1.

NH LNG TNG VI SINH VT HIU KH

A. MC CH Kim tra tng s vi khun hiu kh trong thc phm, v sinh cng nghip. B. PHNG PHP PHN TCH 1.1.Phm vi p dng Phng php ny tham chiu NMKL: 86-2006 thch hp cho vic xc nh vi sinh vt hiu kh c th pht trin trong thc phm. Phng php ny c th c p dng cho tt c cc loi thc phm. 1.2.nh ngha Vi sinh vt hiu kh l nhng vi sinh vt sinh trng iu kin hiu kh khi php th c thc hin theo ng phng php th. Vi sinh vt a lnh l vi sinh vt hiu kh c kh nng pht trin t 0-100C Tng s khun lc hiu kh l s vi sinh vt hiu kh c th pht trin trn 1g thc phm c tm thy t php th theo phng php c m t. 1.3.Nguyn tc ng nht mu bng my dp mu vi dch pha lang. To cc pha lang thp phn t dch mu c ng nht ny. Chuyn 1 ml dch mu pha long vo a petri, sau trn u mu vi mi trng thch khng chn lc mu trong iu kin hiu kh 300C trong 3 ngy, hoc 200C trong 3 ngy, hoc 6.50C trong 10 ngy tnh s vi sinh vt hiu kh pht trin c trn 1gam mu t s khun lc trn a petri. 1.4. Mi trng nui cy v dch pha lang. Dch pha lang (SPW) Mi trng PCA 1.5.Thit b v vt dng T 300C10C B iu nhit 450C 10C Chun b mu Thc hin theo hng dn HD03/QT1 1.6. Quy trnh phn tch 1.6.1.Cy mu: Chuyn 1ml dch mu nng thch hp cho vo a petri v trng (trong thi gian u nn lm 1 nng /2 a) vo mi a 15-20ml mi trng nui cy PCA nhit 450C. Sau khi , trn u dch mu theo hnh s 8 khang 5-10 ln vi sinh vt phn b u trong mi trng. t cc a trn mt phng nm ngang. mt thch ng kh v em

Ch : qu trnh cy mu khng c qu 20pht 1.6.2. a: Lt ngc cc a Petri, trong 726h nhit 3010C 1.6.3.c a: Nu c th m nhng a c s khun lc nm trong khang 25-250. Dng my m khun lc m. 1.6.4.Tnh kt qu. Nu ch c mt a c s khun lc nm trong khang 25-250, da trn h s pha long tnh ton s khun lc trn gram hay ml mu. Nu hai a c s khun lc nm trong khang t 25-250, vic tnh ton kt qu da trn cng thc sau: S lng N vi khun c trong mu th c tnh theo phng php sau: C N= V (n1 + n2)d Trong : C: l tng s khun lc trn cc a 2 pha long k tip nu cc a u c s khun lc nm trong khang 25-250 V: l th tch dch mu cy vo mi a (ml). Trng hp trn V = 1 n1:l s a pha long th nht n2: l s a pha long th hai d: nng tng ng vi pha lang th nht.Trong trng hp mi nng tin hnh trn 2 a. Ta tnh s khun lc trong bnh ca hai a ca cng mt nng c s khun lc nm trong khang 25-250 v tnh kt qu theo cng thc trn. Bo co kt qu vi sinh vt hiu kh trn 1g mu (hay trn 1ml mu) bng cch s dng hai ch s c ngha (v d 7,9 x 104).

Chun b mu ng nht hoc pha long mu c pha long 10-1 ,10-2 ,10-3 ..

Chn 2 nng pha long thch hp, chuyn 1ml mu vo a petrivoo trng (mi nng cy 2 a)

Rt vo mi a 10- 15ml mi trng PCA c lm ngui na 450C, lc mu phaan tn u vo mi trng, 300C trong 72h

Chn cc a c s khun lc trong khong 25-250 khun lc/a m

Tnh kt qu:tng vi sinh vt hiu kh trong mu (CFU/g hoc CFU/ml)

