TRNG I HC CNG NGHIP THNH PH H CH MINH

VIN CNG NGH SINH HC V THC PHM

GVHD: BI HNG QUN LP: HTP5 NHM 5 SVTH: L Th M Vn MSSV: 09078531

Thnh ph H Ch Minh, thng 11 nm 2011 1

MC LC Bi 1: Thit b, dng c phng th nghim v cc phng php tit trng vi sinh vt ............................................................................................... 3 Bi 2: Phng php pha ch mi trng nui cy vi sinh vt ............................... 6 Bi 3: Phng php nui cy vi sinh vt ............................................................. 10 Bi 4: phng php quan st vi sinh vt bng knh hin vi quang hc ............... 16 Bi 5: Phng php nhum mu vi sinh vt ....................................................... 19 Bi 6: Phng php phn lp vi sinh vt ............................................................. 20 Bi 10: Kho st c tnh sinh ha ca vi sinh vt .............................................. 23 Bi 13: nh lng vi sinh vt bng phng php m trc tip ....................... 35 Bi 14: nh lng tng vi khun hiu kh bng phng php m khun lc ................................................................................ 37 Bi 15: nh lng Coliform bng phng php MPN ...................................... 39 Bi 19: Phng php nh lng E.coli trong thc phm ................................... 41 Bi 20: Phng php phn tch Salmonella spp. trong thc phm ..................... 43 Bi 21: Phng php phn tch nh lng Bacillus cereus trong thc phm .... 46 Bi 22: Phng php phn tch nh lng Staphilococcus aureus trong thc phm .................................................................................................. 48 Bi 24: Phng php phn tch Clostridium perfringens trong thc phm ........ 50

BI 1: THIT B, DNG C PHNG TH NGHIM VI SINH VT V CC PHNG PHP TIT TRNG VI SINH VT Bo co thc tp 1. Trnh by yu cu ca vic bao gi dng c nui y vi sinh vt? Yu cu ca vic bao gi dng c nui cy vi sinh vt: Phn giy bao bn ngoi phi cht v kn Bao bng giy du vi dng c hp t Bao bng giy bo vi dng c sy kh khi kh trng t.

- Vi cc dng c nh pipet, que trang phi dng giy bao kn ton b. C th dng hp nhm ng cc dng c trn kh trng. 2. Cng dng v cch s dng cc dng c, thit b phng th nghim vi sinh vt? Cng dng v cch s dng cc dng c, thit b phng th nghim vi sinh vt: a. Ni hp t (autoclave): Thit b ny cp nhit bng hi nc p sut cao, c s dng hp kh trng m trng, mt s nguyn liu v dng c th nghim. b. Knh hin vi: Cng dng: dng nghin cu, quan st t bo vi sinh vt v c im hnh thi, sinh l nh vo kh nng phng i ca knh. c. Cc dng c th nghim Dng c thy tinh c nhiu loi vi nhiu kch c khc nhau nh bnh tam gic, ng nghim, a petri, lam knh, a thy tinh, que trang, ng ong, cc ong, bnh nh mc vt. Phin knh (lame): dng lm tiu bn quan st hnh thi, sinh l t bo vi sinh

- L knh (lamelle): dng y ln vt bi trn tiu bn c nh vi sinh vt trong qua trnh nghin cu. - a petri: dng nghin cu cc c im hnh thi, c im nui cy v phn lp ca t bo vi sinh vt. 3

Que tri: phn lp vi sinh vt theo phng php tri a. Que cy:

+ Que cy u trn: dng thao tc vi sinh trn i tng n bo nh vi khun, nm men. + Que cy nhn: dng cy su trn mi trng rn. + Que cy mc: dng ly khun ty hay mt on t nm. Micro pipette: s dng khi cn ht mt lng chnh xc mi trng.

4.Trnh by phng php tit trng dng c v mi trng nui cy vi sinh vt? Phng php tit trng dng c v mi trng nui cy vi sinh vt: a. Phng php l hc: Nhit kh: - i vi dng c cy kim loi, i khi c thy tinh, phng php thng dng l t trc tip trn ngn la n cn. - i vi dng c thy tinh c th gi v sy 1600C trong 1-2 gi hoc 1800C trong 30 pht. Nhit m: - Phng php luc: cho vt kh trng vo nc si, nhit s thm nhanh vo mu vt lm cho protein ng kt, dn n git cht vi sinh vt. Ch dit t bo sinh dng, bo t vn cn. - Phng php Pasteur: ch dit vi khun gy bnh, khng dit bo t v vi khun hoi sinh. Phng php ny thng dng nhit 70-750C trong thi gian 10-15 pht. - Phng php Tyndall: un cch thy nhiu ln nhit 70-800C, mi ln 3060 pht v lin tip trong ba ngy lin. - Phng php hi nc bo ha p sut cao: dng autoclave. Thng dng nhit 1210C trong 15-30 pht. Dit trng bc x: Tia t ngoi hay UV: ch st trng b mt, khng xuyn su vo mu vt.

- Tia m cc dng trong tit trng dng c gii phu, thuc, thc phm. Vt kh trng phi bao gi knh. 4

Dit trung bng cch lc: - Dng c lc thng l nhng mng xp bng s, aminate, cellulose, c kch thc l lc t 0,2-0,45m, thng dng lc nhng vt phm lng khng kh trng bng nhit c. - i vi kh trng khng kh th thit b kh trng l mt my lc kh c trang b mng lc hay hp ph vi khun. b. Phng php ha hc. Ch st khun ngoi da: x phng, cn, iode, phm mu. Cht dit khun v ty u: phenol, formol, hp cht clor

BI 2: CCH PHA CH CC LOI MI TRNG I. TR LI CU HI

1. Trnh by khi nim mi trng v phn loi mi trng nui cy vi sinh vt Khi nim mi trng dinh dng Mi trng dinh dng l hn hp cc cht dinh dng v cc cht ny c nhim v duy tr th oxy ha kh, p sut thm thu ca t bo v s n nh ca pH trong mi trng. trong cc cht dinh dng l nhng hp cht tham gia vo qu trnh trao i cht ni bo. Phn loi mi trng dinh dng o Theo ngun gc

Mi trng t nhin: c thnh phn l cc sn phm t nhin nh: trng, sa, khoai ty, dch chit nm men, ng, cm. Mi trng tng hp: cha cc cht ha hc m thnh phn ca chng c xc nh v nh lng mt cch c th v chnh xc. V d nh: Czapeck, Hansen, EMB Mi trng bn tng hp: cha c cc cht ha hc ln cc sn phm t nhin, v d nh: PGA, gi u ng o Theo trng thi vt l

Mi trng lng: thnh phn mi trng ny khng cha agar v thng c s dng nghin cu qu trnh tng hp ca vi sinh vt. Mi trng c: c 1000ml mi trng c 15 20 Agar Mi trng bn lng: cha khong 0,3 0,7% agar o Theo cng dng

