tìm hiểu một số phương pháp phân tích vi sinh vật gây bệnh có trong thực phẩm

8/11/2019 Tm hiu mt s phng php phn tch vi sinh vt gy bnh c trong thc phm

1/79

BGIO DC V O TO

TRNG I HC KTHUT CNG NGHTP. HCM

KHA LUN TT NGHIP

TM HIU MT SPHNG PHP PHN TCH VI SINH VTGY BNH C TRONG THC PHM

Ngnh: MI TRNG V CNG NGHSINH HCChuyn ngnh: CNG NGHSINH HC

Sinh vin thc hin : HUNH QUANG VINH

MSSV: 0811110104 Lp: 08CSH1

TP. HCh Minh, 2011

WWW.DAYKEMQUYNHON.UCOZ.COM

WWW.FACEBOOK.COM/DAYKEM.QUYNHONEmail: [email protected]

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2/79

LI CAM OAN

Em xin cam oan tt cni dung trong bi vit l gm nhng ti liu do em

ttng hp c v nhng ti liu c gio vin hng dn cung cp, em

khng sao chp bi ca ngi khc di bt khnh thc no. Nu c gian

di em xin hon ton chu trch nhim.



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3/79

LI CM N

Em xin trn thnh cm n qu thy c tn tnh dy d, hng dn cho em

trong sut thi gian hc tp em c thi c n ngy hm nay. Em

cng xin cm n cc thy c to iu kin tt nht cho em c thhc tp

v nghin cu trong qu trnh hc. Cui cng em xin cm n gio vin

hng dn tn tnh hng dn cho em hon thnh kha lun ny. Ngoi

ra em cng xin cm n gia nh em lun ng vin v nhc nh, to iu

kin cho em c thyn tm hc tp.



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4/79

MC LC

Danh mc hnh nh ......................................................................................... 5

Li mu ...................................................................................................... 6

CHNG 1: CC CHTIU VI SINH VT THNG C KIM

SOT TRONG THC PHM

1.1. Samonella ................................................................................................. 7

1.2. Campylobacter ......................................................................................... 7

1.3. Clostridium perfringens ........................................................................... 8

1.4. Clostridium botulinum ............................................................................. 9

1.5. Staphylococcus aureus ........................................................................... 10

1.6. Vibrio spp .............................................................................................. 11

1.7. Escherichia coli ...................................................................................... 12

1.8. Shigella spp ........................................................................................... 13

1.9. Coliforms............................................................................................... 141.10. Listeria monocytogenes

1.11. Bacillus cereus

1.12. Aspergillus

1.13. Virus

CHNG 2: CC PHNG PHP KIM NGHIM VI SINH VT

TRUYN THNG2.1. Samonella ............................................................................................... 20

2.1.1. Tng quan ........................................................................................... 20

2.1.2. Nguyn tc .......................................................................................... 20

2.1.3. Mi trng v ha cht ........................................................................ 21

2.1.4. Quy trnh phn tch nh nh tnh Salmonella trong thc phm ......... 22



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2.2. Vibrio ..................................................................................................... 27

2.2.1. Tng quan ........................................................................................... 27

2.2.2. Nguyn tc .......................................................................................... 28

2.2.3. Mi trng v ha cht ........................................................................ 28

2.2.4. Quy trnh phn tch .............................................................................. 29

2.3. Listeria monocytogenes .......................................................................... 31

2.3.1. Tng quan ........................................................................................... 31

2.3.2. Nguyn tc .......................................................................................... 32

2.3.3. Mi trng v ha cht ........................................................................ 32

2.3.4. Quy trnh phn tch .............................................................................. 33

2.4. Staphylococcus aureus ............................................................................ 35

2.4.1. Tng quan ........................................................................................... 35

2.4.2. Nguyn tc .......................................................................................... 35

2.4.3. Mi trng v ha cht ........................................................................ 362.4.4. Phn tch nh tnh Staphylococus aureus ............................................ 36

2.4.5. nh lng S.aureus bng phng php m khun lc ...................... 37

2.4.6. nh lng S.aureus bng phng php MPN ..................................... 39

2.5. Bacillus Cereus ....................................................................................... 39

2.5.1. Tng quan ........................................................................................... 39

2.5.2. Nguyn tc .......................................................................................... 40

2.5.3. Mi trng v ha cht ........................................................................ 40

2.5.4. Quy trnh phn tch .............................................................................. 41

2.6. Clostridium ................................................................................................ 46

2.6.1. Tng quan ........................................................................................... 46

2.6.2. Nguyn tc .......................................................................................... 47



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2.6.3. Mi trng v ha cht ........................................................................ 48

2.6.4. Quy trnh phn tch .............................................................................. 48

2.6.5. Bo co kt qu ................................................................................... 49

2.7. Coliform v E.Coli ................................................................................. 49

2.7.1. Tng quan ........................................................................................... 49

2.7.2. nh lng Coliforms, Coliforms chu nhit, Coliforms phn v E.coli

bng phng php MPN ................................................................................ 49

2.7.3. nh lng Coliforms, Coliform phn bng phng php m khun

lc ..................................................................................................................... 52

2.7.4. nh lng E.coli bng phng php m khun lc ........................... 55

2.8. Tng nm men nm mc ........................................................................... 57

2.8.1. Tng quan ............................................................................................... 57

2.8.2. Nguyn tc.............................................................................................. 58

2.8.3. Mi trng v ha cht .......................................................................... 582.8.4. Quy trnh phn tch nh tnh ................................................................. 59

2.8.5. Quy trnh phn tch nh lng .............................................................. 59


KHNG TRUYN THNG

3.1. Phng php pht quang sinh hc ATP trong gim st vsinh ............... 61

3.2. Phng php ELISA (Enzyme-Linked ImmunoSorbent Assay) ............. 63

3.3. Phng php lai phn t(Hybridization) ................................................ 65

3.4. Phng php PCR (Polymerase Chain Reaction) ................................... 66

3.5. Mt sphng php thnhanh khc ...................................................... 70

3.5.1. Kthut phn tch v tng mt ....................................................... 70



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7/79

3.5.2. Kthut mng lc pht hunh quang trc tip (Direct Epifluorscent

Technique DEFT) v mng lc li knc (Hydrophobic Grid

Membrane) ....................................................................................................... 71

3.5.3. Kthut mng petri (Petrifilm) .............................................................. 71

3.5.4. Kthut Redigel .................................................................................... 72

3.5.5. Kthut dn in, trkhng (conductance/impedance) .................. 72

3.5.6. Kthut o vi lng calorie (Microcalorimetry) .................................. 73

3.5.7. Kthut o mc phng x(Radiometry) .............................................. 73

KT LUN ..................................................................................................... 75TI LIU THAM KHO ............................................................................. 76



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8/79

DANH MC HNH NH

Hnh 1.1 Samonella.

Hnh 1.2 Campylobacter.

Hnh 1.3 Clostridium perfringens.

Hnh 1.4 Clostridium botulinum.

Hnh 1.5 Staphylococcus aureus.

Hnh 1.6 Vibrio cholerae.

Hnh 1.7 V.vulnificus.

Hnh 1.8 V.parahaemolyticus.

Hnh 1.9 Escherichia coli.

Hnh 1.10 Shigella.

Hnh 1.11 Coliforms.

Hnh 1.12 Listeria monocytogenes.

Hnh 1.13 Bacillus cereus.Hnh 1.14 Aspergillus flavus.

Hnh 1.15 A.parasiticus.

Hnh 1.16 Virus.



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9/79

LI MU

1.

Tnh cp thit ca ti

Ngy nay ngi tiu dng la chn thc phm th ngoi nhng yu cu vs

bdng, hp khu v, gi cphi chng cn c an ton thc phm cng l mt

trong nhng yu cu hng u. Svngc thc phm cng tlbnh ung th

c cho l do n phi cc cht gy nhim ngy cng gia tng. Trong s,

nhim khun chim tlkh cao ang l mi lo ca nhiu nc trn thgii.

Vit Nam l nc ang pht trin thuc vng nhit i.Trnh sn xut cn

thp cng vi iu kin kh hu nng, m, to iu kin cho nhiu loi vi sinh

vt gy hi thc phm pht trin. Tnh hnh ngc thc phm lin tc xy ra.

V vy, vic tm hiu quy trnh, phng php phn tch vi sinh vt gy bnh c

trong thc phm l rt cn thit nhm pht hin thc phm nhim khun, km

cht lng, bo vsc khe ca ngi tiu dng v cho cng ng. Xut pht t

yu cu trn sinh vin tin hnh ti: TM HIU MT SPHNG PHPPHN TCH VI SINH VT GY BNH C TRONG THC PHM.

2.

Mc ch nghin cu

Ni dung bo co l gii thiu vc trng v phng php xc nh mt s

vi sinh vt gy ngc thng xut hin trong thc phm. Qua gip cho mi

ngi bit c c nhng mi nguy no trong vic sdng thc phm v

phng php kim nghim nhng thc phm nghi ngc nhim vi sinh vt.

3.Kt cu ca kha lun tt nghip

Kha lun gm 3 chng:

Chng 1: Cc chtiu vi sinh vt thng c kim sot trong thc phm.

Chng 2: Cc phng php kim nghim vi sinh vt truyn thng.

Chng 3: Cc phng php kim nghim vi sinh vt khng truyn thng.



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10/79

CHNG 1: CC CHTIU VI SINH VT THNG C KIM

SOT TRONG THC PHM

1.1. Salmonella

Salmonellac thgy ngc thc phm khi hin din n mc ctriu

tbo trong mt gram thc phm. Cc triu chng do Salmonellagy ra thng

l tiu chy, i ma, bun nn. Thi gian bnh ktkhi tiu ththc phm b

nhim cho n khi cc triu chng c biu hin l 12 36 gi. Triu chng

ngc thng ko di t2 7 ngy. Khng phi tt cmi ngi khi tiu th

thc phm bnhim Samonellau bngc. Cc loi thc phm c nguy c

bnhim Samonellacao l tht gia cm, sn phm tht, trng v cc sn phm t

trng, thy sn. Ngun gy nhim cc loi thc phm ny thng l phn ngi

v ng vt, c nhim gin tip hay trc tip. c bit nguy him cho ngi l

cc loi Samonella typhi, S.paratyphiA, B, C gy st thng hn.

