1 g = 10
-6
g )

Thnh phn c bn chnh mng xelluloza ca t bo vi khun
Nguyn t
Dng tn ti cho vi sinh vt s
dng
Thnh phn trung bnh
(%)
C
Cht hu c
CO
2

50
O Cht hu c 20
N NH
4
+
,NO
3
-
,NO
2
-
14
H Cht hu c 8
22

2.3.1. Phn loi mi trng
Mi trng c th c phn thnh cc loi khc nhau theo bn cht ca thnh
phn ca mi trng, theo tnh cht vt l v theo cng dng.
- Theo bn cht
- - Mi trng t nhin: c thnh phn c sn xut t cc sn phm c ngun
gc ng thc vt do vy thnh phn chnh xc ca mi trng l khng xc
nh.
- - Mi trng tng hp: c iu ch t cc ho cht tng hp tinh khit vi
hm lng xc nh
- - Mi trng bn tng hp: l mi trng tng hp m trong thnh phn ca
mi trng c b sung mt s thnh phn c ngun gc t nhin.
- Theo trng thi vt l
- - Lng (Broth): khng cha agar trong thnh phn mi trng. Mi trng lng
dng nui cy tng sinh, th nghim c tnh sinh l, sinh ho.
- - Rn: trong thnh phn mi trng c t 20-25% agar. Mi trng rn dng
phn lp khun lc n, nghin cu c im hnh thi khun lc, nh lng
mt vi sinh vt.
- - Bn lng (bn rn): c dng ln men vi sinh vt trong cng nghip
- Theo mc ch
- - Mi trng tin tng sinh: l mi trng lng dng tng sinh khng chn
lc vi sinh vt. V d: nc pepton.
- - Mi trng tng sinh: l mi trng lng dng tng sinh chn lc i
tng vi sinh vt cn kim nghim. V d: mi trng tetrathionate dng tng
sinh chn lc Salmonella
- - Mi trng chn lc: l mi trng rn dng phn lp chn lc cc khun
lc ca vi sinh vt mc tiu. Tc nhn chn lc trong mi trng chn lc:
- + Cht khng sinh (polymixin B, ampicillin, moxalactam, novobioxin,
oxytetracycline, D-cycloserine, vancomycin, trimethiprim, cycloheximide);
- + Cht ch th mu nh brilliant green, sodium selenite, bile salts, potassium
tellurite, sodium lauryl sulfate;
- + anaerobiosis bng cch to iu kin k kh hoc b sung nhng ha cht c
ch qu trnh h hp nh sodium azide, potassium cyanide (c ch vsv sinh
catalase); ph mt lp thch ln trn mt thch (vd trong phn tch VK lactic);
P H
2
PO
4
2-
, HPO
4
2-
, PO
4
3-
3
S SO
4
2-
1
23
- + pH, hot ca nc, nhit .
- - Mi trng phn bit :l mi trng rn chn lc cha cc thnh phn ch th
gip cho s pht hin d dng i lng vi sinh vt mc tiu. Tc nhn phn
bit trong mi trng phn bit:
- + Ch th pH: Ln men ng to acid v lm gim pH, decarboxyl amino acids
v thy phn urea to ra ammonia amine khin tng pH. Ch th pH thng
dng l phenol red, methyl red v brom cresol purple.
- + Ch th H
2
S: Mt s vsv phn gii amino acid to H
2
S. H
2
S c pht hin
bng cch s dng mui st nh st citrate, ferric ammonium citrate to ra
FeS c mu en.
- + Phn ng lng trng trng: Mt s vsv to lypolytic enzymes khin mi
trng c cha lng trng trng xung quanh khun lc tr nn trong hn.
- + Phn ng phn hu hng cu mu: phn bit mt s VK c hi nh
Staphylococcus aureus, Streptococcus pyogenes, Listeria monocytogenes.
- - Mi trng th nghim sinh ha: l mi trng dng xc nh mt hoc vi
c im sinh ho ca chng vi sinh vt phn lp v lm thun, to c s
nh danh chng.
- Ch th mu mi trng
- Trong rt nhiu cc phn ng sinh ho dng trong qu trnh phn tch vi
sinh vt, thnh phn mi trng ca cc th nghim ny thng c cha nhng
cht ch th mu. Cc cht ch th mu hot ng theo nhiu nguyn tc khc
nhau nh lm cho t bo vi sinh vt, khun lc ca chng c mu, lm cho mi
trng c mu hoc thay i mu.... Trong cc nguyn tc , th s thay i
mu theo s thay i ca pH mi trng l ph bin nht cc phn ng sinh
ho. Mi mt cht ch th mu thay i mu theo nhng gi tr pH khc nhau.
Do , mi phn ng, cn cn c vo c tnh ca vi sinh vt khi phn tch,
mi trng nui cy, c im cn nhn din m b sung nhng cht ch th
mu cho ph hp.

Bng. Mt s cht ch th mu v ngng pH thay i mu ca chng

-
24
-
-
-
-
-
-
-
-
-
-
-

2.3.2. Pha ch, chun b mi trng
Hin hay hu ht cc phng kim nghim vi sinh vt u s dng mi trng
ng kh thng phm ca cc hng chuyn nghip MERCK, OXOID, HIGH-
MEDIA pha ch mi trng nhm hn ch bin ng thnh phn mi
trng cc ln kim nghim khc nhau. Vic pha ch mi trng nui cy t
cc mi trng ng kh ny tng i n gin vi cc bc cn, ho tan,
chnh pH v hp kh trng. Phng php pha ch mi trng ny c thc
hin nh sau:
- Cn, ong ng lng mi trng ng kh theo ch dn
- B sung mt th tch nc ct hoc nc khong bng na dung tch cn thit.
Lc k, b sung phn nc cn li. Nu mi trng c agar hoc gelatin, cn
phi gia nhit lm tan mi trng.
- iu chnh pH mi trng. iu chnh pH dng NaOH 1N hoc HCl 1 N
- Phn phi mi trng vo cc dng c cha thch hp, lm nt y cho cc
dng c cha.
- Tit trng mi trng bng ni hp p lc
- Kim tra v trng ca mi trng nui cy
Mt s im cn lu khi pha ch mi trng:
- Bo qun sai quy nh (nhit , m, oxy ha) dn n mi trng b
hng
- S dng dng c cha (thy tinh) b nhim cht ty ra hoc ha cht khc
- Trn khng u, ha tan cha hon ton
- un qu nng hoc tit trng qu lu: thy phn agar, caramel ha ng,
gim pH, tng hoc gim cc cht c ch do cht mu trong mi trng chn
lc b mt, to thnh cc cht c ch mi.
- Xc nh pH sai, dn n cho qu nhiu kim hoc acid
25
- Mi trng, ha cht cha cht c mu cn bo qun trnh nh sng (gi
phng ti, dng c cha c mu, bc bng giy nu hoc giy nhm)
2.4.KH TRNG
2.4.1. Kh trng dng c, mi trng nui cy
Cc tc nhn vt l hay ha hc nh nhit cao, mt s dng bc x hay ha cht
chuyn dng c th dng kh trng vt dng nui cy
Nhit cao c th tiu dit vi sinh vt do kh nng lm hng hay bin tnh cc thnh
phn quan trng tham gia vo hot ng sng ca t bo nh protein v acid nucleic.
C th kh trng dng c bng phng php nhit kh (sy 170
o
C trong 2 gi) hay
nhit t (un 60
o
C trong 30 pht, 72
o
C trong 15 pht, hoc 100
o
C trong 10 pht)
dit th dinh dng ca vi sinh vt. Cng c th kh trng dng c bng hi nc
cao p 121
o
C trong 15-30 pht dit c cc th sinh dng ln cc bo t-nu c-
ca mi vi sinh vt
Cc bc x in t vi bc sng ngn (nh tia X hay tia gamma) c kh nng ion ha
nhiu loi phn t to nn nhng gc t do vi hot tnh hot ha cc mnh c th ph
hy cc thnh phn cng nh cu trc quan trng nh acid nucleic, mng lipoprotein,
cc protein trong t bo. Tia t ngoi tuy khng c kh nng ion ha nhng li l mt
tc nhn dit khun mnh do kh nng tc ng ln acid nucleic to t bin gy cht
t bo. Sng viba (microwaves, dng sng in t c bc sng t khong 1-1000nm)
cng c dng ph bin nh kh nng sinh nhit do kch thch cc phn t bt i
xng v in tch dao ng vi tn s cao (trn 900 triu ln/giy).
Cc dch lng c th c kh trng bng cch lc qua mng lc c kch thc gii
hn (< 0,75 m), cho php dch lng i qua v gi li tt c cc vi sinh vt (tr
mycoplasma c ng knh ch khong 0,2-0,3 m v virus). Mt s ha cht nh
ethylen oxyde, triethylglycol, cht dit khun, khng sinh c th kim sot s tng
trng ca vi sinh vt thng c dng kh trng hoc c ch s sng cng nh
tng trng ca vi sinh vt.
2.4.2.Kh trng dng c, mi trng sau nui cy
Mi trng sau nui cy vi sinh vt v cc dng c b nhim cn c kh trng, tiu
hy hoc lm sch ph hp. Ni chung vic kh trng bng nhit ng vai tr quan
trng trc khi lm sch hoc loi b cc vt liu sau nui cy. Vic kh trng bng
ha cht thng ch c thc hin vi cc dng c c kch thc v cu to nh gn
(nh pipet, lam knh)hoc trong vi trng hp c bit.
i vi cc dng c thy tinh c dng lm nhiu ln (nh bnh tam gic, ng
nghim) c th kh trng bng autoclave 121
o
C trong 30 pht. Vt liu nui cy
trong cc dng c cha dng mt ln (nh plastic) cng c th kh trng nhit
tng t trong cc bao plastic c im nng chy cao. Sau khi vi sinh b dit c th
loi b cc a Petri hoc bao b nha cng cc th bn trong.

2.5.K THUT GIEO CY V PHN LP VI SINH VT
2.5.1. K thut gieo cy vi sinh vt
Mc ch ca vic gieo cy VSV nhn ging vi sinh vt; pht hin s c mt ca vi
sinh vt trong cc mu (thc phm, bnh phm, dc phm, m phm, nc, t...)
26
cn phn tch; nghin cu cc c tnh hnh thi, sinh l, sinh ha ca i tng VSV
mc tiu.
Cy ging t mi trng lng sang ng nghim cha mi trng lng
Dng que cy vng v lm theo trnh t cc bc nh sau:
- ng nghim c cm tay tri, tay phi cm que cy, ngn t v lng bn tay ca
tay phi dng m v gi nt bng. Sau khi kh trng que cy, m nt bng, kh
trng ming ng nghim bng cch h qua ngn la.
- a que cy kh trng vo bn trong ng nghim hay bnh tam gic v nhng
vo canh trng ly mt t canh trng cha vi sinh vt m khng u que cy
chm vo thnh v ming ng nghim.
- u que cy c dnh VSV c gi khng gian v trng gn ngn la n cn.
- Dng tay tri ly ng nghim mi, m nt bng, kh trng ming ng nghim, ri
a que cy vo bn trong ng nghim nhng vo canh trng v khuy nh que
cy.
- Rt que cy ra, t ming ng nghim v nt bng ri y li.
- cc ng nghim vo gi, t que cy trn n cn ngay trc khi tr v gi
que cy.
- Dn nhn ln ng mi trng mi c gieo cy: ghi tn vi sinh vt v ngy gieo
cy.
Ch : Khi cy chuyn t ng nghim ny sang ng nghim kia c th cm hai ng
nghim cng mt lc trn cng bn tay tri.
Cy ging t ng nghim thch nghing hay mi trng lng sang ng nghim
thch nghing
Thng s dng que cy vng. Cc bc tin hnh ging k thut cy ging t mi
trng lng sang mi trng lng, nhng ch khc bc 4. Sau khi ly c sinh
khi vi sinh vt vo u que cy ri, ta tin hnh a u que cy vo trong ng
nghim v cy ria theo ng dzch dzc t y ng ln.
Cy m su trn ng nghim thch ng
Ngi ta s dng que cy thng m su vo trong mi trng thch ng. Cc
bc tin hnh cng tng t nh mc 1.1 v 1.2, nhng c im khc nh sau:
- Quay ngc ng mi trng cho ming ng nghim xung di trnh vic
nhim khun lc gieo cy.
- a ng nghim c vi sinh vt m su vo dc theo ng thch cho n gn y
ng nghim.
Lu : Khi cm que cy vo cng nh khi rt que cy ra, ch phi gi que cy
thng, nh nhng v khng lm cho mi trng b nt n.
2.5.2.Phn lp vi sinh vt
VSV tn ti trong t nhin dng qun x gm nhiu qun th cc loi VSV. Vic
phn lp 1 loi nht nh trong qun x VSV da vo kh nng phn tch cc t bo ra
thnh ring l trn mi trng chn lc.
Hu ht cc phng php phn lp v thun chng VSV u da trn mt s k thut
pha long phn tch t bo VSV kt hp iu kin nui cy chn lc to u th
tng trng cho chng quan tm. Hin nay, c cc k thut ph bin sau:
27
2.5.2.1.K thut hp ria (streak plate)
L k thut phn tch hn hp VSV bng cch ria trn b mt a thch sao cho cc t
bo ring r nhau ra. Sau khi c trong mt thi gian, mi t bo s tng trng
thnh mt khun lc ring bit. Khun lc ny hnh thnh do s sinh sn, phn chia t
mt t bo ban u.
Trong k thut ria, c nhiu cch ria khc nhau v khng c k thut no hon ho
tuyt i. Hiu qu ca k thut phn ln ph thuc vo ngi cy. Sau y l mt s
k thut ria thng dng:

K thut ria ch T (T streak) K thut ria lin tc (continuous streak)

K thut ria 4 gc (quadrant streak) K thut ria tia (radiant streak)
Hnh 3.1 Cc kiu ria trn mi trng thch
Ch : Sau mi vng ria, ngi ta phi tit trng que cy trn ngn la n cn.
2.5.2.2.K thut hp tri : Mi trng c chun b trc trong cc a Pettri. Yu
cu b mt mi trng phng, khng t. Nh 0,1 hoc 0,2 ml dch mu c pha
long vo chnh gia a mi trng. Dng que trang v trng tri u mu khp b
mt a. Sau thi gian , mi mt t bo s pht trin thnh mt khun lc.

2.5.2.3.K thut hp : Ht 1 ml dch mu c pha long vo chnh gia a.
15-20 ml mi trng tit trng, ngui khong 45-50
o
C vo gia a. Dng
tay xoay nh sang tri v xoay nh sang phi vi ln dch mu ha vo mi
trng. yn a trn b mt phng cho ng. Sau thi gian , mi mt t bo s
pht trin thnh mt khun lc.

2.6.PHNG PHP NH LNG VI SINH VT
2.6.1. NH LNG TRC TIP VI SINH VT BNG PHNG PHP O MT
QUANG ( C)
T bo vi sinh vt l mt thc th khi c trong mi trng s lm cho mi trng
tr nn c. c ca huyn ph t l thun vi s lng t bo vi sinh vt c trong
28
mi trng. Trong mt gii hn nht nh, mi quan h gia s lng vi sinh vt trong
mi trng t l tuyn tnh vi c ca mi trng. Do c th nh lng vi sinh
vt trong mi trng bng my o c hay my so mu bc sng 550 610nm.
Trong trng hp ny cn xy dng biu tng quan tuyn tnh gia c v s
lng vi sinh vt c trong mi trng bng phng php m khun lc trc tip.
Phng php tin hnh theo hai bc:
1. Pha long huyn ph cha vi sinh vt cn kim nghim sao cho cc dung dch
thu c khi o trn my so mu bc sng 610nm c cc tr s OD gn 0,1; 0,2;
0,3; 0,4; 0,5.
Dng phng php m trc tip xc nh lng t bo vi sinh vt c trong 1ml
k hiu N/ml.
Tnh gi tr log (N/ml) cho tng mc nng pha long. V th biu din log
(N/ml) (trc tung) theo cc gi tr OD t trn trc honh. Xc nh khong tuyn tnh
gia log (N/ml) vi OD.
2. Xc nh lng t bo theo c:
- o c ca huyn ph ca t bo vi sinh vt cn xc nh.
- T tr s OD o c i chiu trn th tng quan gia mt t bo v
OD xc nh lng t bo c trong mu nghin cu. Lu N/ml = 10
a
vi a=log
(N/ml).

