Định Tính Salmonella ..

B CNG THNG
TRNG I HC CNG NGHIP THC PHM THNH PH H CH MINH

KHOA: CNG NGH THC PHM
MN: PHN TCH VI SINH THC PHM

Tn ti:
NH T NH SAMONELLA TRONG
THC PHM
GVHD: Nguyn Th Kim Oanh
Lp: 02HTP1
Nhm: 4
TN THNH VI N THAM GI A:
1. Nguyn Th T Linh 2005110252
2. Nguyn Th Hin 2005110167
3. Ging Th Mng Thu 2005110532
4. Th M Duyn 2005110087
5. L Th Hng 2005110104

Tp.HCM , ngy 14 thng 5 nm 2014

ti: nh tnh Samonella trong thc phm
2

MC LC
BNG PHN CNG CNG VIC ................................................................................... 4
M U ............................................................................................................................. 5
NI DUNG .......................................................................................................................... 6
1.1. Lch s pht hin .................................................................................................... 6
1.2. Phn loi Salmonella theo loi v theo kiu huyt thanh. ..................................... 7
1.3. c im ................................................................................................................ 9
1.3.1. c im chung v c im nui cy............................................................ 9
1.3.2. Tnh cht ha sinh ......................................................................................... 10
1.3.3. Cu trc ca Salmonella ................................................................................ 10
1.3.4. Yu t c lc ............................................................................................... 12
1.3.4.1. Ni c t - Endotoxin ........................................................................... 12
1.3.4.2. c t ng rut ................................................................................... 13
1.3.4.3. c t t bo ........................................................................................... 14
1.3.5. C ch gy bnh ............................................................................................ 14
1.3.5.1. C ch gy bnh thng hn .................................................................. 15
1.3.5.2. C ch gy nhim khun v nhim c thc n ..................................... 17
1.3.6. Ngun gc ly nhim .................................................................................... 17
1.3.7. Tnh hnh nhim Salmonella trong nc v trn th gii ............................. 18
1.3.7.1. Trn th gii ........................................................................................... 18
1.3.7.2. Trong nc ............................................................................................. 19
1.3.8. Cc ch tiu v Salmonella trong thc phm ................................................ 19
2.1. Phng php truyn thng : ................................................................................. 20
2.1.1. Giai on tin tng sinh (pre enrichment) .................................................. 21
2.1.2. Giai on tng sinh chn lc (selective enrichment) .................................... 21
2.1.3. Giai on phn lp (isolation) ....................................................................... 22
2.1.4. Khng nh bng cc phn ng sinh ha ...................................................... 23
3

2.2. Phng php hin i : ........................................................................................ 23
2.2.1. Phng php PCR ......................................................................................... 24
2.2.2. Phng php ELISA ..................................................................................... 27
2.2.3. Cc bin php kim sot Salmonella trong thc phm ................................. 28
3.1. Nguyn tc ........................................................................................................... 29
3.1.1. Dng c v thit b ........................................................................................ 30
3.1.2. Mi trng v ha cht ................................................................................. 30
3.1.3. So snh mi trng SPW v BPW ................................................................ 32
3.2. Phng php thc hin ........................................................................................ 33
3.2.1. Tng sinh ....................................................................................................... 34
3.2.2. Tng sinh chn lc ........................................................................................ 34
3.2.3. Phn lp v nhn din ................................................................................... 35
3.2.4. Khng nh .................................................................................................... 36
3.2.5. Khng nh Salmonella bng khng huyt thanh .......................................... 40
TI LIU THAM KHO ................................................................................................. 41

4

BNG PHN CNG CNG VIC
H v tn Cng vic nh gi Ch k
1. Nguyn Th T Linh
- Tm kim ti liu
- Lm word
- Thuyt trnh
Tch cc

2. Nguyn Th Hin
- Tm kim ti liu
- Lm word
- Thuyt trnh
Tch cc

3. Ging Th Mng Thu
- Tm kim ti liu
- Lm powerpoint
- Thuyt trnh
Tch cc

4. Th M Duyn
- Tm kim ti liu
- Thuyt trnh
Tch cc

5. L Th Hng
- Tm kim ti liu
- Thuyt trnh
Tch cc

5

M U
Trong nhng nm gn y, vn ng c thc phm ngy cng tr nn cp thit,
cc bo co cho thy phn ln cc v ng c thc phm l do vi sinh vt. c nhiu
cnh bo, nhng tnh trng ng c thc phm vn ang leo thang v ngy cng nghim
trng.
C rt nhiu vi sinh vt gy ng c thc phm, v d nh Clostridium butolinum,
Escherichia Coli, Listeria monocytogenes trong , Salmonella l loi vi sinh vt gy
ng c rt nguy him. Salmonella thuc h Enterobactriaceae, gy ra bnh thng hn,
nhim trng huyt v nhiu bnh nghim trng khc.
Xut pht t nhu cu tm hiu v c t, kh nng gy bnh v cch pht hin
cng nh cch phng phng chng bnh vi khun Salmonella, chng ti thc hin nghin
cu ti nh tnh samonella trong thc phm c ci nhn tng quan hn v vi
khun Salmonella v mt s chng vi sinh vt khc.

6

NI DUNG
CHNG I : TNG QUAN V SALMONELLA
1.1. Lch s pht hin
Nm 1885 Slamon v Smith (M) tm c Salmonella t ln mc bnh dch t
v gi tn l Bacilus cholerasuis, hin nay gi l Salmonella. Nhng sau Schweinittz
v Dorset 1903 chng minh bnh dch t l do mt loi vi rt gy nn v xc nh
S.choleraesuis l vi khun gy bnh ph thng hn.
Nm 1888 A.Gartner phn lp c mm bnh t tht b v lch ngi bnh, ng
gi vi khun ny l Bacillus enteritidis v ngy nay vi khun ny c gi l S.enteritidis.
Vi khun ny cng c gi bng nhiu tn khc nh: Bacterium enteritidis, Bacillus
gartner
Nm 1889 Klein phn lp c S.gallinarum v Rettger cng phn lp c
S.pullorum nm 1909. Trc y ngi ta cho rng y l hai loi vi khun gy ra hai
bnh khc nhau ln gi chung l bnh ph thng hn g (Typhus avium) v cn bnh
c tn chung l S.gallinarumpullorum.
Nm1896 C.Archard v Rbensauded phn lp c vi khun S.paratyphi equi
v S.paratyphi bacilus. Ngy nay vi khun ny c gi tn l S.paratyphi Bv n nm
1898,S.paratyphi A tm c do N.Guyn v H Keyser.
Vi khn Salmonella

7

1.2. Phn loi Salmonella theo loi v theo kiu huyt thanh.
- V phn loi khoa hc Salmonella c
xp vo:
Gii : Bacteria
Ngnh: Proteobacteria
Lp: Gramma Proteobacteria
B: Enterobacteriales
H: Enterobacteriaceae
Ging:Salmonella lignieres1900

Cc loi in hnh:
Salmonella typhi: Gy bnh thng hn
H V u Salmonella typhi
Salmonella cholera_suis: Gy bnh nhim trng mu

