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    TCVN 7924-2-2008 - Ecoli

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    ynhifood

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    TCVN TIEU CHUAN QUOC GIA TCVN 7924-2:2008 ISO 16649-2:2001 Xuait ban in 4 VI SINH VAT TRONG THUC PHAM VA THUC AN CHAN NUOI! — PHUONG PHAP BINH LUGNG ESCHERICHIA COLI DUONG TINH %GLUCURONIDAZA - PHAN 2: KY FHUAT DEM KHUAN LAC G 44°C SU DUNG 5-BROMO-4-CLO-3-INDOLYL #-D-GLUCURONID Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of /-glucuronidase-positive Escherichia coli - Part 2: Colony-count technique at 44°C using 5-bromo-4-chloro-3-indoly! /+-D-glucuronide HA NOI - 2008 TCVN 7924-2:2008 Léi noi ddu TCVN 7924-2:2008 hoan toan tucng ducng voi ISO 16649-2:2001; TCVN 7924-2:2008 do Ban ky thuat tiéu chudn quéc gia TCVN/TC/F13 Phuong phap phan toh va Kéy m&u bién sean, Téng cue Tiéu chudin Bo ting Chat long 4 nghi, BO koa hoc va Cong nghé cong b6. TCVN 7924:2008 (ISO 16649) Vi sinh vat trong thue phém va thie an chan nudi — Phuong phap dinh lueng Escherichia coll dirsng tinh P-glucuronidaza, bao gém cac phan sau: TCVN 7924-1:2008 (ISO 16649-1:2001) Vi sinh vat trong thue phdm va thie an chan ‘nui ~ Phurang phap dinh luong Escherichia coli dung tinh (eglucuronidaza ~ Phéin 1. Ky thuat dém khudin lec 6 44 °C sit dung mang loc vi 5:bromo-4-clo-3-indoly:f-D- aglucuronid; TCVN 7924-2:2008 (ISO 16649-2:2001) Vi sinh vat trong thue phém va thie an chan nu6i- Phung phap dinh luyng Escherichia coli dusng tinh P-glucuronidaza ~ Phan 2: kj thuat d6m khudin lac d 44°C sit dung 5-bromo-4-clo-3-indolyt-/+D-glucuronid; — TCVN 7924-3: 2008 (ISO/TS 16649-3:2005) Vi sinh vat trong thytc phim va thite an ‘chan nudi - Phuong phap dinh luring Escherichia coli duang tinh f-glucuronidaza — Phan 3: Kj thuét tinh s6 c6 xac sudt lon nhat sit dung S-bromo-4-clo-3-indolyt -D- @ canara TCVN 7924-2:2008 Lai gidi thigu Do tinh da dang ciia thyc phém va thie an chan nudi nén phuong phap nay c6 thé khéng thich hap dén timg chi tiét cho timg sin phdm cu thé. Trong trudng hyp nay, c6 thé sir dung cac phutng phap khic ac trimg cho ting san phdm, nu hoan toan chi vi ly do ky thudt. Tuy nhién, can c6 gang ap dung pphuong phap nay khi c6 thé, hi tibu chun nay duge soat xét thi can phai tinh an moi thong tin lin quan één pham vi ma phutsng php dém dia nay phi tuan theo va cdc nguyén nhan gay sai léch so véi phucng phap trong truing hop cde sén phém cu thé. Vigo hai hoa ee phuong phap thir c6 thé khéng thye hién duge ngay va di véi mot vai nhom san phat c6 thé t8n tai cdc tidy chudn quéc 16 vahodc tiéu chudn quéc gia ma khong phi hgp voi tieu chuan nay. Thong thuing khi cde tigu chudn quéc 16 nhu vay duge sodt xét, thi ching phdi duge sta 446i a8 phi hop v6i tidu chun nay, sao cho cu6i cing chi cdn