Hnh 1.1 Samonella

1.2. Campylobacter

Campylobactergy bnh vim nhim ng rut, hin din khp ni, c

bit trong hvi sinh vt ca nhiu loi ng vt v chim. Tuy nhin cc dng



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11/79

Campylobactergy ngc thc phm thuc loi a nhit bt buc, khng th

pht trin nhit thp hn 30oC. Cc triu chng ngc do vi sinh vt ny

gy ra l au nhc, tiu chy, st, au u, kh chu, chut rt, lnh cng, m

sng, thnh thong c nhng biu hin bnh ging nhcm cm. Thi gian

bnh t2 11 ngy. Cc sn phm sa, tht gia cm, nc l ngun c thgy

nn ngc do Campylobacter. Campylobacterc thhin din trong cc loi

thc phm ht chn khng. Cc loi ny rt nhy cm vi nhit v acid nn

c thbi tit trng hon ton bng phng php thanh trng Pastuer v khng

tng trng c trong thc phm c tnh acid.

Hnh 1.2 Campylobacter

1.3. Clostridium perfringens

C.perfringensc tm thy trong t, trong phn ngi. Trc y ngi

ta cho rng chcc dng C.perfringens khng nhit, to bo tv khng lm tan

mu mi c thgy ngc thc phm. Ngy nay c nhng ghi nhn cc v

ngc thc phm gy ra bi cc dng C.perfringens nhy cm vi nhit, khng

lm tan mu. Cc triu chng ngc do c tca vi sinh vt ny gy ra l au

tht vng bng, tiu chy. Thi gian bnh t12 24 gi. Ngun thc phm c



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12/79

thcha cc vi sinh vt ny thng l tht gia cm, nht l gia cm ng lnh

su, tht trong cc hm cha v cc loi thc phm khc. Bo tca

C.perfringens c tnh bn i vi nhit nn chng c thsng st qua qu trnh

nu chn, c bit l khi un nu thc n nhit thp v thi gian ngn. Cc

bo tsng st ny khi gp iu kin thch hp sny mm, tng trng.

Hnh 1.3 Clostridium perfringens1.4. Clostridium botulinum

Loi vi sinh vt ny phn bkhp ni trong t, nc, trong ng rut

cc gia sc v cc loi thy sn. Vi sinh vt ny sinh c tbotulin gy ngc

tht lm thc phm cho ngi. Triu chng lm sng khi ngc l i ma,

bun nn, sau c nhng biu hin ri lon thn kinh nhchong vng, ri

lon thgic, ri lon cc ccv ming, au vng ngc, kh thv t lit,

c thdn n tvong. Cc triu chng trn biu hin 12 36 gisau khi tiu

ththc phm nhim c tv ko di 2 6 ngy ty theo mc nhim c v

tnh trng sc khe ca tng bnh nhn. Cc loi thc phm nhtht, rau qu

khng c bo qun ng quy nh, thc phm nhim t, phn ng vt c

chbin khng nhit trc khi dng, cc sn phm ng hp khng ng



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13/79

quy cch c nguy cgy ngc bi vi sinh vt ny rt cao. iu kin thch hp

cho stng trng v to c tca vi sinh vt ny l iu kin kkh, pH trung

tnh, khng c cc vi sinh khc cnh tranh. c tbotulin do C.botulinum dng

E thng c ngun gc tc v cc sn phm thy sn. Dng vi sinh vt ny

thng phn lp c tcc mu bn y ti cc ca sng.

Hnh 1.4 Clostridium botulinum1.5. Staphylococcus aureus

Trong tnhin Staphylococcus aureus thng c tm thy trn da, mi,

tc hay long ca cc loi ng vt mu nng. Staphylococcus aureus sn sinh

mt sloi c tng rut enterotoxin bn nhit, khng bphn hy 100oC

trong 30 pht. Khi n phi thc phm c cha cc c tny, sau 4 6 gi

ngi bngc c cc triu chng tiu chy, nn ma ko di t6 8 gi. Cc

loi thc phm c cha nhiu mui nhjambon, kem tng hp, nc sp (t khi

c xl nhit cao hn 40oC) v cc loi thy sn, thc phm ng hp

thng hay nhim cc loi vi sinh vt ny. Con ng ly nhim chyu thng

qua tip xc tnh bp, qu trnh chbin.



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14/79

Hnh 1.5 Staphylococcus aureus

1.6. Vibrio spp

Cc loi Vibriothng hin din trong nc bin do chng cn ion Na+

pht trin, mt sloi c khnng gy bnh cho ngi l Vibrio cholera,

V.parahaemolyticus, V.vulnificus, V.hollisae, V.furnsii, V.mimicus, V.fluvialis,

V.alginolyticus, V.cholerael tc nhn gy nn cc vdch ttrn ton thgii.

Loi vi sinh vt ny c chia thnh hai kiu huyt thanh chnh l O1v khngO1(non-O1). Kiu huyt thanh O1li gm ba kiu huyt thanh phl Ogawa,

Inaba v Eltor (cn c gi l kiu O139). Hai kiu huyt thanh Inaba v

Ogawa ngy nay chcn c tm thy ti cc nc thuc khu vc chu .

Trong khi , cc vdch ttrn thgii li gy ra bi kiu Eltor. Dch tgy ra

bi V.choleraethng c lan truyn rt nhanh qua ng nc, gy nhim

thc phm v truyn nhim qua con ngi khi iu kin vsinh km. V.cholerae

to ra c ttcholera-toxin, c c tnh mnh: chcn 5g gy nhim qua

ng ming c thgy ra tiu chy cho ngi trng thnh. Ngoi c tt,

loi ny cng tit ra mt sc ttng tshiga-toxin. Ngun thc phm c

nguy cnhim v lan truyn dch tl nc ung, nc tri cy, rau qu, sa v



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15/79

cc sn phm sa, cc loi thy sn ti sng (khng qua gia nhit hay gia nhit

nh).

Mt loi gy ngc thc phm quan trng khc l V.parahaemolyticus

to c themolysin bn nhit c phn ng khng nguyn Kanagawa. Tuy

nhin, nhng nm gn y ngi ta pht hin ra rng nhiu dng

V.parahaemolyticusc phn ng Kanagawa m tnh cng c thgy bnh. Triu

chng ngc l au tht vng bng, vim nhim ng rut v tiu chy nh

xut hin 2 96 gisau khi n phi thc phm bnhim. Thi gian bnh ph

thuc vo mt tbo vi khun xm nhim v thtrng ca tng bnh nhn.

Loi vi khun ny hin din v tng trng trong mi trng c hm lng mui

cao, thng phn lp c tcc sn phm thy sn, trong cc vng nc m

ven bin.

Cc loi Vibrio khc khi xm nhim vo trong thc phm cng c thgy

nn cc bnh ng rut v c biu hin bnh l tng tnhhai loi trn.Ring loi V.vulnificuskhng gy cc triu chng bnh ng rut m gy

nhim trng mu cho ngi.

Hnh 1.6 Vibrio cholerae Hnh 1.7 V.vulnificus Hnh 1.8 V.parahaemolyticus

1.7. Escherichia coli



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16/79

E.colil vi sinh vt hiu kh ty hin din trong ng rut ca ngi v

cc loi ng vt mu nng. Hu ht cc dngE.colikhng gy hi v ng vai

tr quan trng trong vic n nh sinh l ng rut. Tuy nhin c 4 dng c th

gy bnh cho ngi v mt sloi ng vt l EnteropathogenicE.coli(EPEC),

EnterotoxigenicE.coli(ETEC), EnteroinvasiveE.coli(EIEC) v

EnterohaemorrhagicE.coli(EHEC)/VerocytoxinE.coli(VTEC) hayE.coli

O157: H7. Cc loiE.colihin din rng ri trong mi trng b nhim phn

hay cht thi hu c, pht trin v tn ti rt lu trong mi trng. Gn y

ngi ta cn chng minh c rngE.colicng hin din nhng vng nc

m, khng b nhim cht hu c. Do sphn brng ri trong tnhin nn

E.coliddng nhim vo thc phm tnguyn liu hay thng qua ngun cc

triu chng ri lon ng tiu ha. Biu hin lm sng thay i tnhn rt

nng, c thgy cht ngi phthuc v mc nhim, dng gy nhim v kh

nng p ng ca tng ngi.

Hnh 1.9 Escherichia coli

1.8. Shigella spp

Ging Shigellathuc hvi khun ng rut (Enterobacteriaceae) gm 4

loi khc nhau l S.dysenteriae, S.sonnei, S.plexneriv S.boydii. Cc loi



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17/79

Shigellac tnh chuyn bit k chcao, chxm nhim v tng trng trong

ngi v cc loi linh trng. Trong mi trng nc cc loi ny c thtn ti

hn 6 thng. Cc vngc thc phm do Shigellagy ra chyu tp trung

cc nc km pht trin, ni vic sn xut chbin thc phm c thc hin

trong iu kin vsinh khng m bo. Shigellacng c thly nhim trc tip

tngi qua ngi. Shigellachyu gy nn cc triu chng l(bnh ltrc

trng) trong khong 1 7 ngy sau khi sdng thc phm bnhim. Biu hin

bnh l c ththay i tdng nhnhtiu chy nhn mc nghim trng

(c bit l trem v ngi gi) nhi tiu ra mu, c nhng mnh nim mc

rut, mt nc, st cao v bco rt thnh bng. Cc triu chng trn c thko

di t12 14 ngy thm ch lu hn. Hng nm c khong na triu ngi t

vong do vi sinh vt gy bnh ny. Vi khun Shigellaly nhim chyu qua nc

v thc phm. Thc phm bnhim Shigellatnguyn liu hay thng qua tip

xc bmt trong qu trnh sn xut, chbin thc phm.

Hnh 1.10 Shigella

1.9. Coliforms

Coliformv Feacal coliform (coliform phn)l nhm cc vi sinh vt dng

chthkhnng c shin din ca cc vi sinh vt gy bnh trong thc phm.



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18/79

Nhm Coliformgm nhng vi sinh vt hiu kh v kkh ty , Gram m ,

khng sinh bo t, hnh que, ln men ng lactose v sinh hi trong mi trng

nui cy lng. Da vo nhit tng trng, nhm ny li c chia thnh hai

nhm nhl Coliformv Coliform phnc ngun gc tphn ca cc loi ng

vt. Trn thc tkim nghim Coliform phn c quan tm nhiu hn

Coliform. Coliform phn c ngun gc trut ngi v cc ng vt mu nng

bao gm cc gingEscherichia, KlebsiellavEnterobacter. Khi Coliform phn

hin din slng ln trong mu th mu c khnng bnhim nc nhim

phn v c khnng cha cc vi sinh vt gy bnh hin din trong phn. Trong

cc thnh vin ca nhm Coliform phnthE.colil loi c squan tm nhiu

nht vvsinh an ton thc phm.

