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Trng i hc Cng nghip thc phm TP.

HCM

2010

THC HNH PHN TCH VI SINH THC PHM

ThS. L Thy Linh


Lu hnh ni b
0

Trng i hc Cng nghip thc phm TP.HCM

NI DUNG Bi 1: nh lng Coliforms v E. coli 1.1. Tm tt kin thc c bn 1.2. Quy trnh nh lng Coliforms v E. coli bng phng php MPN 1.3. Cch tin hnh th nghim 1.4. Cch tnh kt qu Bi 2. nh lng Staphylococcus aureus 2.1. Tm tt kin thc c bn 2.2. Quy trnh nh lng Staphylococcus aureus bng phng php MPN 2.3. Cch tin hnh v cch tnh kt qu Bi 3. nh tnh Salmonella 3.1. Tm tt kin thc c bn 3.2. Qui trnh nh tnh Salmonella trong thc phm Bi 4. nh lng Bacillus cereus 4.1. Tm tt kin thc c bn 4.2. Quy trnh nh lng Bacillus cereus bng phng php m khun lc Bi 5. nh lng tng vi khun hiu kh v tng nm men-nm mc 5.1. Tm tt kin thc c bn 5.2. Quy trnh phn tch Ti liu tham kho

Trang 2 2 3 4 10 10 10 10 11 14 14 14 20 20 20 24 24 26

1. Trn Linh Thc, Phng php phn tch vi sinh vt trong nc, thc phm v m phm, NXB Gio dc, 2006 2. http://www.microbelibrary.org 3. http://www.chromagar.com 4. http://www.rapidmicrobiology.com 5. http://www.marietta.edu

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Trng i hc Cng nghip thc phm TP.HCM

Bi 1: nh lng Coliforms v E. coli 1.1 Tm tt kin thc c bn 1.1.1 nh ngha


Hnh 1: pht hin Coliforms v E. coli trong thc phm hay nc bng mi trng CHROMagar ECC

Coliforms l nhng trc khun gram m khng sinh bo t, hiu kh hoc k kh ty , c kh nng ln men lactose sinh acid v sinh hi 370C trong 24 48 gi. Coliforms c xem l nhm vi sinh vt ch th: s lng hin din ca chng trong thc phm, nc hay cc loi mu mi trng c dng ch th kh nng hin din ca cc loi vi sinh vt khc. Nhm Coliforms gm 4 ging l: E. coli, Citrobacter, Klebsiella, Enterobacter. Tnh cht sinh ha c trng ca nhm ny c th hin qua cc th nghim Indol (I), Methyl Red (MR), Voges-Proskauer (VP) v Citrate (iC) thng c gi l IMViC. Coliforms chu nhit l nhng Coliforms c kh nng ln men lactose sinh hi trong khong 24h khi 440C trong mi trng canh EC. Coliforms phn (Faecal Coliforms) l Coliforms chu nhit c kh nng sinh indole khi khong 24h 440C trong canh Trypton. E. coli l Coliforms phn cho kt qu th nghim IMViC l ++-- (Indol +, Methyl Red +, Voges-Proskauer -, Citrate -) 1.1.2 Phng php Most Probable Number (MPN) y l phng php nh lng da trn kt qu nh tnh ca mt lot th nghim c lp li mt s pha long khc nhau. Thng thng, vic nh lng ny c thc hin lp li 3 ln 3 pha long bc 10 lin tip, tng cng 3 x 3 = 9 ng nghim. Quy trnh thc hin nh sau: Pha long mu phn tch 3 nng pha long bc 10 lin tip. nhit v thi gian thch hp Kt qu biu kin chng minh s tng trng ca vi sinh vt cn kim nh Ghi nhn s lng ng nghim dng tnh tng pha long. Tra bng Mac Crady
Mt vi sinh vt MPN/100 ml hay MPN/1g mu

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Trng i hc Cng nghip thc phm TP.HCM

Ch : a s cc bng MPN cung cp s liu dng s MPN trong 100ml dung dch c s dng phn tch cc liu lng l 10ml, 1ml, 0.1 ml. Trn thc t phn tch, ngi ta thng dng 1ml cho mu pha long 10-1, 10-2, 10-3. Lng mu vi cc pha long ny tng ng vi 1/100 lng mu s dng theo bng MPN/100ml tnh theo lng mu l 10ml, 1ml, 0.1 ml nu trn. Vy s liu ca bng MPN/100ml tnh theo lng mu l 10ml, 1ml, 0.1 ml tng ng vi MPN/ml mu cha pha long khi 1ml ca cc dung dch c pha long 10-1, 10-2, 10-3 c dng phn tch. Trng hp mt vi sinh vt qu cao, cn phi s dng lot nng pha long tip theo cn phi nhn tr s tra bng MPN nu trn vi h s pha long. V d: s dng 1ml cho mi pha long 10-2, 10-3, 10-4 tc l ch s dng 1/10 lng mu mi nng so vi trng hp dng 1ml mu cc pha long 10-1, 10-2, 10-3. Do vy, tr s tra bng MPN cn nhn thm h s pha long l 10. 1.2 Quy trnh nh lng Coliforms v E. coli bng phng php MPN Chun b dch ng nht hoc pha long mu c pha long 10-1, 10-2, 10-3 Chuyn 1ml dung dch 10-1, 10-2, 10-3 vo ng 10ml canh LSB, mi nng c 3 ng lp li, 370C, 48 gi Ghi nhn cc ng LSB (+) (sinh hi) mi nng pha long

