TIU CHUN VIT NAM TCVN 7138:2002

THT V SN PHM THT NH LNG PSEUDOMONAS SPP. Meat and meat products Enumeration of Pseudomonas spp. Li ni u TCVN 7138 : 2002 hon ton tng ng vi ISO 13720 : 1995; TCVN 7138 : 2002 do Ban k thut tiu chun TCVN/TC/F8 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 m t phng php nh lng Pseudomonas spp. c trong tht v sn phm tht, k c tht gia cm. 2. Tiu chun vin dn TCVN 6404 : 1998 (ISO 7218) Vi sinh vt trong thc phm v trong thc n gia sc Nguyn tc chung v kim tra vi sinh vt. TCVN 6507 : 1999 (ISO 6887 : 1983) Vi sinh vt hc Hng dn chung v chun b cc dung dch pha long kim tra vi sinh vt. 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. 3. nh ngha Trong tiu chun ny p dng nh ngha sau: 3.1 Pseudomonas: Vi khun thuc loi Pseudomonas 250C to thnh cc khun lc trong thch xetrimit, fuxidin v xephacloridin (CFC) khi php th c tin hnh theo tiu chun ny. 4. Nguyn tc 4.1 Cy ln b mt mi trng nui cy chn lc c, s dng cc cp a, mt lng qui nh ca mu th nu sn phm dng lng hoc mt lng qui nh ca huyn ph ban u nu sn phm dng khc. Di cc iu kin tng t, cy cc dung dch pha long thp phn ca mu th hoc ca huyn ph ban u, dng hai a cho mi pha long. 4.2 cc a 48 h nhit 250C trong iu kin a kh. 4.3 Tnh lng Pseudomonas trong mt mililit, hoc trong mt gam mu t s khun lc in hnh v/ hoc khng in hnh thu c trn cc a cc mc pha long c chn sao cho kt qu c ngha v c th khng nh bng php th oxidaza v cho pht trin trn thch Kligler 5. Dch pha long, mi trng nui cy v thuc th 5.1 Khi qut V thc hnh phng th nghim hin hnh, xem TCVN 6404 : 1998 (ISO 7218). 5.2 Dch pha long Xem TCVN 6507 : 1999 (ISO 6887). 5.3 Thch xetrimit, fuxidin v xephacloridin (CFC)[1] 5.3.1 Mi trng c s 5.3.1.1 Thnh phn

Gelatin pepton Casein hydrolysat Kali sunfat (K2SO4) Magie clorua (MgCl2) Thch Nc 1) Ty thuc vo sc ng ca thch. 5.3.1.2 Chun b

16,0 g 10,0 g 10,0 g 1,4 g 12,0 n 18,0 g1) 1 000ml

Ha tan cc thnh phn trn hoc mi trng hon chnh kh trong nc bng cch un si. Chnh pH sao cho sau khi kh trng pH l 7,2 0,2 250C, nu cn. Phn chia mi trng thnh cc lng 100 ml vo cc bnh hoc cc chai c dung tch thch hp. Kh trng mi trng trong ni hp p lc (6.1) 15 pht 1200C. 5.3.2 Cc dung dch c ch Khng gi cc dung dch qu 7 ngy trong t lnh. 5.3.2.1 Dung dch Xetrimit: Dung dch A 5.3.2.1.1 Thnh phn Xetrimit1) Nc 0,1 g 100 ml

1) Hn hp gm ch yu l tetradexyltrimetylamoni bromua vi cc lng nh hn ca dodexyltrimetylamoni bromua v xetrimoni bromua. 5.3.2.1.2 Chun b Ha tan xetrimit trong nc. Lc kh trng. 5.3.2.2 Dung dch fuxidin: Dung dch B 5.3.2.2.1 Thnh phn Fuxidin (C31H47NaO6) Nc 5.3.2.2.2 Chun b Ha tan fuxidin trong nc. Lc kh trng. 5.3.2.3 Dung dch xephacloridin: Dung dch C 5.3.2.3.1 Thnh phn Xephacloridin (C19H17N3O4S2) Nc 5.3.2.3.2 Chun b Ha tan xephacloridin trong nc. 0,1 g 100 ml 0,1 g 100 ml