Hnh 1.11 Coliforms

1.10. Listeria monocytogenes

Listeria monocytogenestrthnh tc nhn gy bnh nguy him trem,

phnmang thai hay ngi gi trong nhng nm gn y. Mc d

L.monocytogeneschhin din vi slng nhtrong thc phm, nhng khi

c a vo cth, chng tn ti, khi c iu kin thun li stng trng

nhanh xm nhim vo cc m su hn v gy bnh. Triu chng bnh thng



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19/79

bt u l tiu chy, st nh, sau l nhim trng mu, tn thng hthn kinh

trung ng, tim, mt, gy nn sy thai, non hay nhim trng thai nhi phn

mang thai.L.monocytogenesa lnh, tng trng nhit t2 44oC, thng

c phn lp tph mai, sa, tht c, rau quv trong nc. Loi ny c th

nhim vo thc phm mi cng on trong quy trnh chbin thc phm, sa

hay rau qu. c bit, trong thi gian bo qun thc phm nhit thp, loi

ny c chi tng trng thnh slng ln. Cc sn phm c thanh trng

bng phng php Pastuer v c bo qun trong cc tlnh c nguy cnhim

vi sinh vt ny rt cao.

Hnh 1.12 Listeria monocytogenes

1.11. Bacillus cereus

B.cereusl trc khun Gram dng, sinh bo t, kkh ty , tng trng

c trong khong nhit t5 50oC, ti u 35 40oC, pH dao ng t4,5 -

9,3, dto bo tv bo tny mm rt ddng. Vi khun ny hin din trong

t, bi, cc loi thc phm (sa, tht, rau qu, hn hp gia v, sn phm kh).

Vi khun c thtit ra hai loi c tchnh l diarrhoeal toxin gy tiu chy v

emetic toxin gy nn ma. Ngc thc phm gy ra biB.cereuskhi thc

phm c chun bm khng c gitrong iu kin lnh trong vi gitrc

khi sdng. Thc phm cha vi khun mt trn 106tbo/g gy ng



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20/79

c. Triu chng ngc phbin gy ra biB.cereusl au bng, tiu chy,

khng st, bt u 4 16 gisau khi n thc phm bnhim v ko di trong 12

24 gi. Mt dng triu chng ngc khc l bun nn v nn sau 1 2 gin

phi thc phm nhim khun, khng btiu chy, ko di khong 24 gi.

Hnh 1.13 Bacillus cereus

1.12. Aspergillus

Aspergillus flavusvA.parasiticusl hai loi nm mc quan trng trong

cc loi nm mc c khnng to c tmycotoxin gy ngc thc phm.c tmycotoxin c thc to ra bi nhiu chng ca hai loi ny c gi

chung l aflatoxin c khnng gy ung th. Cc nm mc ny c thpht trin

nhit t7 40oC v ti u 24 - 28oC, do vy c khnng tng trng tt

trn cc loi ngcc, nng sn (go, la, min, u phng, bp, la m) khng

c phi sy tt, c bit l cc nc nhit i vi iu kin m v nhit

khng kh cao. Tcc nguyn liu ny, nm mc v c tc thnhim vo

thc phm gy c cho ngi. cc chng c khnng sinh c t, sau khi tng

trng 1 3 ngy c tc to ra v tit vo ccht. Bn loi aflatoxin c

to ra biA.flavusvA.parasiticusl aflatoxin B1, B2pht hunh quang mu

xanh (blue) v G1, G2pht hunh quang mu lc (green). Khi n choc thc n

cha aflatoxin B1hoc B2, b sa c thchuyn ha chng thnh cc c t



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21/79

aflatoxin M1, M2hin din trong sa b. Do aflatoxin l cht gy ung thmnh

nn c kim tra nghim ngt. Hu ht cc nc u c tiu chun quc gia v

hm lng ti a cho php shin din ca c tny trong nng sn, thc

phm, thng l 10ppb (mt phn t).

Hnh 1.14 Aspergillus flavus Hnh 1.15 A.parasiticus

1.13. Virus

Cc vdch bnh gy ra do tc nhn virus tthc phm cho n nay vnl vn b n. Cc nghin cu vvirus thc phm. c im sinh l ca virus

ng rut, phng php nui cy, pht hin virus trong thc phm cho n nay

vn cn nhiu hn ch. Tuy nhin, bng cc kthut sinh hc phn tnhlai

phn t, kthut PCR ngi ta c thpht hin virus c hi cho con ngi

trong thc phm. Slan truyn virus qua ngi thng qua ng ming c

bit n tnm 1950. Cc virus gy bnh ng rut cho ngi chyu c

ngun gc tcc sn phm thy sn. Trong khong hn 100 loi virus ng

rut c bit hin nay chmt vi loi c khnng gy bnh cho ngi

(Hepatitis type A virus, HAV, Norwalk virus, Calicivirus, Astrovirus, Virus Non

A, Virus Non B). Virus tn ti thkhng hot ng khi bn ngoi tbo,

khng thtnhn ln trong nc hay trong thc phm. Chng xm nhim vo



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22/79

thc phm hon ton do qu trnh chbin, tnc b nhim. Cc loi nhuyn

thn lc nhnghu, s c khnng tch ly nhiu virus trong nc lm mt

virus trong nhuyn thcao hn rt nhiu so vi mi trng nc xung quanh.

Liu lng gy nhim ca virus tthc phm c ththp hn rt nhiu so vi vi

khun. Virus hin din trong cth ngi v cc loi ng vt v c tm thy

vi slng ln trong phn ca nhng ngi bnhim, tn ti tnhiu ngy n

nhiu tun. Nc bnhim phn l con ng ly nhim gin tip hay trc tip

ca virus vo thc phm. Khnng sng st ca virus trong mi trng hay

trong thc phm phthuc vo cc yu t: nhit , nng mui, cng

bc xmt tri hay shin din ca cc thnh phn hu ckhc. Tt cvirus

ng rut u c tnh khng vi acid, cc enzyme thy phn, mui mt c trong

ng tiu ha. Mt svirus c thkhng nhit nhvirus vim gan siu vi A

(HAV), hoc khng phenol, ethanol zn v chlorine l nhng tc nhn c

thlm bt hot mt vi loi virus ng rut. ngn nga cc bnh do virustthc phm, cc loi thc n phi c nu chn khvirus trc khi dng,

cc loi nhuyn thn lc phi c khai thc t, nhng vng nc khng

nhim virus hay c nui trong cc vng nc sch trc khi tiu th.

Hnh 1.16 Virus



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23/79


TRUYN THNG

2.1. Samonella

2.1.1. Tng quan

L trc khun Gram m,hiu kh v kkh ty nghi, khng sinh bo t, c

tin mao, ln men glucose v mannitol sinh acid nhng khng ln men

saccharose v lactose, khng sinh indole, khng phn gii ure, khng c kh

nng tch nhm amine ttryptophane, hu ht cc chng u sinh H2S.Cho n

nay xc nh c 2339 serotype (kiu huyt thanh) thuc ging Salmonella.

Cc serotype ny c chia theo hthng ca Kaffmann White da trn cng

thc khng nguyn O (khng nguyn somatic) v khng nguyn tin mao H

(flagella).

2.1.2. Nguyn tc

Salmonellac thc pht hin (phn tch nh tnh) bng mt quy trnhgm 4 bc l tng sinh, tng sinh chn lc, phn lp v khng nh Salmonella

thng c mt trong mu vi slng nh, btn thng v cng hin din

chung vi mt slng ln cc loi vi khun khc thuc hEnterobateriaceae

c tnh cnh tranh mnh v c chstng trng ca Salmonella.

- Bc tng sinh: ty theo c tnh thnh phn ha hc ca mu cn chn

quy trnh tng sinh ph hp. Thng thng tlgia mu v mi trng tng

sinh l 1:9, tuy nhin ty trng hp cthtlny c ththay i.

- Bc tng sinh chn lc: cc mi trng tng sinh chn lc thng dng

pht hin Salmonella trong cc mu thc phm l Rappaport Vassiliadis

(RV), Selenite Cystein Broth, Tetrathionate Mueler Kauffmann Broth (TT). Cc

nghin cu gn y ca AOAC cho thy mi trng RV c ththay thcho cc



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24/79

mi trng khc phn tch nhiu loi mu khc nhau. Tuy vy, mi trng TT

thng c dng phn tch cc loi mu cha tht ti sng, cc mu c mt

nhim cao, cc loi thc n gia sc Hin nay ngi ta khuyn khch vic s

dng t nht 2 loi mi trng tng sinh chn lc tng cng khnng pht

hin c tt ccc serotype Salmonellanu c hin din trong mu.

- Bc phn lp: nhm tch v nhn dng Salmonellakhi cc qun thvi

sinh vt khc trong mu. Nhiu loi mi trng rn khc nhau c sdng

phn lp Salmonella, mi mi trng gip nhn dng cc loi thuc ging ny

da trn mt vi c tnh. Hin nay, cc tiu chun vsinh an ton thc phm

khuyn khch vic sdng t nht hai loi mi trng phn lp khc nhau

tng cng khnng pht hin tt ccc dng Salmonella, c bit mi trng

XLD c khuyn khch sdng.

- Bc khng nh: nhm xc nhn li cc khun lc c trng cho

Salmonellaxut hin trn mi trng phn lp. Bc ny da trn vic sdngcc thnghim sinh ha v thnghim huyt thanh c trng cho Salmonella.

Cc thnghim c khuyn khch sdng l thnghim KIA/TSI, indol, LDC

(lysine decarboxylase), ODC (ornithine decarboxylase), urea, ln men mannitol,

sorbitol, cc thnghim huyt thanh O v H a gi. Thng thng cc phng

phn tch vi sinh thc phm khng c khnng xc nh n loi phhay

serotype m chxc nhn n Salmonella spp. Vic phn tch chnh xc serotype

nhim vo thc phm thng cthc hin ti cc trung tm vsinh phng

dch hay cc phng th nghim chuyn nh danh vi sinh vt.

2.1.3. Mi trng v ha cht

- Nc pepton m (Buffered Peptone Water, BPW).

