TCVN TIEU CHUAN VIETNAM TCVN 10780-3:2016 ISO/TR 6579-3:2014 Xuat ban Hin 1 VI SINH VAT TRONG CHUOI THU'C PHAM - PHU'ONG PHAP PHAT HIEN, DINH LUONG VA XAC DINH TYP HUYET THANH CUA SALMONELLA - PHAN 3: HU'O'NG DAN XAC BINH TYP HUYET THANH CUA SALMONELLA SPP Microbiology of the food chain - Horizontal method for the detection, enumeration and serotyping of Salmonella - Part 3; Guidelines for serotyping of Salmonella spp HA NOI - 2016Loi noi dau TCVN 10780-3:2016 hoan toan tong duong véi ISO/TR 6579-3:2014; TCVN 10780-3:2016 do Ban kf thuat liéu chudn quéc gia TCVN/TCIF 13 Phuong phép phén tich vé lay mau bién soan, Tang cyc Tiéu chudn Bo luéng Chat lugng thém dinh, B6 Khoa hoe va Céng nghé cing bé. Bd ti8u chudn Vi sinh vét trong thye phém va the én chén nudi nay gdm cé cdc phan sau: TCVN 4829:2006 (ISO 6579:2002, Cor 1:2004) Vi sinh vat trong thye phém va thie &n chén nudi ~ Phuong phdp phét hign Salmonella trén dia thach va Siva 481 1:2008 TCVN 4829:2005 (ISO 6579:2002, ‘Amd. 1:2007) Vi sinh vat trong thy phém va thie an chain nudi ~ Phuong phép phat hign Salmonella spp. trén dfa thach - Sita d8i 1: Phu luc D: Phat hién Salmonella spp. trong phan dong vat va trong m&u méi trudng tir giai doan sin xudt ban dau; ‘TCVN 10780-2:2015 (ISO/TS 6579-2:2012) Vi sinh vat trong thyc phém vai thie &n chan nudi— Phuong phdp phdt hién, dinh iuong va xéc dinh kidy huyét thanh cia Salmonella ~ Phan 2: Binh luong bang ki thudt sé dém o6 xdc suét lon nhdt durge thu nbd; TTCVN 10780-3:2016 (ISO/TR 6579-3:2014) Vi sinh vét trong chudi thy phém — Phuong phép phét hién, dinh luong va xéc dinh typ huyél thanh cia Salmonefia - Ph&n 3: Hurting dén xéc dinh typ huyét thanh cia Salmonella spp. TCVN 10780-3:2016TIEU CHUAN QUOC GIA TCVN 10780-3:2016 Vi sinh vat trong chuéi thye pham - Phuong phap phat hién, dinh lwong va xac dinh typ huyét thanh cua Salmonella - Phan 3: Huong dan xac dinh typ huyét thanh cua Salmonella spp. Microbiology of the food chain ~ Horizontal method for the detection, enumeration and serotyping of Salmonella - Part 3: Guidelines for serotyping of Salmonella spp. CANH BAO ~ D8 bao vé sire khée cia nhan vién phéng thi nghigm, chi thts nghigm dé phat hign ‘Salmonella trong phang thir nghigm dugc trang bj phd hep, dudi sy kim soat ca ngwdi phan tich vi sinh vat ¢6 Kinh nghigm va rdt cn than trong qué trinh xi ly tt ca cac vat ligu d4 U, Ngwdi str dung tiéu chudn nay phai thanh thgo cdc thao tac théng theéng trong phong thir nghi§m. Tiéu chudn nay khdng dé cfip dén tit cd cfc van ad an toan, lién quan dén vigc six dung tiéu chudn, Ngudi sip dyng tiéu chudn phai ty thiét lp céc thao tac an toan strc khée thich hyp va dam bao tuan thi cc quy dinh hién hanh, 4 Pham vi 4p dung Tiéu chudn nay dua ra hwéng dn ve vige x4c dinh typ huyét thanh cdc serovar Salmonella va 6 thé 4p dung 48 xdc dinh typ huyét thanh ctia cdc ching c4y Salmonella spp. thuan khiét, phy thudc vao ngudn ma ching duge phan lap. 2 Tailigu vign dain Cac tai ligu vin dan sau rdt cdn thiét cho viée ap dung tiéu chudn nay. Bi véi cac tai ligu vién dn ghi nm c6ng bé thi 4p dung phién ban dugc néu. Odi voi cac tai ligu vién d&n khéng ghi n&m céng bé thi 4p dung phién ban méi nhat, bao gdm ca cdc siva déi, bd sung (néu cd). aTCVN 10780-3:2016 TCVN 6404 (ISO 7218), Vi sinh vat trong thye phém va thie an chén nudi — Yéu edu chung va huéng an kiém tra vi sinh vét TCVN 8128 (ISO 11133), Vi sinh vét trong thyc phdm, thie an chan nudi va nude - Chudn bj, dn xuét, bao quén va kiém tra hiéu néng cua mi trong nudi cy. ISO 6579-1, Microbiology of the food chain — Horizontal method for the detection, enumeration and serotyping of Salmonella — Part 1:Horizontal method for the detection of Salmonella spp. 3. Thuat ng@ va dink nghia Trong tiéu chun nay sir dyng céc thuat ngt va dinh nghia sau: 34 Salmonelia (Salmonetia) Tryc khuan gram 4m, oxidase Am tinh, ky khi thy y, khdng sinh bao ti, thuéng tao thanh cae khuan lac dung kinh tir 2 mm dn 4 mm trén mbi trréng dic chon ge va c6 cde dc tinh sinh hda va huyét thanh dién hinh khi phép thir dug thye hién theo tiéu chuan nay. 32 XAc dinh typ huyét thanh Salmonelia (serotyping of Salmonelia) Xéc dinh sy cé m&t hay khéng ob mat cdc kh4ng nguyén-O, khang nguyén-H va khéng nguyén-Vi ou thé trong ching vi khudn phan lap 64 khng dinh I Salmonetia (3.1). 33 Céng thirc khang nguyén (antigenic formula) Sy két hop ca cdc con sé va chi cai thé hign céc khang nguyén O-, H- va Vi- eda ching vi khuan phan 4p duoc khang dinh la Salmonella (3.1). 4 Nguyén tic 88 xac inh typ huyét thanh cla Salmonelfa spp., cac khang nguyén sau day duoc xac dinh cho cdc chiing vi khudn phan lap 64 duge khang dinh sinh héa !a Salmonella spp.: Khang nguyén-O, khéng nguyén-H va khdng nguyén-Vi CHU THICH: Co thé si dung cae quy trinh khae 48 khang dinh ching vi khudn phan itp duge la Salmonella spp. voi éidu ign sy phi hop cia quy trinh thay thé 48 dur xéic nhgn [xem TCVN 6404 (ISO 7218)}.TCVN 10780-3:2016 5 Mai treong nudi cdy va huyét thanh 5.41 Quy dinh chung ‘6i voi thyc hanh trong phdng thir nghiém hién tai, 4p dung TCVN 6404 (ISO 7218). ‘Bi voi phép thi hiéu nang oda méi trrdng audi cdy, theo TCVN 8128 (ISO 11133). 