MC LC

M U............................................................................................................................................... 1 I. TNH HNH NG C THC PHM TRN TH GII V VIT NAM................................... 2 II. MT S VI SINH VT C TH GY NG C THC PHM .............................................. 6

2.1 Salmonella ...................................................................................................................... 6 2.2 Campylobacter ............................................................................................................... 6 2.3 Clostridium perfringens ................................................................................................. 7 2.4 Clostridium ..................................................................................................................... 7 2.5 Staphylococcus ............................................................................................................... 8 2.6 Vibrio spp ....................................................................................................................... 9 2.7 Escherichia coli ............................................................................................................ 10 2.8 Shigella ......................................................................................................................... 10 2.9 Listeria monocytogenes ................................................................................................ 11 2.10 Cc virus gy bnh trong thc phm ......................................................................... 12 2.11 Coliforms .................................................................................................................... 13 2.12 Nm men v nm mc................................................................................................ 13
III. CC PHNG PHP NH LNG VI SINH VT............................................................... 15

3.1 nh lng vi sinh vt bng phng php m trc tip............................................. 15 3.1.1 m bng bung m hng cu ............................................................................ 15 3.1.2 K thut m Breed............................................................................................... 17 3.1.3 m bng knh hin vi hunh quang..................................................................... 18 3.2 Cc k thut nh lng vi sinh vt trong mu bng phng php nui cy............... 19 3.2.1 Phng php m khun lc ................................................................................. 20 3.2.2 Phng php c on s lng vi sinh bng k thut MPN (Most Probable Number): ........................................................................................................................ 28 3.2.3 Phng php lai khun lc .................................................................................... 29 3.3 Phng php o c ................................................................................................ 31
IV. P DNG I VI MT S VI SINH VT GY BNH TRONG THC PHM ............... 32

4.1 nh lng Fecal Coliform v E. Coli bng phng php m a ............................ 32 4.2 nh lng Vibrio Parahaemolyticus .......................................................................... 33 4.3 nh lng Coliforms, Coliforms chu nhit, E. Coli gi nh (Fecal Coliforms) bng phng php MPN ............................................................................................................. 35 4.4 nh lng Enterococci ng rut trong nc bng phng php mng lc ........... 37 Thit b: .......................................................................................................................... 37 4.5 nh lng Clostridium kh sunfite trong nc bng phng php tng sinh trong mi trng cy lng................................................................................................................... 38
KT LUN ........................................................................................................................................ 40

TI LIU THAM KHO .................................................................................................. 41 PH LC ........................................................................................................................... 42

M U
Cc vi sinh vt gy bnh nhim trong thc phm thng gp gm cc vi khun Salmonella Shigella, E.coli, Bibrio choterea, Clostridium botulinum, Clostridium perfringens; cc vius Hepatis, virus Hepatis virus A, Rotavirus; cc k sinh trng Amip, sn l gan, sn b, trng lng; cc nm mc v nm men Aspergillus, candida, Furanium... Vi sinh vt gy bnh nhim trong thc phm bng 4 con ng chnh: qua sc vt, qua mi trng, ch bin v bo qun. Mc nguy him v triu chng ca bnh c th gy nn do c t ca vi sinh vt tit vo thc phm hay do chnh t bo ca chng gy nn. c th gy ng c thc phm, vi sinh phi hin din vi s lng t bo ln v ph thuc liu lng ca tng chng loi nhim vo, thc phm phi c cc u kin l ho thch hp cho vi sinh vt pht trin, nhit v thi gian phi thch hp cho qu trnh tng trng ca chng t khi chng nhim vo cho n khi tiu th vi sinh vt nhn ln n liu lng hay sn xut lng c t gy hi. Khi nhim thc phm, vi sinh vt gy h hng lm thc phm b i mu, i v, c mi. Tuy nhin cng c mt s loi gy nhim thc phm nhng khng lm thay i mu, mi, v hay hnh dng bn ngoi ca thc phm. V vy rt kh nhn bit bng cm quan. Do , vic xc nh v nh lng cc vi sinh vt gy bnh nhim trong thc phm l iu rt cn thit.

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I. TNH HNH NG C THC PHM TRN TH GII V VIT NAM

cc nc pht trin c ti 10% dn s b ng c thc phm v mc bnh truyn qua thc phm mi nm; vi cc nc km pht trin t l ny cao hn nhiu. Nhiu nc c quy nh bo co nhng ch t 1% s ca b ng c thc phm. Ng c thc phm M chim 5% dn s/nm (>10 triu ngi/nm), trung bnh 175 ca/100.000 dn, mi nm cht 5.000 ngi; Anh: 190 ca/100.000 dn; Nht: 20-40 ca/100.000 dn; c l 4,2 triu ca/nm. Thc trng vi phm v sinh an ton thc phm nc ta rt ng bo ng. Ng c thc phm cp tnh trong nhng nm qua vn c chiu hng gia tng c v s v v quy m mc. T l mc/100.000 dn trung bnh t nm 2001 2005 l 5,48. C nhiu nguyn nhn gy ra cc v ng c thc phm trong ton quc nh thc phm nhim, mi trng nhim; thc phm c c; iu kin sn xut, ch bin thc phm khng bo m an ton, nhn thc hnh vi ng v phng chng ng c thc phm ca cng ng cn nhiu hn ch Trung bnh mi nm c 202.2 v ng c thc phm xy ra vi 5.525,1 ngi mc v 55.2 ngi cht. S v ng c xy ra nhiu nht l t thng 4 - 7 v thng 9 11. T l mc ng c trung bnh l 7.14/100.000 dn, t l cht l 0,06/100.000 dn/nm. S v ng c ln ( 30 ngi) chim 26,8% vi s mc chim 83,2%. V ng c nh v va (<30 ngi) chim 73,2% vi s mc chim 16,8%. T l mc ng c chim 18.7% tng s i tng cng n chung ba n; t l cht l 0,8% tng s i tng b mc ng c thc phm. Mi la tui u c th b ng c, tui nh (0 - 4 tui) v tui cao 50 c nguy c mc v cht cao do ng c Biu hin chung trong cc v ng c l bun nn (81,0%), nn (83,9%), au bng chim 79.0%, a chy 72.2 %, au u chim 53.2%, chng mt 43,4%, st 26,3%. C s nguyn nhn ng c ch yu l gia nh (54,6%), bp n tp th (15,6%), m ci/gi (16,6%), thc n ng ph (5,4%), bp n trng hc (4,0%). Thc n nguyn nhn ch yu l thc phm hn hp (40,0%); thu sn 14,1%; nm 13,2%; ng cc v cc sn phm l 7,8% Nguyn nhn ng c ch yu l nguyn nhn vi sinh vt (34,0%), c t t nhin (24,0%), ho cht (10,0%). Cn 32,0% s v khng xc nh c nguyn nhn. Vi sinh vt gy ng c l Salmonella, Streptoccocus, E.coli, Staphylococcus aurerus v Vibrio parahaemolyticus; c t t nhin ch yu l c t ca nm c (13,2%).

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Thi gian bo co trung bnh ca v ng c l 9.74 ngy. Vi v ng c thc phm 30 ngi l 8.53 ngy (0-31 ngy), vi v ng c <30 ngi l 10,20 ngy (1-37 ngy). Ly mu xt nghim nguyn nhn v ng c t t l rt thp: Mu thc phm l 47,3%, mu bnh phm l 23,9 %; dng c, bao gi t t l thp 1,5 % cc v ng c.
Bng 1: S lng cc v ng c thc phm ton quc t nm 2000 2008 Kt qu iu tra Nm V ng c (v) 2000 2001 2002 2003 2004 2005 2006 2007 2008 Trung bnh/nm Tng cng 213 245 218 238 145 144 165 247 205 202.2 (247 - 144) 1.820 S mc (ngi) 4233 3901 4.984 6.428 3.584 4.304 7.135 7.329 7.828 5.525,1 (7.828 - 3.584) 49.726 Cht (ngi) 59 63 71 37 41 53 57 55 61 55.2 (71 31) 497

Bng 2. Tnh hnh ng c ti H Ni trong 3 nm gn y S v ng c Nm S v 2 8 8 S ngi mc 41 137 354 S ngi cht 0 0 0 Vi sinh vt 2 7 8 Nguyn nhn gy ng c Ha Thc cht c t phm tn d t bin trong nhin cht thc phm 0 0 0 0 1 0 0 0 0

Nguyn nhn khc 0 0 0

2006 2007 2008

Cng

18

620

17

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Bng 3: Nguyn nhn trong cc v ng c thc phm trong nm 2007

TT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Nguyn nhn ng c Salmonella Streptoccocus E.coli Staphylococcus aurerus Vibrio parahaemolyticus Thuc dit chut Ho cht c t ca c nc c t con so c t ca con sam c t trong tht cc c t Histamin c t trong mm tp c t trong ru c t ca L ngn Nm c Khng r nguyn nhn Tng cng

Kt qu iu tra S lng 3 2 4 7 1 0 1 8 2 2 2 3 0 5 2 27 136 205 T l (%) 1.5 1.0 2.0 3.4 0.5 0.0 0.5 3.9 1.0 1.0 1.0 1.5 0.0 2.4 1.0 13.2 66.3 100,0

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Salmonella

Shigella

E. coli

Vibrio

Staphylococcus

Campylobacter

Clostridium

Listeria

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II. MT S VI SINH VT C TH GY NG C THC PHM

