TRNG I HC CNG NGHIP TPHCM

VIN SINH HC-THC PHM(((TIU LUNMon:

e tai: TPHCM, thng 5 /2011

MC LCI.

LI M U2II.NI DUNG

Chng 1: Staphylococcus aureus v nhng tnh cht c trng

1.1. Gii thiu3

1.2. Hnh dng v c tnh4

Chng 2: Bnh v triu chng lm sng

2.1. c t rut (enteroxin)7

2.2. Cc c t khc ca Staphylococcus aureus8

2.3. C ch gy c ca SEB10

2.4. Bnh v triu chng10

Chng 3: Phng php phn lp v xc nh

3.1. Mi trng chn lc Baird Parker (B-P agar)12

3.2. Phng php ELISA13

Chng 4: Lin h vi thc phm

4.1. Staphylococcus aureus trong thc phm16

4.2. Quy trnh xc nh Staphylococcus aureus trong thc phm17III.KT LUN19

Ti liu tham kho20I. LI M UAn ton v sinh thc phm ang l ch nng ca x hi. Ng c thc phm c rt nhiu nguyn nhn nh do: ha cht, bn cht thc phm cha sn mt s cht c, Nhng quan trng hn ht vn l t vi sinh vt, trong c Staphylococus aureus-mt trong nhng nguyn nhn chnh. iu ng lu v quan tm y l chng ny c kh nng tit ra mt s c t bn vi nhit v kh b phn hy nhit cao. Hn na, chng li c kh nng khng khng sinh, khi gp iu kin thun li cn c th ly lan v gy nn nhng cn bnh nguy him.

V th m nhm em thc hin ti: Vi sinh vt gy bnh trong thc phm-Staphylococcus aureus nhm mc ch tm hiu ngun gc pht sinh, nhng tc hi m Staphylococus aureus gy ra cng nh cc bin php phng nga v cha tr... c bit l tm hiu cc phng php phn tch nhn bit v pht hin chng, y ng thi c th xem l mt bin php hu hiu kim tra an ton ca thc phm.

Bi tiu lun cn nhiu thiu st, chng em rt mong c c gp kin hon thnh ti mt cch tt nht.II. NI DUNGCHNG 1: Staphylococcus aureus v nhng tnh cht c trng

1.1. Gii thiu:

T cu khun (staphylococci) c m t ln u tin bi nh phu thut ngi Scotland, ng Alexander Ogston v nguyn nhn ca mt s hin tng sinh m (hnh thnh m) truyn nhim ngi. Nm 1882, ng t tn cho chng l staphylococcus (ting Hy Lp: straphyle, chm nho; coccus, ht la hay trng c) v hnh dng chng xut hin di knh hin vi.

M t u tin ca ng c thc phm do t cu khun c cho l ca Vaughan v Sternberg, nhng ngi kho st mt dch bnh ln Michigan tin rng nguyn nhn l bi pho mt b nhim t cu khun. S lin kt r rng ca cc sinh vt vi ng c thc phm phi i cho n khi Barber (1914) chng minh rng t cu khun c th gy ra ng c bi sa tiu th t mt con b b vim v do t cu khun. Nm 1930, Dack ch ra rng ng c thc n nhim t cu khun c gy ra bi mt enterotoxin (c t rut) c kh nng lc.

Hin nay c 27 loi v 7 phn loi ca th h Staphylococcus; s sn xut enterotoxin ch yu c i lin vi loi Staph. aureus, mc d n cng c sn xut bi cc loi khc bao gm Staph. intermedius v Straph. hyicus.Nh mt loi bnh tng i nh, thi gian ngn, ng c thc phm nhim t cu c l nhiu kh nng khng c bo co nh cc loi bnh khc. a s nhng trng hp bo co lin quan n nhng s bc pht v mt vi trng hp ri rc c pht hin. M gia nm 1983 v 1987, t cu khun chim 7.8% (47) ca 600 v bc pht ng c thc phm nhim khun m c ghi li. Con s tng ng cho Anh v x Wales trn cng thi k l 1.9% (54) trong tng s 2815 v. Nhng cn ng c thc phm nhim t cu khun Anh ln ti nh im trong nhng nm 50 ca th k hai mi mc 150 v mi nm nhng t suy gim xung cn 5-10 v mi nm trong thi k 1990-1996 v trong giai on 2000-2005 l 1 v trn mt nm.

