Thc tp vi sinh i cng

Nhm 3_Lp 071160C

BI 1:QUY TRNH KIM TRA TNG S VI SINH VT HIU KH 1.nh ngha: Vi sinh vt hiu kh l vi sinh vt tng trng v hnh thnh trong iu kin c oxy phn t 2. ngha ca vic kim tra tng s vi sinh vt hiu kh: Tng s vi sinh vt hiu kh hin din trong mu ch th mc v sinh ca thc phm,nh gi cht lng ca mu v vi sinh vt,nguy c h hng ,thi hn bo qun ca sn phm,mc v sinh trong qu trnh ch bin v bo qun thc phm S tng trng vi sinh vt trong thc phm dn n bin i cht lng : 106 t bo/g(ml) l ranh gii phn bit thc phm c du hiu h hng hay khng.Mt vi trng hp vsv=106 t bo/g(ml) cha c du hiu h hng r rng v mt ha hc.c bit sa khi c 105 t bo/g(ml) sa b chua: 106-107t bo/g(ml) sa c mi hi;108 t bo/g(ml) tt c thc phm c mi hi khng chp nhn c;109-1010t bo/g(ml) thc phm thay i cu trc 3.Quy trnh kim tra:

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Thc tp vi sinh i cng 1ml(10-1)+ 9ml nc nc v trung 1ml(10-1)+ 9ml nc nc v trung

Nhm 3_Lp 071160C

Stomacher .10gr thc phm + 90ml NaCl 0.85% (10-1)

10-2

10-3

Bc 1: ng nht mu Cho 10 g thc phm vo 90 ml NaCl 0.85% v trng(hoc H20 v trng )vo bao PE v trng.Tin hnh ng nht bng my stomacher 30 thu c nng 10-1 Bc 2 : pha long mu Chun b cc ng nghim v pipet v trng.Dng pipet 10ml v trng ly 9 ml NaCl v trng vo cc ng nghim .Dng micropipet 1ml v trng ly 1ml t nng 10-1 a vo ng nghim th nht . Tin hnh rung lc ng nghim bng my rung ng nghim votex, thu c nng 10-2 .Lm tng t vi cc nng k tip Lu khi pha long mu:

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Nhm 3_Lp 071160C

-Nng pha long ph thuc vo tnh trng v sinh ca thc phm,thi gian bo qun, iu kin bo qun,kinh nghim ngi kim nghim. -Tin hnh thao tc bn ngn la n cn.Cc ng nghim,pipet phi v trng.S dng 1 micropipet 1ml/ 1 nng pha long mu trnh hin tng sai s -Mi ln ly mu phi lc u Bc 3: nui cy dch mu Nui cy t nht 3 nng pha long mu lin tip Dng micropipet 1ml v trng ly 1ml dch mu cho vo a,t nht 2 a/nng Chun b mi trng tit trng, ngui 450C, mi trng vo cc a cha 1ml dch mu .Xoay nh a theo vng trn nhm trn u dch mu vi mi trng thu c nhng khun lc tch ri.Khi mi trng ng li,lt ngc a, trong trong t n nhit Memmert Nu mun kim tra tng s vi khun hiu kh,dng mi trng P.C.A hoc N.A Nu mun kim tra tng s men, mc dng mi trng P.D.A hoc Sabouraund Thi gian vi khun hiu kh 24-48h/30-370C.Thi gian men,mc 72h/30-370C Bc 4: m s khun lc /a/cc nng (25-250 khun lc/a) Kt qu th nghim Nng S khun l c
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10-1 a 1 a 2 48 130

10-2 a 1 a 2 113 125

10-3 a 1 a 2 105 120

Thc tp vi sinh i cng

Nhm 3_Lp 071160C

Bc 5: tnh kt qu theo cng thc n = c/(n1+0.1n2)*d n : tng s t bo / ml (g) mu thc phm(cfu/g(ml)) c : tng s khun lc m c n1 : s a m c nng th nht n2 : s a m c nng th hai d : nng pha long th nht c m =>n=
(113 + 125 + 105 + 120).100 =21045 21000 cfu/g(ml) (2 + 0,1.2)

4.Mi trng s dng 4.1.Mi trng Nutrient Agar Peptone Meat extract Agar Nc ct :5.0g :3.0g :12.0g :1000ml

pH sau thanh trng : 7.0 0.2 Hp thanh trng 1210C/15 pht 4.2.Mi trng Plate Count agar Peptone Meat extract D(+) glucose Agar :5.0g :2.5g :1.0g :14.0g

Nc ct va :1000ml pH sau thanh trng : 7.0 0.2 Hp thanh trng 1210C/15 pht 4.3.Mi trng Sabouraund
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Thc tp vi sinh i cng

