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.
2.
3.
4.
5.

Phng php m trc tip

Phng php m khun lc
Phng php m khun lc trn mng lc
Phng php o c
Phng php MPN (most probable number)

y n vi tnh:

t bo/ml
CFU/ml
MPN/ml

t bo/g
CFU/g
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
y
y
y
y

qun mt 45oC trong b iu nhit

Ht 1ml mu vo a trng (chn nng thch hp)
vo a cy 10 15ml mi trng, lc u
ngui mi trng
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
lc

My m khun
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
{ Khng

thch hp cho vic phn tch cc

mu thc phm rn

Khun lc vsv mc trn mng

1A

1B

1C

1D

E. coli trn mi trng ID Coli agar

2A

2C
.
Coliforms

2B

2C
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

2.1

2.2

2.3

2.4

2.5

3.1

3.2

3.3

3.4

3.5

1 - Chun b cc
ng nghim c cha
mi trng thch
hp cho s tng
trng ca i
tng VSV cn
nh lng

1ml

1.1

1.2

1.3

1.4

1.5

10-1
1ml

10-2

2.1

2.2

2.3

2.4

2.5

3.1

3.2

3.3

3.4

3.5

1ml

10-3

2 - Cy mt th
tch chnh xc
dung dch mu
3 nng
pha long bc
10 lin tip

1.1

1.2

1.3

1.4

1.5

2.1

2.2

2.3

2.4

2.5

3.1

3.2

3.3

3.4

3.5

3 em ng
nghim iu
kin thch hp

1.1

S
i
mu
ca
mi
trng

1.2

1.3

1.4

1.5

2.1

2.2

2.3

2.4

2.5

3.1

3.2

3.3

3.4

3.5

4 Quan st cc
biu hin chng
minh s pht trin
ca vsv cn kim
nh

1.1

1.2

1.3

1.4

1.5

5
+

2.1

2.2

2.3

2.4

2.5

3.2

3.3

3.4

3.5

2
3.1

1
+

5 Ghi nhn s
lng
cc
ng
nghim dng tnh
tng pha long

5 Tra bng Mac Crady suy ra mt

VSV
5

Tra
2 bng

Kt
qu

S vsv: 70 MPN/g

NH LNG VSV BNG PP MPN

1ml

1.1
10-1

1.2

1.3

1.4

1.5
5

1ml

2.1

2.2

2.3

2.4

2.5

10-2

Tra Bng

1ml

3.1
10-3

3.2

3.3

3.4

3.5
1
S vsv: 70 MPN/ml