1.2. Hnh dng v c tnh:

Staphylococcus aureus l mt khun cu Gram dng (+) m c dng hnh cu nhng t bo dng trng c ng knh khong 1m. S phn chia t bo mt mt phng to thnh nhng khi t bo khng ng u ging nh chm nho (hnh 7.10)

Hnh 7.10. Staphylococcusaureusbm trn thp khng r (nh M.Lo)Staphylococci l nhng catalase - dng tnh, oxidase - m tnh, vi sinh vt k kh ty . Kh nng ln men glucozo ca chng c th c s dng phn bit chng vi Micrococcus ging h hp hon ton, mc d c nhng loi c 2 dng trn m s phn bit ny khng r v s sn xut acid thp bi mt vi Staphylococci v sn xut s lng nh acid di iu kin khng c khng kh bi mt s micrococci. Vic sn xut Enterotoxin b nh hng mt cch bt li bi nhng iu kin khng c khng kh nhiu hn s tng trng.

Bng 7.8. Cc yu t nh hng n s pht trin v sn xut enterotoxin bi Staphylococcus aureus

Staphylococcus aureus l mt mesophile in hnh vi nhit tng trng khong gia 7 v 480C v nhit ti u ti 370C vi nhng iu kin ti u khc. Phm vi nhit trn enterotoxin c sn xut ra nh hn mt vi v c mt ti u 35-400C (Bng 7.8). Sinh vt c kh nng chu nhit khng c ngoi l vi D62 ca 20-65s v D72 ca 4.1s khi c o trong sa s nui vi khun bng que cy. S chu ng nhit c th hin a dng ng k v nhng gi tr D c tm thy tng gp 3 ln khi s nui cy c th nghim.S tng trng xy ra ti u gi tr pH t 6-7 vi ti thiu v gii hn ti a ln lt l 4 v 9.8 10. Khong pH trn enterotoxin sn xut ra l hp hn vi s sn xut c t di pH = 6.0 nhng m cng vi s pht trin nhng gi tr chnh xc s thay i vi trng thi chnh xc trung bnh.

Mt c trng ca Staph. Aureus m lmtc bitquan trng xem xttrongmt sloi thc phm l s chu ng mui v aw c bin i. N tng trng d dng trong cc mi trng c 57% NaCl v mt vi chng c kh nng tng trng mi trng ln n 20% NaCl. N s pht trin xung aw ca 0.83 ni m n c thi gian vng i 300 pht. Mt ln na phm vi m s sn xut enterotoxin xy ra c gii hn hn mt mc ti thiu l aw 0.86.

Mi trng ch yu ca Staphylococcus l da, nhng tuyn da,cc mng nhy ca ng vt mu nng. Mt vi loi th c lin kt vi vt ch c th: Staph. Hyicus vi heo v Staph. gallinarum vi g. Staph. Aureus l ph bin hn nhng xy ra thng xuyn hn ng vt linh trng bc cao. con ngi th n c bit lin quan n ng mi ni m n c tm thy vi 20-50% ca c th khe manh. N c th c phn lp t phn v mt cch ri rc t phm vi rng ca nhng ni khc nh t,nc bin v nc ngt, b mt ca cy,bi v khng kh. Mc d l mt ng vt k sinh v hi trn b mt c th con ngi ni m n ng mt vai tr hu ch cho qu trnh chuyn ha nhng sn phm trn da v c th ngn chn nhng mm bnh trn da. Staph. Aureus c th gy nn nhng vt r nh trn da nh l vt lot do nhim trng v nghim trng hn l mt mm bnh c hi khi hng ro da b chc thng hoc sc chu ng ca vt ch thp.

CHNG 2: Bnh v triu chng lm sng2.1. c t rut (enteroxin):- c sn xut phn ln bi cc chng Staph.aureus. Cc c t ny l nhng protein tng i bn vi nhit, khng b ph hy khi un nu, c trng lng phn t 28000-30000 Da gm 6 loi c k hiu A, B, C, D, E, F. Nm 2008 tm ra c hai loi c t mi l SES v SET cng nm trong nhm c t rut do Staph.aureus sinh ra.

