CHNG 2

CC PHNG PHP NH LNG

VI SINH THC PHM

I. PHNG PHP M TRC TIP

- Dng xc nh cc loi vi sinh vt n bo c kch thc ln: nm men, to n bo, bo t mc - Quy trnh cho php xc nh nhanh chng s lng vi sinh vt

-Khng phn bit c t bo sng v cht

-Khng phn bit c cc t bo vi sinh vt v ht vt th -Hn ch i vi huyn ph c mt thp

Bung m vi sinh vt

m trc tip bng bung m Goriep

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

Qui trnh xc nh s lng t bo vsv trc tip bng bung m hng cu Quy trnh:

- Pha long mu (5-10 t bo/ nh)

- Np mu vo bung m

- m s lng t bo hin din trong 5 ln

Tnh mt : Mt t bo: N/ml = 0,25a x 106 t bo/ml a: trung bnh cng s lng t bo trong 5 ln

II. PHNG PHP M KHUN LC

- Dng xc nh cc loi vi sinh vt sng trong mu - nhy cao, nh lng vi sinh vt mt thp - C th nh lng cc vi sinh vt kch thc nh, di ng

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

8,5g 1,0g 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

Cc bc tip theo tng t nh pha long mu lng

Quy trnh:
- Pha long mu (25-250 khun lc/a) - To hp - Nui , m s 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 (thch ng c) em nhit thch hp

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 150300 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

Bt m khun lc

Cc thit b h tr m khun lc

My m khun lc

My m khun lc t ng

PHNG PHP M KHUN LC

Tnh kt qu:

N (CFU / ghayCFU / ml )

(n1vd1 ... ni vdi )

Vi : N: s t bo (n v hnh thnh khun lc) vi khun trong 1g hay 1ml mu C: Tng s khun lc m c trn cc a petri chn ni : S hp petri cy mu ti pha long th i di: nng pha long th i v: Th tch mu cy vo trong mi a

NH LNG TNG VI SINH HIU KH TRONG MU THC

10 g mu + 90 g Salin Pepton Watter

ng nht bng stomacher / 30 giy

Dung dch 10 -1 Pha lang 10-2, 10-3, 10-4,.. Cy 1ml/petri 3 pha long lin tip 2 petri/ pha lang Cho 15-20 ml PCA/petri. Lc u

 ng c
Lt ngc a, m 301oC/ 726h Khun lc

Chn a c 25 250 khun lc m

Tng VSV hiu kh/g (TPC) N TPC = n1V1F1 + .+ niViFi N: Tng KL; n: s a cho mi nng V: Th tch cy (=1) ; F: pha lang

III. PHNG PHP DNG MNG LC

- nh lng vi sinh vt c mt thp - nh lng thng qua s lng khun lc hoc theo phng php MPN

- Kch thc l lc: 0,45 m hoc 0,2 m

- Thng dng mng lc k nc, in cc vung ngn cn s mc lan ca khun lc

B lc v mng lc v trng

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

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 lc

1A

1B

2A

2B

1C

1D

E. coli trn mi trng ID Coli agar

2C . Coliforms

2C
trn mi trng ID Coli agar

QUI TRNH XC NH S LNG VSV BNG PP MNG LC

Quy trnh: - Kh trng dng c lc - Lc chn khng - Nui cy mng lc trn mi trng dinh dng - m s lng khun lc trn a petri Tnh mt : A (cfu/ml)= N*d/V

Trong : V: th tch mu th tch mu th (50 or 100ml)

N: tng s khun lc trn mng lc d: pha long mu

Vi khun pht trin trn mng lc

IV. PHNG PHP DNG PETRIFILM

- K thut dng kim tra tng vi khun hiu kh, coliform, coliform phn, nm mc, nm men

- K thut d thao tc
- Tit kim khng gian , bo qun, thi hn s dng lu - Khng cn hp kh trng mi trng

V. PHNG PHP MPN (MOST PROBABLE NUMBER)

- nh gi s lng vi sinh vt theo s lng vi sinh vt c xc sut ln nht hin din trong mt n v th tch mu - L phng php nh lng da trn kt qu nh tnh ca mt lot th nghim c lp li mt s pha long khc nhau - Kt qu nh tnh: sinh hi, i mu, ho c ca mi trng nui cy dng tnh - chnh xc ph thuc vo s ng nghim lp li mi pha long (3 hoc 5 ln) - Phng php dng nh lng mi nhm vi sinh vt nui trn mi trng chn lc, cho kt qu biu kin.

