TIU CHUN VIT NAM TCVN 5667:1992

THT V SN PHM THT PHNG PHP XC NH TNG S VI KHUN HIM KH Meat and meat products. Enumeration of total aerobic bacteria Tiu chun ny quy nh phng php xc nh tng s vi khun hiu kh trn 1cm b mt sn phm v trong 1g cho cc loi tht v sn phm ch bin t tht tr hp tht. 1. Xc nh tng s vi khun hiu kh trn 1cm2 1.1 Nguyn tc Phng php xc nh tng s vi khun hiu kh trn 1cm2, b mt sn phm c p dng nh gi nhim khun trn b mt cc sn phm tht bng phng php m s khun lc vi khun hiu kh c trn 1cm mu th c chuyn tng ng vi 1ml dung dch huyn ph ban u di cc iu kin th c xc nh trong tiu chun ny. 1.2 Ly mu theo TCVN 5153 90 v TCVN 4886 89. 1.3 Thit b v dng c Giy thm v khun, kch thc 2 x 2 hoc 5 x 5 cm; Khung bng kim loi khng r v khun c kch thc trong l 2 x 2 hoc x 5 cm; Bi thu tinh v khun, c ng knh ph hp; Tm bng; Kp gn kim loi; ng nghim 18 x 180mm; Cc ong cc loi; Bnh tam gic cc loi; Bnh nh mc dung tch 100, 500 v 1000 ml; Pipt 1,0 v 100ml, chia ti 0,1ml; a petri c ng knh trong 90 100mm; Phch duy tr c nhit t 0 n + 20C; T m iu chnh c 30 10C; T sy kh; T hp t; Bp cch thu iu chnh c t 30 n 560C; Cn phn tch; My o pH; Knh lp phng i t 3 n 5 ln; Hp m khun lc; Bp in; Bung cy v khun.

1.4 Ho cht v mi trng 1.4.1 Ho cht - Nc ct - Natriclorua, tinh khit phn tch (TKPT)

- Dinatri hydro phot phat (Na2HPO4), TKPT; - Kalidihydro photphat (KH2PO4), TKPT; - Thch dng cho vi sinh vt - Trypton dng cho vi sinh vt - Glucaza.TKPT - Cao men dng cho vi sinh vt - Pepton dng cho vi sinh vt . 1.4.2 Mi trng a) Nc mui sinh l - Thnh phn: Natriclorua 8,5g; - Nc ct, 100ml; - Cch pha ch: Ho tan mui trong nc, tit khun trong ni hp 1200 C trong 15 pht. b) Dung dch m pepton - Thnh phn: Pepton 10g; Natriclorua 5g; Dinatrihydro phophat 9g; Kalidihydro phophat 1,5g; Nc ct, 1000ml. - Cch pha ch: un si ho tan cc cht trn, ngui. iu chnh pH bng natrihydroxit 0,1N sao cho sau khi tit khun pH 250C l 7,0 0,2. Rt 100ml mi trng vo bnh dung tch 250ml v 10 ml vo mt ng nghim. Tit khun trong ni hp nhit 120 10C trong 15 pht. Nu khng dng ngay, mi trng cn c bo qun ni kh ro, ti nhit t 0 n + 50C v khng qu 30 ngy. c) Thch trypten glucoza - Thnh phn: Trypton - Cao men - Glucoza - Thch - Nc ct, - Cch pha ch: Va un nh la va khuy nh ho tan cc cht n khi dung dch si. iu chnh pH sao cho sau khi tit trng pH 250 l 7,0 0,2. Rt mi trng vo bnh vi lng mi trng khng qu 1/2 dung tch mi bnh. Tit khun trong ni hp nhit 120 10C trong 15 pht. ngui trong ni cch thu ti 45 10C s dng. Nu cha dng ngay cn bo qun mi trng ni kh ro, ti, 0 n + 50 v khng qu 30 ngy. Trc khi dng, un cch thu cho chy mi trng v ngui trong ni cch thu t 45 10C. d) Thch mng - Thnh phn: Thch - Nc ct, - Cch pha ch: 12 18g 100ml 5g; 2,5g 1g 12 18g 100ml