Mi trng phn lp 6

Mi trng tng sinh Mi trng nui gi ging: ngho dinh dng Mi trng th nghim sinh ha 2. Trnh by qui trnh pha ch mi trng nui cy vi sinh vt Bao gm cc bc sau: Chun b dng c Cn ha cht Phi ch to mi trng nui cy iu chnh pH ca mi trng Phn phi mi trng vo dng c Kh trng mi trng Lm thch nghin, thch ng, thch vo a petri Kin tra v trng v bo qun 3. Yu cu ca mi trng trong a petri, ng nghim thch nghing v thch ng Lm thch nghing: Cn tin hnh ngay sau khi kh trng mi trng v mi trng cha ng c. t ng nghim c mi trng ln gi t nghing v khng c mi trng chm vo nt bng. yn cho n khi mi trng ng c. Yu cu mt thch phi thng, nhn v lin tc Lm thch ng: t cc ng nghim c mi trng l thch ng vo gi, yn cho mi trng ng c mi trng vo a petri: 7

Ton b qu trnh thch vo a petri c thc hin trong t cy v trng v gm cc thao tc sau:

o M bao giy gi cc a petri o Mt tay cm dng c cha mi trng o Tay cn li m nt bng v h ming bnh trn ngn la n cn. o M h np a petri. Nghing bnh v rt mi trng vo a petri. o y np a li, xoay trn a mi trng c phn phi u bn trong a. o yn cho mi trng ng c. o Lt ngc a li v bo qun. 4. Gii thch ti sao khng phn phi mi trng vo a petri trc khi kh trng V s lm nhim cc vi sinh vt khng mong mun, c th nhim mt s tp cht v vy sau qu trnh nui cy kh c th xc nh c kt qu. C th trnh c hi nc tip xc vo mi trng nui cy v v sau khi cy phi yn mi trng cng mi trng 5. a petri cha mi trng trc khi cy vi sinh vt nn p hay nga? Ti sao? Nn nga bi v lm kn khu vc nui cy, trnh ly nhim cc vi sinh vt khc v cng trnh tip xc vi hi nc. 6. ngha ca vic pha ch mi trng? Chng ta phi pha ch mi trng cho vi sinh vt v mi trng dinh dng l hn hp gm cc cht dinh dng v cc cht c nhim v duy tr th oxy ha- kh, p sut thm thu ca t bo v s n nh pH ca mi trng vi sinh vt c th sinh trng v pht trin mt cch ti u nht c th. Lm mi trng thc hin vic phn lp, nhn ging, gi ging vi sinh vt, ng thi nui cy v nghin cu cc c im sinh hc ca chng 7. Yu cu ca mt mi trng dinh dng nui cy? 8

Yu cu ca mi trng dinh dng: c cht dinh dng cn thit, c pH ph hp. c nht nht nh, khng cha cc yu t c hi, hon ton v trng, m bo s pht trin n nh ca vi sinh vt. 8. Nu ngha ca cc thnh phn trong mi trng nui cy? Peptone chit xut cao tht b dng lm ngun cacbon, nng lng v nit. Cao tht b cha cc axit amin, peptit, nucltit, axit hu c, vitamin v mt s cht khong. Cao nm men l ngun phong ph cc vitamin nhm B cng nh ngun nit v cacbon. 9. C bao nhiu loi peptone? C 3 loi peptone o T ng vt: chit xut t tht, cao tht b, dch thu phn mt phn ca tht b, cazin, dng lm ngun cacbon, nng lng v nit. Cao tht b cha cc axit amin, peptit, nucltit, axit hu c, vitamin v mt s cht khong. o T thc vt: chit xut t u nnh,. o Nm men: cao nm men, Cao nm men l ngun phong ph cc vitamin nhm B cng nh ngun nit v cacbon). 10. C ch lm trong nc ca lng trng trng Cch lm trong nc bng lng trng trng: lng trng trng: nc t l 1:1 nh tan ni bt cho vo 1 lt mi trng un si khong 5 pht ngui lng cn lc. C ch: cc protein trong lng trng trng nh albumin, ovalbumin di tc ng ca s khuy trn v gia nhit b bin tnh khng thun nghch, cc lin kt trong protein c ko dn lm l ra nhiu nhm chc hnh thnh lc ht tnh in vi cc ion, bi bn c trong nc sau khi lng, lc nc trong hn.

BI 3: PHNG PHP NUI CY VI SINH VT PHN LP SINH VT I. Nguyn tc 1. Mc ch ca vic nui cy cu. Tin hnh vic phn ging cc vi sinh vt mt cch nhanh chng. Bo tn cc ging vi sinh vt thun khit. Nghin cu cc c tnh sinh hc v s pht trin tng loi ca vi sinh vt. Pht hin s c mt ca vi sinh vt trong cc nguyn liu vt phm cn nghin

2. Nguyn tc nui cy vi sinh vt Mi thao tc nui cy u phi thc hin trong iu kin v trng trnh tp nhim cc vi sinh vt khng mong mun t mi trng ngoi. Mi trng v dng c nui cy u phi kh trng trit . Duy tr tt cc iu kin nui cy vi sinh vt pht trin thun li. II. Dng c, mi trng v ha cht III. Tin hnh th nghim Cy truyn trn thch a 1. Phng php cy truyn thch (mi trng thch). 1.1. C th cy trn a ptri theo 1 trong 2 cch sau: * Dng que cy u trn v thc hin theo trnh t sau: - a ptri ln bn. - Dng que cy ly canh trng vi sinh vt theo th t v yu cu phng php chung. - Tay tri h m np a ptri va cho que cy vo. - Nh nhng v nhanh chng lt que cy ln mt thch theo mt trong cc kiu sau: + Theo hnh ch chi trn ton b mt thch (hnh 3.3a) + Theo nhng ng song song (hnh 3.3b) + Theo 4 hnh ch chi 4 gc (hnh 3.3c) 10

Hnh 3.1 Cc kiu cy trn thch a

Hnh 3.2: Cch dn vi sinh vt ln b mt mi trng A que gt B dn bng que gt; C: S sinh trng ca VSV sau khi dn u bng que gt; D : S sinh trng ca VSV sau khi dn bng que cy 1.2. Phng php cy trn thch nghing S dng que cy u trn tin hnh cc thao tc theo ng trnh t ni trn Thc hin vic cy ging vo ng thch nghing bng cc thao tc tip 11 Phng php ny dng cy truyn cc vi sinh vt hiu kh.

theo: + Ho ging u que cy vo git nc y ng nghim. + Nh nhng lt que cy trn mt thch theo cc kiu (hnh 3.1) Hnh ch chi Hnh vng xon Hnh vch ngang song song

Hnh 3.2: Mt s kiu cy trn ng thch nghing 1.3. Phng php cy trn thch ng: cy sinh vt k kh