8

H V u Salomonella cholera_suis
Salmonella entertidis: Gy ri lon tiu ha

H V u Salmonella entertidis

Lc u, cc loi Salmonella c t tn theo hi chng lm sng ca chng nh
S.typhi hay S.paratyphi A, B, C (typhoid = bnh thng hn, para = ph), hoc theo vt
ch nh S.typhimurium gy bnh chut,v sau ngi ta thy rng 1 loi Salmonella c
th gy ra nhiu hi chng v c th phn lp c nhiu loi khc nhau. V nhng l
do m cc chng Salmonella mi pht hin c t tn theo ni m n c phn lp
nh S.teheran,S.congo,S.london.
Hin nay theo phn loi ca h thng Vin Pasteur th gii, Trung tm Kim sot
v ngn nga dch bnh ca M CDC (Control and Prevent Disease Center), ging
Salmonella c chia lm hai loi: S. enterica v S. bongori. Trong S. bongori gm tt
c cc kiu huyt thanh (serotype) ca loi ph s V, v S. enterica c chia lm 6 loi
ph nh s: I, II, IIIa, IIIb, IV v VI .Cc loi v loi ph ny c th phn bit c
bng phn ng sinh ha. Trong mi loi ph c nhiu kiu huyt thanh.
Cho n nay xc nh c trn 2463 kiu huyt thanh thuc ging Salmonella.
Theo h thng ca Kauffman White, cc kiu huyt thanh ny c chia da trn khng
nguyn thn O, khng nguyn tim mao H v khng nguyn b mt Vi. Phn ln cc kiu
huyt thanh c t tn theo cng thc khng nguyn, mt s khc c t tn ring
nh Enteritidis (S. enteritidis), Typhi (S. typhi), Paratyphi (S. paratyphi), Typhimurium
(S. typhimurium)
Bng phn loi Salmonella theo loi v theo kiu huyt thanh
9

Loi Loi ph S kiu huyt thanh
S. enterica

S. bongori
S. enterica enterica (I)
S. enterica salamae (II)
S. enterica arizonae (IIIa)
S. enterica diarizonae (IIIb)
S. enterica houtenae (IV)
S. enterica indica (VI)

(V)
1454
489
94
324
324
12

20

Loi ph S. enterica I hu nh chim a s (99%), n c tm thy hu ht
cc ng vt mu nng. Loi ph ny chim 59% (1454/2463) trong tng s kiu huyt
thanh ca Salmonella c kh nng xm nhp v gy nhim cao cho ngi v ng vt
mu nng, trong khi cc loi ph khc hu nh ng vt mu lnh v mi trng.
Nh vy c ti 41% (1009/2463) s kiu huyt thanh khng c phn lp t bnh phm
ca ngi, y l mt t l rt c ngha cc c quan chc nng xem xt v sa i
cc tiu chun hin hnh v Salmonella trong thc phm to iu kin thun li cho cc
nh sn xut tiu th thc phm.
1.3. c m
1.3.1. c im chung v c im nui cy
Salmonella l trc khun Gram m, hiu kh v k kh ty kch thc trung bnh
t 0,5-3 m, di chuyn bng tin mao tr S. gallimarum v S. pullorum, khng to bo t,
chng pht trin tt nhit 6
0
C 45,6
0
C, thch hp nht 37
0
C, pht trin trong pH
4.1- 9.0. nhit t 18
0
C 40
0
C vi khun c th sng n 15 ngy.
Salmonella pht trin c trn cc mi trng nui cy thng thng. Trn mi
trng thch hp, vi khun s pht trin sau 24 gi. C th mc trn nhng mi trng c
cht c ch chn lc nh DCA (deoxycholate citrate agar) v XLD (xylose lysine
deoxycholate).Trong mi trng XLD t cht c ch hn nn thng c dng
phn lp Salmonella.
Khn lc c trng ca Salmonella trn mi trng ny l trn, li, trong sut, c
tm en, i khi tm en ln bao trm khn lc,mi trng xung quanh chuyn sang mu
.
10

1.3.2. Tnh cht ha sinh
Salmonella ln men glucose v mannitol sinh acid nhng khng ln men
saccharose, lactose, salicin v inositol, khng sinh indole, khng phn gii ure, khng c
kh nng tch nhm amine t tryptophane, hu ht cc chng u sinh H
2
S,s dng c
citrate mn trng Simmons.
Tuy nhin khng phi loi Salmonella no cng c nhng tnh cht trn, cc ngoi
l c xc nh l S.typhi ln men ng glucose khng sinh hi, khng s dng citrate
trong mi trng Simmon, hu ht cc chng S.paratyphi v S.Cholerasuis khng sinh
H
2
S, khong 5% cc chng Salmonella sinh c t sinh bacteriocin chng li E.Coli,
Shigella v ngay c 1 s chng Salmonella khc.
1.3.3. Cu trc ca Salmonella
Salmonella c ba loi khng nguyn, l nhng cht khi xut hin trong c th
th to ra kch thch p ng min dch v kt hp c hiu vi nhng sn phm ca s
kch thch , gm: khng nguyn thn O, khng nguyn tin mao H v khng nguyn v
K. Vi khun thng hn (S.typhi) c khng nguyn Vi (Virulence) l yu t chng thc
bo gip cho vi khun thng hn pht trin bn trong t bo bch cu.
- Khng nguyn vch t bo (khng nguyn thn O).
Thnh phn c bn l vch t bo c cu trc phc tp gm 2 lp. Trong cng l
mt lp peptidoglycan mng, cch mt lp khng gian chu cht v ti lp mng ngoi
(outer membrane) l phc hp lipidpolysaccharide gm lipoprotein v lipopolysaccharide.
Bao bn ngoi lp peptidoglycan l lp phospholipid A v B (quyt nh c t
ca Ni c t), sau l hai lp polysaccharide khng mang tnh c hiu. Khng
nguyn ca ni c t c bn cht ha hc l lypopolysaccharide (LPS). Tnh c hiu
ca khng nguyn O v LPS l mt, nhng tnh min dch th khc nhau : khng nguyn
O ngoi LPS cn bao gm c lp peptidoglycan nn tnh sinh min dch ca n mnh hn
LPS. Mng ngoi c cu trc gn ging t bo cht nhng phospholipid hu nh ch gp
lp trong, cn lp ngoi l lipopolysaccharide dy khong 8- 10 nm gm 3 thnh
phn : Lipid A, polysaccharide li, khng nguyn O. Mng ngoi cn c thm cc protein:
Protein c cht: porin vi khun cn gi l protein l xuyn mng vi chc nng cho
php mt s loi phn t i qua chng nh dipeptide, disaccharide, cc ion v c. Protein
mng ngoi: chc nng vn chuyn mt s phn t ring bit v a qua mng ngoi.
Lipoprotein: ng vai tr lin kt lp peptidoglycan bn trong vi lp mng ngoi .
11

Khng nguyn tin mao (khng nguyn H)
- Khng nguyn H: Ch c cc Salmonella c lng. Hu ht Salmonella u c
lng ch tr S.galilarum, S.pulorum gy bnh cho gia cm. Khng nguyn H l mt loi
khng nguyn c bn cht l protit, km bn hn khng nguyn O. Khng nguyn H rt
d b ph hu nhit cao hoc x l bng cn, axit yu.
Khng nguyn H chia lm 2 phase :
Phase 1: C tnh cht c hiu gm c 28 loi khng nguyn lng c biu th
bng cha s La tinh thng: a, b, c
Phase 2: Khng c tnh cht c hiu, loi ny c th ngng kt vi cc loi khc
i khi thnh phn ny c th gp E.coli. Pha 2 gm c 6 loi c biu th bng cha
s Rp 1-6 hay cha s La Tinh e, n, x
Khng th khng khng nguyn H ngng kt vi khun bi cc roi ca chng. S
ngng kt ny s to thnh nhng mng kt t, chng c th b tch bi cc yu t c kh
nng ct roi ca vi khun.
Khng nguyn H khng c tc dng gy bnh, khng gy min dch, n c hiu
cho cc loi vi khun da vo khng nguyn H phn loi bng phng php huyt
thanh.
- Khng nguyn v K
Khng nguyn K: l khng nguyn nm ngoi khng nguyn O v l ni biu hin
c t, c cu to bi polysaccharide ca v ngoi vch t bo, thng kt hp vi
tnh gy c chuyn bit cho vt ch. N c ch s biu hin ca khng nguyn O khi n
pht trin nhiu, n nh vi nhit , cn v HCl.
Khng phc tp, c mt khng nguyn v l khng nguyn Vi v cng c 2 type
huyt thanh S.typhi v S.paratyphi. Khng nguyn Vi-angtigen c Felix v cc cng s
pht hin nm 1935. Khng nguyn Vi gy hin tng ngng kt chm v xut hin cc
ht v, khng nguyn Vi l khng nguyn v bao bc bn ngoi khng nguyn O, khnh
nguyn Vi khng tham gia vo qu trnh gy bnh.