5.2 Moi trong nudi cdy va thudc thir Xem Phylyc A. 6.3 Khang huyét thanh Khang huyét thanh-O, Khang huyét thanh-H va khang huyét thanh-Vi o6 ban sn tir c4c nha cung c4p khac nhau. Théng tin vé cdc khang huyét thanh da gia va khang huyét thanh don gia duc néu trong Phy iyo B. 6 Thiét bj, dung cy Cé thé sir dung cac dung oy thiy tinh dung mét fn 48 thay cho cdc dyng cy ding nhidu tan, néu chung ¢6 cc quy dinh ky thuat tuong ty. ‘Sip dung cac thiét bi. dyng cu théng thudéng cia phong thir nghiém vi sinh va cy thé nhu sau: 6.1 Ta dm, a4 nuéi cay va phan lap Saimonelia, o6 kha nang duy tl nhiét 46 trong khodng tir 34 °C. én 38 °C. ‘CHU THICH: Trong tiéu chudn nay, nhigt 46 d kh&ng phai ia mét thing #4 t6i han. Cc chiing phan Mp dug nudi cdy 8 thu urge Gy 4ii sinh khdi vi khudn thudn khiét, 68 thye hien ofc phép this. Vi vay, bude nubi cdy durge thyc hién & nhiét 49 phat trién ti wu. Bdi vei Salmonella nhiét 4 thudng tir 34 °C dén 38 °C. 6.2. Ta sdy (48 Khir tring kh6) hodc ndt h4p (48 khir triing wot). Xem TCVN 6404 (ISO 7218). 6.3 Ti lanh (4€ bao quan méi trvdng nudi cdy 44 chudn bi), 66 kha naing duy tri nhiét 46 & 5 °C43°C. 64 Lam kinh. 6.5 Dung cu cdy v6 tring, vi dy kim cdy, que cdy hod vong oy (vi dy 4 nl). 6.6 Ong nghiém va binh edu vé tring, c6 dung tich thich hyp. Co thé st’ dyng binh hode chai va ‘ng nghiém cé nap bang kim loai hodic bang chat déo (nip vain) khong déc. 6.7 Dia Petri vd trang, cd dudng kinh khodng 55 mm dén 90 mm.TCVN 10780-3:2016 6.8 Ndi cach thuiy, ¢6 kha nang duy tri nhiét 6 & 47 °c dén 50 °C. 6.9. Nbi cach thay (hogc td Am), cé kha nang duy trl nhigt do & 50°C 22°C. 7 Lay mau igu quan trang [a phéng thir nghiém lam vie voi chiing cy thuan khiét d2 duge khang dinh sinh hoa la Salmonelia spp. 8 Phan loai Salmonella 81 Téng quan Ct khoang 7 n&m, Trung tam nghién ctu vd chuan Saimonetia cla WHO (Vidn Pasteur, Paris) s6 cong bé ban cap nhat vé "Cong thir khang nguyén cla cdc serovar Salmonella", a6 la co sé 48 dinh danh cdc tén typ huyét thanh serovar va céng thire déi véi cae chiing vi khuan phan lap Salmonella spp. Théi diém cong bé phién ban mdi nhét cla so 43 White-Kauffmann-Le Minor la nm 2007 (Tai ligu tham khao {9)). CHU THICH: Nhiing bd sung cho so 4 White-Kauffmann-Le- Minor d& durge céng bé trong cudn Aghién cou Vi sinh, mot 4n phd aia Vign Pasteur (tute day goi la Annales de Mnsttat Pasteur/Microbiologie). Vi dy, bd sung 36 47 48 duge etng ‘bd nim 2010 va diva ra cdc serovar méi G8 phat hign tir nirn 2003 dén nim 2007 (Tai Eu tham khdo [10). Tiéu chudn nay dua ra hudng dan vé xac dinh typ huyét thanh ciia céc serovar Salmonella. 82 Danh phap (cdch dat tn) Cae danh phap khac nhau 44 dugc st dung (hod van dang duge sty dung) cho cdc ching ‘Salmonella: — Ban dau, Kauffmann (Tai ligu tham khdo [12]) da coi méi serovar Salmonella \a mot loai ring biét; ~ Cac kiéu loai hac nhau d& dugc str dung: S. enterica vs.$. choleraesuis, mdi loai co mot ching Khao; ~ Mét s6 ching Salmonetia “quan trong” (nhu Salmonella Typhi va Salmonella Paratyphi) urge coi la 8c loai va khong phai “chi” cac serovar clia cac loai. Hoi ding trong tai cia Tidu ban kg thuat quéc t& va hé thdng ty déng nghién civu vé sinh vat nhan Aguyén thdy (Procaryotes) cho thay rang cé thé si dung nhidu tir ddng nghia trong viéc goi tén ‘Salmonella (Tai ligu tham khdo [22]). Trong tiéu chudn nay, cdc tén gai hién tai 44 dug chép nhan sir dung réng rai, da duoc Trung tam nghién ciru vé chudn Salmonella cilia WHO [Tai liéu tham khdo 9], Higp hdi vé Vi sinh vat otia MY (Tai fiu tham khdo [20)), Trung tam kiém soat va ngdin ngiva bénh tat 8TCVN 10780-3:2016 (Tai ligu tham khdo [3]) va S8 tay Bergey's (Tal ligu tham khdo [17]) ch4p nh&n. Theo danh phap hi¢n hanh, cde chi Salmonella thuge ho Enterobacteriaceae va chi bao gdm hai loai S. enterica va S. bongort. S. enterica dye chia thanh sau phan loai: S. entefica subsp: enterica, S. enterica subsp. salamee, S. enterica subsp. arizonae, S. enterica subsp. diarizonae, S. enterica subsp. houtenae va S. enterica subsp. indice. Cac serovar Salmonella thude S. enterica.subsp. enterica thuémg phan lap dec nhiéu nhat (irén 99,5 % trong s6 cde chiing Salmonella phn lap dug) va chiing dugc dat tén, thugng lién quan dén vi ti dia ly nei serovar duge phan lap lan dau. Cac serovar thugc cdc phan loai khae cla S. enterica va cla S, bongori dug dat tén theo céng thie khang nguyén cila ching. Do cd sy két hop cilia cdc phan lodi va nhigu serovar, nén tén day dui r&t dai (vi dy: Salmoneta enterica subsp. Enterica serovar Typhimurium). Do 46, cdch gol ngén hon thuréng st? dung dé chi tén cia cdc. serovar cla cc phan lodi enterica. Hé thong White-Kauffmann-Le-Minor dua ra ggi y cdc tén goi rit gon sau day: S, enterica serovar Typhimurium hogs Salmonefia ser, Typhimurium. Theo Tai ligu tham khdo [3], @ trich dln dau tién olla serovar trong tén chi cdn theo sau la tl “serovar” viét tét la "ser." va sau dé tén la tén cla serovar. Sau a6, tén cé thé duce viét bang tén chi theo sau [a tén serovar (vi dy ‘Saimonelta Typhimurium). Gach ggi ten serovar Salmonelfa nay cing dug ch4p nhan & hau hét céc tap chi [vi dy: Tap chi etia Higp h6i Vi sinh vat My (ASM)] va cling durge st dung trong tigu chudn nay. Tom Iai, goi tén