S dng que cy hnh kim Sau khi ly ging vi sinh vt, dng que cy ny m su vo phn khi thch hnh tr. m st y ng nghim v m thnh 3 ng: 1 ng chnh gia, 2 ng st thnh ng tu yu cu. ng cy phi thng, nh nhng khng gy nt, v mi trng IV. 1. Tr li cu hi. Cc phng php gieo cy vi sinh vt Cy truyn vi sinh vt trn mi trng lng: cy truyn bng pipette Pasteur, Cy truyn trn mi trng lng: cy truyn trn ng truyn thch nghing,

cy truyn bng que cy vng. trn ng nghim thch ng, cy truyn trn thch a. 2. Cc phng php gi ng vi sinh vt 12

a. Phng php cy truyn nh k trn mi trng mi u im: phng php ny n gin, d lm, thi gian bo qun khng lu. Nhc im: tn mi trng, cng sc v thi gian. D b tp nhim v d dn n mt chng ging gc. Mt hay nhm ln nhn hiu gia cc chng trong qu trnh bo qun. Phi nghin cu v theo di thi gian cy truyn thch hp cho cc chng bo qun. Ging gc c th b mt do sai st khi dng mi trng cy truyn khng thch hp. Chng vi sinh vt b thay i cc c tnh sinh hc do t bin xut hin sau mi ln cy truyn. b. Phng php gi ging trn mi trng thch c lp du khong u im: n gin nhng hiu qu cao nh kh nng lm chm qu trnh bin dng v h hp nn vi sinh vt pht trin chm li. Mi trng khng b mt nc v kh. c. Phng php gi ging trn t, ct, ht u im: d bo qun cc chng bo t tim sinh, thi gian bo qun c th ko di n 1 nm. Nhc im: kh trng mi trng trc d. Phng php lnh ng u im: nhanh, thun li cho vic bo qun mt s lng ln mu. vi sinh vt c bo qun t 10-20 nm, tit kim cng scv sai st nhn mc, tp nhim. Nhc im: gi thnh thit b, n nh ca cc chng vi sinh vt bo qun theo cc t bo qun l khc nhau. Trc khi em ra s dng phi c hot ho trn mi trng thch hp mt s ln m bo phc hi cc c tnh sinh hc ca chng vi sinh vt. e. Phng php bo qun lnh su u im: thch hp cho nhiu i tng vi sinh vt khc nhau nh nm men, vi khun, nm mc, vut, to v cc dng t bo ng vt. thi gian bo qun lu.

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Nhc im: u t kinh ph cho thit b, in qu cao, ri ro chy n, khng thch hp cho cc chng vi sinh vt hay dng n thng xuyn. 3. Trnh by nguyn tc v mc ch ca qu trnh phn lp. Nguyn tc: tch ri cc t bo vi sinh vt, nui cy cc t bo trong mi Mc ch: phn tch cc chng vi sinh vt trong mi trng t nhin v c lp trng dinh dng to khun lc ring r. chng nhm chn la ging vi sinh vt thun khit cho nhng mc ch khc nhau. 4. Mun phn lp nm men, nm mc, vi khun th ngun mu cn chn l g? Tr li: Phn lp vi khun: ngun mu l c kh ct nh. Nm mc: ngun mu l cm ngui, xi lm mc tng, bnh m kh Nm men: ngun mu l b mt tri cy, dch p nh to, l hoc trong ru

np, bia, nc ma. 5. So snh s ging v khc nhau ca cch phn lp vi sinh vt hiu kh v k kh. Tr li: Ging nhau: nhm phn tch cc chng vi sinh vt trong mi trng t nhin

v c lp chng nhm chn la nhng ging vi sinh vt thun khit.Khc nhau:

Phn lp vi sinh vt hiu kh - Ht dch mu pha long cho vao da petri c mi trng thch hp. - Dng que gt thy tinh phn phi dch mu tri u khp mt thch. - Tip tc dng que gt mu cho u khp mt thch a th 2 ri a th 3. - m nhit thch hp sau mt thi gian nht nh ta s nhn c cc khun lc ring r t cc a th 2, 3 Phn lp vi sinh vt k kh - Dng mi trng c trong ng nghim em chng cch thy loi b khng kh. - ngui mi trng cn 45-50oC - Ht dch nghin cu cho vo ng mi trng, y nt li, lc trn quanh trc ng nghim. - Rt nhanh, y nhanh trnh cho ng nghim c khng kh. - Dng parafin hn kn phn tip xc gia 2 a petri v nhit thch hp - Sau khi vi sinh vt pht trin, chn cc khun lc ring r trong mi trng, dng que cy ct c khi mi trng ri cy vo mi trng lng thch hp.

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6. Ti sao khi i cn ho cht ch cn 2-3 ngi? Do dng c trong phng ho cht c hn, i cn ho cht cn 2-3 ngi m bo v thi gian,hiu qu lm vic

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BI 4: PHNG PHP QUAN ST KNH HIN VI BNG KNH HIN VI QUANG HC 1. Trnh by cu to v nguyn tc hot ng ca knh hin vi quang hc a. Cu to Knh hin vi quang hc gm c hai phn: H thng c hc: gi knh gm c chn knh, thn knh, tr v xoay th knh, gn v xoay vt knh, bn knh, thanh trt di chuyn tiu bn, c di chuyn thanh trt, kp gi tiu bn, c di chuyn t quang knh, c iu chnh th s cp, c iu chnh th cp (vi cp). H thng quang hc gm: th knh, vt knh, t quang knh, mng chn sng, ngun chiu sng (n in chiu sng hoc knh chiu sng). Ngoi ra, knh dng ngun chiu sng l n in th c thm b phn cung cp in nh phch cm, dy in, cu ch, mch in (trong chn knh) v nt iu chnh cng chiu sng. b. Nguyn tc hot ng Vt knh l h thng quang hc phc tp gm mt s thu knh, trc tip phng i mu vt. Cc thu knh sp xp theo th t, thu knh nh ngoi cng hng vo tiu bn c phng i ln nht. phng i ca knh ph thuc vo tiu c, tc bn knh cong ca thu knh. Thu knh cng cong, tiu c cng ngn th kh nng phng i cng ln. C hai loi vt knh Vt knh kh phng i nh nh x4,x10,x15,x40. Vt knh du c phng i ln nh x90,x100. Th knh cng c phng i phc tp, gm hai thu knh, mt hng v pha mt ngi xem, mt hng v vt knh. Th knh phng i mt ln na nh do vt knh thu vo, lm nh to ln, xem r hn. phng i ca knh = phng i ca vt knh x phng i ca th knh.

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V d: phng i ca vt knh x100, phng i ca th knh x10. Nh vy phng i ca knh : 100x10=1000 ln. Nng sut phn ly ca knh quan trng hn phng i, v l tiu chun chnh chn knh hin vi. 2. C bao nhiu loi knh hin vi? c im ca tng loi? C 5 loi knh hin vi. Knh hin vi vin en

nh sng thng chiu t di ln, qua ra ca t quang nn en, chiu ht vo xung quanh tiu bn, nhng tia sng ny b tiu bn lm khc x ri a vo vt knh. Tiu bn c soi sng rc trn nn en, ging nh trong phng ti c mt tia sng chiu vo, gip ta thy r tng ht bi trong khng kh b tia sng chiu vo. Chc nng: quan st hnh thi v c tnh ca mt s vi khun m knh hin vi thng kh quan st. Knh hin vi i pha

nh sng thng b i pha v bin dao ng bi cu trc c bit ca t quang knh, vt knh v th knh. Chc nng: dng quan st r nt cc cu trc nh nh tin mao, cc lp mng. Knh hin vi hunh quang

Chm tia t ngoi chiu vo cc tiu bn nhum mu bng cc cht hunh quang. Trong t bo, cc cu trc khc nhau s pht quang vi mu sc khc nhau. Chc nng: quan st v phn bit cc cu trc khc nhau trong t bo vi sinh vt. Knh hin vi in t