Hnh 13. My o c

Phng php xc nh mt t bo theo c c th c dng so snh
mc tng trng ca hai hay nhiu chng vi sinh vt trong mi trng lng. Trong
trng hp khng cn thit gi tr tuyt i ca t bo vi sinh vt th khng cn phi
xy dng biu tuyn tnh gia mt vi sinh vt v gi tr OD.
Phng php ny cho kt qu nhanh, thng c dng theo di hoc nghin
cu c trng tng trng ca cc chng vi sinh vt trong phng th nghim hoc
trong sn xut, tuy nhin khng thch hp cho ng dng trong kim nghim vi sinh vt
29
2.6.2.NH LNG GIN TIP VI SINH VT BNG PHNG PHP M
KHUN LC
Phng php ny cho php xc nh s lng t bo vi sinh vt sng trong mu.
Nhng t bo sng mi c kh nng pht trin thnh khun lc trn mi trng thch
hp.
Phng php ny cho php nh lng chn lc vi sinh vt tu thuc vo mi
trng nui cy.
Phng php ny thc hin nh sau:pha long mu sao cho s lng khun lc
xut hin trn mt da petri khong 25 250. Di mc 25 s kh chnh xc trn mc
250 rt kh phn bit cc khun lc khi m. Mu c th c cy bng k thut cy
ria hay cy tri.
Vi mi loi vi khun i hi phi c thi gian nui cy ti u. Sao cho cc t
bo sng thi gian pht trin thnh khun lc. Kt qu thu c l s trung bnh ca
2 3 a petri vi cng iu kin nui cy. Kt qu cng thng c trnh by di
dng CFU (Colony Forming Unit) thay v s t bo/ml, v rng c nhiu t bo hnh
thnh chung mt khun lc.

Hnh 11. Khun lc vi khun v dng c m khun lc
Mc d c mt s nhc im song y vn l phng php tt nht xc nh
s lng t bo vi sinh vt. Phng php ny c nhy cao, nn thng c dng
trong kim nghim vi sinh vt trong nc, thc phm, bnh phm.
Quy trnh chi tit ca phng php m khun lc: mu c pha long theo dy
nng nh sau: 10
-1
, 10
-2
, 10
-3
, 10
-4
, 10
-5
V d: ly 1ml mu, pha vo 9ml dung
dch, pha long trn u, ri tip tc ly 1ml ca dung dch 1 cho vo 9ml dung dch
pha long ta c dung dch 2 vi pha long l 10
-2
.
30
1ml mau
o
pha
loang 10
-1
10
-2
10
-3
10
-4
10
-5

1ml 1ml 1ml
1ml
1ml
9
ml
9
ml
9
ml
9
ml
9
ml

C tip tc, s c cc dung dch vi pha long tng thm 10 ln cho n khi
tm c pha long thch hp, cho phng php m khun lc.
Nguyn tc chung l 1 phn mu + 9 phn dung dch pha long cho nn nu l
0,1ml cn b sung 0,9ml dung dch pha long.
Chun b mi trng thch hp trn a petri
Cch tnh kt qu :
V d: kt qu s khun lc trung bnh t 2 a petri thu c l 35, pha long
10
-4
, s lng mu a vo a petri l 0,1ml ta c: 35.10.10000 = 3500000 CFU/ml
Cch biu din kt qu:
- Biu din kt qu ph hp phng php ang p dng
- Lm trn s kt qu c c, ch gi li 2 s c ngha.
- Biu th kt qu di dng thp phn gia 1,0 v 9,9 nhn vi 10
n
(n l s
m thch hp ca 10).
V d:
- Kt qu 3500000 CFU/ml vit thnh 3,5.10
6
CFU/ml
- Kt qu 3572000 CFU/ml vit thnh 3,6. 10
6
CFU/ml
- Kt qu 3532000 CFU/ml vit thnh 3,5. 10
6
CFU/ml
2.6.3.NH LNG GIN TIP VI SINH VT BNG PHNG PHP MPN
(MOST PROBABLE NUMBER) (S C KH NNG CAO NHT SCKNCN)
Quy trnh nh lng theo phng php ny nh sau:
Cho cc ng nghim cha mi trng dng lng thch hp vi vi sinh vt cn
xc nh vi 3 mc nng 1/10, 1/100, 1/1000 mi mc 3 ng nghim. nhit
vi thi gian thch hp. Da vo kt qu nhn thy chng minh s tng trng ca vi
sinh vt cn kim nh (thng l nhng hin tng nh:sinh hi, i mu, c) ghi
nhn s lng cc ng nghim dng tnh tng pha long. S dng cc s liu
ny v da vo bng Mac Crady suy ra mt vi sinh vt c trnh by di dng
MPN/100ml hay s MPN/gam.
chnh xc ca tr s MPN ph thuc vo s lng ng nghim lp li trong
mi pha long.

Bng. Bng tra MPN cho lot 3 ng
31
S ng dng tnh

MPN/100 ml

S ng dng tnh
MPN/100 ml
S ml mu s dng
(ml)
S ml mu s dng
(ml)
10 1 0,1 10 1 0,1
0 0 0 - 2 0 0 9
0 0 1 3 2 0 1 14
0 0 2 6 2 0 2 20
0 0 3 9 2 0 3 26
0 1 0 3 2 1 0 15
0 1 1 6 2 1 1 20
0 1 2 9 2 1 2 27
0 1 3 12 2 1 3 34
0 2 0 6 2 2 0 21
0 2 1 9 2 2 1 28
0 2 2 12 2 2 2 35
0 2 3 16 2 2 3 42
0 3 0 9 2 3 0 29
0 3 1 13 2 3 1 36
0 3 2 16 2 3 2 44
0 3 3 19 2 3 3 53
1 0 0 4 3 0 0 23
1 0 1 7 3 0 1 39
1 0 2 11 3 0 2 64
1 0 3 15 3 0 3 95
1 1 0 7 3 1 0 43
1 1 1 11 3 1 1 75
1 1 2 15 3 1 2 120
1 1 3 19 3 1 3 160
1 2 0 11 3 2 0 93
1 2 1 15 3 2 1 150
1 2 2 20 3 2 2 210
1 2 3 24 3 2 3 290
1 3 0 16 3 3 0 240
1 3 1 20 3 3 1 460
1 3 2 24 3 3 2 1100
1 3 3 29 3 3 3 -

2.6.4. NH LNG GIN TIP VI SINH VT BNG K THUT MNG LC
Phng php ny c dng nh lng vi sinh vt trong cc mu nc vi
mt vi khun thp. V vy phng php ny gm bc lc tp trung vi sinh vt
c trong mu nc trn mng lc. Vic xc nh s lng t bo vi sinh vt da vo s
khun lc m c. Sau khi t mng lc ln trn mi trng tht c thnh phn dinh
dng thch hp. Theo nguyn l mt khun lc l mt t bo vi sinh vt.
Mng lc c kch thc 0,2 0,45 m c ch to t cc si thy tinh hay si
polypropylene.
Ngoi mng lc thng thng, ngy nay cn dng mng lc k nc
(Hydrophobic Grid Membrane). Cc vch chia lm bng vt liu ngn cn s mc
lan ca cc khun lc. S vi sinh vt c tnh theo s khun lc mc trong cc .
32
- T s cc vung c khun lc suy ra mt vsv trong mu theo dng s c
xc xut ln nht (MPN): MPN = N. ln(N/(N-x)); trong N l tng s cc vung, x
l s c khun lc mc.

Hnh 12. B lc v trng v mng lc

2.6.5. PHNG PHP M TRC TIP
Cc loi vi sinh vt n bo c kch thc ln nh nm men to C th nh
lng trc tip trong bung m hng cu qua knh hin vi. Quy trnh m trc tip
cho php xc nh nhanh chng s lng vi sinh vt trong mu. Tuy vy phng php
ny c cc nhc im sau:
- Khng phn bit c t bo sng v t bo cht
- Khng phn bit dc t bo vi sinh vt, cc ht vt th khc ln trong mu
- Khng thch hp cho huyn ph vi sinh vt c mt thp.
C ba phng php m trc tip:
1. m trc tip bng bung m hng cu
Bung m hng cu l mt dng c bng thu tinh. Din tch bung m l
1mm
2
, chiu cao l 0,1mm. Din tch 1mm
2
c chia lm 400 nh v th tch ca
400 nh s bng hoc 25 vung ln, tng ng th tch ca vung ln l
1/250mm
3
.
33
Trc khi quan st cho thm vi git formalin vo trong mu. Pha long mu
sao cho trong mi vung nh c khong 5 10 vi sinh vt. Mun t c yu cu
trn cn phi c lng s vi sinh vt trong mu, hay phi lm th mt vi ln xc
nh c cng thc pha long ti u.

Hnh 10. Bung m hng cu
Mu c pha long bng dung dch c thnh phn:
Thnh phn dung dch pha long.
- Pepton 0,1%
- Laurylsulphate 0,1%
- Methyl blue 0,01%
Cc dung dch pha long sau khi chun b phi lc qua trc khi s dng.
iu chnh knh phng i 400 ln ri tin hnh m. m s lng t bo
trong 5 ln nm trn ng cho ca bung m.
Cch tnh s vi sinh vt. Gi x
1
, x
2
, x
3
, x
4
, x
5
l s lng vi sinh vt trong tng
ln ta c :
X =
5
5 4 3 2 1 x x x x x + + + +

Ta bit th tch ca 1 ln bng 1/250 mm
3

Do s lng t bo vi sinh vt co trong 1 ml mu s l:
S vi sinh vt (trong 1 ml mu) = X .250.1000 = X .250000
2. m trc tip bng ng m Breed
Bung m Breed c din tch 1cm
2
c nh du. Dng bm tim cho chnh
xc 0,01ml dung dch mu dn trn mt bung m. Sy kh bng cch h nh trn
n cn bng methyl blue.
Khi m pht mt lp du trn b mt cn m. S lng vi sinh vt /ml c
tnh theo cng thc:
N/ml=4a.10
4
/[d
2

a: s lng t bo vi sinh vt trong vng m
d: ng knh ca vng m
34
3. m trc tip bng knh hin vi hunh quang
Khi nhum vi sinh vt bng mt s thuc nhum pht hunh quang nh:
- Acridin Cam (AODC)
- 4, 6 dianidino 2 phenyl indol (DAPT)
- Fluoresecin isothio cyanate (FITC)
Mt vi sinh vt c th xc nh trc tip di knh hin vi hunh quang.
S m nhn c bng phng php ny c th cao gp 2 ln phng php
m khun lc. S sai khc ny do cc t bo cht hoc tn thng khng c kh nng
pht trin thnh khun lc.
Kt qu s m vi sinh vt bng knh hunh quang phn nh s vi sinh vt c
tht trong mu.
2.7. TH NGHIM SINH HA
Trong k thut phn tch vi sinh vt, sau khi phn lp, vic nh danh vi sinh
vt l mt vn rt quan trng. Vic nh danh ny c thc hin da v cc c
im v kiu hnh, c bit l cc phn ng sinh ho thc hin bi cc chng vi sinh
vt. Thng thng thc hin cc phn ng sinh ho c th tin hnh theo mt trong
ba cch l cch truyn thng, s dng b KIT v dng thit b t ng.
Trong kim nghim, phn tch, cc kt qu th nghim sinh ho biu th quy
c bng cc k hiu nh: (+): dng tnh; (-): m tnh; (+/-): khong trn 70% l
dng tnh v (-/+): khong trn 70% l m tnh.
Trong cc th nghim sinh ho, m bo chnh xc trong vic c kt qu,
ngi ta thng thc hin th nghim trn cc chng i chng. Mt s chng i
chng cho cc th nghim sinh ho thng dng c trnh by trong bng sau:
Tn chng M s NCTC M s ATCC
Acinetobacter calcoaceticus
Aci. lwoffi
7844
5886
15308
15306
Aeromonas hydrophila 8049 7966
Alkaligenes faecalis 415 19018
Bacillus cereus
Bac. subtilis
10876
6633
7464
10400
Clostridium histolyticum
Clos. Perfringens
19401
13124
503
8237
Edwardsiella tarda 10396 19547
Enterobacter aerogenes
Ent. cloacae
13048
10005
10006
-
Enterococcus faecalis 29212 -
35
Tn chng M s NCTC M s ATCC
Escherichia coli 25922 10418
Mycobacterium fortuitum
Myco. kansasii
Myco. phlei
Myco. terrae
10349
10268
8151
10856
6841
14471
19249
15755
Norcadia brazilensis
Nor. otitidicarcaviarum
11274
1934
19296
14629
Proteus mirabilis
Pro. rettgeri
10975
7475
-
-
Pseudomonas aeruginosa 10662
Serratia marcescens 13880 10218
Staphylococcus aureus
Staph. epidermidis
25923
12228
6571
-
Streptococcus agalactiae
Strep. pneumoniae
Strep. salivarius
13813
6303
8681
8181
-
7073

Cc th nghim sinh ho quan trng thng s dng trong phn tch vi sinh vt
bao gm nh sau:
1. Th nghim kh nng ln men
Th nghim kh nng ln men l th nghim dng xc nh kh nng s
dng mt ngun carbon nht nh bi vi sinh vt tng trng theo con ng ln
men. Ty theo vi sinh vt v ngun c cht mun kim tra c th b sung vo mi
trng nui cy nhng ngun c cht khc nhau. Sn phm to thnh t qu trnh sng
ny s lm gim pH ca mi trng nui cy v t lm thay i mu ca cht ch
th mu c b sung vo mi trng.
Cht ch th mu thng s dng trong th nghim l phenol red.
2. Th nghim kh nng oxy ho-ln men
Th nghimkh nng oxy ho ln men nhm mc ch xc nh vi sinh vt
bin dng ngun carbon hydrat theo con ng ln men hay h hp. Phn ng c
xc nh da trn sn phm to thnh t qu trnh sng ca vi sinh vt v c th lm
chuyn mu ca cht ch th mu b sung vo mi trng theo nhng cch khc nhau.
Th nghim tin hnh trn mi trng Hugh-Leifson cha ch th pH l
bromothymol blue
36
3. Th nghim kh nng bin dng citrate
Th nghim dng xc nh kh nng s dng citrat lm ngun carbonhydrat
duy nht ca vi sinh vt. nhng chng c c tnh ny , s c kh nng dng mui
amoniu. Th nghim c thc hin trn mi trng Simmon citrate c ngun carbon
duy nht l citrae vi s hin din ca cht ch th mu bromothymol blue.
4. Th nghim kh nng bin dng malonate
Th nghim dng xc nh kh nng s dng malonat lm ngun carbon duy
nht. nhng chng c c tnh ny, s c kh nng dng mui amonium. Th
nghim c tin hnh trn mi trng malonate broth vi cht ch th mu
bromothymol blue.