céc sal Ich véi phuang phsp dém fia nay 18 cA ly do ky thudt duge thira nan TIEU CHUAN QUOC GIA TCVN 7924-2:2008 wat ban tn 1 Vi sinh vat trong thyc pham va thc an chan nudi Phuong phap dinh lugng Escherichia coli dung tinh B-glucuronidaza — Phan 2: Ky thuat dém khudn lac 6 44°C sit dung 5-bromo-4-clo-3-indoly! B-D-glucuronid Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of p-glucuronidase-positive Escherichia coli ~ Part 2: Colony-count technique at 44 °C using 5-bromo-4-chloro-3-indolyl f-D-glucuronide 1. Pham vi ap dung Tiéu chun nay qui dinh phutong phap dinh lugng Escherichia coll dung tinh Pglucuronidaza trong thue phim hoc thie 8n chan nu6i. Phuang phap nay sit dung ky thuat dém khuan lac 6 44°C trén mai tiutng dc 66 chita thanh phan tao sc dé phat hign enzym B-glucuronidaza. CANH BAO - Mét sé chiing Escherichia coli khong phat trién duge & 44 °C va cu thé la cae Escherichia coli &m tinh p-glucuronidaza nhur Escherichia coll 0157 sé khong phat hign duge. 2 Ti vign dan Cac tai ligu vign dln sau la rat cén thiét cho viéc 4p dung tiu chun. B6i véi cae tai liéu vién dn gbi nam céng bé thi 4p dung phién ban dugc nu. Bd vai cac tal ligu vien dn khong ghi nam céng bé thi ap dung phién ban méi nhét, bao gém cé ca sita di. TCVN 6507-1 (ISO 6887-1), Vi sinh vét trong thuc phém va thie in chan nudi ~ Chuéin bj méu tht, dung dich huyén phi ban du va cdc dung dich pha long thap phan dé kiém tra vi sinh vat ~ Phin 4 (Cac nguyértéc chung 46 chuéin bi huyén phi ban @éu va cée dung dich pha loaing thap phan, TCVN 6404 (iSO 7218), Vi sinh vat trong thyc phim va thé &n chan nudi ~ Yeu cdu chung va huéng dan kiém tra vi sinh vat. TCVN 7924-2:2008 3. Thuat ngit va dinh nghia Trong tiéu chun nay sit dung cae thuat ngit va dinh nghifa sau day: 34 Escherichia coll duong tinh B-glucuronidaza (f-glucuronidase-positive Escherichia coll) Vi khudin & nhigt 66 44 °C hinh thanh cd¢ khudin lac mau xanh dign hinh trén moi truéing trypton-mat- alucuronid (BX) trong cae aiéu kign duge qui dinh trong tidu chudin nay. 32 Dinh lugng Escherichia coll dang tinh #}-glucuronidaza (enumeration of (-glucuronidase-positve Escherichia colt xc dinh s6 long dan vi hinh thanh khuén tac (CFU) cla Escherichia coli duong tinh fi-glucuronidaza duge tinh trén millit hoc trén gam mu, khi phép thir va vige tinh toan duge thye hign theo quy dinh trong tisu chun nay 4 Nauyén tac 4.1 Cay mot luong mau thit xée dinh hode véi mbt Iugng xéc dinh huyén phil ban du lén cdc dia kép chita mi trugng trypton-mat-glucuronid (TBX). ‘Sif dung cdc dung dich pha loding thap phan cia mu this ho¥e cfia huyén phi ban du ey vao hai da cho méi dung dich pha long, rong cling mét diéu kién, (Cac dia nay duge U4 44°C + 1 °C trong 18 h dén 24 h 16 kiém tra dB phat hign sy cb mat cba cac khuéin lac dic tring duge coi la Escherichia coli duong tinh p-glucuronidaza, 4.2. Tinh 86 lugng don vj hinh thanh khuain lac (CFU) cila Escherichia coli duung tinh (-glucuronidaza tran gam hose trén millit mu (xem diéu 10). 