Quy trnh xc nh tng vi khun vi sinh vt hiu khtrong thc phm

2.NH LNG COLIFORM CHU NHIT, E.COLI

A. MC CH nh lng coliform chu nhit v E.coli trong thc phm v v sinh cng nghip. B. PHNG PHP PHN TCH 2.1 PHM VI P DNG Phng php ny tham chiu NMKL. NO 125, 4rd edition, 2005, c dng pht hin coliform chu nhit, E.coli trong thc phm. Phng php ny c th p dng cho tt c cc loi thc phm v thc n gia sc. c bit, phng php ny thch hp cho vic pht hin nhng vi khun b tn thng. 2.2 NH NGHA 4.2.1 Coliform chu nhit L nhng vi khun sinh trng to khun lc c trng trn mi trng Violet red bile 0 44 C/24h. Sau , vi khun c khng nh li bng cch pht hin kh nng sinh hi (gas) trong mi trng lng cha lactose 440C. 4.2.2 E.coli l vi khun coliforms chu nhit to indol 440C 2.3 NGUYN TC S lng vi khun coliforms chu nhit c xc nh bng cch a mt lng mu bit trn mi trng thch khng chn lc. Sau khi s b 20-250C (nhit phng) trong 1-2h. Sau , ph ln mt lp mi trng chn lc cha lactose. cc a 440C/24h. Sau khi , m nhng khun lc in hnh. Sau chn nhng khun lc khng nh trn mi trng ph hp. 2.4 DCH PHA LONG V MI TRNG 4.4.1 Saline peptone Water (SPW) 4.4.2 Tryptone soya agar (TSA) 4.4.3 Violet red bile agar (VRB) 4.4.4 Tryptone broth 4.4.5 Thuc th kovacs 4.4.6 EC broth 2.5 THIT B 4.5.1 Thit b ng nht mu 4.5.2 B iu nhit 450C 10C 4.5.3 T 440C 0,50C 4.5.4 T 370C 10C 2.6 QUY TRNH 2.6.1 Chun b mu: Thc hin theo hng dn HD03/QT1. 2.6.2 a

Chuyn 1ml mu pha long sang 1 a ptri. Thm khang 5ml TSA (4.4.2) 450C 10C. Trn u ngay mi trng v mu. s b cc a 20-250C (nhit phng) trong 12h. Sau , ph khang 12-15ml mi trng VRB (4.4.3). T s dy gia 2 lp trn v di t nht l 2:1 2.6.3 Nui Lt ngc cc a, 440C 0.50C/243h 2.6.4 c kt qu Nu c th, chn cc a cha t 10-100 khun lc in hnh v nghi ng in hnh. Khun lc in hnh thng sm ng knh ti thiu 0.5mm c ta bao quanh. Nhng a cha s lng ln khun lc, th khun lc s nh v khng c trng. 2.6.5 Khng nh 2.6.5.1 Coliform chu nhit Chn i din nhng khun lc nghi ng. Nu c th, chn t nht 5 khun lc mi loi, chuyn sang cc ng nghm cha mi trng lng EC (4.4.6). cc ng nghim 440C 0.50C/243h. Sau khi , thm 2-3 git thuc th Kovacs. c phn ng to mu. Mu lp trn cho thy indol c to t c cht, l phn ng dng tnh. Mu cam hay vng c to thnh trong 10 pht c xem l phn ng m tnh. E.coli l vi khun coliforms chu nhit to indol. 2.7 BO CO KT QU Tnh s khun lc E.coli hay coliform chu nhit c trn mi a bng cng thc bc bnc a = ( x Cc + x Cnc ) x F Ac Anc Trong : bc: s khun lc in hnh th nghim khng nh nghm ng vi phn ng sinh ha Ac: s khun lc in hnh th nghim khng nh. Cc: tng s khun lc in hnh m c trn a. bnc: s khun lc khng in hnh th nghim khng nh nghim ng vi phn ng sinh ha. Anc: s khun lc khng in hnh th nghim khng nh Cnc: tng s khun lc khng in hnh m c trn a F: nghch o ca nng pha long

PHNG PHP TNH TON 2.7.1 Nhng a cha 10 -100 khun lc Tnh s khun lc N E.coli hay coliform chu nhit c trong 1ml hoc 1g sn phm bng

cng thc tnh sau: a N= V (n1 + 0.1n2) d Trong : a l tng s khun lc E.coli hay coliform chu nhit c tnh tan sau khi khng nh trn tt c cc a ca 2 nng pha long lin tip. V: l th tch cy cho mi a tnh bng ml n1 : l s a m nng pha long u n2: l s a m nng pha long k tip. d: l nng pha long u tin. Lm trn kt qu ti 2 con s c ngha. Tnh kt qu: s E.coli hay coliform chu nhit c trong 1ml (mu lng) hoc 1g sn phm (mu rn), c trnh by t 1,1 n 9,9 x 10 ly tha. 2.7.2 Kt qu c lng i vi a cha < 10 khun lc Nu a nng pha long u (i vi sn phm lng nng pha long u 100, i vi sn phm khc nng pha long u 10-1) nh hn 10 th s khun lc sau khi khng nh c thc hin nh trn gi tr l y. Vy kt qu trong trng hp ny c tnh theo cng thc: S E.coli hay coliform chu nhit c trong 1ml hoc 1g sn phm y NE = d xV y: s khun lc trn a nng pha long u c khng nh. d: l nng pha long u tin V: l th tch cy cho mi a tnh bng ml 2.7.3 Nu a nng pha long u khng c cha khun lc no: Tnh kt qu theo cng thc: 1 < E.coli hay coliform chu nhit/g hoc ml, dxV trong : d: l nng pha long u tin