- Cu trc phn t ca Staphylococcal enterotoxin B (SEB): SEB l mt trong cc ngoi c t c sinh ra bi vi khun Staph.aureus. Thng thng khng b ly nhim vo c th, SEB s tc ng ch yu ln cc h vn chuyn ion v nc ca rut, do c gi l enterotoxin.

+ SEB c hnh thnh khi Staph.aureus sng trong iu kin khc nghit nh: nhit mi trng gia tng t ngt, thiu oxy, s mt cn bng trong p sut thm thu.

+ ng vai tr l mt trong nhng ni c t quyt nh ca vi khun Staph.aureus nn SEB c nghin cu kh chi tit. Trnh t a.a ca SEB c xc nh t nm 1970.

- Ging nh cc cu trc protein ngoi bo khc ca Staph.aureus thng c tm thy trong mi trng nui cy hay trong thc phm b nhim, protein SEB bao gm mt trnh t tnh hiu gm 27 a.a u N. on trnh t tn hiu ny c chc nng dn SEB ra ngoi mi trng nui cy, sau trnh t ny s b ct bi protease v tr nht nh. SEB dng hot ng trong mi trng ngoi t bo gm 239 a.a rong mt chui polypeptide n c khi lng phn t khong 28,336 KDa. dng hot ng protein SEB c cu gm: 7 cu trc xon anpha v 14 phin gp np beta v mt cu ni disuifide ni cystein v tr 120 v 140. SEB c kh nng chng li s tc ng ca protease trysin v chymotrysin v papain c trong rut.

- Nm 1986, Christopher v cng s xc nh thnh cng trnh t SEB ca chng Staph.aureus S6. Trnh t ca mt gen hon thin c tnh t codon m u ATG v tr nucleotide 244, sau l vng khung c m gm 798 nucleotide v kt thc ti codon TGA ti v tr nucleotide 1042.

2.2. Cc c t khc ca Staphylococcus aureus:- Hemolysis: gm 4 loi (alpha, beta, gamma, delta), mang bn cht protein gy tan mu beta, tc ng khc nhau ln cc hng cu khc nhau. C kh nng sinh khng, gy hoi t da ti ch v git cht sc vt th nghim.

+ Alpha-hemolysis: lm h hng mng t bo mnh nht, c kh nng c ch thm thu ca mng, lin kt vi cc t bo nhy cm nh tiu cu, bch cu c kh nng phn hy hng cu tn thng hng cu. Alpha-hemolysin tit ra s gn vo mng ca t bo nhy cm, s gn kt to thnh mt mng y nc to iu kin thm nc khng kim sot c cc ion v cc phn t hu c nh. Khi cc phn t quan trng i qua nh ATP, ion th khng th o ngc thm thu dn n ph v thnh t bo gy ra ci cht cho t bo ch.

+ Beta-hemolysis: l mt trong nhng exotoxins c sn xut bi hu ht cc chng Staph.aureus, l protein c kh nng gy thoi ha sphingomyelin gy ng c cho nhiu t bo k c hng cu ngi.

+ Delta-hemolysis: l mt peptide rt nh sn xut bi hu ht cc chng Staph.aureus, l mt protein hot ng b mt v c th d dng chn thm chnh n vo cu trc mng k nc v cc knh ion.

+ Gamma-hemolysis: nhy cm vi cc loi hng cu ca th, cu, ngi, chut, b, nga. Gy ra hoi t nh da th, chut, c th gy cht th.

- c t gy hi chng sc nhim c TSST ( toxic shack syndrome toxin): thng gp nhng ph n c kinh dng bng bng dy, bn hoc nhng ngi nhim trng vt thng. Kh phn bit c t ny vi enterotoxin F. TSST kch thch gii phng TNF (Tumor neerosis factor, yu t hoi t u) v cc interleukin I,II. C ch gy sc ca n ging nh c t rut.

- c t exfoliatin hay epidermolitic: L cc men ph hy thng b. Men ny gy tn thng da to cc bng nc, Gy hi chng phng rp v chc l da tr em. 85% cc polypeptide, loi A bn vi nhit 1000C/20 pht, cn loi B th khng. Khng th c hiu c tc dng trung ha c t ny.

- Alpha toxin: bn cht l protein gy tan cc bch cu a nhn v tiu cu, t gy ra p xe, hoi t da v tan mu. c t c tnh khng nguyn nhng khng th ca n khng c tc dng chng nhim khun.