Nhm 3_Lp 071160C

Peptone Glucose Agar Nc

:20g :40g :20g :1000ml

Hp thanh trng 1210C/15 pht BAI 2: KIM TRA TNG S COLIFORM 1.Nhng kin thc chung v COLIFORM Coliform la nhom trc khun gram- , khng bao t , hiu khi hoc ki khi tuy y, co kha nng ln men ng lactose, sinh hi 37oC /24 48h. Coliform va Feacal coliform (coliform phn) la nhom vi sinh vt dung chi thi kha nng co s hin din cua cac vi sinh vt gy bnh trong thc phm. Nhom coliform gm nhng vi sinh vt hiu khi va ki khi tuy y, gram - , khng bao t , hinh que, ln men ng lactose va sinh hi trong mi trng long, da vao nhit d tng trng, nhom nay c chia thanh 2 nhom nho la coliform va coliform phn co ngun gc t phn cac loai ng vt. Trn thc t kim nghim coliform phn c quan tm nhiu hn coliform. Coliform phn co ngun gc t rut ngi va cac ng vt mau nong bao gm cac ging escherichia, kebsiella, enterobater. Khi coliform phn hin din mt s lng ln trong mu thi mu co kha nng cha cac vi sinh vt gy bnh hin din trong phn.Ch tiu tng coliform khng thch hp lm ch tiu ch th cho vic nhim bn ngun nc bi phn. Tuy nhin vic xc nh s lng Feacal coliform c th sai lch do c mt s vi sinh vt (khng c ngun gc t phn) c th pht trin nhit 44oC. Do s lng E. coli c coi l mt ch tiu thch hp nht cho vic qun l ngun nc. Trong cac thanh vin nhom coliform phn thi E.coli la loai c quan tm nhiu v v sinh an toan thc phm.
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Thc tp vi sinh i cng

Nhm 3_Lp 071160C

Loai vi sinh vt nay phn b moi ni, co trong rut ngi va cac ng vt mau nong. *mt s hinh anh v coliform:

Fecal Coliform 375 x 294

Chromocult Coliform Agar ES 297 x 300

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Nhm 3_Lp 071160C

2. ngha ca vic kim tra ch tiu Coliform c xem la nhom vi sinh vt chi thi. S lng hin din cua chung trong thc phm, nc c dung chi thi cho kha nng hin din cua cac vi sinh vt gy bnh khac. S lng coliforms cao thi kha nng hin din cua vi sinh vt gy bnh khac cao. Colifrorm chiu nhit( coliform phn): La thanh phn trong h vi sinh vt ng rut ngi va cac c xem la vi sinh vt chi thi mc v sinh trong qua trinh ng vt mau nong. ch bin, bao quan , vn chuyn thc phm, nc ung cung nh chi thi s nhim phn trong mi trng.

Ln men ng lactose trong mi trng E.C 44.5oC. Co kha nng sinh indol 24h/44.5oC. Kt qua sinh hoa nghim phap imvic la + + -

3.Quy trnh kim tra Phng phap MPN : Nguyn tc: Mu c pha loang thanh mt day thp phn , 2 nng k tip nhau khac nhau 10 ln. Mu c u trong mi trng thich hp co ng durham. Mi nng pha loang c lp lai 3 ng. Theo doi s sinh hi trong tng ng nghim. Xac inh ng dng tinh mi nng pha loang va da vao bang MPN suy ra s lng nhom vi sinh vt tng ng hin din trong 1g hoc 1ml mu ban u

S quy trnh kim tra :

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Thc tp vi sinh i cng 1ml(10-1)+ 9ml nc nc v trung

Nhm 3_Lp 071160C 1ml(10-2)+ 9ml nc v trung

Stomacher 10gr thc phm 10-2 10-3

+ 90ml NaCl 0.85%( 10-1 )

hinh 1.1 Bc 1:

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Nhm 3_Lp 071160C

10gr thc phm + 90ml NaCl 0.85% hoc la nc v trung, stomacher thu c nng 10-1. muc ich ng nht la phn b u vi sinh vt. Bc 2: Pha loang mu giam s lng vi sinh vt co trong mu ban du. Ly 1ml mu nng 10-1 cho vao ng nghim th nht, sau o cho 9ml nc v trung. Ta c ng nghim co nng 10 -2. Sau o ly 1ml mu nng 10-2 cho vao ng nghim th hai + 9ml nc v trung thu c ng nghim co nng 10-3 . tng t nh trn ta thu c cac ng nghim co nng 10-4, 10-5.. Bc 3: Nui cy dich mu trong mi trng Laury Tryptose (LT) 3 nng lin tip(10-1,10-2,10-3) nh sau : Mi nng ly 3 ng nghim. Cho vao mi ng nghim 1 ng durham, cho mi trng LT vao 9 ng nghim sao cho ngp ng durham. Ghi 3 nng lin tip bn ngoai ng nghim(mi nng 3 ng nghim). Tip n cho 1ml dung dich mu nng 10 -1 vao 3 ng nghim co ghi nng 10-1(cha mi trng LT),tng t cho cac ng nghim nhng nng tip theo. (hinh 1.1) u 37oC/24h Nhng lu khi tin hnh nui cy vi khun ln mi trng Laury Trytose (LT): -Tin hnh gn ngn la n cn trnh nhim khun. - Nh lc mu trc khi tin hnh nui cy vi khun trn mi trng Laury Trytose. - Khng lc nhng ng c ng durham Bc 4: oc kt qua cac ng Laury Tryptose . Co 3 trng hp xay ra:
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Nhm 3_Lp 071160C

