Phng php nh lng

GV: NGUYN VN HNH

 Phng php nh lng l g ?

y L cc phng php xc nh s lng vsv trong mu.

y Tr li cho cu hi: Hm lng vi sinh vt trong mu l bao
nhiu ?
y Cc phng php nh lng
1. Phng php m trc tip
2. Phng php m khun lc
3. Phng php m khun lc trn mng lc
4. Phng php o c
5. Phng php MPN (most probable number)

y n vi tnh: t bo/ml t bo/g

CFU/ml CFU/g
MPN/ml MPN/g
 Phng php m trc tip

y Mt vsv n bo c kch thc ln nh nm men,

men, to c th c xc nh trc tip bng bung
m trn knh hin vi.
m trc tip bng
bung m hng
cu

Pha long mu cn
m sao cho trong mi
nh ca bung m c
khong 5 - 10 t bo
Cch m t bo trong bung m
 u im v nhc im

u im: cho bit c s lng VSV

Nhc im: d nhm ln, chnh xc khng cao,

khng thch hp cho huyn ph vsv c mt thp

Khc phc nhc im ca pp m trc

tip ????
m trc tip bng knh hin vi hunh quang

Cc cht nhum pht hunh quang

- Acridin cam (AODC)
- 4,6-dianidino-2-phenyl-indol (DAPI)
- Fluorescein isothiocyanate (FITC)

u im:
- Loi b sai s do cc cht vn
- Kt qu phn nh ng vi sinh khi
 Phng php m khun lc

u im:

Cho php xc nh s t bo sng

nh lng chn lc vsv

Phng php:
- Chun b dch pha long mu
- Chun b cc chui pha long mu
- Cy mu vo mi trng, mu
- m s khun lc hnh thnh
 Chun b dch pha long mu

Khng s dng nc ct v nc mui sinh l

pha long mu thc phm
 Cc dung dch pha long mu

y Saline peptone water (SPW)

NaCl 1g

Peptone 8,5g

Nc ct va 1 lt

y Buffered peptone water (BPW)

NaCl 5g

Peptone 10g

Nc ct va 1 lt
 Chun b cc chui pha long mu

pha long mu lng theo dy thp phn

i vi cc mu rn hay bn lng:
10g mu + 90ml dung dch pha long => pha long
101
Film

Cc bc tip theo tng t nh pha long mu lng

Cy mu vo mi trng

-Phng php cy b mt
- Phng php a
 Phng php a

y Chun b a petri v trng

y Mi trng c chun b - hp kh trng v c bo
qun mt 45oC trong b iu nhit
y Ht 1ml mu vo a trng (chn nng thch hp)
y vo a cy 10 15ml mi trng, lc u
y ngui mi trng
y em
 Phng php a

u im
Cy c th tch mu ln (1ml)
Xc nh c cc VSV cn dinh dng tip
xc t nhiu pha
Cho php m c mt VSV cao, khong
150-300 khun lc
Nhc im
Khng nh lng c nhng VSV qu nhy
nhit
Khng xc nh c hnh dng khun lc
nht nh
Kh lm thun mt dng VSV
 Phng php cy b mt

y Mi trng phi c chun b trn a trc

1-2 ngy kh mt
y Phng php
- Cy 0.1 0,3ml vo a mi trng
- Tri u trn mt bng que trang tam gic
- nhit phng 15-20 pht cho kh mt

Film
 Phng php cy b mt

u im
- nh lng c cc VSV nhy nhit
- C th nhn dng c dng khun lc c trng
- D dng lm thun chng VSV mc tiu
Nhc im
- Ch cy c th tch mu nh
- Ch cho php m c s lng khun lc thp
Cc thit b h tr m khun lc

