1.

Tng s vi sinh vt hiu kh

1.1 Nguyn tc
Tng vi sinh vt hiu c m bng cch v trong iu kin hiu kh
30 C/726 gi
0

1.2 Mi trng v thit b

- Dch pha long: Saline Peptone Water (SPW)
- Mi trng nui cy: Platecount agar (PCA)
- T m: 30 1 C
0

1.3 Quy trnh.

- a: chuyn 1ml dung dch dung dch mu sau khi ng nht hot
pha long nng thch hp vo a petri v trng, m nng mt
a. Trong 15 pht, vo mi a 15-20ml mi trng nui cy (PCA)
c lm ngui n 45 C . Sau trn iu mu v mi trng nui cy
- Nui : Cc a c lt ngc v trong 726 gi 301 C
0

1.4 c kt qu
c kt qu c th dng mt thng hoc qua knh lp c phng i
2,3 ln m s khun lc mc trn petri (ch m a petri c s
khun lc 30-350 khun lc) sau nhn vi h s pha long.

2. Coliforms
- Phng php: tham chiu theo phng php NMKL 44 n bn ln 4,
1995
2.1 Nguyn tc
Da vo s ln ln men ng lactose mi trng thch hp (thch
Violet red bile) 37 C trong 24 gi. Sau c khng nh li trong mi
trng canh Brilliant Green Bile Salt. Coliforms s sinh kh trong mi
trng ny 37 C trong 24 gi.
0

2.2 Mi trng v thit b

- Dung dch Salin Pepton
- Thch Violet red bile (VRBL)
- Canh Brilliant Green Bile Salt (BGBL)
- Thch Tryptone Soya (TSA)
- T n 371 C
0

2.3 Quy trnh

- a: chuyn 1ml dung dch dung dch mu sau khi pha long cho
vo a petri v trng, s dng hai nng pha long lin tip. vo

mic a khon 5ml mi trng TSA 45 C. Sau cho mi trng ng

hon ton thn 10-15ml mi trng thch VRBL 45 C
- Nui : Cc a c lt ngc v trong 243 gi 371 C
0

2.4 c kt qu
m cc a c s khun lc di 100 sau 24 gi nui cy. Khun lc
coliforms c mu ta, ng knh 0.5mm, i khi c bao quanh bi
mt vng hi do ta. Tnh gi tr trung bnh t cc pha long qui
v s coliform trong mt g mu.
2.5 Khng nh.
Cy ring t nht 5 khun lcn nghi ng ca mi loi vo cc ng nghim
cha mi trng canh BGBL c ng Durham , 370C trong 24 gi.
Phn ng c coi l dng tnh khi c s to kh d ch mt trong 5 ng
nghim trn.

3. Escherichia coli
- Phng php: tham chiu theo TCVN 5287, n bn ln 2, 1994
3.1 Nguyn tc
Cy mt lng dch mu vo mi trng tng sinh (canh Brilliant green
bile lactose), la cc khun lc in hnh trn mi trng chn lc (EMB)
cc php th sinh ha ph hp (nghim php IMViC)
3.2 Mi trng v thit b
- Dung dch Saline (SPW).
- Canh Brilliant green bile lactose BGBL.
- Thch Eosin Methylene Blue Lactose (EMB)
- Canh Methyl Red Voges Proskauer (MR-VP)
- Canh Tryptone (hoc peptone)
- Thch Simmons Citrat
- Thuc th Methyl red 0,2%, -naphtol 5%, kovacs.
- Dung dch KOH 40%
- T m 371 C, 441 C
0

3.3 Quy trnh

- Tng sinh: Cy 1ml dch mu nng 10-1 vo ng nghim cha 5ml mi
trng tng sinh (BGBL), 44,00,50C 24 gi.
- Cy phn lp: Sau khi tng sinh cy dch mu t ng nghim c phn
ng dng tnh (mi trng chuyn c v sinh hi) sang mi trng
EMB, 371/24 gi. Trn mi trng EMB: khun lc mu tm, nh kim,
trn, b iu, ng knh khong 0,5mm
3.4 Khng nh

- Chn ch nht 2 khun lc in hnhtreen mi trng chn lc sang mi

trng thch khng chn lc (TSA) 3710C trong 19-24 gi.
- Kt quae th nghim sinh ha E. coli ph hp: indol(+), methyl red(+),
voges proskauer(-), khng s dng citrat(-)
3.5 c kt qu & bo cao
Pht hin hay khng pht hin

4. Salmonella
- Phng php: tham chiu theo phng php NMKL 71 n bn ln 5.
Nm 1999
4.1 Nguyn tc
- Phng php ny ch dng nh tnh pht hin hay khng pht hin
- Quy trnh kim tra Salmonella bt buc phi qua bn gia on: tin tng
sinh, tng sinh, phn lp v khng nh.
4.2 Mi trng v thit b
- Canh Peptone m (BPW).
- Canh Rappaport-Vasiliadis soy peptone.
- Thnh Brilliant Green Phenol Red Lactose Sucrose (BPLS)
- TSI, TSA
- Canh Lysine decarboxylase, Urea phenol red, Manitol Phenol red be
- B iu nhit 0.2 C
- T m 371 C
0

1.3 Quy trnh

- Ly mu: Ly mu vng b mt cng rng cng tt
- Tin tng sinh: trn 25g mu vi 225ml nc m peptone m, nh
nht nht bng my dp mu. 370,1 C t 18 n 24 gi
- Phn lp: tu mi trng sinh cy chuyn khun dch ln b mt mi
trng phn lp XLD sao cho co th to c nhng khun lc tch ri.
Lt ngc a 370,20C trong 243 gi. Trn mi trng XLD khun
lac Salmonella in hnh trong, hi nhum do s thay i ca cht ch
th trong mi trng, phn ln c tm en. Bao gi cng nhn thy mt
vung mi trng ln hay nh.
0