Salmonella nhu sau: Ho:Enterobacteriaceae (chtr c4l du tién viét hoa, chitin nghiéng) Chi:Salmonelta (chi cai du tién viét hoa, chit in nghiéng) Loai:enterica (khong vidt hoa, chirin nghiéng) Phan loal:enterica (khéng viét hoa, cht in nghiéng) Serovar (typ huyét thanh hoac ser.): vi du: Typhimurium (ch cai dau tién viét hoa, khéng in nghiéng) Phan loal: salamae arizonae diarizonae houtenae indica Loai: bongort Trong phan bé sung cho hé théng White-Kauffmann-Le- Minor (Tai ligu tham khdo [10)) 66 trén 2 600 serovar Salmonella duge 4 cap dén va sé lugng dugc ting déu, nhur théng ké trong Bang 1.TCVN 10780-3:2016 Bang 1 - Sé lugng serovar Salmonella qua cac nam Bé sung Loaifphain loal 1998" 2001? l ‘2007° ‘$6 lugng serovar |saimonelia enterica 243 | 2502 2687 ‘subsp. enterica 1454 yaoz | 1847 _| fsubsp. salamae 489 500 513 Isubsp. arizonae 4 95 100 lsubsp. diarizonae 324 331 3at subsp. houfenae 70 71 [7 subsp. indica 12 13 13 |satmonella bongor! 2» 2 23 |Téng sé serovar (chi Salmonelia) 2483 2523 2610 }* Tai figu tham khdo (18). P Tai figu tham kndo [19]. [_Talliw them ko [10}, qua céc nim 2003 aén 2007. 8.3 Cac dac tinh sinh héa Cac loai va phan loai Salmonella duge xc dinh dya trén cdc phép thir sinh héa khdc nhau. Trong Bang 2 dua ra cc dc tinh khac nhau. Xem Tai ligu tham khdo [9] va Phy luc © dG bidt thém chi tidt. Bang 2— Céc dc tinh sinh héa cia cAc loai va phan loai Salmonella (Tai ligu tham khdo [9]) Loai S. enterica S. bongori Phan toai enterica | salamae } arizonas houtenae | indica Dutcitol + + = 4 + ONPG" (2h) - - + | a + Malonate - + + 7 - Gelatinase - + + + + + - T Sorbitol + + + + + 7 + —LTCVN 10780-3:2016 (Bang 2 ~ k&t thac) Loal 8. enterica 8. bongori Phat trién voi KCN? - - - - + - + Us)-tartrate® + - - - - - . Galacturonate - + - + + + + +y-Glutamyitransferase + + - + + + + -Ghucuronidase 4 qd - + - d - Mucate + + + (70 %) - + + Selicin - - - - + 7 7 Lactose - - | -c75%)] 475%} q - Dung gidi boi pha O1 + + - + - + 4 290 % phan ung durong tinh; f 2 80 % phan ong am tinh; cdc phan ng khée nhau cia c&¢ serovar khdc nhau; ‘o-Nilvopheny|-8-D-galactopyranoside (phép thi (B-galactosidase]. P Kali xyanua; = D-Tarrat, Paratyphi 8:-, Paratyphl B blovar Java:+ fF = Typhimusium:d, Dubtin:-. 8.4 Dc tinh khang nguyén 8.4.1 Yéu clu chung Cac éac tinh khang nguyén quan trong cla Salmonella d6i voi cdc phép tht huyét thanh duge chia thanh ba loai chin nhu sau: ~ Khang nguyén-O, edn goi Ia khéng nguyén than; = Khang nguyén-H, cén ggi ld khang nguyén léng; = Khang nguyén-Vi, cén gol la khang nguyén ve. 4TCVN 10780-3:2016 Cée céng thie khéng nguyén cla Salmonella spp. gdm c6 ba typ khang nguyén sau day: khang nguyén-O, khang nguyén-Vi (néu cé): khéng nguyén-H olla pha thiy nhdt: khang nguyén-H cia pha thir hai. Vi dy: cng thie khang nguyén ciia Salmonella Paratyphi C la:6.7[Vik-c:1.5, voi khang nguyén-O:6 va 0:7; voi khang nguyén-Vi, c6 thé c6 mat hoe khéng cd mat (ky hiéu bdi cdc d4u ngo’ic vusng), voi khang nguyén-H Hic eda pha thir nhat, véi cdc khéng nguyén-H H:1 va H:6 cla pha tht hai 84,2 Khdng nguyén-O (khang nguyén than) Khang nguyén nay bao gdm thanh phan véch té bao va cdc chat chinh Ia polysaccharid, protein va phospholipid. Khang nguyén-O rat b&n va 06 thé chiu duge nhiét 46 dén 100 °C trong 150 min, xt ly bang etanol 95 % thé tich hoac axit loding (Tai ligu tham khdo [16)). Phan ing cla khang nguyén-O voi khang huyét thanh fam ngung két hat. Trude day, trong tai ligu cla Kauffmann-White (Tai ligu tham khdo [9}) cdc khang nguyén-O dug phan loai thanh c&c nhém khang nguyén-O. Cac nhém nay duge at tén voi ch cdi La Ma bat dAu véi nhém A, trong dé gdm cb khang Aguyén 0:2 dén nhém Z od chtra khang nguyén 0:60. VI cé nhidu céc khang nguyén-O, nén cde khang nguyén cén Iai khéng duge dua vao nhém, nhung da durgc dat tén thanh cdc khdng nguyén-0 tir 0:51 dén 0:67. Ngay nay, méi nhém O st dung O-ydu té dién hinh dug ding nhidu hon. Cac chi cai nay 64 duge git Iai va dugc dua vao trong ddu ngo&c don, vi dy:0:4 [B] (Tai ligu tham khdo (9]). Trong Béng 3 dua ra céctén goi oi va moi. Bang 3 ~ C4c nhém huyét thanh Salmoneifa (tén goi ci} va cac khang nguyén-O cé lién quan (tén ggi mol) Nhém Khang nguyén-O| Nhom —Khéngnguyén-O| © Nhom —_ Khang nguyén-O A 2 GrGe 2 Q 39 B 4 H 6.14 R 40 cuca 67 1 16 s a Ca Cs 8 J 7 T 42 o 9 K 18 u 43 0; 9,46 L 2 v “4 Os 9,46,27 M 28 w 45 E,( Ez, Ea)? 3,10 N 30 x 47 & 1,3,19 ° 35 Y 48 F "1 Pp 38 Zz 50 G4 03 dirac gop vao Cr [ Enya En68 duge gop vao Es. 12TCVN 10780-3:2016 8.4.3 Khang nguyén-H (khang nguyén l6ng) Khang nguyén nay nam trén sqi lng va cac thanh phan chinh [a protein. Khang nguyén nay khéng b&n ‘b&ng khang nguyén-O, d8 bj phan hiy trong ancol, axit va nhiét 46 trén 60 °C, nhung ob khé nding chiu urge dung dich formalin 0,5 % thé tich (Tai figu tham khao [16)). Phan tng eda khdng nguyén-H voi khang huyét thanh fam ngung két bong. Nhiéu Salmonelia spp. cd hai pha khang nguyén-H, nhung cac bién thé mét pha va ba pha cha dugc biét, Pha dau tién duoc oi la pha dac higu va pha thy hal dug goi la pha khéng dic higu. Pha ddu duge ky higu bang chi thuréng tir a dén z, Tuy nhién, ti khi nhan biét khang nguyén-z, thi nhidu khang nguyén-H mdi da duge phat hign va durge 