Chm tia in t bc sng rt ngn, nng sut phn li rt ln nn phn gii cao, gip phn bit hai im rt gn nhau. Chc nng: quan st vut, cu trc phn t ca t bo. 17

Knh hin vi quang hc

Dng bc sng 500 nm 560 nm trong chm nh sng thng to nng sut phn li ln gip phn bit hai im cch nhau khong 0.2 m tr ln. H thng phng to ca knh hin vi quang hc gm hai b phn: vt knh v th knh. Mi b phn l mt h thng thu knh hi t phc tp. Chc nng ca knh hin vi quang hc l dng quan st t bo vi sinh vt, k sinh trng, t bo ng vt, thc vt. 3. Ti sao khi quan st vt knh 100 phi cho 1 git du soi knh? Do chit sut nh sng ca khng kh nh hn thu tinh, nn tia sng khi i qua tiu bn thu tinh s b khc x mt phn. Phn pha ngoi cu tia sng do b khc x nn khng i vo c vt knh. Vt knh c phng i cng ln th ng knh ca thu knh cng nh, lng tia sng i vo vt knh rt t nn khng nhn r nh. hn ch nhc im ny ta dng du soi knh c chit sut nh sng gn bng thu tinh. Thu tinh v du soi c xem l mt mi trng ng nht. nh sng i qua khng b khc x nn tp trung y vo vt knh, gip xem r nh.

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BI 5. PHNG PHP NHUM MU VI SINH VT Bo co thc tp 1. Sau khi nhum, vi khun Gram dng c mu xanh en hay tm, Gram m c mu vng hay ta. Gii thch nguyn nhn? Sau khi nhum Gram, vi khun Gram dng s c mu xanh en hay tm, cn vi khun Gram m c mu vang (nhum safranin O) hay ta (Fuchsin Ziehl). S d c mu nh vy l do vi khun Gram dng v vi khun Gram m c s khc bit v thnh phn ha sinh ca thnh t bo vi khun. Thnh t bo vi khun Gram dng th c nhiu peptidoglycan (phc cht protein-ng) v cc peptidoglycan ny c lin kt cht ch vi nhau t c th gip cho t bo khng li cht ty mu nn sau khi ra bng cn th crystal violet khng b ra tri v vn gi c mu tm, sau nhum b sung bng dung dch safranin O hoc Fuchsin Ziehl, nhng khng c s thay i mu nhiu. Thnh t bo vi khun Gram m c thnh phn lipid nng cao cho nn chng c th ha tan trong cht kh mu (aceton, alcohol,) v b ra tri cng vi crystal violet (mu tm), sau khi nh dung dch ty mu, ta nhum them bng dung dch safranin O hoc Fuchsin Ziehl, lc ny vi khun Gram m c mu vng hay ta. 2. Nu khng nhum b sung thuc th safranin hoc fuchsin, vi khun Gram m c mu g? Ti sao? Nu khng nhum b sung thuc th safranin hoc fuchsin, vi khun Gram m s khng c mu. Bi v giai on ty mu bng cn ra tri crystal violet (c mu tm) nn sau giai on ty mu vi khun Gram m b mt mu ca crystal violet c nhum trc . Hoc trng hp vi khun Gram m s c mu tm cng c th giai on ty ra, ta khng ty mu cho n khi git dung dch cui cng chy ra khi phin knh l khng mu.

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BI 6. PHNG PHP PHN LP VI SINH VT 1. Trnh by nguyn tc v phng php phn lp vi khun thun khit? Nguyn tc: - Gieo cy vi khun pha long trn mi trng dinh dng c trng (2% thch hay cn gi l agar). - Nui dng trong iu kin thch hp cho mc cc khun lc tch bit nhau. - Cy tch t khun lc mc tch bit sang ng mi trng dinh dng thch nghing thu nhn chng vi khun thun khit. Phng php phn lp vi khun thun khit: 1.1. Phng php to khun lc n cc vi sinh vt hiu kh:

C nhiu k thut ria khc nhau thc hin hp ria v to khun lc n. Mt s k thut ria thng dng: k thut ria ch T , k thut ria bn gc ,k thut ria tia ,k thut ria lin tc Thao tc k thut to khun lc n c thc hin nh sau: a. K thut hp ria: Dng que cy vng thao tc v trng thu ging. Ria cc ng trn a ptri cha mi trng thch hp (ria ch T v ria bn

gc). Sau mi ng ria, t kh trng u que cy v lm ngui trc khithc hin ng ria tip theo. Bao gi a ptri, nhit v thi gian thch hp trong t m.

b. K thut hp tri: Dng pipetman v u tp v trng, thao tc v trng chuyn 0,1 ml dch cha

ging vi sinh vt ln b mt mi trng trong a ptri. Nhng u thanh gt (que tri) thu tinh vo cn 70 , h qua ngn la kh
0

trng. u thanh gt ngui trong khng gian v trng ca ngn la. M a ptri, t nh nhng thanh gt ln b mt thch ca a petri. Dng u thanh gt xoay, tri u dch ging ln b mt thch. Trong khi tri, 20

thc hin xoay a mt vi ln, mi ln khong 1/2 chu vi a to iu kin cho thanh gt tri dch ging u khp b mt mi trng. - Rt thanh gt khi a, y a, gi v nhit v thi gian thch hp trong t m. c. K thut hp : Dng pipetman v u tp v trng, thao tc v trng chuyn 1 m dch cha

ging vi sinh vt ln b mt mi trng trong a ptri. khong 15 - 20 ml mi trng un chy v ngu n 45 - 55 C vo

0

a petri cy mu. Xoay nh ia petri cng chiu v ngc chiu kim ng h vi ln dung

dch ging c trn u trong mi trng cy. y np a ptri, ng t nhin. Phng php to khun lc n cc vi sinh vt hiu kh:

1.2. -

Dng mi trng c trong ng nghim em chng cch thu loi b khng

kh trong mi trng. ngui mi trng cn 45 - 50 C. Ht 0,1 ml dch nghin cu cho vo ng mi trng, y nt li, lc trn quanh
0

trc ng nghim. Rt nhanh mi trng ng nghim vo np di ca a ptri v y tht

nhanh np trn li, sao cho gia mt np v mi trng khng cn khng kh. - Dng parafin hn kn phn tip xc gia 2 np ca a petri v nhit thch hp. - Sau khi vi sinh vt pht trin, chn cc khun lc ring r trong khi mi trng, dng que cy ct c khi mi trng ri cy vo mi trng lng thch hp. 2. Nguyn tc ca vic nui tch ly? 21

Nguyn tc ca vic nui tch ly: trng hp s vi khun c trong mu t th phi nui tch ly bng cch mu, b sung cht dinh dng v cc iu kin l ha cn thit hoc b sung cc cht c ch sinh trng ca cc vi sinh vt i km. 3. Th no l chun vi khun thun khit (chng sch)? Chun vi khun thun khit (hay cn gi l chng sch) c hiu l th h con, chu, dng c ngun gc t mt t bo ring l. Cc chng vi sinh vt c thu nhn t khun lc phn lp ln u cha chc thun khit v mt khun lc c th c hnh thnh bi mt hoc nhiu t bo, bo t, v th phi lm sch nhiu ln mi thu c chng vi sinh vt thun khit.