- Canh Rappaport Vasiliadis Soya Pepton (RV).



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25/79


26/79

- i vi mu gia cm ti sng: t gia cm vo mt bao nha ln, thm

1 lt mi trng tng sinh BPW. Lc bng my lc khong 30 giy mi trng

thm c vo trong ton bmu.

- i vi sa kh: cho 25g mu vo trong ti PE v trng, bsung 225ml

mi trng BPW, yn 60 pht nhit phng. Sau lc cho n khi sa

tan hon ton.

- i vi cc gia vhay cc loi thc phm cha nhiu gia v: ng nht

mu pha long 1/100 trong BPW (thay v 1/10 nhbnh thng) trc khi

nui tng sinh. Bin php ny nhm lm gim nng cc hp cht c chs

tng trng ca Salmonellatrong bc tng sinh.

- i vi cc loi mu nhcasein, pho mt, bv cc sn phm tng t

khc: thc hin ng nht mu trong mi trng tng sinh c lm m n

40oC.

- i vi sn phm cha cocoa: ng nht mu trong mi trng SkimMilk Broth c lm m 40oC.

- i vi da, cc sn phm ca da v cc mu c hm lng cht bo

cao ng nht mu vi BPW, sau thm vo 2 3 git Triton X-100 trc khi

tng sinh.

2.1.4.2.Tng sinh chn lc

Lc trn u dch tng sinh, v chuyn 0,1ml sang 10ml mi trngtng sinh Rappapport-Vassliadis Soya Pepton c m n 42oC. 42

0,2oC trong 18 24 gi. Khi cn thit c thko di thi gian thm 24 gi.

Mt smi trng tng sinh chn lc khc nhSelenite Cysteine Broth,

Tetrathionate Muller-Kauffman cng c dng. Mi loi mi trng chc tc

dng chn lc trn mt c im pht trin ca Salmonella, mt sdng



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27/79

Salmonella tng trng c trong mi trng ny nhng li khng tng trng

c trong mi trng khc. V dSalmonella typhikhng tng trng c

trong mi trng Rappaport Vassiliadis v Tetrathionate Muller-Kaufmann,

Salmonella Dublin khng tng trng c trong mi trng Rappaport

Vassiliadis. Do vy, tng khnng pht hin tt ccc dng Salmonellahin

din trong mu thc phm cn phi dung t nht hai loi mi trng tng sinh

chn lc khc nhau cho cng mt mu. Ngoi ra, mi mi trng chn lc c

mt nhit nui cy khc nhau nhmi trng RV c 42oC, mi

trng TT v SC c 37oC trong 22 24 gi.

2.1.4.3. Phn lp v nhn din

Dng que cy vng thc hin kthut cy phn lp khun lc n vi

ging tdch tng sinh chn lc ln a mi trng chn lc phn bit c trng

cho SalmonellanhXLD, HE, BS Cng chn lc v mc phn bit

thhin hnh thi khun lc ca Salmonellatrn tng mi trng cng khcnhau. chn lc v nhn dng c tt ccc dng Salmonellaphi dung ti

thiu 2 mi trng chn lc phn bit khc nhau cho cng mt mu. Cc biu

hin ca Salmonellatrn tng mi trng khc nhau nhsau:

- Mi trng XLD: khun lc c mu hng trong sut, c hay khng c

tm en. Mt sdng Salmonellac thc tm en bong rt ln c thchim

gn ht khun lc.

- Mi trng HE: khun lc Salmonellac mu thay i txanh dng

n mu xanh lc, c hay khng c tm en. Mt sdng Salmonellac tm en

bng rt ln c thchim gn ht khun lc.



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28/79

- Mi trng BS: khun lc Salmonellac mu nu xm hay mu en,

thnh thong c xut hin nh kim tm. Mi trng chung quanh khun lc

chuyn thnh mu nu v sau chuyn thnh en nu ko di thi gian .

- Mi trng BPLS: c khun lc Salmonellamu hng nht, trong sut,

xung quanh khun lc mi trng chuyn mu .

Tt ccc a mi trng sau khi cy c 37oC trong 22 26 gi. auk hi

, chn cc khun lc c c im ca Salmonella nhtrn thc hin bc

khng nh bng cc thnghim sinh ha v thnghim khng nguyn.

2.1.4.4. Khng nh

Tmi mi trng chn lc phn bit chn v cy chuyn t nht 5 khun

lc nghi ngsang mi trng khng chn lc v dnhTSA. 37oC trong 18

24 gi. Cc khun lc xut hin trn mi trng ny c sdng cho cc th

nghim sinh ha v huyt thanh.

Cc thnghim sinh ha chnh cn thc hin cho Salmonellal ln menglucose, lactose, saccharose, H2S, urea, indol, VP, ODC, LDC, sdng

mannitol, sorbitol. Cc chng Salmonellacho cc kt quthnghim sinh ha

nhsau:

- Thnghim sinh H2S (TSI hay KIA): Salmonellachln men c

ng glucose trong cc mi trng trong cc mi trng trn v thphn

nghing ca mi trng c mu , phn su c mu vng. a scc dng

Salmonellau c khnng sinh H2S nn c xut hin cc vt mu en trong

mi trng ny. C ththy hin tng sinh hi qua hin tng l vthch mi

trng hoc mi trng by ln trn to ra mt khong khng bn di y

ng nghim.



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29/79

- Thnghim LDC (+): sau khi nui cy mi trng chuyn thnh kim,

mu mi trng c chuyn thnh mu nhban u (mu tm hay xanh).

- Thnghim urea (-): Salmonellakhng phn gii urea nn khng lm

thay i pH mi trng, sau khi nui cy mi trng vn ginguyn mu cam.

- Ln men mannitol, sorbitol (+): mi trng sau nui cy bacid ha v

chuyn thnh mu vng.

- Thnghim Indol v VP (-).

Cc thnghim khng nguyn cn c thc hin song song vi mu

chng khng bng dung dch nc mui sinh l loi trkhnng ngng kt

gi. Hai loi khng huyt thanh a gi cn dng l Salmonella Polyvalent Ov

Salmonella Polyvalent H. Phn ng (+) khi chng thnghim to ngng kt vi

khng huyt thanh nhng khng c hin tng ngng kt vi nc mui sinh l.

2.1.4.5. Bo co kt qu

Quy trnh kim nghim ny chcho php phn tch nh tnh Salmonella,gip kt lun c hay khng c Salmonella hin din trong 25g mu, khng cho

php phn bit c cc dng Salmonellahin din trong mu. khng nh

c tn chng Salmonellaphn lp c cn phi tin hnh thnghim ngng

kt vi cc loi khng huyt thanh n gi khc nhau hay cn gi chng

Salmonellaphn lp c n cc phng th nghim chuyn nh danh vi sinh

vt.

Cn lu Salmonellal nhm cha cc dng gy bnh nguy him. Do vy

cn tun thtrit cc bin php an ton khi lm vic vi cc chng

Salmonella dng lm i chng (+) cng nhkhi thao tc trn cc chng phn

lp c. Cc mi trng hay mu vt sau nui cy cn phi hp khtrng cn

thn trc khi ra.



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30/79

2.2. Vibrio

2.2.1. Tng quan

Vibriol vi sinh vt gram m, hnh que hai u khng u nhau to thnh

hnh du phy, di ng, sng kkh ty , c phn ng catalase v oxidase (+),

ln men glucose nhng khng sinh hi, khng sinh H2S, nhy cm vi

Vibriostaticum O/129. TrV.choleraehin din vng nc ngt, tt ccc

loi Vibriokhc u cn mui tng trng v thng xuyn c phn lp

c tcc vng nc ven bin. Ging ny c 4 loi l tc nhn gy bnh cho

ngi gm: V.cholerae, V.parahaemolyticus, V.vulnificusv V.alginolyticus

c phn bit da trn cc c im sau y:

V.choleraec phn ng oxidase (+) tng trng c trong mi trng

canh trypton 42oC, arginiae dehydrolase (-), lysine decarboxylase (+), ln men

c sucrose (saccharose), khnitrate thnh nitrite, c thtng trng c

trong mi trng cha 0 3% NaCl, khng pht trin c trong cc mi trngcha 6, 8, 10% mui.

V.parahaemolyticusc phn ng oxidase (+), pht trin c trong canh

trypton 24oC, phn ng ADH (-), LDC (+), c khnng khnitrate thnh

nitrite nhng khng ln men sucrose, sdng c mt scarbohydrate khc

ln men nhng khng sinh hi, khng tng trng c trong mi trng khng

c mui, tng trng tt trong mi trng c n 8% mui nhng bc ch

trong mi trng cha 10% mui.

V.vulnificuskhc vi hai loi nu trn l khng ln men sucrose, khng

tng trng c trong mi trng khng c mui, bc chtrong mi trng

c 8 10% mui.



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31/79

V.alginolyticusc khnng ln men ng sucrose, khng tng trng

c khi trong mi trng khng c mui nhng c thpht trin trong mi

trng cha n 10% mui.

2.2.2. Nguyn tc

Mt lng mu xc nh c tng sinh trong mi trng chn lc c

trng. Cy phn lp tmi trng tng sinh sang mi trng phn bit chn lc

c trng. Cc khun lc nghi ngtrn mi trng phn lp c khng nh

bng cc thnghim sinh ha v huyt thanh hc. Mi trng tng sinh chn lc

cho Vibrio cholera, V.alginolyticusv V.vulnificusl nc pepton kim. Trong

khi mi trng Colistine Polymicine Broth thng c dng tng sinh

V.parahaemolticus. Mi trng thng dng phn lp Vibrio l TCBS

(Thiosulphate Citrate Bile Sucrose Agar). Cc vi sinh vt ln men c sucrose

trong mi trng ny scho khun lc mu vng v lm acid ha mi trng

bn di khun lc. Nu vi sinh vt khng ln men c ng sucrose schokhun lc mu xanh.


- Canh Pepton kim Alkaline Peptone Water (APW).

- Canh Colistine Polymicine Broth (Colistine).

- Thch Thiosulphate Citrate Bile salt Sucrose (TCBS Agar).

- Canh Tryptone.

- Thch Kligler Iron (KI).

- Canh Hugh Leifon Glucose (HLG).

- Canh Carbohydrate tm.

- Mi trng thnghim Decarboxylase (Moeller).

- Dung dch oxidase.



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32/79

- Dung dch thnghim String (sodium desoxycholate 5%).