5 Dich pha loding va mai trudng cay [B61 vai cc phing this ngbiém hién hanh, xem TCVN 6404 (ISO 7218). 5.1. Dich pha loang Xem TGVN 6607-1 (|SO 6867-1) hode tu chuéin cy thé lin quan dn sn phim cd xe dinh 5.2 M@i truéng nudi cay: Thach trypton-mat-glucuronid (TBX) TCVN 7924-2:2008 5.2.4 Thanh phan ‘San phém thuy phan casein bing enzym 2006 Musi mit to 150 ‘Axi 5-bromo-4-clo-3-indolyl-D-glucuronid (BCIG) 144 umol* Dimety!sutfoxit (OMSO}* 3m Thach 99den 189° Nube 4.000 mi * Vidu: 0,075 g musi xyclohoxylamoni * Dimety sulfoxide khi bit hoe tip xa ph, Cin sit dung trong 10 ut Kh. Vidi tinh 146 nén nha sin xudt khuyén edo ding dung dich pha loing, * Phy thude vio sae déng eta thech, 5.22 Chuan bi Hoa tan BCIG trong dimetyt sulfoxit hoa trong dung dich long theo khuyén cdo cla nha sn xual Hoa tan tat ¢8 cc thanh phan trén trong nude va dun én s6i Chinh pH a8 sau khi khir tring pH phai la 7,2 + 0,26 25°C, néu cn. Knit tring mdi trudng 15 min 8 nhigt 4 121 °C trong néi hap ap lye. Lam ngudi ngay moi trubng tren Indl céch thuy (6.3) dén khoding tu 44 °C dén 47°C. 6 Thiét bj va dung cy thuy tinh ‘StF dung ce thiét bi, dung eu phong thif nghiém vi sinh thong thuting [xem TCVN 6404 (ISO 7218)] va ou thé nhu sau 6.4. Thiét bj ki trung kno (t6) hodc khir tring uét (ni hap 4p lye). 6.2. Ti dim, c6 thé duy tr nhigt a6 6 44°C + 1 °C. 6.3. Néi cach thuj, c6 thé duy tr duge nhiet a6 ti 44 °C 68n 47 °C 6.4 Binh, éng nghi¢m hoac chai, c6 dung tich thich hop. 6.5 Pipet oac micropipet, xa hét (d4u théi), miéng rong va c6 dung tich danh dinh tis 1 ml d&n 10 mi, ‘duce chia vach 0,1 mi dén 0,5 mi twang tng. 6.6. Dia Petri, dung kinh khong 90 mm. TCVN 7924-2:2008 6.7 May do pH, 06 49 chinh xac dén + 0,1 don vi pH. Nguéng do tai thiéu cila may do pH phal fa 0,01 dan vi pH. May do pH cé thé duce gin véi hé théng can bang nhiét ty dong hoe diéu chinh bang tay. 7 Lay mau igu quan trong la mu git dén phang thir nghiém phi la mBu dai dign, Mau khéng bi hu héng howe thay di thanh phan trong qué trinh van chuyén va bao quan | \Viée ly mau khéng qui dinh trong tiéu chudn nay. Nén ky mau theo tiéu chudn cu thé lin quan dén ssn phim, Néu khéng c6 tiéu chudn cu thé lién quan dn sn phidm thi cdc bén ty thod thuan vé vain i 8 nay. 8 Chuan bj mau thir CChudin bj mu thir theo tiéu chun ou thé lin quan dén sin phidm. Néu khéng c6 tigu chudin cy thé Wan quan dén san phdm thi cdc bén tu thod thuan vé véin dé nay. 9 Cach tién hanh 9.1 Phén mau thi, huyén phi ban déu va cée dung dich pha lodng tiép theo Xem TCVN 6507-1 (ISO 6887-1) hodc tiéu chudn cu thé lién quan dén s4n phém can xac dinh. 92 ciyvaa 9.2.4 Ding pipet ho’e micropipet