3t t8n 21, 22, 25.0.0 Cée vi dy vé cde serovar mét pha [a: = Salmonella Paratyphi A: 1,2,12:a:[1,5] voi Ha déi voi pha du tién, trong dau ngode vudng la pha thi hai (H:1,5) 06 thé o6 mat hoe khéng ¢6 mat; — Salmonella Typhi: 9,12,[Vij:d:-; voi H:d déi voi pha du; - Solmonelia Derby: 1,4,[5], 12:f.9:11,2]; vei H:f.g 46i véi pha Au tién, khi d3 pha thir hai (H:1,2) 08 thé cd hodic khong od mat; - Salmonelfa Enteritidis: 1,9,12:9,m:-; véi H:g,m di voi pha dau. Ngoai cdc H:g,m, mét s6 ching cd. thé cé Hip, hodc H:f, hod Hit, Céc ching ac biét o6 thd cé khdng nguyén H:1,7 14 pha th hal; = Salmonella Dublin: 1,9.12,[Vi:9,p:~ véi H:g,p d6i voi pha dau. CHU THICH 1: Cac yéu 160 gach chan durge xac dinh bang bién trang thyc khudn. Ching chi od mét néu ching cay dugc dung gidi bang thé thire Khun bién trang ttrong dng (Tai bu thar khdo [2)). CHU THICH 2: Cac yéu 18-0 hoc -H trong ngode vutng ob thé e& mat hoc khéng cd mt, ma Khong ign quan dn thye ‘khudin thé bidn trang (Tal iu them khdo (2). CHU THICH 3: Céc ching Salmonella Derby v8 Salmonella Entertidis r&t hidm gap. C6 thd cén chuyén ébi pha 8 phat hién ‘cic ching hiém gdp hay. Tuy nhién, didu nay chT cdn thiét voi nog trong hep nhat dinn {vi dy trong trréng hep sai lech ngudn vashoBc trxég hop 06 di chuyén (6c bleh). 8.4.4 Khang nguyén-Vi (khang nguyén vd) Khang nguyén nay la khang nguyén vé va co thé che khuat céc khang nguyén-O lam cho cac vi khudn khong ngung két voi khang huyét thanh-O. Thanh phan chinh ctia khang nguyén-Vi la polysaccharid. Céc ching Saimonetia cé khang nguy&n-Vi d}c hon so véi cdc ching khéng c6 khang nguyén-Vi. Khang nguyén-Vi oé thé ch? ¢6 mat trong ba Saimanelia serovar.TCVN 10780-3:2016 — Salmonetia Typhi: 9,12, [Vil:d:-; - Salmonella Paratyphi C: 6,7,{\il:o:1.5; - Salmonella Dublin: 1,9,12, [Vil:g,p:-. Trong cac ddu ngoac vusng cho thay cé thé 66 hode khéng c6 mat khang nguyén-Vi. CHU THICH: Sy cé mat cdc khang nguy8r-Vi trong cdc ching phan lap cla Salmonefia tir mu thye phdm hoje mu thi y ta rat hidm gép. Néu ob m&t khang nguyan-Vi, nb 88 che khudt cic khéng nguyén-O. B& phat hign cdc khéng nguyén-O, 06 thé cin lam néng huyan phi mu phan Ip (vi ductrong dung dich nutse mudi sinh if) & 100 °C trong 60 min, hose & 120 *C trong 18min, 9 Quy trinh xc dinh typ huyét thanh Salmonella 9.1 Yéucdu chung ‘Truéc khi bat dau x4c dinh typ huyét thanh, can khang dinh sinh héa rang ching phan lap duge thuée chi Salmonella (nh quy dinh trong ISO 6579-1). Mac dir cdc khang nguyén-H [4 dae higu cho Salmonella, thi mt s6 khang nguyén-O throng gap & cc chi kh4c nhau cua Enterobacteraceae (vi dy: Safmonelia, Citrobacter, Hatnia). ‘CHU THICH: Co tné str dung c&c quy trinh khac 6 khdng dink rang ching phan Kap dugc thudc chi Salmons, voi dibu kien {8 quy trnh thay thé ny 48 durge xc nisin fxemn TCVN 6404 (180 7218). Mi nha cung c4p cdc khang huyét thanh san xudt ra céc b6 khang huyét thanh riéng, kém theo hung dn str dung. Do d6, khéng th8 dua ra mét bd huréng d&n cho viéc xac dinh typ huyét thanh, vi can phdi thy hién theo ding huéng dan cla nha cung cép dé cé dug két qua téi uu. Mét sé nha san xuat cung cép cac khang huyét thanh (hdn hyp cla nhiéu khang huyét thanh-O hode khang huyét thanh-H) rt htru ich cho vige xéc dinh typ huyét thanh chura biét. Khi chiing ngung két véi khang huyét thanh hdn hop, thi cé thé tiép tuc thir nghiém véi nhém khang huyét thanh vashode chi véi loai khang huyét thanh hén hop duong tinh. Khi cin nhén manh cdc serovar nhat dinh va kha naing dé chi ra cac ‘serovar khdc theo Salmonella spp. thi 6 thé tin hanh ngung két ngay chi voi khang huyét thanh don gid cy thé cda serovar 6 lién quan. Trong Phy luc B dva ra quy trinh chung vé viéc x4e dinh typ huyét thanh chura biét cia ching phan lap Salmonella 9.2. Vi dy vé quy trinh xéc dinh typ huyét thanh cho nam serovar Safmonella anh huéng dén strc khée cng ding 14TCVN 10780-3:2016 9.2.4 Yéu cdu chung ‘Trong vi dy dui day mé td quy trinh xac dinh typ huyét thanh cho nam serovar Salmonella anh hwdng én size khée cOng dang (xem Phy luc D). Trong Bang 4 dua ra cdc ching nay voi céng thire khang nguyén cia ching. Trong cdc phan sau, mé ta viée ngung két cdc ching phan lap Salmonella, quy trinh nay thuéng dues thye hién nhiéu nhét. Tuy nhién, vin 06 ede quy trinh khde, vi dy: phuong phap dia vi giéng (xem Phy ye). Bang 4~ Céng thisc khang nguyén cia nam serovar Salmonella Anh hweng dén sic khde cng ddng Khang nguyén-H Ten Khdng nguyén-0” Phat Pha2 |Satmonetta Typhimurium AAsgy2 i 42 \Salmonetta Enteritidis* 19.12 gm : \Satmonetta infantis e718 ' 18 \Safmonetta Virchow 67.14 ' 12 \safmoneta Hadar 68 20 enx Nagoai cdc yeu t6 Higam, mot 68 ching 06 thé c6 yu tb Hip hode Hi hoe H:t. Trir cae ching 06 thd cb Khang! Prouyén H:1,7 4 pha the 2 (Tai fu tham khdo (9). P ae yeu %6 0 gach chan duge xée dinh bang bién trang thye Khun. Ching chi cb mat néu ching cy duoc dung| Jpidi bling thé thy khudn bién trang trang Geng (Tai ligu tham khdo {2). 9.2.2 Chon cac khudn lac nghi nge a Salmonella Cay mét khudn lac tl ching cdy thuan khiét nghi ngé [a Salmonella theo diac tinh sinh héa. Ste dung méi truéng nudi cdy va cdc phong phdp do nha sdn xuat quy dinh a4i voi khang huyét thanh duge sir dung. Néu khong ¢6 sn thong tin, thi cé thé si dung mai trvdng thach khéng chen loc nhu thach dinh dung (vi dy: xem A.2). U cae dia thach dinh duéng 64 cy tir 34 °C dén 38 °C (6.1) "qua dém" {khoang 18 h). 