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 c u

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 Peptone Nc ct va 1g 8,5g 1 lt

y 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
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 i m

- 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

thch hp cho vic phn tch cc mu thc phm rn

y Nhc im
{ Khng

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)

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 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

1ml

NH LNG VSV BNG PP MPN

1.1 1.2 1.3 1.4 1.5 5

10-1

1ml

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