Cy vo ng canh BGBL, 370C 10C, 48 gi S ng (+)(sinh hi ) mi pha long

Cy vo ng canh EC, 44.50C 0.20C, 24 gi S ng (+) mi pha long Cy ln thch EMB, 370C, 24 gi

Coliforms

Xem trang sau

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Trng i hc Cng nghip thc phm TP.HCM

Tip theo

Chn khun lc (trn, dt hnh a v c nh kim xanh), cy vo Trypton, MR-VP, SC Citrate, 44.50C 0.20C, 24 gi

Th nghim IMViC

m s ng canh EC (+) v IMViC ++--, tra bng MPN

E. coli 1.3 Cch tin hnh th nghim Mu phn tch: nc ma nguyn cht (dng 10ml cho mt t) 1.3.1 Pha mi trng v kh trng dng c Bc 1: pha mi trng Tn mi trng SPW (Saline Water) Thnh phn Tng th tch Ghi ch 500 ml nc Phn phi cho mi t 27ml ct SPW (9ml/ng nghim) trc khi kh trng. (bui 1) 1000 ml nc Phn phi 90ml LSB cho ct mi t (10ml/ng nghim) pH 6.8 0.2 trc khi kh trng. (bui 1)

NaCl : 42.5g Pepton Pepton: 5g

Tryptose: 20g LSB (Lauryl Sulphate Lactose: 5g Broth) Sodium chloride: 5g KH2PO4: 2.75g K2HPO4: 2.75g Lauryl sulphate: 0.1g BGBL Peptone: 10g

1000 ml nc Phn phi 90ml BGBL cho


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Trng i hc Cng nghip thc phm TP.HCM

(Brilliant Bile Broth)

Green Lactose: 10g Lactose Mt b: 20g Brilliant green: 0.0133g

ct pH 7.4

mi t (10ml/ ng nghim c cha ng Durham) trc khi kh trng. (bui 1)

EC (E.coli medium)

Trypticase or tryptose: 1000 ml nc Phn phi 90ml BGBL cho 20g ct mi t (10ml/ ng nghim c Mui mt No.3: 1.5g pH 6.9 cha ng Durham) trc khi Lactose: 5g kh trng. (bui 1) KH2PO4: 1.5g K2HPO4: 4g NaCl: 5g

Pancreatic digest of 1000 ml nc Phn phi 100ml EMB cho EMB (Eosin Methylene gelatin: 10g ct. mi t (20ml/petri) (bui 2) Blue Agar) Lactose: 10g pH 7.1 0.2 K2HPO4: 2g Eosin Y: 0.4g Methylene blue: 65mg Agar: 15g Tryptone water Tryptone trypticase:10g hoc 1000ml nc Mi t ch dng 20ml, phn ct phi 10ml/ng nghim. pH 7.2 0.2 (bui 3) Mi t ch dng 30ml, phn phi 10ml/ng nghim. Kim tra li mi trng cung cp no pha mi trng. (bui 3)

1000ml nc MR-VP Both Mi trng 1: (Glucose Buffered peptone-water ct. Phosphate) powder: 7g pH 6.9 0.2 Glucose: 5g K2HPO4: 5g Mi trng 2: Casein Pancreatic Digest: 3.5g Peptic digest of animal tissue:3.5g Dextrose: 5g Potassium phosphate: 5g Bin son: L Thy Linh
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Trng i hc Cng nghip thc phm TP.HCM

pH 6.9 0.2 Mi trng 3: Peptone: 5g Glucose: 5g Phosphate buffer: 5g pH 7.5 0.2 Sodium citrate: 2g SCA (Simmons Citrate NaCl: 5g Agar) K2HPO4: 1g NH4H2PO4: 1g MgSO4: 0.2g Bromothymol 0.08g Agar: 15g 1000ml nc Mi t ch dng 50ml, phn ct. un nng phi 10ml/ng nghim. nh v thnh (bui 3) thong lc. un si 1-2 blue: pht cho n khi ha tan ht. pH 6.8 0.2

Bc 2: kh trng dng c v mi trng pha. Bao gi dng c v phn phi mi trng vo dng c, gn nt v bao gi. Hp tit trng 1210C trong 15 pht. 1.3.2 Cch tin hnh: Bc 3: pha long mu
1ml 1ml 1ml 1ml 1ml 1ml

ng mu

9ml 10-1 10-2 10-3 10-4 10-5 10-6 pha long


Hnh 2: quy trnh pha long mu

Pha long mu thnh 3 nng pha long 10-1, 10-2, 10-3. Bin son: L Thy Linh
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Trng i hc Cng nghip thc phm TP.HCM

Bc 4: Cy mu vo canh LSB Chuyn 1ml dung dch 10-1, 10-2, 10-3 vo ng 10ml canh LSB, mi nng c 3 ng lp li, 370C, 48 gi.
Hnh 3: S cy mu t cc pha long

10-1

10-2

10-3

Bc 5: nh lng Coliform v E. coli nh lng Coliform Ghi nhn s ng c sinh hi (+) nh lng E. coli Ghi nhn s ng c sinh hi (+)