12

V tr ca cc khng nguyn ca Salmonella
1.3.4. Yu t c lc
Vi khun Salmonella c th tit ra 2 loi c t: Ngoi c t v ni c t.
- Ni c t ca Salmonella rt mnh gm 2 loi: Gy xung huyt v mn lot, c
t rut gy c thn kinh, hn m, co git.
- Ngoi c t ch pht hin khi ly vi khun c c tnh cao cho vo ti colodion
ri t vo bng chut lang nui, sau 4 ngy ly ra, ri li cy truyn nh vy t 5
n 10 ln, sau cng em lc, nc lc c kh nng gy bnh cho ng vt th nghim.
Ngoi c t ch hnh thnh trong iu kin invivo v nui cy k kh. Ngoi c t tc
ng vo thn kinh v rut.
1.3.4.1. Ni c t - Endotoxin
Mng ngoi t bo vi khun gram m ni chung v vi khun Salmonella ni ring,
c cu to bi thnh phn c bn l lipopolysaccharide (LPS). LPS c cu to phn t
ln, gm 3 vng ring bit vi c tnh v chc nng ring bit: Vng a nc, vng li
v vng lipit A.
Vng a nc bao gm mt chui polysaccharide cha cc n v cu trc khng
nguyn O. Vng li c bn cht l acid heterooligosaccharide, trung tm ni khng
nguyn O vi vng lipit A. Vng lipit A m nhn chc nng ni c t ca vi khun.
Cu trc ni c t gn ging cu trc ca khng nguyn O. Cu trc ni c t bin i
s dn n s thay i c lc ca Salmonella.
13

Ni c t thng l lipopolysaccharide (LPS) c phng ra t vch t bo vi
khun khi b dung gii. Trc khi th hin c tnh ca mnh, LPS cn phi lin kt vi
cc yu t lin kt t bo hoc cc receptor b mt cc t bo nh: T bo lm ba cu B,
lm ba cu T, t bo i thc bo, tiu thc bo, t bo gan, lch.Rt nhiu cc c quan
trong c th chu s tc ng ca ni c t LPS: Gan, thn, c, h tim mch, h tiu ho,
h thng min dch; vi cc biu hin bnh l: Tc mch mu, gim trng lc c thiu
oxy m bo, toan huyt, ri lon tiu ho, mt tnh thm n
Ni c t tc ng trc tip ln h thng min dch ca c th vt ch, kch thch
hnh thnh khng th.
- LPS tc ng ln cc tbo tiu cu, gy st ni c t, theo c ch :
Gii phng cc cht hot ng mnh nh: Histamin.
Ngng kt cc tiu cu ng mch.
ng vn, tc mch qun.
- LPS tc ng ln qu trnh trao i gluxit: LPS lm tng cng hot lc ca cc
men phn gii glucoz, cc men phn gii glycogen, lm gim hot lc cc men tham gia
qu trnh tng hp glycogen
1.3.4.2. c t ng rut
V c ch min dch v di truyn cc Enterotoxin ca Salmonella c quan h gn
gi vi Choleratoxin, nn c gi l Choleratoxin like enterotoxin (CT). Cn v c
tnh sinh hc Enterotoxin ca Salmonella khng ch vi ging CT m cn ging vi
Enterotoxin ca E.Coli.
c t ng rut ca vi khun Salmonella c hai thnh phn chnh: c t thm
xut nhanh Rapid permeability facto (RPF) v c t thm xut chm Delayed
permeability facto (DPF).
RPF gip Salmonella xm nhp vo t bo biu m ca rut, n thc hin kh
nng thm xut sau 1-2 gi v ko di 48 gi v lm trng cc t bo CHO (Chinese
Hamster Ovary cell). c t thm xut nhanh c cu trc, thnh phn ging vi c t
chu nhit ca E.Coli, c gi l c t chu nhit ca Salmonella. ST c kh nng chu
c nhit 100
0
C trong 4 gi, bn vng nhit thp, c th bo qun nhit -
20
o
C. Cu trc phn t gm mt chui polysaccharide v mt chui polypeptide.
RPF kch thch co bp nhu ng rut, lm tng s thm thu thnh mch, ph hu
t chc t bo biu m rut, gip vi khun Salmonella xm nhp vo t bo v pht trin
14

tng nhanh v s lng. Vi khun tch cc tng cng sn sinh c t lm ri lon cn
bng trao i mui, nc v cht in gii. Qu trnh bnh l ng rut v hi chng
tiu chy cng thm phc tp v nghim trng.
DPF ca Salmonella c cu trc, thnh phn ging c t khng chu nhit ca vi
khun E.Coli, nn c gi l c t khng chu nhit ca Salmonella. N thc hin chc
nng phn ng chm t 18-24 gi. LT b ph hu 70
0
C trong vng 30 pht v 56
0
C
trong vng 4 gi. LT c cu trc gm 3 chui polypeptid.
DPF lm thay i qu trnh trao i nc v cht in gii, dn n tng cng bi
xut nc v cht in gii t m bo vo lng rut, cn tr s hp thu, gy thoi ho lp
tbo villi ca thnh rut, gy tiu chy.
1.3.4.3. c t t bo
Khi c th ngi v ng vt b tiu chy th km theo hin tng mt nc v
mt cht in gii l hin tng hng lot cc t bo biu m rut b ph hu hoc b tn
thng cc mc khc nhau. S ph huhay tn thng l do c t t bo
caSalmonella gy nn, theo c ch chung l:c chtng hp protein ca t bo
Eukaryotic v lm trng t bo CHO.
t nht 3 dng c t ca t bo:
- Dng th nht: Khng bn vng vi nhit v mn cm vi trypsin. Dng ny c
pht hin rt nhiu serovar Salmonella; c trng lng phn t trong khong t 56 n
78 kDa; khng b trung ho bi khng th khng c t Shigella toxin hoc Shigella - like.
c t dng ny tc ng theo c ch l c ch tng hp protein ca t bo Hela v lm
teo t bo.
- Dng th hai: C ngun gc t protein mng ngoi t bo vi khun c cu trc v
chc nng gn ging cc dng c t t bo do Shigella v cc chng (ETEC) sn sinh ra.
Dng c t ny cng ph bin hu ht cc serovar Salmonella gy bnh.
- Dng th ba: C trng lng phn t khong 62 kDa; c lin h vi c t
Hemolysin. Hemolysin lin h vi cc c t t bo c s khc bit vi cc Hemolysin
khc v trng lng phn t v phng thc tc ng ln t bo theo c ch dung gii
cc khng bo ni bo.
1.3.5. C ch gy bnh
Salmonella c th gy ng c thc phm khi hin din n mc c triu t bo
trong mt gram thc phm. Cc triu chng do Salmonella gy ra thng l tiu chy, i
15

ma, bun nn. Thi gian bnh k t khi tiu ththc phm b nhim cho n khi cc
triu chng c biu hin l 12 36 gi. Triu chng ng c thng ko di t 2 7
ngy. Khng phi tt c mi ngi khi tiu th thc phm b nhimSamonella u b ng
c. Cc loi thc phm c nguy c b nhimSamonella cao 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 loiSamonella typhi, S.paratyphi A, B, C gy st thng hn.