- c t bch cu (Leucocidin): l nhn t git cht bch cu ca nhiu loi ng vt, bn cht l protein, khng chu nhit. T cu gy bnh c th b thc bo nh t cu khng gy bnh nhng li c kh nng pht trin bn trong bch cu. Gm hai mnh F v S c th tch ri nhau, trng lng phn t l 32000, 38000 Dalton. Nu hai mnh ny tch ri nhau th chng s mt kh nng gy c. Chng gy ra nhim trng da v hoi t.

- Ngoi c t sinh m (pyrogenic): c t ny tng t nh c t sinh m ca lin cu. Protein ngoi c t ny c tc dng sinh m v phn bo lymphocyte, ng thi n lm tng nhy cm vi ni c t nh gy shock, hoi t gan v c tim. Gm 3 loi k hiu A, B, C. Ba loi ny khc nhau v trng lng phn t v v tnh c hiu khng nguyn, ging nhau v kh nng sinh m v phn bo.

- Dung huyt t (hemolycin staphylolycin): ph hng cu (tan mu) v gy cht cc t bo ht cng nh i thc bo.

- Fribrinolysin (enzyme Staphylokinase): Nhiu chng ca Staph.aureus th hin mt hot ha plasminogen gi l staphylokinase. N lm ph hy fibrin. L mt enzyme c trng cho cc chng gy bnh ngi trong cc cc mu v gy v cc cc mu ny to nn tc mch. C ch ny ging vi Steptokinase, c s dng trong y hc iu tr bnh nhn b huyt khi ng mch vnh.- Coagulase: lm ng huyt tng ngi v th, chng ng vi citrate natri v oxalate natri. Coagulase lm dnh t huyt vo b mt vi khun v do cn tr s thc bo. Men ny gn vi prothrombin trong huyt tng v hot ha qu trnh sinh fibrin t tin cht fibrinogen. Enzyme ny cng vi yu t kt cm v mt enzyme vch vi khun gip S. aureus to kt ta fibrin trn b mt ca n.

- Hyaluronidase: men ny c kh ph hy cht c bn ca t chc, gip vi khun c th pht tn trong t chc. Thy phn axit hyaluronic ca m lin kt.

- Beta- lactamase: men ny ph hy vng beta lactam, cu trc c bn ca cc khng sinh nh penicilline G, Ampicilline v Uredopenicilline lm cho cc khng sinh ny mt tc dng.2.3. C ch gy c ca SEB:- SEB l trung gian kch thch cc lympho T h min dch ca cc vt ch cc c t lin kt trc tip n phc hp protein (MHC) lp II trn b mt t bo ch sao kch thch gia tng s lng ln cc lympho T. SEB c xem l mt siu khng nguyn ca vi khun v c th to thnh mt cu ni gia MHC lp II ca cc t bo trnh din khng nguyn v vng Vbeta ca cc th th t bo T nh: CD4, CD7. T kch thch hot ha cc t bo T biu hin cc on gen Vbeta m khng cn thit phi c mt qu trnh ch bin v trnh din thng thng. - iu ny gy ra s sn sinh mt s lng ln ca cytokine v interleukin (IL-2), cc yu t hoi t khi u beta (TNF-beta) v cc interferon. Nu n thc phm c SEB bnh nhn c cc triu chng nh: chn n, bun nn, tiu chy Cc triu chng ny xut hin l do cc cytokine trong cc t bo T ca lng rut c sinh ra t.

- SEB d ha tan trong nc, tnh cht ha hc tng i n nh, chu c cc tc ng c hc va phi, chu c nhit si.

2.4. Bnh v triu chng:Ng c thc phm bi Staph. aureus c c trng bng thi k bnh ngn, in hnh l t 2-4h. Bun nn, i ma, co tht bao t v kit sc l nhng triu chng ch yu, v hi phc hon ton bnh thng trong vng 1-2 ngy. Trong trng hp mt nc nghim trng, xanh xao r rt c l cn phi iu tr bng cch truyn dch vo tnh mch.

