Phng php nh lng

GV: NGUYN VN HNH

Phng php nh lng l g ?

L cc phng php xc nh s lng vsv trong mu. Tr li cho cu hi: Hm lng vi sinh vt trong mu l bao nhiu ? 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)

n vi tnh:

t bo/ml CFU/ml MPN/ml

t bo/g CFU/g MPN/g

Phng php m trc tip

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

Saline peptone water (SPW)
NaCl Peptone Nc ct va 1g 8,5g 1 lt

Buffered peptone water (BPW)

NaCl Peptone Nc ct va 5g 10g 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
Chun b a petri v trng Mi trng c chun b - hp kh trng v c bo 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

Mi trng phi c chun b trn a trc 1-2 ngy kh mt 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

m tt c khun lc n l mc trn mi trng Thng chn nhng a c s khun lc khong 30 300 Dng bt m cc khun lc m 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

Kch thc l lc 0,47m hay 0,22m ng knh v hnh dng mng lc ph thc vo ng knh phu lc ng knh mng thng l 45mm

Thit b lc nhiu phu

Cc b phn ca thit b lc VSV

Phng php lc VSV

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

Phng php mng lc

u im
Xc nh c mt VSV c th trong mt th tch mu ln: 10ml, 100ml

Nhc im
Khng thch hp cho vic phn tch cc mu thc phm rn

Khun lc vsv mc trn mng

1A

1B

2A

2B

1C

1D

E. coli trn mi trng ID Coli agar

2C . Coliforms

2C
trn mi trng ID Coli agar

NH LNG VSV BNG PP MPN (Most Probable Number)

Phng php MPN da trn nguyn tc xc sut thng k s phn b VSV trong cc pha long khc nhau ca mu. Mi pha long c nui cy lp li nhiu ln (3 10 ln) Cc 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. S 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 10-1
1ml

1.2

1.3

1.4

1.5

10-2
1ml

2.1

2.2

2.3

2.4

2.5

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

3.1 10-3

3.2

3.3

3.4

3.5

1.1

1.2

1.3

1.4

1.5

2.1

2.2

2.3

2.4

2.5

3 em ng nghim iu kin thch hp

3.1

3.2

3.3

3.4

3.5

1.1

1.2

1.3

1.4

1.5

S i mu ca mi trng

2.1

2.2

2.3

2.4

2.5

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

3.1

3.2

3.3

3.4

3.5

1.1

1.2

1.3

1.4

1.5

5
+ + + + +

2.1

2.2

2.3

2.4

2.5

2
+ +

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

3.1

3.2

3.3

3.4

3.5

1
+

5 Tra bng Mac Crady suy ra mt VSV 5

Kt qu

Tra 2 bng

S vsv: 70 MPN/g

NH LNG VSV BNG PP MPN

1ml

1.1 10-1
1ml

1.2

1.3

1.4

1.5 5

2.1 10-2
1ml

2.2

2.3

2.4

2.5 2
Tra Bng

3.1 10-3

3.2

3.3

3.4

3.5 1
S vsv: 70 MPN/ml