TIU CHUN VIT NAM TCVN 7135:2002

THT V SN PHM THT - NH LNG E.COLI - K THUT M KHUN LC 440C S DNG MNG LC Meat and meat products - Enumeration of Escherichia coli - Colony - count technique at 44 C using membranes Li ni u TCVN 7135 : 2002 hon ton tng ng vi ISO 6391 : 1997; TCVN 7135 : 2002 do Ban K thut tiu chun TCVN/TC/F 8 Tht v sn phm tht bin son, Tng cc Tiu chun o lng Cht lng ngh, B Khoa hc v Cng ngh ban hnh. 1. Phm vi p dng Tiu chun ny quy nh phng php nh lng Escherichia coli c trong tt c cc loi tht v sn phm tht, k c tht gia cm. Phng php ny cho php pht hin Escherichia coli (tip sinh hc 1) v cc bin th khng ln men lactoza hoc sinh trng k kh (xem tham kho [1]). Ch thch 1 - Phng php ny c th khng thch hp cho vic kim tra tht ch bin c nhiu vi khun sinh bo t hiu kh. Ch thch 2 - Mt s chng Escherichia coli gy bnh c th khng pht hin c bng phng php ny. 2. Tiu chun vin dn TCVN 6507 : 1999 (ISO 6887 : 1983), Vi sinh vt hc - Hng dn chung v cch pha ch cc dung dch pha long kim tra vi sinh vt. TCVN 6404 : 1998 (ISO 7218 : 1996), Vi sinh vt trong thc phm v trong thc n gia sc Nguyn tc chung v kim tra vi sinh vt. 3. nh ngha Trong tiu chun ny p dng nh ngha sau y: 3.1. E.coli (Escherichia coli): Vi khun hnh thnh cc khun lc indol dng tnh (mu hng) 440C trn mng xelulo axetat t trn mi trng thch mt trypton di cc iu kin quy nh trong tiu chun ny. 4. Nguyn tc Nhn chung, vic pht hin Escherichia coli cn n ba giai on lin tip di y. 4.1. Hi phc Cy mt lng quy nh ca huyn ph ban u ln mng xenlulo axetat c t trn mi trng thch glutamat khong ci tin v nhit 370C khong 4h. Ch thch - Quy trnh ny cho php hi phc li cc E.Coli b h hi do bo qun ng lnh, do lm kh hoc do cc iu kin lnh hoc b h hi do x l nhit hoc do ha cht. ng thi cng lm gim nng ca tt c cc cacbon hidrat c th ln men nu chng c mt trong mu th vi nng cao, iu c th gy cn tr vic sinh indol trong giai on phn lp tip theo. 4.2. Phn lp Chuyn cc mng trn mi trng thch glutamat khong ci tin giai on hi phc sang mi trng thch mt trypton. nhit 440C trong thi gian t 18h n 20h. 4.3. Pht hin