Dng que cy vng cy chuyn dch mu Dng que cy vng cy chuyn dch mu t cc ng LSB (+) sang cc ng c cha t cc ng LSB (+) sang mi trng canh canh BGBL v 370C 10C, 48 gi EC, 44.50C 0.20C, 24 gi Ghi nhn s ng c sinh hi (+) ng vi Ghi nhn s ng c sinh hi (+) ng vi mi pha long mi pha long Tnh kt qu (xem 1.4) Dng que cy vng ria dch mu t cc ng (+) trn canh EC sang mi trng thch a EMB. 370C, 24 gi. Chn khun lc ng knh ln hn 1mm (trn, dt hnh a v c nh kim xanh) (xem hnh 4), cy vo Trypton, MR-VP, SC Citrate, 44.50C 0.20C, 24 gi

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Trng i hc Cng nghip thc phm TP.HCM

Hnh 4: E.coli nui trn mi trng thch EMB cho khun lc mu kim xanh (theo Naowarat Cheeptham, Thompson Rivers University, Kamloops, BC, Canada)

Bc 6: th nghim IMViC

Hnh 5: Kt qu IMViC ca Escherichia coli sau 24gi 37C. (Anne Hanson, University of Maine, Orono) ng A: dng tnh th nghim indole trn mi trng tryptone. Xut hin lp mu trn b mt mi trng sau khi nh thuc th Kovcs. ng B: dng tnh th nghim methyl red xut hin mu ca methyl red. ng C: m tnh th nghim Voges-Proskauer biu hin qua s khng thay i mu sau khi cho thuc th Barritts A v Barritts B. ng D: m tnh th nghim citrate biu hin qua s thay i mu sc v khng c khun lc trong ng nghim.

a. Th nghim Indol (I): Nguyn tc: Tryptophan l mt acid amin c th b oxi ha bi mt s vi sinh vt c h enzyme tryptophanase to nn cc sn phm cha gc indol. Vic pht hin indol c thc hin bng phn ng ca phn t ny vi cc thuc th cha pDimethylaminbenzaldehyde (p-DMABA) to nn mt phc cht dng quinone c mu . Phng php tin hnh: Mi trng s dng cho th nghim ny l nc tryptone. Sinh khi chng thun c cy vo mi trng lng tryptone, nhit 370C trong 24-48 gi. B sung 1ml ether hoc xylene vo ng nghim, lc u chit Bin son: L Thy Linh
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Trng i hc Cng nghip thc phm TP.HCM

tch indol ln lp dung mi hu c. Nh 5 git thuc th vo ng dch nui cy, yn vi pht, theo di s to mu trong lp dung mi hu c. c kt qu: (+) xut hin ca lp mu trn b mt mi trng. (-) lp mu vng ca thuc th. i khi c s xut hin ca mu cam do skatol, l tin cht ca methyl ha ca indol, to ra.
Hnh 6: th nghim Indol

b. Th nghim Methyl Red (MR): Nguyn tc: ch th methyl red gip phn bit nng H+ hin din trong mi trng sau khi vi sinh vt ln men glucose. Ch th ny thay i mu nh sau: pH<4.4 ; pH 5.0-5.8 mu cam; pH>6.0 mu vng. Phng php tin hnh: Mi trng s dng cho th nghim ny l mi trng lng Glucose Phosphate (MR-VP Broth). Dng que vng cy mt lng nh khun lc trn mi trng MR-VP, 370C trong khong 2-5 ngy. Thm vo vi git thuc th methyl red (0.02% trong hn hp cn nc c t l 3:2, bo qun 40C) vo trong ng nghim dch nui cy, c kt qu ngay. c kt qu: (+) mi trng c mu sau khi b sung thuc th. (-) khi c mu vng. Trng hp mu cam, cn tip tc ng mi trng c ging trong 4 ngy v thc hin li
Hnh 7: th nghim MR

th nghim.

c. Th nghim Voges-Proskauer (VP) (xem mc 3.3, bi 3) d. Th nghim Citrate (iC) Nguyn tc: s bin dng citrate bi vi sinh vt s to ra CO2 lm km ha mi trng. Mt khc mi VSV c kh nng s dng citrate lm ngun carbon duy nht u c kh nng dng mui ammonium lm ngun m duy nht. S phn gii ca mui ammonium dng lm ngun m trong mi trng s sinh NH3 lm kim ha mi trng. S gia tng gi tr pH ny c ch th bng s i mu ca ch th pH trong mi trng. Bin son: L Thy Linh
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Trng i hc Cng nghip thc phm TP.HCM

Phng php tin hnh: Mi trng s dng cho th nghim ny l mi trng lng SCA. Dng que vng cy ria mt lng nh khun lc ln mi trng SCA rn trong ng nghim thch nghing, 350C trong khong 24-48 gi. c kt qu: (+) khi xut hin khun lc v mi trng chuyn sang mu xanh dng. (-) khi khng c khun lc v mi trng gi nguyn mu xanh lc. 1.4 Cch tnh kt qu
Hnh 8: th nghim Citrate

T s lng cc ng nghim c E. coli (+) mi pha long ca mu, dng bng MPN lot 3 ng nghim 3 nng pha long lin tip tnh ra mt vi sinh vt trong mu v biu din di dng tr s MPN/ml mu ban u cha pha long.

