TCVN TIEU CHUAN QUOC GIA TCVN 9049 : 2012 Xuat ban lan 1 THU’C PHAM - XAC BINH CLOSTRIDIUM BOTULINUM VA BOC TO CUA CHUNG BANG PHUONG PHAP VI SINH Foodstuffs — Determination of Clostridium botulinum and its toxins by microbiological method HA NOI - 2012TCVN 9049:2012 Loi ndi dau TCVN 9049:2012 dugc x4y dung dya trén co sé AOAC 977.26 Clostridium botulinum and Its Toxins in Foods. Microbiological Method; TCVN 9048:2012 do Cyc An toan vé sinh thyc phdm t8 chic bién soan, 86 Y té 48 nghi, Téng cuc Tiéu chudn Bo lvéng Chét lugng thm éinh, B6 Khoa hoc va Céng nghé cong bé.TIEU CHUAN QUOC GIA TCVN 9049:2012 Thyc pham — X4c dinh Clostridium botulinum va déc t6 cla ching bing phuwong phap vi sinh Foodstuffs — Determination of Clostridium botulinum and its toxins by microbiological method — 1 Pham vi ap dung Tiéu chudn nay quy dinh phuong phép xac dinh Clostridium botulinum va cde doc té cla ching trong ‘thyc phdm bang phuong phap vi sinh. 2 Nguyén tic Dich chiét tur thye phdm chira mot lidu tdi thiéu gay chét (MLD) tl dc t6 botulinum duge tiém vao phic mach chudt, con vat s8 chét trong vong 48 h sau khi co biéu hién tuan ty cdc trigu chimg nhiém doc botulinum. Khéng 4c t6 trong déng s& bao vé chudt khdi cdc trigu ching 6, trong khi cdc khang doc t6 khdc lai khong, dya vao a6 48 xc dinh typ huyét thanh. Ce bao tir séng trong thy phm sé phat trién trong méi trréng nubi cdy thich hop va sinh dg t6, cde de t8 nay dugc phat hién va dinh typ. 3 Thudc thir va mdi truvéng Chi str dung thuéc thé loai tinh khit phan tich va nude c&t hoc nude da Khir khodng hoac nude c6 46 tinh Khiét tong durong, tr khi cé quy dinh khéc, 3.4 Canh thang ti thit va tir gan, v6 tring 3.4.4. Canh thang gan Nghién nhé 600 g gan bé twei trong 800 mi nude, Bun dén séi va dé trong 1 h. 88 ngudi, chinh pH én 7,0 va dun s6i trong 10 min. Loc qua vai tha, ép lai bé phan chét léng. Cho vao canh thang nay 10 g pepton, 1 g dikali hydrophosphat (K,HPO,) va 1 g tinh bét hda tan. Chinh dén pH 7,0 va pha lofing bang nude één 1 lit. Loc qua gidy thé. Canh thang nay cé thé bao quan riéng ré trong tl lanh 48 sir dung sau 5TCVN 8049:2012 nay, néu can, Phan phéi vao cdc éng nghiém kich thse 18 mm x 150 mm ho&e 20 mmx 150 mm, Jugng gan voi chidu cao tir 1 om dén 2 om va chat lang tir 10 ml dén 12 mi. Hp Ap lye 20 min & 121 °C. 3.4.2. Ganh thang thit Cé thé str dung canh thang cé ban s&n trén thi trudng cd thanh phan sau: 454 g tim bd, 20 g pepton proteoza, 2 g dextroza va 5 g natri clorua, Hda 12,5 g mdi truéng nay vao 100 mi nude lanh. Tron ky va dé yén cho dén khi cdc hat 4a th4m ky nude (khodng 15 min). Cach khdc, thém 1,25 g mdi trudng ac vao céc éng nghi¢m, thém 10 ml nuéc lanh va tron kj cho dén khi céc hat thm uot nuéc. Hp 4p lye 15 min & 121 °C. pH cudi cling phai la 7,2 + 0,1. 