5. Th nghim catalase
Th nghim dng phn bit vi sinh vt hiu kh v vi sinh vt k kh. Vi
sinh vt hiu kh c enzym catalase, c kh nng phn gii H
2
O
2
(cht rt c i vi
t bo) thnh H
2
O v O
2
. Phn ng c nhn din nh s si bt ca git H
2
O
2
khi
O
2
bay ln.
6. Th nghim decarboxylase
Th nghim dng phn loi v nh danh cc loi vi khun ng rut, da
trn loi enzym decarboxylase xc tc phn ng phn gii mt acid amin c trng,
to CO
2
lm tng pH mi trng.
7. Th nghim coagulase
Th nghim thng dng nh danh Staphylococcus. Loi ny c kh nng
tit enzym coagulase c tc dng lm kt t cc thnh phn huyt tng, to khi ng
huyt tng.
8. Th nghim urease
Th nghim dng xc nh kh nng to enzym urease ca mt s chng vi
sinh vt, nht l nhm Proteus. Enzym ny xc tc phn ng phn gii ur thnh NH
3

v CO
2
lm tng pH ca mi trng.
9. Th nghim gelatinase
Th nghim dng nh gi kh nng tit enzym gelatinase phn gii gelatine
thnh polypeptid v acid amin ca cc i tng vi sinh vt.
10. Th nghim kh nng sinh H
2
S
Th nghim dng xc nh kh nng phn gii cc acid amin cha lu
hunh (cystein, cystin, methionin) sinh H
2
S nh enzym desulfuahydrase.
11. Th nghim kh nng sinh idol
Th nghim dng xc nh kh nng chuyn ho cc sn phm trung gian
ca qu trnh oxy ho thnh indol. Sn phm indol to thnh c xc nh nh phn
ng vi thuc th p-dimethylaminobenzaldehide to phc hp dng qiunon c mu
.
12. Th nghim KIA, TSI
37
Th nghim KIA hay TSI l th nghim c thc hin ng thi trn mi
trng KIA, TSI dng kim tra kh nng s dng cc ngun carbon khc nhau
(glucose, lactose, sucrose) v kh nng sinh H
2
S ca vi sinh vt.
13. Th nghim nitratase (kh nitrate)
Th nghim dng kim tra c tnh s dng enzym nitratase kh nitrat
thnh nitrite v cc sn phm khc. Nitrite to thnh s c nhn bit nh phn ng
vi sulphanilamide v N-napthylethylenediamide hydrochloride pH acid cho phc
cht mu hng.
14. Th nghim oxidase
Th nghim nhm xc nh s hin din ca h enzym oxydase vi sinh vt.
Hot tnh oxydase c pht hin nh thuc th p-phenylenediamin, nu c s hin
ca enzym, thuc th b oxy ho mt hp cht indolphenol c mu xanh dng.
15. Th nghim ONPG
Th nghim dng xc nh hot tnh enzym |-galactosidase tham gia vo
qu trnh ln men lactose vi sinh vt. Mi trng th nghim l ONPG broth cha o-
nitrophenyl-D-galactopyranoside. Sn phm to thnh l o-nitrophenol c mu vng.
16. Th nghim MR (Methyl Red)
Th nghim nhm phn bit vi sinh vt da vo s khc bit trong qu trnh to
v duy tr cc sn phm c tnh acid t s ln men glucose. Vi sinh vt ln men
glucose to v duy tr sn phm acid, lm i mu thuc th methy red trong mi
trng. Nu sn phm acid tip tc bin i thnh cc sn phm trung tnh, khng lm
i mu thuc th.
17. Th nghim VP (Voges-Proskauer)
Phn ng dng phn bit cc loi trong h vi khun ng rut
Enterobacteriaceae, da vo s oxi ho acetoin c to ra t 2,3-butanediol thnh
diacetyl. Diacetyl c xc nh da vo phn ng kt hp vi nhn guanidine ca
pepton to phc .
18. Th nghim CAMP
Phn ng nhm phn bit cc nhm Streptococcus. Phn ng CAMP l thc
hin gia nhn t CAMP do Streptococcus nhm B tit ra v |-hemolysin c tit ra
bi Staphylococcus aureus lm tng hot tnh ca |-hemolysin gy ph v hng cu
(tan huyt)
19. Th nghim tnh di ng
L th nghim dng xc nh c tnh di ng nh tim mao ca vi sinh vt.
Th nghim c thc hin trn mi trng bn lng. Kt qu c xc nh da vo
kch thc ca vt cy vi sinh vt.

B KIT sinh ho nh danh vi sinh vt
Ngy nay, cc th nghim sinh ho truyn thng nh danh vi sinh vt c
th thc hin cc thuc th dng a giy, que giy. Ngoi ra, cn c cc b KIT
cho php thc hin hng lot th nghim sinh ho theo mt quy trnh hai la chn
38
nh danh vi sinh vt. Cc b KIT ny c sn xut di dng thng phm c u
im l tit kim thi gian, cng sc; tuy nhin cng c nhc im v d nh mi b
KIT ch cho php nh danh mt s vi sinh vt, chi ph th nghim t tin hn

Hnh 24. Cc dng KIT sinh ho nh danh vi sinh vt

CHNG III
QUY TRNH PHN TCH VI SINH VT THEO PHNG PHP TRUYN THNG

1. PHN TCH TNG S VI KHUN HIU KH

1.1.Nguyn tc phn tch tng s vi khun hiu kh
Tng s vi khun hiu kh l ch th mc v sinh ca thc phm. Ch s ny
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 mt khun lc l mt sinh khi pht trin t
mt 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.
1.2.Dng c, thit b, mi trng, ha cht
1.2.1.Dng c v thit b
Bng 1.1.. Dng c v thit b ch tiu 1
39
Dng c Thit b
ng nghim (C16 mm) T cy v trng
a petri (C100 mm) Ni hp
Cc thu tinh (100 ml, 250 ml) T m
Bnh tam gic (250 ml) My dp mu (Stomacher)
ng ong (100 ml) My trn mu (vortex mixer)
u tip Pipetman (1000, 5000 l) Cn phn tch
Que tri pH k
Kp inox B n nhit
n cn Pipetman (1000, 5000 l)
L viba

1.2.2.Mi trng v ha cht
Bng 2.2.. Mi trng v ha cht ch tiu 1
Mi trng Ha cht
Saline Pepton Water (SPW) Cn 90
o
v 70
o

Mi trng Cao tht pepton HCl 10%
plate count agar (PCA) NaOH 10%

1.3.Thc hnh
40
1.3.1.Quy trnh phn tch

S quy trnh nh lng tng s vi khun hiu kh
1.3.2.Cc bc tin hnh
1. nh lng mu:
Bc 1. Cn 10,0 g (hay 25 g) hay ht 10,0 ml (hay 25 ml) mu a vo cc thu
tinh v trng (nu l mu rn, phi dp mu)
2. ng nht v pha long mu:
Bc 2. mu vo bnh tam gic c 90 ml (hay 225 ml) SPW.
Bc 3. Mu lng th lc u, mu rn th ng nht mu bng my dp mu
(stomacher)
Bc 4. Lc u mu trong bnh tam gic t nht 2 pht ta c mu c pha
long 10
-1
1ml 1ml 1ml
10
-1
10
-2
10
-3
10
-4
9ml 9ml 9ml
SPW SPW SPW
90 ml SPW + 10g mau

Hnh 1.1. K thut pha long
nh lng mu
10 g hoc 10 ml
ng nht v pha long mu
Cy mu v rt mi trng vo a
Petri

m khun lc
Chn a c 25 -250 khun lc
250 khun lc
mu
30 1
o
C trong 72 gi
41
Bc 5. Dng pipetman vi u tip v trng (hay pipet 1ml v trng) ht 1ml
dch mu 10
-1
vo ng nghim cha 9 ml dch SPW c pha
long 10
-2
. Lm tng t c cc pha long cao hn (theo Hnh
4.1)
Bc 6. Trn mu trong ng nghim cho u bng bng my vortex mixer hay
lc mnh bng tay.
3. Cy mu v rt mi trng vo a petri:
Bc 7. Chn cc pha long thch hp (d kin c 25 - 250 TB vi khun/1ml
mu). i vi mu l, khng c on c lng vi sinh vt nhiu hay
t phi cy nhiu nng pha long t thp n cao.
Bc 8. Dng pipetman ht 1 ml mu vo a petri v trng (mi pha long
02 a)
Bc 9. vo mi a petri 15 - 20 ml mi trng
Bc10. Trn u dch mu vi mi trng bng cch xoay trn a petri xui v
ngc chiu kim ng h 3 - 5 ln.
4. mu:
Bc11. t cc a trn b mt ngang cho thch ng li. Lt ngc v trong t
m 30 1
o
C trong 72 gi.
5. m khun lc
Bc 12. Chn cc a c 25 - 250 khun lc m.
1.3.3.Kt qu
Mt tng vi khun hiu kh trn 1 g mu c tnh nh sau:
... Vf n Vf n
N
A(CFU/g)
2 2 1 1
+ +
=

Trong : A l s t bo vi khun /1g mu
N l tng s khun lc trn cc a chn
n
i
l s lng a cy ti mt pha long
V l th tch dch cy vo petri
f
i
l pha long tng ng
V d: Phn tch tng s vi khun hiu kh trn 1 gram tht heo sng qua bo
qun 3 ngy 10
o
C. Kt qu:
pha long (f
i
) 10
-3
10
-4

a s 1 (n
1
) 224 20
a s 2 (n
2
) 235 26
p dng cng thc, ta c kt qu:
(CFU/g) 10 2,3
10 1 1 10 1 2
26 235 224
A
5
4 3
=
+
+ +
=

Cc kt qu ca tng s vi khun hiu kh thng c biu din di dng s
m ca c s thp phn.
42

Petri c > 250 khun lc Petri c 25 250 khun lc Petri < 25 khun lc Petri c khun lc mc loang
Hnh 1.2. S khun lc m trn a Petri
Trng hp khun lc vi sinh vt mc loang, mi vt loang c tnh l mt
khun lc.
Nu pha long cao nht, s khun lc m c trn 1 a > 250, v d
nng 10
-4
s m > 250 th kt qu ghi: > 2,5 x 10
-6
CFU/g.
Nu pha long thp nht, s khun lc m c trn 1 a < 25, v d
nng 10
-1
s m nh hn 25 th kt qu ghi: < 2,5 x 10
-2
CFU/g.

1.4.Cu hi n tp
1. Tai sao moi trng nuoi cay tong vi khuan hieu kh c coi la
moi trng dinh dng?
2. Ke ten mot so thng hieu moi trng nuoi cay vi sinh vat ma
em biet?

2. NH LNG TNG S NM MEN-NM MC

2.1.Nguyn tc phn tch tng s nm men nm mc
Mt nm men v nm mc c xc nh di
dng tng nm men v nm mc bng k thut pha
long, tri hp v m khun lc trn mi trng
Dichloran 18% Glycerol Agar (DG18) hay
Dichloran Rose Bengal Chloramphenicol Agar
(DRBC). Mi mt khun lc c pht trin t
mt t bo hay bo t.


Hnh 2.1. Nm men v nm mc (DRBC)
43
Bng 2.1. Dng c v thit b ch tiu 2
Dng c Thit b
ng nghim (C18 mm) T cy v trng
Bnh tam gic (250 ml) My dp mu (Stomacher)
ng ong (100 ml) My trn mu (vortex mixer)
u tip Pipetman (1000, 5000 l) Cn phn tch (4 s l)
Que tri pH k
n cn Pipetman (1000, 5000 l)
L viba

2.2.2Mi trng v ha cht

Bng 2.2. Mi trng v ha cht ch tiu 2
Mi trng Ha cht
Saline Pepton Water (SPW) Cn 90
o
v 70
o

DG18 HCl 10%
DRBC NaOH 10%

2.3.Thc hnh


S quy trnh nh lng tng s nm men - nm mc

2.3.2.Cc bc thc hin
Bc 1. nh lng mu 10 g hay 10 ml bng cn k thut chnh xc 0,01 g
nh lng mu
10 g hoc 10 ml
ng nht v pha long mu trong dung dch
SPW
Tri 0,1 ml dung dch mu ln mi trng DRBC

m khun lc
Chn a c 25 - 250 khun lc
mu
30 1
o
C trong 3 7 ngy
44
Bc 2. ng nht mu bng my dp mu (mu dng mm) hay xay mu (mu
dng cng, dai). Sau pha long theo cp 10
-n
.
Bc 3. Chn pha long ph hp v ht 0,1 ml dch VSV tri ln b mt thch
ca a petri. Mi nng cy 2 a.
Bc 4. mu 30
o
C t 3 7 ngy.
Bc 5. Chn m a c t 25 250 khun lc.
2.3.3. Tnh kt qu
Tng t cch tnh tng s vi khun hiu kh.

2.4. Cau hoi tho lun
1. Em cho biet ngoai moi trng DRBC, cac moi trng nao khac
c s dung e nuoi cay tong so nam men, nam moc?
2 .Tai sao a so cac vi khuan khong moc c trong moi trng
nuoi cay DRBC?

45

3. NH LNG TNG COLIFORMS BNG PHNG PHP TNH S SC
XUT LN NHT (MPN)

3.1.Nguyn tc
Phng php phn tch trong bi ny c da trn quy trnh MPN bng s dng canh
Lauryl Sulphate Broth (LSB) vo test on chng (presumptive test), sau dng
canh Brilliant Green Lactose Bile Broth (BGLB) v m 37
o
C/24-48 gi.
Phng php MPN l phng php da trn nguyn tc mu c pha long thnh 1
dy thp phn (2 nng ni tip nhau nhng khc nhau 10 ln); mi nng pha
long c t 3 n 5 ng lp li c ng Duharm. Theo di s c mi trng v
sinh hi 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 1g
(hoc 1ml) mu ban u.
3.2.Dng c, mi trng, ha cht v thit b
Dng c Thit b
Ti dp mu T cy v trng
u tip Pipetman (1000, 5000 l) Pipetman (1000, 5000 l)
Que trang My dp mu (Stomacher)
ng Durham My trn mu (vortex mixer)
n cn Cn k thut
ng nghim
Pipette 1 ml, 10 ml

Mi trng Ha cht
Dung dch Saline Peptone Water
(SPW)
Cn 90
o
v 70
o

Lauryl Sulfate Tryptone Broth. HCl 10%
Mi trng BGBL (Brilliant Green
Lactose Bile Salt)
NaOH 10%

46
3.3.Thc hnh


3.3.2.Cc bc thc hin
Bc 1. Cn 10 g mu cho vo ti dp mu (loi ti dng cho Stomacher). 90 ml
dung dch SPW v trng vo v ng nht mu trong 1 pht.
Bc 2. Tun t cy 1 ml dch mu pha long 10
-1
( v d dng lot pha long lin
tip 10
-1
, 10
-2
, 10
-3
) vo 3 ng nghim ging nhau, mi ng cha 10 ml
canh LSB, mi ng nghim cho 1 ng Durham. Thc hin tng t vi dch
mu pha long 10
-2
, 10
-3
.
Bc 3. 37
o
C trong 48 gi.
Bc 4. Ghi nhn s ng sinh hi v canh trng c. Kt lun cc ng LSB (+)
Bc 5. Cy chuyn 1 ml t cc ng LSB (+) sang canh trng BGBL.
Bc 6. 37
o
C trong 48 gi
Bc 7. Ghi nhn s ng sinh hi v canh trng c. Kt lun cc ng BGBL (+)
3.3.3.Kt qu
Da vo s ng BGBL (+) tra bng MPN, suy ra s lng coliform tng

Chun b dch ng nht hoc pha long mu c cc
pha long 10
-1
, 10
-2
, 10
-3

Chuyn 1ml dung dch 10
-1
, 10
-2
, 10
-3
vo ng
10ml canh LBS, mi nng 3 ng lp li,
37
0
C, 48 gi
Ghi nhn cc ng LSB (+) mi nng pha long
Chuyn 1 ml dung dch pha long ba nng lin tip vo
ng nghim c 10 ml canh LSB, mi nng lp li 3 ng,
37 1
o
C, 48 gi
Chuyn 1 ml t LSB (+) sang canh BGBL, 37 1
o
C,
48 gi
Ghi nhn ng BGBL (+) mi long
Tra bng MPN suy ra s Coliform tng
47
3.4.Cu hi tho lun
1.Trong Coliforms tng c cc ging vi sinh vt no?
2. Nu nguyn tc ca phng php MPN? Vit tt ca MPN?
3. Nu vai tr ca cc thnh phn trong mi trng LSB v BGB