+ ng durham khng thay i + ng durham ni ln trn + ng durham sinh hi. oc kt qua cac ng LT dng tinh, sau o cy chuyn cac ng LT dng tinh nay vao cac ng mi trng BGBL 2% bng cach ly que cy vong nhung vao mi trng LT dng tinh trn,ri cho vao ng co mi trng BGBL. Ghi nng trn san phm. U 37oC /24h. ng LT + : mi trng uc va ng durham ( ng chung) ni hoc co bot khi trong ng chung (th tich bot khi trong ng chung =1/10 th tich ng chung). ng LT - : khng co hin tng gi xay ra Bc 5: oc kt qua ng BGBL dng tinh. Lp ty l cac ng BGBL dng tinh 3 nng lin tip. Tra bang Mac Crady tim s MPN tng ng + ng BGBL dng tinh: mi trng uc va ng durham ( ng chung) ni hoc co bot khi trong ng chung( th tich bot khi =1/10 th tich ng chung). + ng BGBL m tinh: khng co hin tng gi xay ra. Bc 6: Tinh kt qua: Tng s coliform (cfu/g hoc cfu/ml)= s MPN 10n n la s nguyn dng cua nng pha loang u tin c nui cy. Bc 7: T kt qua tinh c, so sanh vi tiu chun v an toan v sinh thc phm. 4. Mi trng s dng:
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Thc tp vi sinh i cng

Nhm 3_Lp 071160C

Mi trng BGBL 2% : la mi trng dung phat hin va m coliforms, coliforms phn, E.coli trong sa, thc phm, nc. Nguyn tc: Mt bo ( bile) va brilliant green c ch hu ht cac vi khun gram + va vi khun gram

khng phai coliform. Brilliant green co nng c hiu

nhm ngn vi khun ki khi ln men lactose sinh trng 440C , Tranh c hin tng dng gia, luc nay coliform phat trin lam uc mi trng va sinh khi trong ng durham do ln men lactose. 5.Cc thit b ,dng c s cho th nghim Binh erlen, en cn, que cy vong, ng nghim ,ni autoclave, tu sy, ng durham , micropipet, mu la tng t. 6.Kt qu th nghim

_ 10-1

+
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Thc tp vi sinh i cng

Nhm 3_Lp 071160C

10-2

_ 10-3

Ty l cua BGBL dng tinh 3 nng (10 -1:10-2:10-3) = (0,2,0) Tra bang Mac Crady ta c s MPN =6.2N/g

coliform = 6.2 101=62 (cfu/ml)

BI 3: KIM TRA TNG S E.COLI Escherichia coli l dng coliform c ngun gc t phn, pht trin c 44C, sinh indol (phn ng ind+), sinh acid (phn ng MR+), khng sinh aceton (phn ng V.P-) v khng s dng citrate lm ngun cacbon (phn ng cit-).L trc khun gram-, c kh nng gy bnh tiu chy v sinh ni c t.c coi l vi sinh vt ch th cho s nhim phn v cht lng v sinh thc phm. Cc chng E.coli c kh nng gy bnh ngi : - Cc chng truyn thng gy tiu chy tr s sinh v tr em.
-

Cc chng ym kh khng bt buc gy tiu chy khng thng xuyn c quan h gn gi vi h vi sinh vt bnh thng ng rut.

- Cc chng sinh c t bn hoc khng bn vi nhit hoc c hai.