22

BI 10: CC PHN NG SINH HA 1. Phn ng to indol Mc ch: Pht hin cc VSV c kh nng sinh indol cc VSV c h emzym tryptophanase S :
Chng Vi Sinh Vt

Mi Trng: NB v Tripton

Thuc th Kovacs

37oC 24h

Kt qu, dng tnh (mu hng) v m tnh (mu vng)

37 C / 24h

m tnh

Chng Vi Sinh Vt

Mi Trng Dng tnh m tnh

23

C ch php th: Vi sinh vt tit enzyme tryptophannase chuyn ha tryptophan trong mi trng thnh Indol. Indol s kt hp vi para-dimethylaminbenzaldehyd trong thuc th kovacs to thnh phc cht c mu hng cnh sen. ngha: Php th cho bit cc chng loi vi sinh vt c kh nng tit enzyme tryptophannase, gip ta nh danh c mt s chng loi vi sinh vt. L phn ng gip phn bit E. coli (+) vi Klebsiella (-) Proteus mirabilis (-) vi Proteus khc (+) Bacillus alvei (+) vi Bacillus khc (-) (+) Proteus rettgeri (-) Serratia marcescens 2. Phn ng MR (methyl red) Mc ch: xc nh vi sinh vt sn xut v duy tr cc acid bn trong qu trnh ln men glucose.
Chng vi sinh vt

i chng

Mi trng MR-VP

Methyl Red

24h, 37oC

Kt qu, mu (Dng tnh), mu vng: (m tnh)

24

2-4 ngy

Chng Vi Sinh Vt C ch:

Mi Trng

Dng tnh

m tnh

C s sinh ha: Cht ch th pH: methyl red

Di 4,4

5,0 5,8

Trn 6,0

MR (+) cng ko di thi gian nui cy mi trng cng acid MR (-) cng ko di thi gian nui cy cc cht c tnh acid b chuyn ha mi trng dn trung tnh

Thi gian 2 5 ngy 37oC C ch: mt s vi sinh vt c kh nng chuyn ha Glucose qua qu trnh ng phn to thnh acid pyruvic, ri t Acid pyruvic chuyn ha thnh acetyl coenzyme A, i qua chu trnh Kreps to ra cc acid: acid citric, acid succinic, acid fomic, a cid malic, a cid oxalic hoc t Acid pyruvic chuyn trc tip thnh cc acid lactic, acid fomic, Trong mi trng MR-VP c PH= 6.90.2 c K2HPO4 (m phosphat) gip n nh PH, khi c mt cc acid hu c mnh s tn cng v ph v th PH n nh lm cho PH gim mnh, PH<4.3, Vi s c mt ca thuc th MR (Methyl Red) s lm mi trng chuyn sang mu (Phn ng dng tnh)

25

 ngha: nh danh mt s vi sinh vt c kh nng chuyn ha Glucose (thng l gram -). 3. Phn ng VP Mc ch: Pht hin vsv to sn phm trung tnh (acetoin) trong qu trnh ln men glucose. Chng vi sinh vt

Mi trng MR-VP KOH 10% (NaOH 40%) 24h, 37oC -naphthol

Kt qu,mu hng (Dng tnh), khng i mu (m tnh)

Quy trnh ha hc:

m tnh

Dng tnh

26

 ngha: nh danh vi sinh vt to sn phm trung tnh (acetoin) trong qu trnh ln men glucose, thng l gram (-) 4. Th nghim Citrate Mc ch: Xc nh kh nng vi sinh vt s dng ngun citrat nh l ngun cacbon duy nht. C s sinh ha: VSV s dng citrate, sinh ra CO2 lm kim ha MT VSV s dng mui ammonium l ngun m duy nht to ra NH3 lm kim ha MT Mi trng Simmon citrate agar Ammonium dihydrogen phosphate Dipotassium hydrogen phosphate NaCl 1.0g 1.0g 5g 27

Sodium citrate MgSO4 Bromothymol blue Agar

2g 0,2g 0,08g 13g

Chng vi sinh vt

Mi trng Simmon citrate

24h, 37oC Kt qu, mu xanh dng (Dng tnh), mu xanh lc (m tnh)

i chng trng

P m tnh

P dng tnh

C ch: Mt s vi sinh vt c enzyme citrate permease c kh nng s dng citrate trong mi trng c Na. Citrate l ngun cacbon duy nht.

28

Khi s dng citrate, chng gii phng ra mi trng ion Na+ lm tng PH ca mi trng, ng thi nhng vi sinh vt c kh nng s dng citrate lm ngun cacbon th cng c kh nng s dng mui amonium v c NH4H2PO4 lm ngun Nito to ra NH3 cng lm kim ha mi trng, s thay i PH ca mi trng c nhn bit nh ch th mu Bromothymol blue. ngha: nh danh c mt s vi sinh vt c kh nng s dng citrate nh mt ngun cacbon duy nht. 5. Th nghim KIA/TSI Mc ch: pht hin kh nng S dng cc ngun cacbonhydrate Sinh H2S To hi (gas)

Mi trng KIA ch c hai loi ng 0.1% glucose, 1% lactose. Mi trng TSI c thnh phn ging KIA, c b sung thm 1%sucrose
Chng vi sinh vt

Mi trng KIA/TSI 24h, 37oC

Kt qu

29

370C/24h

Quan st: Phn nghing / phn su / hi / H2S

C ch: C ba trng hp xy ra: Ch s dng glucose, sau 18-24h nui cy phn nghing (b mt) tr nn c PH

kim v phn ng (phn su trong ng nghim) c PH acid. Do Glucose trn b mt ca mi trng c vi sinh vt oxy ha hon ton thnh CO2 v nc thu ly nng lng, p ng nhu cu nng lng trong tng trng. Vi sinh vt tip tc d ha peptone qua gii phng NH3 lm phn b mt ca mi trng c PH kim. Trong khi , phn sau trong mi trng c iu kin oxy khng y , glucose c ln men k kh sinh cc acid hu c lm PH mi trng gim. S dng c glucose v Lactose, sau 18-24h, ton b mi trng u tr nn c PH acid v s bin dng ng thi c hai loi ng gip vi sinh vt nng lng tng trng m cha cn s dng n peptone. Nu ko di thi gian nui cy qu 24h b mt PH s tr nn kim do ht ngun cacbon v vi sinh vt pha s dng n peptone. Khng s dng glucose, lactose: vi sinh vt s bin dng peptone thu ly nng lng v vt cht cho s tng trng. tuy nhin do peptone ch c bin dng trong iu kin hiu kh nn hin tng kim ha ca mi trng ch din ra trn b mt mi trng. Kh nng sinh H2S do trong mi trng sodium thiosunfat, vi sinh vt kh sunface c th kh cht ny, nh c enzyme thiosunfat reductase gii phng H2S, H2S s phn ng vi Ion Fe2+ ca ch th amonium citrate to kt ta mu en F2S. Mt s trng hp c th xy ra khi dng mi trng KIA/TSI 30