Cc chng Salmonella v cc biu hin bnh thng gp

1.3.5.1. C ch gy bnh thng hn
Bnh thng hn do S.typhi v S.paratyphi A, B, C gy ra. Cc yu t c lc
chnh ca vi khun thng hn l kh nng bm v xm nhp vo t bo ch, kh nng
nhn ln trong i thc bo v ni c t. Khng nguyn Vi c mt S.typhi v
S.pararatyphi C l yu t c lc quan trng, nhng chng vi khun gy bnh thng hn
khng c khng nguyn Vi th s lng cn thit gy bnh cao hn rtnhiu so vi
nhng chng c khng nguyn ny.
Vi khun xm nhp vo c th qua ng tiu ha do thc n hay nc ung b
nhim bn, s lng cn thit gy bnh vo khong 10 5 n 10 7
u tin, vi khun thng hn phi vt qua mi trng axit ca d dy, mc d
chng c kh nng khng vi axit nh c gen art (acid response tolerance), nhng
ngi bnh thng, vi khun thng hn khng th tn ti lu, nui cy dch d dy m
16

tnh sau 30 pht. Sau khi vt qua c ro cn d dy, vi khun di chuyn xung rut
non ri nhn ln , nhng trong tun u s c 1 t vi khun o thi theo phn, cy
phn dng tnh trong 5 ngy khng c ngha l bnh thng hn s xy ra. T rut non,
vi khun thng hn i vo hch mc treo rut nh t bo M, mt i thc bo ca mng
Peyer. Sau theo ng bch huyt v mu gy nhim trng ton thn. Sau khong mt
tun, nhim khun huyt th pht xut hin. Vi khun theo gan qua ng mt li tip tc
xm nhp vo rut non, tip tc nhn ln cc mng Peyer. T mu, vi khun ti lch v
cc c quan khc. Trong rut non, vi khun cht v gii phng ra ni c t. Ni c t
kch thch dy thn knh giao cm rut gy ra hoi t chy mu v c th gy thng rut,
v tr gy tn thng thng nm cc mng Peyer. y l bin chng hay gp c
bnh nhn n sm khi cha bnh phc, nht l n cc thc n cng. Ni c t theo mu
ln h thn kinh v kch thch trung tm thn kinh thc vt no tht ba. Giai on ton
pht bnh, bnh nhn st cao, biu thn nhit tng ln theo thi gian. Thn nhit tng
nhng nhit khng tng gy ra hin tng mch v nhit phn ly.

Thi k cha c iu tr bng khng sinh, khon mt tun bnh, din bin in
hnh ca bnh thng hn tri qua 4 giai on, mi giai on khong mt tun, Tun th
nht thn nhit tng cao, tun th hai th au bng, gan v lch to dng thi c s xut
hin ca cc m hng trn da, tun th 3 c th xut hin thm cc bin chng nh xut
huyt, thng rut, tun th 4 s xut hin thm cc bin chng nguy him dn n t
vong, nu khng bnh nhn sbnh phc.
17

Bnh nhn thng au u, bun nn hoc nn, chng bng, tiu chy, khong
50% bnh nhn s thy gan v l lch di sn. Nhng trng hp nng thng c du
hiu ly b, hn m, try tim mch, khng cha tr kp thi c th dn n t vong. Nhng
bin chng khc nh vim phi cp, vim mng no, vim gan, vim ty xng cng c
th gp.
Nhng bnh nhn qua khi c khong 5 10% vn tip tc thi vi khun qua phn
trong qu trnh hi phc v 1- 4% tr thnh ngi mang vi khun lu di do vi khun vn
tn ti trong ti mt. Tnh trng ny c th ko di n nhiu nm v h tr thnh ngun
mang bnh rt nguy him.
1.3.5.2. C ch gy nhim khun v nhim c thc n
Bnh thng xy ra do n phi thc n b nhim Salmonella, nhng cng c th
ly truyn trc tip. Cn nguyn thng do vi khun S.enteritidis v S.typhimurium.
Vi khun xm nhp qua rut non nh cc t bo M ca mng Peyer. Trn b gen
ca Salmonella c cc tc nhn gyc ch cc cht khng khun c trong lysosome, bin
i t bo ch m bo cho s tn ti ca vi khun, lm cn tr hot ng ni bo nh
gim lng NADH oxidase rt cn thit cho vic sn xut cc hpcht khng khun.
S hy hoi ca i thc bo v cc t bo biu m ln cn, s cht ca vi khun
gii phng ni c t gy nn tn thng cho c th vt ch v gy ra cc triu chng.
Thi gian bnh trung bnh t 10 48 gi. Sau thi gian bnh, ngi nhim
thng c biu hin nh st, nn, au bng v tiu chy. Mc st khc nhau ty vo
th trng tng ngi, tiu chy phn thng khng c mu. ngi ln thng ch dn
n tnh trng ri lon ng tiu ha, nhng tr s sinh thng gy ra nhim trng rt
nng, c th dn n tnh trng nhim khun huyt, vim mng no v vim xng.
nhng ngi khe mnh, nhim trng do nhim c thc n thng t khi sau 2-5 ngy
ri t khi.
1.3.6. Ngun gc ly nhim
Cc sn phm tht ni chung, nht l tht gia cm v tht ln. Tt c cc thc n
ti sng c ngun gc ng vt u c th l ngun vi khun Salmonella. Vi khun ny
sng t do trong rut ng vt v c trn lng. Gia cm c nhiu Salmonella nht, tip
theo l cc ng vt nui trong nh v ng vt hoang (vt, ra, ch, ch, chim mng
bin, loi gm nhm, rn). Vi khun ny c th c trong thnh phn dn xut cc cht t
18

ng vt nh gelatin hoc nc bt ng vt, bi cn trng, loi gm nhm, chim hoc
cc sn phm tht nhim khun gy nhim vo thc phm.
Ngoi ra c th b nhim t ngi khe mnh c mang vi khun ny. Thc phm
c ngun gc thc vt t c nguy c nhim khun. V l do b c ch bi pH < 4 v c mt
vi khun lactic nn cc sn phm ln men t b nhim. Trng v cc sn phm trng v d
nh bt nho, nc st mayonnaise, protit ng t tch t sa, gia cm l ngun mang
nhiu Salmonella, nn trng ca n cng b nhim v vi khun ny c th xuyn qua v
trng v sinh sn trong lng trng.
Cc sn phm sa nh sa khng thanh trng, phomt t sa ti, kem cht bo
sa, v cc sn phm t sa ni chung c ch bin t cc nng tri, cc thit b u c
th gy nhim vo nguyn liu, to mi trng thun li choSalmonella, v t gy
nhim c cho sn phm sa. Nu tin hnh axit ha chm th vi khun d dng sinh sn
trong phomt nhng n b ph hy vi pH < 4,5. Nhng sn phm c sa phi c gim
st cht ch bi chng khng c thanh trng na, v vy nu cSalmonella trong sa
bt th chng vn c th sinh sn c bi chng c kh nng tn ti iu kin kh hn
v ly nhim sang cc sn phm khc
1.3.7. Tnh hnh nhim Salmonella trong nc v trn th gii
1.3.7.1. Trn th gii
T l nhim Salmonella chu u gim u n t nhng nm 1990 tr li y,
trong nm 2007 c khong 152.000 ca nhim Salmonella trn ngi c pht hin, s
sai lch ca bo co ny l rt ln, s lng thc t rt c th gp 10 ln nh th. M,
tnh trng c kh hn, n nh mc 15 ca trn 100.000 ngi t nm 2001 do kim sot
tt Salmonella trong thc phm t nm 1990, bao gm cc thc phm sa, trng, nc
tri cy, sn phm ti sng, rau, bnh ko, v c bit l tht. Mt t dch gn y
M gy ra bi S.typhimurium nhim trong b u phng gy nh hng n hn 700
ngi trn khp nc M
T thng 7/2009 ti nay, s ca nhim khun Salmonella c pht hin M
tng ln 184 ngi, thuc 38 bang khc nhau. C quan y t ca nc ny vn cha xc
nh nguyn nhn chnh xc khin s ca nhim khun Salmonella tng nhanh nh vy.
Tuy nhin, mi y cc chuyn gia y t ca bang Oregon cho rng ngun ly lan vi khun
Salmonella t cc sn phm xc xch.Va qua, cc c quan iu tra M thu hi hn
560 tn xc xch do nghi bnhim vi khun Salmonella, tt c s lng ny u do Cng
ty Daniele International sn xut.Tuy nhin, ng Jason Maloni, pht ngn vin ca cng
19