Thi k bnh ngn l c trng ca mt s nhim c ni m bnh tt l kt qu ca vic n vo mt c t c hnh thnh trc trong thc phm. Staph. aureus sn xut t nht 11 enterotoxin c nh r t SEA ti SEJ. thm vo mt cht rc ri v gy nhm ln cho ngi thiu thn trng, khng c SEF v c ba bin th ca SEC. Loi c t A v D, d n c hay trong kt hp, u thng xuyn c lin quan nht trong nhng v ng c thc phm. Anh , loi A chu trch nhim cho 52% v, loi D chim 6%, loi A v D kt hp chim 19%, v loi C v D chim 9%. Tnh nhy cm khc nhau gia nhng c th c nh gi rng, trong nhng v ng c i hi t hn 1mg c t thun gi ra nhng triu chng. Nhng c t th nh (Mr 2630 kDa) mi mc xch polypeptide chia s ng k amino acid ng ng. Ngoi tr SEI, mi ci cha mt lin kt n disulfide gn phn t trung tm. V cu trc chc chn ca n, c c th khng c li nhng protease trong rut v chu nhit, b bt hot ch bi un si ko di.

Mc d c m t nh l nhng enterotoxin nhng nhng c t Staph. aureus chnh xc l neurotoxin. N gy ra phn ng bun nn bng cch tc ng trn c quan th cm trong rut, kch thch trung tm nn e trong no thng qua dy thn kinh v dy thn kinh giao cm. nu nhng dy thn kinh ny b ct t vy s nn e khng xy ra. Khng bit lm th no c t gy ra tiu chy nhng n c chng t rng khng phi l tc ng ca adenylate cyclase.

Nhng enterotoxin Staph. aureus hin nay cng c bit l nhng siu khng nguyn, cc phn t c kh nng kch thch mt t l cao hn nhiu ca nhng t bo T so vi nhng khng nguyn thng thng. Ci ny c l ng mt vai tr g trong bnh ng tiu ha, m khng c bit n.

CHNG 3: Phng php phn lp v xc nh3.1. Mi trng chn lc Baird Parker (B-P agar):Mi trng chn lc bao bc Staph. aureus hiu qu v ph bin nht c Baird-Parker xut nhng nm u thp nin 60. Phng php ny l s kt hp gia mi trng c mc chn lc cao, phn ng nhn bit c trng v kh nng phc hi cc t bo b tn thng. Liti clorua v khong cht l cc yu t chn lc, cn lng trng g v piruvate h tr s phc hi cc t bo b tn thng. Ta c th nhn bit t cu khun qua c im khun lc en huyn, sng bng, mi trng bao quanh sch s do phn ng thy phn protein lipovitellenin trong lng trng g. Khun lc cng thng c ng vin trng bn trong do s kt t ca cc acid bo. Khun lc xut hin trong mi trng Baird-Paker (B-P) agar chng t s c mt ca t khun cu, vi sinh vt thng c s dng trong cc th nghim sn xut coagulase v nuclease chu nhit.

Coagulase l mt cht ngoi bo lm ng t huyt tng ca ngi v ng vt trong mi trng khng c canxi. Enzim ny khng c trng cho Staph. aureus v Staph. intermedius v Staph. hyicus cng to c enxim ny. Staph. intermeius khng s dng khong cht nn ch hnh thnh khun lc trng trn B-P agar, cn Staph. hyicus, vi khun c tm thy trn da ln v gia cm, cn nhiu th nghim sinh ha hn phn bit mt cch chn chn hn vi Staph. aureus.

S c mt ca coagulase c th kim tra bng phng php s dng huyt tng th c x l EDTA trong ng nghim coagulase. Hin nay c rt nhiu cc b dng c (kit) th nghim nhanh chng, da trn s pht hin mi lin h gia coagulase (cn gi l yu t vn cc) vi protein A, cht phn ng vi Fc, mt phn ca phn t IgG. Pht kin c tm thy bi s ng t ca hng cu, ht nha bc fibrinogen v huyt tng khi tip xc vi khun lc t mi trng nui cy chn lc. iu ny c th kim chng trc tip, khng cn thng qua cc th h vi khun trung gian. S hnh thnh coagulase c th c nhn bit thng qua s thay i lng trng trng trong mi trng agar c cha huyt tng ln v th.

Nuclease chu nhit c th pht hin bng agar toluidine xanh/DNA, bng khun lc b nu si b mt nui cy hoc bng lp ph khun lc c x l nhit trn B-P agar.

c 4 chng (biotype) Staph. aureus c pht hin nhng ng dng ca chng vn cn hn ch v hu ht cc ging m con ngi tm c u thuc chng A. Staph. aureus c s dng cng vi phage; hu ht cc ging gy c thc phm thuc v nhm huyt thanh III.