10

11

ngha: nh danh cc loi vi khun c kh nng: s dng cc ngun cacbon khc nhau v c kh nng sinh H2S, kh nng sinh hi. Thng c dng nh danh cc vi khun gram (-), vi sinh ng rut. 6. Phn ng LDC Mc ch: dng nh danh cc loi vi khun c kh nng sinh enzyme Lysine decarboxylase, thng dng nh danh salmonella C ch: Trong mi trng c NH2-CH2-(CH2)3-CH2-NH2-COOH, trong mi trng c vi sinh vt c kh nng tit enzyme Lysine decarboxylase, enzyme Lysine decarboxylase ct t gc COOH ra khi hp cht NH2-CH2-(CH2)3-CH2-NH2COOH, to thnh CO2 v gc NH2-CH2-(CH2)3-CH2-NH2, gc ny s lm cho PH ca mi trng tng ln. Glucose s b phn gii thnh acid lm PH ca mi trng gim cho mi trng c mu vng, nhng sau 3-4 ting, glucose s cn kit v PH tng ln theo lysine. Kt hp hai qu trnh trn s to ra mi trng c pH ln. Cht ch th: Bromocresd purple c mu vng vi pH<2.2, c mu tm khi 5.2< pH <6.8, v c mu tm khi pH>6.8. 31

Cresol red c mu vng tm khi pH<7.2, c mu tm khi 7.2<pH<8.3 v mu tm khi pH>8.3 V sau khi vi khn phn hu NH2-CH2-(CH2)3-CH2-NH2-COOH v Glucozo mi

trng c pH ln nn mi trng c mu tm ca thuc th Bromocresd purple v Cresol red. ngha: nh danh cc loi vi khun c kh nng sinh enzyme Lysine decarboxylase. 7. Phn ng di ng Mc ch: Xc nh kh nng di ng ca vi sinh vt Mi trng: mi trng bn lng NA, MIU,SIM ng. C ch: Mt s loi vi khun c tim mao c th di ng trong mi trng bn lng, lm c mi trng hay mc ging r cy xung quanh ng cy. Cch thc hin: Vi sinh vt Dng que cy thng

Mi trng 37oC, trong 24-48h c kt qu Cch c kt qu: vi sinh vt c tim mao c kh nng mc lan ra khi ng cy, nu khng c kh nng mc lan th ch c th pht trin trn ng cy

32

ngha: dng nh danh mt s loi vi sinh vt c tim mao hoc khng c tim mao,. Thng dng dnh danh nhng con cu khun khng c tim mao v khng to bo t. 8. Phn ng Coagulase test C ch: mt s vi sinh vt c kh nng tng hp enzym coagulase, enzyme ny c kh nng lm ng huyt tng ngi hoc th. Khi cy vi sinh vt v mi trng, nu vi sinh vt c kh nng tit enzyme coagulase, enzyme coagulase c kh nng phn ct fibrinogen to thnh fibrin v prothromibis, to thnh cch khi ng. Mi trng: Huyt tng ngi hay th ng kh dng thng phm. Cch c kt qu:

33

(+) khi xut hin khi ng t huyt tng (-) khng xut hin khi ng t, dung dch ng nht. ngha: Phn ng coagulase test thng c dng nh danh vi sinh vt c kh nng tit enzyme coagulase, thng l cc vi khun gy bnh (thng dng nh danh Staphylococus). 9. Phn ng sinh H2S C ch: Vi sinh vt c kh nng phn gii mt s cht cha lu hunh, ta c th dng Na2S2O3, Na2SO3, peptone hay trptone, to thnh kh H2S. Cch c kt qu: B sung vo ion Fe2+ hoc Fe3+ to thnh kt ta en. Dng giy ch axetat trn ng nghim.

10. Phn ng phn hu Nitrate thnh Nitrit. Mt s vi sinh vt c kh nng to thnh enzyme reductase c kh nng phn hu NO-3 thnh NO2-. kim tra NO2- c trong dung dch, ta dng: Cress A c A.sulfanilic Cress B c -naphthilamine

Cress A v Cress B c kh nng kt hp vi NO2- to thnh phc cht c mu nu hng (dng tnh). Cy vi khun vo mi trng c cha Nitrate => cc ng nghim 37oC trong 24h => nh thuc th Cress A v Cress B=> c kt qu.

34

BI 13: NH LNG VI SINH VT BNG PHNG PHP M TRC TIP Bo co thc tp 1. Nguyn tc v cch tin hnh ca phng php nh lng vi sinh vt bng phng php m trc tip a. Nguyn tc Da trn s quan st v m trc tip s lng t bo VSV bng knh hin vi v bung m. Phng php ny dng xc nh s lng cc loi VSV n bo c kch thc ln nh: nm men, to n bo, bo t mc d quan st trn knh hin vi. Quy trnh m n gin, cho php xc nh nhanh chng s lng VSV. Tuy nhin, do quan st v m bng mt trn knh hin vi nn c mt s tr ngi Khng phn bit cc t bo VSV sng v cht trong mu. Khng phn bit cc t bo VSV v ht vt th. Hn ch i vi huyn ph c mt thp do lng dd em m nh Ty mc c mt ca VSV trong mu ban u, dd em m cn c pha long n mc ph hp ( s tb trong mt nh khng qu 10 tb). Nguyn tc pha long: Mu phi c pha long. Ty theo mc nhim khun ca mu ta c s c lng pha long 1/5, 1/10, 1/20 hay 1/10, 1/100, 1/1000 Dung dch pha long mu thng l NaCl 9% hay phosphat m c v trng. Cch pha: + Mu 1/5: 1ml mu+4ml NaCl 9% + Mu 1/10: 1ml mu+9ml NaCl 9%\ T mu 1/10 ly 1ml +9ml NaCl 9% ta c pha long 10-2, c nh vy ta c cc pha long tip theo 10-3, 10-4, b. Cch tin hnh t lamelle sch ph ln khung m.

35

Dng ng nh git ht dd nm men pha long, bm nh v rnh bung m, dd thm vo k bung m v lamelle. Dung dch chy trn t t vo cc rnh, lan ta lp y khp lamelle. Nu b bt mc li trong lamelle th phi lm li. t bung m ln bn kp ca knh hin vi, dng kp c nh bung m. Thao tc knh hin vi, dng vt knh x10 iu chnh s b trc, sau dng vt knh x40 m. m s TB trong 5 ln (4 gc, 1 gia), m ln lt tng nh trong 1 ln. Trng hp 1 ln c 16 nh: m tt c 80 nh trong 5 ln. Trng hp 1 ln c 25 nh: m tt c 125 nh trong 5 ln.

2. Phng php nh lng vi sinh vt bng cch m trc tip thng p dng cho nhng i tng vi sinh vt no? Gii thch i tng: nhng vi sinh vt c kch thc ln nm men, to n bo, bo t mc d quan st trn knh hin vi. Do quan st bng knh hin vi vt knh x40, ngha l phng i ln 400 ln nn ch c th m c nhng vi sinh vt c kch thc ln.