4.NH LNG E.COLI BNG PHNG PHP MPN
4.1.Nguyn tc
T Coliforms c Blachstein s dung au tien vao nam 1893
e noi ve nhng vi khuan Bacilli co ac iem hnh thai giong E.
coli.
Coliforms tong cong la nhng trc khuan ng ruot, gram (-),
khong sinh bao t, hieu kh hoac k kh tuy tien, co kha nang len
men lactose sinh acid va sinh hi 35
o
C - 37
o
C trong 24-48 gi. Nhom
Coliforms hien dien rong rai trong t nhien, trong ruot ngi, ong
vat. So lng Coliforms trong mau c dung e ch th kha nang
hien dien cua cac vi sinh vat gay benh khac, mac du ieu nay
con nhieu tranh cai. Nhom Coliforms gom bon giong la: Escherichia
vi mot loai duy nhat la E. coli; Citrobacter; Klebsiella va Enterobacter.
Coliforms chu nhiet la nhng Coliforms co kha nang len men
lactose sinh hi khi u 44
o
C/24h trong moi trng EC.
Coliforms phan (Faecal Coliforms hay E. coli gia nh) la Coliforms
chu nhiet co kha nang sinh indole khi u 44.5
o
C/24h trong tryptone.
Escherichia coli lan au tien c Escherich phan lap t phan
ngi vao nam 1855. ay la vi khuan c chu y nhieu va
nghien cu ky lng nhat. Chung c tru ng ruot cua ngi
va ong vat mau nong. Chung co the la vi khuan gay benh c
hoi. E. coli la vi sinh vat ch th tieu bieu nhat. S co mat cung
nh so lng cua chung ch ra mc o ve sinh trong qua trnh che
bien, bao quan, van chuyen thc pham, nc uong cung nh s
o nhiem phan trong moi trng. T nam 1982 a phat hien thay
4 loai E. coli co kha nang gay benh co nguon goc thc pham
(Enteropathogenic-EPEC; Enteroinvasive-EIEC; Enteroinvasive- EIEC;
Enterotoxigenic ETEC, trong o co chung O157:H7). Bai thc hanh
nay ch gii han viec xac nh E. coli nh mot vi sinh vat ch th
thc pham.
Phng phap phan tch trong bai nay c da tren quy trnh
MPN bang s dung canh thang Lauryl sulphate tryptose (LST) vao test
oan chng (presumptive test), sau o dung canh thang BGLB (Brilliant
Green Lactose Bile Broth) va u am 37
o
C/24-48 h (e kiem tra
Coliforms). Tiep theo chuyen sang moi trng canh EC (E. coli medium)
u 44.5
o
C e kiem tra Coliforms chu nhiet. Sau o xac nh
Coliforms phan (E. coli gia nh) bang cach cay sang moi trng
EMB (Eosine Methylene Blue Agar), chon khuan lac ien hnh va th
kha nang sinh Indol (I). e xac nh E. coli can th ba phan ng
sinh hoa tiep theo la Methyl Red (MR), Voges-Proskeur (VP) va Citrate
48
(iC). E. coli la Coliforms phan va cho ket qua th nghiem IMViC la +
+ - -.


Dng c Thit b
Ti dp mu T cy v trng
u tip Pipetman (1000, 5000 l) My dp mu (Stomacher)
ng Durham My trn mu (vortex mixer)
n cn Cn k thut
ng nghim
Micropipet

Bng 4.2.. Mi trng v ha cht ch tiu 4
Mi trng Ha cht
Dung dch Saline Pepton
Water(SPW)
Cn 90
o
v 70
o

Lauryl Sulfate Tryptone Broth. HCl 20%
Mi trng BGBL (Brilliant Green
Lactose Bile Salt)
NaOH 20%
Moi trng Tryptic Soya Agar
(TSA)
Thuc th Kovacs
Moi trng EMB (Eosine
Methylen Blue agar)/ Endo
Thuc th Metyl Red
Moi trng MRVP (Metyl Red
Voges Prokauer
Dd creatine 0,5 %
Moi trng thach Simons Citrat

Dung dch o- naphtol 5%
Moi trng Trypton Water (TW) KOH 40%

Thuoc th Kovac
HCl 10%
49
NaOH 10%

4.3.Thc hnh

Chun b dch ng nht hoc pha long mu c cc pha long
10
-1
, 10
-2
, 10
-3

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

Chuyn 1 ml dung dch pha long ba nng lin tip vo ng nghim c 10
ml canh LSB, mi nng lp li 3 ng, 37 1
o
C, 48 gi
Chuyn 1 ml t LSB (+) sang canh EC, 44,5 0,2
o
C, 24gi
Ghi nhn s ng (+) mi long
Cy ln thch EMB, 37 1
o
C, 24 gi
Chon khuan lac det, hnh da, co anh kim tm, ng knh 1mm
e cay sang TSA/BHI, 37 1
o
C, 24 gi

Th nghim IMViC
m s canh EC (+) v IMViC(++--), tra bng MPN
E.coli
50

4.3.2.Cc bc thc hin
Bc 1.Cn 10 g mu cho vo ti dp mu (loi ti dng cho Stomacher). 90 ml
dung dch SPW v trng vo v ng nht mu trong 1 pht.
Bc 2.Tun t cy 1 ml dch mu pha long 10
-1
( v d dng lot pha long lin
tip 10
-1
, 10
-2
, 10
-3
) vo 3 ng nghim ging nhau, mi ng cha 10 ml canh LSB,
mi ng nghim cho 1 ng Durham. Thc hin tng t vi dch mu pha long 10
-
2
, 10
-3
. 37
o
C trong 48 gi.
Bc 3.Ghi nhn s ng sinh hi v canh trng c. Kt lun cc ng LSB (+)
Bc 4.Cy chuyn 1 ml t cc ng LSB (+) sang canh trng EC. 44,5 0,2
o
C,
24gi.
Bc 5.Ghi nhn s ng sinh hi v canh trng c.
Kt lun cc ng EC (+)

Bc 6.Dng que cy vng ria dch mu t cc ng (+) trn mi trng canh EC sang
mi trng thch a EMB/ Endo . cc a 37
o
C, 24gi.
Nhan dang khuan lac E.coli:
- Tren moi trng EMB: khuan lac mau tm, anh kim, tron, b
eu, ng knh khoang 1mm
- Tren moi trng Endo: Khuan lac mau o, anh kim, tron, b
eu, ng knh khoang 1mm
Bc 7. Chn khun lc E.coli gi nh trn EMB/Endo cy vo TSA/BHI, qua m
Bc 8.Cy chuyn t mi trng TSA/BHI vo cc mi trng th nghim sinh ha
Trypton broth, MR- VP, SC. 44,5 0,2
o
C, 24gi.
Bc 9. Th phan ng sinh hoa .Th nghim IMViC
Phan ng Indol: Cay khuan lac nghi ng vao ong nghiem
cha 5ml Trypton broth, u 37
0
C trong 24gi. Cho them 0,5ml
thuoc th Kovac bang cach cho chay doc theo thanh ong
nghiem

Hnh 4.2 : E.coli trn mi trng EMB

Hnh 4.1 : Cc ng EC (+)

51
+ Moi trng chuyen mau o: indol (+)
+ Moi trng khong oi mau: indol (-)
Phan ng MR (metyl red): Cay khuan lac nghi ng vao 2
ong nghiem, moi ong cha 5ml moi trng MRVP e
37
0
C t 48 96 gi. Mot ong th phan ng Methyl red,
mot ong th phan ng VP.
Cho vao mot ong nghiem vai giot metyl red pH trung tnh
hoac kiem yeu
+ Moi trng chuyen mau o: MR (+)
+ Moi trng khong oi mau: MR (-)
Phan ng VP (Voges Proskauer) Cho vao ong nghiem cha
moi trng MRVP con lai khoang 0,6ml dung dch o- naphtol
va 0,2ml dung dch kalihydroxyt 40%, lac eu, e yen trong 2h.
+ Moi trng oi sang mau o eosin hoac co vet o eosin
phat trien trong vong 15 phut : VP (+)
+ Moi trng khong chuyen mau o: VP
(-)
Phan ng citrat: Cay ria khuan lac nghi
ng len mat nghieng cua moi trng
simon citrat, e 37
0
C trong 24h.
+ Moi trng oi mau luc sang xanh
dng va co khuan lac: citrat (+)
+ Moi trng khong oi mau, khong
moc khuan lac: citrat (-)

4.3.3.Kt qu
ng nghim cho kt qu EC (+), v khun lc E.coli gi nh trn mi trng
EMB cho kt qu th nghim IMViC (++-- ) l ng nghim c E.coli (+). T s
lng cc ng c E.coli (+) mi pha long ca mu tra bng MPN tnh
ra mt vi sinh vt trong mu v biu din di dng tr s MPN/g hay
MPN/ml

5. NH TNH SALMONELLA TRONG THC PHM

5.1.Nguyn tc
Salmonella c phat hien t 1880, ai a
so la vi khuan gay ngo oc thc pham
va gay benh (sot thng han). Salmonella
thuoc ho Enterobacteriaceae, trc trung gram
am, ho hap tuy tien, co kha nang di

Hnh 4.3. Phn ng
citrat

E.coli trn Simoncitrat

Hnh 5.1. Salmonella

52
ong, khong tao bao t, len men glucose va mannitol sinh acid nhng
khong len men saccharose va lactose, khong sinh indole, khong phan
giai urea, khong co kha nang tach nhom amine t tryptophane, hau
het cac chung eu sinh H
2
S. Cac loai thc pham eu yeu cau
phai kiem nghiem Salmonella vi tieu chuan khong phat hien/25
g(ml)
Nh vay viec xac nh thong thng ch dng mc o nh
tnh, ten chung ch c xac nh trong nhng trng hp quan
trong. Can lu y Salmonella la nhom cha cac dong gay benh
nguy hiem, do vay can tuan thu triet e cac bien phap an toan
khi lam viec vi cac chung oi chng (+) cung nh khi thao tac
tren cac chung phan lap c. Cac moi trng hay mau vat sau
khi nuoi cay can phai hap kh trung can than trc khi ra. Do
trong thc pham Salmonella thng ton tai so lng t, te bao co
the b ton thng do o quy trnh xet nghiem thong thng gom 4
bc: tang sinh, tang sinh chon loc, phan lap va khang nh.
5.2.Dng c, thit b, mi trng v ha cht
5.2.1..Dng c v thit b

Dng c Thit b
ng nghim T cy v trng
Que trang, que cy vng My dp mu (Stomacher)
Kp inox My trn mu (vortex mixer)
n cn Cn phn tch
Pipette 1 ml, 10 ml L viba

Bng 5.2.Mi trng v ha cht ch tiu5
Mi trng Ha cht
Dung dch Buffered Peptone Water (BPW) Cn 90
o
v 70
o

Canh Rappaport-Vassiliadis Soya Pepton (RV) Dd creatine 0,5 %
Selenit xystin / Tetrathionat (TT)
Thch Xylose Lysine Desoxycholate (XLD) KOH 40%
Mi trng Hektoen Entric Agar (HE) Thuoc th Kovac
Moi trng Trypticase Soy Agar (TSA) HCl 10%
Moi trng Lysine Decacboxylase broth
(LDC)
NaOH 10%
53
Moi trng KIA hoac TSI

5.3.Thc hnh

5.3.2.Cc bc tin hnh
Bc 1. Tng sinh
- Can 25g mau a nghien, hoc ht 25 ml mu cho vao bnh a
cha san 225ml moi trng BPW, lac eu e 37
0
C trong 24 gi.
Bc 2. Tng sinh chn lc
- Hut 1ml canh khuan BPW sang moi trng RV, nuoi trong 24
gi 42
o
C.
- Hut 1ml canh khuan BPW sang moi trng Selenit-cystin/TT,
nuoi trong 24 gi 37
o
C.

Bc 3. Phn lp v nhn din
- Dung que cay vong cay ria canh trng t moi trng
tang sinh chn lc len a petri cha moi trng phan lap. Can cay
tha e tao khuan lac rieng biet. e 37
0
C trong 24 gi. Tren cac
Phan lp khun lc n trn t nht 2 mi trng
chn lc phn bit XLD, HE, BS, SS
-1
, 10
-2
, 10
-3
vo ng
37
0
C, 48 gi
Th nghim sinh ha cho kt qu:
- KIA/TSI: /vng, c /khng H
2
S, sinh hi/khng
- Urea (-), Indol (-), VP (-)
- LDC (+),ONPG (-)

Chn cc khun lc c trng cy sang canh BHI
hoc thch TSA, qua m, 37
o
C

5

Th nghim ngng kt khng nguyn huyt thanh:
( ngng kt vi t nht mt trong hai khng huyt thanh a gi OMA,OMB)
Cy 1 ml canh khun BPW sang RV, 42
o
C, 18 -
24 g; v sang Selenit cystein/TT , 37
0
C /24g

ng nht 25 g mu trong 225 ml dung dch BPW
37
o
C, 18 24 gi
Salmonella dng tnh/m tnh trn 25g(ml) thc
phm

54
moi trng phan lap. Tren moi trng XLD, Salmonella tao cac
khuan lac o hong, co hoac khong co tam en. Tren moi trng
HE khuan lac Salmonella ien hnh co mau xanh lam, co hoac khong
co tam en

\

Bc 4. Th phn ng sinh ha
Th nghim TSI/KIA):
Mi trng KIA c s dng th
nghim kh nng s dng cc ngun
carbon khc nhau (glucose, lactose) v kh
nng sinh H
2
S.
Cay am sau phan cot thach va
cay ria len phan thach nghieng vi
khuan t nhng khuan lac ien
hnh/nghi ng len ong thach
nghieng cha moi trng KIA.
37
0
C trong 24 gi.

Dien giai s bien oi moi trng
nh sau:
Cot thach:
- Mau vang: glucose dng tnh (len men glucose)
- Mau o hoac khong oi mau: glucose am tnh (khong len
men glucose)
- Mau en: co tao thanh sunfua hidro
- Co bot kh hoac co vet nt : sinh kh t glucose
Be mat thach nghieng:
- Mau vang: lactose/saccharose dng tnh (co s dung
lactose/saccharose)
- Mau o hoac khong oi mau: lactose/saccharose am tnh

Th nghim LDC
Ly mt que cy y khun lc trn mi trng TSA cy ngay di b mt ca mi
trng lng LDC, 37
0
C trong 24 gi

Hnh 5.2. Khun lc Salmonella trn mi trng XLD (tri), HE (phi)

1. Khng c VSV
2. Ln men lactose v glucose (mi trng mu vng)
3. Sinh kh
4. Ch ln men lactose ( trn b mt, vng di )
5. Kh lu hunh, sinh H
2
S (c mu en)
Hnh 5.3. Th nghim KIA
55

Hnh 5.6.Th nghim indol

TW ID(-) ID(+)
cha cy
Hnh 3.6. Th nghi INDOL

Tren moi trng LDC, Salmonella phat trien se lam uc va chuyen
mau moi trng sang tm hay o ta[LDC (+)], LDC (-) khi moi trng
co mau vang.

Th nghim urea
Thc hin trn mi trng urea lng RSU cha ch th
phenol, chuyn mu t vng sang (vng chuyn mu l pH
6,8 - 8,4). C th s dng mi trng urea thch c bn c b
sung urea.
Ly y mt que cy vng t khun lc trn mi trng TSA
vo ng cha 3ml-5ml mi trng RSU v trng, lc nh
ng trn u vi khun v 37
o
C trong 48 gi.
Salmonella khng phn gii urea nn khng lm thay i pH
mi trng; nu phn ng dng tnh, vic phn gii ure s
gii phng NH
3
lm i mu phenol red sang mu hoa hng v
sau chuyn sang mu tm.

Th nghim VP
Ha mt vng que cy y t khun lc trn mi trng TSA vo
cc ng mi trng MR-VP 37
o
C trong 24 gi.
Sau thi gian , ly 0,2 ml dch cy vo 01 ng nghim, thm 02
git creatin, 06 git dung dch 1- naphthol trong cn, sau thm
02 git KOH 40%, vic xut hin mu hng n mu ti
trong vng 15 pht l phn ng dng tnh
Salmonella cho phn ng m tnh vi th nghim VP, khng i
mu trn b mt mi trng. Th nghim VP (+) khi c mu
trn b mt mi trng.

Th nghim indol
Cy khun lc nghi ng vo ng nghim cha 5 ml mi trng TW,
nui 37
o
C trong 24 gi.
Sau nui, thm 1 ml thuc th Kovacs. Phn ng dng tnh khi c
vng trn b mt mi trng.