3.2 Canh thang dich chiét ndm men trypticaza-pepton-glucoza (TPGY) hogc TPGY véi trypsin (TPGYT) Chi sir dung TPGYT dé thay thé khi vi sinh vat nghi ngy thude ching clia typ B, E, hoa F khéng phan gidi protein. Héa tan 50 g trypticaza, 5 g bacto-pepton, 20 g chat chiét ndm men, 4 g dextroza va 19 natri thioglycollat trong 1 lit nude va phan phéi voi céc lugng 15 ml vao cée éng nghiém dyng moi trang kich thude 20 mm x 150 mm hoae cdc lurong 100 ml vao cae chai dung quy dinh. Hép 4p Ive 10 min 66i voi dng nghiém hose 15 min didi voi chai dyng & 121 °C. pH cudi cng phai [a 7,0 + 0,1. Bao quan trong tli lanh va loai bd néu khong durge str dung trong véng 2 tudn. Ngay trude sir dung, ‘un sdi hoac hap 4é loai bé oxi va lam ngudi nhanh, sau dé bang cach v6 tring thém 1,0 mi dung dich trypsin cho méi 15 ml canh thang. Chuan bj dung dich trypsin bang céch héa tan 1,5 g trypsin trong 100 ml nuéc. Khiy tring bing cach loc qua mang loc c& 18 0,45 um hod trong duong va a8 trong td lanh. 3.3 Thgch long 4d tréeng-thit-gan ba hofe thach long dé trieng kj khi 3.34 Thach lang 46 tréeng-thit-gan bé (LVEY) Riva sach 2 hoac 3 qua tring bang ban chai cing va lam réo née, Ngam trémg trong dung dich thay ‘Agan(|l) clorua (HgCk) 0,1 % (khéi lgng/thé tich) trong 1 h. & réo dung dich thay ngan(li) clorua va ng&m trimg trong etanol 70 % khoang 30 min. Lay tring ra, bang cach vé triing dap vo v6 va logi bd long trang. Dung xyranh ldy léng 43 cho vao vat dyng v6 tring va thém mot Ivgng thé tich tuong ty ca dung dich natri clorua 0,85 % (khdi lurgng/thé tich), trOn kf. Ci 500 ml thach gan-thit bé khé 4a khiy tring ¢6 ban s&n trén thi truéng & 50 °C thi thém 40 mi huyén phu léng é trézng-natri clorua. Trén kj va dé dia. Lam kh6 cdc dia nay 2 ngay & mhigt 46 phdng ho&e 24 h & 35 *C. Loai bd cdc dia bi nhi&m ban va bao quan cdc dla v6 tring nay trong td lan.‘TCVN 9049:2012 3.3.2. Thach trong kj khi Hoa tan 5 g chdt chiét nm men, 5 g trypton, 20 g pepton proteoza, 5 g natri clorua va 20 g thach trong 1 lit nude. Chinh pH dén 7,0, phan phéi cdc lueng 500 mi vao binh cau 4 lit va h4p 4p lye 20 min & 121 °C. ‘Cho 40 ml huyén phi [dng 46 trieng-natri clorua 4a chudn bj nhur trong 3.3.1 vao 500 mi thach da lam tan hay dén khodng tir 45 °C dén 50 °C. Tron va 68 dia ngay. Lam khé dia va bao quan nhw trong 3.3.1 3.4 Dung dich am gel-phosphat, pH 6,2. Hea tan 2 g gelatin va 4 g dinatri hydrophosphat (NazHPO,) trong 1 lit nuéc eb dun néng nhe. Phan ph6i vao cac chai 100 mi.,Hdp dp lve 20 min & 121 °C. 3.8 Cac ché phdm khang dgc t6 Clostridium botulinum Cac typ tir A dén F hoc khang thé da dong tir A-F. CHU THICH: Céc ché phdm sinh hgo oé thd mua & Biological Products Branch, Office of Sclentifc Services, Centers for Disease Control an Prevention, Atlanta, GA 20233, USA. 3.6 Dung djch natri hydroxit, 1M. 3.7 Dung dich axit clohydric, 1 M. 3.8 Dung dich trypsin bao hoa Héa tan 1 g trypsin vao 10 mi nude trong éng nghiém sach. 3.9 Dung dich natri clorua, 0,85 %. 