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Nhm 3_Lp 071160C

- Cc chng gy ly nhim ng rut. 1.Mc ch th nghim: Xc nh E.coli trong mu thc phm. y, nhm tin hnh kim tra tng s E.coli trn mu tng t. 2. Nguyn tc: mu c pha long thnh mt dy thp phn, hai nng k tip nhau khc nhau 10 ln. Mu c trong mi trng thch hp c ng durham. Mi nng pha long c lp li 3-5 ng. Theo di s sinh hi trong tng ng nghim. Xc nh cc ng dng tnh mi nng pha long v da vo bng MPN suy ra s lng nhm vi sinh vt tng ng hin din trong 1g hoc 1ml mu ban u. 3.Dng c v ha cht: 3.1.Thit b v vt liu: - T m - B iu nhit - My lc - Cn - Pipet tit trng - Cc bnh cha mu v khun - Cc l phc v cho mc ch pha long, c nt y - Mu thc phm kim tra - ng durham

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Nhm 3_Lp 071160C

3.2.Mi trng v ha cht: - Mi trng E.C broth - Mi trng Lauryl tryptose( LT) - Mi trng endo agar
-

Mi trng E.M.B agar (eosin methylene blue lactose sucrose agar).

- Mi trng N.A (nutrient agar) 4.Tin hnh th nghim: Bc 1: Ly mu Ty loi vt phm cn xc nh m ta ly mu vi s lng v khi lng khc nhau cho ph hp. Khi ly mu cn m bo : - Mu phi c tnh i din. - Lng mu va phi, phn tch cc c tnh l ha,sinh hc. - Dng c ly mu, cha mu phi v khun. - Mu ly xong phi phn tch ngay khng qu 24 gi. - Mu phi c nhn ghi k hiu, ghi li nhng c im ca mu v ni thu mu. Ly 10g tng t + 90ml NaCl 0.85%, stomacher thu c nng 10-1

Bc 2: Pha long mu S tin hnh pha long mu lng:

10ml 1ml 1ml Trang 14 1ml

Thc tp vi sinh i cng

Nhm 3_Lp 071160C

Mu

90ml NaCl v trng 10-1 (nc v trng)

10-2 9ml NaCl v trng

10-3 9ml NaCl v trng

10-4 9ml NaCl v trng

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Thc tp vi sinh i cng

Nhm 3_Lp 071160C

S tin hnh pha long mu rn:

10ml
1ml

1ml

1ml

10g mu Stomacher

90ml NaCl v trng 10-1 (nc v trng)

10-2 9ml NaCl v trng

10-3 9ml NaCl v trng

10-4 9ml NaCl v trng

Tin hnh tng t cho cc mu pha long khc Nhng iu lu khi pha long mu: - Khi pha long mu nn dng nc mui v trng nhm to mi trng dinh dng cho vi sinh vt, nu ch dng nc ct v
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Thc tp vi sinh i cng

Nhm 3_Lp 071160C

trng vi sinh vy s cht khi lu bn ngoi m khng c mi trng dinh dng. - Tin hnh gn ngn la n cn trnh nhim khun. Cc dng c phi c v trng. - Khuy trn u dung dch mu trc khi pha long vi sinh vt phn tn u trong mu. - T bo cc pha long khc nhau cn c ly vi cc pipet khc nhau nhm trnh cc t bo pha long trc ln vi cc t bo pha long sau. - Nng pha long cn ph thuc: c im ca thc phm(thi hn s dng, iu kin bo qun, quy trnh ch bin...). Ngi kim phm ( kinh nghim, thao tc....). Bc 3 : Nui cy vi khun ln mi trng Laury Trytose ( LT) Tin hnh nui cy dch mu trong mi trng Laury Trytose 3 nng lin tip, 3 ng/ nng , 1ml dch pha long/ ng LT. 37C/24h.

10-2

10-3

10-4

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Thc tp vi sinh i cng

Nhm 3_Lp 071160C

Mi trng Laury Trytose( LT)

Ri yn cho thch ng li, em a trong t m. 37oC trong 24h Sau khong thi gian trn ly a ra v m khun lc, tnh kt qu. Nhng lu khi tin hnh nui cy vi khun ln mi trng Laury Trytose (LT): - Tin hnh gn ngn la n cn trnh nhim khun. - Nh lc mu trc khi tin hnh nui cy vi khun trn mi trng Laury Trytose. - Khng lc nhng ng c ng durham Bc 4 : c kt qu cc ng Laury Tryptose dng tnh( +), cy chuyn cc ng LT(+) vo cc ng mi trng E.C. 44C/24h. E.coli pht trin 44C, nhit ny mt s vi khun khc b hn ch kh nng sinh trng .Hn na mi trng E.C mui mt c ch c vi khun Gr + v cc vi khun khng thuc nhm vi khun ng rut

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Thc tp vi sinh i cng

Nhm 3_Lp 071160C

ng LT + : mi trng uc va ng durham ( ng chung) ni hoc co bot khi trong ng chung (th tich bot khi trong ng chung =1/10 th tich ng chung). ng LT - : khng co hin tng gi xay ra T cc ng E.C (+), cy phn lp trn mi trng endo agar v E.M.B agar. 37C/24h Nhng lu khi tin hnh nui cy vi khun ln mi trng endo agar v E.M.B agar: Tin hnh gn ngn la n cn trnh nhim khun.