36

BI 14: NH LNG TNG VI KHUN HIU KH TRONG THC PHM BNG PHNG PHP M KHUN LC Nguyn tc Tng s vi khun hiu kh l tng s nhng vi khun thuc nhm vi khun hiu kh tn ti trong mi trng. Tng s cc nhm vi khun ny trong thc phm c th xc nh bng phng php nui cy tri ln b mt thch hoc bng phng php to hp . Thng qua s lng khun lc m c trn cc a peptri cho php xc nh c lng vi sinh vt cn c kh nng sinh trng trong mi trng trong mu ban u m kt qu chnh xc th s vi khun trong a phi trong gii hn 25-250 khun lc. Nu vt qu gii hn th phi tin hnh pha long v chn nhng pha long ln hn. Bo co thc tp 1. Trnh by phng php nh lng tng vi khun hiu kh trong mu bng phng php m khun lc. C 2 phng php l m trc tip v m gin tip Cch m trc tip: i vi nhng vi sinh vt c kch thc ln to, nm

men th ta c th m trc tip bng bung m hng cu. Phng php ny nhanh, tuy nhin khng chnh xc v ta khng th phn bit s lng t bo cht vi t bo sng, ngoi ra cn c s nhm ln do cc vt th khc trong mu. Cch m gin tip bng cch m s lng khun lc mc trn mi trng thch: phng php ny c trnh by trong qu trnh tin hnh trn. 2. So snh u nhc im ca phng php to hp v phng php cy tri Phng php cy tri: u im: ng u cao (khun lc mc u) nn d nhn dng cc khun lc c trng 37

 Nhc im: o Thi gian tri lu o Th tch mu cy nh Phng php to hp : u im: th tch mu cy ln, mt vi sinh vt cao Nhc im: D lm vi sinh vt cht nu mi trng cha h xung nhit 450C. Khng xc nh c hnh dng khun lc nht nh

38

BI 15: NH LNG COLIFORMS BNG PHNG PHP MPN Bo co thc tp 1. c im ca nhm vi khun Coliform v tc hi ca chng Coliform l nhng trc khun, Gram (-), khng sinh bo t, k kh ty tin, c kh nng ln men lactose sinh acid v sinh hi 370C trong 24-48h. Pht trin pH t 4,4-9,0, nhit rt rng -2:500C, Nhm Coliform hin din rng ri trong t nhin, trong rut ngi, ng vt. S lng coliform trong mu c dng ch th kh nng ca cc vi sinh vt gy bnh khc, ch th cho mc v sinh ca mi trng hay mt sn phm. Nhm coliform gm 4 ging l : E.coli, Citrobacter, Klebsiella v Enterobactor Tc hi: vi khun c a vo thc phm t nc c nhim phn hay t nguyn liu thc phm nhim phn. Triu chng ng c: thi gian bnh t 2-20h. Bnh pht t ngt, au bng d di, t nn ma, i phn lng, thn nhit bnh thng hoc st nh, c trng hp st cao, chn co qup. Bnh thng sau 2-3 ngy s khi, b nng s lu hn. 2. Trnh by nguyn tc v phng php nh lng coliform trong thc phm theo phng php MPN. a. Nguyn tc Dng phng php MPN xc nh copliform trong thc phm. Phng php MPN( phng php c xc sut cao nht, s ti kh) cn c gi l phng php pha long ti hn hay phng php chun . y l phng php dng nh gi s lng VSV theo s lng VSV c xc sut ln nht c mt trong 1 n v th tch mu hay ni cch khc MPN da trn nguyn tc xc sut thng k s phn b VSV trong cc pha long khc nhau ca mu. y l phng php nh lng da trn kt qu nh tnh ca 1 lot cc th nghim c lp li 1 s pha long khc nhau. Thng thng vic nh lng ny c lp li 3 ln 3 pha long bc

39

10 lin tip, tng cng 9 ng nghim. cc pha long c la chn sao cho cc ln lp li c 1 ln dng tnh v 1 m tnh. T cc kt qu ca th nghim nh tnh, da vo bng Mac Crady suy ra mt c on s lng VSV c trong mu, c trnh by di dng s MPN/100ml hay s MPN/g mu. chnh xc ca phng pho MPN ph thuc vo s lng ng nghim lp li trong mi pha long: s lng ng nghim lp li cng cao th chnh xc ca tr s MPN cng ln b. Phng php nh lng
Mu

ng nht mu ( ht 1ml mu trong 9 ml nc mui sinh l) Pha long mu ( pha long thnh 10-2, 10-3, 10-4) Tng sinh ( chn 3 nng lin tip thch hp cy 1 ml mu vo 10ml BGBL, mi nng cy 3 ng, 370C/48h)

Ghi nhn s ng dng mi nng pha long

Chn ng (+) cy vo ng canh EC 44oC /24h

Tra bng MPN

Chn ng (+) cy sang mi trng nc tryptone 440C/24h

Th phn ng Indole bng thuc th Kowacs

Tra bng MPN, tnh mt E.coli

40

BI 19: PHNG PHP PHN TCH NH LNG ESCHERICHIA COLI TRONG THC PHM Bo co thc tp 1. Trnh by c im ca E.coli L trc khun, gram(-), khng to bo t. Pht trin nhit t 7-500C, t0opt l 370C,pHopt l 4,4. E.coli sng trong rut gi ca ngi v ca ng vt. E.coli theo phn ngi v phn gia sc ra thin nhin 2. Trnh by quy trnh phn tch nh lng Ly 1ml mu nc ma cho vo 9ml nc mui sinh l, c 10-1 Pha long mu 10-2, 10-3,10-4. Chuyn 1ml dung dch 10-2, 10-3, 10-4 vo 10ml canh BGBL, nng 3 ng lp li, 370C, 36h Chn ng (+), cy sang EC, 440C trong 24-48h

Xc nh ng (+) mi nng , cy sinh khi VSV t cc ng (+) sang a mi trng EMB phn lp E.coli

a. C ch ca cc th nghim sinh ha Phn ng Indol Trong mi trng NB/ tryptone s c Tryptophan Nu trong mi trng c vi khun E.Coli th s sn sinh ra Emzyme Tryptophanase Tryptophanase s kh Tryptophan th Indol

41

 nhn bit c Indol trong mi trng hay khng th chng ta s nh thuc th kowacs vo. Nu sinh ra hp cht c mu hng cnh sen th c mt ca Indol Suy ra phn ng Indol (+)

Th nghim MR Nguyn tc: pht hin cc vi khun ln men glucose to sn phm acid hu c Trong mi trng c h m phosphat ( K2HPO4) nhm n nh pH (6,9)mi trng. E.coli c kh nng ln men glucose to ra acid hu c bn th n s ph v h m lm pH gim mnh xung khong 4 4,3. Nh MR nu pH <4,3 th mi trng c mu th (+) cn mu vng th (-) Kt qu MR(+)

Th nghim VP Nguyn tc: cng ging nh vi MR nhng n s khng to ra acid hu

c m to thnh acetoin Sau nh thm KOH 10% v cho n 10 pht Phn ng (+): c mu hng Phn ng (-): mi trng c mu vng hoc mu nu t =>E.coli khng c kh nng sinh acetoin nn phn ng cho kt qu m tnh. napthol. yn i trong vng 30s