Bi 2. nh lng Staphylococcus aureus 2.1. Tm tt kin thc c bn Staphylococcus aureus l vi khun hiu kh hay k kh ty , hnh cu, gram dng, c th nghim coagulase, phn ng DNAse, phosphatease (+) c kh nng ln men v sinh acid t mannitol, trehalose, sucrose. Tt c cc dng S. aureus u mn cm vi novobiocine, c kh nng tng trng trong mi trng cha n 15% NaCl. Mt s dng c kh nng tng trng lm Hnh 9: S. aureus trn BPA tan mu trn mi trng thch mu. S. aureus c th nhim vo thc phm qua con ng tip xc vi ngi thao tc trong qu trnh ch bin thc phm. S hin din vi mt cao ca S. aureus trong thc phm ch th iu kin v sinh v kim sot nhit km ca qu trnh ch bin. 2.2 Quy trnh nh lng Staphylococcus aureus bng phng php MPN Phng php ny c dng nh lng S. aureus trong mu c mt S. aureus thp nhng mt vi sinh vt cnh tranh cao.

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Trng i hc Cng nghip thc phm TP.HCM

ng nht mu v pha long mu thnh cc dy nng thp phn 10-1, 10-2, 10-3 Chuyn 1ml dung dch 10-1, 10-2, 10-3 vo ng 10ml canh MSB, mi nng c 3 ng lp li, 370C, 48 gi Chn ng (+) c mi pha long Ria ln a thch BPA, 370C, 48 gi Chn khun lc c trng (trn li, en bng, c qung trong v qung sng xung quanh (i khi ch xut hin 1 trong 2 qung)) Cy vo TSA, 370C 10C , 24 gi Th nghim ngng kt coagulase Ghi nhn s coagulase (+) mi pha long

Tra bng MPN

Mt S.aureus (MPN/g hoc MPN/ml)

2.3 Cch tin hnh v tnh kt qu Mu phn tch: tht heo sng, khi lng 50g cho 1 lp Bin son: L Thy Linh
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Trng i hc Cng nghip thc phm TP.HCM

Bc 1: pha mi trng Tn mi trng Thnh phn SPW (Saline Water) MSB (Mannitol Broth) Cao tht: 1g Salt Polypeptone: 10g NaCl: 75g Mannitol: 10g Phenol red: 0.025g BPA (Baird Agar) Mi trng c bn Parker Tryptone:10g Cao tht: 5g Cao nm men: 1g Sodium pyruvate: 10g Glycine: 12g Lithium chloride.6H2O: 5g Agar: 20g Mi trng hon chnh 5ml Bacto EY tellurite enrichment (45-500C) vo 95ml mi trng c bn c lm nng chy. Mi trng hon chnh phi c. TSA (Tryptone Agar) Trypticase peptone: 15g Soya Phytone peptone: 5g NaCl: 5g Agar: 15g NaCl : 42.5g Pepton Pepton: 5g

Tng th tch Ghi ch 500 ml nc Phn phi cho mi t 27ml ct SPW (9ml/ng nghim) trc khi kh trng. (bui 2) 1000 ml nc Phn phi 90ml MSB cho ct mi t (10ml/ng nghim) pH 7.4 0.2 trc khi kh trng. (bui 2)

1000 ml nc Phn phi 100ml BPA cho ct cho mi mi t (20ml/petri) (bui 2) trng c bn pH 7.0 0.2

1000 ml nc Phn phi 50ml BGBL cho ct mi t (25ml/petri) (bui 3) pH 7.3 0.2

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Trng i hc Cng nghip thc phm TP.HCM

Bc 2: kh trng dng c v mi trng pha. Bao gi dng c v phn phi mi trng vo dng c, gn nt v bao gi. Hp tit trng 1210C trong 15 pht. Bc 3: ng nht mu v pha long mu Cn 10g mu trong ti PE v trng, thm 90ml dung dich SPW, ng nht mu bng my dp mu khong 30 giy. Pha long mu (xem bi 1, mc 1.3.2) Bc 4: Cy mu vo canh MSB Chuyn 1ml dung dch 10-1, 10-2, 10-3 vo ng 10ml canh MSB, mi nng c 3 ng lp li, 370C, 48 gi. (xem bi 1, mc 1.3.2). Chn cc ng (+) c vi sinh vt pht trin lm c mi trng tin hnh phn lp khun lc n trn BPA Bc 5: phn lp khun lc n trn BPA Dng k thut cy ria ln a thch BPA, 370C, 48 gi. Chn khun lc c trng :trn li, en bng, c qung trong v qung sng xung quanh; i khi ch xut hin 1 trong 2 qung (xem Hnh 10: hnh dng S. aureus trn BPA hnh 10). Chn khun lc c trng ca S. aureus trn BPA th nghim sinh ha coagulase Bc 6: th nghim sinh ha coagulase Nguyn tc: cc loi thuc ging Staphylococcus c kh nng tit ra mi trng enzyme coagulase c tc dng lm kt t cc thnh phn ca huyt tng, to thnh cc khi ng lm ng huyt tng. Phng php tin hnh: Huyt tng ngi hay th ng kh thng phm c dng cho th nghim ny. Cho vo ng nghim 0.5ml huyt tng ngi hay th khng pha long, b sung 0.5ml dch nui cy chng thun hoc mt lng ln sinh khi khun lc. Xoay nh ng trn u VSV. ng th nghim 370C trong 4 gi. Quan st, ghi nhn s ngng kt mi 30 pht. Tip tc n 24 gi nu khng thy xut hin khi kt t. c kt qu: (+) c s kt t; (-) khng c s kt t, hn hp vn ng nht
Hnh 11: th nghim coagulase. ng trn (+); ng di (-)

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Trng i hc Cng nghip thc phm TP.HCM