2.1 Salmonella Salmonella Trc nm 1983 cc nh khoa hc chia Salmonella ra lm 3 loi da theo phn ng sinh ha m chng tham gia : S.typhi, S.choleraesuis,S. enteridis. Salmonella l trc khun garm (-), hiu kh v k kh ty , c kh nng di ng khng to bo t, ln men glucose v manitol sinh acid nhng khng ln men sacarose v lactose, khng sinh indole, khng phn gii ur khng c kh nng tch nhm amin t tryptophane, hu ht cc chng u sinh H2S. pH ti u cho chng pht trin nm trong vng trung tnh, tuy nhin i khi pht trin trong vng pH t 4 9, khng c kh nng pht trin nng mui cao. Vi khun Salmonella tn ti trong c th nhiu loi ng vt v c th gy bnh cho ngI. Vi khun Salmonella c th gy bnh truyn nhim cho ng vt nh ph thng hn b, ph thng hn ln, bnh sy thai cu v nga, bnh bch l g, bnh vim rut b, Nhng bnh k trn do Salmonella gy ra ngi ta gi l bnh truyn nhim nguyn pht, ngoi ra c th ph hin c Salmonella trong cht bi tit ca ng vt. S lng Salmonella gy ng c l khi chng hin din c triu t bo trong mt gam thc phm. Cc triu chng do Salmonella gy ra thng l tiu chy, i ma, bun nn. Thi gian bnh cho n khi cc triu chng biu hin thng sau 12-36 gi k t khi tiu th thc phm b nhim. Triu chng thng ko di t nht t 2-7 ngy. Khng phi tt c mi ngi khi tiu th thc phm b nhim Salmonella iu c biu hin bnh, ngc li mt s ngi khng c triu chng lm sng khi tiu th phi thc phm nhim vi sinh vt ny khi chng c bi tit ra ngoi. Cc loi thc phm c nguy c b nhim Salmonella nh tht gia cm, sn phm tht, trng v cc sn phm ca trng, thy sn. Ngun nhim vi sinh vt vo cc loi thc phm thng c ngun gc t ng rut ca ngi v cc loi ng vt, chng c th c nhim gin tip hay trc tip. Salmonella gy nn bnh st thng hn thuc cc serotype Salmonella typhi, Salmonella paratyphi A, B, C. cc dng ny thng khng gy bnh cho cc loi ng vt. 2.2 Campylobacter y l vi sinh vt gy nn bnh vim nhim ng rut, bng cc phng php phn lp chng minh vi sinh vt ny hin din khp ni. Campylobacters l mt trong nhng h vi sinh vt ca nhiu loi ng vt v chim. Nhng cc dng c kh nng gy ng c thc phm khng th pht trin khi nhit thp hn 300C, y l vi sinh vt a nhit bt but. Sn phm
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sa v tht gia cm l nhng ngun c th gy nn ng c do vi sinh vt ny. Nc cng l mt trong nhng ngun mang bnh ny. Campylobacters l vi sinh vt rt nhy vi nhit , chng b tiu dit hon ton bng phng php thanh trng Pasteur, chng khng th sng st trong thc phm c mi trng acid. Chng khng th pht trin trong thc phm bo qun trong iu kin hiu kh m ch pht trin trong cc loi thc phm ht chn khng. Khi xm nhim Campylobacter, thi gian bnh thng t 2-11 ngy. Cc triu chng do vi sinh vt ny gy nn nh au nhc, tiu chy, st, au u, kh chu, chut rt, lnh cng, m sn. Thnh thong c nhng biu hin bnh ging nh cm cm. 2.3 Clostridium perfringens Quan nim v s ng c thc phm do Clostridium perfringens gy ra c nhng thay i trong nhng nm gn y. Theo nhng quan nim trc y cho rng cc dng C.perfringens khng nhit, to bo t v khng lm tan mu mi c th gy ng thc phm. Nhng trong nhng nm gy y cc dng nhy cm vi nhit, khng lm tan mu cng c tm thy trong cc v ng c do vi sinh vt ny gy nn. V cc bo t ca C. perfringen khng nhit nn chng thng sng st qua qu trnh nu chn. Tuy nhin cng ph thuc vo thi gian tip xc vi nhit. Nu nhng bo t sng st, khi gp iu kin thch hp chng s ny mm v nhn ln. Khi un nu thc n nhit thp v thi gian ngn c th lm cho cc dng khng nhit tn ti v th chng s gy ti nhim sau khi bo qun. Cc ngun thc phm c th gy ng c vi cc vi sinh vt ny thng l tht gia cm, nht l cc loi gia cm ln ng lnh su, tht trong cc hm cha. C. perfringens cng c tm thy trong t, trong phn ngi v trong cc loi thc phm khc. Cc triu chng do vi sinh vt ny gy ra thng l au tht vng bng, tiu chy. Thi gian bnh t 12-24 gi. Cc triu chng lm sng gy nn do c t ca chng. 2.4 Clostridium Clostridium l cc vi khun garm (+), hnh que, k kh sinh bo t , khng di ng, c th thy gii saccaride v protein trong cc hot ng thu nhn nng lng to ra cc sn phm nh acid acetic, butiric, ru, to ra cc mi kh chu trong sn phm. Clostridium pht trin mnh nhit 55oC, nhit ti u l 43 47oC. Nhit 15 20oC lm chm hoc lm ngng s pht trin ca vi khun ny. Khng pht trin pH hn 5,0 hoc trn 9,0, b c ch bi 5% NaCl. Clostridium hin din trong t, mt s loi trong nhm ny gy bnh cho ngi v ng vt, mt s loi khc gy h hng thc phm, kh sunphit thnh sunphur to ra mu em v gy mi kh chu.

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Clostridium botulinum l vi sinh vt sinh c t gy bnh ng c tht cho ngi (botulism). Bnh biu hin rt nghim trng ngi. Bnh gy ra do c t c hnh thnh bi C.botulinum nhim trong thc phm. Triu chng lm sng ca bnh l i ma, bun nn, sau c nhng biu hin ri lon thnh kinh nh chong vng, ri lon th gic, ri lon cc c c v ming, au vng ngc, kh th v t lit, c th dn n t vong. Cc triu chng trn biu hin sau 12-36 gi sau khi tiu th thc phm nhim c t. Cc triu chng thng ko di 2-6 ngy tu theo tnh trng nhim c v sc kho cng tng bnh nhn. Cc loi thc phm nh tht, rau qu khng c bo qun ng qui nh hay ly nhim t t, phn ng vt hay do ch bin khng nhit trc khi dng, cc sn phm ng hp khng ng qui cch cng c nguy c nhim vi sinh vt ny ry cao. iu kin thch hp cho vic hnh thnh c t ca vi sinh vt ny iu kin mi trng k kh, pH trung tnh, khng c cc vi sinh vt khc cnh tranh. c t botuline do C. botulinum tit ra gm mt s loi khc nhau nh A, B, C1, C2, D, E, F, G. cc c t ny l nhng protein c trng lng phn t ln khong 1 triu danton. Nhng nhng dng c tc ng mnh n con ngi l A, B, v E. y cng l mt trong nhng loi c t sinh hc c cng mnh nht. Trong nhng nm gy y, cc v ng c botulism gy ra do C.botulinum dng E thng c pht hin khi tiu th c v cc sn phm thy sn. Dng vi sinh vt ny thng xuyn phn lp c t cc mu bn y ti cc ca sng. 2.5 Staphylococcus Staphylococcus l loi cu khun gram (+), cc t bo ca chng lin kt thnh hnh chm nho. Khi pht trin trong mi trng, Staphylococcus c kh nng to ra sc t t mu trng n vng sm, nhit 20 25oC thch hp nht cho chng to mu. Staphylococcus khng di ng, khng to bo t, nhit thch hp cho s pht trin l 37oC, chu c s kh hn, hi nng ( nhit 50oC chng vn sng trong 30 pht), c kh nng sng nng mui 9 10%. Staphylococcus pht trin pH rt rng (pH = 4,0 9,8). Khong pH ti u ca chng l 6 7 v aw ti u khong 0,83 0,86. Staphylococcus c kh nng ln men v sinh acid t manitol, trehalose, sucrose. Staphylococcus c phn b khp ni nhng ch yu c phn lp t da v mng nhy ca ngi v ng vt mu nng. Staphylococcus c th nhim vo trong 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 Staphylococcus trong thc phm ch th iu kin v sinh v kim sot nhit km ca qu trnh ch bin. Staphylococcus aureus l VSV c kh nng sn sinh mt s loi c t ng rut bn nhit, khng b phn hu khi un 100oC trong khong 30
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pht. Khi vi sinh vt ny xm nhim vo trong thc phm, chng tit c t vo trong sn phm v gy c. Khi con ngi tiu th loi thc phm c cha c t ny, sau 4-6 gi bnh s bc pht cc triu chng lm sng nh tiu chy, nn ma, cc triu chng ny ko di t 6-8 gi. Cc loi thc phm c cha hm lng mui cao thng c nguy c nhim vi sinh vt ny nh jambon, kem tng hp, nc soup v cc loi thc phm ny t khi c x l nhit cao hn 40oC. Cc loi thu sn hay thc phm ng hp cng thng hay b nhim loi vi sinh vt ny. Cc ngun ly nhim vo thc phm ch yu t cc khu ch bin trong nh bp. Trong t nhin cc vi sinh vt ny thng tnh thy trn da, mi, tc hay lng ca cc loi ng vt mu nng. 2.6 Vibrio spp Cc loi Vibrio c ngun gc t bin, chng cn ion Na+ pht trin. Ging Vibrio c mt s loi c kh nng gy bnh cho ngi nh V. cholerae, V. parahaemolyticus, V. vulnificus, V. hollisae, V. furnsii, V. mimicus, V. fluvialis, V. alginolyticus. V. cholerae l tc nhn gy nn cc v dch t trn ton th gii. Loi vi sinh vt ny c chia thnh hai kiu huyt thanh chnh l O1 v non-O1, kiu huyt thanh O1 bao gm ba kiu huyt thanh ph nh sau: Ogawa; Inaba (hai kiu ny c gi chung l kiu c in Classic) v kiu Eltor (kiu Eltor cn c gi l kiu O139). Hai kiu huyt thanh Inaba v Ogawa ngy nay ch cn c tm thy ti cc nc thuc khu vc chu A. Trong khi cc v dch t trn khp th gii gy ra do kiu Eltor. Khi c cc trn dch do V. cholerae gy ra thng lan truyn rt nhanh vo trong nc, gy nhim vo thc phm, nu iu kin v sinh km, vi khun s lan truyn qua con ngi v dch bnh cng thm nghim trng. Vi sinh vt ny sn sinh c t cholaratoxin, y l loi c t ng rut c cng mnh, ch cn 5g gy nhim qua ng ming c th gy tiu chy cho ngi trng thnh. Mt s c t khc cng c vi sinh ny tit ra nh hemolysine c c tnh tng t tetrodotoxin (c t c nc) hay c t tng t shiga-toxin. Cc loi thc phm c th lan truyn V. cholerae nh nc ung, nc tri cy, rau qu, sa v cc sn phm sa, thm ch bia cng c kh nng nhim vi sinh vt ny. Cc loi sn phm thu sn ti sng, khng qua gia nhit, gia nhit nh hay do s nhim cho sau khi gia nhit cng c khuyn cc l c nguy c mang V.cholerae kh nghim trng. V. parahaemolyticus l loi vi sinh vt tn ti v pht trin trong mi trng c hm lng mui cao, chng thng xuyn c phn lp t cc sn phm thy sn, trong cc vng nc m ven b bin. Chng sn sinh c t hemolysine bn nhit, cht ny chu trch nhim cho c tnh khng nguyn Kanagawa. Nhng trong nhng nm gn y cc dng V.parahaemolyticus c phn ng Kanagawa m tnh cng c th gy bnh. Triu chng biu hin ca
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bnh c th xut hin trong khong 2-96 gi sau khi tiu th thc phm b nhim, thi gian ny ph thuc vo liu lng xm nhim v th trng ca tng bnh nhn, loi thc phm tiu th v hm lng acid trong d dy. Cc biu hin bnh l khi vi sinh vt ny xm nhim v au tht vng bng, vim nhim ng rut v tiu chy nh. Cc loi Vibrio khc khi xm nhim vo trong thc phm cng c th gy nn cc bnh ng rut v c biu hin bnh l tng t nh hai loi trn. D nhin tu tng loi v liu lng m c nhng biu hin bnh nng nh khc nhau. Ch ring loi V. vulnificus khng gy cc triu chng bnh ng rut m chng gy nhim trng mu cho ngi. 2.7 Escherichia coli E.coli thuc h Enterbacteriaceae, catalose (+), oxidase (-), gram (-), trc khun ngn, khng to bo t, chu c nhit, khng b hy khi un nng 100oC trong 2 gi. E.coli c th khng cn, khng b hy khi tip xc vi cn 50%, b hy bi focmol 5%, c kh nng pht trin nhit t 30 50oC, nhit pht trin ti u ca chng l 37oC, pht trin pH ti u l 4,4 v aw ti u l 0,95. E. coli l vi sinh vt hiu kh ph bin trong ng tiu ho ca ngi v cc loi ng vt mu nng. Hu ht cc dng E. coli tn ti mt cch t nhin v khng gy hi trong ng tiu ho, ngc li chng cn ng vai tr quan trng trong vic n nh sinh l ng tiu ho. Tuy nhin c t nht 4 dng sau y c th gy bnh cho ngi v mt s loi ng vt: Enterobathogenic E. coli (EPEC) Enterotocigenic E. coli (ETEC) Enteroinvasive E. coli (EIEC) Enterohaemorrhagic E.coli (EHEC)/ verocytocin E.coli (VTEC) hay Ecoli O157: H7 Cc dng E. coli gy bnh khi chng xm nhim vo ngi qua con ng thc phm c th gy nn cc bnh ri lon ng tiu ho, cc biu hin lm sng bin ng c th t nh n rt nng, c th e do mng sng ca con ngi ph thuc vo liu lng, dng gy nhim v kh nng p ng ca tng ngi. 2.8 Shigella Ging Shigella cng l mt thnh vin ca h vi khun ng rut Enterobacteriacae, chng gm c 4 loi sau: S. dysenteriae, S. sonnei, S. plexneri, S. boydii. y l ging vi sinh vt c t bo ch c hiu, chng ch thch nghi v pht trin trong t bo ch l ngi v cc loi linh trng. S hin din ca chng trong mi trng l do s nhim phn ca ngi v cc loi mang vi sinh vt ny. Shigella c th tn ti hn 6 thng trong mi trng
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nc. Cc v ng c thc phm do Shigella gy ra ch yu tp trung cc nc km pht trin, ch bin thc phm trong iu kin km v sinh. Bnh cng c th truyn trc tip t ngi qua ngi. Shigella ch yu gy nn cc bnh l trc trng. Thi gian bnh sau khi tiu th thc phm b nhim l 1-7 ngy. Cc biu hin triu chng bnh c th nh, biu hin khng r, ch thong qua nh tiu chy nh, nhng i khi cng c nhng biu hin nghim trng nh i tiu ra mu, c nhng mnh nim mc rut, mt nc, st cao v b co rt thnh bng. Cc triu chng trn c th ko di 12-14 ngy hay lu hn. i vi ngi ln, cc trng hp t vong do Shigella him khi din ra, nhng bnh biu hin rt nghim trng i vi tr em v ngi gi. Hng nm c khong na triu ngi t vong do vi sinh vt gy ra trn khp th gii. S ly nhim vi khun Shigella ch yu ng ming. Nc l mt mi trng truyn bnh quan trng, c bit nhng ni km v sinh. Tuy nhin cc loi thc phm cng l nguyn nhn gy nn cc bnh do Shigella. Vi sinh vt ny nhim vo thc phm qua nguyn liu hay qu trnh ch bin. i khi nhim bnh do v sinh c nhn km. 2.9 Listeria monocytogenes Trong nhng nm gn y L. monocytogenes ni ln nh mt mt tc nhn gy bnh nguy him. i tng gy bnh ca vi sinh vt ny l tr em, ph n mang thai hay nhng ngi gi. i vi nhng vi sinh vt gy bnh g c thc phm khc, chng bc pht bnh khi con ngi hp thu liu lng, sau thi gian bnh cc triu chng lm sng biu hin. Ngc li L. monocytogenes hin din vi mt s lng nh trong thc phm, khi c a vo c th, chng tn ti v ch c hi. Khi c iu kin thun li, chng nhn ln xm nhim vo cc m su v gy bnh. Cc bnh do vi sinh vt ny gy nn bt u t ng tiu ho nh tiu chy, st nh. Sau chng xm nhim vo cc i thc bo gy nn bnh nhim trng mu, tc ng ln h thn kinh trung ng, tim mt, v c th xm nhp vo bo thai gy nn sy thai, non hay nhim trng thai nhi. L. monocytogenes thuc loi vi sinh vt a lnh, chng c th pht trin nhit t 2-44oC. Chng thng c phn lp t cc loi thc phm nh phomat sa, tht c rau qu v thm ch phn lp c t trong nc mt. Trong tt c cc cng on ch bin thc phm, sa hay rau qu u c kh nng xm nhim vi sinh vt vt ny. t bit trong cng on bo qun cc sn phm nhit thp, vi sinh vt ny c c hi pht trin thnh s lng ln. Cc sn phm thanh trng Pasteur v c bo qun nhit thp trong cc t lnh c nguy c nhim vi sinh vt ny rt cao.