Lc kh trng. 5.3.3 Mi trng hon chnh 5.3.3.1 Thnh phn Mi trng c s Dung dch A Dung dch B Dung dch C 5.3.3.2 Chun b Di cc iu kin v trng, b sung cc dung dch cht c ch vo mi trng c s, lm tan chy, duy tr 470C v trn cn thn. 5.3.4 Chun b cc a thch CFC Rt khong 15 mm mi trng hon chnh vo cc a Petri v trng (6.9). yn cho c li. Ngay trc khi s dng, lm kh cc a thch, tt nht l m np ra v lt ngc a thch xung di, t vo t sy (6.2) nhit t 350C n 550C, cho n khi cc git nc bin khi mt ca mi trng. Tip theo khng lm kh thm na. Cc a thch cng c th c lm kh trong t cy v trng an ton khong 30 pht khi a c m mt na np hoc a y np th qua m. Nu a c chun b trc th cc a thch cha c lm kh khng c lu qu 1 thng nhit t 00C n 50C. 5.4 Thch dinh dng 5.4.1 Thnh phn Cao tht Pepton Natri clorua (NaCl) Thch Nc 1) Ty vo sc ng ca thch 5.4.2 Chun b Ha tan cc thnh phn kh hoc mi trng hon chnh kh trong nc bng cch un si. Chnh pH sao cho sau khi kh trng pH l 7,0 0,2 250C, nu cn. Chia mi trng thnh cc lng 100 ml vo cc bnh hoc cc chai dung tch khng ln hn 500 ml. Kh trng trong ni hp p lc (6.1) 20 pht 1210C. 5.4.3 Chun b cc a thch dinh dng Chuyn cc phn mi phn khong 15 ml mi trng nui cy mi chun b sang cc a Petri ng knh 90 mm (6.9) v cho ng c li. Ngay trc khi s dng, lm kh cc a thch nh m t trong 5.3.4. Nu c chun b trc, th cc a thch cha c lm kh khng c lu qu 1 thng nhit t 00C n 50C. 5.5 Thch Kligler 3,0 g 5,0 g 5,0 g t 12,0 n 18,0 g 1) 1 000 ml 100 ml 1 ml 1 ml 5 ml

5.5.1 Thnh phn Cao tht b Cao men Pancreatic casein pepton Natri clorua (NaCl) Lactoza Glocoza St (II) amoni sunfat ngm 6 phn t nc [(NH4)2SO4.FeSO4.6H2O] Natri thiosunfat ngm 5 phn t nc (Na2S2O3.5H2O) phenol Thch Nc
1)

3,0 g 3,0 g 20,0 g 5,0 g 10,0 g 1,0 g 0,5 g 0,5 g 0,025 g t 12,0 g n 18,0 g1) 1 000 ml

Ty thuc vo sc ng ca thch

5.5.2 Chun b Ha tan cc thnh phn kh hoc mi trng hon chnh kh trong nc bng cch un si. Chnh pH sao cho sau khi kh trng pH l 7,4 0,2 250C, nu cn. Phn chia mi trng thnh cc lng 10 ml vo cc ng th nghim (6.7). Kh trng trong ni hp p lc (6.1) 15 pht 1210C. yn trng thi nghing sao cho on u mt ngp su c 2,5 cm. Ch Khng chun b mi trng ny qu 7 ngy hoc 8 ngy trc khi s dng. Mt khc, mi trng ny phi c lm tan chy v phi c phc hot trong ni cch thy un si, sau ng c li trng thi ng ca n. 5.6 Thuc th pht hin oxidaza 5.6.1 Thnh phn N,N,N,N-Tetrametyl-p-phenylenediamin dihidroclorua Nc 5.6.2 Chun b Ha tan thuc th trong nc ngay trc khi s dng. 6. Thit b v dng c thy tinh Xem TCVN 6404 : 1998 (ISO 7218). Tt c cc dng c thy tinh phi bn vi vic kh trng nhiu ln. Xem TCVN 6507 : 1999 (ISO 6887). S dng cc thit b, dng c phng th nghim vi sinh thng thng v c bit l: 6.1 Thit b kh trng kh (t sy) hoc thit b kh trng t (ni hp p lc). Xem TCVN 6404 : 1998 (ISO 7218) 6.2 T sy hoc t thng gi ( lm kh cc a thch), c th duy tr c nhit t 350C 10C n 550C 10C, hoc t cy v trng. 6.3 T m, c kh nng duy tr nhit 250C 10C. 1,0 g 100 ml