Bi 3. nh tnh Salmonella 3.1 Tm tt kin thc c bn


Hnh 12: Salmonella trn thch XLD

Salmonella l trc trng gram m, hiu kh v k kh ty , c kh nng di ng khng to bo t, ln men glucose v mannitol sinh acid nhng khng ln men saccharose v lactose, khng sinh Indole, khng phn gii ure, khng c kh nng tch nhm amine t tryptophane, hu ht cc chng u sinh H2S. Salmonella c th phn tch nh tnh bng m quy trnh gm 4 bc: tng sinh, tng sinh chn lc, phn lp v khng nh. Salmonella thng c mt trong mu vi s lng nh, b tn thng v cng hin din chung vi mt s lng ln vi cc loi vi khun khc thuc h Enterobacteriaceae c tnh cnh tranh mnh v c ch s tng trng ca Salmonella. 3.2 Quy trnh nh tnh Salmonella trong thc phm ng nht 25g mu trong 225ml mi trng tng sinh BPW, 370C, 18-24 gi Cy 0.1ml dch tng sinh sang mi trng tng sinh chn lc RV, 420C, 18-24 gi Phn lp khun lc n trn t nht 2 mi trng chn lc c bit (XLD, HE, BS, SS), 370C, 24 gi Chn cc khun lc c trng cho Salmonella, 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 (+)

Salmonella dng tnh/ m tnh trong 25g mu Bin son: L Thy Linh
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Trng i hc Cng nghip thc phm TP.HCM

3.3 Cch tin hnh v tnh kt qu Mu phn tch: c hoc rut c, khi lng 25g cho 1 lp Bc 1: pha mi trng Tn mi trng Thnh phn Tng th tch Ghi ch Pepton: 10g BPW (Buffered Pepton NaCl : 5g Water) Na2HPO4: 3.5g KH2PO4: 1.5g RV Mi trng c bn (RappaportTryptone:5g Vassiliadis Soya NaCl: 8g Pepton) KH2PO4: 1.6g Nc ct: 1000 ml Dung dch MgCl2 MgCl2.6H2O: 400g Nc ct: 1000 ml Dung dch Malachite green oxalate Malachite green oxalate: 0.4g Nc ct: 100 ml Mi trng hon chnh Mi trng c bn: 1000ml Dung dch MgCl2: 100ml Dung dch Malachite green oxalate: 10ml Cao nm men: 3g XLD (Xylose Lysine L-lysine: 5g Desoxycholate) Xylose: 3.75g Lactose: 7.5g Sucrose: 7.5g Bin son: L Thy Linh
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1000 ml nc Phn phi cho mi t 20ml ct BPW trc khi kh trng. pH 7.2 0.2 1110 ml pH 5.5 0.2 (bui 2) Phn phi 30ml RV cho mi t (10ml/ng nghim) trc khi kh trng. (bui 2)

1000 ml nc Phn phi 50ml XLD cho ct mi t (25ml/petri) (bui 3) pH 7.4 0.2 un si mi trng.
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Trng i hc Cng nghip thc phm TP.HCM

Sodium deoxycholate: 2.5g Ferric ammonium citrate: 0.8g Sodium thiosulfate: 6.8g NaCl: 5g Agar:15g Phenol red: 0.08g HE (Hektoen Agar) Proteose Peptone: 12.0g Entric Yeast Extract: 3.0g Bile Salts No. 3: 9.0g Lactose: 12.0g Saccharose: 12.0g Salicin: 2.0g Sodium Chloride: 5.0g Sodium Thiosulfate: 5.0g Ferric Ammonium Citrate : 1.5g Agar: 14.0g Bromthymol Blue: 65.0mg Acid Fuchsin : 0.1g Trypticase peptone: 15g Soya Phytone peptone: 5g NaCl: 5g Agar: 15g Polypeptone peptone: Iron 20g Lactose: 20g Dextrose: 1g NaCl: 5g

Khng Khng

hp. gi

qu 1 ngy

1000 ml nc Phn phi 50ml HE cho mi ct t (25ml/petri) (bui 3) pH 7.5 0.2 un si mi trng. Khng hp.

TSA (Tryptone Agar) KIA (Kliger Agar)

1000 ml nc Phn phi 50ml TSA cho ct mi t (25ml/petri) (bui 4) pH 7.3 0.2 1000 ml nc Phn phi 20ml KIA cho ct mi t (10ml/ng nghim) pH 7.4 0.2 (bui 5)

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Trng i hc Cng nghip thc phm TP.HCM

Ferric ammonium citrate: 0.5g Sodium thiosulfate: 0.5g Agar: 15g Phenol red: 0.025g MR-VP Both Mi trng 1: (Glucose Buffered peptone-water Phosphate) powder: 7g Glucose: 5g K2HPO4: 5g Mi trng 2: Casein Pancreatic Digest: 3.5g Peptic digest of animal tissue:3.5g Dextrose: 5g Potassium phosphate: 5g pH 6.9 0.2 Mi trng 3: Peptone: 5g Glucose: 5g Phosphate buffer: 5g pH 7.5 0.2 RSU (Rustigian-Stuart Urea Broth) Dipotassium Phosphate: 9.5g Yeast Extract: 0.1g Urea: 20g Monopotassium Phosphate: 9.1g Phenol Red: 0.01g 1000ml nc Mi t ch dng 30ml, phn ct. phi 3ml/ng nghim. pH 6.8 0.2 (bui 5) 1000ml nc Mi t ch dng 40ml, phn ct. phi 10ml/ng nghim. pH 6.9 0.2 Kim tra li mi trng cung cp no pha mi trng. (bui 5)