Th nghim -galactosidase
Ha mt vng que cy y t khun lc nghi ng vo ng nghim c 0,25ml nc
mui 0,85%, th mt a giy ONPG vo ng nghim, t ng nghim vo ni cch
thy
37 1
o
C, yn vi pht, c kt qu. Phn ng dng tnh i mu mi trng sang
vng

VP (-) VP
(+)
Hnh 5.5. Th nghimVP

Urea (-) Urea
(+)
Hnh5.4. Th nghim
Urea
56
Th nghim khng huyt thanh
+ Kim tra s t dnh kt ca cc chng;
Cho mt git dung dch nc mui sinh l ln mt phin knh ra sch. Dn u
khun lc cn th nghim trong git dung dch sao cho thu c mt th huyn ph
c ng nht. Lc nh phin knh trong 30-60 giy.
Quan st kt qu trn nn ti, tt nht nn dng knh lp.
Nu cc vi khun vn li thnh cc n v t nhiu c kt dnh th chng ny c
coi l t kt dnh v khng cn phi th tip v vic pht hin khng nguyn khng
th thc hin c.
+ Kim tra khng nguyn O:
S dng mt khun lc thun khit khng t ngng kt, tin hnh nh phng
php kim tra s t kt dnh trn, bng cch thay mt git khng huyt thanh O
thay cho dung dch nc mui.
Nu xut hin dnh kt, phn ng c coi l dng tnh. S dng tun t mt
khng huyt thanh a gi v n gi.
+ Kim tra khng nguyn Vi:
Tin hnh nh phng php kim tra s t kt dnh bng cch thay mt git huyt
thanh Vi thay cho dung dch nc mui.
Nu xut hin dnh kt, phn ng coi l dng tnh.
+ Kim tra khng nguyn H:
Cy t 1 khun lc khng t dnh kt thun khit ln thch dinh dng th na c
(phn dnh km), m 18-24 gi, 371
o
C.
Dng ging ny kim tra khng nguyn H, tin hnh nh phng php kim tra
s t dnh kt, nhng s dng mt git khng huyt thanh H thay cho dung dch
nc mui sinh l.
Nu xut hin dnh kt, phn ng coi l dng tnh.
LU
C th s dng khng huyt thanh a gi OMA, OMB pht hin s c mt ca
cc khng nguyn Salmonella. Trn 98% cc chng Salmonella cho phn ng ngng
kt vi mt trong hai khng huyt thanh OMA, OMB. Do vy khi thy c s dnh kt
vi mt trong hai khng huyt thanh a gi trn c th kt lun l Salmonella.
5.3.4.Kt qu
Vi quy trnh kim nghim ny cho php kt lun c hay khng c Salmonella c
pht hin trong 25g mu

57
5.4.Cu hi n tp
1. Nu cc bc trong quy trnh phn tch Salmonella
2. Vai tr ca mi trng tng sinh BPW ?
4. Mi trng tng sinh chn lc dng trong quy trnh phn tch Salmonella l g?
5. Vai tr ca mi trng tng sinh chn lc?
6. Gii thch ti sao khun lc Salmonella c th c tm en?
7. Trnh by cc phn ng sinh ha dng trong phn tch Salmonella: tn mi trng,
ha cht, kt qu, gii thch kt qu.

6. PHN TCH STAPHYLOCOCCUS AUREUS
( PHNG PHP M KHUN LC)

6.1.Nguyn tc
Staphylococcus aureus l cu khun Gram dng, khng
sinh bo t, khng di ng, thng t thnh chm c kh
nng ln men v sinh acid t succrose, mannitol v sinh sc
t vng, chu mn (7,5% NaCl). v c kh nng ng t
huyt tng (phn ng coagulase (+)).
Staphylococcus aueus c xc nh trn c s cc c
im tng trng trn mi trng Baird Parker Agar bng
k thut hp tri v phn ng lm ng huyt tng. Da
vo s khun lc c trng cho phn ng Coagulase (+) suy
ra mt vi khun ny trong thc phm.
6.2.1. Dng c v thit b
Dng c Thit b
Phien knh, la kinh T cy v trng
Que trang My dp mu (Stomacher)
n cn Cn phn tch
ng nghim

6.2.2. Mi trng v ha cht

Hnh 6.1. Sta. aureus trn BPA

58
Mi trng Ha cht
Dung dch Saline Peptone Water
(SPW)
Cn 90
o
v 70
o

Moi trng Baird-Parker HCl 10%
Moi trng Trypticase Soy Agar
(TSA)
NaOH 10%
Brain heart broth (BHI) KH
2
PO
4

Huyt tng th

6.3.Thc hnh

6.3.2.Cc bc tin hnh
Bc 1. Pha long mu theo nng pha long thp phn: Cn/ht 10g/10ml mu
thc phm c chun b vo chai/bnh tam gic c cha sn 90ml dung
dch m, lc u mu 2-3 pht c nng pha long 10
-1

Bc 2. Chuyn 1 ml dung dch mu th 10
-1
sang ng nghim cha sn 9 ml dung
dch m, lc u mu 2-3 pht c nng pha long 10
-2
. Tip tc lm
tng t, thu c mu th tng ng 10
-3
, 10
-4
....
Bc 3. Chuyn 0,1 ml dung dch mu th cc nng pha long thu c vo
chnh gia a mi trng BPA c chun b trc (b mt cc a mi
Mu th pha long (10
-1
, 10
-2
, 10
-3
...)

-1
, 10
-2
, 10
-3
vo ng
37
0
C, 48 gi
Canh BHI/thch TSA
(37,0 1,0
o
C, 24 gi)

Baird-Parker (37,0 1,0
o
C; 24 48 gi)
Chn 5 khun lc c trng, 5 khun lc khng c trng

5

Phn ng coagulase (37,0 1,0
0
C)
(0,1ml dch BHI; 0,3 ml huyt tng th)

ng nht mu
30 giy
nh lng 10 g hoc 10 ml mu thc phm
trong dung dch SPW

59
trng ny khng c t), dng que trang tri u mu trn mt thch. Cy
vo 2 a mi nng .
Lu : Nu s lng Staphylococcus aureus dng tnh thp, c th nng nng
dch mu ln 10 ln, ngha l cy 1ml dch mu s khi (mu nguyn nu l
cht lng, hoc mu 10
-1
nu l cht rn) vo 3 a. Kt qu 3 a ny c
x l gp nh mt a khi c, khng nh v bo co kt qu
Bc 4. Lt ngc cc a v 37,0 1,0
0
C trong 24 3 v 48 4 gi
Bc 5. Sau 24 3 gi, trn mi trng thch Bair parker khun lc Staphylococcus
aureus dng tnh c ng knh 1-1,5mm, mu en sng, li. Mi khun lc
c qung sng rng 1-2mm bao quanh. Nhng khun lc in hnh c nh
du trn mt sau ca a v tip tc thm 24 gi na. Sau 48 4 gi, khun
lc Staphylococcus aureus dng tnh c ng knh 1,5-2,0mm, mu en,
sng v li, quanh khun lc c mt vng c m hp, tip l vng sng,
trong rng 2-4 mm. Ngoi ra cn c cc khun lc khng to vng sng bao
quanh, khng to vng c st khun lc, l cc khun lc khng in
hnh. Cc khun lc khng in hnh ny cng c nh du mt sau ca
a.
Bc 6. Cy chuyn 5 khun lc in hnh v 5 khun lc khng in hnh t Baird
Parker Agar sang ng nghim cha 5 ml mi trng BHI. 37,0 1,0
o
C
trong 24 3 gi.
Bc 7. Chuyn 0,1ml dch nui cy trong mi trng BHI
vo 0,3 ml huyt tng th (hoc theo hng dn ca
nh sn xut) trong cc ng nghim nh tit trng
c kch thc 10mm x 75mm, t m 37,0 1,0
o
C.
Ch lm mu blank( mu chng). Kim tra s ng
vn ca huyt tng sau 4, 6, 8, 24gi. Phn ng lm
ng t huyt tng c coi l dng tnh khi th
tch ng vn chim 3/4 ton b th tch chn la
6.3.4.Ket qua
Mat o Staphylococcus aureus trong 1g mau (CFU/g) =
2 1
) ( 10
F F
H N H N
a a t t
+
+

Trong :
- F
1
v F
2
: 2 pha long lin tip
- N
t
: tng s khun lc c trng trn mt a cc pha long F
1
, F
2

- N
a
: tng s khun lc khng c trng trn mt a cc pha long F
1
, F
2

- H
t
: t s gia khun lc c trng cho th nghim khng nh (+) so vi s
khun lc c trng
- H
a
: t s gia khun lc khng c trng cho th nghim khng nh (+) so
vi s khun lc khng c trng

6.4. Cu hi n tp
1. Ti sao phi b sung Egg Yolk Tellurite emulsion vo mi trng BPA c
bn?
2. Mi trng BHI c vai tr g trong quy trnh phn tch Staphylococcua aureus?

Hnh 6.2. ng t huyt tng th
60
3. Hy nu hnh thi khun lc c trng ca Staphylococcua aureus trn BPA.
4. Gii thch ti sao Staphylococcua aureus c kh nng ng t huyt tng th

7. KIEM TRA VI KHUAN BACILLUS CEREUS
7.1. Nguyen tac:
B.cereus la nhng trc khuan, Gram dng, hieu kh, k kh tuy y,
di ong, tao noi bao t, len men glucose sinh hi, phan ng VP(+).
Co kha nang s dung nitrat. B.cereus co the tiet ra hai loai oc to
chnh la diarrhoeal toxin gay tieu chay va emetic toxin gay non ma.
Loai nay tang trng c trong khoang nhiet o t 5-50
0
C,
toi u 35-40
0
C, pH dao ong t 4,5-9,3, de tao bao t va bao t
nay mam rat de dang. Tren moi trng chon loc loai nay tao
khuan lac rat to, moc lan, ra nhan. Vi khuan nay hien dien trong
at, bui, cac loai thc pham (sa, tht, rau qua, hon hp gia v, san
pham kho...).
B.cereus c phat hien va nh lng bang moi trng thach
chon loc Manitol-Egg Yolk-Polymycin (MYP) hoac Cereus Selective Agar
(Mossel). Khuan lac B.cereus co hnh thai ac trng tren cac moi
trng nay. Cac khuan lac nay c tiep tuc khang nh da tren
cac th nghiem sinh hoa va cac ac iem nh len men glucose
sinh acid trong ieu kien k kh, kh nitrat thanh nitrit, th nghiem
VP(+), thuy phan L-tyrosine, tang trng c trong 0,001% lysozym
Dng c Thit b
Micropipet My dp mu (Stomacher)
Que trang, que cy vng My trn mu (vortex mixer)
Kp inox Cn k thut
n cn L viba
Pipette 1 ml, 10 ml

61
Mi trng Ha cht
Nc m Salin Pepton Water (SPW) / Buffered
Petone Water (BPW)
Cn 90
o
v 70
o

Dd creatine 0,5 %
Manitol-Egg Yolk-Polymycin (MYP) /Cereus
Selective Agar (Mossel)
Nh lng trng (EGG YOLK Emulsion),50% KOH 40%
Canh Phenol Red Glucose
Thch Tyrosine HCl 20%
Canh Lysozyme NaOH 20%
Mi trng MRVP
Griess Ilowvays reagent
MANNITOL MOBILITY NITRATE
Bi km
Thch dinh dng Nutrient Agar/ Tryptic Soya
Agar (TSA)
Ha cht nhum Gram
Nutrient broth
polymyxin B 0,1%

Tyrosine 5%, lysozyme 0,1%

7.3.Thc hnh

7.3.2. Cc bc thc hin
Bc 1.Pha long mu
10 g mu + 90 ml dung dch m 10
-1

Tip tc pha long mu n 10
-2
10
-3
10
-4

......

Cy tri 0,1ml mu ln mi trng MYP, 30
0
C, 24-48 gi
Cy 05 khun lc nghi ng ln TSA, 30
0
C qua m
Th sinh ha cc khun lc nghi ng:
Th kh nng chuyn ha nitrat thnh nitrit (+),th VP(+), th
ln men glucose(+), th nghim tyrosin(+), th nghim
Lysozyme(+)

62
Mau a ve phong th nghiem, m ra cho vao khay vo trung.
Dung keo va kep vo trung lay ai dien 10g mau cho vao bao PE
vo trung trong ieu kien vo trung. Mau c ong nhat vi 90ml
dung dch em phosphat bang may dap mau e co dung dch pha
loang 10
-1
.
Dung pipet vo trung hut 1ml dung dch pha loang 10
-1
sang ong
nghiem cha 9ml dung dch em phosphat e c dung dch pha
loang 10
-2
. Tuy theo mc o nhiem ban cua mau ma tien hanh
pha loang cac o pha loang cao hn.
Bc 2. Cay mau len moi trng
MYP

Chon 2 nong o pha loang thch hp,
hut 0,1ml dch pha loang vao moi a
MYP (moi nong o 2 a). Dung que gat
trai can than chat nuoi cang nhanh
cang tot. U 30
o
C, 24 48 gi.

Bc 3. Khang nh Bacillus cereus bang cac phan ng sinh
hoa

Chon va em cac a co t 15 150 khuan lac ac trng cua
Bacillus aureus (det, ng knh 2 3mm, b hnh rang ca, mau o
hong, xung quanh co vong uc). Chuyen 5 khuan lac nghi ng nay
len moi trng phuc hoi TSA , u 30
o
C qua em trc khi tien
hanh cac th nghiem sinh hoa.
- Th nghiem kha nang len men glucose: cay vi khuan t moi
trng TSA sang moi trng canh glucose, u 35
o
C trong 24 gi. Bacillus
cereus cho phan ng dng tnh lam oi mau moi trng t o
sang vang.
- Th nghiem kha nang chuyen hoa nitrat thanh nitrit: cay vi
khuan t moi trng TSA sang moi trng nitrat, u 35
o
C trong 24
gi. Bacillus cereus co phan ng dng tnh nen sau khi nho thuoc
th vao canh khuan chuyen sang mau o cam trong 10 phut.
- Th nghiem Voges proskauer: cay vi khuan t moi trng
TSA sang moi trng canh MR - VP, u 35
o
C trong 48 gi. Bacillus
cereus cho phan ng dng tnh: moi trng chuyen sang mau hong
khi cho thuoc th o-Naphthol vao (trc khi nho o-Naphthol vao phai
lam kiem hoa moi trng nuoi cay bang KOH 40%).
- Th nghiem Tyrosin: cay vi khuan t moi trng TSA sang
ong thach nghieng Tyrosin, u 35
o
C trong 48 gi. Khuan lac Bacillus
phan huy tyrosin tao khoang trong xung quanh khuan lac.

Hnh 7.1. Bacillus aureus trn
MYP
63
- Th nghiem Lysozyme: cay vi khuan t moi trng TSA sang
ong nghiem cha moi trng Nutrient Broth vi 0,001% lysozyme, u
35
o
C trong 24 gi. Bacillus phat trien lam moi trng uc eu.
7.3.3. c ket qua:
So Bacillus cereus tren 1g mau =
f V n
R N

N: tong so khuan lac em c
n: so lng a em
f: o pha loang tng ng
V: the tch dch mau cay vao trong moi a
R: t le xac nhan = t le gia so khuan lac nghi ng cho th
nghiem dng tnh so vi so khuan lac nghi ng.