ty ny khng nh: Cha c bng chng no chng minh cc sn phm xc xch ca
chng ti b nhim vi khun Salmonella. S bng n ca s ca nhim khun Salmonella
khu vc ty bc Thi Bnh Dng khin cc c quan iu tra nghi ng rng ngun
gy bnh cho dch ny l t cc sn phm xc xch sau khi h pht hin rt nhiu ngi
n xc xch mua ti cc ca hng khu vc ny b nhim khun Salmonella. Cc nhn
vin iu tra ti bangWashington cng cho bit 14 bnh nhn b nhim khun Salmonella
bang ny, tng n xc xch ca hng Daniele. Ngoi ra, cc chuyn gia y t khng
nh h kim tra v pht hin khun Salmonella c trong cc mu xc xch ca cng ty
ny. Hin ti, cc nhn vin iu tra lin bang M vn ang lm vic tm ra nguyn
nhn chnh xc gy ra dch nhim khun Salmonella nc ny. V th, h vn cha th
kt lun sn phm xc xch ca Cng ty Daniele c phi l ngun ly bnh chnh hay
khng. V th, nhng sn phm xc xch ca cng ty ny b thu hi l do kim tra
bnhim khun Salmonella.
1.3.7.2. Trong nc
Theo ng Nguyn Cng Khn, Cc trng Cc ATVSTP cho bit, hin nay, mng
li kim nghim ATVSTP c hnh thnh rng khp trong c nc nhng thc t
nng lc kim nghim nhiu ch tiu v an ton thc phm ti cc a phng vn rt hn
ch. Trong 330 mu hoa qu c ti 2,7% s mu c t l tn d thuc bo v thc vt cao,
nht l to , qut v l. c bit, trong s 1.416 mu tht v sn phm t tht pht
hin ti 40,9% s mu nhim khun Salmonella gy ra cc bnh v ng tiu ha. Hn
na, khu vc TP H Ch Minh v ng Nai l nhng a phng c t l mu thc phm
nhim Salmonella cao nht, chim t 84- 95% mu c gim st.
1.3.8. Cc ch tiu v Salmonella trong thc phm
Hu ht cc tiu chun an ton v sinh thc phm ca Vit Nam cng nh cc
nc trn th gii u khng cho php s c mt ca Salmonella trong thc phm. V
vy cc phng php phn tch Salmonella l quy trnh nh tnh trong mt lng mu
thc phm nht nh. S hin din ca chng c kim tra gt gao bi cc c quan chc
nng. Theo tiu chun cho php vi sinh vt hin din trong thc phm ca B Y t
4/1998, quy nh khng chp nhn s hin din ca Salmonella trong 25 g hay 25 ml i
vi tt c cc loi thc phm. Theo thng t ban hnh s 01/2000/TT BYT ca B Y
t i vi thc phm sn xut trong nc th ch tiu Salmonella bt buc phi kim tra
i vi tt c cc loi thc phm .y Ban Chu u quy nh khng c pht hin
Salmonella /25 g trong bt k loi thc phm no.
20

Theo phng php truyn thng vic pht hin Salmonella bao gm nhng bc
nh tin tng sinh, tng sinh chn lc, cc th nghim sinh ha v cui cng l th
nghim huyt thanh, tnh chung mt khong 7 ngy c kt qu.Hin nay c nhiu
n lc rt ngn thi gian pht hin Salmonella bng nhng phng php nh th
nghim ELISA, v th nghim DNA. Vic khng nh kt qu m tnh trong vng 48 gi
l chuyn bnh thng. Tuy nhin mt khi mu c kh nng dng tnh, cn tin hnh
cc thao tc truyn thng khng nh s c mt ca Salmonella.
Trong cc yu cu v vi sinh i vi thc phm, Salmonella l mt ch tiu c
bit nhy cm, do tnh cht gy bnh ca mt s kiu huyt thanh ca Salmonella.
Yu cu ca kim tra bt buc i vi Salmonella trong cc loi thc phm theo Thng
t 01/2000/TT-BYT ca B Y t
CHNG : CC PHNG PHP PHT HIN
SALMONELLA
2.1. Pg pp truyn thng :
Pht hin Salmonella bng phng php nui cy: y l phng php nh tnh
v kt qu c bo co l c hay khng pht hin Salmonella trn lng mu c kim
nghim. Do quy nh khng cho php c mt trong thc phm nhng li kh pht hin,
cho nn mu ly ti thiu phi 25g v quy trnh kim tra Salmonella bt buc phi c
thm giai on tin tng sinh t hiu qu cao. iu ny cn thit v Salmonella
21

thng c mt trong mu vi s lng nh, b tn thng nng qua qu trnh bo qun
ch bin v s tn ti mt s lng ln cc vi khun khc thuc h Enterobacteriaceae,
nhng vi khun ny s cnh tranh hay c ch s pht trin ca Salmonella.
pht hin Salmonella cn tin hnh bn giai on: tin tng sinh, tng sinh
chn lc, phn lp v khng nh. Vic khng nh da trn cc kt qu th nghim sinh
ha v khng huyt thanh ph hp.
2.1.1. Giai on tin tng sinh (pre enrichment)
y l khu quan trng khng th thiu trong quy trnh kim nghim i vi mu
kim nghim khng phi l mu bnh phm. Ngi ta thng s dng mi trng tin
tng sinh khng chn lc, giu dinh dng, khng cha cc cht c ch c hiu to iu
kin cho s phc hi sc sng v tng trng ca nhiu vi sinh vt hin din trong mu
b tn thng trong qu trnh ch bin v bo qun thc phm.
V d: nc peptone m Buffered Peptone Water (BPW) l mi trng tin tng
sinh c khuyn khch s dng trong mi trng kim tra nhiu loi vi sinh vt gy
bnh thuc h Enterobacteriaceae; ring FDA ca M th thng s dng Lactose broth
cho mt s nhm thc phm.
Ty theo c tnh thnh phn ha hc ca mu cn chn quy trnh tin tng sinh
ph hp. Thng thng t l gia mu v mi trng tin tng sinh l 1:9, tuy nhin ty
trng hp c th t l ny c th thay i.
2.1.2. Giai on tng sinh chn lc (selective enrichment)
Giai on ny lm tng mt Salmonella so vi cc vi sinh vt khc bng cch
c ch hoc ngn chn s sinh trng ca mt s nhm vi sinh vt khc, to thun li
cho vic pht hin vi khun ny trong bc phn lp.
Cc mi trng tng sinh chn lc cho Salmonella thng c dng l
Rappaport Vassiliadis (RV), Tetrathionate Mueler Kauffman Broth (TT), Selenite
Cystein Broth (SC) Thng thng mi trng TT c dng phn tch cc loi mu
c thnh phn chnh l nhng loi tht ti sng, cc mu c mt nhim vi sinh vt cao,
cc loi thc n gia sc, hay cc loi thc phm khc.
Mt vi nghin cu gn y ca AOAC (Association of Official Analytical
Communities - Hip hi cc nh phn tch ca M) cho rng mc d mi trng Selenite
Cystein Broth c cha selenite c ch mnh cc vi khun khc ngoi tr Salmonella
22