4 Thiét bj, dung cy ‘Sir dung céc thiét bi, dung cu cilia phéng thir nghigm vi sinh thang thang va cu thé cdc thiét bi, dung cy Sau: 44 Dyng cy me hép. 4.2. Binh kj khi”. 43 Bia Petri, dudng kinh 100 mm. Lam khé cc dia thach 44 chudn bj khoding 24 h & 35°C true Khi cd. Co te sir dung san phdm GasPak (Becton Dickinson Microbiology Systems, 7 Lovton Circe, Sparks, MD 21152, USA, No 249511) hobe tong dong. Thong tn nay to thu fi cho ngudi sir yng ti chun va ibu chun ny khéng bn inh phat 24 dung sin phd néu tren‘TCVN 9049:2012 44 May Il tam lanh, t6c 66 cao. 4.8 Xyranh, dung tich 1,0 ml hode 3,0 mi, c6 mAi kim 15,9 mm 48 tiém chudt. 4.6 Pipet, v6 tring. 4.7 Binh djnh méec, dung tich 1 lit. 4.8 Bép dign. 4.9 Ong nghigm, kich thuéc 15 mm x150 mm va 20 mm x150 mm. 4.10 Céiva chay, v6 tring. 4.11. Kep, v6 tring. 4412 Vong cdy, vd tring. 5 Laymau ‘Mau gai dén phéng thr nghiém phai dting la mu dai dign. Mau khéng bi hu héng hoac thay 64i trong ‘sudt qué trinh van chuyén ho&c bao quan. 6 Cach tién hanh 6.1 Chuan bj mau thir 6.1.1 Kiém tra so bo Gir céc mau & trang thai ééng lanh. Bi v6i cdc loai thyc phém déng hop chua mé, trv khi 4 bj phdng va ¢é nguy co bj nd, thi khéng can bao quan lanh. Ghi lai m& sé va tinh trang cla vat chira. Lam sach va nhén biét céc vat chira mau. 6.1.2 Thye phdm dang ran ‘Chuyén mét cach vé triing phan mau sang c6i gid v6 triing. Thém mot lugng tueng ty dung dich dém gel-phosphat (3.4) va nghién bang chay v6 tring (4,10). Cach khdc, ding kep v6 tring (4.11) cy cdc miéng mau thir nhé tryc tiép vao canh thang tang sinh. 6.1.3. Thyc phdm dang ling ‘Ding pipet v6 tring (4.6) chuy€n tryc tiép mau vao canh thang tng sinh.TCVN 9049:2012 6.1.4 Thye phdm d6ng hop Chuan bi, khir tring bang dung dich cin iét va mé hép. Vige mé hdp duge thy hién trong phéng vé tring. Théo bd nhan hép, kiém tra hop va khuyét tat bén goal va ghi Iai. Rika hop bang x4 phdng ho&c bing chdt ty riva va nude ri ding khan gidy sach d& tau khé. Ding ngon ita dét phan hép khong 6 ma sé cho dén khi bay hét hoi nuée ngung ty rdi dat lai vao vi tri cG. Lau sach dao me hép bang khan giy thdm cbn 70 %. Ding nggn lira di a8 digt hét vi sinh vat trén np hop. Dung dao.cat mot 18 cé duéng kinh khoang 4 cm & mat hop vira khiF tring xong. Néu hop bj phdng thi dat.hép sao cho mép doc cua hdp khong huréng vé ngudi thao tac. Néu hop cé ktida ‘mé thi lam lanh truée khi mé va khi str dung ngon lira phai cn than a8 tranh lam nd hop. 6.4.5 Kidm tra cam quan hi lai két qua quan sat bén ngoai, mil va bang chéng v8 sv phan hiy, néu cé. CHU Y: Khéng thir ném san phdm. 