9.2.3 Kiém tra phan tng ty ngung két Vi dy vd phép thir kiém tra phan tng ty ngung két dug mé ta sau day. Cé thé str dung cdc phyong phap khac, Thy hign theo huéng d&n cia nha sén xuat, — Cho mét giot nvéc mudi (06 thé thay ddi ti” NaCl 8,5 g/l dén NaCl 35 g/l, ndng 4 cao hon cé thé nhay hon) trén lam kinh (6.4).TCVN 10780-3:2016 Chuyén mét lgng nhé chiing cay vi khudn (vi dy: 06 thé ly mét lgng 1 pl baing que cdy dung mét ln) sang lam kinh va tron véi giot nuéc musi. ~ Nghiéng nhe lam kinh qua Iai. Tay thudc vao nha san xudt vashodc néng 66 mudi, throng khoang 5 § én 60s, ~ Dénh gia huy&n phi. Khi cd mat cdc het trong huyén phi ching td cd sy tyr ngung két. Cae ching, 66 phan Gng dong tinh trong phép thi ty ngung két rét khé kiém tra thém 68 xdc dinh typ huyét thanh. Béi véi cdc ching ty ngung két, khéng thé thyc hién dug phép thir khang nguyén-O. Tuy nhién, doi khi van o6 thé didu tra vé cc khéng nguyén-H. Néu mé6t chiding cho thy tu ngung két, cd thé thi trén mét hodc hai céch dudi day trén cing mot khudn lac vayhoae trén cdc khuan lac khac. = Hoa khuan lac trong nuréc vé triing thay vi trong dung dich nude muéi va tién hanh quy trinh ty ngung két nhy trén. ~ Cy cac ching trén méi trréng thach ban d&c nh méi truéng thach Rappaport Vassiliadis cai bién (MSRV) {theo quy dinh trong [SO 6579-1) va sir dung khuan lac tir thach ban dac dé thyrc hign quy trinh ty ngung két nhu trén, CHU THICH: Céo ching ty ngung két con due gol 18 cdc chiing "rp". 9.2.4 Ngung két voi khang huyét thanh-O- C4 huréng dan sir dyng eda cde Khang huyét thanh c6 thé khdc nhau gitra cdc nha san xudt Do 66, didu quan trong [a luén tuan thd c&c huéng dn cia nha sdn xudt. Hau hét cdc nha san xuat str dyng phueng phap ngung két lam kinh d@ phat hién cdc khdng nguyén- O. Trong phuong phap nay, tron mét gigt huyét thanh véi huyén phi vi khudn {try tiép ti dia, ng hoc canh thang) trén lam kinh. Nghiéng nhe lam kinh qua Iai, Sau 4, quan sat su ngung két trén lam Kinh. Su c6 mat cdc hat chieng td phan Gng dung tinh. 6i véi cdc nha san xudt khde nhau, 06 thé c6 nhtng thay i trong cach tién hanh nhu sau: ~ Kich tho cita giot trén tam kinh (vi dy: 25 yl hoe mét "giot"]; - Céch trén khang huyét thanh véi vi khuan trén fam kinh (vi khudn durge ldy tryc tiép tir mai trvéng thach hodc huyén phi, khang huyét thanh duge cho tryc tiép trén lam kinh hay dirgc cho vao huyén pho vi khuan); — Thdi gian nghiéng trot lam kinh (c6 thé dao déng tir 5 s dén 60TCVN 10780-3:2016 . = C&ch quan sét sur ngung két (phéng dai hay khéng, nan téi hodc anh sang binh throng); = Gidi thich cac két qua (doc cdc chu thich lién quan én nhémg han ché ctia quy trinh} 9.2.5 Ngung két voi khang huyét thanh-H ‘Sau khi ngung két véi khang nguyén-O, tién hanh ngung két véi khang nguyén-H. Salmonel/a thudng ¢6 hai loai khang nguy&n-H (pha 1 va pha 2). Néu mét pha-H dugc tim thy am tinh voi cde chiing hai pha, thi cAn thy hign phuong phép dao pha. Pha-H chinh bi khéng ché bang phuong phép do pha. Bang cach khéng ché pha-H chinh, thi pha-H thir hai c6 thé dugc thé hién va xde dinh. Phuong phdp thudng duc st? dyng d& dao pha la phuong phap ota Sven Gard, xem 9.2.7. B6i voi viéc nay, khang huyét thanh d&o pha cy thé duge cho vao méi trueng thach va ching Salmonella dug céy thanh diém trén dia. Méi tnréng thach phdi dd mém d8 cho Salmonella di chuyén dé dang trong méi trong. Nang dd thach trong méi trvdng cé thé thay ddi ti 0,5 % dén 1 % khéi lvong (tly thude vao ndng @6 gel iia thach). Vi dy sy di chuyn trén bé mat méi trong due dua ra trong A.3. Cac vi dy khde vé do pha Guryc diva ra trong Phy luc F. é kiém tra v8 sw ngung két, thyre hign theo hudng dain cia nha san xudt. 9.2.6 Phép thi ngwng két 44 xac dinh typ huyét thanh cia ndm serovar Salmonella anh hudng dén strc khée e6ng ddng 9.2.6.1 Yéucdu chung 4 phdt hién nm serovar Salmonella G8 c4p trong Bang 4, edn dén cdc khang huyét thanh-O va khang huyét thanh-H sau day: 0:4, 0:5, 0:6 (0:61 ho&e 0:6,7 hoc 0:6, 14,24), 0:7. 0:8, 0:9 va 0:46, Hii, H:2, H:G hoe H:g (don gia), Him, H:q, His, Hit, Hr, H:5, H:Zie Va Hix. é x4c dinh cdc typ huyét thanh cia cac chiing Salmonella theo thir ty néu trong Bang 4, thuc hién 2 Gdn 9.2.6.5 (xem so 4 tai Phy luc D). quy trinh quy dinh trong 9. 