6.4 Ni cch thy, c kh nng duy tr 470C 20C. 6.5 pH-met, c chnh xc n 1 n v pH 250C. 6.6 Que cy vng, bng platin/iridi, niken/crom hoc bng cht do v trng, ng knh khong 3 mm v dy cng cht liu hoc a thy tinh. Ch thch 1 Khng s dng que cy vng bng niken/crom trong php th oxidaza (xem 9.4.2.1). 6.7 ng v chai th nghim, c dung tch thch hp. 6.8 Pipet chia x ht, c hiu chun ch dng cho kim tra vi khun, dung tch danh nh 1 ml, c chia vch 0,1 ml v c l x ng knh t 2mm n 3mm. 6.9 a Petri, lm bng thy tinh hoc cht do, ng knh t 90 mm n 100 mm. 6.10 Que gt, bng thy tinh hoc bng cht do, th d cn cm bng a thy tinh di 20 cm v c ng knh khong 3,5 mm, mt u di khong 3 cm c un vung gc v u mt c lm nhn bng nhit. 7. Ly mu iu quan trng l phng th nghim phi nhn c ng mu i din v khng b h hng hoc gim cht lng trong sut qu trnh vn chuyn hoc bo qun. Vic ly mu khng qui nh trong tiu chun ny. Nn ly mu theo TCVN 4833-1 : 2002 (ISO 3100 1). Bo qun mu trnh b h hng v thay i thnh phn, nu cn. 8. Chun b mu th Chun b mu th theo TCVN 4833 2 : 2002 (ISO 3100 -2). 9. Cch tin hnh 9.1 Phn mu th, huyn ph ban u v cc dung dch pha long Xem TCVN 6507 : 1999 (ISO 6887). 9.2 Cy v 9.2.1 Ly hai a thch CFC (5.3.4). Dng pipet v trng (6.8) chuyn vo mi a 0,1 ml huyn ph ban u. Ly hai a CFC khc. Dng pipet v trng (6.8) chuyn vo mi a 0,1 ml dung dch pha long thp phn th nht ca huyn ph ban u (10-2). Lp li cc thao tc trn i vi tng pha long tip theo, dng pipet v trng mi cho mi pha long. 9.2.2 Dng que gt mu v trng (6.10) dn u cht lng trn b mt a thch cho n kh. 9.2.3 Lt ngc cc a chun b v lm mt n 250C ri trong t m (6.3) 48 h nhit 250C. 9.3 m v chn cc khun lc Sau khi kt thc thc thi gian qui nh, m s khun lc trn tng a v gi li cc a cha t 15 khun lc n 300 khun lc. T mi a c gi li chn nm khun lc mt cch ngu nhin. 9.4 Th khng nh 9.4.1 Cy truyn Ria cy t mi khun lc chn ln cc a thch dinh dng (5.4.3) th khng nh.