6.1.6 Phan miu thir dy phdng ‘Sau khi cy xong, bang cach v6 tring l4y phan mau thir cho vao binh vé tring dé ding cho cac phép thar tiép theo, néu cn. 6.2 Phat hign C. botulinum séng 624 Tang sinh Loai oxi héa tan ra khdi mdi trudng truéc khi nudi edy bang cach dun cach thy céc éng dyng canh thang tt 10 min dén 15 min va lam ngudi nhanh, khong lac éng. Cay vao 2 éng nghiém, cir 15 mi ‘canh thang thit (3.1) thi ly tr 1 g dén 2 g mau dang rén hoac tir 1 mi dén 2 mi mau dang léng hod dich chiét, dura chdt cAy tir tiv vao ngay dudi bé mat can thang. U & nhiét é 35 °C. Tong ty, ody vao 2 6ng nghiém dyng canh thang TPGY (3.2) va d & mhiét 4 26°C. 622 Kidmtra ‘Sau 5 ngay, kiém tra cac éng nghigm 8 cdy v8 GG duc, sinh khi, phn hily cia cdc hat thit va mii. Ngoai ra, cdn kiém tra tiéu ban sau khi nhuém Gram, tim tinh thé, hoge xanh metylen bang kinh hién vi ¢6 vat kinh dau. Quan sat hinh thé cla cdc vi sinh vat va ghi Iai oé mat clia cdc té bao clostridia aién hinh, kich thuéc va sé lvgng twong d6i cia cdc bao tt, vi tri clia cac bao ti trong cdc té bao.TCVN 9049:2012 6.23 Xi litlép theo Théng thudng, sau 5 ngay nudi cdy thi doc t6 s8 duge sinh ra va c6 ndng a9 cao nht, cling nhy sy hinh thanh bao tt. Git lai dich cdy trong ti lanh 48 phan lap khudn lac thuan khiét. Néu sau 5 ngay khéng thay phat trign thi G tiép 10 ngay 48 phat hign kha nding sinh bao ti cham cia C, botulinum truréc khi loi bd dich ody. 6.3 Phan lap khudn lac thuan khiét Néu hinh thanh bao tie t6t thi C. botulinum d& dang phan lap hon tir hé vi sinh hn hgp trong mai trudng tang sinh hoae tir m&u géc. 6.3.4 Xirlise bo Thém mot thé tich turong ty cla cdn tuyét di dé ioc a8 kh tring vao khoding tir 1 mi dén 2 ml dich cdy hoac phan mau thir dyng trong én§ nghiém vé tring o6 nap van. Tron kj va d & nhiét 4 phdng trong 1 h. Céch khée, dun tir 1 ml dén 2 mi dich cAy a8 tang sinh & 80 °C trong khodng tir 10 min dén 15 min 48 diét cc té bao sinh dudng. Khong xiP I nhiét 44] véi typ C. botulinum khOng phan gidi protein. 6.3.2 Riacdy Ding vang cAy vd tring (4.12) Idy mot hoc hai véng day dich cdy 48 xir ly bang cdn hode nhiét, pha long néu edn, ria chy vdo cdc dia thach ling 48 trimg kj khi (3.3.2) hode thach ling 43 trimg-thit-gan bé (3.3.1) khd, sao cho thu duge cac khudn lac phan lap tét. U cac dia nay trong binh kj khi (4.2) & 35°C trong khong 48 h 6.3.3 Chon khudn lac Cac khudn lac dién hinh la cde khudn lac Idi hodc det, tran hoe nham, thuéng moc lan téa va cb mép Khong d&u. Trén mdi trudng thach long dé trizng, cdc khudn lec throng co bé mat ong dnh khi Cuge kiém tra bang 4nh sang xién. Ving éng anh nay giéng mhu “Iép ngoc trai". Vung nay thuréng me rong va duéng vidn cia khudn lac khéng déu. Ngoai ving nggc trai, cc khudn lac typ C, D va E thong durgc bao quanh bdi