TIU CHUN VIT NAM TCVN 5153:1990

THT V SN PHM THT PHNG PHP PHT HIN SALMONELLA Meat and meat products Detection of Salmonella Trn c s tiu chun TCVN 4829-89 hng dn cibm v cc phng php pht hin Salmonella. Tiu chun ny quy nh phng php pht hin Salmonella trong tht v sn phm ca tht dng lm thc phm cho ngi v thc n gia sc. 1. c tnh chung Vi khun hnh gy, mp, ngn, gram m, kch thc 0,5 - 0,7 x 1 - 3 micrmt, hai u hi trn, khng c gip m, khng c nha bo, di ng.Vi khun c nhng c tnh sinh ho v huyt thanh c quy nh kim nghim trong tiu chun ny. 2. Nguyn tc Cn c vo tnh cht sinh ho v khng nguyn ca vi khun kt lun. 3. Ly mu 3.1. Ly mu trung bnh trong tng l hng ng nht. 3.2. L hng ng nht l mt lng tht hoc sn phm ca tht c cng mt tn gi, cng loi sn phm, cng mc cht lng, c ng trong bao b cng kiu, cng kch thc, cng khi lng, c sn xut, ch bin, ng gi, bo qun trong cng mt c s sn xut, theo cng mt quy trnh cng ngh, trong khong mt thi gian lin tc, c giao nhn cng mt ln, kim tra cng mt lc v khng qu 20 tn vi tht ti hoc lnh ng, 5 tn vi tht ch bin ng hp hoc ng trong bao b. 3.3. Mu trung bnh l lng sn phm bng 0,1 - 0,2% khi lng mt l hng nhng khng t hn 3 ti i vi tht lnh ng, 10 hp i vi cc loi sn phm ch bin v c ly cc v tr khc nhau bng phng php ngu nhin. 3.4. Trc khi ly mu trung bnh phi kim tra cc giy t, xc nh tnh ng nht, iu kin bo qun, . . . loi b nhng kin khng ng tiu chun. 3.5. Mu th trung bnh l lng sn phm bng 80% mu trung bnh c dng nh sau: - 20% kim tra cm quan. - 20% kim tra l ho, kim loi nng, tn d thuc tr su, thuc khng khun. - 40% kim tra vi sinh vt. - 20% lu mu. 3.6. Mu lu do c quan kim dch gi th li khi cn thit v c bo qun theo ng quy nh vi tng loi sn phm; tht ti, tht lnh ng nhit khng ln hn -15 oC, cc sn phm khc khng ln hn 4oC. 3.7. Thi gian lu mu khng qu 4 thng i vi hp thanh trng v khng qu 1 thng i vi cc sn phm khc. 3.8. Nu kt qu cn nghi ng th c quan kim dch phi thng bo cho ch hng bit v ly mu th li ln hai vi s lng gp i. 3.9. Khi cn thit c quan kim dch c php gi mu n cc c quan chuyn trch trong v ngoi nc nh kim nghim.