S c mt E.Coli trn mng c biu hin bng s sinh indol t mi khun lc. 4.4. Tnh ton Tnh s lng E.Coli c trong mt mililit hoc trong mt gam mu t s khun lc indol dng tnh thu c trn mng cc pha long chn thu c cc kt qu c ngha. 5. Dch pha long, mi trng nui cy v thuc th 5.1. Khi qut V thc hnh ti phng th nghim hin hnh, xem TCVN 6404 : 1998 (ISO 7218). 5.2. Dch pha long chun b cc dung dch pha long, xem TCVN 6507 : 1999 (ISO 6887). 5.3. Mi trng hi phc: Thch glutamat khong ci tin 5.3.1. Thnh phn Natri glutamat Lactoza Natri fomat L(-)-xystin Axit L(-) aspactic L(+)-aginin Thiamin Axit nicotinic Axit pantothenic Magie sunfat (MgSO4, 7H2O) St (III) amoni xitrat [ti thiu l 15% Fe (m/m)] Canxi clorua (CaCl2.2H2O) Dikali hidro phosphat Amoni clorua Thch Nc 1) Ty vo sc ng ca thch 5.3.2. Chun b Ha tan amoni clorua trong nc. B sung cc thnh phn khc vo v un si. Chnh pH sao cho gi tr pH sau khi kh trng l 6,7 250C. Chuyn vo mi dng c cha thch hp (6.5) 100 ml mi trng ny v kh trng trong ni hp p lc (6.1) nhit 1210C trong 10 pht. 5.3.3. Chun b cc a thch Rt vo cc a Petri v trng (6.6) t 12 ml n 15 ml mi trng, c lm ngui n 470C trong ni cch thy (6.10), v cho ng c li. Cc a ny c th bo qun trong vng mt tun nhit t 00C n + 50C. Ngay trc khi s dng, lm kh cc a thch ny mt cch cn thn [th d: trong bung sy hoc t sy (6.4) 500C], tt nht l m np v mt thch hng xung di, cho n khi cc git nc nh trn mt thch bin mt. Khng tip tc lm kh thm. 6,35 g 10,0 0,25 g 0,02 g 0,024 g 0,02 g 0,001 g 0,001 g 0,001 g 0,100 g 0,010 g 0,010 g 0,90 g 2,5 g 12 g n 18 g 1) 1000 ml

Ch thch - Mi trng thch cn c lm kh sao cho 1 ml dch mu cy c hp th hon ton ln mng/thch trong vng 15 pht (xem 9.2.3). 5.4. Mi trng chn lc: Thch mt trypton (Xem tham kho [3]) 5.4.1. Thnh phn Trypton (thy phn pepsin ca casein) Mui mt (tinh ch) Thch Nc 20,0 g1) 1,5 g 2) T 12 g n 18 g 3) 1 000 ml

1) Nn s dng cc loi bn sn thun li cho vic hnh thnh indol. 2) Mui mt Oxoid No.3 l mt th d v sn phm thch hp c bn sn. Thng tin ny a ra to thun tin cho ngi s dng tiu chun v t chc Tiu chun ha quc t khng n nh phi s dng sn phm ny. 3) Ty thuc vo sc ng ca thch. 5.4.2. Chun b Ha tan cc thnh phn trn trong nc v un n si. Chnh pH sao cho sau khi kh trng gi tr pH l 7,2 250C. Chuyn tng lng 100 ml mi trng vo cc dng c cha thch hp v kh trng trong ni hp p lc (6.1) nhit 1210C trong 15 pht. 5.4.3. Chun b cc a thch Rt vo cc a Petri v trng (6.6) t 12 ml n 15 ml mi trng, c lm ngui n 470C trong ni cch thy (6.10), v cho ng c li. Cc a ny c th bo qun trong vng 4 ngy nhit t 00C n + 50C. Ngay trc khi s dng, lm kh cc a thch ny mt cch cn thn (th d: trong bung sy hoc t sy (6.4) 500C], tt nht l m np v mt thch hng xung di, cho n khi cc git nc nh trn mt thch bin mt. Khng tip tc lm kh thm. 5.5. Thuc th pht hin Indol (Thuc th Vracko v Sherris) 5.5.1. Thnh phn p-Dimetylaminobenzaldehyt A xit clohydric c(HCl) = 1 mol/l 5.5.2. Chun b Ha tan thuc th trong axit clohidric v bo qun nhit phng khng qu mt thng. 6. Thit b v dng c thy tinh Thit b thng thng ca phng th nghim phn tch vi sinh v c bit l: 6.1. Thit b kh trng kh (t sy) hoc kh trng t (ni hp p lc) Xem TCVN 6404 : 1998 (ISO 7218). 6.2. T m, c kh nng duy tr nhit 350C 10C hoc 370C 10C. 6.3. T m, c kh nng duy tr nhit 440C 0,50C. 6.4. Bung sy hoc t sy, c thng gi bng i lu, c kh nng duy tr nhit 500C 10C. 6.5. ng nghim, c kch thc 18 mm x 18 mm v bnh hoc l c dung tch t 125 ml n 300 ml, dng kh trng v bo qun mi trng nui cy. 6.6. a Petri, bng thy tinh hoc cht do, ng knh t 90 mm n 100 mm. 5g 100 ml