ving két tila mau vang dudng kinh ti’ 2 mm dén 4 mm. Cac khudn lac typ A va B thuréng cho thay c6 vung két tta bao quanh nhé han. Chi cé mot sé loai Clostridium 6 cac dc tinh hinh thai dién hinh nhung khéng sinh doe 16, 6.3.4 Chung cdy Dang ving cy v6 tring (4.12), cdy 10 khuan lac dug chon vao éng dyng mai truéng v6 tring nhu sau: = canh thang TPGY (3.2) d6i véi C. botulinum typ E, U 5 ngay & 26 °C; ~ canh thang thit v6 trung (3.1) &6i véi cdc typ adc té khdc, d 5 ngay & 35°C. ‘Sir dung cae chiing cdy nay 48 khang dinh, phat hién va nhan dang adc 16 10TCVN 9049:2012 6.3.5 Khang ajnn Cy ria dich cdy thu duge trong 6.3.3 lap lai hai ln trén céc dia thach Ing 43 tring (3.3), & mét cia trong binh kj khi va mdt dfa trong binh hiéu khi & 35 °C. Néu tim théy cdc khudn lac dign hinh cia C. botulinum trén fa kj khi va khéng thdy phat trién tran dla hidu khi thl 66 c6 thé [a dich cy thuan chting. Viéc phan lap khéng dung C. botulinum tir mot trong cac khuan lac duge chon c6 thé cho thay rng 86 lugng vi khuan trong nudi cdy tang sinh Ia th4p. Vie lap lai céc bude tng sinh (6.2.1) bd sung ¢é thé cho di s6 Ivong d& phan lap. Bao quan ching cy thudn khiét 6.3.3 trong tl lanh trén cdc vién thily tinh hoac bao quan bang phuong phap dong khé. 6.4 Phat hién dc t6 6.4.1 Chuan bj mdu thir Chiét thy phdm dang ran bing mét thé tich tuong duong cla dung dich dém gel-phosphat (3.4), nghién nhé bang c6i va chay v6 tring (4.10). Li tm cht chiét va thye phAm dang lang chia cdc hat chat rn bang may li tam lanh (4.4). Lay vai mililit dung dich 48m gel-phosphat dé trang hop dyng ring ma cé thé chira dc t6 thyc ph&im. Str dyng mét long nha nhét 48 khéng lam long doc t6. 6.4.2 Xtrli biing trypsin Néu co matt céc typ doc t6 khéng phan gidi protein thi cAn hoat héa trypsin dé phat hién-Khong xt ly bang trypsin voi chét cdy TPGYT 6 chia san trypsin. Ngoai ra, viéc xt Hi nay cé thé lam gidm dc t6 68 hoat héa cé mat trong chat cdy. Lay phan chat l6ng néi phia trén (6.4.1) ca mu thyc phdm dang ling ho&c dich cAy trong canh thang thit v6 trang. Chinh pH bling dung dich natri hydroxit 1 M (3.6) hoc axit clohydric 1 M (3.7) dén khi pH at 6,2. Tron 0,2 mi dung dich trypsin bao héa (3.8) voi 1,8 ml chat lng c&n thi’ nghiém. U & 37 °C trong 4 h, thinh thoding Ide éng. 6.4.3 Phép thir dgc tinh Tién hanh méi phép thir lp lai hai |An, nghfa [a thyre hién trén céc mau thi chua xi li va da xi li bang trypsin. Pha loging cac phan chat ldng ni phia trén, thyre phdm Iéng hodc dich cdy chua xi Il va 48 xu Ii-v6i dung dich dém gel-phosphat theo cac ti Ié tong ting 1:2, 1:10 va 1:100, Dung xyranh (4.5) tiem vao phic mac cdc cp chubt riéng ré o6 trong lvgng khoang 15 g dén 20 g cdc dung dich géc va dung dich 68 pha long, 43 xi Ii va chura xi I, Dun néng 1,5 ml dung dich géc