3.10. Trong qu trnh kim tra, ly v lu mu cn c h s ghi chp y . 4. Thit b v dng c 4.1. Thit b 4.1.1. Phng cy v khun (thanh trng khng kh trong phng cy bng n cc tm 1,5 - 2,5 ot/1cm3 trong 3 gi hoc xng phc mn, phenol 5%, ...). 4.1.2. T m c iu chnh nhit n nh 35 - 37 - 43oC. 4.1.3. T sy kh. 4.1.4 T hp t. 4.1.5. Ni hp cch thu. 4.1.6. T lnh 0 - 4oC. 4.1.7. T lnh -35oC . 4.1.8. Ni cch thy c iu chnh nhit n nh t 35 - 56oC . 4.1.9. Cn phn tch c chnh xc 0,01g v 0,1g. 4.1.10. Cn thin bnh 50 - 100g. 4.1.11. My o pH c chnh xc hiu chnh l 0,1 n v pH 25oC. 4.1.12. My li tm 3.000- 15.000 vng/ pht. 4.1.13. Knh hin vi c vt knh du. 4.1.14. Ci chy s c trung bnh. 4.1.15. My xay nghin 10.000 vng/ pht tr ln. 4.1.16. Dao m, ko ct, pince kim loi. 4.1.17. Que cy c vng trn u ng knh khong 3 mm v que cy thng. 4.1.18. Khay men. 4.1.19. n cn. 4.1.20. Phu lc Seitz, Sephadex G 25. 4.2. Dng c thu tinh. 4.2.1. Bnh tam gic v bnh cu y bng 150, 200, 250 v 500 ml. 4.2.2. Cc ong hnh tr 20, 50, 100, 500 ml. 4.2.3. Pipt c khc 1,5 v 10 ml. 4.2.4. ng nghim 18 x 160 mm. 4.2.5. a petri ng knh 150 mm, chiu cao 15mm, mt a phng v song song vi y. 4.2.6. Phin knh . 5. Mi trng, thuc th v khng huyt thanh. 5.1. Phi dng cc ho cht tinh khit phn tch. Nn s dng nhng thnh phm c bn hoc ch sn; khi dng phi tun theo ch dn ca ni sn xut. Mi trng, thuc th phi bo qun ni ti, nhit 4oC, khng qu 1 thng. nh k kim tra li mi trng, thuc th bng cc ging vi khun tiu chun. 5.2. Mi trng v thuc th (ph lc B). 5.2.1. Mi trng tetrationat (Muller - Kauffman).

5.2.2. Mi trng Salmonella - Shigella (S.S). 5.2.3. Mi trng thch deoxycholatxitrat. 5.2.4. Mi trng thch st ng trisaccarit (TSI). 5.2.5. Mi trng nc tht. 5.2.6. Mi trng thch dinh dng. 5.2.7. Mi trng thch ur (Christensen). 5.2.8. Mi trng lysin decarboxylas. 5.2.9. Mi trng nc pepton (th phn ng Indol). 5.2.10. Mi trng nc pepton glucose (th phn ng V.P.). 5.2.11. Thuc th B - Galactosidas (O.N.P.G.). 5.2.12. Thuc th Indol (dung dch Kowacs). 5.2.13. Thuc th Voges - Proskauer (dung dch Barritt). 5.2.14. Nc mui ng trng. 5.2.15. Khng huyt thanh Salmonella a gi, n gi, Vi v H. 6. Cch tin hnh. 6.1. Yu cu chung: Cc dng c, mi trng, thuc th, 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. Lm phong ph vi khun trong mu th: Cn 25g (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 225 ml mi trng tetrationat, 43 oC t 20 - 24 gi. 6.4. Gim nh tnh cht sinh ho. 6.4.1. Ly mt vng que cy canh trng tetrationat (6.3) ria ln mt thch SS. v thch deoxycholat-xitrat mi loi 2 a, hoc dng mt trong hai loi mi trng, cn loi th hai c s xt nghim c ty chn nh thch Endo, Macconkey, E.M.B, Istrati, v.v..., p sp a mi trng, 37oC trong 24 gi, c kt qu. Khun lc Salmonella hnh trn, ng knh t 1-2 mm, ra gn, mt li, khng mu, bng lng(dng S). Lm phin nhum gram, gim nh bng knh hin vi vi vt knh du: Vi khun gram m, c hnh dng nh mc 1. 6.4.2. Chn 5 khun lc in hnh, ria cy 2 - 4 a thch, 37oC t 18 - 24 gi gim nh tip. 6.4.3. Trn a mi trng thch st ng trisaccarit (TSI): Dng que cy u thng, ria cy vi khun trn mt thch nghing sau cm su xung y theo ng thng ng, 37 oC trong 24 gi, c kt qu. Salmonella th hin: Trn mt thch nghing c mu (khng phn gii lactose v sucrose); thn v y c mu vng, c bt kh hoc vt nt (phn gii glucose, sinh hi), c mu en (H 2S) . 6.4.4. Trn mi trng thch ur (Christensen): Ria cy vi khun trn mt thch nghing, 37oC trong 24 gi, c kt qu. Salmonella c phn ng m tnh: khng chuyn mu (dng tnh: mu ). 6.4.5. Trn mi trng lysin decacboxylas:

Cy vi khun xung di b mt mi trng hoc sau khi cy vi khun nh parafin lng v khun, dy khong 4 - 5 mm ln mi trng, 37 oC trong 24 gi, c kt qu. Salmonella c phn ng dng tnh: mu tm. (m tnh: mu vng). 6.4.6. Th B - Galactosidas (O.N.P.G): Ho tan mt vng que cy khun lc t mi trng T.S.I trong ng nghim c 0,25 ml nc mui ng trng v khun. Nh 1 git toluen, lc u, cch thu 37 oC trong 15 pht; nh tip 0,25 ml thuc th, lc u, tip tc cch thu 37oC trong 24 gi; c kt qu. Salmonella c phn ng m tnh: khng chuyn mu. (dng tnh: mu vng) 6.4.7. Th phn ng Indol: Cy vi khun vo mi trng nc pepton, 37 oC t 24 - 48 gi, sau nh 1 ml thuc th, c kt qu. Salmonella c phn ng m tnh: mu vng nht (dng tnh: mu ). 6.4.8. Th phn ng Voges- Proskauer: Cy vi khun vo mi trng nc pepton glucos, 37oC trong 48 gi, chuyn 1 ml canh trng sang mt ng nghim khc, ri nh 0,6 ml dung dch cn -naptol, 0,2ml dung dch 40% Kalihydroxyt, lc u, c kt qu sau 5 - 15 pht. Salmonella c phn ng m tnh: khng mu hoc vng nht (dng tnh: mu ). 6.4.9. Nhn nh tnh sinh ho c hiu: Tnh cht sinh ho TSI lactos TSI sucros TSI glucos TSI glucos sinh hi TSI hydro-sulfua Phn gii ur Lysin decacboxylas B- Galactosidas (O.N.P.G) Phn ng V.P Phn ng Indol Dng hoc m tnh + + + + T l % vi vi khun Salmonella 99,2 99,5 100 91,2 91,6 100,0 94,6 98,5 100,0 98,2

6.5. Gim nh huyt thanh hc bng phn ng ngng kt trn phin knh 6.5.1. Loi b nhng vi khun t ngng kt (6.4.2) bng phng php nh 1 git nc mui ng trng ln phin knh dng que cy ly mt lng vi khun va trn u thnh huyn dch, c kt qu. Nu c nhng ht ngng kt th loi b, nu huyn dch vn c u th tin hnh gim nh. 6.5.2. Vi khng huyt thanh O a gi. Phng php lm theo 6.5.1. ch khc l dng khng huyt thanh O a gi thay cho nc mui. Nu l Salmonella th xut hin nhng ht ngng kt, tu theo mc ngng kt nhiu hay t, ht ngng kt to hay nh, nc c hay trong m nh gi t mt n bn cng. Huyn dch vn c u l m tnh. 6.5.3. Khi dng tnh vi O a gi th lm ngng kt tip vi O n gi v H. Vi khng huyt thanh O n gi. Phng php lm v c kt qu theo 6.5.2. Ch khc l dng khng huyt thanh O n gi thay cho O a gi.

Vi khng huyt thanh H: Cy chuyn vi khun vo mi trng nc tht, 37 oC t 24 -48 gi, ly ra ly tm 3.000 vng/pht, trong 5 pht, cht ly cn. Phng php lm v c kt qu theo 6.5.2, ch khc l dng khng huyt thanh H thay cho O a gi. 6.5.4. Trng hp m tnh vi khng huyt thanh O a gi th th lm ngng kt vi khng huyt thanh Vi. Nu c phn ng dng tnh th cy chuyn vi khun vo 2 ng mi trng thch nghing v 2 ng mi trng nc tht, 37oC t 24 - 48 gi. 6.5.4.1. Dng nc mui ng trng thu hoch vi khun trn mt thch nghing, un si cch thu 20 pht lm li phn ng ngng kt vi khng huyt thanh O. Phng php lm v c kt qu theo 6.5.2. v 6.5.3. 6.5.4.2. Ly tm canh khun (mi trng nc tht) cht ly cn lm phn ng vi khng huyt thanh H. Phng php lm v c kt qu theo 6.5.3. 7. Tnh ton kt qu 7.1. Cn c vo kt qu gim nh tnh cht sinh ho. 7.2. Cn c vo kt qu gim nh huyt thanh nh nhm v sero-tp.