Nguyn tc
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.

 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 9 ng
10ml mi trng

H thng 15 ng

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
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 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

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

IV. PHNG PHP MPN (MOST PROBABLE NUMBER)

Phng php MPN (lot 5 ng)

Bng tra MPN (lot 3 ng)

S lng ng dng tnh S ml mu s dng 10 1 0,1 0 0 0 0 0 1 0 0 2 0 0 3 3 3 0 3 3 1 3 3 2 3 3 3

S MPN/100 ml
3 6 9 240 460 1100 -

Bng tra MPN (lot 5 ng)

Quy trnh:

- Pha long mu bc 10,chn 3 nng lin tip

- Cy vo lot ng nghim (9 hoc 15 ng) cha mi trng chn lc mt lng mu xc nh t 3 nng chn

- Nui , xc nh s ng dng tnh 3 nhm

- Tra bng Mac Cradytr s MPN (a) Tnh mt :

Vi: a: tr s tra t bng Mac Crady

V2 S MPN/100ml (MPN/1g) = a x xd V1

V1: th tch mu s dng (thng l 1)

V2: th tch mu theo bng Mac Crady lot ng u tin (10ml)

d: pha long lot ng u tin

VI. PHNG PHP O C

PHNG PHP O C
- c ca huyn ph t l thun vi s lng t bo vi sinh vt c trong mi trng. Trong mt thi gian nht nh, mi quan h gia s lng vi sinh vt trong nc t l tuyn tnh vi c ca mi trng. - nh lng vi sinh vt trong mi trng bng my o c hay my so mu bc sng 550- 610nm. - Trong phng php ny cn xy dng biu tng quan tuyn tnh gia c v s lng vi sinh vt trong mi trng bng phng php m khun lc trc tip

PHNG PHP O C

Phng php tin hnh:

Pha long huyn ph cha vi sinh vt cn kim nghim sao cho cc dung dch thu c khi o trn my so mu bc

sng 610nm c cc tr s OD gn 0,1; 0,2; 0,3; 0,4; 0,5.

Dng phng php m trc tip xc nh s lng t bo vi sinh vt c trong 1ml k hiu N/ml

Tnh gi tr log(N/ml) cho tng nng pha long, V th

biu din log(N/ml) theo cc gi tr OD. Xc nh khong tuyn tnh gia log(N/ml) vi OD.

Tnh mt
T tr s OD o c i chiu trn th tng quan gia mt t bo v OD xc nh s lng t bo c trong mu. N/ml = 10a vi a=log (N/ml).

VII. PHNG PHP ELISA (Enzym linked immunosorbent asay)

- ELISA (Phng php hp th min dch dng
enzyme) da trn nguyn tc phn ng kt hp gia t bo (khng nguyn) vi mt khng th c hiu.

- Tn hiu phn ng min dch c nhn bit qua

s ngng ta, kt dnh khng nguyn khng th

hoc bng nhng khng th c nh du (pht

hunh quang ng v phng x hay enzyme)

a ging ELISA

H THNG ELISA

My ra

My c

QUI TRNH NH LNG CC CH TIU VI SINH TRONG THC PHM

CC CH TIU VI SINH TRONG THC PHM

1. Tng vi sinh hiu kh 2. Tng nm men, nm mc 3. Coliform tng s 4. E.coli 5. Sallmonella 6. Staphylococcus aureus 7. Bacillus aureus .