Va un nh la va khuy nh ho tan thch n khi si, iu chnh pH sao cho sau khi tit khun pH 250C l 7,0 0,2. Rt mi trng vi lng 4ml vo mi ng nghim v 100 ml vo mi bnh tit khun, s dng v bo qun ni trng nh mc C. 1.5 Trnh t xc nh 1.5.1 Chun b mu a) Dng giy thm v khun (1.3) c lm t trc bng nc mui sinh l v c tng din tch l 100cm (gm 4 mnh 5 x 5 cm hoc 25 mnh 2 x 2 cm) dn ln cc v tr khc nhau ca b mt mu (1.2) cn dm sao cho ton b din tch giy thm tip xc trc tip vi b mt mu. Sau 2 pht, chuyn ton b s giy thm trn vo ng nghim cha sn 10 ml dung dch m pepton. C th chun b mu theo cch: p cc khung kim loi v khun c kch thc tng t (1.3) ln b mt mu cc v tr khc nhau vi tng din tch l 100cm 2 ( 4 ln vi khung 5 x 5 cm hoc 25 ln vi khung 2 x 2cm). Nh nhng qut u tm bng c thm m bng nc mui sinh l ln ton b din tch c khung kim loi gii hn. Cchuyn ton b s ln u tm bng trn vo ng nghim cha sn 10ml dung dch m pepton. Cc ng nghim cha mu c th c bo qun 0 n + 20 trong vng 6h. b) Chuyn ton b lng mu trong ng nghim vo bnh tam gic dung tch 100 ml cha khong 10 vin bi thu tinh v khun. Trng ng nghim cha mu bng dung dch m pepton v c vo bnh cha mu, lc nh cho giy thm hoc bng tan vn, sau cho thm dch dch m pepton ti khong 100ml c) lng hoc tin hnh ly tm dung dch mu khong 5 pht tc 200 vng/ pht. Gn ly lp dng dch trong vo bnh nh mc dung tch 100ml v cho thm dung dch m pepton ti vch mc 100ml (tng ng vi 100cm2 b mt mu c chun b). Dung dch ny c gi l dung dch huyn ph ban u. Cn tin hnh kim tra ngay sau khi chun b xong dung dch, nu cha kim tra, dung dch cn c bo qun 0 n + 50C nhng khng qu 1h. 1.5.2 Pha long mu Dng dung dch m tepton ( mc b iu 1.4.2) pha long dung dch huyn ph ban u theo t l 1/9 ti cc pha long thp phn khc nhau (10 -1, 10-210n). Cn cn c mc nhim khun ca mu tin hnh pha long dung dch mu ti cc pha long ph hp. 1.5.3 Tin hnh nui cy a) Vi mi mu phi nui cy t nht 3 pha long lin tip, mi pha long nui cy 2 a, phi dng pipet ring trong mi pha long. b) Dng pipet khc nhau ly 1 ml dung dch mu cc pha long khc nhau cho vo gia cc a petri. Rt vo mi a khong 15 ml mi trng thch trypton glucoza (mc c iu 1.4.2). Lc vng trn 5 ln theo chiu kim ng h v 5 ln ngc lai trn u mi trng v dung dch mu. t cc a cha mi trng trn mt phng ngang sao cho mi trng ng t nhin. Thi gian t khi pha long mu (1.5.2) n khi nui cy xong khng qu 20 pht. c) Nu nghi trong sn phm c cha cc vi sinh vt m khun lc ca chng c thm mc lan trn b mt mi trng th sau khi mi trng ng rt thm 4 ml thch mng ln b mt a. d) Khi mi trng ng, lt p cc a peptri v t vo t m duy tr 30 10C trong 72 h. 1.6 Tnh kt qu 1.6.1 C sau 24h m s b s khun lc mc v sau 72h m chnh thc tnh kt qu. Ch tnh kt qu khi s phn b khun lc trn cc a l hp l vi mi tng quan nghch gia pha long v s khun lc mc trn . Dng mt thng hoc knh lp m s khun lc mc trn mi a petri, ch m nhng a c s khun lc mc ring bit v t 15 n 300 khun lc. m tt c s khun lc mc trong mi a, trong trng hp s lng khun lc ln v phn b u, c th phn y a theo ng knh thnh cc phn u nhau c bi s l 2 m mt phn sau nhn kt qu vi tng s phn chia.