PH LC A
PHC KIM NGHIM

PH LC B
PHA CH MI TRNG, THUC TH V THUC NHUM B1. Mi trng Tetrationat (Muller-Kuafman) B1.1. Mi trng c bn: - Nc tht b - Pepton 1.000 ml 10 g

- Natri clorua - Canxi cacbonat

5g 45 g

un si nc tht, ho tan cc thnh phn cn li. Hp 121oC trong 20 pht. Ch thch: C th thay nc tht b bng 5g bt cao tht ho tan trong 1.000 ml nc ct. B1.2. Dung dch Natri-thiosulfat: - Natri-thiosulfat (Na2S2O3.5H2O) - Nc ct Ho tan. Hp 121oC trong 20 pht. B1.3. Dung dch Iod: - Iod - Kali iodua (KI) - Nc ct 20 g 25 g 10 ml 50 g 100 ml

Ho tan trc Kali iodua trong 5 ml nc; ri cho iod vo, tip tc lc mnh cho tan ri cho thm phn nc cn li. Bo qun trong l mu nt kn B1.4. Dung dch lc sng (Brillant green): - Lc sng - Nc ct 0,5 g 100 ml

Ch thch: C th thay dung dch lc sng bng phenol, dung dch 0,2% trong nc (phenol red) B1.5. Dung dch mt b: - Mt b kh - Nc ct Ho tan mt b trong nc si. Hp 121 B1.6. Mi trng hon chnh: - Mi trng c bn (B1.1) - Dung dch Natri-thiosulfat (B1.2) - Dung dch Iod (B1.3) - Dung dch lc sng (B1.4) - Dung dch mt b (B1.5) 900 ml 28 ml 20 ml 2 ml 50 ml 10 g 100 ml
oC

trong 20 pht.

Ln lt ho ln cc dung dch theo th t t trn xung, chia mi trng vo cc ng vi dung tch thch hp cho kim nghim. B2. Mi trng thch S.S - Pepton - Lactos - Mui mt - Natri-xitrat - Natri-thiosulfat - St xitrat 5g 10 g 10 g 12 g 12 g 0,5 g

- Bt cao tht - Thch

5g 18-20 g 4,5 ml 0,33 ml 1.000 ml

- trung tch, dung dch 0,5% trong nc (neutral red) - Lc sng, dung dch 0,1% trong nc (brillant green) - Nc ct

Tr dung dch trung tnh v lc sng ra, ln lt ho tan cc thnh phn cn li trong nc si. ngui, chnh pH 7,2 - 7,4 ho ln dung dch trung tnh v lc sng vo. un si, ngui xung cn 55oC. Chia vo cc a petri v khun, mi a t 15-20 ml B3. Mi trng thch deoxycholat-xitrat: - Bt cao tht - Pepton - Thch - Natri deoxycholat - Natri- xitrat: Na3C6H5O7. 11H2O hoc Na3C6H5O7. 2H2O - St xitrat - Lactos - trung tnh, dung dch 1% trong cn (neutral red) - Nc ct 25 g 20 g 1g 10 g 2 ml 1.000 ml 5g 5g 18-20 g 5g

Ho tan cao tht, pepton, thch trong 1.000 ml nc ct, un si. Chnh pH 7,4 ln lt ho tan cc thnh phn cn li, chnh pH 7,4. un si cch thu hoc hp cch thu t 20-30 pht. Chia ra cc a petri, mi a t 15-20 ml B4. Mi trng thch st, ng trisaccarit: - Pepton - Natri clorua - Lactos - Saccaros - Glucos - St xitrat - Natri-thiosulfat - phenol - Thch - Nc ct 20 g 5g 10 g 10 g 1g 0,3 g 0,3g 0,025g 18-20 g 1.000 ml

Ln lt ho tan cc thnh phn trn trong nc si chnh pH 7,4 chia vo cc ng nghim, mi ng 10 ml. Hp 121oC t 10-15 pht. t ng nghim nm nghing thn mi trng c chiu cao 2,5 cm B5. Mi trng nc tht: - Nc tht b 1.000 ml

