TIU CHUN VIT NAM TCVN 5156:1990

THT V SN PHM CA THT PHNG PHP PHT HIN V M S STAPHYLOCOCCUS AUREUS Meat and meat products Detection and enumeration of Staphylococcus aureus Trn c s tiu chun TCVN 4830-89 hng dn cibm v Staphylococcus aureus. Tiu chun ny quy nh phng php pht hin v m s Staphylococcus aureus trong 1 gam tht v trn 100 cm2 tht v cc sn phm ca tht dng lm thc phm cho ngi v thc n gia sc. 1. c tnh chung Vi khun hnh trn, gram dng, kch thc 0,5 x 1 micr mt, khng c gip m, khng c nha bo, khng di ng, t thnh chm nho hoc ghp i, hp thnh chui ngn. Sinh trng trong mi trng him kh, ym kh c 7,5% mui; phn gii ng glucose v mannit, ng vn huyt tng, tan hng cu, sc t mu vng. 2. Nguyn tc Cn c vo c tnh sinh ho xc nh vi khun. Pha long mu th cc m khc nhau, ria cy trn mi trng chn lc m v tnh s vi khun. 3. Ly mu: Theo TCVN 5153-90. 4. Thit b v dng c: Theo TCVN 5153-90. 5. Mi trng, huyt tng: 5.1. Phi dng cc loi ho cht tinh khit phn tch, nn s dng cc thnh phn c bn hoc ch sn; khi dng phi theo ch dn ca ni sn xut. Mi trng phi bo qun ni ti, nhit 4 oC, khng qu 1 thng. nh k kim tra li mi trng bng ging vi khun tiu chun. 5.2. Mi trng, huyt tng (ph lc B) 5.2.1. Mi trng thch telurit - glycin (mi trng thch T.G). 5.2.2. Mi trng nc tht. 5.2.3. Mi trng nc tht B.H.I. 5.2.4. Mi trng Chapman. 5.2.5. Dung dch chng mu ng. 5.2.6. Huyt tng. Khi cng huyt tng ng kh phi theo ng ch dn ca ni sn xut. Nu ch huyt tng th ly mu th cho vo bnh ng sn dung dch chng ng theo t l 9 phn mu mt phn dung dch ngn mu ng; ly tm 15-20 pht hng cu lng xung y ng nghim, thu khi nc trong mu vng nht (huyt tng). Cng nh mi trng, trc khi dng phi kim tra li hiu lc bng chng Step. aureus tiu chun. 6. Cch tin hnh 6.1. Yu cu cibm: Cc dng c, mi trng, v.v... v thao tc phi v khun. 6.2. Chun b mu th: Tht lnh ng phi gii ng t nhin nhit trong phng cho n khi t 13 - 15oC, hp phi c ra sch v ngoi, lau kh, 37oC t 5 - 7 ngy. 6.3. Ch huyn ph ban u.

6.3.1. m s vi khun trong 1 gam sn phm: cn 100 - 225 gam (khng ly m, ly c cht lng nu c) nghin trc trong ci s, nghin nhuyn tip trong my xay tht t 2 - 3 pht vi tc 10.000 vng/pht, va nghin va b sung dn 9 phn mi trng nc tht hoc B.H.I (10 -1) 6.3.2. m s vi khun trn 100 cm2 sn phm: Dng nhng ming giy thm v khun c kch thc 5 x 5 cm c lm t bng nc mui ng trng, ri dn ln b mt sn phm cc v tr khc nhau t 4 - 8 ming (100 - 200 cm2). Sau 2 pht chuyn ton b giy thm vo bnh ng sn 10 - 20 ml mi trng nc tht hoc nc tht B.H.I. v 10 - 15 vin bi thu tinh. Lc cho tan giy, tu theo khi lng mu th cn s dng m pha long thnh 1:1 (1ml tng ng vi 1 cm2) 6.4. m s vi khun: T huyn ph ban u 6.3 tip tc pha long 10 ln (10-1, 10-2, 10-3 ...) (ty mc nhim khun m quyt nh). Mi m cy vo 2 a mi trng thch Telurit glycin mt khi lng bng nhau trong khong t 0,1 - 1 ml (ty mc nhim khun m quyt nh), dn u trn mt thch hoc trn u trong thch nng lng 45 oC, t p a mi trng, 37 oC t 24 - 48 gi, c kt qu. Khun lc Stap. aureus trn, mt nhn, vin mp gn, mu en, c vng bao quanh mu trng c, sau 48 gi chuyn thnh vng trng rng khong 3 mm, cn cc t cu khun khc mu xm nht. Cn c vo hnh dng v mu sc trn m m. Nu nghi ng th phn thnh 2 nhm: in hnh v khng in hnh; chn trong mi nhm 5 khun lc gim nh phn ng ng vn tuyt tng m xc nh t l (theo ph lc B, TCVN 5155-90). 6.5. Gim nh tnh cht sinh ho: 6.5.1. Trn mi trng Chapman: Cy (6.4) vo 2 a mi trng, 37 oC trong 24 gi, c kt qu. Khun lc Stap. aureus trn, hi c, mt nhn, vin mp gn, mu vng. 6.5.2. Th phn ng ng vn trn phin knh: Nh 1 git nc mui ln phin knh, dng que cy ly vi khun va (6.4) trn thnh huyn dch c u, nh tip 1 git huyt tng vo v thm th nghim i chng m tnh (1 git nc mui v 1 git huyt tng). c kt qu bng knh lp hoc knh hin vi. 6.5.3. Th phn ng ng vn trong ng nghim: Ch lm khi 6.5.2. m tnh. Cy vi khun 6.4. vo mi trng nc tht B. H.I , 37 oC t 20 - 24 gi. Sau chuyn 0,1 ml canh khun vo ng nghim c 10 x 75 mm cha sn 0,3 ml huyt tng pha long theo t l 1 phn huyt tng, 3 phn nc. Lm thm th nghim i chng m tnh (0,1ml nc mui v 0,3 ml huyt tng pha long) trn u, 37 oC; c kt qu sau 2, 4, 6, 8 v 24 gi. Ch thch: Nu dng huyt tng ng kh thng phm th theo hng dn ca ni sn xut 7. Tnh ton kt qu 7.1. Cn c vo kt qu gim nh tnh cht sinh ho 7.2. Cn c vo cng thc di y tnh s Staphylococcus aureus trong 1 gam hoc trn 100 cm2 sn phm (theo ph lc B, TCVN 5155-90)