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2.10 Cc virus gy bnh trong thc phm Cc t dch bnh gy ra t thc phm do tc nhn virus cho n nay vn l vn b n. Nhng mt s tc gi vn tin rng virus trong thc phm l tc nhn gy nn cc bnh him ngho. Nhng tin b trong cc nhin cu v virus thc phm vn cn hn ch. Cho dn nay cc c im sinh l ca virus ng rut vn cn bit rt hn ch. Cho n nay cc phng php nui cy pht hin virus trong thc phm cho n nay vn cha th thc hin c. Nhng vi cc tin b v k thut sinh hc phn t nh k thut lai phn t, k thut PCR c th pht hin c cc virus c hi cho con ngi trong thc phm. S lan truyn virus cho ngi qua con ng thc phm c bit t nhng nm 1950. Cc virus gy bnh ng rut cho ngi ch yu c ngun gc t cc sn phm thu sn. Cho n nay c bit c khong hn 100 loi virus ng rut. Nhng ch mt vi loi trong s c kh nng gy bnh cho ngi. Theo Kilgen v Cole (1991) cc loi virus sau y c th gy nguy him cho ngi. Hepatitis type A (HAV) Virus Norwalk Calicivirus Astrovirus Virus NonA v Non B. Virus tn ti th khng hot ng khi bn ngoi t bo, chng khng th t nhn ln trong nc hay trong cc sn phm thc phm cho d bt k iu kin ho l nh th no. Chng xm nhim vo thc phm hon ton do qu trnh ch bin, t nc b nhim. Cc loi nhuyn th n lc c kh nng tch lu nhiu virus trong nc. Hng ngy mt con nguyn th c th lc 1500 lt nc, theo mt s lng ln virus c th vo c th ca con vt ny v tch lu ti . V th mt virus trong c th nhuyn th cao hn rt nhiu so vi mi trng nc chng ang sinh sng. Liu lng gy bnh ca virus c th thp hn rt nhiu so vi vi khun khi con ngi tiu th thc phm b nhim. Liu lng gy nhim ti thiu ca mt s loi vurus ng rut tng ng vi s lng hin din trong thc phm m cc phng th nghin c th pht hin c bng phng php nui cy. C th ngi v cc loi ng vt l ngun cha cc virus ng rut. Virus c tm thy vi s lng ln trong phn ca nhng ngi b nhim v tn ti trong nhiu ngy n nhiu tun. S nhim phn vo trong thc phm bng con ng gin tip hay trc tip l con ng xm nhim virus vo thc phm. S sng st ca virus trong mi trng hay trong thc phm ph thuc vo yu t nh nhit , nng mui, cng bc x mt tri hay s hin din ca cc thnh phn hu c khc. Virus ng rut cng c kh nng tn
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ti nhiu thng trong nc bin nhit < 10oC thm ch c th lu hn. V th y cng l nhn t ch th nhim phn. Tt c cc virus ng rut u khng vi acid, cc enzym thy gii, hay mui mt c trong ng tiu ho. Mt s virus c th khng nhit nh Hepatits type A, mt s khc c th khng vi cc cht ty u nh phenolic, ethanol . Ozon v chlorine l nhng tc nhn c th lm bt hot mt vi loi virus ng rut. ngn nga cc bnh do virus t thc phm, cc loi thc n phi c nu chn,kh virus trc khi tiu th, cc loi nhuyn th n lc phi c khai thc tronng nhng vng nc khng nhim virus hay c nui trong cc vng nc sch trc khi tiu th. 2.11 Coliforms Coliforms l nhng trc khun hnh gy, gram m (-), khng sinh bo t, hiu kh hoc k kh ty . Chng c kh nng pht trin nhit rt rng t -2oC n 50oC, pH trong khong 4,4 9,0. Coliforms c kh nng ln men lactose sinh acid v sinh hi 37oC trong 24 48gi. Coliforms hin din rng ri trong t nhin trong rut ngi, ng vt. Khi s Coliforms ca thc phm cao th kh nng hin din ca cc vi sinh vt gy bnh khc cng cao. Coliforms chu nhit l nhng Coliforms c kh nng ln men lactose sinh hi trong khong 24 gi khi c 44oC tromg mi trng canh EC. Coliforms phn l Coliforms chu nhit c kh nng sinh indole khi c khong 24 gi 45,5oC trong canh Trypton. Coliforms phn l mt thnh phn ca h vi sinh vt ng rut ngi v cc ng vt mu nng khc v c s dng ch th mc v sinh trong qu trnh ch bin, bo qun, vn chuyn thc phm, nc ung. Cn c vo nhit vi sinh vt c th pht trin chia nhm Coliform thnh hai nhm. Nhm Coliform c ngun gc t phn ca cc loi ng vt v, nhm ny c gi l Coliform phn v nhm khng c ngun gc t phn ng vt. Trn thc t, cc phng php kim nghim ch xc nh Coliform phn l xc nh nhm coliform c ngun ngc t rut ngi v cc ng vt mu nng bao gm cc ging nh Escherichia; Klebsiella v Enterobater. 2.12 Nm men v nm mc Nm men v nm mc l nhm vi sinh vt rt a dng, y l nhm vi sinh vt nhn tht c vch t bo v lp v chitin, c nhn v cc bo quan khc. Tt c cc loi nm men v nm mc u thuc nhm vi sinh vt d dng. Chng ch c kh nng nhn cc cht dinh dng dng ha tan. Trong qu trnh trao i cht xy ra t bo chng li c kh nng chuyn ha cc cht ha tan thnh cc cht khng ha tan nh lignocellulose,. Ngoi ra, chng cn c th to ra cc cht c, cc cht c ca chng c gi chung l
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c t vi nm (mycotoxins). Tiu biu c Aspegillus flavus, Aspegillus parasiticus, Aspegiluus moninus, Penicillium italicum, Penicillium digitatum, Penicillium roquefortii, Penicillium cammenbertii. Trong thc phm nm mc v nm men hin din c th tng trng lm thay i mu ca thc phm, lm pht sinh mui hay v l, lm h hng hay thay i c cu ca thc phm, mt s c th to c t gy ng c thc phm.

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III. CC PHNG PHP NH LNG VI SINH VT

3.1 nh lng vi sinh vt bng phng php m trc tip S lng Vi sinh vt trong mu c th c xc nh bng cch m trc tip trn knh hin vi. Qui trnh n trc tip cho php xc nh c s lng vi sinh vt vi kt qu cao nht. Mt d phng php m trc tip bng knh hin vi cho php c lng nhanh chng s lng vi sinh vt c trong mu. Tuy vy phng php ny c nhng im hn ch nht nh nh khng phn bit c s lng t bo sng v s lng t bo cht, d nhm ln t bo vi sinh vt vi cc mnh v nh ca mu v khng cho php tm hiu cc c im khc ca ca vi sinh vt c c quan st. 3.1.1 m bng bung m hng cu i vi cc vi sinh vt c kch thc ln nh nm men, nm mc, to v protozoa, phng php m trc tip d dng thc hin vi cc loi bung m. C rt nhiu loi bun m vi sinh vt khc nhau ph hp vi tng th tch v kch thc ca tng nhm vi sinh vt. V d c th s dng bung m hng cu Petroff Hauser m vi khun. i vi cc mu nc v cc mu khc, bung m Holber c s dng rng ri nht. Bung n Holber l mt lam knh dy 2-3mm c mt vng a m nm gia lame knh vng ny c bao quanh bi mt rnh. a m thp hn b mt ca lame knh khong 0,02mm, c hnh trn v th khi ph ln trn bng knh y vt th su ca a m s ng u nhau. Vng a m c in tch 1mm2 v c chia thnh 400 vung nh hn, mi c din tch 0.0025mm2. th tch ca mi l 0,02 x 0,0025 mm2, th tch ny tng ng vi 0,00005ml. Thm vi git formalin vo trong nhng l cha mu, trn u. Pha long mu cn m sao cho trong mi nh ca bung m c khong 5 10 t bo vi sinh vt. t c pha long nh vy cn phi c lng c s lng vi sinh vt trong mu, ng thi phi th vi ln trong qu trnh pha long. Mu phi c pha long bng dung dch pha long cha 0,1% pepton v 0,1% laurylsulphat v 0,01% methyl blue. Tt c cc dung dch pha long u phi cn lc trc khi s dng.