8. KIEM TRA VI KHUAN CLOSTRIDIUM PERFRINGENS
8.1.Nguyen tac:
Giong Clostridium la cac vi khuan Gram dng, hnh que, k kh,
sinh bao t, phan ln di ong. Clostridium perfringens la loai k kh
khong bat buoc, tao cac khuan lac mau en trong moi trng
phan lap quy nh
64
Clostridium perfringens l vi khun ch s nhim do phn ngi, ng vt, bn t,
nc cng rnh. Vi khun c bo t v c sc khng cao. Nu thc phm ch bin
khng m bo v sinh, nhit kh khun, un nu khng dit bo t, vi khun
vn cn tn ti v pht trin, lm h hng thc phm v gy ng c thc n ( nht l
thc phm hp)
Do tnh cht ca Clostridium perfringens l pht trin trong mi trng k kh v c
kh nng phn gii Sodium sulfit hoc phn gii protein to H
2
S.
Cy trn mi trng c Sodium sulfit v phn st ( mi trng thch Tryptose
Sulfide Cycloserin:TSC), to khun lc mu en, th tnh cht sinh ha nh danh


Dng c Thit b
Kp inox Cn k thut
n cn L viba
Pipette 1 ml, 10 ml
Bnh k kh

Mi trng Ha cht
TRIPTOSE SULPHIT CYCLOSERINE AGAR
BASE (TSC)
Cn 90
o
v 70
o

MANNITOL MOBILITY NITRATE Dd creatine 0,5 %
THIOGLYCOLATE Dung dch o- naphtol 5%
GELATINE-LACTOSE KOH 40%
IRON SULPHITE AGAR (ISA) Thuoc th Kovac
HCl 10%
NaOH 10%

C.selective supplement
Anaerobic indicator

Ti k kh
Griess Ilowvays reagent
65
Bi km

8.3.Thc hnh
8.3.1. Quy trnh phn tch

8.3.2. Cc bc thc hin

Bc 1: Chun b dch ng nht hoc pha long mu c cc pha long 10
-1
,
10
-2
, 10
-3

Bc 2:Dng pipet v trng ly mi nng pha long 1 ml dung dch huyn
ph ban u hoc 1 ml mu th ( nu mu l cht lng) cho vo gia a Petri, mi
nng 02 a.
Bc 3: Rt 15-20 ml mi trng TSC vo a Petri v trn u bng cch xoay nh.
Ly 01 ml mu vo a, rt khong 20ml mi trng TSC,
xoay u, i ng, ph thm ln b mt a 10ml mi trng
TSC, 37
0
C 1
0
C, 20 gi trong bnh k kh

Chn 10 khun lc in hnh t cc a m. Cy ria cc
khun lc ny ln mi trng thch TSC c bn, ph thm 10
ml mi trng TSC c bn, 37
0
C 1
0
C, 20 gi trong bnh k
kh
Th sinh ha cc khun lc in hnh:
Th nghim tnh di ng, th nghim s c mt ca nitrit, th
nghim s ha lng geletin, ln men lactose

10 g mu + 90 ml dung dch m 10
-1

Tip tc pha long mu n 10
-2
10
-3
10
-4

......

Vi sinh vt sinh ra cc khun lc en mi trng TSC, khng
di ng, kh nitrat thnh nitrit, sinh acide v kh t lactose, ha
lng geletin trong 48 gi c coi l Clostridium perfringens
66
Bc 4: Khi mi trng ng cht, rt thm mt lp 10 ml mi trng TSC ph
ln trn.
Bc 5: ng v xp cc hp Petri np pha trn vo bnh k kh, Bc 6: m
v ghi li s lng khun lc in hnh cc a. Khun lc Clostridium perfringens
c mu en
Xc nhn
Chn tng s 10 khun lc in hnh t cc a m. Nu khng c kh nng tch
ring r tt cc khun lc in hnh th nui 10 khun lc in hnh trong mi
trng lng thioglycolate, nui dng trong iu kin ym kh 37
0
C 1
0
C, 18-24
gi.
Cy ria cc khun lc ny trn mi trng thch c bn TSC, ph thm mt lp 10 ml
mi trng TSC c bn. ng v nui ym kh 37
0
C 1
0
C, 18-24 gi.
Tch mi a t nht mt khun lc in hnh v d tch bit. Xc nhn tnh sinh ha
Th nghim tnh di ng : Lm mi trng thch ng Mannitol Mobility
Nitrate. Cy su khun lc phn lp vo mi trng . Nui dng trong iu kin
ym kh 37
0
C 1
0
C, 18-24 gi.
Kim tra ng mi trng xem dng sinh trng dc theo ng cy su. S
sinh trng khuych tn vo mi trng t vt cy su th hin r tnh di ng
ca vi sinh vt nui cy
Th nghim s c mt ca nitrit: Thm 0,2-0,5 ml thuc th pht hin nitrit
vo mi ng mi trng nitrat di ng (tin hnh trong t HOP). S hnh thnh
mu xc nhn phn ng kh nitat thnh nitrit. Nu trong 15 pht m mu
khng hnh thnh th thm mt lng nh bt km. yn 10 pht, nu mu
c hnh thnh sau khi thm bt km th khng c phn ng nitrat thnh nitrit.
Xc nh s ha lng geletin:
Cy cc khun lc phn lp vo ng nghim cha mi trng gelatin-lactose.
Nui dng trong iu kin ym kh 37
0
C 1
0
C, 18-24 gi. Kim tra ng
mi trng xem c s sinh kh v c chuyn mu vng khng ( to acide) cho
bit s ln men ca lactose. Lm lnh 1 gi 5
0
C v kim tra s ha lng ca
geletine. Nu mi trng vn rn, nui cy thm 24 gi v kim tra s ha lng
ca geletine.

8.3.3. c kt qu
Vi sinh vt sinh ra cc khun lc en mi trng TSC, khng di ng, kh nitrat
thnh nitrit, sinh acide v kh t lactose, ha lng geletin trong 48 gi c coi l
Clostridium perfringens. Nu nhiu hn hoc bng 80% s khun lc in hnh m
c c xc nh l Clostridium perfringens th ton b s khun lc m c
c coi l Clostridium perfringens
Cc trng hp khc kt qu c tnh ton da vo t l phn trm ca cc khun lc
c xc nhn l Clostridium perfringens so vi s khun lc m c
67

Tham kho quy trnh phn tch Clostridium( khng dng bnh k
kh)

9. NH TNH VI BRI O CHOLERAE TRONG THC PHM

9.1. Nguyn tc
Vibrio cholerae thuc ging Vibrio, h Vibrionaceae l phy khun gram m,
k kh tu nghi, c kh nng di ng, phn ng oxidaza dng tnh, pht trin c
nhit 42
0
C, ln men ng sacaroza, kh lysin; pht trin c mi trng khng
c mui nhng khng pht trin c mi trng c nng mui ln hn hoc
bng 6 %; nhy vi hp cht c ch phy khun O/129 Vibriostat.
ong nhat mau va pha loang mau theo day
thap phan, x ly mau 80
0
C trong 15-20 phut,
Cay 1ml mau vao trong ong nghiem vo
trung, o 10-15 ml moi trng ISA 45
0
C ,
lac eu
o lp ISA th hai cao 1-2 cm sau khi phan
di ong ac, u 37
0
C, 24-48 gi
em tat ca cac khuan lac mau en xuat
hien trong ong
Tnh ket qua so Clostridium trong 1ml/1g mau
phan tch
68
Pht hin Vibrio cholerae bng cch tng sinh trong mi trng lng chn lc
v cy chuyn phn lp trn mi trng rn chn lc. Nhng khun lc nghi ng c
khng nh bng th nghim sinh ho v khng huyt thanh.
9.2.1. Dng c v thit b
Bng 9.1. Dng c v thit b ch tiu9
Dng c Thit b
Kp inox Cn k thut
n cn L viba
Pipette 1 ml, 10 ml My nghin ng th mu
B iu nhit
My lc ng nghim

9.2.2. Mi trng v ha cht
Mi trng Ha cht
Ancalin Peptone Water (APW)
Cn 90
o
v 70
o

Thch Thiosunfat-Citrat-Bile-Salt-Sacaroza
(TCBS thch).Ch thch: khng hp kh trng mi trng
HCl 10%
Triple Sugar Iron agar TSI/ Klingler Iron Agar -
NaOH 10%
69
KIA

Hugh - Leifson Glucoza hoc O/F Glucoza

Trypton
NaCl
Canh thang mui trypton ( trypton 1%) c di
nng NaCl l: 0; 1; 3; 6; 8 v 10 % NaCl (k
hiu ln lt l: T
1
N
0
; T
1
N
1
; T
1
N
3
; T
1
N
6
; T
1
N
8
v
T
1
N
10
).Ch thch: cc ng mi trng phi c nt cht
trong qu trnh bo qun trnh bc hi nc lm thay i
nng mui

Que th Oxidase
RSU Cc a 10 mg v 150 mg O/129
Vibriostat
Mi trng th decarboxylaza (Mi trng c
bn) (LDC)

Du khong v trng
(Rt khong 20 - 50 ml du khong vo bnh c
np xoy. Hp du nhit 121
0
C trong 30
pht)

Moi trng Tryptic Soya Agar (TSA) 2%
NaCl
a th ONPG
Canh thang bromcresol ( Mi trng c bn)
(symolcitrat)

Dung dch nc mui sinh l

9.3. Thc hnh

25 g mu + 225 ml nc APW, nghin, dp mu 37,0
0
C
1,0
0
C trong 6 - 24 gi
70

9.3.2.Cc bc thc hin
Bc 1. Chun b mu thc phm
Cn v trng 25 g mu thu sn vo mt ti PE v trng. Ct cc mu ln
thnh cc mnh nh ri thm 225 ml nc pepton kim APW vo ti, nghin bng
my nghin ng th trong 2 pht.
Mu kim tra thng v d: mu hu
Ly phn tht ca 10 - 12 con hu (k c nc) ri ct nh, trn u. Sau ,
nghin bng my nghin ng th 50 g mu hu trong 450 ml nc pepton kim APW
(4.2.1) trong 2 pht. Chia dung dch mu ny vo 2 bnh v trng, mi bnh cha 225
ml ri pha long thnh hai dy bng cch trn u 10 ml dung dch mu trong 90 ml
Cy ria vo mi trng TCBS thch . 37,0
0
C 1,0
0
C trong
18 - 24 gi
Ria cy 03 khun lc nghi ng sang mi trng TSA 2% NaCl,
37,0
0
C 1,0
o
C trong 18 - 24 gi
Th nghim sinh ha:
Th nghim sinh ha s b:TSA/KIA, th nghim canh thang
trypton mui(+), th nghim ln men, oxy ha(+), th nghim
oxidaza(+), nhum Gram(-)
Th nghim sinh ha khng nh: th pht trin 42,0
0
C(+), th
nghim cacbonhydrat, th nghim decacboxylaza, th nghim
urea(-), th nghim ONPG(+), th nghim tnh nhy cm vi hp
cht c phy khun O/129 Vibriostat(-)
Th nghim khng huyt thanh:

Th nghim khng huyt thanh

Pht hin hoc khng pht hin c V. cholerae trong 25 g mu.

71
nc pepton kim APW. mu hai ch nhit l 37,0
0
C 1,0
0
C v 42,0
0
C
0,2
0
C .
Bc 2. Tng sinh
a. i vi mu thng thng
cc bnh cha mu chun b (k c mu chng dng v mu chng m)
nhit 37,0
0
C 1,0
0
C trong 6 - 8 gi. Sau , phn lp nh qui nh , phn cn li
tip 16 - 24 gi ri phn lp li.
b. i vi mu hu
1 dy pha long 10
-1
n 10
-2
nhit 37,0
0
C 1,0
0
C trong 6 - 8 gi v 16 -
24 gi.
1 dy pha long 10
-1
n 10
-2
nhit 42,0
0
C 0,2
0
C trong 6 - 8 gi.
Bc 3. Phn lp v c kt qu trn a
Sau khi nui cc ch nhit v cc khong
thi gian trn , khng lc, dng khuyn cy ly dch
mu trn b mt cy ria vo mi trng TCBS thch .
cc a TCBS thch nhit 37,0
0
C 1,0
0
C trong
18 - 24 gi.
Trn mi trng TCBS thch, khun lc V.
cholerae trn, hi dt, bng, mu vng (do vi khun
ln men sacaroza), gia m v c vin m xung
quanh.

Bc 4. Th nghim sinh ho v khng huyt thanh
a. Th nghim sinh ho s b
Chn t nht 3 khun lc nghi ng trn TCBS thch cy chuyn sang mi
trng khng chn lc TSA 2% NaCl . nhit 37,0
0
C 1,0
o
C trong 18 - 24 gi.
Khun lc mc trn mi trng ny c s dng thc hin cc th nghim sinh
ho.
* Th nghim trn mi trng TSI/KIA
Cy chuyn cc khun lc t mi trng TSA vo cc ng thch nghing TSI (
hoc KIA . Ni lng cc np ng to iu kin hiu kh. Nui nhit 37,0
0
C
1,0
0
C trong 18 - 24 gi.
Trn mi trng TSI, V.cholerae lm phn thch nghing v thch ng ca
mi trng chuyn mu vng, khng sinh hi v khng sinh H
2
S. Trn mi trng
KIA, phn thch nghing c mu v thch ng mu vng, khng sinh hi v
khng sinh H
2
S.
* Th nghim vi cc canh thang trypton mui
Cy khun lc t mi trng TSA vo cc ng canh thang mui trypton c di
nng NaCl l: 0; 1; 3; 6; 8 v 10 % . Nui nhit 37,0 1,0
0
C trong 18 - 24
gi, ri li cc ng m tnh sau 18 - 24 gi na. V. cholerae pht trin c trong
ng canh thang c nng NaCl l 0%, 1%, 3% v lm c mi trng.

Hnh 9.1.V. cholerae trn TCBS

Hnh nh V. cholerae trn mi trng TCBS
72
* Th nghim ln men - oxy ho
Cy khun lc t mi trng TSA vo 2 ng mi trng bn lng Hugh-Leifson
glucoza hoc O/F glucoza. Ph khong 1 - 2 ml du khong v trng vo mt trong 2
ng. cc ng nghim nhit 37,0 1,0
0
C trong 1 - 2 ngy. V. cholerae ln men
glucoza lm mi trng Hugh - Leifson t mu tm chuyn thnh vng v mi trng
O/F t mu xanh thnh vng.
* Th nghim oxidaza
t giy lc ln mt np a petri, nh khong 2 - 3 git thuc th oxidaza /que
th oxidaza. Dng que cy bch kim (platin) hoc que cy nha dng mt ln; hoc
que g v trng ly cc khun lc t mi trng TSA ria ln v tr nh thuc th
oxidaza. V. cholerae lm vt ria c mu tm thm hoc xanh thm sau 10 giy .
Ch thch: khng dng que cy bng crm hoc bng st trong th nghim ny.
* Nhum gram
V. cholerae l vi khun gram m, hnh cong dng du phy.
b. Th nghim sinh ho khng nh
* Th nghim pht trin nhit 42
0
C
Cy vi khun t TSA vo mt ng canh thang trypton 1% NaCl . nhit
42,0
0
C 0,2
0
C trong 18 - 24 gi. V. cholerae pht trin c nhit 42
0
C lm mi
trng b c.
* Th nghim cacbonhydrat
Cy vi khun t TSA vo cc ng canh thang bromcresol c cha mt trong cc
loi cacbohydrat: sacaroza, lactoza, D-cellobioza, arabinoza, D-manniton, D-mannoza
. cc ng canh thang nhit 37,0
0
C 1,0
0
C. Phn ng dng tnh khi mi
trng c mu vng, m tnh khi mi trng gi nguyn mu .
V. cholerae cho phn ng sacaroza, manniton, mannoza dng tnh v lactoza,
cellobioza, arabinoza m tnh.
* Th nghim decacboxylaza/dehydrolaza
Cy vi khun t mi trng TSA vo cc ng canh thang ornithin, lysin, arginin v
ng i chng khng c axit amin. Mi ng c ph 1 - 2 ml du khong v trng .
cc ng nhit 37,0
0
C 1,0
0
C trong 18 - 24 gi. Phn ng dng tnh khi mi
trng gi nguyn mu tm hoc hi m hn. Phn ng m tnh khi ton b ng canh
thang c mu vng n nh trong 4 ngy (tng t mu ca ng i chng).
V. cholerae cho phn ng lysin v ornithin dng tnh, phn ng arginin m
tnh.
* Th nghim ur
Cy vi khun t TSA vo canh thang ur , nhit 37,0
0
C 1,0
0
C trong 18
- 24 gi. V. cholerae cho phn ng m tnh nu mi trng gi nguyn mu tm hng.
* Th nghim ONPG
C th dng a ONPG thay cho thuc th ONPG. Ho tan khun lc nghi ng
vo ng nghim cha 0,2 ml nc mui sinh l. t a ONPG vo y ng ri m
v c kt qu nh trn. V. cholerae cho phn ng ONPG dng tnh khi dung dch c
mu vng.
73

* Th nghim tnh nhy cm vi hp cht c ch phy khun O/129 Vibriostat
Dn vi khun ln a thch TSA 2% NaCl . t cc a O/129 c cha 10 mg
hoc 150 mg Vibriostat vo cc v tr khc nhau. Sau , lt ngc a ri nhit
37,0 1,0
0
C trong 18 - 24 gi. V. cholerae khng pht trin c do b c ch bi
Vibriostat to mt vng v khun xung quanh cc a O/129.
* Th nghim khng huyt thanh
Nh 1 git khng huyt thanh ln lam knh sch v mt git nc mui sinh l
ln mt v tr khc ca lam. Dng que cy v trng khc nhau chuyn vi khun t TSA
ln hai git dung dch trn ri phn tn u vi khun. Kt lun dng tnh khi c hin
tng ngng kt git c khng huyt thanh v khng c hin tng ngng kt git
nc mui. S dng chng chng dng v chng chng m trong qu trnh thc
hin.
Tm tt th nghim khng huyt thanh V. cholerae theo Bng 1 v Bng 2.