Chun b ng nht hoc pha long mu c pha long 10-110-2,10-3 .

Cy 1ml dung dch mu vo da petri, b sung 5ml mi trng TSA,lc u, yn 1-2h

Rt vo mi a 10-15ml mi trng thch VRB, ng , 37oCtrong 24-48h

m cc khun lc mu dn m,c vng ta mui mt ,ng knh >=0,5mm:chn 5 khun lc

Cy vo ng canh BGBL, 370C,24-48h m s ng canh(+)(sinh hi):tnh t l khng nh coliforms

Cy vo ng canh EC, 440c,24-48h

Chn ng canh(+) (sinh hi)

Mt coliform

Th nghim Indol

Cy vo ng canh Tryptone,MR-VP,thch SC citrate, 440C,24 h Th nghim IMVIC

m ng canh EC(+)v indol(+),tnh t l coliforms phn

Mt coliforms phn

m s ng canh EC(+) v IMVIC++--,tnh t l khng nh Ecoli Mt ecoli

3.PHNG PHP PHT HIN SALMONELLA

A. MC CH

Kim tra Salmonella trong thc phm. B. PHNG PHP PHN TCH 3.1 PHM VI P DNG Phng php ny tham chiu theo NMKL 71-1999, m t quy trnh pht hin Salmonella trong thc phm. Phng php ny c th p dng cho tt c cc loi thc phm. 3.2 NH NGHA Ging Salmonella c khong 2300 serotype v thuc h Enterobacteriacea Salmonella c nhng c im sau: Gram m, hnh que, k kh ty nghi Di ng bng tin mao nhng nhng dng t bin c th khng di ng v c mt serotype (Gallinarum) l khng di ng. Cc chng Salmonella to acid t glucose v manitol nhng khng to acid t sacchorose v lactose. Chng khng to indol v khng phn ct urea. Phn ln cc chng sinh H2S v c enzyme Lysine decarboxylase v Ornithine decarboxylase. Ging Salmonella c chia thnh nhiu serotype theo xp loi Kauffmann White, da theo khng nguyn thn (somatic) O v khng nguyn H (flagella). Mt s serotype nh Enteritidis, Typhi, Paratyphi v Typhimurium c xp loi theo phage. 3.3 NGUYN TC Phng php ny ch nh tnh v kt qu c bo co l: pht hin/khng pht hin Salmonella trong lng mu c ly. pht hin Salmonella cn 4 bc ring bit. Nhng bc ny l cn thit do Salmonella thng c mt s lng thp, i khi b tn thng v thng c s lng ln nhng vi khun khc thuc h Enterobacteriacea 3.3.1 Tin tng sinh Mu c tng sinh trong mi trng khng chn lc (buffered peptone water) 37 0C trong khong 18h. 3.3.2 Tng sinh Mu sau khi tin tng sinh, c chuyn sang mi trng lng chn lc (RappaportVassiliadis soy peptone broth) v c 420C trong khong 24h. 3.3.3 Cy a Mu sau khi tng sinh, c cy chuyn sang hai mi trng thch chn lc (Xylose lysine desoxycholate agar v chn thm mt mi trng thch chn lc na). Cc a c 370C trong khong 24h. 3.3.4 Khng nh