nhng mui sodium biselenite c th gy ng th khi tip xc vi c th ngi nn hn
ch s dng v thy rng mi trng RV c th thay th cho cc loi mi trng trn
phn tch mu. Canh RV c tc dng c ch vi khun khc nh vo MgCl
2
bng cch tc
ng ln thnh t bo vi khun v nh vo mu xanh malachite c ch cc vi khun
Gram dng .
Cn lu l khng mi trng tng sinh chn lc no l mi trng ti u chung
cho tt c cc dng Salmonella. Mi loi mi trng tng sinh chn lc c thit lp
da trn mt s c im pht trin khc nhau ca Salmonella, mt s dng Salmonella
tng trng c trong mi trng ny nhng li khng tng trng c trong mi
trng khc. V d: S. typhi khng pht trin c trong mi trng RV v TT; S. dublin
khng pht trin c trong mi trng RV. Ngoi ra, mi mi trng chn lc c
mt nhit nui cy khc nhau nh mi trng RV c 42
0
C, mi trng TT v SC
c 37
0
C trong 22 24 gi.
Ngy nay, cc yu cu v v sinh thc phm u khuych khch cc phng th
nghim s dng t nht hai loi mi trng tng sinh chn lc Salmonella tng cng
kh nng pht hin tt c cc serotype Salmonella trong mu.
2.1.3. Giai on phn lp (isolation)
y l bc tch bit Salmonella trong canh khun ra khi qun th ca chng.
Mt s mi trng phn lp nh Xylose Lysine Desoxycholate Agar (XLD), Bismuth
Suffite Agar (BSA), Brilliant Green Phenol Red Lactose Sucrose Agar (BGLS), Hektoen
Entric Agar (HE)Cng chn lc v mc phn bit th hin hnh thi khun
lc ca Salmonella trn tng mi trng cng khc nhau.
Mt s c im chnh ca cc mi trng trn l vi s hin din ca nm men,
ng, peptone to thun li cho s tng trng Salmonella v Shigella yu t. S sn
sinh ra H
2
S cng c th xy ra nh s hin din ca st thiosufate v st citrate th
hin bi nhng khun lc c tm en do s to nn sulfua st. Trn cc mi trng phn
lp khc nhau Salmonella s cho cc kiu khun lc c trng khc nhau. Trn mi
trng XLD: khun lc mu hng trn, trong sut, c hay khng c tm en. Mt s
dng Salmonella c kh nng sinh H
2
S, tr S. typhi th khun lc trong sut v khng c
tm en.
Trn mi trng BSA: khun lc Salmonella c mu nu xm hay en, c hay
khng c nh kim tm trn b mt khun lc, mi trng xung quanh c mu nu chuyn
sang en khi tng thi gian m, gy kh khn cho vic nhn bit khun lc vi khun
23

Salmonella. Mt s chng c th sinh khun lc mu lc vi mi trng xung quanh t
nhiu c mu en.
Trn mi trng BPLS: khun lc Salmonella in hnh trong sut v hi trong
mi trng, c mu hng xung quanh khun lc. Mt s Salmonella xut hin khun
lc mu lc trong sut nu bao quanh l vi khun ln men lactose hoc glucose gy ra
vng mu vng xanh hoc xanh; di 1% Salmonella khng in hnh, chng ln men
ng lactose v xut hin khun lc vng lc hoc lc. [9, 37] Trn mi trng HE:
khun lc Salmonella c mu xanh dng n mu xanh lc, c hay khng c tm en.
Mt s loi Salmonella c th pht trin thnh khun lc c cc chm mu en bng, ln
hay phn ln cc khun lc hon ton c mu en. Mt cch in hnh, c m s t loi
Salmonella sinh ra khun lc mu vng c hoc khng c tm en. [9, 20] Cng nh
bc tng sinh chn lc, vic s dng hai hay nhiu mi trng phn lp s cho php gia
tng s pht hin s lng cc kiu huyt thanh khc nhau trong mu, trong mi
trng XLD c khuyn khch s dng. Hn na, qua cc bc tin tng sinh, tng sinh
chn lc v phn lp th t bo Salmonella b gi v suy thoi, v vy cn phi phc hi
chng bng mt trong cc mi trng nh Tryticase Soya Agar (TSA) hay Brain Heart
Infusion (BHI) nhn sinh khi v c dng tip cho cc phn ng sinh ha v phn
ng ngng kt khng huyt thanh v sau
2.1.4. Khng nh bng cc phn ng sinh ha
y l bc xc nhn cc dng vi khun nghi ng Salmonella trn mi trng
phn lp bng cc th nghim sinh ha v huyt thanh c trng. Cc biu hin sinh ha
ca Salmonella nh sau: glucose (+), lactose (-), indol (+), lysine decarboxylase (+),
ornithine decarboxylase (+), urea (-), mannitol (+), sorbitol (+). Nu cc biu hin sinh
ha ph hp, chng phn lp c khng nh li bng cc th nghim ngng kt vi
khng huyt thanh O a gi v H a gi.
Vi khun sau khi phc hi c cy chuyn sang cc mi trng th nghim sinh
ha nhm xc nh cc khun lc nghi ng l Salmonella.
2.2. Pg pp hi i :
Phng php truyn thng mt kh nhiu thi gian (5 ngy) cho ra kt qu, do
ngi ta pht trin rt nhiu phng php th nhanh pht hin Salmonella trong
cc mu thc phm v mu nc trong thi gian ngn, t c bin php x l kp thi
i vi cc mu nhim. Xu hng s dng PCR v IMS thng c s dng. Phng
php PCR gip pht hin nhanh; tuy nhin d xy ra hin tng m tnh gi, i hi cc
24

thao tc phi thnh tho. Multiplex PCR gip tit kim thi gian, s lng mu v chi ph;
tuy nhin li c nhy km hn. Ngi ta pht trin thm phng php Real Time
PCR t PCR truyn thng. Phng php ny c nhy rt cao, cho kt qu sm hn,
khng cn phi chy in di sau phn ng khuch i. Phng php min dch t phn
tch l mt la chn tt hn. N c nhy cao (< 104 CFU/ml), thi gian ngn (th
nghim c thc hin trong 4-5 gi).
Bn cnh nhng k thut Sinh hc phn t c bn pht hin v nh danh
Salmonella, cn c nhng phng php khc, nh: phng php min dch (huyt thanh
hc, k thut ELISA, Western blot). V i khi tng hiu qu, tin cy cho xt
nghim ngi ta cn kt hp nhiu phng php vi nhau, v d: IMS-PCR assay
(Immunomagnetic Separation and Polymerase Chain Reaction) pht hin Salmonella
trong thc phm.
2.2.1. Phng php PCR
- Nguyn tc :
Phng php nh tnh ny da vo vic xc nh on ADN ch c c
khuch i hay khng. Qu trnh xc nh c thc hin bng cch in di sn phm
khuch i trn gel agarose, nhum mu ADN bng etyl bromua v quan st bng n
UV c bc sng l 302 mm. Nu trong mu c gen ch, trn bng gel in di s xut
hin sn phm khuch i c kch thc ph hp vi di ca on ADN ch nh.
Nu trong mu khng c gen ch, trn gel din di khng xut hin sn phm khuch i
hay sn phm khuch i c kch thc khng ph hp vi on ADN ch.
- Thit b, dng c, ho cht v mi trng
Thit b, dng c :
+ T m 37
0
C.
+ My lun nhit.
+ My ly tm dng cho ng eppendorf 1,5 ml.
+ My lc ng nghim.
+ Thit b in di ngang v b ngun in di c in th hot ng t 80 150 volt.
+ Hp n soi UV c knh lc 302mm.
+ B chp nh trn n UV.
+ ng Eppendorf 0,2. 0,5 ml; 1,5 ml chuyn dng cho PCR.
Ho cht, mi trng:
Mi gm hai mi invA1 v invA2 c thit lp cch nhau 520 bp trong gen invA
c vai tr trong qu trnh xm nhim Salmonella vo thnh rut ng vt v ngi. Trnh
t ca hai mi nh sau :
25

invA1 :5' -TTGTTACGGCTATTTTGACCA -3'
invA2 : 5' -CTGACTGCTACCTTGGCTGATG -3'
Nng mi s dng trong phn tch c pha long thnh 6 pM trong
m TE. m TE c thnh phn nh sau : 10 mM Tris-HCl (pH = 8), 1 mM EDTA.
Mi trng nui cy vi sinh vt v cc loi ho cht khc : Khuyn khch s dng
mi trng nui cy dng bt kh v cc vt liu dng cho phn ng khuch i PCR
c tng hp thnh kit ang c lu hnh trn th trng c thnh phn ph hp nh.
Qu trnh pha ch, bo qun, s dng phi tun th theo hng dn ca nh sn xut.
Trong trng hp s dng cc vt liu ho cht ring l, tinh khit ca cc thnh phn
v nc pha ch phi m bo cht lng dng cho vi sinh v sinh hcphn t.
Thang DNA :Nn s dng thang o ph hp c th c lng c on khuch
i 520 bp. C th s dng sn phm khuch i c kch thc 520 bp bit trc lm
thang o trong phng php ny.
Agarose : S dng trong kthut ny l loi dng in di ADN c kch thc
nhhn 1000 bp.