chua xiii trong 10 min & 100°C 48 kiém tra, D& ngudi va tiém vao hai chudt, mBi con 0,5 ml dung dich 44 dun néng. Céc chuot nay 86 khdng chét vi doc t6 botulinum, néu cd mat, cling bj bat hoat khi xi Ii nhiét Quan sat chu@t sau 48 h, ghi lai cdc triéu ching va théi gian chudt chét. Cac trigu ching dién hinh ca ng6 doc botulinum thuréng bat dau trong vong 24h, con vat xil I6ng sau dé khé thé, chan run va cudi "1TCVN 9049:2012 cling ligt toan than, thd g4p va chét do suy hd hp. Chuét chét ma khong o6 cdc trigu ching ngd doc botulinum la khdng 44 bang chteng vé dung dich tiém c6 chira dc td botulinum. Con vat chét c6 thé do cdc hda chat c6 mat trong chat lng hodc do ch4n thong. Néu sau 48 h, t&t cd chudt duge tiém adu chét thi lap tal phép thi dc tinh, str dung c&c dung dich pha long hon. Can phai cd cdc dung dich pha loang d8 giét chét vat thi nghigm cting nhur céc dung dich pha lo’ing khéng giét chét 46 thiét lap mét diém két thuic hod lidu gay chét t6i thiu (MLD) cing nh: 6c tinh lugng adc t6 cb mat. MLD ¢é chia trong dung dich pha loang nhAt gidt chét cd hai con chudt (hode tat 8) a4 tiém. Tinh fidu gay chét t6i thidu theo milli. 6.5 Xéec dinh typ cia dc té Pha long cdc khang doc t don déng typ A, B, E va F trong dung dich natri clorua 0,85 % (3.9) én nding 66 1 1U/0,5 ml. Chuan bi di khang doc té pha long 46 titm vao 2 chugt, mdi con 0,5 mi déi voi mi dung dich pha loang 44 chuan bj 48 thir nghigm. ‘St dung ché phim doc td cho lidu gay chét tdi thiéu (MLD) cao nhat, ke cd da xir If ho&c chua xi li, N6u chua xiF Ii thi 66 thé chun bj tung ty nhu trong phép thir vé dc tinh. Néu dung ché phim 4 xi Ul bing trypsin ma gay chét nhidu nhét thi chun bj dung dich via méi due xu if bang trypsin, vi hoat. tinh cda trypsin ¢6 thé pha hiy doc tb. Chudn bj cdc dung dich pha long bao trim it nhat dai lidu gay chét 10 MLD, 100 MLD va 1000 MLD thép han aiém két thdc cla de tinh 48 xéc dinh true 46. Khodng tir 30 min dn 60 min truéc khi cdng cudng déc bang ché phim adc td, tiém vao phic mac mot s6 nhém chu@t, mBi con nhan dug 0,5 ml cla mét trong céc khéng Gdc tb pha long. Tiém méi 49 pha loang cia ché phdm déc tb vao phiic mac cdc di chudt dug tiém phdng sau khi tiém khang doc t6 don dong. Bdng thdi tiém cho mt 6bi chudt chura durge phéng (chwa tiém khang Gc 16) mdi dung dich dc t6 kam i chizng. Quy trinh nay c&n dén 30 chudt, 3 c&p ding cho mét trong 4 loai khang dc té don dong [A, B, E va F], méi cap nhan dug litu cudng d9c ca mot trong ba dung dich pha long cia ché phdm ade t6 [2 x 3 x 4 = 24] céng véi mét cp chura tigm phdng ding cho mdi dung dich pha loang ciia dgc té 48 lam déi chirng [2 x 3 = 6]. ‘Quan sat chudt trong 48 h vé cac trigu chien nhi&m 6c va ghi lai thoi gian chét. Néu két qua cho thay chat dc bj trung héa thi Ip Iai