v6 tring (6.5) chuyén 1 mi mu thit (ndu mu thi 6 dang feng) hoc @ ‘1 ml dich pha lodng ban du (10°) (ndu sn phém 6 dang khac) vao dia Petri v6 tring (6.6.) Cay vao hai dia & mai d6 pha lodng. Lap lai quy trinh trén cho cde dung dich pha lodng thap phan tig theo, si dung mét pipet mdi vo tring ‘cho mai 46 pha long, néu cn. 9.2.2 R6t vio mdi dia Petri khoding 15 mi méi tru’ng TBX (5.2) ma trube 46 43 duse lam ngudi déin khong tir 44 °C dn 47 °C tr8n ndi cach thuy (6.3) ‘Trén cn than dich cay vai méi truéng va a8 yén cho hén hop déng lai, dé cac dia Petri trén mat phdng mat nm ngang Thai gian tinh tt ki phan phéi dich ey vao da dn khi rot méitrung Khong dude qué 15 min TCVN 7924-2:2008 9.2.3 Lat gue cac dia (9.2.2) va 46 vao tii Am (6.2) 48 6 44°C trong khoang tis 18 h dén 24 h. Téng thdi gian U khong duge qua 24 h, CANH BAO - Néu nghi ngé 6 mat cac té bao bj Uc ché thi i giai doan déu khoang 4h 6 37 °C sau do ting nhigt do lén 44 °C di trong khong 18 h dén 24 h. Nhigt do. khong duge vugt qua 45°C. 9.3. Dém cac dan vj hinh thanh khuan lac Sau giai doan a 4m quy dinh (9.2.3), dém céc CFU dién hinh cla Escherichia coli duong tinh {i-glucuronidaza trén mdi dia thach chia it hcn 150 CFU din hinh va ft hon 300 CFU téng s6 (dién hin va khéng dign hinh). Néu dich edy khéng thé tach riéng va khdng thé quan sat duge cdc khudn lac dign hinh, thi cae da chita 0 CFU din hinh cn duce xem xét theo cae phutong phdp tinh khdc nhau nhu trong digu 10, 10 Biéu thi két qua 10.1. Yéu céu chung Vic tinh todn trong 10.2 cén tinh dén cae trudng hdp thutng gp nha khi tién hanh theo thye hanh phong thir nghiém t6t. Going 06 mdt s6 trusng hap dic biét nhung cing him khi xdy ra (vi dy nhs sé lugng CFU rt khac nhau givfa hai dia ti ciing mot dung dich pha long, hoc cdc tf Ié khdic nhau nhigu tir mot h8 $6 pha lo’ng gidta cae dia tr hal do pha long lién tip). Cac két qua dm cn due nguot phan tich c6 nang luc kiém tra, gi thich va két qua 46 cing c6 thé bi loai bé 10.2 Tinh toan DE co dién inh, qua dung, cdn dém cac CFU dién hinh trén it nhat mot dia chifa it nhét 15 CFU mau xanh Tinh N, 86 iutgng CFU cla Escherichia coli duong tinh f-glucuronidaza co mat trong mau thiltrén mililit hoac trén gam sén phém tir hai 46 pha loting lién tiép sit dung cOng thie (1): ze a Vx (0, +010, )xd trong 66 ‘2 la téng s6 cfc Khun lac dm duge trén tat c& ede dia duge git lai sau hai dd pha losing én tip, 66 it nhiit mot dia chia t6ithidu 15 CFU mau xanh; ny 1a86 dia duge git al tai do pha loang thir nha; TCVN 7924. 2008 V thé tich dich cdy da ding trén m6i dia, tinh bang mililit: n'a 86 dia duge git lai tai d6 pha long thir hai; d-“hé 86 pha long tuong img voi dé pha loang thir nhat duge gif lai [d = 1 trong truéing hop (cae mau 8 dang l6ng) khi mau thi duge cy truc tip) Lam tron ed két qua dn hai chit $6 c6 nghia [xem TCVN 6404 (ISO 7218) Lay két qua la 86 lung Escherichia coli duong tinh fi-glucuronidaza trén mili (s4n phdm dang Kong) hod trén gam (san phdm dang Khac) duge bigu thi theo 96 nguyén dén hai chi 86 66 nghia (du6i 100) hove theo s6 tis 1,0 dén 9,9 nhan luy thi cla 10. 