Bng - Phn bit V. cholerae O1 v V. cholerae non - O1
Khng nguyn Khng huyt
thanh a gi O1
Dung dch mui
sinh l
Kt lun
Chng c kt qu sinh ho
tng t V. cholerae
+ - V.cholerae O1
tng t V. cholerae
- - V.cholerae non- O1
tng t V. cholerae
+ + Chng khng c hiu
vi khng nguyn O
nhm 1
Bng - Phn bit cc kiu huyt thanh chng V.cholerae O1
Khng nguyn Khng huyt
thanh Inaba
Khng huyt
thanh Ogawa
Dung dch
mui sinh l
Kt lun
V. cholerae O1 + - - Chng Inaba
V. cholerae O1 - + - Chng Ogawa
V. cholerae O1 + + - Chng Hikojima
V.cholerae O1 - - - Chng khng c hiu
9.3.3. c kt qu
Pht hin hoc khng pht hin c V. cholerae trong 25 g mu.

74

10. NH TNH SHI GELLA TRONG THC PHM
10.1.Nguyn tc
Shigella l trc khun Gram m, hiu kh v k kh ty tin, cho th nghim
catalase (+) (tr Shigella dysenteriae), oxidase (-), ln men glucose khng sinh hi,
hu ht cc loi khng ln men v to acid t latose, dulcitol, khng sinh H
2
S,
khng c enzyme lysine decarboxylase.
Shigella c th c pht hin bng cch cy mt lng mu xc nh vo mi
trng lng khng chn lc, sau c chuyn vo mi trng tng sinh chn lc.
Dch khun sau khi tng sinh chn lc c cy phn lp trn t nht 2 loi mi
trng thch a vi mc chn lc khc nhau. Khun lc nghi ng c kim tra
bng th nghim sinh ha v khng huyt thanh.
75
Dng c Thit b
n cn Cn phn tch

Bng 10.2.Mi trng v ha cht ch tiu 10
Mi trng Ha cht
Dung dch Trypton Soya (TSB) Cn 90
o
v 70
o

Canh Gram Negative (GN) Dd creatine 0,5 %
Tergitol-7 Agar (T7A)
Thch Xylose Lysine Desoxycholate (XLD) KOH 40%
Mi trng Hektoen Entric Agar (HE)
Deoxycholate Citrat Agar (DCA) HCl 10%
Thch Mac Conkey (MAC) NaOH 10%
Moi trng Trypticase Soy Agar (TSA) Thuc th catalase
Moi trng Lysine Decacboxylase broth
(LDC)
Que th oxydase
Moi trng KIA hoac TSI
RSU

10.3.Thc hnh


Cy 0,1 ml dch tng sinh sang 10 ml mi trng GN,
37
0
C , 16 24 gi
ng nht 25 g mu trong 225 ml TSB, 37
0
C ,
16 24 gi
76

10.3.2.Cc bc tin hnh
Bc 1. Tng sinh
- Can 25g mau a nghien, hoc ht 25 ml mu cho vao bnh a
cha san 225ml moi trng TSB, lac eu , 37
0
C , 16 24 gi
Bc 2. Tng sinh chn lc
- Chuyn 0,1 ml dch tng sinh sang 10 ml canh tng sinh GN, 37
o
C
trong 10-20 gi.

Bc 3. Phn lp
- Dung que cay vong cay ria canh trng t moi trng
tang sinh chn lc len a petri cha moi trng phan lap. Can cay
tha e tao khuan lac rieng biet. e 37
0
C trong 24-48 gi. Nn s
dng t nht 2 mi trng phn lp khc nhau. Tren moi trng XLD, Shigella
tao cac khuan lac o,trong sut. Tren moi trng HE khuan lac
Shigella ien hnh co mau xanh nht, trong sut. Tren moi trng T7A
khuan lac Shigella ien hnh co mau xanh nht (mi trng c nh sa, c
Chn t nht 05 khun lc nghi ng cy ln TSA ,
37
0
C trong20-24gi

Phn lp khun lc n trn t nht 2 mi trng chn
lc phn bit (T7A,MAC,XLD,HE,DC), 37
0
C , 24
48gi
Th nghim sinh ha:
KIA/TSI : /vng, H
2
S (-), gas (-)
Khng di ng
Oxydase (-)
Th nghim sinh ha khng nh
Kt lun: Shigella dng tnh/m tnh trong 25 g mu
77
mu xanh hi vng nht). Tren moi trng MAC khuan lac Shigella ien
hnh co mau nht (mi trng c mu cam, hi c)

Th nghim sng lc trn TSI/KIA):
Mi trng KIA c s dng th
nghim kh nng s dng cc ngun
carbon khc nhau (glucose, lactose) v kh
nng sinh H
2
S.
Cay am sau phan cot thach va
cay ria len phan thach nghieng vi
khuan t nhng khuan lac ien
hnh/nghi ng len ong thach
nghieng cha moi trng KIA.
37
0
C trong 24 gi.

Dien giai s bien oi moi trng
nh sau:
Cot thach:
- Mau vang: glucose dng tnh (len men glucose)
- Mau o hoac khong oi mau: glucose am tnh (khong len
men glucose)
- Mau en: co tao thanh sunfua hidro
- Co bot kh hoac co vet nt : sinh kh t glucose
Be mat thach nghieng:
- Mau vang: lactose/saccharose dng tnh (co s dung
lactose/saccharose)
- Mau o hoac khong oi mau: lactose/saccharose am tnh

Th nghim khng nh :
Cc c trng sinh ha ca Shigella c tm tt theo bng :

Th nghim sinh ha Kt qu Th nghim sinh ha Kt qu
Simmon citrate - Saciline -
Arginine decarboxylase - Xylose -
Lysine decarboxylase - Cellobiose -

1. Khng c VSV
2. Ln men lactose v glucose (mi trng mu vng)
3. Sinh kh
4. Ch ln men lactose ( trn b mt, vng di )
5. Kh lu hunh, sinh H
2
S (c mu en)
Hnh 5.3. Th nghim KIA
78
Urea - Adonitol -
Malonate - Dulcitol -
MR + Inositol -
VP -

Th nghim LDC
Ly mt que cy y khun lc trn mi trng TSA cy ngay di b mt ca mi
trng lng LDC, 37
0
C trong 24 gi
Tren moi trng LDC, Shigella phat trien se lam uc va chuyen
mau moi trng sang tm hay o ta[LDC (+)], LDC (-) khi moi trng
co mau vang.

Th nghim urea
Thc hin trn mi trng urea lng RSU cha ch th
phenol, chuyn mu t vng sang (vng chuyn mu l pH
6,8 - 8,4). C th s dng mi trng urea thch c bn c b
sung urea.
Ly y mt que cy vng t khun lc trn mi trng TSA
vo ng cha 3ml-5ml mi trng RSU v trng, lc nh ng
trn u vi khun v 37
o
C trong 48 gi.
Shigella khng phn gii urea nn khng lm thay i pH mi
trng; nu phn ng dng tnh, vic phn gii ure s gii
phng NH
3
lm i mu phenol red sang mu hoa hng v sau chuyn sang mu
tm.

Th nghim VP
Ha mt vng que cy y t khun lc trn mi trng TSA vo
cc ng mi trng MR-VP 37
o
C trong 24 gi.
Sau thi gian , ly 0,2 ml dch cy vo 01 ng nghim, thm 02
git creatin, 06 git dung dch 1- naphthol trong cn, sau thm
02 git KOH 40%, vic xut hin mu hng n mu ti
trong vng 15 pht l phn ng dng tnh
Shigella cho phn ng m tnh vi th nghim VP, khng i
mu trn b mt mi trng. Th nghim VP (+) khi c mu

VP (-) VP
(+)
Hnh 5.5. Th nghimVP

Urea (-) Urea
(+)
Hnh5.4. Th nghim
Urea
79
trn b mt mi trng

10.3.4.Kt qu
Vi quy trnh kim nghim ny cho php kt lun c hay khng c Shigella c pht
hin trong 25g mu

10.4.Cu hi n tp
1. Nu cc bc trong quy trnh phn tch Shigella
2. Nu hnh thi cc khun lc c trng Shigella trn cc mi trng phn lp khc
nhau

11. NH TNH FAECAL STREPTOCOCCUS TRONG THC PHM
11.1.Nguyn tc
Khi c nui cy trong mi trng cha acid tetrazolium cha triphenyl
tetrazolium chloride, khun lc Streptococcus phn c mu hng n mu m do
s kh triphenyl tetrazolium chloride. Cc loi ny khng c catalase, khng c
cytocrom c, c th pht trin c trong mi trng cha 6,4% mui cha NaCl, pH
9,6 45
o
C.


80
Dng c Thit b
n cn Cn phn tch

Bng 11.2.Mi trng v ha cht ch tiu 11
Mi trng Ha cht
Dung dch Trypton Soya (TSB) Cn 90
o
v 70
o

Enterococcus agar Dd creatine 0,5 %
BHI Brain heart infusion 6,5% mui NaCl
BHI Brain heart infusion pH 9,6 KOH 40%
Moi trng Trypticase Soy Agar (TSA)
HCl 10%
NaOH 10%
Thuc th catalase
Que th oxydase

11.3.Thc hnh

m tt c khun lc c trng
Cy 5 khun lc c trng vo TSA, 37
o
C, 24 gi

Tri ln MT Enterococcus agar, 44
o
C, 48 gi

Chun b dch pha long 10
-1
, 10
-2
, 10
-3
..

81

11.3.2.Cc bc tin hnh
Bc 1. Mu c pha long thp phn thnh cc nng ph hp 10
-1
, 10
-2
, 10
-3

Bc 2. Phn lp
Cy tri 0,1 ml mu ln b mt mi trng chn lc Enterococcus agar, tri khp b
mt a bng que tri thy tinh. Lt ngc a v 44
0
C trong 2 ngy.
Bc 3. m tt c cc khun lc c mu hng n mu m, kch thc khong
0,5-3mm, c th c vng khng mu xung quanh khun lc.
Chn t nht 5 khun lc c trng, chuyn qua TSA, qua m 37
0
C. Th sinh
ha qua mi trng BHI 6,5% NaCl, BHI pH 9,6, th nghim catalase, oxydase.
Enterococcus phn c phn ng catalase (-), oxydase (-), pht trin c trong mi
trng BHI 6,5% NaCl, BHI pH 9,6.

11.3.4.Kt qu
Tnh t l khng nh v mt theo CFU/g hay CFU/g

11.4.Cu hi n tp
1. Nu cc bc trong quy trnh phn tch Streptococcus phn
2. Nu cc phn ng sinh ha khng nh Streptococcus phn

Khng nh (+):
Chu mui 6,5%, chu pH 9,6 (+)
Catalase (-); Oxydase (-)

Mt Streptococcus phn
( CFU/g hay CFU/ml)

82

CHNG IV
QUY TRNH PHN TCH VI SINH VT THEO PHNG PHP HIN I

4.1. PHNG PHP PHT QUANG SINH HC ATP
Phng php pht quang sinh hc ATP c ng dng trong 3 lnh vc: gim st
v sinh, kim tra nhng loi cht lng nh nc ra lm sch h thng, nh gi cht
lng vi sinh ca thc phm. Phng php ny da trn nguyn tc pht quang sinh
hc om m di xc tc ca enzym luciferase.
Phn t ATP hin din tt c cc t bo sinh vt sng, do , s pht hin ATP
l du hiu nhn bit vt cht sng ang tn ti. ATP c th c pht hin mt
cch nhanh chng bi lng nh sng pht ra thng qua s kt hp vi enzym
luciferase nh mt my o sng. K thut ny c th pht hin c 1pg ATP (10
-12
g)
tng ng vi khong 1000 t bo vi khun (10
-15
g ATP/t bo). Thng thng
trong phn tch, ATP ca t bo vi sinh vt s c tch chit ra khi t bo (thng
dng trichloacetic acid 5%) v c nh lng da vo cng nh sng pht ra.
S phn tch c thc hin nhanh chng ch din ra trong vi pht. C ch phn ng
pht quang ca phng php xy ra nh sau:
Luciferase + Luciferin + ATP Luciferase-Luciferin AMP + PPi
Luciferase-Luciferin AMP + O
2
Oxyluciferin + AMP + CO
2
+ hv (pht
quang)
4.2. PHNG PHP ELISA (Enzyme-Linked immunosorbent Assay)
Phng php ELISA (Phng php hp ph min dch dng enzyme) da trn
nguyn tc l phn ng kt hp gia mt t bo (khng nguyn) vi mt khng th c
hiu. Tn hiu ca phn ng min dch c th nhn bit thng qua s ngng ta hay kt
dnh ca khng nguyn-khng th hoc bng nhng khng th c nh du (bng
cht nhum pht hunh quang, ng v phng x hay enzym).
ELISA c m t ln u tin vo nm 1971 v t tr thnh mt phng
php c s dng ngy cng rng ri v quan trng hn trong nghin cu, chn on
v xt nghim bi v n c kh nng pht hin nhy bn vi mt lng vt cht rt
nh.
ELISA thay th mt s k thut huyt thanh c in phc tp, cng knh
tn nhiu thi gian hn v m rng phm vi phng php pht hin virus cng nh
cc marker lin quan n s nhim ca chng.
Xt nghim ELISA c th c tin hnh vi mt s phng php nh ELISA
trc tip, gin tip, sandwich v cnh tranh.
Nguyn tc c bn ca phng php ELISA l khng nguyn ho tan trong
dung dch m thch hp c th ph ln b mt plastic (nh polystyrene). Qu trnh
ny c th l trc tip hoc thng qua mt khng th. Khi huyt thanh c thm
vo, cc khng th c th kt hp vi khng nguyn pha c (solid phase).
Xt nghim ELISA c thc hin trong a plastic kch thc 8cm x 12cm,
cha 8x12 ging. Mi ging c chiu cao khong 1cm v ng knh l 0,7cm.
83

Hnh 15: a plastic s dng tin hnh xt nghim ELISA
Cc khng th s dng trong phng php ELISA c gn vi enzyme bng
lin kt ng ho tr. Khng nguyn c gn vi ging plastic v khng th lin kt
vi enzyme c gn vi khng nguyn. Khng th khng gn khng nguyn
s b ra tri i. Enzyme c gi li v v vy lng khng th gn enzyme
c pht hin bng cch cho thm vo mt c cht lm thay i mu do hot tnh ca
enzyme. mu to thnh l t l vi lng enzyme bm ging plastic, t suy ra
lng khng th, sau tip tc suy ra lng khng nguyn.
Tnh nhy ca ELISA l do s khuych i bi hot tnh enzyme.
Mi mt phn t enzyme bm vo khng th c th to ra hng ngn phn t mu do
hot tnh enzyme. Trc khi cc khng th gn enzyme c th c s dng rng ri,
cc khng th phng x c s dng trong k thut min dch phng
x (radio immuno assays-RIA). K thut RIA nh l mt t ph c ngha v ngi
sng to ra n l Rosalyn Yalow c nhn gii thng Nobel Sinh l v Y hc vo
nm 1977.