phép thi, st dyng cdc khang déc té don dong typ C va typ D, cng voi nhém khang doc t da dang typ A dén typ F. 7 Din gial két qua ‘Doc t6 trong the phdm, nghTa 1a khi diing thyc phim khéng dug xi If nhiét kj cé thé gay ngd doc botulinum. Khi cé mat 4c té trong thyc phdm can nghT ngay dén gc t6 cia botulinum. Vi khudn . botulinum séng, nhung chura han da la doc t6, khi phat hin 66 C. botulinum trong thre pham thi 46 12TCVN 9049:2012 ling chva di bang ching vé ngd déc thyc phdm. CAc sinh vat dn phai dc t6 ob thé tin thdy duge trong duéng tiéu héa, nhung dc té thi khdng thé sinh sdi va phat trién trong co thé. ‘Sy c6 mat dic té botulinum va/hoae cae vi sinh vat trong 4 hép cb mdi trurdng axit thdp (pH > 4,6) thi d6 1a bang chirng vé san phdm chua duge ché bién kj ho&c bj nhiSm ban do sy rd ri sau ché bién, ‘Cac hép bj phdng c6 nhiéu kha nang han so véi hop phang vé viée nhi&m déc té botulinum béi vi vi Khudn sinh khi trong qué trinh phat trién. Sy cd mat cia dc 16 trong hOp phang 6 thé oé thé do mi ghép cia hép bj hd nén khi thodt duge ra ngoai. Déc t6 trong thyc phdm déng hép thudng [a typ A hoc typ B phan gidi protein, vi cdc bao tl phan gidi protein c6 thé 1a cdc bao tir cla vi khudn chiu nhigt, Céc bao tir khéng phan gidi protein typ 8, E va F, thudéng it chju nhigt hon va thuéng khong séng dug ngay ca khi xi? {i & nhiét d6 thép. Néu chugt duge bao vé truéc doc t6 botulinum va chét béi mét trong sé céc khang déc t6 botulinum don déng thi khang dinh sy cé mat cis 6c t6 botulinum va xac dinh typ huyét thanh cla dc t6 trong mau thi, Néu chudt khéng duge bdo vé bing mét trong cdc khéng dc t6 don dong thi cé thé cé nhidu d6c t8 botulinum trong mau thir, cé thé cé mot vai typ doc t6 66 mat, hode chudt bi chét cd thé do mat vai niguyén nhan khdc. Khi dé, c&n thi nghigm Iai & cdc ndng 49 pha loang hon alla dung dich kiém tra va cn stv dung cdc hn hgp khang dc té thay cho khang huyét thanh don dang. Néu chuét van khéng duge bao vé thi 06 thd do mt s6 chdt doc khdc khéng bén nhiét, néu cA chat ling 68 xi Ii nhigt va chat 16ng chua xt Ii nhigt du gay chét, Didu nay cling c6 thé la c mot chat dc chiu nhiét khac c6 thé che ldp dc td botulinum, 8 Bao cdo thir nghigm Bao cdo thir nghigm phai ghi 6: a) tdt cd cdc thong tin cn thiét 48 nhan biét day au vd mau thir; b) vin dan tiéu chudn nay hoac phuong phép ldy mAu duge str dung; ©) ngay va thdi gian lay mau (néu biét); d)_ngay nhan mau; e) _ngdy thir nghigm; f)_két qua va éon vj bidu thj két qua; 9) cdc diém dic biét quan sat dugc trong qua trinh thir nghiém; h) moi thao téc kh6ng quy dinh trong tiéu chun nay ho&c tuy chon ma c6 thé nh huéng dén két qua, 13TCVN 9049:2012 Thw myc tai ligu tham khao. [1] AOAC 972.44 Microbiological Method. [2] J.AOAC 60, 541(1977). [3] J.AOAG 73, 168(1990).