Khi nghi ng Salmonella, c cy chuyn sang mi trng thch thch hp v thm tra bng th nghim sinh ho v huyt thanh. 3.4 MI TRNG NUI Cy 3.4.1 Buffered peptone water (BPW) 3.4.2 Rappaport- Vassiliads soy peptone broth (RV) 3.4.3 Brilliant green phenol red lactose sucrose agar (BLPS) 3.4.4 Xylose lysine desoxycholate (XLD) agar 3.4.5 Triple sugar iron agar 3.4.6 Tryptic soy agar 3.4.7 Urea broth 3.4.8 Lysine decarboxylase broth 3.4.9 Ornithine decarboxylase broth 3.4.10 Manitol broth 3.4.11 Succrose broth 3.4.12 Lactose 3.4.13 Tryptone broth 3.4.14 Simmon citrate 3.4.15 Kovacs indole reagent 3.4.16 Motility 3.5 THIT B 9.5.1 B iu nhit 420.20C 9.5.2 T : 3710C 3.6 QUY TRNH *Chun b mu: Thc hin theo hng dn HD03/QT1 3.6.1 Tin tng sinh ng nht mu trong khong 30 giy. 370C 10C trong 18-24h 3.6.2 Tng sinh Trn u dch mu sau khi tin tng sinh, chuyn 0,1ml sang 10ml RV broth c lm m trc. Nhit rt quan trng tng sinh ti u. 420.2 0C (trong b iu nhit) trong 243h. 3.6.3 Phn lp v c kt qu Sau khi tng sinh, dng que cy vng, cy ria dch mu sang b mt mi trng thch XLD v BLPS. Lt ngc cc a v 370C 10C trong 243h Trn mi trng XLD: Khun lc Salmonella c trng c vng trong, hi nhum do cht ch th mu thay i trong mi trng, v c tm en. Trn mi trng green phenol red lactose sucrose: khun lc Salmonella in hnh hi trong c mu hi trong mi trng. 3.6.4 Khng nh

Nhng khun lc nghi ng Salmonella phi c thm tra bng cc th nghim sinh ho. Ly t nht 5 khun lc nghi ng t mi a mi trng thch XLD v BPLS cy sang mi trng thch khng chn lc TSA. 3710C trong 18-24 Nhng th nghim sinh ha c dng khng nh Salmonella Stt Th nghim m tnh (-) Dng tnh (+) Salmonella 1 Indole Khng xut hin mu xut hin mu b b mt mt 2 Mannitol Vng + 3 Ure Khng thay i mu cnh sen 4 ODC Vng Ta + 5 LDC Vng Ta + 6 Citrate Khng thay i mu Xanh dng 7 Sucrose Vng 8 Lactose Vng 9 Di ng Mu xut hin dc Mu xut hin khp theo ng cy mi trng 10 TSI mt nghing: , y ng: vng; H2S (); kh (+) 11 LIA mt nghing: ta, y ng: ta, H2S (); kh (-) 3.7 BO CO KT QU Pht hin hoc khng pht hin Salmonella trn 25gam mu

ng nht 25g mu trong 225ml mi trng tng sinh (BPW), 370C ,18-24h

Cy 0,1ml dch tng sinh sang mi trng tng sinh chn lc RV, 420C,18-24h

Phn lp khun lc n trn t nht 2 mi trng chn lc phn bit (XlD,HE,BS,SS..), 370c,24h

Chn cc khun lc c trng cho samonnella, cy sang BHI hay TSA : qua m

Th nghim sinh ha cho kt qu Trn KIA/TSI: /vng, c/khng H2S,sinh hi/khng Urea:indol(-),VP(-),LDC(+) ODC(+),Manitol:(+),sorbitol:(+)

Th nghim ngng kt khng huyt thanh:poly:O,poly:H

Kt lun:samonella dng tnh /m tnh trong 25g mu

4.TNG NM MC ,NM MEN

A.Mc ch: Kim nh nh nm men nm mc trong thc phm B. PHNG PHP PHN TCH 4.2 NH NGHA V NGUYN TC Nm men v nm l nhm vi sinh vt rt a dng n nay co hn 400.000 loi nm men v nm mc c miu t. y l nhm vi sinh vt nhn tht,co vch t bo l lp v chitin , co nhn v bo quan khc, tt c cc loi nm men v nm mc u thuc nhm vi sinh vt d dng, chng cn co ngun cacbon hu c d cung cp t mi trng been ngoi. Co th phn bit nm men v nm mc theo khi nim sau :nm mc l vi nm dng si sinh sn bo t hoc khun ty :nm men l nhng t bo n tinhsphats trin theo kiu ny chi, thnh thong c tn ti dng khun ty gi trong cc t bo kt vi nhau thnh chi .

3.4 MI TRNG NUI CY Dung dch pha long (nc pepton 1%) Mi trng thch Dichloran Glycerol Agar (DG18) Mi trng thch Dichloran rose bengal Chloramplenicol Agar(DBRC) Mi trng thch Malt Extract Agar (MEA) Mi trng Potato Dextrose Agar PDA Mi trng thch Sabouraud Agar SDA Mi trng thch Sabouraud Dextrose SDB.

5 THIT B 3.6 QUY TRNH Chun b mu: 3