- Phng php tin hnh
Ly mu : Cn chnh xc 25 g mu (hoc mt khi lng chnh xc tutheo yu
cu) ri cho vo bnh tam gic hoc bao PE v trng.
Tng sinh : Nhm lm tng s lngSalmonella trong mu, cc t bo b suy yu
hay tn thng cng c phc hi v pht trin. Giai on ny c tin hnh trong mi
trng khng chn lc v trn nguyn tc c mt phn khi lng mu sb sung9 phn
khi lng mi trng tng sinh. Nu ly 25 g mu, phi b sung 225 g mi trng tng
sinh dung dch pepton m. mu c mi trng tng sinh nhit 37,0 o C 1,0 o C
trong khong 18 gi 2 gi.
X l mu gii phng DNA : Giai on ny nhm thu nhn sinh khi sau khi tng
sinh, ra sch mi trng sau khi nui cy, ph v t bo gii phng DNA. Cch x l
nh sau:
Lc u canh khun tng sinh. Ht 0,5 ml vo trongng eppendorf c th tch 1,5
ml, ly tm vi tc 10 000 vng/pht trong 5 pht ri loi b phn mi trng lng bn
trn. Ra sinh khi bn di vi nc ct v trng ri tip tc ly tm vi ch nh trn
loi b phn nc.
Huyn ph sinh khi trong ng vi 0,5 ml nc ct v trng. un si cch
thutrong 10 pht. Ly tm huyn dch sau khi un vi tc 10 000 vng/pht trong 5
pht lng cc mnh v t bo xung y. Phn dch trong bn trn c coi l khun
DNA tin hnh phn ng khuch i.
Khuch i
26

Giai on ny nhm lm tng s lng bn sao on DNA cha trn my lun
nhit (thermocycler) bng hai mi c trng. Qu trnh khuch i c tin hnh
trong khong 30 chu k.
Chun b ng khuch i :
+ Ht 45 ml hn hp khuch i PCR 1,1 x cho vo trong ng nghim PCR c th
tch 0,2 hoc 0,5 ml, thm vo 1 ml mi mi invA1 v invA2 c nng 6 pM v 3 ml
mu khun ADN. Tng th tch trong mt ng khuch i l 50 ml.
+ i chng dng: thay dch khun DNA mu bng dch ADN ca Salmonella
chun bit.
+ i chng m: thay dch khun DNA mu bng nc ct v trng.
Chng trnh khuch i Cc ng khuch i c t vo trong my lun nhit.
+ Chng trnh khuch i nh sau: Duy tr nhit 95
0
C trong 5 pht lm bin
tnh hon ton cc si DNA trong mu. Tip theo l 35 chu k, mi chu k c 3 bc nh
sau: 95
0
C/60 giy; 54
0
C/45 giy v 72
0
C/60 giy. Sau khi kt thc 35 chu k, mu c
gi nhit 72
0
C trong 10 pht, sau gi n nh nhit 20
0
C cho n khi in di.

in di sn phm khuch i
27

+ Chun b gel in di agarose 1%
+ Gel agarose 1 % pha trong m TAE 1x c un chy hon ton v vo khay
in di c sn cc lc to ging. Gel in di phi c dy khong 3 - 4 mm. Gel
sau khi chun b c ngm chm hon ton trong m TAE.
+ Chun b dch in di
+ Mu sau khi khuch i c nhum vi 10 ml m ti mu 6x ri trn tht u.
+ in di:Mt ging trong gel in di c s dng cho thang DNA chun hay mu
i chng dng. Np 10 ml dch in di chun b vo trong gel agarose. Tin hnh
in di trong 60 pht hiu in th100 vn.
Nhum DNA, quan st sn phm khuch i
+ Chun b dung dch nhum mu :Pha dung dch nhum DNA nh sau: cho 0,2 ml
etyl bromua 10 mg/ml vo 0,5 lt nc, pha vo trong khay cha c ming rng hn bn
gel in di.
+ Nhum gel : Ngm bn gel in di vo dung dch nhum trong 10 pht. Ra
gel bng nc trong khong 3 - 5 pht loi b phn etyl bromua d.
+ Quan st, chp hnh :Cho bn gel nhum ln hp n soi UV, ng knh bo v,
bt n ri quan st cc vch sng ca DNA xut hin trn bn gel. Sau , chp hnh
lu tr kt qu.

- c v gii thch kt qu
Kt qu phn tch ch c xem xt kt lun khi mu i chng dng tnh c sn
phm khuch i DNA vi kch tc 520 bp v mu i chng m khng c sn phm
ny.
Mu c kt lun l dng tnh Salmonella khi c sn phm khuch i 520 bp
trn bn gel.
Mu c kt lun l m tnh khi khng c sn phm khuch i, haysn phm
khuch i c kch thc khc hn 520 bp.
2.2.2. Phng php ELISA
ELISA c s dng rng ri di dng cc kiu thng mi v c th ci tin
t ng ha. ELISA c th s dng pht hin v nh lng vi sinh thc phm trong
thi gian vi gi sau khi tng sinh. K thut ny c nhy khong 10
6
CFU/ml. tuy
nhin vn phi thc hin bc tng sinh v tng sinh chn lc trc khi thc hin cc
phn ng min dch
28

Transia Salmonella (Diffchamb AB, Sweden) l 1 b kit pht hin nhanh
Salmonella spp. da trn nguyn tc E sanwich. Khi hn hp tng sinh chn lc c cho
vo ging, nu c s hin din ca vi sinh vt mc tiu, khng nguyn tim mao ca vi
sinh vt s to phc hp vi khng th c nh trn ging v khng th t do c gn
enzyme peroxydase to thnh 1 phc hp kp (sanwhich). Sau cc khng th c gn
enzyme dng t do khng to phc hp sanwhich s b ra khi phn ng. Phc hp
sanwihch s c pht hin nh s b sung c cht ca phn ng (ure peroxide v
teramethylbenzidine).Enzyme peroxide s thy phn c cht to phn ng mu xanh
dng. Phn ng kt thc bng cch bt hot enzyme bng dung dch kt thc lm acid
ha mi trng v lm mi trng chuyn t mu xanh sang mu vng.S hnh thnh
mu vng chng minh s hin din ca khng nguyn hay vi sinh vt mc tiu v mt
mu ca vi sinh vt c th xc nh c bng cch o cng mu bng my so mu
2.2.3. Cc bin php kim sot Salmonella trong thc phm
i vi gia sc v gia cm: Trong chn nui cn ch phng bnh tt cho
chng. Phi kim tra th y khi git sc vt, iu ny cng lm tt th cng t c c hi bn
hoc xut ra cc loi tht nhim Salmonella. Trong khi git tht phi m bo tnh
ring r, trnh s ly lan ca vi khun, ch ti cc loi dng c dng khi git tht .
Trong bo qun thc phm, m bo thi gian ct gi thc phm ch bin v
cc nguyn liu. Ch tht bm, pat... tht nghin m khng p lnh ngay sau s to
iu kin cho ton b khi nguyn liu nhim trng mau chng.
un si thc phm trc khi n l bin php tt nht. Tht p lnh thi gian
un nu phi ko di hn bnh thng, khi un phi m bo nhit si c bn trong
ming tht (t nht l 5 pht). Thc phm cn tha, thc phm d tr phi un li trc
khi n. Vi trng c th b nhim Salmonella rt sm ngay khi mi (trng vt, ngan,
ngng...), v vy phi ch bin chn hon ton, tuyt i khng n sng hoc hng
o.Bo m v sinh ni n, trnh rui, nhng, chut.
Thc hin nghim ngt ch khm tuyn trc khi vo v khm nh k (1
nm/1 ln) i vi ngi tip xc trc tip vi thc phm nht l thc phm nu chn.