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t mt git mu c pha long vo trong a m trn bung m, y bng knh y vt. Tt c cc bung m v knh y vt phi c lau tht sch. Th tch mu cha trong bung m l khong khng gian gia a m v knh y vt, khng mu trng ra bn ngoi rnh ca a. Sau khi t mu, yn khong 5 pht n nh v tr ca cc t bo trong bung m. m ngu nhin khong 50-100 nh. Tnh trung bnh s lng vi khun trong tt c cc m. Sau nhn vi 20 000 v vi pha long mu trc khi m c c s lng t bo trong 1ml. Lp li hai ln hay nhiu hn ly gi tr s m trung bnh. i vi cc vi sinh vt t thnh tng cm, khng th phn bit tng t bo th mi cm c xem l mt vi khun. Vi nhng kinh nghim ca mi c nhn v pha long, thao tc k thut thnh tho c th nhn c cc s m chnh xc. Tuy vt kt qu c chnh xc hay khng cn ph thuc vo s sch s ca bung m, knh y vt nhm m bo khng c vi khun khc khng phi t mu hin din trong bung m v trong knh y vt. 3.1.2 K thut m Breed K thut ny rt hu ch trong vic xc nh tng s vi sinh vt bng phng php m trc tip. Lame knh Breed c mt vng c din tch 1cm2 c nh du trn lame. Dng bm tim, hay que cy vng cho 0,01ml mu cn m vo trong vng ny, sy kh bng ngn la sau nhum vi methyl blue. Khi m, cho du nhng ln din tch vng cn m. Thng thng vng ny c ng knh 0,16 mm. Din tch vng m l pr2 hay 3,14 x 0,08 x 0,08 = 0,02mm2. Vi din tch ny chim 1/5000 trong tng din tch t mu l 100mm2 hay th tch trong vng m l 1/5000 x 0,01ml. Nh vy nu c 1 vi sinh vt trong vng m th tng ng vi 5000/0,01ml hay 500 000 t bo/ml. S m ca cc vi sinh vt trong cc vng khc nhau ca vng qui c c tnh bng cng thc nh sau:

Nx4x104 C= d2 d: ng knh va vng m N: s lng t bo trong 1 vng C: s t bo trong 1ml Khi s dng knh hin vi hunh quang vi cc cht nhum khc nh acridin cam (AODC), 4,6-dianidino-2-phenyl-indol (DAPI), fluorescein isothiocyanate (FITC) cng c s dng rng ri m s lng vi khun. Cc nghin cu ca K.G. Porter v Y.S. Feig cho thy rng khi s dng DAPI m s lng vi khun trong nc cho kt qu tt hn khi s dng cht nhum AODC khi trong mu nc c nhiu cht dinh dng v nhiu phiu sinh vt khc. V bn tnh pht hunh quang ca DAPI cng cao hn
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AODC. Hai tc gi trn cng chng minh rng khi nhum vi DAFI c th bo qun trong ti n 24 tun m cng pht quan khng thay i. Trong khi AODC cng v s lng pht quang b gim khi bo qun trong vng 1 tun cng iu kin. 3.1.3 m bng knh hin vi hunh quang Mt k thut khc da trn nguyn tc nhum mu c hiu DNA c gi l Hoechst 33258 cng c s dng nhum v m s lng vi sinh vt trn knh hin vi hunh quang. Phng php ny nh sau: Bisbenzumide kt hp vi vng DNA giu adenine v thyamine sau khi gn phc hp ny s pht hung quang. Hoechst 33258 c s dng nhiu trong vic nh lng vi sinh vt trn b mt. t bit trn b mt c gn cc acridine orange. Mt s thun li khc khi s dng phng php nhum mu bisbenzimide cho php m s lng vi sinh vt trong cc dng dch cht ty ra, trong mui dng cho cc phng th nhim hay trong cc ch phm sinh hc khc m khng c s hin din ca DNA. Phng php m s lng vi sinh vt bng phng php pht hunh quang c ng dng rng ri trong vic phn tch cc mu mi trng nh nc t . S dng du nhng pht quang thp l mt pht minh quan trng trong vic hn ch s pht hunh quang ca cc mng lc. Mng carbonate Nuclepore l mt mng cellulose cao cp dng trong vic m trc tip vi khun bi v chng c kch thc l ng nht v b mt mng phng, chng c th gi li tt c cc vi sinh vt trn b mt ca chng. Mng Nuclepore c th nhum vi thuc nhum Irgalan en hay cc loi thuc nhum ph hp khc to nn mt nn mu en tng phn vi mu pht quang ca vi sinh vt v th vic m c thc hin d dng. Khi s dng thuc nhum l acridine cam vi khun v cc vi sinh vt khc pht quang mu xanh hay mu cam. Mu pht quang xanh hay cam l ph thuc vo tnh trng sinh l ca tng vi sinh vt. Nhng kh nng phn bit t bo ang sng hay t bo cht bi mu pht quang i khi a n s nhm ln. S dng ADPI nhum DNA ca t bo vi khun chng a n s pht quang mu xanh dng c cho l ti u hn so vi vic s dng acridin cam nhum cc vi khun c kch thc nh. S m nhn c t phng php m trc tip trn knh hin vi pht hunh quang lun cho kt qu cao tng ng hai ln so vi kt qu nhn c bng k thut nui cy khc. Nhng vi khun c kch thc nh, c hnh dng bt thng khc cng u c th nhn thy bng phng php pht hunh quang. Mt s dng vi sinh vt khng th nui cy trong cc mi trng tng hp trong phng th nghim. Hin tng khng th nui cy ca cc vi sinh vt cho n nay vn cha r l do chng thoi ho, khng th nhn ln trong cc mi trng nhn to hay do khng p ng c cc iu kin sinh l cn thit
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cho s pht trin ca chng. Trong mt s trng hp, s tng quan cao gia phng php m trc tip bng phng php pht hunh quang v phng php m khun. Tuy nhin trong mt s trng hp khc s tng quang ny rt km. S khc bit gia phng php m trc tip v phng php m khun lc phn nh s chn lc ca mi trng v iu kin nui cy, mt phn t bo b cht hay b thng tn v s loi c th trong mu.

Gi tr ca s m trc tip bng phng php pht hunh quang trn knh hin vi tng ng vi vi s lng vi sinh vt tht s c th c vi tt c cc loi khc nhau. iu cho php c on c c s lng tht s trong nc bin, trong nc t nhin, trong trong mt loi mu no , mt d cc loi ny c s khc bit ln v s lng ca tng chng loi, khc bit v c im sinh l din ra trong cng iu kin ca mu. S m trc tip thng phn nh ng vi sinh khi, v th thng c dng c lng sinh khi vi sinh vt c trong mu. Tuy nhin c c s m chnh xc bng knh hin vi pht hung quang cn phi m nhiu ln v nhiu v tr khc nhau trn mu. Thm vo nu m nhiu t bo ang phn chia c th cho php c on tc pht trin ca vi sinh vt trong mu. Tn sut ca t bo ang phn chia c nhn thy c mi lin h vi ch s o khc ca tc pht trin vi sinh vt nh tc tng hp RNA c o bi s phng thch ca adenine c nh du. Tng xut ca t bo ang phn chia c tnh bng thi gian gia lc bt u s tht ca t bo n khi t bo phn chia l mt hng s. Con s ny khng phi l bt bin. Tht kh khn nhn ra s phn chia t bo khi s dng knh hin vi quang hc m khng s dng knh hin vi pht hunh quang. 3.2 Cc k thut nh lng vi sinh vt trong mu bng phng php nui cy C hai cch xc nh s lng vi khun bng phng php nui cy: phng php m khun lc v phng php c on s lng vi khun (MPN Most probable number). Tt c cc phng php nh lng vi khun
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trong mu bng phng php nui cy u yu cu cc t bo phi c tch ri nhau, qua qu trnh nui cy cc t bo ny pht trin thnh cc dng ring bit. Tt cc qui trnh nh lng bng phng php nui cy nhm chn lc mt hay nhiu nhm vi khun nht nh no , mc chn lc ph thuc v tng qui trnh qui trnh c th. xc nh tng s vi sinh vt trong mu phi chn qui trnh gim ti thiu kh nng chn lc. Nhng d mc chn lc mc ti thiu cng ch nh lng c s lng vi sinh vt c th nui cy. Cn mt s lng ln vi sinh vt khc khng th pht trin c trong qu trnh nui cy th khng th nh lng c bng phng php ny. C cc phng php nh lng nui cy nh sau nh sau: 3.2.1 Phng php m khun lc Phng php m khun lc trn a c s dng rng ri nh lng vi sinh vt, c bit l vi khun, nhng php ny c nhng khuyt im v c nhng cch nhn nhn khc nhau v mt khoa hc. Khuyt im ca phng php ny nm trong s lm dng biu t sai cc kt qu. Tt c mi ngi u mc sai lm khi nhn ra rng phng php ny khng th dng thu c kt qu tng s vi sinh vt. Phng php m khun lc s dng nhiu loi mi trng v cc iu kin nui cy khc nhau. Agar l cht thng c dng lm c cc mi trng nui cy v hu hr cc vi sinh vt u mt cc gen tng hp enzym phn gii agar. Cc mu c pha long tri ln b mt mi trng agar (phng php tri) hay khuyt tn vo trong ni trng trc khi lm c (phng php a). Mt vn cn phi cn nhc l cc vi sinh vt cn xc nh c th tn ti v pht trin trong mi trng agar hay khng. Mt s vi sinh vt c th b cht khi tri trn b mt mi trng trong qui trnh cy tri b mt. Mt s loi vi khun khc khng th sng trong iu kin nhit duy tr trng thi lng ca agar trong qui trnh a. Bi v agar ch l tc nhn lm rn mi trng nui cy trong qui trnh nh lng vi sinh vt v th trong mt s trng hp agar c th c thay th bng cc tc nhn lm rn khc. Trong mt s trng hp cc vi sinh vt c cc nhu cu dinh dng c bit khc, cc cht nh silicagel c th c thay th lm rn mi trng. Nh ng v chun b mi trng silicagel kh khn hn chun b mi trng agar nn ch dng mi trng silicagel khi khng th thay th c mi trng agar. Phng php ng cun c dng nh lng cc vi sinh vt k kh bt buc. y l mt phng php ci bin ca phng php a. Cc a hay cc ng sau khi cy vi khun c trong iu kin nhit c bit trong mt thi gian nht nh cho sinh vt pht trin thnh cc khun lc c th nhn thy bng mt trng, s lng khun lc xut hin trn a s c m. S lng khun lc s c gn cho s t bo n l trong mu ban u. s lng khun lc phn nh c s lng t bo vi khun, mu phi c phn
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tn u vo trong mi trng hay trn b mt mi trtng rn. Nhng a c qu nhiu khun lc xut hin th khng th cho s lng m chnh xc bi v chng c th chng lp ln nhau. Nhng a c qu t khun lc cng phi b i v theo nguyn tc ca xc sut. Nhng vn chnh yu cn xem xt trong qui trnh m khun lc l thnh phn mi trng, iu kin v thi gian nui . Mi trng nh lng cc vi sinh vt d dng khng th c nh nit phi cha cc ngun nit, carbon, phosphate d s dng v cc cation, anion cn thit nh st, mangie, calci, natri, clo v sulphate. Thnh phn ca cc hm lng khng nht nh, chng t th cho tng loi mi trng v tng loi mu nht nh c th nhn c s lng vi sinh vt mc tiu cao nht. Nh vy mi trng nui cy phi c cc thnh phn dinh dng ph hp vi tng yu cu ca loi vi sinh vt, bao gm c cc nhn t cn thit khc cho s pht trin ca mt h vi sinh vt nht nh. Mc ch chung ca mi trng l hm lng dinh dng phi cao hn cc thnh phn c trong cc mu ang phn tch. Hm lng dinh dng trong cc loi mi trng m tng s vi sinh vt phi cao, v c tnh ca mi trng i vi sinh vt mc thp nht. nng cao hiu qu ca qui trnh nh lng vi sinh vt bng phng php m khun lc, cc iu kin cn phi iu chnh sao cho ch c cc thnh vin vi sinh vt cn pht hin theo nh ngha c m. Cc mi trng m a c chn lc hay phn bit vi cc thnh vin vi sinh vt khc. Qui trnh m a chn lc c thit k cho thch hp vi s pht trin ca vi sinh vt mong mun. S pht trin ca cc nhm vi sinh vt khc c loi b bi cc thnh phn ca mi trng hay iu kin nui . Mi trng phn bit l mi trng khng loi b cc nhm vi sinh vt khng mong mun m ngc li cho php chng pht trin cng trn mi trng nhng c th bit phn bit cc nhm vi sinh vt mong mun vi cc nhm khc bng cc c im nhn dng. Mi trng cng c th c thit lp chn lc cc loi nm mc. Mt d k thut m khun lc khng phi l phng php c chn cho vic xc nh s lng nm mc trong cc mu thc phm v trong mi trng. Bi v k thut ny ch thch hp cho vic nh lng cc loi vi sinh vt khng c si hay bo t. D nhin phng php m khun lc cng thch hp cho vic nh lng cc loi nm men. V s lng ca vi khun lun nhiu hn s lng ca nm mc nn ngn cn s pht trin ca vi khun trong cc khun lc ca nm mc, cc tc nhn c ch vi khun c thm vo trong cc mi trng nui cy. Cc thuc nhum bengal rose v cc khng sinh nh streptomycine, neomycine thng c s dng l nhng tc nhn c ch vi khun. Mt bin php n gin c ch s pht trin ca vi khun l h thp pH ca mi trng nui cy xung khong 4,5 - 5,5. Hu ht cc nm mc u khng b tc ng khi pht trin trong khong pH ny nhng khi vi khun b c ch.
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Mi trng chn c xc lp cng thc c ch s pht trin ca nhm vi sinh vt v cho php mt nhm vi sinh vt khc pht trin. V d mi trng Saubouraud Dextrose Agar c dng nh lng nm mc da trn nguyn tc pH thp v ngun carbohydrate. B sung vo mi trng cc cht khng sinh khc nh peniciline hay methyl bule l nhng tc nhn c ch vi khun gram m. Cc vi sinh vt khng khng sinh cng c th c nh lng trn mi trng bng cch thm vo mt liu lng khng sinh. Mi trng phn bit c thit lp da trn nhiu cch. Cc thuc th c thm vo trong mi trng cho php nhn ra cc s khc bit ca nhng vi sinh vt mong mun. C th thm thuc th vo mi trng sau khi nui cy nhn ra cc loi vi sinh vt c trng cn tm. Cha kho nng cao kh nng phn bit ca mi trng l qui trnh nui cy cho php mi trng phn bit mt cch tt nht vi vi sinh vt cn nh lng v cc vi sinh vt khc c trong mu. Mi trng Eosin methyl blue (EMB) v MacConkey agar l nhng mi trng c s dng rng ri trong vic phn tch vi sinh vt trong nc. Nhng mi trng ny bao gm tnh chn lc v tnh phn bit. Chng chn lc cho s pht trin ca cc vi khun gram m bi trong c thm vo cc tc nhn c ch cc vi khun gram dng. Mi trng ny c th phn bit cc vi khun c th s dng lactose bi s hnh thnh cc khun lc c th phn bit bng mu sc. Trn mi trng EMB, vi khun thuc nhm coliforms l nhm gram m to nn nhng khun lc c mu tm nh kim. nh lng s lng coliform bng cch m s lng khun lc c cc c im ny. Cch ny thng xuyn c s dng trong vic phn bit cc vi sinh vt ch th trong nc v trong thc phm. Vi k thut n khun lc, cc mu cn phn tch phi c pha long thnh chui pha long lin tip v c xc nh mt vi nng pha long nht nh cy vo cc mi trng chn ph hp vi cc i tng cn xc nh. Mi khun lc c hnh thnh c gn cho mt n v hnh thnh khun lc (cfu-colony forming units). K thut thng qua cc bc nh sau: Dung dch pha long: mt s dung dch dng pha long c th lm cht t bo nh: nc mui, nc ct. Cc dung dch pha long khng c dng ngay khi ly ra t t lnh c th gy sc v lm cho vi sinh vt khng th pht trin c. Dung dch pepton water l dung ch pha long c s dng nhiu nht v c xem l dung dch pha long cho tt c cc loi mu. Nu trong mu cn c mt t cc cht kh trng th phi thm vo dung dch pha long tc nhn gim thiu kh nng kh trng ca chng, v d: nu trong mu cn d lng ca cc hp cht amonium (NH4-) th c th thm vo Tween 80 hay lecithin. Ngc li nu trong mu c cha cc hp cht chlorin hay iodin th cn thm vo cc mui nh sodium thiosulphate.
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Chun b cc chui pha long mu Bm chnh xc 9ml dung dch pha long vo trong ng nghim c np y. Nu s dng cc ng nghim c np khng ng chc c th phi kh trng ng nghim, sau bm dung dch pha long vo, cc thao tc ny phi c thc hin mt cch v trng. Phn phi dung dch pha long vo cc ng sau khi kh trng cn c ngha m bo th tch dung dch pha long khng b mt do qu trnh kh trng. Cc thao tc pha long tu thuc vo tng loi mu. C th nh sau: - Nu pha long cc mu l cht lng, trc khi pha long mu phi c lc k, cm u pippette su vo trong mu khong 1 ht ra 1ml, cho vo trong ng cha dung dch pha long u tin, lc k ng nghim cha mu, b pippette va s dng, dng mt pipptte mi thc hin li thao tc nh trn vi cc pha long tip theo. C tip tc nh vy cho n khi t c pha long mong mun. Mi pha long ch c php s dng 1 pippette. Hm lng mu trong 1ml dung dch cc pha long ca dy nh sau: ng s T l pha long Th tch mu ban u (ml) pha long 1 1/10 0,1 10-1 2 1/100 0,01 10-2 3 1/1000 0,001 10-3 4 1/10000 0,0001 10-4