84

Hnh 16. Nguyn tc ca phng php ELISA

Hnh 17. Quy trnh thc hin phng php ELISA
Trong phng php ny, cc khng th n dng c ph bn ngoi nhng a
ging. Nu c s hin din ca khng nguyn mc tiu trong mu, khng nguyn ny
s c gi li trn b mt ging. Cc khng nguyn ny s c pht hin bng cch
s dng khng th th cp c gn vi enzym nh horseradish peroxidase hay alkaline
phosphatase. Khi b sung mt c cht c hiu ca enzym vo ging, enzym xc tc
phn ng thu phn c cht to ra cc sn phm c mu hay pht sng. Thng qua
vic theo di s i mu, c th pht hin s hin din v nh lng khng nguyn.
ELISA c s dng rng ri di dng cc b ho cht (kit) thng mi. Hin
nay, c cc b kit dng cho Salmonella, E. coli gy bnh, Listeria, Staphylococcus.
nhy ca cc b kit ny khong 10
4
CFU/ml, v vn cn thc hin tng sinh hoc
tng sinh chn lc khi thc hin phng php ny. Ngoi ra cn c cc sn phm khc
nh TECRA (Australia) da trn nguyn tc ny nh danh c t ca
Staphylococci trong tht .

85

Hnh 18. My ra v my c trong quy trnh ELISA
4.3.PHNG PHP LAI PHN T (Hybridization)
Phng php lai phn t cn c gi l phng php mu d, probes. Phng php
s dng mu d pht hin vi sinh vt trong thc phm c da trn s pht hin
mt on gen c trng ca vi sinh vt. C s ca vic s dng mu d l qu trnh lai
phn t . Qu trnh ny bao gm s tch ri hai mch i ca chui xon kp DNA khi
nhit vt qu nhit nng chy (T
m
) ca phn t DNA v s ti bt cp gia cc
on DNA c trnh t nucleotide b sung khi nhit tr li bnh thng. Mt trong
hai mch DNA b sung (gi l DNA mc tiu l DNA ca t bo vi sinh vt) c c
nh trn mt gi th rn hoc nm ngay trn t bo hay m. S lai phn t xy ra khi
on mi c trnh t nucleotide b sung vi mt vng trnh t trn DNA mc tiu gp
nhau do chuyn ng nhit v khi nhit mi trng thp hn T
m
t nht vi . S
lai phn t cn c th xy ra gia DNA v RNA. Qu trnh lai phn t chu nh hng
rt nhiu bi cc yu t: nng DNA ca mi trng, nhit v thi gian phn
ng, kch thc cc trnh t lai v lc ion ca mi trng.

4.4. PHNG PHP PCR (Polymerase Chain Reaction)
Phng php PCR (polymerase chain reaction) l mt phng php in vitro
tng hp DNA da trn khun l mt trnh t ch DNA ban u, khuch i, nhn s
lng bn sao ca khun ny thnh hng triu bn sao nh hot ng ca enzym
polymerase v mt cp mi c hiu cho on DNA ny. K thut ny c Karl
Mullis (M) pht minh vo 1985.
Phng php PCR cho php tng hp rt nhanh v chnh xc tng on DNA
ring bit. y thc s l phng php hin i v thun tin cho vic xc nh s c
mt ca mt gen no trong t bo vi chnh xc cao.
Phng php ny da trn s khm ph hot tnh sinh hc nhit cao ca
DNA polymerase c tm thy trong cc sinh vt a nhit (vi khun sng trong cc
sui nc nng). Phn ln cc DNA polymerase ch lm vic nhit thp. Nhng
nhit thp, DNA xon cht v vy DNA polymerase khng c nhiu kh nng lm
bin tnh phn ln cc phn ca phn t. Nhng cc polymerase chu nhit
ny hot ng nhit rt cao, c th ln n 100
o
C. nhit ny DNA (dng
thng) s b bin tnh.
4.4.1. Cc thnh phn ch yu ca phn ng PCR
a. DNA mu (DNA template)
y l mu DNA sinh hc m chng ta mun khuych i. Phn ng PCR ti
u xy ra trn DNA tht tinh sch nhng phn ng PCR vn cho kt qu tt vi DNA
thu nhn trc tip t dch chit t bo. Lng mu DNA s dng c khuynh hng
gim khi s dng cc enzyme DNA polymerase cho hiu qu cao (<100ng).
86
Lng DNA mu nu cao qu phn ng PCR s khng xy ra. PCR cn cho
php khuych i c nhng mu DNA khng c bo qun tt, cc mu DNA b
phn hy tng phn nh cc vt mu lu ngy, tinh dch kh, tc, mng tay
ca ngi cht....
b. Mi (primer)
Mi l nhng on DNA si n ngn v cn thit cho vic xc tin phn ng
dy chuyn tng hp DNA . Chng nhn ra phn DNA cn c nhn ln, bt cp b
sung vi mt u ca DNA mu v to ra v tr bt u ti bn. Cc mi ny c chiu
ngc nhau, bao gm mt mi xui (forward primer) v mt mi ngc (reverse
primer).
Mi l yu t quan trng nht ca phn ng PCR, quyt nh hiu qu ca phn
ng. Do vic thit k mi cn c tun th mt s nguyn tc nht nh:
+ C hai mi trong mt phn ng PCR phi c nhit nng chy (Tm) gn
nh nhau bi v chng c cng mt nhit .
+ Kch thc ti thiu ca mi l 18 base (thng thng l 18-24 base) qu
trnh lai xy ra tt hn.
+ Mi phi c hiu c ngha l n phi c trng cho trnh t DNA cn c
khuych i, mt mi ch bm vo mt v tr nht nh trn gen.
+ Khng c nt ci tc (hairpin loop): trong mi mt mi cn trnh trnh t i
xng bc hai (palindromic sequences) c th lm tng cu trc si bn trong n nh
do hn ch vic mi bm vo DNA mu.
+ Trnh s b sung gia hai mi: s b sung gia hai mi s lm tng sn phm
primer dimer (Hnh 3.7).
+ Trt t cc base cng nh hng n s n nh vic bm ca mi di nhit
cao. Hai trong ba base u 3 ca mi nn l G hoc C, v G v C c 3 lin kt
hydro do s polymer ho s tt hn.
+ Khong cch ti u gia hai mi: y l mt ng dng rt c trng, nhng
i vi phn ln cc th nghim PCR chn on, tt nht khong cch gia hai mi
khi bm vo DNA khun l 150-500 base.
c. Enzyme polymerase chu nhit
Enzyme ss dng trong phn ng PCR l enzyme DNA polymerase t
Thermus aquaticus (Tag-polymerase) khng bin tnh sau mi chu k. DNA
polymerase chu nhit s dng cho phn ng PCR ln u tin c bn trn th
trng l Tag-polymerase. Hin nay c nhiu enzyme chu nhit khc c s dng l:
Vent- polymerase (Tli-polymerase), Pfu- polymerase, rTth...Cc hot tnh ca chng
c trnh by bng:
Enzyme

Hiu sut
tng i
(Relative
efficiency)
Tn s li
(Error
rate)

Tn s
m rng
(Extention
rate)
Exo
3-5
Exo
5-3

Taq-polymerase
Vent-
88
70
2x10
-4

75
67
Khng
C
C
Khng
87
polymerase
Pfu- polymerase
rTth
60
Khng X
4x10-5
7x10-7
Khng X
Khng X
60

C
Khng

Khng
C

d. Cc loi nucleotid
Trong phn ng PCR, bn loi nucleotid thng c s dng dng
deoxynucleotid: dATP, dCTP, dGTP, dTTP vi nng cn bng trong mt phn ng
(200M/loi nucleotid).
e. Nc
Nc s dng cho phn ng PCR phi tht tinh khit, khng cha ion no,
khng cha DNAase, RNAase, enzyme ct hn ch... Ni cch khc l khng cha bt
k mt thnh phn no khc.
f. Dung dch m
Dung dch m 10X (100mM KCl, 100mM (NH4)
2
SO
4
,
200mM Tris-
Cl pH 8.8, 20mM MgSO
4
, 1% (w/v) Triton X-100)
g. Ion Mg
2+

Nng ion Mg
2+
cng l mt yu t nh hng mnh n phn ng PCR v
n tu thuc vo tng phn ng. Nng ion Mg2+ ti u l 150-200 M. Ngi ta
thy rng nu nng DNA qu cao th enzyme polymerase s gy ra nhiu li hn.
4.4.2. Ba giai on trong mt chu k ca phn ng PCR
C 3 giai on chnh trong phn ng PCR v chng c lp i lp li nhiu
ln (chu k) (thng t 25 n 75 chu k).
a. Giai on bin tnh (denaturation)
Trong giai on ny phn t DNA mu b bin tnh nhit cao (thng l t
94-95
o
C, ln hn nhit nng chy ca phn t) trong vng 30 giy n 1 pht, tt
c cc lin kt hydro gi hai mch ca phn t b b gy v to thnh cc DNA si
n.
b. Giai on lai (hybridization)
Nhit c h thp ( thng t 40-70
o
C, thp hn nhit nng chy ca
mi c s dng khong t 3-5
o
C) cho php cc mi bm vo cc phn t DNA si
n, nh du phn DNA cn c khuych i. Giai on ny ko di t 30 giy n
mt pht (cn c gi l giai on ).
Nu nhit qu thp th cc mi s gy nn nhiu li v kt qu l s to nn
nhiu sn phm ph. Nu nhit qu cao th phn ng s khng c kt qu.
Cng thc xc nh nhit nng chy (Tm) mt cch tng i l:
Tm=4(G+C) + 2(A+T)
c. Giai on ko di (elongation)
88
Nhit c tng ln n 72oC gip cho DNA polymerase xc tc tng hp
DNA tt nht. Cng vic ca DNA polymerase l di chuyn dc theo DNA si n v
s dng n lm khun tng hp si DNA mi b sung vi DNA mu bng cch
ko di cc phn c nh du bi cc mi. Thi gian ca giai on ny ph thuc
vo kch thc ca DNA mu, thng ko di t 30 giy n nhiu pht.
giai on ny ca chu k cui cng, thi gian c tng thm vi pht cc
si DNA cha c sao chp xong hon thnh qa trnh tng hp.Sau mi chu k, s
bn sao ca DNA mu li c tng gp i. y l s nhn bn theo cp s nhn.
Nh vy 1 phn t DNA mu, sau phn ng PCR vi n chu k to thnh 2n bn sao
phn t DNA.

Hnh 19. Ba giai on ca ca phn ng PCR
Chu k ba bc lp li nhiu ln, sau 30-40 chu k s to ra 10
6
bn sao. Sau
phn ng PCR, ADN c nhum bi ethidium bromide v c quan st qua in di
sn phm PCR trong gel agarose v quan st di tia UV

89

Hnh 20. th biu din mi quan h gia thi gian v nhit trong mt chu k ca
phn ng PCR

Bng . S bn sao ca phn t DNA mu to thnh qua cc chu k ca phn ng PCR

Chu k

S bn sao

1
2
4
10
15
20
25
30
...
n
21
=2

22
=4

2
4
=16
2
10
=1.024
2
15
=32.768
2
20
=1.048.576
2
25
=33.554.432
230=1.073.741.824
...
2n
90

Hnh 21. Phn ng PCR vi lng sn phm tng theo cp s nhn
u im ca phng php PCR
+ Thi gian (6-48 gi)
+ Pht hin vsv kh nui cy
+ Ha cht d tm v d bo qun hn so vi huyt thanh hc
+ C th t ng ha

Hnh 22. M hnh my PCR (Polymerase chain reaction) v my PCR realtime ca
Bio-Rad
Nhc im
+ Thc phm cha cht c ch enzyme polymerase
+ Mt vsv trong mu thp, i khi cn tng sinh
+ Khng phn bit c t bo sng v cht, c th dn n dng tnh gi (tuy
vy, c th pht hin bo t, t bo cht ca vsv gy c)
+ Ngy cng c quan tm nhiu hn c bit pht hin virus
Thng thng khi thc hin phng php PCR, trnh t c tin hnh nh sau:
- Tng sinh: vi sinh vt c nui cy tng sinh trn mi trng chn lc (TSB)
10-20 gi.
- Tch chit DNA vi sinh vt
- Thc hin phn ng PCR
- in di trn gel agarose 1,5%, c kt qu trn n UV.

4.5. PHNG PHP DNG PETRIFILM
Petrifilm l mng qut ln mt lp cht dinh dng v tc nhn to gel. Khi tin
hnh xt nghim, 1ml mu c cy vo Petrifilm v . Cht mu c bit trn a
91
khin cc khun lc c mu c trng d dng phn bit bng mt thng. Cc ng
k gip cho vic m s lng khun lc d dng hn. Cc phng php Petrifilm
c th nghim v c AOAC cng nhn. K thut ny c dng trong cc ng
dng kim tra tng vi khun hiu kh, s coloform, coliform phn, nm mc, nm men.
u im ca k thut ny l d thao tc, tit kim khng gian v bo qun, thi gian
s dng lu v khng cn hp kh trng mi trng

Hnh 14. Quy trnh phn tch vi sinh vt dng Petrifilm

CHNG V
NH GI V SINH CNG NGHIP
QUY NH KIM SOT VI SINH
MI TRNG, THIT B, V SINH TAY, BAO B
iem kiem soat Cach lay mau kiem tra
Ch tieu kiem
tra
1. Moi trng:
-Phong rot san
pham

- at mau a moi trng v tr
tai bat ky mot trong cac may
rot trong 15 phut.

- TPC 10kl
- Nam men/ moc:
2kl
92

TI LIU THAM KHO

[1]. Bi ging: Thc hnh phn tch vi sinh thc phm
-Trn Quc Huy, Nguyn thy Hng,2010
[2]. L nh Hng, Nguyn c Hng, Hunh L Tm, S tay kim nghim vi sinh
thc phm thy sn, NXB Nng nghip H Ni, 2004
- Phong Vi sinh - at mau a moi trng tai
v tr ban th trong 15 phut
- TPC 2kl
- Nam men/ moc
: khong co
2. Thiet b:
- Bon cha
-ng ong rot
san pham
- au rot san phm

- Bon cha: Dung bong tiet
trung swab mot vung ben trong
bon, dien tch 100cm
2
ngay sau khi
ket thuc ve sinh
- ng ong rot:Dung bong tiet
trung swab vung ben trong ong,
chieu cao 10cm ngay sau khi ket
thuc ve sinh
- au rot: Dung bong tiet trung
swab vung ben trong ong, chieu
cao khoang 5-10cm tuy theo au
rot san pham ngay sau khi ket
thuc ve sinh

- TPC 10kl
- Coliform: khong
co
- Nam men/ moc
: khong co

3.Ve sinh tay:
-Nhan vien phong
rot san pham
-Nhan vien che
bien /phan chia thc
an

- Dung bong tiet trung swab ban
tay
-Coliform: khong
co
- E.coli: khong co
4.Bao b sau tiet
trung
- Dung nc tiet trung trang
ben trong bao b cha san
pham
- Dung bong tiet trung swab
vung ben trong bao b
- TPC : khong
co
- Coliform: khong
co
- Nam men/ moc
: khong co

5.Bao ho lao
ong:
Quan/ao/non
- Swab 1 vung dien tch100cm
2
tren
quan /ao /non ngay sau khi hoan
tat cong oan say kho
- Coliform: khong
co
- E.coli: khong
co
- Nam men/ moc
: khong co
93
[3]. Trn Linh Thc, Phng php phn tch vi sinh vt trong nc, thc phm v
m phm, NXB Gio dc, 2006
[4]. TCVN 4830-1:2005(ISO 6888-1:1999) Vi sinh vt trong thc phm v thc n gia
sc - phng php nh lng Staphylococci c phn ng dng tnh vi
coagulase (Staphylococcus aureus v cc loi khc) trn a thch. Phn 1: K
thut s dng mi trng thch Baird-Parker.
[5]. Thomas A. Mc Meekin, Detecting pagothens in food, Woodhead
Publishing Limited (ISBN 1-85573-704-3) and CRC Press LLC (ISBN 0-
8493-1756-8), 2003
[6]. Shabbir Simjee, Foodborn Diseases, Humana Press (ISBN: 978-1-
59745-501-5), 2007

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