Nh lc mu trc khi tin hnh phn lp vi khun trn mi trng endo agar v E.M.B agar

Sau khong thi gian trn ly ra em nhn din khun lc in hnh: - Mi trng endo agar: khun lc trn, bng, c nh kim loi - Mi trng E.M.B agar: khun lc trn, bng, c tm en, c nh kim. Bc 5 : T cc khun lc nghi ng, cy truyn sang mi trng N.A. 37C/24h. Buc 6 : lm nghim php imvic vi vi khun cy bc 5 I : indol M :methyl red V : V.P C : simon citrat Phn ng sinh ha kim tra s c mt ca trc khun ng rut Gr c kh nng s dng simon citrat
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Nhm 3_Lp 071160C

Kt qu : + + - - : E.coli type I - + - - : E.coli type II BI:4 KIM TRA VI KHUN STAPHYLOCOCCUS AUREUS 1. c im ca Stapylococcus aureus: 1.1. lch s: Staphylococcus c Ogston pht hin nm 1881 trong vt thng c m.Nm 1884, Rosenbach tip tc nghin cu. 1.2. Phn b:

c phn b rng ri, c nhiu trong sn phm ng vt nh tht, sa ngi thng c trn da, tc, khoang mi. B ly nhim t ngi ch bin, ng vt b nhim bnh. c xp vo nhm vi khun c hi(opportunist type) v s c mt rng ri v thng xuyn trong m v ch i iu kin thun li xm nhp.

1.3. Hnh dng t bo: L vk gram+, hnh cu khng bo t, hiu kh hoc k kh tu . Trong vt thng v mu thng thy hnh dng ging chm nho 1.4. c im v iu kin sinh trng: Pht trin tt cc mi trng tng hp, c bit pht trin tt mi trng thch mu hoc huyt thanh. Nhit ti thch l 37oC , pHop=7.2. Trn mi trng thch ,khun lc c dng trn trn bng,c vun m. mi trng lng, t bo dng cn, vng nhn m trong ng nghim b mt mi trng .
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Nhm 3_Lp 071160C

1.5. c im sinh ho:

Ln men ng glucose, lactose, maltose, saccharose, glycerol, manitol Khng ln men salicin, raffinose,inulin. C kh nng chu mn cao Lm ng t sa Sinh beta hemolysis trong mi trng thch mu. Phn ng indol-,NH3-, thu phn gelantine, ng huyt tng.

1.6. Kh nng gy bnh: Gy ng c thc phm: Bnh gy ra do vi khun tit c t vo thc phm, ngi n thc phm v b ng c. Khng cn c s hin din ca vi khun cn sng trong thc phm m ch cn c c t ca chng. Loi ny thng c tnh cht cc b, t c kh nng truyn nhim Khi xt nghim phn ngi bnh b ng c thc phm khng thy c vsv gy bnh. Sinh ngoi c t(vi khun tit c t vo thc phm, ngi n thc phm b ng c). Bn cht ca ngoi c t l protein nn chng km chu nhit (tr c t ca Staphylococcus aureus) v kch thch c th sinh ra khng th c hiu(tnh khng nguyn mnh) do vy c th pht hin c bng phn ng khng nguyn khng th. c t gy vim d dy, vim rut. Type A v D gy ng c thc phm cho ngi. Triu chng bnh: Khi n phi thc phm c cha cc c t ny, sau 30- 6h (ph thuc vo c th ngi bnh) t lc n ngi b ng c c triu chng
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Nhm 3_Lp 071160C

au tht bng, tiu chy, nn ma ko di t 6 8h, kit sc mc nghim trng, au u tot m hi, bn rn tay chn.S phc hi xy ra sau 24 72 h, nn nhn khng cht nhng rt au n do cc phn ng cc k d di. t thy vi khun trong phn ngi b ng c. L c t bn nhit, bin php nu nng khng lm bt hot c t ny. 100oC/30; 137oC/9c t ny vn cn hot lc. Cc loi thc phm c cha nhiu mui nh Jambon, kem tng hp, nc sp (t khi c s l nhit >40oC) v cc loi thu sn, thc phm ng hp thng hay nhim loi vsv ny. Con ng ly nhim ch yu thng qua tip xc t nh bp, qu trnh ch bin. Bin php phng nga:

Kim tra sc kho cng nhn. Khng ly sa t ng vt b vim v. Tr lnh thc phm < 8oC, ngn nga kh nng sinh c t. H pH ca thc phm c ch vi khun pht trin. toC = 60oC/ 0.5h ph hu cc t bo, mt s b phn hu to= 80oC /0.5h. Ho cht: fenol 1%, fenol 2%/15, hgcl 0.5%/1h, formaldehyt 10%/ 10, gentiant violet1:25.000/5 10, x l nhim vt thng trn da ngi v ng vt do Staphylococcus gy ra dng dung dch gentiant violet 2%. Mt s hnh nh v Staphylococcus aureus

