VIEN CNG NGHE SINH HOC & THUC PHAM Tiu Lun Mn: VI SINH THUC PHAM D tai: Phn Tich ColiIorm Va E.Coli trongThuc Phm Giao Vin Huong Dn:Th.SLuu Huvn Trang Nhom : 07 (CDTP8LT-CDTP10TB) 1. D Vn Toan08845384 2. Hoang Thi Nguyt 08835414 3. Nguyn Thi Ha08844884 4. Vuong Thi Thuy MUC LUC LOI MO DAU ....................................................................................................... 3 TONG QUAN VE COLIFORM VA E.COLI ........................................................ 4 I.COLIFORM: ................................................................................................ 4 1.Dinh Nghia ................................................................................................ 4 2.Dc Dim .................................................................................................. 4 II.Escherichia coli ............................................................................................ 4 1.Dinh Nghia ................................................................................................... 5 2.Dc Dim. .................................................................................................... 5 PHUONG PHAP PHN TICH COLIFORM VA E.COLI TRONG THUC PHAM............................................................................................................................... 6 1.QUY TRINH THUC HIEN: ......................................................................... 7 2.QUY TRINH DINH LUONG COLIFORMS VA E. COLIBANG PHUONG PHAP MPN. ...................................................................................... 8 3.TIEN HANH PHN TICH ..........................................................................10 3.1.Pha mi truong va dung cu ....................................................................10 3.2.Khu trung dung cu va mi truong d pha...............................................11 3.3.Tin hanh phn tich. ..............................................................................12 3.4.Cach Tinh Kt Qua. ...............................................................................17 KET LUAN ..........................................................................................................19 TAI LIEU THAM KHAO .....................................................................................20 LI MO DAU Xungquanhchungta,ngoaicacsinhvtlonmachungtacothnhinthy duoc, con covvanvi sinhvtnhobe,munnhin thy chung phai sudung kinh hin vi, nguoi ta goi chung la vi sinh vt. Mn khoa hoc nghin cuu v hoat dng sng cua cac vi sinh vt duoc goi la Vi sinh vt hoc.Visinhvthocphattrinrtnhanhvaddndnvichinhthanhcaclinh vuckhacnhau:vikhunhoc(Bacteriology),nmhoc(Mycology),taohoc (Algology) virus hoc (Virolory),... Vic phn chia cac linh vuc con co th dua vao phuonghuongungdung.Dodochungtathyhinnayconcoysinhvisinhvt hoc, vi sinh vt hoc thu y, vi sinh vt cng nghip, vi sinh vt hoc nngnghip. Vi sinh vt phn b rng ri trong tu nhin: trong dt, nuoc, khng khi, trn co th cac sinh vt khac, trn long thuc, thuc phm va cac loai hang hoa. Chng nhung th, su phn b cua chung con theo mt h sinh thai v cung phong phu, da dang, tu lanh dn nng, tu chua dn kim, tu hao khi dn ki khi,...Do su phn b rngrivadohoatdngmanhmnnvisinhvtcotacdungrtlontrongvic thamgia cacvong tunhoanvt cht trntrai dt cngnhu thamgiavao cac qua trinh san xut nng nghip. Trongthinnhin,visinhvtgiunhungmtxichtrongyutrongsuchu chuyn lin tuc va bt dit cua vt cht, nukhng co vi sinh vt hay vi mt ly do naodomahoatdngcuavisinhvtbingungtrduchitrongthoigianngn,co th no s lam ngung moi hoat dng sngtrn trai dt. Hin nay vi sinh vtla mt mn khoa hoc mi nhontrong cuc cach mang sinhhoc. Nhiuvn d quan trong cua sinhhochin dainhu,ngungc su sng, cochthngtin,cochditruyn,cochdiukhinhocvacactchucsinhvt hoc, vi sinh vt hoc dang co nhung buoc tin vi dai, dang tro thanh v khi sc ben trong tay con nguoi d nhm chinh phuc thin nhin phuc vu dc luc cho san xut va doi sng. TONG QUAN VE COLIFORM V E.COLI I. COLIFORM: 1. Dinh Nghia ColiIormsla nhungtruc khun Gram m khngsinh bao tu,hiu khi hoc ky khi tuy y, co kha nng ln men lactose sinh acid vasinh hoi o 370oC trong 2448 gio. ColiIorms duoc xem la nhom visinh vt chi thi: s luong hin din cua chung trong thuc phm, nuochaycacloai mu mitruongduocdungdchithikha nnghindin cua cac loai vi sinh vt khac. 2. Dc Dim - Hinh que, Gram (-), khng bao tu - Ln men lactose sinh hoi - Nhit d phat trin; (-) 2-50oC - pH: 4.4-9.0 - Ngun nhim: nuoc hoc thuc phm nhim phn - Loai tiu biu: E.Coli, Enterobacter aerogenes, Shigella II. Escherichia coli 1. Dinh Nghia - ThuchoEnterobacteriaceae, catalose (), oxidase (-), Gram(-) , truc khun ngn, khng tao bao tu. - Sng trong rut gia cua nguoi va dng vt, theo phn di ra ngoai - Cokhannglnmennhiuloaiduong,sinhhoi,khunitratthanh nitrit - Kha nng gy bnh da dang: gy nhim khun duong tiu; voi co th yu gy nhim khun mau; gy vim mang no o tre so sinh; gy tiu chay. 2. Dc Dim. - Hinh que, khng tao bao tu - Nhit d phat trin: 7-50oC, nhit d thich hop: 37oC - pH thich hop: 7.0-7.5 - Gy bnh duong rut, tiu chay, nhim khun mau, vim mang no.. PHUONG PHP PHAN TICH COLIFORM V E.COLI TRONG THUC PHAM PHUONG PHAP MOST PROBABLE NUMBER (MPN) THEO TCVN 6505-2:1999 Dylaphuongphapdinhluongduatrnktquadinhtinhcuamtloatthi nghimduoclplaiomtsdphalongkhacnhau.Thngthuong,vicdinh luong nay duoc thuc hin lp lai 3 ln o 3 d pha long bc 10 lin tip, tng cng 3 x 3 9 ng nghim. Quv trnh dnh luong: Cho cac ng nghim chua mi truong dang long thich hop voi vi sinh vt cn xacdinhvoi3mucnngd:b1/10,1/100,1/1000mimuc3ngnghim.Uo nhitdvoithoigianthichhop.Duavaoktquanhinthychungminhsutang truongcuavisinhvtcnkimdinh(thuonglanhunghintuongnhu:sinhhoi, dimau,duc.)ghinhnsluongcacngnghimduongtinhotungdpha long.Su dung cac s liunayva duavao bang Mac Crady suyramt dvi sinh vt duoc trinh bay duoi dang MPN/100ml hay s MPN/gam. DchinhxaccuatrisMPNphuthucvaosluongngnghimlplai trong mi d pha long. Su dungmi truong BGBL o 37oC co th dinhluongnhom ColiIorm, cung mi truong nay o 45oC cho phep dinh luong ColiIorm trong phn. . QUY TRINH THUC HIEN:

Ghi nhn s luong ng nghim duong tinh o tung d pha long. Tra bang Mac Crady U o nhit d va thoi gian thich hop Kt qua biu kin chung minh su tngtruong cua vi sinh vt cn kim Pha long mu phn tich o 3 nng d pha long bc 10 lin tip.

Mt d vi sinh vt MPN/100 ml hay MPN/1g mu Da scac bang MPNcung cp sliu o dang s MPNtrong100mldung dich duoc su dung d phn tich o cac liu luong la 10ml, 1ml, 0.1 ml. Trn thuc t phntich,nguoitathuongdung1mlchomudphalong10-1,10-2,10-3. Luong mu voi cac d phalong nay tuong duong voi 1/100 luongmu su dungtheo bang MPN/100ml tinh theoluongmula 10ml, 1ml, 0.1mlnutrn. Vy sliucuabangMPN/100mltinhtheoluongmula10ml,1ml,0.1mltuong duongvoiMPN/mlmuchuaphalongkhi1mlcuacacdungdichcodpha long 10-1, 10-2, 10-3duoc dung d phn tich. - Truonghopmtdvisinhvtquacao,cnphaisudungloatnngdpha long tip theo cn phai nhn tri s tra bang MPN nu trn voi h s pha long. Vi du:sudung1mlchomidphalong10-2,10-3,10-4tuclachisudung1/10 luong mu o mi nng d so voi truong hop dung 1ml mu o cac d pha long 10-1, 10-2, 10-3. Do vy, tri s tra bang MPN cn nhn thm h s pha long la 10. . QUY TRINH DINH LUONG COLIFORMS V E. COLIBANG PHUONG PHP MPN.