PH LC A
PHC KIM NGHIM

PH LC B
PHA CH MI TRNG B1. Mi trng thch Telurit - glycin ( thch T.G.). B1.1. Mi trng c bn: - Trypton - Bt cao tht - Cht trit men (yeast extract) - Glycin - Lithium clorua - Mannit - Thch - Nc ct - Nu cn th cho thm dung dch sulfamezatin Ch thch 1/ C th thay 1000 ml nc ct v 5g bt cao tht bng 1000 ml nc tht. 2/ Dung dch Sulfamezatin (dng khi mu th nghi c vi khun Proteus): - Sulfamezatin - Natrihydroxyt, dung dch 0,1N - Nc ct va B1.2. Dung dch Telurit: 0,2g 10 ml 100 ml 10g 5g 5g 12g 5g 20g 18 - 20g 1.000ml 27,5ml

Ho tan cc thnh phn trn trong nc si. Chnh pH 7,4 - 7,6. Hp 115oC trong 15 pht.

Ho tan Sulfamezatin trong dung dch Natrihydroxyt. Cho thm nc ct va 100 m.

- Kali telurit - Nc ct B1.3. Dung dch Natri pyruvat: - Natri pyruvat - Nc ct B1.4. Mi trng hon chnh: - Mi trng c bn (B1.1) - Dung dch Telurit (B1.2) - Dung dch pyruvat (B1.3) B2. Mi trng nc tht B.H.I. - Pepton - Bt c b - Bt tim b - Glucos - Natri clorua - Natrihydro photphat ( Na2HPO4) - Nc ct

1g 100ml

Ho tan trong nc un nng. Lc v khun. Bo qun 0 - 4oC. 20 g 100 ml

Ho tan trong nc un nng. Lc v khun. Bo qun 0 - 4oC. 94 ml 1 ml 5 ml

Bng thao tc v khun, chia ra cc a petri, mi a 15 - 20 ml. 10g 12,5g 5g 2g 5g 2,5g 1000 ml

Ho tan cc thnh phn trn trong nc si. Chnh pH 7,6. Chia vo cc bnh thu tinh mi bnh t 10 - 20 m. Hp 121oC trong 20 pht. B3. Mi trng nc tht: - Nc tht b - Pepton - Natri clorua 1000 ml 10g 5g

Ho tan pepton v Natri clorua trong nc tht, un si. ngui, chnh pH 7,4 - 7,6. Chia vo cc bnh thu tinh, mi bnh t 10 - 20 ml. Hp 121oC trong 20 pht. B4. Mi trng Chapman: - Mannit - Bt cao tht - Pepton - Natri clorua - Thch - trung tnh - Nc ct 10g 1g 10g 75g 18 - 20 g 0,025 g 1000 ml

Ho tan cc thnh phn trn trong nc un si. Chnh pH 7,6. Hp 121oC trong 15 pht. Chia vo cc a petri mi a 15 - 20 ml.

B5. Dung dch ngm mu ng: - Natri xitrat - Nc ct 7,8g 100 ml

Ho tan trong nc. Chia vo cc l nh t 3 - 5 ml. Hp 121oC trong 15 pht.