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Nhm 3_Lp 071160C

2. ngha ca vic kim tra vi khun: S hin din vi mt cao ca Sta. Aureus trong thc phm ch th iu kin v sinh v kim sot nhit km ca qu trnh ch bin. 3. Quy trnh kim tra: 3.1. Quy trnh kim tra nh tnh Bc 1: ng nht mu: ly 10ml thc phm (nc Sm ) cho vo 90 ml nc v trng .stomacher. Bc 2: tng sinh: ht 1ml dch ng nht a vo mi trng MSB c mu . 37oC/ 24h. Sau c kt qu: -ng tng sinh m:c mi trng MSB vn gi mu -ng tng sinh dng: C mi trng MSB chuyn t sang vng. Bc 3: t ng tng sinh dng, cy phn lp ln mi trng baird parker agar (hoc MSA). 37oC/24h. Bc 4: nhn din khun lc in hnh.

Trong mi trng Baird Parker: Sta. Aureus c c im trn, li, c tm en, bng vun c vng sng quanh khun lc. Trong mi trng MSA : mi trng chuyn t sang vng, vi khun trn, bng.

Bc 5: T khun lc in hnh lm phn ng pastorex, staphylatex, c kt qu.

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Bc 6: Kt lun. 3.2 Quy trnh kim tra nh lng. Bc 1: ng nht mu: ly 10ml thc phm (nc Sm ) cho vo 90 ml nc v trng .stomacher. Bc 2:pha long mu ti cc nng thch hp. Bc 3:nui cy 0.1 ml dch mu /a. 3 a/ nng , tin hnh thao tc bng que cy trang. Nui cy dch mu trn mi trng baird parker. 37oC/ 48h. Bc 4: nhn din khun lc in hnh. nh du 5 khun lc in hnh v 5 khun lc khng in hnh ri cy chng t mi trng BPA sang mi trng TSA. 37oC/ 24h. Bc 5: cy chuyn VK sang ng nghim c cha 0.3 ml huyt tng th. 37oC. Theo di kt qu phn ng ng huyt tng sau 1,3,6,24h. -Phn ng dng tnh :c khi ng hnh thnh. -Phn ng m tnh: khng c khi ng hnh thnh, dch mu ging nh ban u. Bc 6: tnh kt qu S lng Staphylococci coagulase dng tnh: cs=(f1*nt*ht)+(f2*na*ha) trong cs:s lng Staphylococci coagulase dng tnh. f: nng pha long ca mu nt:tng s khun lc in hnh trn cc a na:tng s khun lc khng in hnh trn cc a ht=s khun lc in hnh th nghim cho kt qu +/ s khun lc in hnh c th nghim
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Nhm 3_Lp 071160C

ha=s khun lc khng in hnh th nghim cho kt qu +/ khun lc in hnh c th nghim Ch : nu s khun lc nm ngoi khong 20 200, m s khun lc in hnh v khng in hnh, sau tnh kt qu nh cng thc trn v ghi r trong biu mu. 3.3 phn ng sinh ho Saulatex Dng nh danh c ging v loi Sta.aureus. Quy trnh: Bc 1:ly dd mui vo tiu bn Bc 2: ly Sta.aureus vo git nc mui trn tiu bn , trn u nhuyn. Bc 3: ly 1 git saulatex vo bn cnh git nc mui . Bc 4: ko 2 git nc li trn u vi nhau . Bc 5: c kt qu: - phn ng dng tnh:c mu trng sa,ln cn nhng ht nh -phn ng m tnh:mu trng sa nh ban u 4. M Rng : 4.1 mi trng s dng: Mi trng Chapman agar(manitol salt agar): L mi trng chn lc dng pht hin, phn lp v nh lng t cu khun gy bnh tronh thc phm nh sa, tht , hi sncng nh cc sinh phm khc. Cc th nghim ca koch cho thy t cu khun c th chu c nng mui cao 7.5%. Sau Chapman xc minh hin tng trn, ng thi ghi nhn t cu khun no gy ng t huyt tng th s hnh thnh khun lc vng trn mi trng chapman agar, mi trng c kh
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Nhm 3_Lp 071160C