4 biét thém thong tin v8 cac phép th tun ty cdc khang nguyén khde nhau, xem Phy lyc B. 9.2.6.2 Néu 0:4 duong tinh Ngung két voi 0:5. Két qua o6 thé [a dureng tinh hay m tinh d6i véi Salmonella Typhimurium, van con ¢6 théng tin c6 thé lién quan dén cac muc dich dich té, Ngung két voi cc khang huyét thanh H:i va H:2 (cé thé can dao pha).TCVN 10780-3:2016 Cac serovar la chang Typhimurium néu cd hai phan ung nay fa duong tinh. Ngung két voi 0:12 Ia khong can thiét d& chieng to ching phan lap Salmonella Typhimurium, vi 0:4 duong tinh chiong t8 cd mat 0:12. Twong ty, viéc phat hién khang nguyén H:1 khéng cé khd nang phan biét va khéng can phai kiém tra bd sung. 9.2.6.3 Néu 0:9 duong tinh Ngung két voi khang huyét thanh H:G (hén hop) hoc véi khang huyét thanh H:g (khang thé don gia), Néu phan tng nay duong tinh, tién hanh ngung két tiép voi khang huyét thanh H:m. Néu két qua dvong tinh, nguing két vii cdc khang huyét thanh H:¢ va H:s 4 kiém chieng am. Néu nhing phan tong nay &m tinh, ngung két tiép voi 0:46 va néu muén, ngung két tiép voi 0:12, Neu 0:46 am tinh (va 0:12 duong tinh), thi serovar cla chiing la Enteritidis. CHU THICH: Ngoai cdc yéu 18 H:g.m, mot sé ching Salmonella Enteritidis 5 ‘thé cho phn eng duvang tinh vei khang huyét thanh H:p hoe H:f hoge H:t. Tuy nhién, cc chiing nay la r&t him. Tre cac chiing dic bigt co thé 6 khang nguyén H:1,7 nhur tapha thera 9.2.6.4 Néu 0:7 dong tinh Ngung két vai khang nguyén H:r, H:2 va H:5 (06 thé can dd pha). Néu Hirva H:2 dong tinh, serovar cia chiing la: Virchow. Néu H:rva H:5 dong tinh, serovar cla ching la: Infantis. Viéc phat hign khang nguyén H:1 khéng c6 khd nang phan biét va khong can phai kiém tra bd sung. 9.2.6.5 Néu 0:8 duong tinh Ngung két vai cac khang nguyén H:zio va H:x. Néu ca hai déu duong tinh, ngung két tiép voi cdc khang huyét thanh 0:6, hoac 0:6,7 hodc 0:6,14,24. Néu 0:6, hod 0:6,7 hode 0:6,14,24 durang tinh thi cdc serovar cila chiing la:Hadar. Mat sé mé khang huyét thanh O:6,7 khdng phan tng véi ching 0:6,8. Khi mua khang huyét thanh nay, ‘can chac chan rang 6 phan tmg véi chting O:6.8 (hdi céc nha san xuat). CHU THICH: Dang bién thé khudn tac 66 thé xdy ra véi cc bidu hign cila khang nguyén 0:6, bai mot sé serovar nhém huyét thanh C: (Tai igu tham kno [11]. VI IY do 6, khong phat ide nao cing co thé nhin biét duoc, vi dy Saimoneve Hadar va ‘Salmonella Istanbul ahu cac serovar r6 rang.TCVN 10780-3:2016 9.2.7 Vi dy vé dao pha si? dung phuong phap Sven Gard Vi dy nay mé ta viée do pha ding cho viée xée dinh typ huyét thanh Salmonella Typhimurium. Nhin chung, phurong phdp nay eting 4p dung duige cho cc serovar Salmonella khac. Khi 0:4 va H:i duong tinh, cdn H:2 4m tinh, thi chun bj mét dia thach di chuy&n (xem A.3 hoac F.1), v6i khang-H:i (vi dy: hn hgp huyét thanh ado pha cé chiva Hii). Chm. ching vi khudn vao gia dia thach. U dia ty 34 °C dén 38 °C (6.1) qua dém {khoang 18 h). Sau khid, [am lai viée ngung két ching voi khang nguyén H:2. Néu H:2 lai am tinh, ngung két tiép véi Hii. Néu H:i am tinh ho&c c6 phan Ung yéu, thi ching nay khéng phdi la Salmonella Typhimurium. Néu H:i cho phan tng duong tinh (manh), thi lap lai viéc 640 pha. Chuan bj lai dla thach v6i khang-H:i va ly vikhudn tir diém xa nht moc lan trén dia thach, ao pha ther nhdt 68 cdy vao dia thir hai, Lap lai quy trinh dao pha nhu trén. CHU THICH 1: BOi Khi cn bd sung khang huyét thanh Hi vao thech (cla Ta dao pha lap lei) 48 thu duce phn Ung tét hon. CHU THICH 2: Cling 6 thé o6 cdc bidn thd mot pha cia Safmanetla Typhimurium, vi dy do thiéu hoge khong thé hign pha H ‘thi hai 1,4,[5]12:i-. Trong khi xéc dinh typ huyét than bién thé nay, co thé phai 8p Iai mot lan hode nhidu lan vide dao pha dé ogi trr sy €6 mt ciia pha thé hai. Ngoal ra, ob th sir dung phuong phap phan ti G8 khang dinh ob phai dién thé cia ‘Salmonetfe Typhimurium hay Khong, 10 Kidm soat chat lugng Huyét thanh dugc str dung a4 ngung két phai trong (triv cdc khang huyét thanh duge str dyng é thy nghiém latex). Luén luén phai kiém tra huyét thanh tre khi sir dung. Trrdng hyp bi duc, the hién theo huéng dn cia nha san xuat. Céc quy trinh kiém sodt chdt Iueng ca quy trinh xc dinh typ huyét thanh nhw sau: ~ Méi tuan lam vie chon hai chiing (ho&c sé Iugng chiing chiém khoang 2 % khéi Iueng chiing phan fap durgc). Mai chiing duge cdy hai Lan, Cac ching cdy chuyén durgc xir ly nhu [a hai ching méi phan lap a8 xéc dinh typ huyét thanh, Sau khi hoan thanh céng viéc, céc két qua cila hai chiing va cae ching cay chuy&n duoc so s4nh vé sy khdc bist. Néu c6 bit ky mat sy khde bit nao, thi sé dure nghién tu tiép. — Phéng thir nghiém lun git? dy ii ching géc cc serovar Salmonella dc higu (vi dy: ti” bd suu tap. chiing cdy hode tir cdc nghién ciru so s4nh lién phéng). Ching Iuu géc nay, dinh ky (vi du: hang tudn) chon mét hoac hai serovar cla 10 serovar thudng gap nhat trong phéng thir nghiém oé kiém tra quy trinh xae dinh typ huyét thanh, Cac serovar due sv dung dé thye hién vig kiém soat chat lugng od 19TCVN 10780-3:2016 thé thay 481 hang tuan 68 dam bao rang trong mét khoang thdl gian céc khang huyét thanh khac nhau abu 43 duge kiém tra, = Phai kiém tra kha ning xdc dinh typ huyét thanh gifra cc phong thi nghigm khéc nhau. D4 thyc hign, nén thudng xuyén tham gia vao cdc nghién ciru so sénh lién phong, khi cé thé. 411 Bao cao két qua ‘Déi véi cdc chling phan lap thude S. enterica subsp. enlerica, thl b4o cdo tén (Bay di) va khi 06 thé/khi ‘dn kém theo cOng thirc khang nguyén. 