Qu trnh x l nhit c th tiu dit nhiu vi sinh vt nhng c t rut vn cn. Do vy vic nhn bit thc phm b nhim c mang tnh cnh bo v mi nguy him hn l phc v cho cng vic m cc t bo vi khun sng st. n nay c mt s k thut xt nghim min dch i vi c t ca t cu khun. Cc phng php c ch ng t trc y nh th nghim khuch tn microslide gel c nhy km, tn nhiu thi gian v cn thao tc cn thn nhn bit c c t. Hin nay, k thut ELISA ngy cng c dng ph bin, phng php ny c th pht hin c t mc 0,1-1,0 ng/g thc phm v c th o ngc s kt t cao su th ng vi nhy 0,5 ng/ml.

3.2. Phng php ELISA: Phng php ELISA: (Enzyme Linked ImmunoSorbent Assay ) l 1 k thut sinh ha pht hin khng th (KT) hay khng nguyn (KN) trong mu xt nghim. Hin nay, ELISA c s dng trong nhiu lnh vc nghin cu nh y hc, nng nghip c bit l trong kim tra an ton trong cc sn phm sinh hc.

C 2 loi phng php ELISA:

+ Phng php ELISA trc tip (direct ELISA): dng pht hin khng nguyn trong mu xt nghim.

+ Phng php ELISA gin tip (indirect ELISA): dng d pht hin khng th chuyn bit trong huyt thanh.

u im: Phng php ny c s dung cho cc loi khng nguyn vi nhy v c hiu cao, c th pht hin c phc hp nh KN-KT, cho php pht hin sm tc nhn gy bnh giai on sm khi mm bnh mi xm nhim.

+ Nhanh, thao tc n gin.+ t tn sinh phm, ha cht, s lng mu ln, rt thch hp vi vic phn tch i vi nguyn liu th.

Nhc im: chnh xc khng cao.

Ngun tc k thut ELISA:

S dng KT n dng (Mabs) ph b mt nhng a ging v s c pht hin bng cch s dng cc khng th th cp nh: horeseradich perosidase hay alkaline phosphatase). Nu c s hin din ca KN trong mu, KN s to phc hp vi KT c nh trn ging v KT t do c gn enzyme to thnh mt phc hp kp (sandwich). Khi b sung c cht c hiu ca enzyme vo ging, enzyme xc tc phn ng thu phn c cht to ra cc sn phm c mu hay pht sng.

Khi b sung cc c cht c hiu ca enzyme vo ging, enzyme s xc tc phn ng thu phn c cht to ra cc sn phm c mu hay pht sng. Bng cch theo di s i mu c th pht hin s hin din ca KN. Cn tng sinh chn lc trc khi thc hin phn ng. K thut ny c nhy pht hin khong 106CFU/ml.

Vt liu v phng php:

Chun b cc KN: S. aureus c nui cy trn mi trng Staph.110 370C trong 24h. Thu chng vi khun vi nc mui m phosphate (pH 6.8) c cha formalin 0.3% v un nng 1000C trong 1h.

Sn xut KT: ba con th c tim dch huyn ph ni trn di da vi lng 0.5;1;1.5 v 2ml khong 4 ngy. Sau 7 ngy tip tc tim 1ml Staph.aureus c nui cy trong mi trng canh tht vo th. Sau 14 ngy tim mi trng ging nh trn, thu nhn mu v huyt thanh khi git th. Tinh ch v c lng Protein: khng th th c phn lp v tinh khit mt phn thng qua phng php kt ta ammonium sulfate v hm lng protein c c tinh bng phng php Biuret sau khi tiu bn theo ng chun ca huyt thanh b albumin. S tip hp: Enzyme peroxidase t u tng (10mg) c tip hp vi mt phn KT tinh khit ca th, s dng bc hai trong phng php glutaradehyde. Lp v KT: 100l KT th (pha long 1/10 trong dung dch m ph) c vo tng ging, polystyrene, tm microtitration gm 96 ging 40C trong 24h. Sau , tm c ra nm ln vi m ra. Cc tm c gi c nh bi 100l PSB vo tng ging v 370C trong 24h. Sau khi , tm c ra sch bng m ra.