42

BI 20: PHNG PHP PHN TCH SAMONELLA SPP TRONG THC PHM Nguyn tc Qu trnh phn tch Samonella gm 4 bc: tng sinh, tng sinh chn lc, phn lp, v khng nh. Bc tng sinh chn lc thng dng cc loi mi trng chuyn bit ty vo ngun mu thu nhn. Bc khng nh nhm xc nhn cc khun lc c trng cho Samonella xut hin trn mi trng phn lp. Bc ny da trn vic s dng cc th nghim sinh ha v th nghim huyt thanh c trng cho Samonella Bo co thc tp 1. Trnh by quy trnh phn tch Samonella trong thc phm

43

Mu

Tin tng sinh( ng nht 25ml mu trong 225ml canh BPW 370C trong 16-24h)

Tng sinh chn lc

Cy 0,1ml dung dch tng sinh sang mi trng RV. 41,50C trong 24

Cy 0,1ml dung dch tng sinh sang mi trng Selenite. 370C trong 24

Phn lp

Cy phn lp trn mi trng Hektoen 370C trong 24

44

2. Ti sao khi phn tch Samonella ch cn phn tch nh tnh Ch tiu Samonella l 1 trong nhng ch tiu rt quan trng trong kim nh cc loi hi sn nh c, mc, tmtheo nhng tiu chun VN quy nh v s c mt ca Samonella trong 25gam cc loi ny l 0, ngha l khng cho php s c mt ca n trong thc phm ny. Nn chng ta ch cn pht hin s c mt ca Samonella khng cn phn tch nh lng a. Gii thch c ch cc phn ng sinh ha xc nh Samonella i vi phn ng MR-VP c trnh by trong bi phn tch nh lng E.coli. Ch gii thch phn ng sinh ha LDC v MIU LDC Trong mi trng LDC cng c glucose nn vi sinh vt s phn gii glucose to thnh cc acid hu c lm gim pH mi trng, do mi trng s c mu vng Khi ngun glucose cn kit th vi sinh vt s s dng Lysine C ch: vi sinh vt c kh nng to ra enzyme lysine decarboxylase phn ct phn t lyzine to thnh diamine Trong diamine c 2 nhm amoni lm pH mi trng tng to mu tm (+) MIU M: th chng ta s dng que cy thng vo mi trng, nu vi khun mc lan khi ng cy thng th VSV c tnh di ng I: Indol c trnh by nh trn bi E.coli Ure: vi sinh vt c kh nng to enzyme urease phn gii ure to thnh NH3 lm pH thay i, dng thuc th phenol red nu c mu hng cnh sen th phn ng dng tnh, mu vng (-)

45

BI 21: PHNG PHP PHN TCH NH LNG BACILLUS CEREUS TRONG THC PHM Nguyn tc Qu trnh phn tch nh lng Bacillus cereus trong thc phm c th c thc hin theo phng php MPN hoc phng php m khun lc. Khun lc ca Bacillus cereus trn mi trng phn lp MYP c xc nh bng cc phn ng sinh ha c trng Bo co thc tp 1. Quy trnh phn tch Bacillus cereus trong thc phm

Mu ng nht mu

Phn lp ( dn u 0,1ml dung dch mu ln 2 a mi trng MYP 300C trong 24h

m s khun lc in hnh

Khng nh

Chn 5 khun lc in hnh trn mi a thch cy vo mi trng thch mu cu

Tnh kt qu

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2. Gii thch c ch ca cc phn ng sinh ha xc nh Bacillus cereus Th nghim MR-VP, c trnh by. Th nghim Tyrosin v Lysozyme khng c tin hnh. Trong bi ny ch gii thch kh nng chuyn nitrat thnh nitric NO3 NO-2 C ch: vi khun to enzyme reductase s kh NO3- thnh NO2 Khi cho thm Gress A v Gress B, nu to ra cc hp cht mu hng l (+), hoc khi thm 2 cht vo, nu kh NO2- thnh N2 v thm vo CH3COOH v Zn m to ra kh H2 th cng l (+). Nu khng mu l (-) 3. Ti sao khi phn tch Bacillus aureus ch cn phn tch nh tnh V trong 1 s tiu chun VN c quy nh s vi khun Bacillus aureus ti a c mt trong thc phm l 102. S lng rt nh nn ch cn kim nghim nh tnh m khng cn kim tra nh lng.

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BI 22: PHNG PHP PHN TCH NH LNG STAPHYLOCOCCUS AUREUS TRONG THC PHM Nguyn tc Nguyn tc xc nh s c mt v nh lng s lng ca Staphylococcus Aureus trong thc phm da vo s xut hin ca khun lc in hnh ( ch nh bi s kh ca kali tellurite leicithin lng trng ) trn mi trng phn lp Baird Parker v phn ng dng tnh coagulase trong dch huyt tng . Bo co thc tp 1. c tnh v c t ca Staphylococcus aureus c tnh: L t cu khun, gram (+), khng di ng v khng sinh bo t Pht trin pH mi trng trung tnh, nhit 370C c t: Chng gy ra nhiu bnh nguy him trn da, trong c th bng cch tit ra c t nh hyaluronidase, hemolysine, leukocidine, exfoloatine, 5 c t enterotocxine A, B, C, D, E. 2. Quy trnh phn tch nh lng Sp.aureus Trong bi thc hnh ny, ch c thc hnh n giai on m khun lc in hnh. rut

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Mu ng nht mu

Pha long Phn lp (Dn u 0,1ml dd mu ln 2 a mi trng BP, 370C, 24h-48h)

m s khun lc in hnh

Khng nh ( chn 5 khun lc in hnh cy vo mi trng)

Coagulase test( cho 0,1ml BHI vo 0,3 ml huyt tng th 370C/4-6h)

ng huyt tng. Mc ng tr ln c xem l dng tnh

Khng hnh thnh khi ng huyt tng( hn hp ng nht

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BI 24: PHNG PHP PHN TCH CLOSTRIDIUM PERFRINGENS TRONG THC PHM Nguyn tc Clostridium perfringens c phn tch bng cch s dng mi trng c cha st v sulphite. Khun lc mc trn mi trng c mu en do phn ng gia S2- v Fe2+ Tr li cu hi 1. c tnh sinh l v gy bnh ca Clostridium perfringen Vi khun Clostridium perfingen l vi khun to bo t, gram (+), khng chuyn ng. a s sng k kh, a nhit. Thy phn mnh protein v chuyn ha acidmin to mi kh chu. Sinh c t trong thc phm v gy bnh hoi t vt thng Triu chng ng c: vim rut v d dy, au bng i ngoi, phn lng hoc ton nc ln mu, thnh thong c nn ma. Thi gian bnh 12-24h 2. Quy trnh phn tch Clostridium perfringens

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Mu

ng nht mu

Pha long mu

Cy mu: chn nng thch hp cy 10ml mu vo 2 ng mi trng thch Wilson Blair b sung thm 0,5 ml Na2SO3 5% v 0,25ml FeSO4 5%. Trn u

un cch thy 750C/15p

Lm ng thch nhanh

Cho 1 ml parafin lng vo ng thch. 370C/18-24h

Khng nh: chn khun lc in hnh, trn,b li, en nhn

Chn khun lc in hnh cy vo mi trng Motility Nitrate. trong iu kin k kh 370C/18-24h

Chn khun lc in hnh cy vo mi trng Lactose gelatine. trong iu kin k kh 370C/1824h

Motility

Nitrate

Lactose

Gelatine

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