- Pepton - Natri clorua

10 g 5g

Ho tan pepton, mui trong nc tht un si. ngui, chnh pH 7,4-7,6. Chia vo cc ng nghim, mi ng 5 ml. Hp 121 oC trong 20 pht B6. Mi trng thch dinh dng: - Mi trng nc tht (B5) - Thch 1.000 ml 18-20 g

Ho tan thch vo mi trng nc tht, un si. ngui, chnh pH 7,4-7,6. Hp 121oC trong 20 pht. Chia vo cc a petri mi a 15-20 ml v vo cc ng nghim, mi ng 5 ml. t ng nghim nm nghing B7. Mi trng thch ur (Christensen): B7.1. Dung dch c bn: - Pepton - Glucos - Natri clorua - Kali dihydro photphat - phenol - Thch - Nc ct B7.2. Dung dch ur: - Ur - Nc ct va Ho tan ur trong nc. Lc v khun B7.3. Mi trng hon chnh: - Dung dch c bn (B7.1) - Dung dch ur (B7.2) 950 ml 50 ml 400 g 1.000 ml 1g 1g 5g 2g 0,012 g 18-20 g 1.000 ml

Ho tan cc thnh phn trn trong nc ct un si. Chnh pH 7,0. Hp 121oC trong 20 pht.

Trong iu kin v khun, un nng dung dch c bn, ngui xung cn 45 oC trn dung dch ur vo. Chia ra cc ng nghim v khun, mi ng 5 ml, t ng nghim nm nghing cho ng li. B8. Mi trng lysin decacboxylas: - L. Lysin monohydro clorit - Cht chit men (yeast extract) - Glucos - Tm bromocresol - Nc ct 5g 3g 1g 0,015 g 1.000 ml

Ho tan cc thnh phn trn trong nc ct un si. Chnh pH 7,0. Chia vo cc ng nghim c ng knh 8 mm, mi ng 5 ml. Hp 121oC trong 10 pht B9. Mi trng nc pepton ( th phn ng Indol)

- Pepton - Natri clorua - Nc ct

10 g 5g 1.000 ml

Ho tan cc thnh phn trn trong nc nng, chnh pH 7,4 - 7,6. Chia vo cc ng nghim, mi ng 5 ml. Hp 121oC trong 20 pht. B10. Mi trng nc pepton glucos ( th phn ng V.P.) - Pepton - Natri clorua - Glucos - Kalihydro photphat (K2HPO4) - Nc ct 10 g 5g 5g 5g 1.000 ml

Ho tan cc thnh phn trn trong nc nng, chnh pH 7,4 - 7,6 chia vo ng nghim, mi ng 3 ml. Hp 121oC trong 20 pht. B11. Thuc th B. Galactosidas (O.N.P.G.): B11.1. Dung dch m: - Natri dihydro photphat (NaH2PO4) - Natri hydroxyt, dung dch 0,1 N trong nc (NaOH) khong - Nc ct va 6,9 g 3 ml 50 ml

Ho tan Natri dihydro photphat trong khong 45 ml nc. Chnh pH 7,0 bng Natri hydroxyt, dung dch 0,1 N. B sung nc va 50 ml. Bo qun lnh. B11.2. Dung dch O.N.P.G. - O-Nitrophenyl - B.D. Galactopyranosid (O.N.P.G.) - Nc ct 37 oC Ho tan trong nc, ngui B11.3. Thuc th hon chnh: - Dung dch m (B11.1) - Dung dch O.N.P.G. (B11.2) Ho ln, bo qun lnh, trc khi dng un nng cch thu 37 oC B12. Thuc th Indol (dung dch Kowacs): - Paradithylaminobenjaldehyd - Cn amyl hoc iso-amyl - Axitclohydric tinh khit B13. Thuc th Voges- proskauer (dung dch Barritt); - Dung dch 5% cn - napthol + - napthol + Cn etanol - Dung dch 40% Kalihydroxyt 5g 100 ml 5g 75 g 25 g 5 ml 15 ml 80 mg 15 ml

Ho tan aldehyd trong cn, nh tng git axit vo, va nh va lc cho tan u.

+ Kalihydroxyt (KOH) + Nc ct - Natri clorua - Nc ct

40 g 100 ml 8,5 g 1.000 ml

B14. Nc mui ng trng:

Ho tan mui, hp 121oC trong 20 pht.