- i vi cc mu rn hay bn lng, cc thao tc c tin hnh nh sau: cn 10 g mu vo trong cc bao dp mu hay cc my xay v trng, thm 90ml
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dung dch pha long v ng nht mu, phi m bo sao cho vi khun phn tn u vo trong dung dch. Sau khi mu v dung dch pha long c ng nht ta c dung dch pha long 1/10. Cc pha long sau c tin hnh tng t nh phn pha long mu lng. Trong 1ml cc dung dch pha long cha hm lng mu nh sau: ng s T l pha long Th tch mu ban u (g) pha long 1 1/10 0,1 10-1 2 1/100 0,01 10-2 3 1/1000 0,001 10-3 4 1/10000 0,0001 10-4

Cy mu bng phng php a Cc mi trng agar c un chy trong cc chai hay trong cc ng nghim c th tch ph hp. c lm mt trong cc b iu nhit 45oC. Ht 1ml mi dung dch pha long cho vo trong a petri v trng. Mi pha long thng c cy vo hai a petri hay nhiu hn. Cng s dng cc pippette sch cho mi pha long. Ch chn cy vo a petri nhng pha long c mt vi sinh vt ph hp, thng trong khong 25-300 t bo/ml. vo mi a cy 10-15ml mi trng c un chy v lm ngui, lc a theo chiu v ngc chiu kim ng h khong 5-6 ln, c a ln mt phng ngang v i cho n khi mi trng trong a ng c. Lt ngc a v trong iu kin nhit v thi gian xc nh. Cy mu trn b mt y l phng php thay th cho phng php a phn tch cc ch tiu vi sinh trong thc phm, nht l cc ch tiu vi sinh vt nhy cm vi nhit nh Pseudomonas, cc vi sinh vt ny s b suy yu khi tip xc vi nhit ca mi trng agar trng thi lng. Phng php ny cng c dng phn tch cc ch tiu m cc dng nhn c phi cy chuyn cc bc tip theo. Cy 0,1ml hay th tch ph hp ln b mt bng pipettes hay bng que cy ln a mi trng c lm kh v tri u bng que cy trang hay que cy vng tri mu trn khp b mt mi trng. U cc a cy iu kin nhit v thi gian xc nh tu theo tng ch tiu phn tch, m cc khun lc c trng hay tt c cc khun lc xut hin trn a. Khi m cc khun lc, trong mt s trng hp xut hin cc khun lc mc lan, thng thng cc khun lc khc cng c nhn thy bn trong cc vt khun lc loang. Khi m ta ch coi khun lc loang l mt n v hnh
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thnh khun lc v n cc khun lc khc trong khun lc loang. Cc khun lc loang ny l do cc vi khun tp trung thnh tng tp on trong qu trnh pht trin. m khun lc trong trng hp cy trang hay a m tt c cc khun lc n l trn cc a cy chn, thng thng chn cc a c s lng khun lc trong khong 30 - 300 khun lc. Thng dng cc bt c th vit ln thu tinh nh du cc khun lc m pha sau a petri. C th dng cc thit b h tr trong qu trnh m s lng khun lc nh my m hay knh lp. Tnh ton s n v hnh thnh khun lc theo cc cng thc thng k da trn th tch mu cy, s lng khun lc trn cc a, pha long cy v s lng a ca mi pha long. Thng thng s n v hnh thnh khun lc trong mt gram mu hay 1ml c tnh ton t nht t hai nng pha long lin tip ca mu. Cc tng trnh kt qu c biu din di dng s n v hnh thnh khun lc/g (ml) (cfu/g). Khng biu din kt qu di dng s vi khun/g(ml).

My m khun lc

My m khun lc t ng

Phng php m khun lc trn ng Thay v dng a petri, c th dng ng nghim c ng knh ln hay cc chai nh c np thay th. Cy mu vo, cho mi trng vo, lc theo chiu ngang cho n khi mi trng trong chai hay ng ng c. Phn phi 2-4 ml mi trng c hm lng agar cao hn cc mi trng dng cho a khong 0,5-1% vo ng nghim c np vi th tch 25ml, mi trng c lm ngui n 45oC, cy vo ng nghim 0,1ml mu c pha long. Lc cc ng theo chiu ngang trong nc lnh cho n khi agar ng c to thnh mt mng film ng nht trn thnh ng nghim. V th k thut ny cn c nhng k nng kho lo. Mt phng php khc c th c dng l s dng mt mu nc , t ln trn mt mnh vi v lm mt ng rnh bng vi ng nghim bng cc xoay trn ng nghim t ngang trn ming bng. Cc ng nghim cy mu v mi trng c xoay trn mnh

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ca ming bng. Vi phng php ny, cc ngi thc hin c th tit nhin c nhiu thi gian v sc lao ng. Cc ng sau khi cy mu c lt ngc trnh s ngng t hi nc gy nn vt loang sau khi . m cc khun lc xut hin trong lp mng fiml agar xung quanh ng, cc khun lc nng su bn trong c th c quan st bng knh lp. Phng php dng k thut ng xoay c s dng nhiu trong vic phn tch cc mu sa v cc sn phm ca sa, cc loi mu thc phm cng nghip. Cho n nay c nhng thit b thay th cho vic xoay ng, m bo cc mng film trn thnh ng ng nht v tit kim c thi gian cng nh cng lao ng. Phng php cy theo ng xon c Phng php ny da trn s h tr ca mt thit b xoay. Thit b ny c th hot ng hon ton t ng v cng c th hot ng khng t ng. Thit b ny s phn phi mt lng mu xc nh ln b mt a ang xoay trn. im tip xc ca mu v a mi trng bt u t trung tm a v di chuyn dn ra bn ngoi theo ng xon c. Sau khi lm kh b mt mi trng, a c trong iu kin xc nh, vi khun s pht trin theo ng xon c. Cng xa tm ca a, mt vi sinh vt s gim dn v s xut hin cc khun lc ring bit. Cn c vo tc xoay ca thit b, th tch mu phn phi v kch thc ca a, mt khun lc xut hin vng xon cui cng s tnh c mt vi sinh vt trong mu. Kt qu tng s n v hnh thnh khun lc c xc nh bng phng php ny c cho l tng ng vi phng php a hay cy tri trn b mt. Cc h thng t ng c thit k da trn nguyn tc cy xoay c thit k vi s h tr ca cc my m khun lc bn tia laser c bn ngoi th trng rt thun tin cho vic phn tch tng s vi sinh vt v cho kt qu chnh xc trong thi gian nui cy ngn. Phng php m khun lc trn mng lc Phng php ny thng c p dng cho cc mu lng nh nc, sa v c nh gi l cho kt qu chnh xc hn so vi phng php c on MPN. Cc mu cht lng c lc qua mng lc c kch thc l lc nh hn kch thc ca vi sinh vt. Vi sinh vt c gia li trn mng lc. Mng c t trn mi trng agar hay trn giy thm c ngm sn mi trng nui cy lng chn v trong iu kin xc nh. Sau khi , m cc khun lc s xut hin trn b mt mng lc. Thit bi lc c th bng thy tinh, bng kim loi hay bng nha, bao gm phu lc gi mng lc, b thit b h tr ht chn khng v bnh cha. Mng lc c lm bng cellulose ester c l lc mm trong khong 0,1-1 m, cc
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loi mng lc dng cho vic phn tch tng s vi sinh vt thng c kch thc l lc l 0,47m, ng knh mng lc c nhiu kch c khc nhau ph hp vi ng knh ca phu lc, b dy ca mng khong 120m. Khi lc tt c cc vi khun u c gi li trn mng. Khi c mng lc sao cho b trn ca mng hng ln pha trn, vi khun khng tip xc trc tip vi mi trng nhng, cc thnh phn ca mi trng d dng thm qua mng v nui vi khun pht trin thnh khun lc. Mt s loi mng lc c gn cc li k nc ch cho php khun lc pht trin trong mi ca li v khng cho lan sang cc khc nhm to iu kin thun tin khi m. Cc b phn ca thit b lc