- Khng huyt thanh polyvalentO dng cho V.cholerae.

- Khng huyt thanh O.

- Dung dch NaOH 1N.

- Dung dch HCl 1N

- Dung dch du phv trng.

- Dung dch Bromocresol tm.

2.2.4. Quy trnh phn tch

2.2.4.1. Tng sinh chn lc

Cn 25g mu vo trong bao PE v trng, thm 225 ml canh thang tng

sinh chn lc (dng nc pepton kim cha 1% mui NaCl cho trng hp

V.cholerae, V.vulnificus, V.alginolyticus, canh Colistine cho trng hp

V.parahaemolyticus). ng nht mu bng my dp mu. 37oC trong 6 8

gi.2.2.4.2. Phn lp

Dng que cy vng ria vng trn bmt mi trng tng sinh chn lc ln

bmt a thch TCBS phn lp khun lc n, 37oC trong 18 22 gi.

Hnh dng khun lc c trng ca cc loi Vibriotrn mi trng ny l nh

sau: khun lc V.choleraev V.alginolyticusln, ng knh khong 2 3mm,

lng, c mu vng, hi phng, tm c v chung quanh khun lc c qung trng

c. Khun lc V.parahaemolyticusv V.vulnificusln, ng knh khong 3

4mm, c mu xanh n xanh dng. Tip theo, cy chuyn sinh khi tcc

khun lc c trng trn mi trng phn lp sang mi trng khng chn lc

hay thch mu, qua m nhit 37oC thu sinh khi sdng cho cc th

nghim sinh ha.



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2.2.4.3. Thnghim sinh ha

Cc thnghim sinh ha quan trng pht hin hay phn bit cc loi

Vibriol quan st tnh di ng trn knh hin vi, quan st sdi ng trn thch

mm v cc thnghim khc. Cc c im sinh ha ca V.cholerael: ONPG

(+), lactose (-), ADH (-), LDC (+), TDA (-), Indol (+), manose (+), sucrose (+),

oxidase (+), tnh a mn NaCl 0% (+), 3% (+), 6% (-), 8% (-), 10% (-). Cc c

im sinh ha ca V.parahaemolyticusl: ONPG (-), lactose (-), ADH (-), LDC

(+), TDA (-), Indol (+), manose (+), sucrose (-), oxidase (+), tnh a mn NaCl

0% (-), 3%(+), 6% (+), 8% (+/-). 10% (-).

2.2.4.4. Thnghim khng huyt thanh:

Thnghim ngng kt huyt thanh c dng xc nh cc dng

Vibrio c cc biu hin khng nguyn chuyn bit. Trng hp V.cholerae c

thphn bit c cc dng V.cholera O1v V.cholerae non-O1(khc O1) nh

sau:- Thnghim vi khng huyt thanh a gi polyvalent O nhm 1: nhmt

git khng huyt thanh O a gi ln phin knh sch v mt git nc mui sinh

l ln mt vtr khc ca phin knh. Dng que cy v trng chuyn vi khun ln

2 git dung dch trn, phn tn u vi khun vo trong git dung dch. Kt lun

(+) khi c hin tng ngng kt.

- Xc nh kiu khng nguyn O1: dng khng huyt thanh n gi Inaba

v Ogawa. Khng huyt thanh Inaba (+) khi khng nguyn chngng kt vi

khng huyt thanh Inaba khng ngng kt vi cc nhm khc hay dung dch

mui sinh l. Khng huyt thanh Ogawa (+) khi khng nguyn chngng kt vi

khng huyt thanh Ogawa v khng ngng kt vi cc nhm khc hay dung dch

mui sinh l. Khng huyt thanh Hikojima (+) khi khng nguyn ngng kt vi



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khng huyt thanh Ogawa v Inaba nhng khng ngng kt vi dung dch mui

sinh l. Phn ng khng nguyn l (-) khi chng khng ngng kt vi cc nhm

khng huyt thanh nu trn v khng ngng kt vi dung dch mui sinh l.

Trng hp ny c thgp do sai lm trong thao tc thnghim hoc l do

chng Vibrionu trn thuc nhm non-O1. Phn ng ngng kt l khng c

hiu khi tt cthnghim khng huyt thanh u cho kt qu(+).

Trng hp V.parahaemollyticusthc hin ngng kt vi hai loi huyt

thanh l huyt thanh O v huyt thanh K. Tuy nhin trong mt strng hp

khng nguyn O bkhng nguyn K che lp, v thcn phi xl chng trc

khi thc hin ngng kt vi khng nguyn O bng cch dng que cy chuyn

sinh khi ca chng trn mi trng thch chn lc vo dung dch nc mui

3%, hp 121oC trong 1 gibiu lkhng nguyn O ra bn ngoi.


Kt quc bo co di dng pht hin hay khng pht hin V.cholerahay V.parahaemolyticustrong 25g mu.

Cn lu Vibriol nhm cha cc dng gy bnh nn cn tun thtrit

cc bin php an ton khi lm vic vi cc chng Vibriodng lm i chng

(+) cng nhkhi thao tc trn cc chng phn lp c. Cc mi trng hay

mu vt sau nui cy cn phi hp khtrng cn thn trc khi ra.

2.3. Listeria monocytogenes2.3.1. Tng quan

L.monocytogenesl vi khun c hnh gy ngn, mnh, gram dng sau khi

nui cy 20 gi, cho thnghim catalase (+), oxidase (-), chuyn ng xoay trn

thnh t trong tiu bn git treo (khi nui cy 20 25oC), c khnng thy

gii esculin v lm tan huyt trn mi trng thch mu, sinh acid rhamnose



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nhng khng xylose, c phn ng CAMP (+) vi Staphylococcus aureusv (-)

viRhodococcus equi.L.monocytogenesl mt stc nhn gy bnh listeriosis

rt nguy him (nhim trng mu, sy thai phn, vim mng no, gy tvong

thai nhi) c tltvong rt cao (30 35% ngi ln v 70% trem). Loi

ny thng c pht hin trong thc n gia sc, nc, nc thi, l loi c bit

nguy him trong cc loi thc phm qua gia nhit. Cc quy nh van ton v

sinh thc phm thng khng cho php shin din caL.monocytogenestrong

25g thc phm qua gia nhit nhng cho php 100 CFU/g i vi thc phm

phi gia nhit trc khi sdng.

2.3.2. Nguyn tc

Quy trnh pht hinL.monocytogenesc thc hin bng qu trnh tng

sinh 2 giai on. Mu c cn vo trong ti PE v trng, ng nht vi mi

trng tng sinh scp. Sau khi 24 gi, dch nui cy c chuyn vo mi

trng tng sinh thcp v c tip 24 gi. Hin nay quy trnh ci tin chophp tng sinh mt giai on nhng thi gian tng sinh l 48 gi. Quy trnh tng

sinh mt giai on c sdng khi cc mu c mt vi sinh vt nhim thp;

quy trnh tng sinh hai giai on c sdng cho cc mu c mt vi sinh vt

nhim cao. Sau bc tng sinh, canh khun c chuyn sang bc phn lp

trn mi trng chn lc c trng. Cc khun lc nghi ngc khng nh

bng cc c tnh hnh thi, sinh l v sinh ha v min dch bng cc th

nghim c trng. Trong pht hinL.monocytogenescn sdng cc chng vi

sinh vt lm i chng nh:L.monocytogenes,L.innocua, S.aureusdung huyt

yu,R.equi.


- Canh tng sinh scp Listeria Enrichment Broth I (LBI).



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- Canh tng sinh thcp Listeria Enrichment Broth II (LBII) c chun

bthnh cc ng nghim 10ml.

- Mi trng Enrichment Broth (EB) ci bin tmi trng LB, c s

dng trong quy trnh tng sinh mt giai on.

- Mi trng thch Oxford Agar.

- Thch mu.

- Mi trng thch mm BHI: BHI cha 5g agar/l, phn phi 5ml vo

trong cc ng nghim.

- Rhamnose Phenol Red Broth (RPR), Xylose Phenol Red Broth (XPR).

- Thuc thcatalase, oxidase.


2.3.4.1. Tng sinh

ng nht 25g mu trong 225ml mi trng tng sinh LBI bng

Stomacher trong 30 giy. 30oC trong 24 gi. Chuyn 0,1ml LBI sang ng10ml LBII tip tc 30oC trong 24 gi. Quy trnh tng sinh mt giai on

c thc hin nhsau: Cn 25g mu, thm vo 225ml canh EB ( c lm

m 45oC nu mu thl cc sn phm ca sa v 30oC i vi cc sn phm

khc) v tin hnh ng nht mu trong 30 giy. 30oC trong 48 gi.

2.3.4.2. Phn lp

Dng tm bng v trng thm dch mu tng tng sinh, tri sang 1/2 a

mi trng Oxford Agar. Tnhng vt cy ny dng que cy vng ria sang 1/2

a cn li. 37oC trong 24 48 gi. Trn mi trng ny khun lcListeria

c mu xm hay nu c bao quanh bi vng en, khun lc lm, nh, ng

knh khong 1mm.

2.3.4.3. Khng nh



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- Thkhnng tan huyt: chn khun lc in hnh trn mi trng thch

Oxford, dng que cy vng cy chuyn sang mi trng thch mu, 37oC

trong 24 48 gi. Trn mi trng thch mu khun lcL.monocytogenesc

bao quanh bi vng tan huyt hp do hin tng dung huyt dng . Trc khi

tin hnh thkhng nh, cy chuyn khun lc nghi ngL.monocytogenes sang

mt mi trng lng khng chn lc, 25oC trong 20 gi.

- Thnghim catalase v oxidase:L.monocytogenes c catalase (+) v

oxidase (-).

- Nhum gram:Listerianhum gram dng.

- Khnng di ng phng php git treo:Listeria sppchuyn ng xoay

trn trong canh trng 25oC. Thtnh di ng trong ng nghim: cy vi trng

bng cch m su vo thch mm trong ng nghim, 25oC trong 40 gihoc

lu hn, kim tra stng trng ca vi khun xung quanh ng cy,Listeria

sppdi ng to hnh chic d cch bmt thch vi mm.- Khnng bin dng ng: cc ng canh trng thkhnng ln men

ng 37oC trong 7 ngy. Kt qu(+) (mu vng) thng quan st c trong

vng 24 48 giL.monocytogenesln men ng rhamnose nhng khng c

khnng ln men ng xylose.