Bin son: L Thy Linh


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Trng i hc Cng nghip thc phm TP.HCM

Bc 2: kh trng dng c v mi trng pha. Bao gi dng c v phn phi mi trng vo dng c, gn nt v bao gi. Hp tit trng 1210C trong 15 pht. Bc 4: Tng sinh i vi cc loi mu thng thng tin hnh cn 25g mu trong ti PE v trng, b sung 225ml dung dch BPW v ng nht mu bng Stomacher trong 15 hoc 30 giy. 370C, 18-24 gi. i vi mt s loi thc phm (mu gia cm ti sng, sa kh, cc gia v hay cc loi thc phm cha nhiu gia v, casein, pho mt, b, sn phm cha cocoa, cc sn phm ca da) c cha cc cht c th gy c hoc c ch s tng trng ca Salmonella cn thc hin quy trnh tng sinh c bit (xem TLTK). Bc 5: Tng sinh chn lc Lc trn u dch tng sinh, v chuyn 0.1ml sang 10ml mi trng tng sinh RV c m 420C. 420C, 18-24 gi. Khi cn thit c th ko di thi gian thm 24 gi. Bc 6: Phn lp v nhn din Dng que cy vng thc hin k thut cy phn lp khun lc n vi ging t dch tng sinh chn lc ln a mi trng chn lc phn bit c trng cho Salmonella nh XLD v HE. Biu hin ca Salmonella trn tng mi trng nh sau: + Mi trng XLD: khun lc c mu hng trong sut, c hay khng c tm en. Mt s dng Salmonella c th c tm en bng rt ln c th chim gn ht khun lc.
Hnh 14: khun lc Salmonella trn HE Hnh 13: khun lc Salmonella trn XLD

+ Mi trng HE: khun lc Salmonella c mu thay i t xanh dng n mu xanh lc, c hay khng c tm en. Mt s dng Salmonella c th c tm en bng rt ln c th chim gn ht khun lc. Bc 7: Khng nh T mi mi trng chn lc phn bit chn v cy chuyn t nht 5 khun lc nghi ng sang mi trng khng chn lc TSA. 370C, 18-24 gi. Cc khun lc xut hin trn mi trng ny c s dng cho cc th nghim sinh ha nh sau: Bin son: L Thy Linh
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Trng i hc Cng nghip thc phm TP.HCM

+ Th nghim sinh H2S trn mi trng KIA (Kligler Iron Agar): Mi trng KIA c s dng th nghim kh nng s dng cc ngun carbon khc nhau (glucose, lactose) v kh nng sinh H2S. V ngun carbon, mi trng KIA cha 2 loi ng l 1% lactose v 0.1% glucose. Khi cy chng vi sinh vt ln mi trng ny c 3 trng hp xy ra i vi s tng trng ca VSV: ch s dng glucose, s dng glucose v lactose, khng s dng c hai ng ny. Salmonella ch ln men c ng glucose v th phn nghing ca mi trng c mu , phn su c mu vng. Thi gian t 18-24 gi. V sinh hi: a s cc dng Salmonella u c kh nng sinh H2S nn c xut hin cc vt mu en trong mi trng ny. C th thy hin tng sinh hi qua hin tng lm v thch mi trng hoc mi trng b y ln trn to mt khong khng bn di y ng nghim.
Hnh 15: th nghim KIA. ng 1: mi trng KIA khng c VSV. ng 2: xut hin mu vng chng t VSV ln men lactose tt (ln men glucose cng tt). ng 3: c sinh kh, chng t ln men c sinh kh. ng 4: mu trn phn thch nghing, mu vng di y chng t VSV ch ln men glucose. ng 5: mu en xut hin chng t c qu trnh kh lu hunh v sinh H2S.

+ Th nghim urea (-): c thc hin trn mi trng urea lng RSU (Rustigian Stuarts Urea Broth), cha ch th phenol, chuyn mu t vng sang (vng chuyn mu l pH 6.8-8.4). Dng que cy vng cy lng sinh khi ln t khun lc ca chng thun vo ng cha 3ml mi trng v trng, lc nh ng trn u vi khun v 370C trong 48 gi. Salmonella khng phn gii urea nn khng lm thay i pH mi trng; sau khi nui cy mi trng vn gi nguyn mu vng cam.

Hnh 16: kt qu th nghim Urea

+ Th nghim VP (-): mi trng c s dng l mi trng lng Clark-Lubs (mi trng MR-VP), pH 6.9. Dng que cy vng cy vo cc ng mi trng MR-VP mt t sinh khi t khun lc ca chng thun 18-24 gi trn mi trng KIA. Sau thi gian , b sung thuc th trc tip vo ng mi trng. Thuc th Barritt gm dung dch A l 5% -naphthol trong cn
Hnh 17: kt qu th nghim VP Bin son: L Thy

Linh

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Trng i hc Cng nghip thc phm TP.HCM

tuyt i, dung dch B l 40% NaOH hay KOH. Trc tin nh 6 git dung dch A, sau nh 2 git dung dch B. c kt qu sau 20 pht hoc chm nht 4 gi. Salmonella cho phn ng m tnh vi th nghim VP c hin tng khng i mu trn b mt mi trng. Th nghim VP (+) khi c mu trn b mt mi trng.