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3.2.2 Phng php c on s lng vi sinh bng k thut MPN (Most Probable Number): Phng php MPN l phng php c th thay th phng php m khun lc xc nh mt vi sinh vt trong mu, phng php ny c da trn nguyn tc xc sut thng k s phn phn b vi sinh vt trong cc pha long khc nhau ca mu. Mi pha long c nui cy lp li nhiu ln trong cc mi trng lng c chn, thng thng phi cy lp li t 3-10 ln ti mi nng pha long. Cc pha long c tin hnh sao cho trong cc ln lp li c mt s ln cho du hiu dng tnh v mt s ln cho du hiu m tnh. S ln lp li cho du hiu dng tnh v m tnh c ghi nhn i chiu vi bng thng k s c gi tr c on s lng vi sinh vt trong mu. Qui trnh MPN cho gi tr s lng vi sinh vt trong mu c ngha thng k theo xc sut phn b vi sinh vt trong mu khi s dng mt s ln lp li, v th khong tin cy l rt ln. Phng php MPN nh lng vi sinh vt cho n nay c nhiu ci tin cho php tin hnh qui trnh d dng v tn t sc lao ng hn v cho gi tr chnh xc cao hn. Chn nng pha long sao cho trong cc ln lp li c mt s ln cho kt qu dng tnh v mt s ln cho kt qu m tnh , s la chn ny rt quan trng. Trong nhiu trng hp qui trnh MPN c thit lp sao cho gia tng s lng ln lp cho kt qu dng tnh bng tn hiu pht trin ca vi sinh vt. Trong mt s trng hp khc, qui trnh c thit lp c nhiu th nghim khng nh sau khi cc ln lp li cc pha long cho tn hiu pht trin ca vi sinh vt nh pht hin protein hay mt emzym no . Cc th nghim sau ny cho kt qu dng tnh th cc ln lp li ban u mi c khng nh l dng tnh. Cng ging nh qui trnh m khun lc, qui trnh MPN cng c s dng cc mi trng chn lc, khng chn lc hay mi trng phn bit. Phng php MPN cng c dng nh lng virus ng rut. Trong phng php ny, mt chui pha long ca mu cn kim tra c cho vo trong cc ng nghim cha t bo ch thch hp c nui cy. Sau khi , cc ng nghim c kim tra hiu ng cytopathic (CPE), l cc biu hin lm cht t bo b xm nhim. S lng virus trong mu cng nhn c t bng MPN bng s tham chiu s lng cc ng nui cy cho hiu ng CPE dng tnh. S lng ca virus cng c th c nh lng bng ch s TCID 50 (Tissue culture infectious dose 50%). Nng pha long thp nht c s hin din ca virus l nng c t s CPE l 50% trong cc ng nghim. Cng ging nh qui trnh m a, trong phng php MPN, mi trng v nng nui cy cng c iu chnh chn lc cho mt nhm vi sinh vt hay phn bit cc nhm vi sinh vt ny vi cc nhm vi sinh vt khc vi cc c im mong mun, r rng rng s kt hp gia iu kin nui cy v mi trng cng c th nh lng nhng nhm vi sinh vt c bit no
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theo nh ngha c th. Mi qui trnh phi c chn lc mt cch c th v cn thn c th thu c kt qu mt cch chnh xc. Theo quan im ca C.H. Collins v Patricia M. Lyne thc cht con s MPN biu din s lng vi sinh vt trong mu l s lng trung bnh sau cc ln lp li. Nu s lng vi sinh vt trong mu ln th s khc bit ca cc mu gia cc ln lp li l nh, kt qu ring l ca tt c cc ln lp gn vi kt qu trung bnh. Nu s lng vi sinh vt trong mu nh, s khc bit ny s ln. Nu trong mt mu cht lng cha 100 vi sinh vt/100 ml, th trong 10 ml mu s cha trung bnh 10 t bo. D nhin c mt s phn mu nhiu hn 10 t bo, thm ch c nhng phn mu c th cha 20 t bo trong 10ml mu v mt s phn mu cha t hn. Nu tt c cc phn mu trn c nui cy trong mi trng v iu kin thch hp, cc vi sinh vt trong mu s pht trin v cho tn hiu dng tnh. Tng t nh vy, 1ml s cha trung bnh 1 t bo vi sinh vt, nh vy c nhng ln ly s c 2 hay 3 t bo v c nhng ln ly s khng c t bo no. Nu cc ln ht ny c nui cy trong mi trng v iu kin thch hp s thu c nhng ng nghim cho kt qu dng tnh v nhng ng cho kt qu m tnh. Nu ht 0,1ml th sau 10 ln ht mi c kh nng nhn c 1 ln ht c 1 t bo, nh vy hu ht cc ln nui cy khi ly 0,1ml th u cho kt qu m tnh. C th tnh ton con s chc chn s lng vi sinh vt trong trong 100ml mu bng s kt hp cc kt qu nhn c t cc chui cy nh trn. Bng gi tr MPN khi s dng h thng cy vi cc dy 5 ng 10ml, 5 ng 1ml v 5 ng 0.1ml c tnh sn v dng cho phn tch cc mu nc hay cc mu pha long. Cho n nay c rt nhiu bng gi tr MPN c s dng vi chnh xc v cc khong tin cy khc nhau v c s dng cho cc mc ch khc nhau. Phng php MPN c dng ch yu phn tch Coliforms v cc vi sinh vt khc khi chng pht trin trong mi trng nui cy lng cho cc tn hiu d dng nhn dng nh sinh hi, lm c mi trng chn lc, thay i pH mi trng V d nm men v nm mc trong nc tri cy hay rau qu, vi sinh vt k kh hay cc bo t Clostridia. 3.2.3 Phng php lai khun lc Lai khun lc l phng php kt hp gia phng php lai phn t v phng php nui cy truyn thng trong vic phn tch cc ch tiu vi sinh vt trong cc mu. Sau thi gian nui cy trn b mt mi trng thch, cc khun lc vi khun c chuyn ln mng lai, cc khun lc ny c ly gii trong mi trng kim hay x l bng emzym, sau tin hnh lai phn t. Phng
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php ny ph thuc vo kh nng pht trin ca vi sinh vt mc tiu trn mi trng, chng khng ph thuc s pht trin cc cc qun th vi sinh vt khc. S pht trin ca vi sinh vt mc tiu trong mi trng lm gia tng s lng bn sao ca cc gen mc tiu, v th chng s gia tng kh nng pht hin ca cc mu d. Nguyn tc ca phng php ny c pht trin u tin bi M. Grunstein v S.D. Hogness (1975) nhm mc ch sng lc trn a c mt cao cho cc dng nui cy thun. Trong qui trnh nui cy khun lc vi khun pht trin trn mi trng rn c chuyn ln mt gi ph hp nh mng nitrocellulose, ly gii tch DNA v bin tnh chng sau c nh chng trn mng. Mng lc c nh DNA c vi mu d. Mu d c tch ra t mt on thng tin di truyn. V c nh du bng cc cc ng v phng x nh 32P hay cc cht pht quang khc nh biotin. Hin tng lai c din ra nu cc trnh t base ca cc t bo c ly gii tng ng vi cc trnh t trn mu d hnh thnh cc on lai mch i. Cc on lai ny c pht hin bng bng cc film phng x t ghi khi cc mu d c nh du bng cc ng v phng x, hay cc film nhy sng khi cc mu d c nh du bng biotin. Bng phng php ny, cc c im di truyn c trng c pht hin mt cch chuyn bit. Nu chn mu d c trng cho mt nhm vi sinh vt, phng php ny c th pht hin v nh lng nhm vi sinh vt trong mu. Mu d c lai vi cc vi sinh vt c ngun gc t mu, hay cc dng thun c phn lp v nui cy th cp. Bng phng php ny c th ci thin c cc bt tin trong qu trnh phn tch bng phng php nui cy thun tu nh: - Trnh c nhng sai lch trong qu trnh m khun lc trn mi trng nui cy chn lc. - Chc chn pht hin c cc dng mang kiu gen mc tiu cn pht hin d cc gen khng hay biu hin rt km trong mt s iu kin nui cy. - C th phn tch c cc vi sinh vt b tress hay khng th nui cy c trn cc mi trng chn lc bng cch thay th bng mi trng khng chn lc hay mi trng dinh dng ti a. - Gim c thi gian phn tch bng cch gim thi gian nui cy, qu trnh nh lng gi nh v thi gian khng nh kiu gen hay kiu hnh. Phng php lai khun lc cng c s dng khng nh cc dng vi sinh vt nghi ng c lm thun. ng dng ch yu ca phng php lai khun lc l pht hin, nh lng v phn lp cc vi sinh vt c kiu hnh v kiu gen c trng. V c s dng c bit trong vic kim sot s hin din v hot ng ca cc dng vi sinh vt
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trong mi trng. Nghin cu s phn b cc vi sinh vt khng khng sinh, khng kim loi nng trong nc trong cc mu mi trng v trong thc phm. 3.3 Phng php o c L phng php gin tip xc nh mt vi sinh vt. nh lng mt t bo thng qua o c bng my so mu cc bc sng t 550 610 nm. Phng php xc nh mt t bo theo c c th c dng so snh mc tng trng ca hai hay nhiu chng vi sinh vt trong mi trng lng. phng php ny cho kt qu nhanh chng v thng c ng dng trong theo di hoc nghin cu c trng tng trng ca cc chng vi sinh vt trong phng th nghim hoc trong sn xut tuy nhin khng thch hp dng trong kim nghim vi sinh vt. N A (CFU/g hay CFU/ml) = Trong : n1Vf1 + + niVfi A: s t bo vi khun 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

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IV. P DNG I VI MT S VI SINH VT GY BNH TRONG THC PHM

4.1 nh lng Fecal Coliform v E. Coli bng phng php m a Coliforms chu nhit: l nhng vi khun sinh trng cho khun lc in hnh trn mi trng thch Violet Red Bile 440C trong 24 gi. Trong trng hp nghi ng, vi khun c khng nh bi s to kh trong mi trng EC broth 44 0C trong 24 gi. E. coli: l Coliforms chu nhit c kh nng sinh indol 44 0C. Nguyn tc: Cy mt lng mu bit trn mi trng rn khng chn lc. Sau , s b nhit phng (20-250C) trong 1-2 gi, ph ln mt lp mi trng rn chn lc c cha Lactose. Cc a c 440C trong 24h, sau m nhng khun lc in hnh v nghi ng cy nhng khun lc chn ln nhng mi trng khng nh thch hp. Mi trng-thuc th -dch pha long: - Dung dch Saline Peptone - Thch Tryptone Soya ( TSA ) - Thch Violet Red Bile ( VRB ) - Canh Escherichia coli (EC broth) - Canh Tryptone - Thuc th Kovacs Chng chng: - Chng chng dng: Escherichia coli - Chng chng m: Salmonella typhimurium Thit b chnh: - T m 44.0 + 0.50C Quy trnh: Chun b mu. a: Cy 1ml mu nng thch hp vo a Petri . Thm vo 5ml mi trng TSA c lm ngui 45.00+1.00C. Trn u dch nui cy v mi trng. s b cc a nhit phng (20 - 25 0C) trong 1-2 gi. thm 10-15ml VRB agar c lm ngui 45.00 +1.00C . T l mi trng gia hai ln t nht l 1:2. Nui : Cc a c lt ngc ln v 44.0 + 0.50C trong 24 3 h.