ColiIormsE.Coli . TIEN HNH PHAN TICH Mu phn tich: Nuoc mia nguyn cht .. Pha mi trung v dung cu Tn mi truongThanh phnTng th tich SPW(SalinePeptonWater) NaCl : 42.5gPepton: 5g 500ml nuocct LSB(LaurylSulphateBroth) Tryptose: 20gLactose: 5gSodiumchloride: 5gKH2PO4: 2.75gK2HPO4: 2.75gLauryl sulphate: 0.1g 1000 ml nuocctpH6.8 0.2 BGBL(BrilliantGreenBileLactose Broth) Peptone: 10g Lactose: 10gMt bo: 20gBrilliant green: 0.0133g 1000ml nuocctpH 7.4 EC(E.coli medium) Trypticaseortryptose: 20g Mui mt No.3: 1.5g Lactose: 5g KH2PO4: 1.5g K2HPO4: 4g NaCl: 5g 1000 ml nuocct pH 6.9 EMB(Eosin MethyleneBlue Agar) Pancreatic digestoI gelatin: 10gLactose: 10gK2HPO4: 2gEosin Y: 0.4gMethylene blue: 65mgAgar: 15g 1000 ml nuocct.pH7.1 0.2 Tryptone waterTryptonehoctrypticase:10g 1000ml nuocctpH7.2 0.2 MR-VPBoth (Glucose Phosphate) trung 1:BuIIeredpeptone-water powder: 7gGlucose: 5gK2HPO4: 5g1000ml nuocct.pH6.9 0.2 trung 2:CaseinPancreaticDigest: 3.5gPepticdigestoIanimal tissue:3.5gDextrose: 5gPotassium phosphate: 5g pH 6.90.2 trung 3:Peptone: 5gGlucose: 5gPhosphate buIIer: 5gpH 7.50.2 SCA(SimmonsCitrateAgar) Sodium citrate: 2gNaCl: 5gK2HPO4: 1gNH4H2PO4: 1gMgSO4: 0.2gBromothymolblue: 0.08gAgar: 15g 1000ml nuocct. Dun nongnhevathinhthoang lc.Dunsi1-2phutchodnkhihoatanht.pH6.80.2 .. Khu trng dung cu v mi trung d pha Bao goi dung cu va phn phi mitruong vao dungcu, gn nut va bao goi. Hp tit trung o 1210C trong 15 phut. .. Tin hnh phn tch. f !ha long mu.

b. v mu vao canh LSB Chuyn 1ml dung dich 10-1, 10-2, 10-3 vao ng 10ml canh LSB, mi nng d co 3 ng lp lai, u o 370C, 48 gio. So d cy mu tu cac d pha long Ong Mu c. nh luong oliform va E.oli Dinh luong ColiIormDinh luong E. coli Ghi nhn s ng co sinh hoi ()Ghi nhn s ng co sinh hoi () Dungque cyvong cychuyn dichmutucacngLSB()sangcacngcochuacanh BGBL va u o 370C10C, 48 gio Dungque cyvong cychuyn dichmutucacngLSB()sangmitruongcanhEC,uo 44.50C0.20C, 24 gio Ghinhnsngcosinhhoi()ung voi mi d pha long Ghinhnsngcosinhhoi()ung voi mi d pha long Tinhktqua(3.4cachtinhkt qua) Dungquecyvongriadichmu tu cac ng() trn canhEC sang mitruongthachdiaEMB.Uo 370C, 24 gio. Chonkhunlacduongkinhlon hon 1mm (tron, dethinhdiavacoanhkimxanh)(xemhinh4),cyvaoTrypton, MR-VP, SC Citrate, u o 44.50C0.20C, 24 gio