Bi 4. nh lng Bacillus cereus 4.1 Tm tt kin thc c bn B. cereus l nhng trc khun, gram dng, hiu kh v k kh ty , di ng, to ni bo t, ln men glucose sinh hi, phn ng VP (+), c kh nng s dng nitrate. Loi ny tng trng c trong khong nhit t 50C 500C, ti u 350C 400C; pH dao ng t 4.5-9.3, d to bo t v bo t ny mm rt d dng. Trn mi trng chn lc loi ny to khun lc rt ln, mc lan, ra nhn. 4.2 Quy trnh nh lng Bacillus cereus bng phng php m khun lc Cn 25g mu, ng nht trong 225ml BPW, ng nht bng Stomacher, pha long 10-1 Pha long n cc pha long 10-2, 10-3 Tri 0.1ml mi pha long ln mi trng thch MYP, 300C, 24 gi Chn 5 khun lc c mu hng eosin, lecithinase dng tnh (ta lecithinase bao quanh khun lc) cy sang thch nghing MYP, 300C, 24 gi

Nhum Gram v th nghim sinh ha nh: ln men glucose, th nghim VP.

Kt lun B. cereus

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Trng i hc Cng nghip thc phm TP.HCM

4.3 Cch tin hnh v tnh kt qu Mu phn tch: tm kh, khi lng 50g cho 1 lp Bc 1: pha mi trng Tn mi trng Thnh phn Tng th tch Ghi ch Pepton: 10g BPW (Buffered Pepton NaCl : 5g Water) Na2HPO4: 3.5g KH2PO4: 1.5g MYP (Mannitol-Egg Yolk-Polymycin) Mi trng c bn Cao tht: 1g Pepton: 10g Mannitol: 10g NaCl: 10g Phenol red (1% trong ethanol 90%): 2.5ml Agar: 15g Nc ct: 900 ml Dung dch Polymixin 0.1% Ha tan 500 000 n v polymixin B sulfate trong 50ml nc ct. Lc v trng v ct trong ti 40C cho n khi dng. Egg yolk emulsion 50% (sn phm thng mi) Mi trng cui cng Mi trng c bn: 225ml ( 500C) Dung dch Polymixin B: Bin son: L Thy Linh
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1000 ml nc Phn phi cho mi t 50ml ct BPW trc khi kh trng. pH 7.2 0.2 (bui 4)

Mi trng Phn phi 100ml MYP cho c bn em mi t (12.5ml/petri). (bui kh trng, 4) ngui 500C. Sau trn cc thnh phn cn li

Trng i hc Cng nghip thc phm TP.HCM

2.5ml Egg yolk emulsion (lng trng): 12.5ml Proteose peptone No. 3: 1000 ml nc Phn phi 30ml PRG cho Red 10 g ct mi t (3ml/ng nghim) Glucose Broth) NaCl: 5 g pH 7.4 0.2 (bui 4) Beef extract (optional): PRG (Phenol 1g Dextrose: 5 g Phenol red (7.2 ml of 0.25% solution): 0.018 g 1000ml nc MR-VP Both Mi trng 1: (Glucose Buffered peptone-water ct. pH 6.9 0.2 Phosphate) powder: 7g Glucose: 5g K2HPO4: 5g Mi trng 2: Casein Pancreatic Digest: 3.5g Peptic digest of animal tissue:3.5g Dextrose: 5g Potassium phosphate: 5g pH 6.9 0.2 Mi trng 3: Peptone: 5g Glucose: 5g Phosphate buffer: 5g pH 7.5 0.2 Mi t ch dng 50ml, phn phi 10ml/ng nghim. Kim tra li mi trng cung cp no pha mi trng. (bui 4)

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Trng i hc Cng nghip thc phm TP.HCM

Bc 2: kh trng dng c v mi trng pha. Bao gi dng c v phn phi mi trng vo dng c, gn nt v bao gi. Hp tit trng 1210C trong 15 pht. Bc 3: Pha long mu Cho 25g mu vo ti PE, b sung 225ml mi trng BPW ng nht c pha long 10-1, ng nht bng Stomacher trong 1 pht. Mu tip tc pha long thnh 10-2, 10-3. Bc 4: Pht hin bng mi trng chn lc Tri 0.1ml mi pha long trn mi trng thch MYP, 30 C, 24 gi. Do B. cereus khng ln men mannitol, to lecithinase v khng polymycin nn trn mi trng ny khun lc B. cereus c mu hng eosin, c bao quanh bi vng c ta chng t lecithinase c to thnh. Chn 5 khun lc (+) cy sang thch Hnh 18: khun lc B. cereus trn MYP nghing chun b cho cc phn ng khng nh.
0

Bc 5: Cc th nghim khng nh
Hnh 19: nhum gram B. cereus

- Nhum Gram: nh 1 git nc ct ln phin knh sch; dng que cy vng ly 1 t sinh khi vi khun chuyn vo git nc trn phin knh, khuy nh. C nh vt bi trn phin knh bng cch h trn ngn la n cn. Nh vi git methyl violet ln vt bi, gi yn 20 giy. Ra sch phm nhum bng nc. Nh vi git dung dch KI/I2 ln vt bi, yn 1 pht. Ra bng cn 95% n va mt mu. Ra li bng nc. Nhum bng dung dch safranin 30 giy. Ra sch bng nc. Thm nc d bng giy lc. Quan st vi khun bng vt knh 100X nhng trong du, Gram dng c mu xanh ta, Gram m c mu hng. Th nghim ln men glucose: cy vi khun vo 3ml canh PRG, 350C, 24 gi trong iu kin k kh. Lc ng nghim tht mnh v quan st s pht trin thng qua c v s chuyn mu t sang vng, chng t s sinh acid t glucose trong iu kin k kh.