ELISA trc tip:

ELISA trc tip c thc hin pht hin KT tip hp enzyme. Cc tip hp enzyme ny pha long trong PBS l 1:100; 1:200; 1:400 v 1: 800. 100l PBS c thm vo mi ging. Huyt thanh th c pha long trong PBS t l 1:10.

l 2 ln tun t pha long t 2 ln n 11 ging, nh 100l c thm vo. Ging 12 c s dng kim sot. Cc a c 370C trong 2h. Sau ra li 5 ln bng dung dch ra. Tip tc pha long 100l mi hng, u tin pha long A+B, th 2 l pha long C+D, th 3 l pha long E+F, th 4 l Pha long G+H trn tm microtitration. 370C trong 2h. Sau khi ra li 5 ln bng dung dch ra. Sau thm 100l guaiaclo (guaicol + H2O2) v 370C trong 20 pht. Tip tc thm 50l H2SO4 1M (thuc dng) vo mi ging. O.D c ghi li ngay lp tc trong cng vi a c ELISA, c bc sng 450nm (Kemney v Challacombe, 1989).

Phn tch thng k: cc d liu c phn tch qua Duncans Multiple Range (DMR) kim tra hon ton theo thit k ngu nhin (CRD) (Steel v Torrie,1984).

Kt qu:

Cc xt nghim huyt thanh khc nhau c s dng cho cc KN pht hin nh l th nghim nhanh chng lm ngng kt, th nghim agar gel pht hin, nhng tt nht l phng php ELISA pht hin KN. N tng ca phng php ELISA l da trn s thay i mu sc, c li th hn cc xt nghim khc nh ngng kt, hunh quang hoc phng x trong phn ng KN-KT c th c o u n khch quan trong sc k n gin. Ngoi ra, s dng cc tm microtitre ELISA cho php mt s lng ln phn ng c c trong thi gian ngn (Dawkins et al,1990).CHNG 4: Lin h vi thc phm4.1. Staphylococcus aureus trong thc phm:Vic tn ti mt lng nh Staph. aureus trong thc phm khng c g l. Chng tn ti t nhin trong tht gia cm v cc loi tht sng khc nh mt thnh phn vi sinh thc vt b mt. Tng t, t khun cu c th c tm thy trong sa ti vi mt nh giai on sinh si ca bnh vim tuyn v. L mt i th cnh tranh km, chng thng khng gy ra vn g nghim trng v chng khng th pht trin mnh v d b tiu dit sau qua trnh nu nng hay tit trng. Tuy nhin vn c s pht trin bng n trong cc sn phm t sa nh sa bt, chocolate sa, ni m t khun cu sinh si v tit ra c t t trong sa ti khng qua tit trng. V d in hnh l s kin Nht Bn nm 2000, nh hng ti 13000 ngi. Mt giai on trong sn xut sa bt phn lp to iu kin cho t khun cu nhn ln v tch ly c t. Lng bt b nhim c c dng ch bin cc sn phm n thng ngy. Mc d bn thn ca chng khng gy nguy him n sc khe con ngi nhng sc c mt ca t khun cu trong tht sng lm cho thc phm ch bin b nhim c cho. S mt v sinh trong quy trnh ch bin cng l mt yu t nhim c thng gp trong tc nhn gy c do con ngi. Bnh vim mi hng do vi khun thng ko theo s xut hin ca chng trn da v thc n cng s b nhim khun theo t phn da b tn thng, do ho v ht hi. Phi cn mt s lng >106 c th trn 1g mi sn sinh lng c t gy bnh. Nhim bn l iu kin cn thit nhng cha phi tt c cho mt s bng n vi sinh vt. c bit, iu kin nhit v thi gian phi ph hp cho s pht trin ca chng.

Nhng nghin cu M v Anh chng minh sn phm tht g cm v tht gia sc ng lnh hoc nu chn l mi trng pht trin tt ca t khun cu. Tht c p mui nh i ln v tht b cng b nguy him bi t khun cu khng b mui c ch nh phn ln thc vt vi sinh khc. Nhng ming tht phc v trong cc ba buffet l mi trng thun li cho s pht trin ca t khun cu bi v thc n phi c ch bin trc mt thi gian v thng c lu tr nhit thng hoc khng c p lnh y . hp cung cp mi trng thch hp, khng c i th cnh tranh cho t cu khun v s nhim bn sau khi hp b r r l c hi cho s nhn ln ca t cu khun.