 cc a ny 24 h nhit 250C. Trn mi a chn ly mt khun lc phn lp tt th khng nh c tnh sinh ha (xem 9.4.2). 9.4.2 Th khng nh c tnh sinh ha 9.4.2.1 Phn ng oxidaza 9.4.2.1.1 Thnh phn N,N,N,N-Tetrametyl-p-phenylenediamin dihidroclorua Nc 9.4.2.1.2 Chun b Ha tan thuc th trong nc. Thuc th phi c chun b ngay trc khi s dng. C th s dng cc a hoc que cy bn sn. Khi , theo ch dn ca nh sn xut. 9.4.2.1.3 Cch tin hnh Lm m mnh giy lc bng thuc th. Dng dy platin hoc bng a thy tinh hoc a bng cht do (dy bng niken/crom cho kt qu dng sai) ly mu vi khun thu c trn mi trng thch cho ln giy lc c lm m. 9.4.2.1.4 Gii thch kt qu Trong trng hp c mt oxidaza, th c mu tm n mu ta trong khong t 5s n 10s. Nu sau 10s m mu sc khng i th php th c coi l m tnh. 9.4.2.2 c tnh chuyn ha ng trong thch Kligler Cy ria xin ln b mt thch (5.5.2) bng cc khun lc tng t trong 9.4.2 v m xuyn xung y ca ng thch. 24h 250C. 9.4.2.3 Gii thch kt qu Cc khun lc cho thy phn ng oxidaza dng tnh v khi cy vo trong thch Kligler ch thy pht trin trn b mt (a kh) c coi l cc khun lc Pseudomonas. 10. Biu th kt qu 10.1 Phng php tnh Sau khi nhn dng, tnh s lng vi sinh vt, a, c kh khng nh trn mi a, dng cng thc sau: a= trong A l s lng khun lc c chn th khng nh (trong trng hp ny l 5); b l s khun lc ph hp vi chun mc nhn dng; C l tng s khun lc m c. Tnh s lng vi sinh vt nhn dng, N, trong mu th l trung bnh ca hai pha long lin tip, theo cng thc sau: N= V (n1 + 0,1n2 )d b C A 1,0 g 100 ml

trong a l tng cc khun lc m c trn tt c cc a c gi li sau khi nhn dng; V l th tch mu cy s dng trn mi a, tnh bng mililit;

n1 l s a c gi li pha long u tin; n2 l s a c gi li pha long th hai; d l h s pha long tng ng vi pha long th nht c gi li. Lm trn cc kt qu tnh c n hai ch s c ngha. Nh vy, nu ch s sau cng nh hn 5 th s ng trc s khng b thay i, cn nu ch s sau cng l 5 hoc ln hn 5 th tng s ng trc ln mt n v. Lm trn tip theo cho n khi thu c hai ch s c ngha. Ly kt qu l s lng vi sinh vt trong mt mililit (sn phm dng lng) hoc trong mt gam (sn phm dng khc), c biu th bng s t 1,0 n 9,9 nhn vi ly tha tng ng ca 10. 10.2 Cc s m c tnh 10.2.1 Nu c hai a mu th gc (sn phm dng lng) hoc huyn ph ban u (sn phm dng khc) cha t hn 15 khun lc, th tnh trung bnh s hc y ca cc khun lc m c trn c hai a. Biu th kt qu nh sau: - i vi sn phm dng lng: s lng vi sinh vt c tnh trong mt mililit l: NE = y - i vi sn phm dng khc: s lng vi sinh vt c tnh trong mt gam sn phm l: NE = y/d trong d l h s pha long ca huyn ph ban u. 10.2.2 Nu trn hai a mu th gc (sn phm dng lng) hoc huyn ph ban u (sn phm dng khc) khng c bt k mt khun lc no th biu th kt qu nh sau: - t hn 1 vi sinh vt trong mt mililit (sn phm dng lng); - t hn 1/d vi sinh vt trong mt gam (sn phm dng khc); trong d l h s pha long ca huyn ph ban u. 10.3 Gii hn tin cy Xem TCVN 6404 : 1998 (ISO 7218). 11. Bo co th nghim Bo co th nghim phi ch r: - phng php ly mu; - phng php s dng; - s ngy ; - kt qu th nghim thu c; - kt qu cui cng thu c, nu kim tra lp li. Bo co th nghim phi ch ra phng php s dng v kt qu thu c. Cng phi cp n tt c cc chi tit thao tc khng qui nh trong tiu chun ny, cng vi cc chi tit bt thng khc c th nh hng ti kt qu. Bo co th nghim cng phi bao gm mi thng tin cn thit nhn bit y v mu th.

PH LC A
(tham kho) TI LIU THAM KHO

[1] MEAD, G.C and ADAMS, B.W.A setective medium for the rapid isolation of Pseudomonas associated with poultry meat spoilage. Br. Poult, Sci, 18,1977, pp. 661-670. [2] TCVN 4833-1 : 2002 (ISO 3100-1 : 1991) Tht v sn phm tht Ly mu v chun b mu th - Phn 1: Ly mu.