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c kt qu: Chn cc a c s khun lc in hnh v khng qu 100 m. Khun lc coliform chu nhit in hnh c mu sm, ng knh t nht l 0,5mm, xung quanh khun lc c vng kt ta . Khng nh: Coliform chu nhit : Chn 5 khun lc nghi ng ca mi loi, cy vo mi trng canh EC, 44.0 + 0.50C trong 243 h. Phn ng c xem l dng tnh khi trong ng Durham c kh. E. coli:Test dng tnh ca Coliform chu nhit c thc hin thm v kh nng to indol trong mi trng canh tryptone bng cch thm 0.3- 0.5 ml thuc th Kovac's trc tip vo trong mi trng Tryptone c 44.0 + 0.5 0C trong 243 h. Phn ng c xem dng tnh khi c mu xut hin trn b mt. Mu vng hay mu cam xut hin trong 10 pht c xem l m tnh. E.coli gi nh cho phn ng indol dng tnh. Bo co kt qu: m kt qu bng cch nhn s khun lc m vi nng pha long v t l khng nh dng tnh. Cc ch v gii hn ca phng php Thuc th Kovacs l mi nguy cho sc khe, v vy cn s dng cc bin php chng c nh eo khu trang v pha thuc th trong t ht. Khng c thanh trng mi trng VRB v un qu nhit. Rt mi trng E.C vo cc ng nghim c ng Durrham o ngc. Cc ng ny s chm xung sau khi hp thanh trng. T l dy gia lp trn v lp di ca mi trng VRB nn l 2:1. 4.2 nh lng Vibrio Parahaemolyticus Nguyn tc: Cy lng mu bit vo mi trng thch chn lc (TCBS). Nhng khun lc V.parahaemolyticus in hnh s c th nghim sinh ho. Mi trng v thuc th: - Thch TCBS (Thiosulphate citrate bile salt sucrose); Thch Motility medium; Thch OF - Canh tryptone c nng NaCl 0%, 6%, 8%, 7% - a ONPG - a O/129 Vibriostaticum cha 150g 2,4- diamino- 6,7diisopropylpteridine - Canh Lysine decacboxylase - Dung dch cc loi ng sucrose, lactose, cellobiose - Canh Ure - Thuc th N,N,N,N-tetramethyl-1,4 phenylenediamine (TMPD)
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Thit b chnh: T m 37,0 1,0 0C Quy trnh: Chun b mu. Cy mu: Cy trang 1 ml dch mu nng thch hp bng que cy tam gic ln 03 a mi trng thch TCBS. Nui : cc a 37,0 1,00C trong 2 ngy, c kt qu sau 1 v 2 ngy. c kt qu:Trn mi trng thch TCBS khun lc V. parahaemolyticus trn, c mu xanh, ng knh 3 5mm. Th nghim sinh ho: T cc khun lc nghi ng trn TCBS cy chuyn sang mi trng TSA c b sung thm 1% NaCl, 37,0 1,0 0C / 1 ngy. Th nghim s b:
Th nghim sinh ho Di ng OF Oxidase KOH Kt qu + ln men, sinh kh + +

Ghi ch: A: Axit, K: Kim. Test khng nh:

Th nghim sinh ho Pht trin cc nng mui: 0%NaCl 6%NaCl 8%NaCl 10%NaCl Sinh acid t: Succrose |%NaCl Mannitol Cellobiose ONPG ADH LDC Ure O/129 10 g O/129 150 g + + R(khng nhy) S(nhy) + + Kt qu

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4.3 nh lng Coliforms, Coliforms chu nhit, E. Coli gi nh (Fecal Coliforms) bng phng php MPN Trong phng php ny: Coliforms l nhng vi sinh vt sinh hi trong mi trng canh Lauryl sulphate v canh Brilliant green lactose bile salt nui 37.0oC 1.00C/ 2 ngy. Coliforms chu nhit l nhng vi sinh vt sinh hi trong mi trng canh EC nui 44.5oC 0.2 0C/1 ngy. E. coli gi nh (Coliforms phn) l Coliforms chu nhit sinh indole trong canh trypton nui 44.5oC 0.2 0C/ 1 ngy Nguyn tc: S lng Coliforms, Coliforms chu nhit, E.coli gi nh (Coliforms phn) v E.coli c xc nh bng phng php MPN, phng php ny da vo nguyn tc mu c pha long thnh mt dy thp phn (hai nng k tip chnh nhau 10 ln) v a vo cc ng nghim c cha mi trng thch hp, v c s ng cho kt qu dng tnh. S ng nghim cho phn ng dng tnh ca cc nng pha long c tra theo bng MPN xc nh tng s vi khun trong 1 g (hoc 1 ml) mu. Mi trng v thuc th: - Canh Lauryl sulphate (Sodium dodecyl sulphate - LSB) - Canh Brilliant green lactose bile salt (BGBL) - Canh EC (E.coli medium) - Canh Tryptone - Thuc th Kovacs Thit b: - B iu nhit 44.5oC 0.2 0C - T m 37.0oC + 1.00C Tin hnh: - Coliforms: Cy 1 ml dch mu pha long 1:9 vo dy 5 ng nghim, mi ng cha 10 ml canh Lauryl sulphate. Thc hin tng t vi dch mu pha long 1:10 v 1:100. Nu nghi ng lng vi sinh vt trong mu qu cao, phi tng pha long mu. 37.0oC + 1.00C/2 ngy. Ghi nhn s ng c sinh hi sau 1 ngy v 2 ngy. Dng khuyn cy cy chuyn dch mu t cc ng LSB dng tnh (ng sinh hi) sang cc ng c cha canh Brilliant green lactose bile salt. 37.0oC 1.00C/2 ngy. c s lng cc ng cho kt qu dng tnh(ng sinh hi). S ng nghim cho phn ng dng tnh ca cc nng pha long c tra theo bng MPN xc nh tng s Coliforms trong 1 g (hoc 1 ml) mu.
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- Coliforms chu nhit: Dng khuyn cy cy chuyn dch mu t cc ng canh Lauryl sulphate c sinh hi sang mi trng canh EC, 44.5oC 0.2 0C/1 ngy. c s lng cc ng cho kt qu dng tnh(ng sinh hi). S ng nghim cho phn ng dng tnh ca cc nng pha long c tra theo bng MPN xc nh tng s Coliforms chu nhit trong 1 g (hoc 1 ml) sn phm. - Coliforms phn: Dng khuyn cy cy chuyn dch mu t cc ng c sinh hi trn mi trng canh EC sang mi trng canh Trypton, 44.5oC 0.2oC/1ngy. Nh thuc th Kovacs vo cc ng nghim Trypton . Phn ng dng tnh khi c mu xut hin trong mi trng trong vng vi pht. c s lng cc ng cho kt qu dng tnh. S ng nghim cho phn ng dng tnh ca cc nng pha long c tra theo bng MPN xc nh tng s Coliforms phn trong 1g (hoc 1ml) mu. Cc ch v gii hn ca phng php: Phng php MPN thng qua phn ng ln men lactose khng nh lng c vi sinh vt thun chng. Kt qu sai c th xy ra do s c mt ca cc vi sinh vt ln men lactose khc. Khng nn cy chuyn cc bt bng c sau qu trnh ng nht mu vo mi trng nui cy. Sau khi chuyn dch pha long mu vo ng Lauryl tryptose nn xoay t t ng nghim dch dnh thnh ng i xung. C th c kt qu m tnh gi do s c mt ca cc Coliforms khng ln men lactose. Nhit b iu nhit cn c duy tr 44.50C v mc nc phi trn mc dung dch trong cc ng nghim. Nhit m rt quan trng, s thay i cht t c th lm thay i loi v s lng vi sinh vt pht trin trong mi trng th nghim. Cc chng loi chun E. coli v Enterobacter aerogenes cy song song vi php th s ch ra s sai lch ca nhit b iu nhit. Nhit cao s dn n s ln men ca E.coli sai. S ln men lactose ca E. aerogenes ch ra nhit thp hn 44.50C. nh lng E.coli bng Lauryl tryptose broth c th xut hin m tnh gi do E.coli b tn thng trong qu trnh ch bin thc phm. Nn s dng chng chng v kim sot mi trng trong sut qu trnh th nghim.