10.3 Use tinh cac sé lugng nhd- 40.3.1 Néu c6 hai dia [ola mau thir (néu sn phdm & dang ling) hode cila huyén phis ban déu (néu ‘sn phdm 8 dang khéc) hode cila 46 pha lofing thir nhat duge cay hoac duce git lai} chifa ft han 15 CFU mau xanh, thi tinh Ne, s6 CFU cila Escherichia coll duong tinh -glucuronidaza co mat trong ‘mu thir trung binh cia hal dia song song theo céng thite: (2) Ce @ Ne= Venxd trong 66 Se ta téng cdc CFU mau xanh dién hinh dém duc trén hai dia; V_ thé tich dich eay da ding trén mdi dia, tinh bing mili; la $6 dia dutge git lai (trong truting hgp nay n = 2), dh s6 pha long tuung (ng vai dung dich huyén phil ban dau hodc do pha loang thi nhat duc cay ho’ duve git lai [d= 1 trong truting hap (sn phdm dang lng) khi mau thir duge cay tru tp]. Lam tron cdc két qua dn hai chit s6 66 nghia [xem TCVN 6404 (ISO 7218)}, BBidu thi kbt qué nhu sau: ~ 86 lutong Escherichia coli duéng tinh (}-glucuronidaza ute tinh trong mét millt (46i vai sin phdm dang ong) hoae trong mot miligam (san phdm dang khéo): Ne = Y. 10.3.2 Néu hai dia cla mu thi [(san phdm dang Iéng) ho&e huyén phi ban du (sn phdm dang hc) cia 46 pha loang thir nhat 48 c&y hode git lal] khéng chéfa bat ky CFU mau xanh nao, thi biGu thi ket qua nhu sau 10 TCVN 7924-2:2008 it hon 1/d Escherichia coll duong tinh B-glucuronidaza trong mili (sn phdm dang ling) hoae trong ‘gam (san phém dang khac); trong d6 d la hé s6 pha long olla huyén phi ban dau hoac do pha loang thif nhat 44 cay hoc git Iai [d= 1] trong truéng hgp (sn phém dang \éng) mau thit dude edy try tiép. 10.3.3 Néu tat cA ce CFU didn hinh hotic khong dign hinh trén hai dia d6 pha loang thir nhat 4, cchifa nhiéu han 300 cé CFU mau xanh ¢6 thé nhin thay, hoe néu trén hai dia 6 dé pha lodng tiép theo 4d, chita it hon 300 Khudn lac, khong cé CFU mau xanh nao cén dém, thi bidu thi két qua nhu sau: ft hon 1/d, va nhiéu hon t/d, Escherichia coli ducng tinh f-glucuronidaza c6 trong mot millit (sn phdmm dang long) hoac trong mét gam (sén phém dang kh) trong 46 d, va d, la cae hé s6 pha long twang tig véi cdc dung dich pha loang d, va a 10.3.4 Néu tat c& cde CFU din hinh hodic khong dign hinh trén hai dia & 49 pha loang thir nhat 4, chit nhidu hon 300, khong cé CFU mau xanh 6 thé nhin thay, ho&ic néu trén hai dia & 4% pha loang tip theo d, chita it han 300 khudn lac, khong c6 CFU mau xanh can dém, thi biéu thi két qua nhu sau: it hon ‘ld, cde CFU ala Escherichia coli dung tinh fi-glucuronidaza ¢6 trong mot miliit (sn phém

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