nng c ch hu ht vi khun khc. Nng mui cao c ch sinh trng hu ht cc loi vi khun tr Staphylococcus. Vi khun ln men ng manitol sinh axit lm i mu ch th (fenol red)ca mi trng t sang vng. Xc nh tt c cc loi khun lc c mu vng hoc mu trng vi mi trng xung quanh c mu vng. Tuy nhin, c th ln cc khun lc trong ging Bacillus nn cn xc nh cc khun lc ny bng cch quan st di knh hin vi. T cu khun gy bnh s to khun lc c sc t vng, ng thi mi trng xung quanh i t sang vng. t cu khun gy bnh thng to khun lc nh c mu v khng lm thay i mu mi trng. Sau 48h nui cy, vi chun cu khun ng rut Baciilus, Micrococcus v Serratia cng c th mc trn mi trng ny. Mi trng Baird parker agar L mi trng dng phn l p v nh lng vi khun .Staphylococcus trong thc phm, dc phm. Cng thc mi trng ny do baird parker khai trin nm 1962 nhm nh lng Staphylocoagulase. Nm 1964, Smith v Baird Parker chng minh rng khi b sung sulfamerthzine vo mi trng s c ch sinh trng ca vi khun proteus. Nm 1971, Tardio v Baer quan st trong 18 mi trng phn lp chn lc th nghim th mi trng B.P t gy c ch hn cc mi trng khc. Thnh phn mi trng c lithium chloride v tellurite c tc dng c ch cc qun th vi khun thng hin din cng Staphylococci trong khi pyruvate v glycine kch thch s pht trin ca Staphylococci .
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Nhm 3_Lp 071160C

Khun lc Staphylococci c hai c im:Khun lc c mu en do kh telurite thnh telurium, dng li v to vng trong xung quanh khun lc,ng knh t 2 5 mm do s thy phn protein. Sau c th xut hin 1 vng c trong vng trong (c tc ng ca lecithinase, mt loi lipase). y l mt c tnh thng thy v c tnh chuyn bit cc t cu khun gy bnh. c tnh ca Staphylococcus aureus s c kim chng qua phn ng coagulase v tt nht bng cc th nghim desoxyribonuclease v phosphatase. Cc loi vsv ngoi l khc c th mc trn mi trng ny nh: Staphylococcus epidermis, Bacillus, Micrococci, nm men. rt nh, mu nu ti en. mu nu sm c, mc sau 48h. trng. Pastorex: L mt th nghim dng nh danh S.aureus c thc hin trc tip vi vi khun trn mi trng nui cy u tin. Bng phn ng ngng kt latex trn knh (latex c mn cm vi huyt tng ngi), th nghim cho php pht hin ng thi 2 yu t: yu t cm clumping factor (mt cht c b mt t bo vi khun v c i lc cao vi fibrinogen) v protein A Kt qu nghin cu vi pastorex vi 228 chng Staphylococci cho thy: nhy cm 97.5%, c hiu 92.5%. BI 5: KIM TRA NH TNH VIBRIO CHOLERA,VIBRIO PARAHAEMOLYTICUS
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Micrococci Bacillus sp nm men

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Nhm 3_Lp 071160C

1.Gii thiu chung 1.1.Lch s Vibrio cholera : nm 1883,Robert Kock vi vai tr l trng nhm nghin cu bnh dch t pht hin ra vi khun ny trong phn bnh nhn v i dich t ln th 5 vng Alexandria,Ai cp V.parahaemolyticus c Fujino pht hin ln u tin vo ma h nm 1951 ti vng ven bin Nht Bn sau cc v ng c do n c ,ho Ngi ta xc nh uc 21 loi thuc ging Vibrio,trong c 4 loi thuc tc nhn gy bnh cho ngi gm : V.cholera,V.parahaemolyticus ,V.vulnificus,V.alginolyticus 1.2.c im phn b V.cholera phn b rng khp vi s lng ln lan ti c chn M,gy bnh dch t cho mt s vng chu ,n v ng Nam .Chng tng gy i dch do ly truyn qua tip xc, nc ,sa,thc phm v cn trng V.parahaemolyticus l vi sinh vt bin,tn ti t nhin trong nc bin,thng gp cc loi hi sn loi nhuyn th v gip st trong nc bin Tr V.cholera tn ti trong nc ngt,cn tt c cc loi Vibrio khc u cn mui tng trng v thng xuyn c phn lp cc vng nc ven bin 1.3.c im hnh thi L vi khun Gram -,hnh que hai u khng u nhau to thnh hnh du phy,khng sinh bo t,di ng nh 1 lng roi 1.4.c im sinh trng v pht trin