1.6.2 Tng s vi khun hiu kh trn 1cm2 mu th ( X1) c quy ra tng s vi khun hiu kh c trong 1ml dung dch huyn ph ban u ca mu th c tnh 2 pha long lin tip, mi pha long gm 2 a theo cng thc sau: X1 = Trong : C - tng s khun lc trn tt c cc a 2 pha long lin tip c m n1 - s lng a pha long th nht c m; nz - s lng a pha long th hai c m; d - h s pha long ng vi pha long th nht c m. Kt qu c lm trn ti s hng nghn v biu th di dng tch ca mt s thp phn hai ch s ( 1,0 .9,9) vi 10n ( n= s ch s nguyn ca kt qa tr 1). Th d: Hai a pha long th nht c m ( 10-2) c s khun lc l 168 v 215; Hai a pha long lin tip th hai c m ( 10-3) c s khun lc l 14 v 25. X1 =

C (n1 + 0,1nz ) xd

C 168 + 215 + 14 + 25 422 = = = 19182 2 (n1 + 0,1nz )d [ 2 + ( 0,1x2) ) x10 0,022

Kt qa 19182 c lm trn thnh 19000 v c biu th di dng 1,9 x 102 khun lc/cm2. 1.6.3 Nu cc a ca hai pha long lin tip c s khun lc t 15 n 30, tnh s khun lc ca mi pha long v kt qu l trung bnh s hc ca hai gi tr thu c. Ly trng hp t s ca gi tr cao so vi gi tr thp ln hn hai ly gi tr thp l kt qu. 1.6.4 Nu hai a cha dch huyn ph ban u u c s khun lc thp hn 15, kt qu l trung bnh s hc ca s khun lc mc trong hai a . 1.6.5 Nu hai a cha dch huyn ph ban u u khng c khun lc no, kt qu l: t hn 1 vi khun hiu kh trn 1cm2 b mt sn phm. 2. Xc nh tng s vi khun hiu kh trong 1 g 2.1 Nguyn tc Tng s vi khun hiu kh c trong 1 g mu c tnh theo phng php m s khun lc vi khun hiu kh quy ra 10ml dung dch huyn ph ban u di cc iu kin xc nh ca tiu chun ny. 2.2 Ly mu, theo TCVN 5153 90 2.3 Thit b v dng c, xem iu 1.3 2.4 Ho cht v mi trng, xem iu 1.4 2.5 Trnh t xc nh 2.5.1 Chun b mu, theo TCVN 4887 89 Khi lng mu c chun b pha long l 10 1g. 2.5.2 Pha long mu, theo iu 1.5.2 2.5.3 Tin hnh nui cy, theo cc mc a, b, c iu 1.5.3 2.6 Tnh kt qu Cch m khun lc, tnh ton v biu th kt qu theo cc iu 1.6.1; 1.6.2 v 1.6.3. 2.6.1 Nu 2 a cha dch huyn ph ban u u c s khun lc thp hn 15, ly trung bnh s hc m ca chng v kt qu X2 c tnh: X2 = m x

1 = 10m d

(d l h s pha long ca dch huyn ph ban u bng 10-1). 2.6.2 Nu 2 a cha dch huyn ph ban u u khng c khun lc no, kt qu X2 c tnh: X2 < 1

1 d

X2 <10