TIU CHUN VIT NAM

TCVN 5375 - 1991

SN PHM ONG - YU CU VI SINH V PHNG PHP KIM TRA
Honey-Bee Products. Micro biological requirements and examination methods
Li ni u
TCVN 5375-1991 do Cc Chn nui v Th y, B Nng nghip v cng nghp thc phm bin
son, Tng cc Tiu chun - o lng - Cht lng ngh v c y ban Khoa hc Nh
nc ban hnh theo quyt nh s 343/Q ngy 11 thng 6 nm 1991.
SN PHM ONG - YU CU VI SINH V PHNG PHP KIM TRA
Honey-Bee Products. Micro biological requirements and examination methods
Tiu chun ny qui nh nhng yu cu vi sinh v phng php kim tra, p dng cho cc sn
phm ong, bao gm mt ong, phn hoa v sa ong cha t nhin.
1. Yu cu vi sinh
Yu cu vi sinh vt i vi sn phm ong c trnh by trong bng sau:
TT

Tn ch tiu

Mc, s khun lc trong 1 sn phm

Tng s vi khun hiu kh khng ln hn

E.Coli

Vi khun k kh sinh H2S

-nt-

Salmonella

-nt-

Staphylococcus aureus

-nt-

Nm mc:
- Mt ong v sa ong cha t nhin

15.000
Khng cho php c

Khng cho php

- Phn hoa: khng ln hn

100

Nm men: khng ln hn

1.000

2. Phng php kim tra:

2.1. Ly mu: theo TCVN 5261-90
2.2. X l mu:
Ti phng th nghim, mu sn phm ong c a vo bung, v trng v pha long trc tip
bng cch dng hai bnh cu, cho vo mi bnh 10 gam ( 0,1 gam) mu sn phm cn kim tra
(vi mu l phn hoa th c th pha long bng cch dng vin bi thy tinh), sau thm vo
mi bnh 90ml nc ct v lc cho tan hon ton (ta thu c nng pha long c s l 1/10).
em mt trong 2 bnh trn un si 10 pht, sau b li phn nc bay hi cho 100ml
dung dch. Bnh ny dng kim tra s c mt ca cc vi khun k kh.
T nng pha long 1/10, tip tc pha long c mt nng pha long na bng cch:
dng pipt v trng ht 1ml dung dch ban u sang ng nghim th 2, cho thm 9ml nc ct
vo v trn u. Nh vy ta thu c nng pha long 1/100.
Bng phng php trn, tip tc pha long c c cc nng tip theo l 1/1.000;
1/10.000

Ch thch: trong trng hp kim tra nh gi cht lng sn phm, phi dng 2 dy mi
trng, mi dy gm 2 ng nghim hoc 2 a petri mi trng cho 1 nng pha long.
2.3. Thit b, dng c:
- Thit b x l khng kh (bung v trng, n t ngoi hoc t Laminar);
- My sy kh, c th duy tr 165-170oC;
- My hp t (Antoclave), c th duy tr 121 2oC;
- T m;
- My o pH;
- T lnh;
- Cn k thut;
- Knh hin vi hai mt;
- Knh lp;
- Cc loi que cy vi trng v nm mc;
- Cc ong 50; 100; 250; 1.000ml;
- Bnh cu y bng 100; 250; 500ml;
- ng nghim thy tinh tiu chun;
- a petri;
- Pipet cc loi 1; 2; 5ml;
- n cn;
2.4. Ha cht v mi trng:
- Natri hydroxyt, dung dch 1N;
- Axit clohydric, dung dch 1N;
- Mi trng thch thng;
- Mi trng canh thang gan;
- Mi trng thch Sabouraud dextroza;
- Mi trng thch Czapex-dox;
- Mi trng Rose-bengal cloramphenicol;
- Dung dch fucxin 4%;
Ch thch:
+ Vt liu ca cc loi dng c thy tinh phi m bo chu nhit trong trng hp hp sy
kh trng nhiu ln khng b nt, v.
+ Tt c cc loi dng c thy tinh trc khi s dng phi c hp kh trng bng ni t
121oC trong 20 pht hoc sy kh trng bng t sy 165-170 oC trong 30 pht.
+ iu chnh pH ca mi trng bng dung dch natrihydroxyt 1N hoc dung dch axit
clohydric 1N.
2.5. Tin hnh kim tra:
Vic kim tra cc ch tiu vi sinh vt i vi sn phm ong c thc hin trong bung v trng
hoc trn t Laminar, nhm m bo cho mu khng b nhim khun.
2.5.1. Kim tra tng s vi khun hiu kh:

T mi nng pha long ca mu sn phm ong cn kim tra (iu 2.2), dng pipet v trng
ht 1ml v nh u vo cc a petri, thm vo mi a mt lng 15 ml mi trng thch
thng c hm nng 45oC. yn cc a petri cho thch ng cng li v trong t
m 37oC trong 24 n 48 gi, sau ly ra m s khun lc trong cc a petri. Tnh tng s
(X) vi khun hiu kh c trong 1 gam sn phm theo cng thc:
X

S khun lc trung
bnh m c

1
Khi lng mu th

1
Bi s pha long

2.5.2. Kim tra E.Coli

Theo TCVN 5155-90
2.5.3. Kim tra Staphylococcus aureus:
Theo TCVN 4830-89 (ISO 6888-1983)
2.5.4. Kim tra vi khun k kh sinh H2S:
Dng pipet v trng ht 2 ml dung dch mu sn phm ong cn kim tra (trong bnh un si v
pha long cc nng khc nhau). Cy vo 3 ng mi trng canh thang gan ( gn b
nc). cc ng vo bp cch thy 80oC trong 15 pht, sau ly ra, ln pha trn cc
ng ny mi ng mt lng l 15ml thch ( hm nng 45 oC) v lm ngui nhanh bng cch
tt c vo t lnh hoc ngm vo nc lnh. cc ng trn trong t m 37 oC trong 72 gi.
nh gi kt qu:
Kt qu dng tnh c xc nh khi cc ng trn c hin tng sinh kh H 2S lm nt thch
pha trn. Mi trng nui cy bin mu en v c mi hi thi.
2.5.5. Kim tra Salmonella:
Theo TCVN 4829-89(ISO 6579-1981)
2.5.6. Nm mc v nm men:
Dng pipet v trng ht 1 ml dung dch mu cn kim tra ( pha long cc nng khc
nhau), nh u vo cc a petri (d: 90 x 120 mm). ln pha trn mt lng 15ml mi trng
thch Savouraud dextroza (pH: 4,5 -5,0) hm nng n 45oC v yn cc a trn n khi
thch ng, lt ngc a petri v trong t m 26-30 oC trong 24 n 48 gi.
nh gi kt qu: sau 48 gi nui cy, ly cc a petri ra v dng knh lp pht hin v m s
bo t nm mc v nm men mc trn cc a mi trng. Cn c vo mu sc ca cc bo t,
kt hp vi vic phit knh nhum bng dung dch fucxin 4% v soi di knh hin vi xem c
im hnh thi v cu to nhn dng cc chng nm sinh c t.
Ch thch: C th dng mi trng thch Czapex-dox hoc thch Rose-bengalcloramphenicol
tip tc phn lp, xc nh cc chng nm mc hoc nm men gy bnh.

PH LC
CC LOI MI TRNG
1. Mi trng thch thng:
- Cao tht

5,0g

- Pepton

15,0 g

- Natri clorua

5,0 g

- Thch

25,0 g

- Nc ct va

1.000 ml

iu chnh pH; 7,4- 7,6, sau hp kh trng bng ni hp t 121 oC trong 30 pht.
2. Mi trng canh thang gan:
- Tht b xay nh

180,0g

- Gan b xay

500,0g

- Axit clohydric 37%

75,0 ml

- Pepsin vayang hiu gi 200 :

10 g

- Nc ct va

9.000ml

cc thnh phn trn trong ni men, gi nhit 40-50oC trong 18-24 gi, sau un trong
30 pht nhit 80oC, iu chnh pH: 7, 4, lc v hp kh trng trong ni hp t 121 oC
trong 30 pht.
3. Mi trng thch Sabouraud dextroza:
- Mycological pepton

10,0 g

- Dextroza

40,0 g

- Thch

15,0 g

- Nc ct va

1.000 ml

iu chnh pH: 5,2-5,5, sau lc cho tan hon ton v hp kh trng bng ni hp t 121 oC
trong 30 pht
4. Mi trng thch Czapex dox:
- Sacaroza

30,0 g

- Natri nitrat

2,0 g

- Natri clorua

0,5 g

- Magie glyxerophotphat

0,5g

- St sunphat

0,01g

- Kali dihydrophotphat

1,0 g

- Thch

20,0 g

- Nc ct va

1.000 ml

iu chnh pH 6,8 0,2 v un si cho tan hon ton sau hp kh trng bng ni hp t
121oC trong 30 pht.
5. Mi trng thch Rosa-bengal cloramphenicol:
- Mycological pepton

5,0 g

- Dextroza

10,0 g

- Dikali hydrophophat

1,0 g

- Magie sunphat

0,5 g

- Rose bengal

0,05 g

- Thch

15,5 g

- Nc ct va

1.000 ml

iu chnh pH 7,2 0,2 un si cho tan ton b, sau hp kh trng bng ni hp t

121oC trong 30 pht. ngui cn 50oC cho thm 100 mg cloramphenicol v lc nh cho ha
tan.