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T0op=370C , pHop=8.6 ,sng k kh ty , c kh nng pht trin tt trn mi trng kim. V.cholera b tiu dit nhit l t0=560C trong 30, trong iu kin kh hn,nh sng v cc cht dit khun 1.5.Cu trc khng nguyn v c t V.cholera sinh c t rut v ni c t trong ng tiu ha,kch thch nghim trng mng nhy to ra dch d dy,gy tiu chy nng ,mt nc,chong,thm ch gy t vong vi t l ln ti 25-50%.Bin php iu tr tt nht l b sung nc v cht in gii thay th Phn ln cc t dch bnh do V.parahaemolyticus gy ra thng vo ma h,khi nc cc thy vc m ln.Cc triu chng nh tiu chy,nn ma,hi n lnh,au u xut hin sau 12 h sau khi n mt lmg ln vi sinh vt sng (105 t bo/g).C triu chng trn tng t nh triu chng do Salmonella gy ra nhng trm trng hn.Salmonella tc dng ln vng bng trong khi V.parahaemolyticus tc dng ln d dy ngi bnh 2.Quy trnh kim tra Bc 1: Tng sinh 25 g thc phm ( mm tm) + 225ml peptone kim, stomacher . 370C/24h Rt kh pht hin Vibrio trong mu,v vy ta s dng lng mu ln l 25g.V cng chnh v kh pht hin nn phi to iu thun li Vibrio sinh trng bng cch tng sinh trong mi trng pepton kim Bc 2 : Phn lp t phn vng ca dch tng sinh ln mi trng TCBS agar.370C/24h Bc 3 : nhn din khun lc in hnh Vi sinh vt
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c im

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Nhm 3_Lp 071160C

Vibrio cholera Vibrio parahaemolyticus Vibrio alginolyticus Vibrio fluvialis,V.vulnificus Pseudomonas,Aeromonas Cc loi vk ng rut khc

Vng,chuyn mu mi trng t xanh sang vng,dp,2-3mm Khng mu,tm xanh l cy m hn mu mi trng,3-4mm Khun lc vng ln Khun lc vng ln Khun lc xanh dng Khun lc nh ,trong sut

Bc 4: t khun lc nghi ng,cy chuyn sang mi trng N.A. 370 C / 24h Bc 5 : ly vi khun trn mi trng N.A lm khnhg huyt thanh Vibrio ,c kt qu phn ng ngng kt hoc lm BIS 14 GNE, ri c kt qu -Huyt tng th b ng li sau 30 pht khi cy vi khun vo.Trn vch t bo bi khun c cha protein A v enzyme - BIS 14 GNE gm 14 phn ng sinh ha nh danh vi khun Gr - ,c 10 ging (ging 8 c 2 phn ng l phn ng sinh H2S v phn g Indol) v 3 phn ng bn ngoi.Trong MOB kim tra kh nng di ng ca vi khun

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MOB+(mc lan nhe

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trn ng cy) 3.Mi trng s dng Mi trng TCBS agar l mi trng chn lc phn lp Vibrio cholera v cc loi Vibrio gy bnh ng rut khc ( c bit l Vibrio parahaemolyticus) trong cc mu bnh phm hoc trong cc mu thc phm ,hi sn Nng cao cc cht thiosulfate v sodium citrate cng vi tnh kim s c ch tng i s pht trin ca vi khun ng rut Mt b v mui mt lm chm sinh trng cu khun ng rut v c ch s pht trin ca vi khun Gram + S acid ha mi trng do Vibrio ln men saccharose lm thay i mu ca ch th bromothymol blue sang vng Hydrogen sulfide sinh ra do s hin din ca thiosulfate v ferric citrate,tt c cc loi Vibrio u khng sinh H2S Cc vi khun khc cng c th mc trn TCBS agar nh : E.coli ,Salmonella typhy, Klebsiella,Shingellanhng nhng khun lc ny khng c mu vng
MC LC

Trang BI 1:QUY TRNH KIM TRA TNG S VI SINH VT HIU KH 1.nh ngha 2. ngha ca vic kim tra tng s vi sinh vt hiu kh 1 3.Quy trnh kim tra
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1 1

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4.Mi trng s dng BAI 2: KIM TRA TNG S COLIFORM 1.Nhng kin thc chung v COLIFORM 2. ngha ca vic kim tra ch tiu 3.Quy trnh kim tra 4. Mi trng s dng 5.Cc thit b ,dng c s cho th nghim 6.Kt qu th nghim BI 3: KIM TRA TNG S E.COLI 1.Mc ch th nghim 2. Nguyn tc 3.Dng c v ha cht 4.Tin hnh th nghim BI:4 KIM TRA VI KHUN STAPHYLOCOCCUS AUREUS 1 c im ca Stapylococcus aureus 18 2. ngha ca vic kim tra vi khun 21 3. Quy trnh kim tra 4. M Rng 24 BI 5: KIM TRA NH TNH VIBRIO CHOLERA,VIBRIO PARAHAEMOLYTICUS 1.Gii thiu chung 26 2.Quy trnh kim tra
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21

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3.Mi trng s dng

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