My m khun
My m khun
lc
lc
t ng
 m khun lc

y m tt c khun lc n l mc trn mi trng

y Thng chn nhng a c s khun lc khong 30
300
y Dng bt m cc khun lc m
y Tnh ton kt qu
(da trn s khun lc m c v pha long tnh
ra s khun lc vsv trong dung dch ban u)
 Phng php mng lc

y Kch thc l lc 0,47m hay 0,22m

y ng knh v hnh dng mng lc ph thc vo
ng knh phu lc
y ng knh mng thng l 45mm
Thit b lc nhiu phu
Cc b phn ca thit b lc VSV
 Phng php lc VSV

y Phu lc, gi mng lc phi c v trng

sau mi ln lc
y Mt VSV trong dch lc thch hp:
<150 khun lc/mng
y Th tch dch lc trong 1 ln: 50 -100ml
y Nu th tch dch lc nh hn th phi pha
long bng dd pha long hay nc ct v trng
 Phng php mng lc

y u im
{Xc nh c mt VSV c th trong
mt th tch mu ln: 10ml, 100ml
y Nhc im
{ Khngthch hp cho vic phn tch cc
mu thc phm rn
 Khun lc vsv mc trn mng

1A 1B 2A 2B

1C 1D 2C 2C
E. coli trn mi trng ID Coli agar .
Coliforms trn mi trng ID Coli agar
 NH LNG VSV BNG PP MPN
(Most Probable Number)

9 Phng php MPN da trn nguyn tc xc sut

thng k s phn b VSV trong cc pha long khc
nhau ca mu.
9Mi pha long c nui cy lp li nhiu ln (3
10 ln)
9Cc pha long c chn la sao cho trong cc
ln lp li c mt s ln dng tnh v c mt s ln
m tnh.
9S ln dng tnh c ghi nhn v so snh vi
bng thng k gi tr c on s lng VSV trong
mu.
 NH LNG VSV BNG PP MPN

Hai h thng MPN

- H thng 9 ng
- H thng 15 ng
c im
- Vi sinh vt mc tiu phi c nhng biu hin c
trng trn mi trng nui cy nh
S to hi: Coliforms
S i mu: S. aureus
- Cho php nh lng c mt VSV thp trong
th tch mu ln
 H thng MPN/g(ml)

H thng 9 ng H thng 15 ng

10ml mi trng
NH LNG VSV BNG PP MPN

1.1 1.2 1.3 1.4 1.5

1 - Chun b cc
ng nghim c cha
2.1 2.2 2.3 2.4 2.5 mi trng thch
hp cho s tng
trng ca i
tng VSV cn
3.1 3.2 3.3 3.4 3.5 nh lng
 1ml

1.1 1.2 1.3 1.4 1.5

10-1
1ml
2 - Cy mt th
tch chnh xc
2.1 2.2 2.3 2.4 2.5 dung dch mu
10-2
3 nng
pha long bc
1ml
10 lin tip
3.1 3.2 3.3 3.4 3.5
10-3
1.1 1.2 1.3 1.4 1.5

3 em ng
nghim iu
2.1 2.2 2.3 2.4 2.5 kin thch hp

3.1 3.2 3.3 3.4 3.5

 1.1 1.2 1.3 1.4 1.5

4 Quan st cc
S
biu hin chng
i
2.1 2.2 2.3 2.4 2.5 minh s pht trin
mu
ca vsv cn kim
ca
nh
mi
trng
3.1 3.2 3.3 3.4 3.5
 1.1 1.2 1.3 1.4 1.5
5
5 Ghi nhn s
+ + + + + lng cc ng
2.1 2.2 2.3 2.4 2.5 nghim dng tnh
tng pha long
2
+ +

3.1 3.2 3.3 3.4 3.5

1
+
 5 Tra bng Mac Crady suy ra mt
VSV

Kt
Tra qu
S vsv: 70 MPN/g
2 bng

1
 NH LNG VSV BNG PP MPN
1ml

1.1 1.2 1.3 1.4 1.5

5
10-1 1ml

2.1 2.2 2.3 2.4 2.5

10-2 2 Tra Bng

1ml

3.1 3.2 3.3 3.4 3.5

10-3 1

S vsv: 70 MPN/ml