Bin son: L Thy Linh


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Trng i hc Cng nghip thc phm TP.HCM

Hnh 20: th nghim ln men glucose. ng a: i chng. ng b: khng ln men glucose. ng c: ln men yu. ng d: ln men mnh.

Th nghim VP (xem bi 3)

Bi 5. nh lng tng vi khun hiu kh v tng nm men-nm mc 5.1 Tm tt kin thc c bn Vi khun hiu kh l nhng vi khun tng trng v hnh thnh khun lc trong iu kin c oxi phn t. Tng s vi khun hiu kh hin din trong mu ch th mc v sinh ca thc phm. Ch s ny c xc nh bng phng php m khun lc mc trn mi trng thch dinh dng t mt lng mu xc nh trn c s xem 1 khun lc l sinh khi pht trin t 1 t bo hin din trong mu v c biu din di dng s n v hnh thnh khun lc (colony forming unit, CFU) trong mt n v khi lng thc phm. Nm mc l vi nm dng si, sinh sn bng bo t hoc khun ty. Nm men l nhng t bo n tnh pht trin theo kiu ny chi, thnh thong c th tn ti dng khun ty gi trong cc t bo kt nhau thnh chui. n v hnh thnh khun lc ca nm mc v nm men l mm to nn mt khun lc khi nui cy trong mi trng. Mm c th l mt bo t, mt t bo hay mt on ca khun ty. Trong thc phm, nm mc v nm men hin din c th tng trng lm thay i mu ca thc phm, lm pht sinh mi hay v l, lm h hng hay thay i c cu ca thc phm.

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Trng i hc Cng nghip thc phm TP.HCM

5.2 Quy trnh phn tch ng nht v pha long mu c pha long 10-1, 10-2, 10-3, 10-4

Xc nh tng s VK hiu kh bng k thut hp

Xc nh tng s nm men nm mc bng k thut hp tri

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

Tri 0.1ml mu ln a DRBC, nga a 250C, 5-7 ngy (mi nng cy 2 a)

Rt vo mi a petri 1015ml mi trng PCA c lm ngui n 450C, lc cho mu phn tn u vo mi trng, 300C trong 72 gi Chn cc a c s khun lc trong khong 25-250 khun lc/a m 5.3 Cch tin hnh th nghim

m khun lc nm mc, nm men

Tnh kt qu: tng s vi sinh vt hiu kh v tng s nm men nm mc trong mu (CFU/g hoc CFU/ml)

Mu phn tch: nc ma nguyn cht (dng 10ml cho mt t) 1.3.1 Pha mi trng v kh trng dng c Bc 1: pha mi trng Tn mi trng SPW (Saline Water) Thnh phn Tng th tch 500 ml nc ct Ghi ch Phn phi cho mi t 27ml SPW (9ml/ng nghim) trc khi kh trng. (bui 5) Bin son: L Thy Linh
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NaCl : 42.5g Pepton Pepton: 5g

Trng i hc Cng nghip thc phm TP.HCM

PCA (Plate Agar)

Casein enzymic Count hydrolysate: 5g Yeast extract: 2.5 g Dextrose: 1g Agar: 15g

1000 ml nc Phn phi 100ml PCA cho ct mi t (15ml/petri). (bui pH 7.0 0.2 5)

Glucose: 10g DRBC (Dichloran Rose Peptone: 5g Bengal KH2PO4: 1g Chloramphenicol MgSO4: 0.5g Agar)

1000 ml nc Phn phi 100ml DRBC ct cho mi t (15ml/petri). pH 5.6 0.2. (bui 5) Lu : trn ln

Rose bengal (dung dch cc thnh phn 5%, w/v): 0.5ml tr Dichloran (0.2%(w/v) chloramphenicol, trong ethanol): 1ml un nng ha Chloramphenicol: 0.1g tan ht agar, hp Agar: 15g kh trng. ngui n 500C, b sung 1ml dung dch khng sinh 100X vo 100ml mi trng.

Bc 2: kh trng dng c v mi trng pha. Bao gi dng c v phn phi mi trng vo dng c, gn nt v bao gi. Hp tit trng 1210C trong 15 pht. Bc 3: pha long mu (xem bi 1, mc 1.3.2) Bc 4: thc hin nh qui trnh

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Trng i hc Cng nghip thc phm TP.HCM

Cch tnh kt qu m tt c cc khun lc xut hin trn cc a sau khi . Chn s a c s m trong khong 25-250 tnh kt qu. Mt tng vi khun hiu kh hay tng s nm men nm mc c tnh nh sau: A (CFU/g hay CFU/ml) = N n1 x V x f1 + + ni x V x i

Trong :

A: s t bo (n v hnh thnh khun lc) vi khun (hay nm mc nm men) trong 1g hay 1ml mu. N: tng s khun lc m c trn cc a chn ni: s lng a cy ti pha long th i V: th tch dch mu (ml) cy vo trong mi a fi: pha long tng ng. Nu pha long cao nht, s khun lc m c trn mt a >250, v d nng 10-5 s m ln hn 250, kt qu c ghi: >2.5 x 107 CFU/g. Nu pha long thp nht, s khun lc m c trn mt a <25, v d nng 10-1 s m nh hn 25, kt qu c ghi: <2.5 x 102 CFU/g.

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