/. Th nghim IMJi KtquaIMViCcuaEscherichiacolisau24giouo37C.(AnneHanson, University oI Maine, Orono%OngA:duongtinhthunghimindoletrnmitruongtryptone.Xuthin lop mau do trn b mt mi truong sau khi nho thuc thu Kovacs.Ong B: duong tinh thu nghim methyl red xut hin mau do cua methyl red. OngC:mtinhthunghimVoges-Proskauerbiuhinquasukhngthay di mau sau khi cho thuc thu Barritt`s A va Barritt`s B. OngD:mtinhthunghimcitratebiuhinquasuthaydimauscva khng co khun lac trong ng nghim. Thu nghim Indol (I): Nguyntc:Tryptophanlamtacidamincothbioxihoaboimtsvi sinhvtcohenzymetryptophanasetaonncacsanphmchuagcindol.Vic phat hin indol duocthuchin bngphanung cuaphn tunay voicacthucthuchuap-Dimethylaminbenzaldehyde(p-DMABA)taonnmtphuccht dangquinoneco mau do. Phuongphap tin hanh:Mitruong su dungchothu nghim nay la nuoc tryptone. Sinhkhichungthunduoc cyvao mitruong longtryptone,uonhitd370Ctrong24-48gio.Bsung1mletherhocxylenevaong nghim, lc du d chit tach indolln lopdung mi huu co. Nho5giotthucthuvao ng dich nui cy, d yn vai phut, theo di su tao mau do trong lop dung mi huu co. Thu nghim Indol. Doc kt qua: () xut hin cua lop mau do trn b mt mitruong.(-) lop mau vang cuathucthu. Di khico su xut hin cua mau cam do skatol, la tin cht cua methyl hoa cua indol, tao ra. Thu nghim Methyl Red (MR): Nguyntc:chithidomethylredgiupphnbitnngdHhindin trong mi truong sau khi vi sinh vt ln men glucose. Chi thi nay thay di mau nhu sau: pH4.4 do; pH 5.0-5.8 mau cam; pH~6.0 mau vang.Phuong phap tinhanh: Mi truong sudung chothunghimnay lamitruonglongGlucosePhosphate(MR-VPBroth).Dungquevongcymtluong nhokhunlactrnmitruongMR-VP,uo370Ctrongkhoang2-5ngay.Thm vaovaigiot thuc thu domethylred (0.02 tronghnhop cnnuoc co ty l 3:2, bao quan o 40C) vaotrong ng nghim dich nui cy, doc kt qua ngay.

Thu nghim MR. Doc kt qua: () mi truong co mau do sau khi b sung thuc thu. (-) khi comauvang. Truonghopmau cam, cn tiptucu ngmitruongcogingtrong 4 ngay vathuc hin lai thu nghim. Thu nghim Citrate (iC) Nguyn tc: su bin dung citrate boi vi sinh vt s tao ra CO2 lam km hoa mi truong. Mt khac moi VSV co kha nng su dung citrate lam ngun carbon duy nhtducokhanngdungmuiammoniumlamngundamduynht.SuphngiaicuamuiammoniumdunglamngundamtrongmitruongssinhNH3lamkimhoamitruong.SugiatnggiatripHnayduocchithi bng su di mau cua chi thi pH trong mi truong. Phuong phap tinhanh: Mi truong sudung chothunghimnay lamitruonglongSCA.DungquevongcyriamtluongnhokhunlaclnmitruongSCArntrongngnghimthachnghing,uo350Ctrongkhoang24-48 gio. Thu nghim Citrate. Docktqua:()khixuthinkhunlacvamitruongchuynsang mauxanh duong. (-) khi khng co khunlac vami truonggiunguynmauxanh luc. .4. Cch Tnh Kt Qua. TusluongcacngnghimcoE.coli()omidphalongcuamu, dung bang MPNloat 3 ng nghim o 3 nng d phalonglintip dtinhra mt d visinh vt trongmu va biu din duoi dang tri s MPN/mlmu ban du chua pha long. Bang Tra MPN dung cho loat 3 ng nghim o 3 nng d pha long lin tip.

KET LUAN

PhuongphapMostProbableNumber(MPN)laphuongphapph binvadathiuquatrongvicxacdinhColiIormvaE.Colicotrong thuc phm. Dy la phuong phap dua trn bang MPN d tim ra co su hin din cua ColiIorm va E.Coli. Phuongphapnaydoihoithoigiantuongdidaivaphuongphap thuchincngtuongdiphuctap.Cacbuocthuchindambaophai dungtranh khngcosunhmlntrongcac buocnukhngtasphai thuc hin lam lai tu du. TI LIEU THAM KHAO

1. GiaotrinhphntichViSinhThucPhm,Ths.LThuyLinh,TruongDai Hoc Cng Nghip Thuc Phm Tp.HCM 2. TCVN6505-2:1999SuavacacsanphmtusuadinhluongE.Coligia dinh bng phuong phap MPN. 3. Google.com.vn 4. Giao trinh Vi Sinh Thuc Phm, truong Dai Hoc Cng Nghip Tp.HCM.