- Ththnghim CAMP: trn a thch dng thCAMP, cy S.aureus

thnh mt ng cy mng, tng tcyR.equito thnh hai ng cysong song cch nhau 4cm.

Cy chng nghi ngListeriagia, gn nhng khng chm vo hai

ng cy song song ca S.aureusvR.equi. C thcy mt hoc nhiu dng vi

khun nghi ngL.monocytogenes(chng thnghim) kim tra trn cng mt

a. Cy chng i chng (+)L.monocytogenesv chngL.innocua. a



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37oC trong 20 36 gi. Phn ng (+) khi xut hin vng cng hng tan huyt

rng (5 10mm) v c hnh dng u mi tn. Phn ng (+) vi S.aureusthng

to vng tan huyt hp (khong 2mm) c dng hnh trn.L.monocytogenescho

phn ng CAMP (+) vi S.aureusv (-) viR.equi. Ngc li,L.innocuac

phn ng CAMP (-) vi chai loi S.aureusvR.equi.


Bo co pht hin hoc khng pht hinL.monocytogenestrong 25g mu.

2.4. Staphylococcus aureus

2.4.1. Tng quan

Thuc nhm tcu khun, Gram dng, hiu kh hay kkh ty nghi, c

thnghim coagulase, phn ng DNAse, phosphatease (+), c khnng ln men

v sinh acid tmannitol, trehalose, sucrose. Tt ccc dng S.aureusu mn

cm vi novobiocine, c khnng tng trng trong mi trng cha n 15%

NaCl. Mt sdng c khnng lm tan mu trn mi trng thch mu. ngknh vng tan mu phthuc vo tng chng nhng u nhhn ng knh

ca khun lc. Sn sinh ra ni c tenterotoxin bn nhit dn n ngc thc

phm,cc tbo thng lin kt thnh hnh cc chm nho, khng to bo t,

khng di ng, c khnng sinh coagulase lm ng huyt tng, khng phn

btrong tnhin, tn ti chyu trn da v tc ca ngi v ng vt.Khi pht

trin trong mi trng, to sc tc mu ttrng ti vng m. Shin din vi

mt cao ca S.aureustrong thc phm chthiu kin vsinh v kim sot

nhit km ca qu trnh chbin.

2.4.2. Nguyn tc



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S.aureusc xc nh trn cscc c im tng trng v phn ng

ng huyt tng ca cc dng thun tcc khun lc c trng trn mi trng

phn lp.


- Mi trng canh Mannitol Salt Broth (MSB): mi trng ny c dng

phn tch nh tnh S.aureushay trong nh lng bng phng php MPN.

- Mi trng thch mu: Mu c sdng l mu cu hay b non di 5

thng tui c loi bcc si mu hay mu c bsung cht chng

ng citrate. Mi trng cn c pha cht nht 2 ngy trc khi sdng

kim tra khnng bnhim.

- Mi trng thch Baird Parker Agar (BPA): thnh phn lng trng

ti v potassium tellurite chc bsung vo mi trng sau khi khtrng v

lm ngui n khong 60oC.

- Mi trng thch Tellurite Glycine Agar (TGA).- Mi trng Brain Heart Infusion (BHI).

- Huyt tng th: huyt tng thc cnh bng 0,1% EDTA hay

sodium oxalate v c phn phi 0,3ml vo cc ng nghim nh.

2.4.4. Phn tch nh tnh Staphylococus aureus:

Ly 2ml dung dch mu vo ng nghim cha 8ml mi trng MSB, trn

u v 37oC trong 24 gi. Dng que cy vng cy ria dch mu tng (+)

(mi trng chuyn tsang mu vng) ln mi trng phn lp l thch TGA

hay thch Baird Parker, 37oC trong 24 gi. Tm khun lc c trng ca

S.aureustrn mi trng phn lp c mu en nhnh, sng, trn, li, ng knh

1 1,5mm c vng sng chung quanh khun lc. Chn khun lc c trng, cy

vo ng mi trng BHI, 37oC trong 24 gi. Cy vo ng nghim nhcha



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khong 0,3ml huyt tng v 37oC trong 24 githphn ng ng kt.

Thc hin song song mt ng i chng khng c cy dch vi sinh vt. Mu

c kt lun l c S.aureuskhi thnghim coagulase ny (+) (c sxut hin

ca khi ng trong khi ng i chng khng c).

2.4.5.nh lng S.aureus bng phng php m khun lc

2.4.5.1.ng nht mu v pha long

Cn chnh xc 10 0,1g mu trong ti PE v trng, thm 90ml dung dch

pha long, ng nht mu bng my dp mu khong 30 giy. Chun bdy pha

long thp phn thch hp ty theo mc nhim ca tng loi mu sao cho khi cy

mt thtch xc nh ln a thch Baird Parker sxut hin khong 10 100

khun lc/a.

2.4.5.2. Phn lp trn mi trng chn lc

Cy 0,1ml mu nguyn hoc pha long vo a mi trng thch Baird

Parker. Dng que cy tam gic thy tinh (thanh gt thy tinh) tri u mu ln bmt mi trng cho n khi kh. Thc hin lp li 3 a mi trng thch Baird

Parker cho mi pha long. Thc hin tng tvi mi trng thch mu. Lt

ngc a, 37 1oC trong 24 48 gii vi mi trng Baird Parker v 24

gii vi mi trng thch mu.

Sau 24 gi, khun lc S.aureustrn mi trng thch Baird Parker c

ng knh khong 0,5 1mm, li, en bng c vng sng rng khong 1 2mm bao quanh. nh du trn mt y ca a cc khun lc c c im nh

trn v tip tc n 48 gi. Sau 48 gikhun lc S.aureusc ng knh

khong 1 1,5mm, mu en bng, li, c vng trng c hp v vng sng rng

khong 2 4mm quanh khun lc. Khun lc mt sdng S.aureusc thkhng



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to cc vng sng quanh khun lc nhtrn. Cn m v nh du chai dng

khun lc.

Trn mi trng thch mu sau 24 giS.aureus cho khun lc bng

long, c, li c mu xm hay vng nht, ng knh khong 1 2mm. Hu ht

S.aureusc vng tan mu, tuy nhin mt sdng khng to vng tan mu ny.

2.4.5.3. Khng nh

Dng que cy vng cy 5 khun lc c trng v 5 khun lc khng c

trng tmi trng Baird Parker ln mi trng thch TSA, 37 1oC trong

24 gi. Cy sinh khi vi khun tmi trng TSA vo cc ng nghim cha

huyt tng r ng, 37 1oC. Theo di kt quphn ng ng huyt

tng sau cc khong thi gian 2, 4, 6, 8 v 24 gi. Tnh tlkhng nh da

trn skhun lc c trng v khng c trng. Thc hin tng tvi cc

khun lc c trng trn mi trng thch mu. Kt quthnghim l (+) khi c

khi ng huyt tng hnh thnh (mi mc ng kt u c xem l (+)).

Kt qul (-) khi khng c hnh thnh khi ng, hn dch vn ng nht nh

ng khng cy.

2.4.5.4. Kt qu

Da vo skhun lc nghi ngl S.aureus m trn cc a petri v

da vo tlsng phn ng coagulase dng tnh trn tng sng thphn

ng, p dng cng thc di y tnh slng S.aureustrung bnh c trong1ml hay 1g mu.

Mt (CFU/g hay CFU/ml) =

Trong :

F: pha long.

Nt: tng skhun lc c trng.



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Na: tng skhun lc khng c trng.

Ht: tsgia khun lc c trng cho thnghim khng nh (+) so vi skhun

lc c trng.

Ha: tsgia skhun lc khng c trng cho thnghim khng nh (+) so

vi skhun lc khng c trng.

2.4.6.nh lng S.aureus bng phng php MPN

Phng php ny c dng nh lng S.aureustrong mu c mt

S.aureusthp nhng mt vi sinh vt cnh tranh cao, kh c thxc nh bng

phng php m khun lc. Dung dch mu c pha long thp phn vi ba

pha long thp phn lin tip (v d10-1,10-2,10-3). Ty theo yu cu v

chnh xc ca kt quphn tch, c thsdng phng php MPN vi hthng

9 ng nghim (3 pha long vi 3 ln lp li mi pha long) hay hthng

15 ng nghim (3 pha long vi 5 ln lp li mi pha long).

Mi trng c sdng l canh MSB hay Tryptose Soy Broth cha 10% muiNaCl. Cy 1ml dch mu c pha long khc nhau vo ng 10ml mi trng,

37oC trong 48 gi. Chn cc ng (+), c vi sinh vt pht trin lm c mi

trng tin hnh phn lp khun lc n trn mi trng thch Baird Parker,

37oC trong 48 gi. Chn cc khun lc c trng trn mi trng Baird

Parker thc hin thnghim khng nh S.aureustng ttrng hp

phng php m khun lc. Cc a cho kt qukhng nh S.aureus(+) c

i chiu vi cc ng nghim trong hthng cc dy ng nghim ban u v ghi

li sng nghim (+) ng vi mi pha long. Tra bng MPN suy ra mt

S.aureustrong mu (sMPN/g hay MPN/ml).

2.5. Bacillus Cereus

2.5.1. Tng quan



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B.cereusl nhng trc khun, gram dng, hiu kh v kkh ty , di

ng, to ni bo t, ln men glucose sinh hi, phn ng VP (+), c khnng s

dng nitrate. Loi ny tng trng c trong khong nhit t5 50oC, ti

u 35 40oC; pH dao ng t4,5 9,3, dto bo tv bo tny mm rt

ddng. Trn mi trng chn lc loi ny to khun lc rt to, mc lan, ra

nhn. Vi khun ny hin din trong t, bi, cc loi thc phm (sa, tht, rau

qu, hn hp gia v, sn phm kh). Vi khun c thtit ra hai loi c t

chnh l diarrhoeal toxin gy tiu chy v emetic toxin gy nn ma. Loi ny

phn bit vi cc loi khc trongBacillusnhm 1 nhB.anthracisgy bnh than

ngi,B.thuringiensisto c tkt tinh gy bnh cho cn trng.