86i voi cdc loai (phan loai) khdc, thi bdo cdo céng thirc khang nguyén tim thdy du¢c. Cac ky higu cla cac céng thirc khang nguyén la nh sau: Khang nguyén-O (cach nhau bang d4u phy), khang nguyén-Vi (néu 6): cdc khéng nguyén-H cla pha dau tién (cach nhau bang déu phay): c&c khéng nguyén-H cla pha thir hai (néu cd; cach bang déu phy); khdng nguyén-H cila pha thir ba (néu cd). Vi du, theo hé théng White-Kauffmann-Le-Minor (Tai figu tham khdo [9]), céng thie khang nguyén day di cba Salmonelta Typhimurium la: 4.4,[5), 12::4,2. Véi cdc khang nguyén-O 0:4,4,I5],12; trong dé yéu té O duge gach duéi dug xac dinh bang thé thyc khudn bién trang va chi xuat hién néu chiing ody duc dung gidi bang thye khudn thé bién trang tong Ung va trong ddu ngoac vudng chi ra rang 08 thé co hoaic khéng o6 mat yéu td lién quan dén thyc khudn thé bién trang (Tai ligu tham khdo [9}) : voi cdc khdng nguyén-H, H:i d6i véi pha thir nhat va H:1,2 d6i voi pha thir hai. Dé bdo cdo chinh thir, céng thir Khéng nguyén ciia chiing phan lap, tét nhat la khdng co ngodc vuéng hoae khong c6 gach dud. Béo céo céng thirc khang nguyén 44 durge xac dinh, vi dy 4,12::1,2. ‘bi véi cdc phn loai khdc clia S. enterica, lodi oO serovar phu hop duge thé hién bang ky higu sau ay (Tai ligu tham kndo [9): 11 66i v6i serovar S. enterica subsp. salamae; Illa 46i véi serovar S. enterica subsp. arizonae; {lb d8i voi serovar S. enterica subsp. diarizonae; IV déivéi serovar S. enterica subsp. houtenae; VI d6i voi serovar S. enterica subsp. indica. Vi dy vé bdo cdo két qua ctia cdc Lodi khdc vdi S. enterica subsp. enterica la S. 11 30:z,0:e.n,x,216. 20‘TCVN 10780-3:2016 Phu tye A (Tham khdo) Thanh phan va chuan bj mai trudng nudi cdy va thude they A‘ Yau cdu chung ‘Trong Phy luc nay dua ra cdc méi truémg nudi cay lién quan dén cdc phép thir sinh héa (xem Phy luc C). Thanh phan cia cdc mdi trudng nudi cy néu trong phy Iyc nay duge [dy lam vi dy. Méi trong nudi cdy 66 cde tén gol trong ty cé thé durge néu trong tai li@u hoc dug ban s3n véi cdc thanh phan hoi kha nhau. Do 6, didu quan trong aéi voi méi phép thi sinh héa can kiém tra caéc phan tng cla moi truég nudi cy véi céc ching kiém ching dong tinh va am tinh dién hinh, DIEU QUAN TRONG - Néu méi trong nudi cdy hoge thudc thir dugc chudn bj tir méi trudng nuéi cdy hoan chinh khé hode thude thir hojc méi truéng nudi cdy 44 chudn bj sin dugye sir dung, thi thyc hign theo huong dan cia nha san xudt vé chudn bj, diéu kin bdo quan, han sie dung va cach str dung. Thdi han siv dung cla cde mdi trvéng nudi cdy néu trong phy luc nay G3 duge thé hin trong mét s4 tai ligu nghién cu. Nguéi si dyng can kiém tra xde nhan didu nay trong cac diéu kién bdo quén eda minh [xem TCVN 8128 (ISO 11133)]. A2 Thach dinh dudng A24 Thanh phin Chat chiét tir thit 3.09 Pepton® 509 Natri clorua (NaCi)(tuy chon) 509 Thach ty 99 dén 18g” Nude 1000 mi © Viaye thdy phan casein bang enzym ° Tuy thudc vao sire déng ciia thach. 24TCVN 10780-3:2016 A22 Chuan bj ‘Bun néng ¢é hda tan cac thanh phan trong nude. Chinh pH 48 sau khi khir tring pH tuong tng 18 7,0 + 0,2 & 25 °C, néu can. Chuyén méi truréng nudi cy vao binh (6.6) cd dung tich thich hgp. Khd triing trong ndi hp dp lye (6.2) & 121 °C trong 15 min. Chuydn khoang 20 ml mdi truéng 6 tan chy vao cac dia Petri vé tring voi eng Kinh 90 mm (6.7). Dé cho déng dac. Bao quan céc dia 8 rot (huéng lén trén), trénh hut Am va dé & noi thi & 5 °C (6.3) dén 2 thang, Néu can, lam khé cdc dffa truréc khi str dung. A3 Mii trudng thach - Sven Gard (vi dy) A.34 Vidu 1 (Tai ligu tham khdo [16]) ‘Str dung thach dinh dwong véi ndng 49 thach cai bin Gan xéc dinh ndng 66 thach cia thach dinh duéng dung cho do pha H trong dia Petri, Nang d6 nay 6 thé thay di tir 0,5 % dén 1% phan khéi lurgng, tly thude vao mé thach duge sir dung. Méi trong thach yéu cdu phai di mém cho Salmonella di chuyén d& dang trén kh4p méi trvéng sau khi G qua Gém tir 34 °C dén 38 °C (6.1), C&c thir nghiém so bd, khéng bé sung khang huyét thanh, do 46 can sir dyng méi trréng thach dinh duéng (pH 7.4) duc chudn bj cho muc dich dé hoa thach dinh dong cua phong thir nghiém théng thueng, dugs lam ban dc bang cach bd sung canh thang voi ty Ié thich hop a€ qua dém. Thé tich c&n cho mét dia Petri 10 cm la 30 mi ‘Viéc di chuyén trén bd mat thach sé dé hon néu bd sung natri desoxycholate (0.3 g/l) vao méi tong (natti desoxycholate kich thich sy di chuy&n), xem A.3.2. A32 Vidy2 A324 Thanh phan Chat chiét tir thit 5.09 Chat chiét nam men 4.09 ‘Canh thang dau tuong trypto-casein 30,0¢ 22TCVN 10780-3:2016 Glucose 1,09 Natri desoxycholat 0,359 Thach tu 5g dén9g* Nude 1.000 mi " Tuy thuge vao sire dong cia thgch. C6 thé cn cai thign d8 xac dinh ndng & cia thach 464i voi mol trong cn thiét cho sy di chuyén ti wu cia Satnoneli, A3.2.2 Chuan bj Hoa tan cdc thanh phan trong nude bang cach dun néng, néu cin. Chinh pH 48 sau khi khir tring pH tong dng la 7.6 £ 0,2 6 25 °C, néu can, Chuyén méi trvéng nudi cdy vao binh (6.6) c6 dung tich thich hep. Khé tring trong ndi hp ap Iye (6.2) duge duy tri 110 °C trong 20 min. Lam ngu@i thach dén khodng tir 47 °C dén 60 °C (6.8), hodc bao quan trong chai day kin & 5 °C (6.3) dén 2 thang. Nagay truréc khi sir dung: — bé sung mét giot khang huyét thanh ¢6 lién quan (SG1 dén SG6) vao méi truéng tan chy va trén au; — rét khodng 10 ml vao cdc dia Petri durong kinh 55 mm (6.7) hoe rét khoang 20 mi vao cdc dia Petri duydng kinh 90mm, A4 Canh thang malonat A44_ ‘Thanh phan (Tai itu tham khdo [14)) ‘Amoni sulfat 209 Dikali phosphat 089 Monokali phosphat 049 Natriclorua 2.09 Natri matonat 309 23‘TCVN 10780-3:2016 Bromothymol xanh 0,025 9 Nuée 1.000 mi A4.2 Chuan bj Héa tan cac thanh phan trong nuéc bang cach dun néng, néu