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4.4 nh lng Enterococci ng rut trong nc bng phng php mng lc Enterococci ng rut l cc vi khun c kh nng phn gii 2,3,5triphenyltetrazolium chloride to thnh formazan v thu phn aesculin 440 C trn mi trng quy nh ti phng php ny. Lc, nui cy v m: m Enterococci ng rut da trn vic lc mt th tch xc nh ca mu nuc qua mt mng lc c kch thc l thch hp (0,45 m) gi li cc vi khun. Mng lc c t vo trong mi trng thch chn lc cha natri azide ( ngn chn s sinh trng v pht trin ca cc vi khun gram m) v 2,3,5-triphenyltetrazolium chlorua (mt cht nhum khng mu). Enterococci ng rut s kh thuc nhum khng mu thnh focmazan . Sau khi nui cy 36.0 2.00C/ 44 4 h, m tt c cc khun lc mc mu , mu ht d hoc mu hng. Khng nh: Chuyn mng lc c cc khun lc vo mi trng bile-aesculin-azide agar, 44.0 0,50C. Enterococci ng rut thu phn aesculin trong mi trng trong 2 gi, sn phm cui cng l 6,7 - dihidroxycoumarin s kt hp vi ion st (III) cho hp cht mu nu vng n en v khuch tn vo mi trng. Mi trng nui cy: - Mi trng Slanetz and Bartley - Thch mt aesculin - azide Thit b: - Thit b mng lc - Mng lc v trng vi kch thc l 0,45m. - T m 36.0 2.00C - T m 44.0 0.50C Quy trnh tin hnh: Chun b: Chun b mu, thit b lc v trng v mi trng nui cy. Mu nn c phn tch ngay sau khi ly. Nu mu bo qun nhit phng (250C) th khng c bo qun qu 6 gi, nu mu bo qun nhit 5 30C th khng c qu 24 gi trc khi phn tch. Lc v nui Lc mt th tch nc thch hp. iu chnh cc dch pha long sao cho cc khun lc c th mc tch bit ra v d dng m c.
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Sau khi lc, tho mng bng kp kh trng v t mng lc ln mt thch Slanetz and Bartley. Nui cc a 36.0 2.00C trong 44 gi 4 gi. Khng nh v m : Sau khi nui, m tt c cc khun lc c mu nu hoc mu hng t trung tm hoc ton b khun lc. Nu trn mng c cc khun lc c trng, chuyn mng lc sang mi trng bile aesculin-azide agar c m n 440 C (khng lt ngc mng), 44.00 C 0.50C trong 2 gi. c kt qu ngay. Tt c cc a cho khun lc c mu t nu n en, hoc mi trng bao quanh c mu nu hay en c coi l phn ng dng tnh. Bo co kt qu: Kt qu biu th bng s vi khun Enterococci ng rut trong mt n v th tch mu v c tnh theo hng dn trong ISO 8199. Cc ch v gii hn ca phng php Bile-aesculin-azide-agar c cha Natri azide. Do Natri azide c c tnh cao nn ch khi thc hin cn trnh tip xc trc tip vi ho cht , c bit khng c ht cc ht bi ca mi trng trong qu trnh pha ch mi trng tng hp. Mi trng c cha azide khng nn pha vi cc axit v c mnh bi v n c th to ra cht c HN3. Dung dch cha azide cng c th hnh thnh nnn hp cht gy n khi tip xc vi cc ng dn bng kim loi. Mng lc c lm t cellulose v c ng knh mt li khong 4750mm. Mng lc phi m bo khng c tnh cht c ch hoc kch thch s pht trin ca vi khun, mc in s dng in cc mt li trn mng lc cng khng c nh hng n s pht trin ca vi khun. Nu cha v trng th chng cn phi c kh trng theo hng dn ca nh sn xut. Vic s dng mng lc ca cc hng khc nhau c th dn n vic hnh thnh mu sc khc nhau ca vi khun trn mng. 4.5 nh lng Clostridium kh sunfite trong nc bng phng php tng sinh trong mi trng cy lng Ging Clostridium thuc h Bacillaceace bao gm cc c im nh sau: sinh bo t, phn ln di ng, gram dng, hnh que, k kh. Hin din trong phn ca ng vt v ngi, trong nc thi v trong t. Cc bo t ca vi sinh vt ny sng st trong nc rt lu v chng bn vng hn cc dng t bo sinh dng i vi cc tc ng ca cc yu t vt l v ha hc. V vy chng c th lm ch th v s nhim nc. Chng thm ch bn vi c chlorin mc thng vn c s dng x l nc.
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Nguyn tc: S lng vi khun Clostridium c xc nh trong mt th tch nc xc nh theo cc bc tng sinh trong mi trng cy lng, nui trong iu kin k kh. Mi trng: - Mi trng phn lp: DRCM nng kp v nng n. - Dung dch pha long- nc mui pepton (saline pepton) Dng c v thit b: - My dp mu. - B iu nhit 7550C. - T m 371.00C. - Bnh k kh vi thit b to iu kin k kh. Tin hnh th nghim: X l mu: Mu c x l nhit bng cch un nng trong b iu nhit o 75 C + 5 trong 15-20 pht k t lc t nhit trn, gim bt th dinh dng ca vi khun. Cy mu v nui m: Cy 50ml mu nc ban u vo 50ml canh DRCM nng kp. Cy 10ml mu nc ban u vo 5 ng nghim, mi ng cha 10 ml canh DRCM c. Cy 1ml mu nc ban u vo 5 ng nghim, mi ng cha 25 ml canh DRCM n. Nu nghi ng lng vi sinh vt trong mu qu cao, phi tng pha long mu. Nu cn, ln mt tt c cc ng cy mt lng canh DRCM n th tch cht lng y ng v m bo rng ch cn li mt th tch nh khng kh, sau gn kn cc ng nghim v cc bnh, hoc em nui trong iu kin k kh, 37.0 + 1.00C/44 + 4 gi. B sung cc on dy thp - c nung t vo mi trng trc khi cy mu vo, c th to iu kin cho sinh vt k kh pht trin. c v bo co kt qu: Cc l trong thy c vt en c coi l dng tnh. Kt qu biu th theo s c xc sut cao nht (MPN) ca Clostridium kh Sulfite trn th tch mu th.

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KT LUN
Hng nm Vit Nam c khong hn 3 triu trng hp nhim c t thc phm, gy thit hi hn 200 triu USD (WHO), hn 70% s ca do vi sinh vt gy nn, do vic xc nh vi sinh vt gy bnh trong thc phm (nh tnh, nh lng) l rt cn thit cho cng tc ngn chn, phng nga v gim thit hi gy ra bi ng c thc phm. C nhiu phng php nh lng vi sinh vt, mi phng php u c nhng u, nhc im ring v c dng nh lng nhng vi sinh vt nht nh, do cn c s nghin cu v c im sinh hc ca vi sinh vt c th p dng ng phng php nh lng, em li hiu qu cao nht. Bi tiu lun s dng s liu ca Cc an ton v sinh thc phm - B y t v cc phng php nh lng ca Cc qun l cht lng nng, lm sn & thy sn - B NN&PTNT.

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TI LIU THAM KHO

1. Nguyn Tin Dng, 2007, Phng php kim nghim vi sinh vt trong thc phm, H KHTN-H QGHCM. 2. Trn Xun Hin, 2001, nh gi cht lng thc phm, H Nng Lm TpHCM. 3. Lm Quc Hng, T Ngc Thanh, 2009, Mt s c im dch t hc cc v ng c thc phm trong ton quc t nm 2002 n nm 2008, Cc An ton v sinh thc phm - B Y t. 4. L Thanh Mai, 2006, Kim tra vi sinh trong cng ngh thc phm, H BKHN. 5. Lng c Phm, 2005, Vi sinh vt hc v an ton v sinh thc phm, NXB Nng nghip. 6. TCVN 17025.053, 2005, Vn phng cng nhn cht lng. 7. S tay Phng php vi sinh, 2009, Cc qun l cht lng nng lm thy sn.

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PH LC
Ph lc 1. Cc ch tiu vi sinh vt trong thc phm c quy nh bi tiu chun ca B Y t (Bng 1) bao gm cc ch tiu sau: tng s vi khun hiu kh, Coliforms, E.coli, Staphylococcus aureus, Salmonella, Vibrio parahaemolyticus, Bacillus cereus, Streptococcus faecalis, Pseudomonas aeruginosa, Clostridium botulinum, Clostridium perfringens, tng s nm men, nm mc Quy nh v gii hn cho php ca cc ch tiu thay i theo nhm v chng loi thc phm. Ph lc 2. Kt qu gim st vi sinh vt trong mt s loi thc phm do Cc An ton v sinh thc phm tin hnh nm 2006 (Bng 2) v 2007 (Bng 3) Ph lc 3. H thng phng php phn tch vi sinh vt Cc Qun l cht lng nng lm thy sn, B NN&PTNT. Ph lc 4. TCVN (7048,7049,7050) i vi cc sn phm tht.

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Nhm thc phm

Bng 1. Tiu chun vi sinh cho php trong thc phm, B Y t 4/1998 Gii hn cho php (CFU/g hoc CFU/ml thc phm) TVKH EC SAL/25 BC CO CP VP NM SF SAU K O g E L E A MO A 106 3. 105 106 105 106 102 3 102 3 10 3 102 10 102 10 102 10 0 0 0 0 0 0 10 102 102 102 10 50 10 102 10 20 102 10 102 10

PA E

CB O

Nhm tht: - Tht ti, tht ng lnh, tht xay nh, tht nghin, tht ch bin. - Sn phm ch bin t tht: tht hun khi, pate, xc xch. Nhm c v hi sn: - C v thy sn ti sng - Sn phm ch bin: tm, c hp nng hun khi, ch c, ch mc, cc loi gip xc, nhuyn th luc hp. - Thy sn kh s ch: c kh Nhm trng: - Trng ti, dch trng ti hoc ng lnh. - Sn phm ch bin t trng ( tit trng bng phng php Pasteur)

105 103

10

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Nhm sa: - Sa kh, sa bt - Sa ti tit trng theo phng php Pasteur. - Sa ti tit trng theo phng php UHT - Sn phm ch bin t sa (b, sa chua, phomat) Sn phm ch bin t ng cc, khoai c, u : - Cn s l nhit trc khi dng - Dng trc tip, khng x l nhit: bnh bt. Nhm nc khong v nc gii kht ng chai: - Nc gii kht c cn - Nc gii kht khng cn - Nc khong ng chai Nhm gia v:

5. 104 5. 104 10 104 106

0 3 0 0 102 3 0 0

0 0

10 10 0 10 102 10 103 10 102 10 0 10 0 0 0 102 10 103 102 0 0 0 0 0 0

0 0 102 10 0 0

0 0

104 10 102 Theo G.M.P 104

102

102

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Nhm nc chm: - Ngun gc ng vt: nc mm - Ngun gc thc vt Nhm thc n kh v cha dinh dng cho tr em, thc n thay th c bit: - Phi x l nhit trc khi s dng - Dng trc tip khng qua x l nhit Kem, nc Nhm hp Nhm du m

104 104 105

0 0 10

3 3 102

0 0 0 102

102 102 10

10 10

10 10

104 5. 104 10
3

0 0 0 3

3 10 0 0

0 0 0

10

10 102 10

10 10 0 0 0 0

TVKHK: tng vi khun hiu kh; ECO: E.coli; SAU: Staphylococcus aureus; SAL: Salmonella; BCE: Bacillus cereus; COL: Coliforms; CPE: Clostridium perfringens; VPA: Vibrio parahaemolyticus; NM MO: tng s nm men, nm mc; SFA: Streptococcus faecalis; PAE: Pseudomonas aeruginosa; CBO: Clostridium botulinum; G.M.P: Goof Manufacturing Practice: quy phm sn xut GMP

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Bng 2 Kt qu gim st ch ng mu vi sinh 2006 Stt 1 2 3 4 5 Tht ti Thy sn kh s ch Thy sn ti Bnh n lin cc loi Nc gii kht cc loi: nc ma, rau m, nc sm, nc sinh t. Tng cng : Tn mu Tng s mu 40 17 13 37 20 127 t 6 8 9 16 0 39 (30,7%) Khng t 34 9 4 21 21 88 (69,3%) Ch tiu khng t Coliforms, E.coli, S.aureus Coliforms, E.coli, S.aureus Coliforms, E.coli, Coliforms, E.coli, S.aureus, Cl. Perfringen,
B. cereus, TSNM- NM

Coliforms, E.coli, S.aureus, Cl. Perfringen,

S. feacalis, TSNM- NM

Nhn xt: 88/127 mu c kim tra khng t cc ch tiu vi sinh trng im Coliforms, E.coli, S.aureus, C. perfringens, B. cereus, S. fecalis, TSBT mc men

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Bng 3 Kt qu gim st ch ng mu vi sinh 2007 STT 1 2 3 4 5 6 7 Tn sn phm Nem chua l chui Nem chua hp Jambon B thnh Nem chua cy Nem b Nem chua Tng Kt s Coliforms qu t mu 3 3 20 14 2 11 20 73 1 1 0 8 0 2 5 17 (23,3%) 2 2 20 6 2 9 15 56 (76,7%) E. coli 1 2 15 1 0 5 8 S. C. aureus perfringens 0 0 1 0 0 1 4 0 0 0 0 0 3 7 10 (13,7%) S. fecalis 0 0 0 0 0 0 0 0 (0,0%)

Tng cng

31 6 (42,5%) (8,2%)

Nhn xt: C 56/73 mu c kim tra (76,7%) khng t ch tiu vi sinh ( xt nghim cc ch tiu vi sinh trng im: Coliforms, E. Coli, S. Aureus, C. Perfringens)

STT 1 2

Tn sn phm Mm thi Mm c cc loi Mm ruc

Tng s mu 16 74

t 6 (37,5%)

Khng t 10 (62,5%)

Ghi ch Ch tiu khng t: C. Perfringens

51 23 Ch tiu khng t: (68,9%) (31,1%) C. Perfringens 15 8 Ch tiu khng t: 3 23 (65,2%) (34,8%) C. Perfringens Mm tm 3 3 Ch tiu khng t: 4 6 chua (50%) (50%) C. Perfringens Mm ba kha 3 Ch tiu khng t: 5 5 8 (37,5%) C. Perfringens (62,5%) Mm tm, 8 3 Ch tiu khng t: 6 11 mm tp (72,7%) (27,3%) C. Perfringens Mm b hc 3 2 Ch tiu khng t: 7 5 xay (60%) (40%) C. Perfringens 89 54 Tng cng 143 (62,2%) (37,8%) Nhn xt: Khng pht hin Vibrio cholerae, V. Parahaemolyticus trong s mu c kim tra. C 54/143 mu c kim tra (37,8%) nhim C. Perfringens.c bit mm ba kha v mm thi c t l nhim trn 62%.

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