Nguyn nhn ca s nhn ln ca t cu khun cn c th do ph mai cng hay ko lnh, custards v bnh ph kem lnh. V d nh Nht Bn l cm nm lm bng tay v Hungary l kem.4.2. Quy trnh xc nh Staphylococcus aureus trong thc phm:- c im quan trng nht ca Staph.aureus c cc nh khoa hc s dng phn bit vi cc Staphylococci khc l kh nng sinh tng hp coagulase va kh nng s dng Mannitol.

- Mt s chng thuc loi Staph.aureus c kh nng sinh tng hp c t rut enterotoxin khi chng nhim vo thc phm. Tuy nhin s lng t bo vi khun phi ln (khng thp hn 106 CFU/g) th mi sinh ra mt lng toxin gy ng c cho ngi s dng. Do vic nh lng Staph.aureus trong thc phm l rt cn thit d on kh nng gy ng c ca sn phm v nh gi tng qut mc an ton v sinh thc phm.

Quy trnh nh lng Staphylococcus aureus trong thc phmIII. KT LUN

Staphylococcus aureus (t cu khun) l vi khun gram dng, khng di ng, khng sinh nha bo, mt s chng Staph.aureus c th to v polysaccharide.

Staph.aureus sinh trng tt trong mi trng tng hp nh thch mu v huyt thanh, c phn ng indol, thy phn gelatin v lm ng huyt tng, chu nhit v mui tt.

Trong mi trng thch, khun lc c hnh trn bng, c m, c th c mu vng m, vng cam hay trng.

Nhit sinh trng ti u 37oC, hiu kh hay k kh ty .

Staph.aureus c kh nng sinh nhiu c t nh: hemolysis, TSST, exfoliatin, epidermolitic, alpha toxin, leucocidin, pyrogenic, hemolycin staphylolycin, fribrinolysin, coagulase, hyaluronidase, beta-lactamase, c bit l c t rut (enterotoxin).

C th xc nh Staph.aureus bng phng php truyn thng trn mi trng thch Braid-Parker Agar (BPA), hay bng cc phng php hin i nh ELISA, lai phn t, b Kit MRSA.

Staph.aureus l mt trong nhng nguyn nhn gy ng c thc phm v gy nn nhng cn bnh nguy him ngi. iu ng lu l khi gia nhit ta ch c th tiu dit c t bo nhng c t ca n li bn vi nhit. V th thc phm cn m bo v sinh t khu ch bin n khu bo qun. Ngi lao ng tip xc vi thc phm phi thn trng gi gn v sinh tt nh eo gng tay, khu trang

Ngoi ra mt s Staph.aureus c c lc mnh cn c kh nng khng khng sinh nh penicillins, methicillin, dicloxacillin, nafcillin, oxacillin, cephalosporins ngi mc bnh c nguy c t vong cao. Nguyn nhn ch yu l do s lm dng qu nhiu khng sinh, cht kch thch tng trng trong chn nui. Cho nn, thc phm trc khi a ra th trng cn c kim nh k lng v cht ch.TI LIU THAM KHO1. Martin R. Adams and Maurice O. Moss, University of Surrey, Guildford, UK, Food microbiology 3rd Edison.

2. http://tcyh.yds.edu.vn/2008/2008%20PB%20T12%20so%204%20-%20YTCC/@YTCC%2008%20Dan%20trang/T%E1%BA%A0O%20KH%C3%81NG%20TH%E1%BB%82%20%C4%90A%20D%C3%92NG%20V%C3%80%20X%C3%82Y%20D%E1%BB%B0NG%20QUI%20TR%C3%8CNH%20ELISA.htm3. www.yduocngaynay.comStaphylococcus aureus

di knh hin vi

Chun b mu

Cy mu ln mi trng thch Baird-Parker hoc mi trng Mannitol

mui v

Quan st v chn khun lc

Tnh ton kt qu

Kim tra Coagulase

Tnh cht ha sinh

Kh nng ln men glucose v mantol

Tnh mn cm vi lysostaphin

Kh nng sinh tng hp thermonuclease