can, Chinh pH dé sau khi Khir tring pH tong dng la 6,7 + 0,2 & 25 °C, néu can, Chuyén méi trudng nudi cy voi cdc lugng § mi vao cac éng cho phép cé dd oxy dé xay ra phan ung malonat. Vi dy, sir dung cae éng kich thud x4p xi 22 mm x 220 mm. {Khié tring trong ndi hdp dp lye (6.2) dee duy tri & 121 °C trang 15 min. Bao quan & noi tbi & 5 °C (6.3) én 2 thang. AS Canh thang Dulcitol (Tai ligu tham khdo [2]) ASA. Méi treéng hoan chinh AS4.4 Thanh phan Chat chiét te thit 3.09 Pepton® 10,09 Nati clorua 5.69 ‘Chat chi thj Andrade (A.5.2) 10 mi Dung dich Dulcito! (A.5.3) 50m! Nuéc 1.000 mi * Vi dy: phan gigi casein béing enzym. A5.1.2 Chuan bj ‘Héa tan céc thanh phan trong née. Chinh pH dé sau khi hi? tring pH [a 7,0 + 0,2 & 25 °C, néu can, Chuyén m6i trrdng nudi cdy voi cac Iugng 5 mi vao cdc éng cé dung tich danh nghta 15 im Khir triing trong ndi hdp 4p liye (6.2) durgc duy trl & 121 °C trong 15 min. 24TCVN 10780-3:2016 Bao quan & noi t6i & 5 °C (6.3) dén 2 thang. A5.2 Chat chi thj Andrade A.5.2.1. Thanh phan (sau Andrade-Penny n&m 1895; trich din trong Tai liéu tham khdo [7]) Fuchsin axit 059 Natri hydroxit (1,0 mol): 059 Nude 100 ml A6.2.2 Chudn bj Héa tan fuchsin trong nude, thém natri hydroxit. Néu sau vai gid ma fuchsin khéng mat mau thi thém 1m! ho&e 2 mi natri hydroxit. Thudc thir nay c6 hen sir dyng dai va cn chun bj mot lugng di tn 68 ding trong nhigu nam. 48.3. Dung dich Dutitol A831 Thanh phan Dutcitol 10g Nese 100 mi A§3.2 Chun bl Hoa tan dutcitol trong nue, Tiét tring bang c&ch loc qua mang loc c& 18 0.22 um, Bao quan 6 § °C-(6.3) dén 2 thang. AS Dung dich ONPG (0,013 3 mol) (Tai liéu tham khao [2)) ABA Mi trong hoan chink A811 Thanh phan o-Nitrophenyi-D-galactoside (ONPG) 0,089 Dung dich mononatri phosphat (NaH,PO.H,0:A6.2) 5 mi Nuoc 15 mi 25TCVN 10780-3:2016 A642 Chuan bj Hoa tan ONPG trong nude & khodng 37 °C. Thém dung dich NaH-PO, 1,0 mol/l (A.6.2.1). Dung dich phai khéng mau. Bdo quan dung dich & § °C (6.3). ‘Trude khi sir dung, lam dm mét Ivong thich hep (40 cho 66 lwng phép thir) dung dich ONPG dén khoang $7 °C. A.6.2 Dung dich mononatri phosphat (1,0 mol!) A624 Thanh phan NaH,PO,.H20 699 Dung dich NaOH, 300 g/l 3 mi Nuoe 45 mi 6.2.2 Chuan bj Haa tan NaH,PO,.H,O trong nuéc. Thém dung dich NaOH 300 gf va chinh pH dén 7,0 + 0,2 (6 25 °C). Thém nude dén 50 ml va bao quén dung dich & 5 *C (6.3) dén 2 thang. AJ Canh thang p-tartrat (axit tartrat hu co) A741 Thanh phan Pepton® 10,09 Kali natri (+) tartrat 100g Dung dich bromothymol xanh, 10 gf 24m Nuoc 4.000 mi * Vi dy: phan gidi casein bang enzym. A.7.2 Chudn bl Héa tan cdc thanh phan trong nuée bang céch dun néng, néu can. Chinh pH 48 sau khi khir tring pH twong (rng Ia 7,4 + 0,2 6 25 °C, néu can. Chuyén m6i truéng nuéi cdy véi cdc lung 5 ml vao cdc éng cd dung tich danh dinh 15 mi, Khir trung trong nbi hap ap lyre (6.2) due duy trl & 115 °C trong 10 min. 26TCVN 10780-3:2016 Bao quan & noi t6i & 5 °C (6.3) dén 2 thang. A.B Dung dich chi axetat A841 Thanh phan Chi axetat khoang 60 g Nuge 10 mi A8.2 Chuan bi Cho nue vo chai cé ndp van thich hep. Bd sung dd fugng chi axetat dé tao dung dich bo héa (nghia la cé thdy cht axetal khéng tan 13 trén day chal). Bao quén & nhiét a9 phong dién 6 thang A.9 Canh tharig tim-ndo (BHI) AQ Thanh phan Bét nao 12,59 Bot tim bo 50g Pepton proteose 10,09 Glucose 209 Natri clurua 5,09 Dinatri phosphat 259 Nuéc 1.000 mi A9.2 Chudn bj Héa tan cdc thanh phan trong nuée bang cach dun néng, néu can. Chinh pH dé sau khi khir tring pH turong tng [a 7,4 + 0,2 & 25 °C, néu can. Chuydn méi tevdng nudi cdy véi cdc lveng 5 ml vao céc éng ¢6 dung tich danh nghia 15 ml. Khi tring trong ndi hap dp Ive (6.2) durge duy trl & 121 °C trong 15 min. 27TCVN 10780-3:2016 Bao quan noi t6i & 5 °C (6.3) dén 2 thang A.10. Dung dich nuéc mui format (1% phan thé tich) A104 Thanh phan Dung dich formaldehyd, 370 gi 40,0 ml Dung dich natri clurua 170 gf 200 mi Nuée Lén dén 4 000 mi A.10.2. Chudn bi ‘Cho dung dich formaldehyd vao dung dich natri clurua. Thém nuéc dén ting thé tich 4 1. A11 Ong Craigie A141 Thanh phan Canh thang dinh duéng* 25.09 Thach 2,759 Nuéc 1000 mi * Bi véi cdc thanh phan, xem A.2 (khéng ob thach). A11.2 Chuan bj Héa tan cdc thanh phan trong nuée bang cach dun néng. Chinh pH 68 sau khi khie tring pH tong ting [a 7,2 + 0,2 & 25 °C, néu can ‘Chuyén mdi truéng nudi cdy véi cdc long 5 ml vo cde éng hoac chai miéng rong ¢6 ndp van ¢6 dung tich danh nghia khoang 15 mi va chtra cdc dng Craigie (céc dng hep ng&n, mé & ca hai dau). Ong Craigie cin cao hon bé mat thach. Khi tring trong ndi hap ap tye (6.2) durge duy trl & 115 °C trong 10 min. Bao quan & nhiét a¢ phdng dén 2 thang. A112’ Gelatin dinh duéng A121 Thanh phan Chat chiét tir thit 3,09 28TCVN 10780-3:2016 Pepton® 5.09 Gelatin 120,09 Nuse 1.000 mt * Vidy: phan gid casein bling enzym. A122 Chuan bj Hoa tan céc thanh phan trong nuéc bang each dun néng. ‘Chinh pH 68 sau khI kht triing pH tvong tng la 6,8 ¢ 0,2 6 26 °C, néu can. Chuyén méi nudi cay truéng v6i cdc lugng 5 ml vao cdc éng 6 dung tich danh nghTa 15 ml. Khi tring trong nbi hdp 4p lye (6.2) durge duy tri 8 121 °C trong 15 min. ‘Bé méi trvang ngudi theo vi tri th3ng dieng va bao quan o 5 °C (6,3) dén 2 thang.