6.7. Mng xenlulo axetat, c c l t 0,45m n 1,2 m v ng knh 85 mm. 6.8. Pipet, c hiu chun dng cho vi sinh vt, c dung tch danh nh 1 ml, c chia 0,1 ml v c ng knh l x t 2 mm n 3 mm. 6.9. Que gt, bng cht do hoc bng thy tinh, th d: que gt lm bng a thy tinh c ng knh khong 3,5 mm, di 20 cm, mt on u di khong 3 cm c un to thnh gc vung v u mt c lm nhn bng nhit. 6.10. Ni cch thy, hoc thit b tng t, c th duy tr nhit 470C 0,20C. 6.11. n cc tm sng di (365 nm), c gn vi b lc thch hp loi bc x UV di 310 nm. 6.12. pH-met, chnh xc n 0,1 n v pH 250C. 7. Ly mu Vic ly mu khng quy nh trong tiu chun ny. Nn ly mu theo quy nh trong TCVN 4833 - 1: 2002 (ISO 3100 - 1 [4]). iu quan trng l phng th nghim nhn c ng mu i din v khng b h hng hoc bin i trong sut qu trnh vn chuyn hoc bo qun. 8. Chun b mu th Chun b mu th theo tiu chun c th thch hp i vi sn phm c lin quan. Nu khng c tiu chun c th th cc bn lin quan cn tha thun vi nhau v vn ny. Phng php ly mu c khuyn ngh nu trong TCVN 4833 - 2 : 2002 (ISO 3100 : 2 [5]). 9. Cch tin hnh 9.1. Phn mu th, huyn ph ban u v dch pha long Xem TCVN 6507 : 1999 (ISO 6887) v tiu chun c th thch hp lin quan n sn phm. 9.2. Quy trnh hi phc 9.2.1. Dng b kp v trng t cc mng xenlul axetat (6.7) trong iu kin v trng ln b mt kh ca tng cp hai a c cha mi trng thch glutamat khong ci tin (5.3.1), ch trnh to bt kh di mng. Dn phng cc mng mt cch nh nhng bng que gt v trng (6.9). Dng pipet v trng (6.8) ly 1 ml huyn ph ban u cho vo chnh gia mi mng. Dng que gt v trng (6.9), dn u phn mu cy ln ton b b mt ca mng, trnh khng trn khi mng. 9.2.2. Dng cc pipet v trng khc nhau (6.8) cy cc th tch ging nhau ca cc dch c pha long khc nhau chun b t huyn ph ban u ln cc mng khc nhau, nh quy nh trong 9.2.1. Thi gian t khi chun b mu th (iu 8) n khi cy dch ln mng khng c vt qu 45 pht, tr khi c quy nh khc trong cc tiu chun c lin quan. 9.2.3. cc a nui cy trong t th nm ngang nhit phng khong 15 pht cho n khi phn mu cy ngp vo trong thch. cc a petri trong t m (6.2) 350C hoc 370C trong 4h 0,5h, cc mng/mt thch hng ln trn. 9.3. Chuyn sang mi trng chn lc 9.3.1. Dng b kp c u t v trng chuyn cc mng, vi phn mu cy hng ln trn, t mi trng thch glutamat khong ci bin (5.3.1) sang mi trng thch mt trypton (5.4). Mng m ni trn phi dnh vo mt thch, trnh to bt kh. Khng s dng que gt v s lm phn tn mi khun lc c mt, lm kt qu nh lng cao hn gi tr thc.