2.5.2. Nguyn tc

B.cereusc pht hin v nh lng bng mi trng thch chn lc:

Mannitol-Egg Yolk-Polymycin (MYP) hoc Cereus Selective Agar (MOSSEL),

Polymycin Elgelb Mannitol Bromothymol Blue Agar (PEMBA). Khun lcB.cereusc hnh thi c trng trn cc mi trng ny. Cc khun lc ny c

thtip tc khng nh da trn cc thnghim sinh ha vi cc c im nh

ln men glucose sinh acid trong iu kin kkh, khnitrate thnh nitrite, th

nghim VP (+), thy phn L-tyrosine, tng trng c trong 0,001% lysozyme.

Ngoi raB.cereuscng c nh lng bng phng php MPN.

2.5.3. Mi trng v ha cht- Nc pepton m Buffered Peptone Water (BPW).

- Thch Mannitol-Egg Yolk-Polymycin (MYP).

- Thch Polymycin Elgelb Mannitol Bromothymol Blue Agar (PEMBA).

- Mi trng thch Cereus Selective Agar (MOSSEL).

- Nhlng trng (Egg Yolk Emulsion), 50%.



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- Canh Trypticase Soy Polymycin (TSP).

- Canh Phenol Red Glucose.

- Thch Tyrosine.

- Canh Lysozyme.

- Mi trng thnghim Voges-Proskauer.

- Canh Nitrate Broth.

- Thch dinh dng Nutrient Agar cho B.cereus.

- Thch mu Trypticase Soy Sheep.

- Mi trng kim tra sdi ng.

- Thuc thnitrate (dung dch A, dung dch B).

- Ha cht nhum Gram.


Cho 25g mu vo ti PE, bsung 225ml mi trng pepton m (BPW)

ng nht c pha long 10-1, ng nht bng Stomacher trong 1 pht. Mutip tc c pha long thnh dy thp phn c cc pha long thch hp.

2.5.4.1.nh lng B.cereus bng phng php m khun lc:

a. Pht hin bng mi trng chn lc

Tri 0,1ml mi pha long ln mi trng thch MYP, 24 gi30oC.

DoB.cereuskhng ln men mannitol, to lecithinase v khng polymycin nn

trn mi trng ny khun lcB.cereusc mu hng eosin, c bao quanh bi

vng c ta chng tlecithinase c to thnh. Trng hp sdng mi

trng MOSSEL, khun lcB.cereusto, mu hng, xung quanh c vng sng.

Chn t5 khun lc (+) cy sang thch nghing chun bcho cc phn ng

khng nhB.cereus.

b. Cc thnghim khng nh



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Nhum Gram: cy ria cc khun lc chn tmi trng MYP hay

MOSSEL sang ng thch dinh dng. 30oC trong 24 gi. Sau tin hnh

nhum Gram, quan st di knh hin vi. Vic nhum Gram c thc thc

hin bng phng php Jensen hay phng php Hucker. Trc tin, to vt bi

vi sinh vt trn phin knh. Chn mt phin knh sch, dng bt ghi trn knh v

1 vng trn ng knh khong 2cm mt di ca phin knh. t vtr

tng ng vi vng nh du mt trn ca phin knh vi git nc ct thnh

mt git ln. Dng que cy vng chuyn mt t sinh khi khun lc vo git

nc trn phin knh, khuy nhbng u que cy c huyn ph ng nht

v bi u trong khu vc ca vng trn. Trng hp chng nm trong dch nui

cy th dng que cy vng chuyn vi vng dch tbo ln vng vng trn

trung tm bmt phin knh, bi u trong khu vc ca vng trn. yn cho

vt bi kh. Dng tay gihai cnh ca mt u phin knh, a phin knh (v

tr cnh vt bi) qua li vi ln trn ngn la n cn hoc n Bunsen cnh vt bi trn knh. Trnh vt bi tip xc trc tip vi ngn la. Thc

hin tng tto mt vt bi chung hai chng vi sinh vt i chng trong

cng mt phin knh khc. SdngE.coli lm chng i chng cho Gram (-) v

Staphylococcus aureuslm chng i chng cho Gram (+).

Theo phng php Jensen, nhvi git dung dch methyl violet ln vt

bi, giyn trong 20 giy. Dng bnh xt nc, bm nc ln vt bi ra sch

phm nhum. Nhvi git dung dch KI/I2ln vt bi, yn 1 pht. Dng bnh

xt cha cn 95%, bm cn ln vt bi, ra phm nhum n va mt mu.

yn vi giy sau ra li bng nc. Nhum bng dung dch safranin 30 giy.

Ra sch bng nc. Thm nc dbng giy lc. Theo phng php ca

Hucker, bng thao tc tng tnhphng php Jensen, nhum vt bi 1 pht



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bng dung dch crystal violet. Ra bng nc. Nhum bng dung dch KI/I2

trong 1 pht. Khmu bng cch xt cn 95% cho n khi sch mu. Ra li

bng nc v nhum bng dung dch safranin trong 2 pht.

Sau khi thc hin xong quy trnh nhum, quan st mu nhum ca tbo

bng vt knh 100X nhng trong du. Tbo nhum Gram (+) c mu ta

(S.aureus), tbo nhum Gram (-) c mu hng (E.coli).B.cereusl trc

khun ln, Gram dng, thng kt hp vi nhau thnh dng chui. Bo thnh

bu dc, khng c dng nang bo t.

Dng que cy vng cy chuyn mt lng sinh khi chng thnghim

trong ng thch dinh dng vo 0,5ml BPW v trng. Huyn ph ha dch ny

sdng cho cc phn ng sinh ha.

- Thnghim ln men glucose: cy vi khun vo 3ml canh Phenol Red

Glucose Broth, 35oC, 24 gitrong iu kin kkh. Lc ng nghim tht

mnh v quan st spht trin thng qua c v schuyn mu mi trngtsang vng, chng tssinh acid tglucose trong iu kin kkh.

- Thnghim khnng khnitrate: cy vi khun vo 5ml mi trng canh

Nitrate Broth, 35oC trong 24 gi. Kim tra shin din ca nitrite bng cch

bsung vi git dung dch A v dung dch B ca thuc thnitrate. Nu c mu

cam xut hin trong vng 10 pht th chng tnitrate bkhthnh nitrite.

- Thnghim VP: Dng que cy vng cy vo cc ng mi trng MR-

VP mt t sinh khi tkhun lc thun 18 24 gitrn mi trng KIA

hoc TSI. yn cc ng mi trng ny 37oC trong 24 48 gihoc n 10

ngy khi cn thit. Thnghim VP l (+) khi c mu trn bmt mi trng,

l (-) khi bmt mi trng khng i mu.



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- Thnghim khnng thy phn tyrosine: cy vi khun vo thch

nghing Tyrosine, 35oC trong 48 gi. Sxut hin ca sinh khi, khun lc

l chthtyrosine bphn hy.

- Thnghim vi canh Lysozyme Broth: cy vi khun vo 2,5ml mi

trng Nutrient Broth cha 0,001% lysozyme. Thc hin tng tvi mi

trng Nutrient Broth khng cha lysozyme. ng nghim 35oC trong 24 gi.

Kim tra stng trng trong mi trng cha lysozyme v mi trng i

chng. nhng ng m tnh thm 24 gitrc khi kt lun kt quthnghim.

c. Cc thnghim phn bit cc loi trong Bacillus nhm 1

phn bit cc loi khc nhau trongBacillusnhm 1 cn tin hnh b

sung cc thnghim sau:

- Thnghim tnh di ng: dng que cy vng cy dch 24 ginui cy

thng vo gia mi trng kim tra di ng choB.cereus. 30oC, t18 24

giv kim tra di nh n kiu mc dc theo ng cy. Loi di ng mckhuch tn vo mi trng theo hng xa ng cy. Loi khng di ng ch

mc trong v dc theo ng cy. Bsung 0,2ml nc ct v trng vo bmt

mi trng thch nghing Nutrient Agar. Cy huyn ph vi khun vo. thch

nghing t6 8 gi30oC. Nhnc v trng ln phin knh v t sinh khi

vo. Quan st ngay di knh hin vi kim tra sdi ng. Hu ht cc chng

B.cereusvB.thuringiensisl di ng,B.anthracisv hu ht cc chng

B.mycoideskhng di ng.

- Shnh thnh rgi: chm nhque cy vng mang huyn ph 24 gi

nui cy ln gia a Nutrient Agar. 30oC trong 48 72 gi. Kim tra s

pht trin ca rgi, c trng bi vic to nhng khun lc c cu trc ging

nhrhoc tc mrng vi centimet tvtr cy.B.cereuskhng to cu trc r



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gi, thng to nhng nhm khun lc x x khc vi cu trc rgic trng

caB.mycoides.

- Thnghim lm tan mu: cy chng ln mi trng thch mu

Trypticase Soy. 35oC trong 24 gi.B.cereuslm tan mu mnh v to vng

tan mu hon ton ( ) 2 4mm xung quanh vng pht trin.B.thuringiensisv

B.mycoidescng tan mu .B.anthracisthng khng lm tan mu sau 24 gi.

- Sto c tprotein dng tinh th: cy huyn ph tbo 24 giln ng

thch nghing Nutrient Agar, 24 gi30oC, sau nhit phng 2 3

ngy. Thc hin nhum bng phm mu fuchsin. Quan st di knh hin vi

nhng tinh thc thnh tgic (dng kim cng) c nhum mu ti nh

hn bo t. Tinh thc tcaB.Thuringiensisxut hin nhiu sau 3 4 ngy

nui cy nhng khng thpht hin c bng kthut nhum cho n khi bo

tnang vra. Do , nu khng quan st c bo ttdo, cn thm vi

ngy nhit phng ri tin hnh kim tra li.B.cereus v ccBacilluskhc

cng nhm khng c tinh thc.

d. Cch tnh kt qu

Tnh stboB.cereustrn 1g mu da vo skhun lc mc mi

pha long v hiu chnh bng tlkhng nh (% khun lc c xc nhn l

B.cereus). V d, skhun lc m c pha long 10-4l 65, v c 4 trong

s5 khun lc c chn xc nhn lB.cereussau khi kim tra bng cc phnng sinh ha. Nhvy, stboB.cereustrong 1g thc phm l

65 4/5 10.000 10 = 5.200.000 (phi nhn vi 10 v chc 0,1ml mu c

sdng tri a).