4.4 Khng nh
Nhng khun lc nghi ng c kim tra khng nh bng th nghim
sinh ha: Lactose (-), Sucrose (-), Glucose (+), Urease (-), Indol (-),

Mannitol (+), VP (-), LDC (+), ODC (+) v amygdaline (-)

4.5 c kt qu & bo co
Pht hin hay khng pht hin

5. Clostridium perfringens
- Phng php: tham chiu theo phng php NMKL 56 n bn ln 3.
Nm 1994
5.1 Nguyn tc
nh lng Clostridium bng cnh cy mt lng mu bit vo mi
trng thch hp c cha ion Fe3+ v ion (S2O3)2- 370C trong 1-2 ngy
5.2 Mi trng v thit b
- Thch iron sulphite
- Dung dich mui peptone pha long
5.3 Quy trnh
- Cy mu: Chuyn 1ml mu nng thch hp vo ng nghim. Sau
, 12ml mi trng thch iron sulphite vo ng trn iu mu trc
khi mi trng ng li. Sau khi mi trng ng thm 2-3ml mi
trng thch iron sulphite ln b mt 371 C trong 28-48 gi
5.4 c kt qu
- m tt c khun lc en, xung quanh c qung en nhn vi nng
pha long
0

6. Virio Cholerae v Virio parahaemolyticus

- Phng php: tham chiu theo s tay phn tch vi sinh FDA , n bn ln
8, nm 1995
6.1 Nguyn tc

- Virio Cholerae v Virio parahaemolyticus c nui trong mi trng lng

chn lc. t y dch khun c cy chuyn sang mi trng rn chn
lc. nhng khun lc ging Virio Cholerae v Virio parahaemolyticus c
th nghim bng cc phn ng sinh ha.
6.2 Mi trng v thit b
- Thch Thiosulphate citrate Bile salt sucrose (TCBS agar)
- Peptone kim cha 1% Nacl (APW)
- TSA + 1,5 %Nacl
- T m 3710C
6.3 Quy trnh
Tng sinh: 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. Dp mu 2 pht, 371 0C trong 6 gi v 16-24 gi
Phn lp v c kt qu: t mi trng tng sinh cy ria vo mi trng
TCBS thch 37,0 1,0 C trong 18 - 24 gi.
Trn mi trng TCBS thch khun lc V. cholerae trn, ln c ng knh
0

2-3mm hi dt, mu vng (do vi khun ln men sacaroza), gia m v

c vin m xung quanh. V. parahaemolyticus trn, ln c ng knh 34mm , mu xanh dng
6.4 Th nghim sinh ha
-Khun lc nghi ng trn TCBS thch cy chuyn sang mi trng khng chn lc TSA 1,5%
NaCl. nhit 37,0 1,0oC trong 18 - 24 gi.

+ Th nghim s b:
Php th
TSI
Tinh duy ng
Oxydase
KOH
+ Th nghim khng nh
Php th
Tnh chu mn
0% NaCl
3% NaCl
6% NaCl
8% NaCl
10% NaCl
Ln men sinh axt t
Sucrose
Lactose
Maninitol
ONPG
ADH
LDC
Urease

Phn ng tiu biu ca

Virio parahaemolyticus
K/A -+
+
+

Phn ng tiu biu ca

Virio Cholerae
K/A -+
+
+

Phn ng tiu biu ca

Virio parahaemolyticus

Phn ng tiu biu ca

Virio Cholerae

+
+
-

+
+
-

+
+
-

+
+
+
+
+

6.5 Bo kt qu
Pht hin hay khng pht hin

7. Staphylococci aureus Coagulase positive

- Phng php: tham chiu theo phng php NMKL 66 n bn ln 3,
1999
7.1 Nguyn tc
nh lng St. aureus Coagulase positive c thc hin bng cnh cy
trang mt lng mu bit ln mi trng thnh Bard Parker, vi khun

cho nhng khun lc t trng. V c xc nh bng phn ng

coagulase
7.2 Mi trng v thit b
- Dung dch Saline Peptone
- Thch Trypton Soya
- Thch Bid Parker
- Canh Brain Heart Infusion (BHI)
- Huyt thanh th
- T n 3710C
7.3 Quy trnh
Cy trang 1ml mu vo 3 a a petri . lt ngc cc a v
3710C trong 243 gi v 484 gi
7.4 c kt qu
- Sau 243 gi, trn mi trng Baird Paker khun lc
Staphylococci aureus Coagulase positive c ng knh 1-1,5mm, mu
en sng v li. Mi khun lc c qung sng rng 1-2mm bao quanh.
Nhng khun lc in hnh s c nh du trn mt sau ca a v tip
tc thm 24 gi na.
- Sau 48 gi Staphylococci aureus Coagulase positive 1,5-2mm c mu
en, sng v li, quanh khun lc c mt vng c m hp, tip l mt
vng sng trong rng 2-4mm .
- Mt vi chng St. aureus khng to qung sng trong bao quanh, vng
c st khun lc cng c th khng c (khun lc khng in hnh). C
hai loi khun lc ny iu c nh du mt sau ca a
7.5 Khng nh
Cy 5 khun lc in hnh v khng in hnh sang mi trng TSA
3710C trong 24 gi. T mi trng TSA cy chuyn vi khun sang cc
ng nghim c cha 0,3ml huyt tng th 371 C. kim tra s hnh
thnh khi ng sau 1,3,6 v 24 gi. Kt qu Dng tinh c s hnh thnh
khi ng. m tnh khng c s hnh thnh khi ng
0