9.3.2. cc a ny t 18 h n 24 h trong t m (6.3) 440C, cc mng/mt thch hng ln trn, khng chng cc a ln nhau qu ba chic. 9.4. Pht hin c tnh sinh indol ca cc khun lc trn mng 9.4.1. Ghi nhn trn np ca mi a nhn bit. 9.4.2. Dng pipet ly 2 ml thuc th pht hin indol (5.5) cho vo np ca a petri lt ngc, t t th nm ngang. 9.4.3. Dng b kp v trng ly mng ra khi mt thch tng ng v cho cc thuc th indol. Nu cn, phi nghing np sao cho tt c b mt ca mng c thm t bng thuc th indol. Sau 5 pht, dng pipet loi b thuc th cn li. 9.4.4. t mng di n cc tm (6.11) trong 30 pht. Cc khun lc indol dng tnh c mu hng. 9.5. m khun lc m cc khun lc indol dng tnh (mu hng) trn cc mng, tt nht l trn cc mng c t 15 khun lc n 150 khun lc mu hng. 10. Biu th kt qu S dng chun c sau y xc nh s lng E.Coli c mt. 10.1. Phng php tnh 10.1.1. Nu mt hoc c hai mng lc ca mt pha long nht nh cha t 15 khun lc n 150 khun lc mu hng, th tnh gi tr trung bnh s hc ca s khun lc m c trn hai mng. Ch gi li hai ch s c ngha, tin hnh nh sau: - nu s nh hn 100, th lm trn n bi s gn nht ca 5; - nu s ln hn 100 v kt thc bng s 5, th lm trn s n bi s gn nht ca 20; - nu s ln hn 100 v tn cng khng phi l 5, th lm trn s n bi s gn nht ca 10. Nhn gi tr ny vi s nghch o ca pha long tng ng thu c s E.Coli trong mt gam sn phm. Biu th kt qu ny di dng tch ca mt s nm trong khong 1,0 n 9,9 vi ly tha c s 10 v s m thch hp. 10.1.2. Nu c cc mng cha t 15 khun lc n 150 khun lc mu hng hai pha long lin tip, th tnh s lng E.Coli theo quy nh trong 9.3.5.1 ca TCVN 6404 : 1998 (ISO 7218 : 1996). 10.1.3. Nu trn cc mng c t hn 15 khun lc mu hng tng ng vi dch huyn ph ban u, th tnh gi tr trung bnh s hc, y, ca s cc khun lc m c trn hai a. Bo co kt qu thu c bng s lng E.Coli tnh trong mt gam NE: NE = y/d Trong d l h s pha long ca huyn ph ban u. 10.1.4. Nu trn cc mng khng c cc khun lc mu hng no tng ng vi huyn ph ban u th bo co kt qu nh sau: - t hn 1/d. E.Coli trong mt gam Trong d l h s pha long ca huyn ph ban u. 10.2. Gii hn tin cy tnh di tin cy, xem TCVN 6404 : 1998 (ISO 7218 : 1996).

11. Bo co kt qu Bn bo co kt qu phi ghi r: - mi thng tin cn thit v vic nhn bit hon ton mu th; - phng php ly mu dng, nu bit; - phng php th p dng, vin dn tiu chun ny; - tt c cc chi tit thao tc khng quy nh trong tiu chun ny, cng vi cc chi tit bt thng no khc c th nh hng ti kt qu; - kt qu th thu c; - nu kim tra lp li, th nu kt qu cui cng thu c.

PH LC A
(tham kho) TI LIU THAM KHO [1] Holbrook, R. and Andreson, J.M. Society of Applied Bacteriology, Technical Series, No.17, Corry. J.E.L., Roberts, D. and Skinner, F.A (eds). Academic Press, London, pp.239-254. [2] The bacteriologycal examination of drinking water supplies. Report No.71, 1982, Her Majestys Stationary Office, London. [3] Delaney, J.E., McCarthy, J.A. and Grasso, R.J. Water Sewage Wks., 1962, pp. 109; 289. [4] TCVN 4833 - 1 : 2002 (ISO 3100 - 1: 1991) Tht v sn phm tht - Ly mu v chun b mu th - Phn 1 : Ly mu. [5] TCVN 4833 - 2 : 2002 (ISO 3100 - 2 : 1988) Tht v sn phm tht - Ly mu v chun b mu th - Phn 2: Chun b mu th kim tra vi sinh vt.