2.5.4.2.nh lng B.cereus bng phng php MPN



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Phng php MPN c sdng nh lngB.cereustrong nhng

mu thc phm khng c cB.cereusnhiu hn 10 tbo/g. Phng php

ny cng c sdng kim tra nhng mu thc phm c tinh bt kh m

phng php m khun lc khng thch hp. Cy 1ml mu c pha long 10-

1, 10-2v 10-3vo ng nghim cha 10ml canh Trypticase Soy-Polymycin. Thc

hin 3 ng nghim lp li cho mi pha long (h9 ng nghim). 30oC

trong 48 2 gi. Kt qul (+) khi c stng trng caB.cereus. Cy ria t

cc ng (+) ln mi trng thch MYP. a 30oC trong 24 48 gi. Chn

nhng khun lc mu hng eosin, lecithinase (+), cy chuyn sang thch nghing

Nutrient Agar tin hnh cc phn ng sinh ha khng nhB.cereus. Da vo

bng MPN tnh trsMPN/g mu da vo sng (+) B.cereus c khng nh:

TSP (+), MYP (+), cc thnghim sinh ha khc.

2.6. Clostridium

2.6.1. Tng quan

Ging Clostridiuml cc vi khun gram dng, hnh que, kkh, sinh bo

t, phn ln di ng, c ththy gii saccharide v protein trong cc hot ng

thu nhn nng lng. Nhng loi thy gii saccharide c thln men cc loi

ng polysaccharide to thnh acetic acid, butyric v ru. Nhiu loi c th

thy gii protein v chuyn ha khng hon ton cc acid amin to thnh mi rt

kh chu trong sn phm. Hu ht ging Clostridiumthuc nhm a nhit va,tuy nhin c mt sloi thuc nhm a nhit v mt sloi khc thuc nhm a

lnh.

Cc loi Clostridiumhin din trong t, mt sloi trong nhm ny gy

bnh cho ngi v ng vt, mt sloi khc gy hhng thc phm, kh

sulphite thnh sulphur to ra mu en v gy mi kh chu. Cc loi gy ngc



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thc phm quan trng l C.botulinumv C.perfringens. Ngoi ra, loi C.tetanil

tc nhn gy bnh un vn, mt sloi khc nhC.novyi, C.perfringens,

C.septicum, C.sordellii gy hoi tcho m bnhim, gy bin chng ti cc

vt thng nhng cho n nay cha xc nh c cc c im bnh l. c

im quan trng ca mt sloi thuc ging Clostridiumthng gp l nhsau:

- C. botulinuml loi sng kkh bt buc, chtng trng c trong

mi trng trung tnh, khng c scnh tranh vi cc vi sinh vt khc. Cc dng

khc nhau trong loi ny c cc c im nui cy khc nhau v 6 kiu khng

nguyn c k hiu tA F. Kiu khng nguyn A, B v F c hot tnh thy

gii protein to nn mt vng phn gii xung quanh khun lc trn mi trng

Willis v Hobbs, trong khi kiu khng nguyn C, D, E khng c cc c tnh

ny. Kiu khng nguyn A thng c tm thy trong cc mu tht trong khi

kiu E chc phn lp tcc mu c.

- C.teranil loi kkh bt buc, bcht ngay khi tip xc vi khng kh.Loi ny c c im to khi kt tkhi c nui cy trong mi trng lng.

Loi ny c tm thy trong t, trong phn cc loi ng vt v ngi, l loi

gy bnh un vn rt nguy him.

- C.perfringensl loi kkh khng bt buc, rt t kh to bo ttrong

cc mi trng nui cy nhn to, nhng c thquan st c bo tkhi nui

cy trong mi trng Ellner, mi trng co bsung mui mt v bicarbonate

hay quinoline. Loi ny c su kiu khng nguyn c k hiu tA F. Kiu

khng nguyn A thng gy hoi tcho cc vt thng v gy ngc thc

phm.

2.6.2. Nguyn tc



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Mt vi khun Clostridiumc xc nh bng cch sdng mi trng c

cha ferri ammonium citrate v disodium sulphite, 37oC trong 1 2 ngy.

Nu nghi ngc Clostridiuma nhit c ththm 50oC. Trn mi trng

ny cc khun lc Clostridiumc mu en do phn ng gia ion sulphide (S2-)

v ion st (Fe2+) c trong mi trng.


- Pepton m Buffered Peptone Water (BPW).

- Iron Sulphite Agar (ISA).

- Perfringens Selective Agar (hay Shahidi Ferguson Perfringens, SFP).


Mu c gii ng nhit khng qu 45oC v c phn tch ngay

sau khi gii ng. Cn 10g (hoc 25g) mu trong ti PE v trng, bsung 90ml

(hoc 225ml) nc pepton m v ng nht mu bng my dp mu. Mu c

tip tc pha long thp phn ty mt hin din ca Clostridiumtrong mu.Trc khi cy, mu c xl nhit 70 80oC trong 20 pht dit bt tbo

sinh dng ca cc vi sinh vt khc.

Cy vo a v trng 1ml dch mu c pha long thch hp vo mt

a petri v trng. 15ml mi trng ISA hoc SFP Agar c m 45oC

vo a, lc u. Sau khi mi trng ng, thm ln trn bmt khong

10ml ISA hoc SFP Agar. Mt phng php khc l cho vo mt ng nghim v

trng 1ml dch mu nng thch hp, thm 12ml ISA hoc SFP Agar

c m 45oC vo ng, trn u mu. Sau khi mi trng ng, thm

ln trn bmt 2 3ml ISA hoc SFP Agar. a hoc ng nghim cy mu

c 37oC trong 24 48 gitrong cc bnh kkh. Nu nghi ng



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Clostridiumchu nhit, thc hin song song 37oC v 50oC. Thng thng,

vic c kt qutrn a l dhn trong ng nghim.

ISA l mi trng khng chn lc nn cc loi vi khun sinh H2S khc

khng phi l Clostridium cng tng trng c v to khun lc mu en trn

mi trng ny. khng nh khun lc l Clostridiumcn thc hin thm

nhng quy trnh tiu chun gip khng nh kt qu.

2.6.5. Bo co kt qu

m tt ccc khun lc en, hoc c thbao quanh bi vng en. Mt

Clostridiumtrong mu c tnh tskhun lc m c nhn vi hs

pha long mu v c trnh by dng CFU/g hay CFU/ml sn phm.

2.7. Coliform v E.Coli

2.7.1. Tng quan

Bao gm mt svi khun Gram m, khng to bo t, hiu kh hoc k

kh khng bt buc, c khnng ln men ng lactoza, sinh hi trong vng 48ginhit nui cy thch hp.

Coliform a nhit (coliform phn): ln men lactoza nhit 44oC.E.Colithuc

nhm Coliform a nhit v l loi coliform rt bn vi phenol 0,085 v sinh

indol nhit 42 44oC.

Xc nh lng ColiformvE.colicho bit mc nhim v tnh trng

vsinh trong phn xng sn xut.

2.7.2.nh lng Coliforms, Coliforms chu nhit, Coliforms phn v E.coli

bng phng php MPN

2.7.2.1. Nguyn tc

Slng Coliforms, Coliforms chu nhit, Coliforms phnvE.colitrong

mu thc phm cha mt thp ca nhm vi khun ny c thc xc nh



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bng phng php MPN (Most Probable Number). Phng php ny da vo

nguyn tc mu c pha long thnh mt dy thp phn (hai nng ktip

nhau khc nhau 10 ln); 3 hoc 5 mu c pha long thp phn lin tip c

trong ng nghim cha mi trng thch hp c ng by kh Durham. Mi

nng pha long c t3 n 5 ng lp li. Theo di ssinh hi v i

mu tnh shin din trong tng ng thnghim; y l cc ng dng tnh.

Ghi nhn sng nghim cho phn ng dng tnh mi nng pha long v

da vo bng tnh MPN suy ra slng nhm vi sinh vt tng ng hin din

trong 1g (hoc 1ml) mu ban u.

2.7.2.2. Mi trng v ha cht

- Mi trng lng Lauryl Sulphate Broth LSB (canh Lauryl Sulphate).

- Mi trng lng Brilliant Green Lactose Bile Salt (canh BGBL).

- Mi trng lng E.coli (E.coli medium, canh EC).

Cc mi trng lng trn c chun btrong cc ng nghim cha ngDurham p ngc. Sau khi khtrng, chsdng cc ng nghim khng c bt

kh bn trong ng Durham.

- Canh Tryptone.

- Mi trng rn Simmon Citrate Agar (thch Simmon Citrate).

- Dung dch nc mui pepton SPW (Saline Pepton Water).

- Thuc thKovacs.

- Thuc thMethyl Red.

- Thuc th -napthol.

2.7.2.3. Quy trnh phn tch

Chun bng nht mu hoc pha long mu c dch mu c pha

long 10-1.



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54/79

a. nh lng Coliforms

Tun tcy 1ml dch mu pha long 10-1vo 3 ng nghim ging

nhau, mi ng cha 10ml canh LSB. Thc hin tng tvi dch mu pha

long 10-2v 10-3. y l trng hp xc nh MPN bng h3 dy nng v 3

ng nghim lp li (h3 3 hay 9 ng nghim). Nu nghi ngslng

Coliformstrong mu qu cao, phi sdng cc mu c bc pha long cao hn.

cc ng nghim 37oC trong 48 gi. Ghi nhn sng c sinh hi. Dng que cy

vng (khuyn cy) cy chuyn dch mu tcc ng LSB (+) sang cc ng c

cha canh BGBL v 37oC 1oC trong 48 gi. Ghi nhn sng cho kt qu

(+) (c sinh hi) ng vi mi pha long.

b. nh lng Coliforms chu nhit

Dng que cy vng chuyn mt vng dch mu tcc ng canh LSB (+)

sang mi trng canh EC 44,5 0,2oC trong 24 gi. m slng cc ng

cho kt qu(+) (sinh hi) mi pha long.

c. nh lng Coliforms phn

Dng que cy vng ria dch mu tcc ng (+) trn mi trng dch EC

sang mi trng thch a EMB. cc a ny 37oC trong 24 gi. Cc khun

lc trn, dt hnh a v c nh kim tm l khun lc ca Coliforms phnhay

E.coliginh. Chn khun lc ng knh ln hn 1mm v cy chuyn vo

canh Trypton, 44,5 0,2oC trong 24 gi. Nhthuc thKovacs vo ccng nghim. ng nghim c sxut hin ca mu trong mi trng trong vi

pht l ng (+). Thc hin tng tcho tt ccc ng (+) trn mi trng EC.

Ghi nhn slng cc ng cho kt qu(+) trn mi trng Trypton tng ng

vi mi pha long.

d. nh lng E.coli

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