29

CHNG : QUY TRNH PHN TCH C TH

3.1. Nguyn tc
Salmonella c th c pht hin ( phn tch nh tnh ) bng mt quy trnh bao
gm 4 bc l tng sinh, tng sinh chn lc, phn lp v khng nh. Salmonella thng
c mt trong mu vi mt s lng nh, b tn thng v cng hin din chung vi mt
s lng ln vi sinh vt thuc h Enterobacteriaceae c tnh cnh tranh mnh v c ch
s tng trng ca Salmonella.
- Bc tg s : ty theo c tnh thnh phn ha hc ca mu cn chn quy
trnh tng sinh ph hp. Thng thng t l gia mu v mi trng tng sinh l 1 : 9,
tuy nhin ty trng hp m t l ny c th thay i
- Bc tg s cn 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 Kauffmanm Broth (TT ) Cc nghin cu gn y
cho thy mi trng RV c th thay th cho cc mi trng khc phn tch nhiu mu
khc nhau. Tuy vy, mi trng TT thng c dng phn tch cc mu tht ti
sng, cc mu c mt nhim cao, cc mu thc n gia sc Hin nay ngi ta khuyn
khch l nn dng t nht hai loi mi trng tng sinh chn lc pht hin tt c cc
serotype Salmonella nu c hin din trong mu.
- Bc phn lp : nhm tch v nhn dng Salmonella khi cc qun th vi sinh vt
khc trong mu. Nhiu loi mi trng rn khc nhau c s dng phn lp
Salmonella, mi mi trng gip nhn dng cc ging ca nhm ny da trn mt vi
c tnh. Hin nay, cc tiu chun v sinh an ton thc phm khuyn khch s dng t
nht 2 loi mi trng phn lp khc nhau tng cng kh nng pht hin tt c cc
dng Salmonella, c bit l mi trng XLD c khuyn khch s dng
- Bc khg nh : nhm xc nh li cc khun lc c trng ca Salmonella
xut hin trn mi trng phn lp. Bc ny da trn cc th nghim sinh ha v cc
th nghim huyt thanh c trng cho Salmonella. Cc th nghim sinh ha c khuyn
30

khch s dng l KIA/TSI, indol, LDC (Lysine decarboxylase), ODC ( Ornithine
decarboxylase ), urea, manitol, sorbitol, cc th nghim huyt thanh O v H a gi.
- p dng tiu chun: TCVN 4829:05
3.1.1. Dng c v thit b
Dng c
Bnh tam gic, cc, ng ong hnh tr 50, 100, 500 ml, ng nghim, hp a petri,
que cy vng, que cy thng, n cn, bao PE v trng, ko ct, pince kim loi, khay kim
loi, lame, v bm ht chn khng.
Thit b
Cn phn tch, my o pH, my dp mu,pipetman, knh hin vi, ni hp autoclave,
t sy v trng, t cy an ton sinh hc, t m 37
0
C, v t lnh
3.1.2. Mi trng v ha cht

Ha cht
Cc loi khng huyt thanh O a gi, hn hp OMA, OMB v khng huyt thanh
n gi nh nhm A, B, C, D, E. Cc loi khng huyt thanh ny do Vin Pasteur Thnh
ph H Ch Minh sn xut. Cc loi carbonhydrate nh sorbitol, dulcitol. Cn 70
o
, cn
96
o
v nc ct.
Mi trng
Bao gm nhng loi mi trng thng dng nui cy, phn lp v khng nh
Salmonella trong thc phm:
Mi trng Buffered Pepton Water (BPW): c s dng tin tng sinh
Salmonella hin din trong mu. Thnh phn mi trng gm: peptone 10 g/l, NaCl 5 g/l,
Na2HPO4.12H2O 9 g/l, KH2PO4 1,5 g/l, hp kh trng 121oC trong 15 pht, pH sau
khi kh trng l 7.
Mi trng Rapparport Vassiliadis broth (RV): c s dng tng sinh chn
lc cho Salmonella. Thnh phn mi trng nh sau: peptone u nnh 4,5 g/l,
MgCl2.6H2O 29 g/l, NaCl 8 g/l, K2HPO4 0,6 g/l, malachite green oxalate 0,036 g/l, pH
5,5 0,2. Phn phi 10ml mi trng vo ng nghim. Hp kh trng 115oC trong 15
pht.
31

Mi trng Xylose Lysine Desoxycholate agar (XLD): c dng phn lp
Salmonella. Thnh phn XLD bao gm: cao nm men 3 g/l, L Lysine HCl 5 g/l, xylose
3,75 g/l, lactose 7,5 g/l, sucrose 7,5 g/l, sodium desoxycholate 1 g/l, NaCl 5 g/l,
Na2S2O5 6,8 g/l, ammonium ferrous (II) citrate 0,8 g/l, phenol red 0,8 g/l, agar 18 g/l,
pH cui l 7,4 0,2. un si mi trng, khng hp. ngui n 50oC. Mi trng
trn a petri v kh trc khi cy.
Mi trng Tryptic Soya Agar (TSA): c s dng bo qun v phc hi
ging vi sinh vt trong qu trnh th nghim. Thnh phn TSA gm: tryptone 15 g/l,
peptone u nnh 5 g/l, agar 15 g/l. un nng ha tan agar trong cc bercher, phn
phi vo cc ng nghim khong 5 7 ml. Hp kh trng 121oC trong 15 pht, pH sau
khi kh trng l 7,3. Sau ngui khong 50 60oC ri thch nghing.
Mi trng Triple Sugar Iron Agar (TSI) gm mi trng 1 c thnh phn nh sau:
peptone 20 g/l, NaCl 5 g/l, lactose 10 g/l, sucrose 10 g/l, glucose 1 g/l,
Fe(NH4)2(SO4)2.6H2O 0,2 g/l, Na2S2O3 0,2 g/l, phenol red 0,025 g/l, agar 13 g/l v
mi trng 2 gm cc thnh phn nh sau: cao tht 3g/l, cao nm men 3g/l, peptone 15g/l,
proteose peptone 5 g/l, glucose 1 g/l, lactose 10 g/l, sucrose 10 g/l, FeSO4 0,2 g/l, NaCl 5
g/l, Na2S2O3 0,3 g/l, phenol red 0,024 g/l, agar 12 g/l. Phn phi vo cc ng nghim,
mi ng 7 ml. Hp kh trng 121oC trong 15 pht, sau i ngui khong 50 60oC
nghing phn thch su y ng cao 2,5 cm.
Mi trng Mannitol phenol red broth: c s dng th nghim kh nng ln
men ng mannitol ca Salmonella. Thnh phn mi trng bao gm: peptone tht 5 g/l,
peptone u nnh 5 g/l, mannitol 10 g/l, NaCl 5 g/l, phenol red 0,018 g/l, pH sau khi kh
trng l 7,4 0,2.
Mi trng Lysine Decarboxylate broth (LDC): c s dng th nghim kh
nng sinh enzyme lysine decarboxylase ca Salmonella. Thnh phn mi trng nh sau:
peptone 5 g/l, cao nm men 3 g/l, dextrose 1 g/l, L-Lysine HCl 10 g/l, ferric (II)
ammonium citrate 0,5 g/l, Na2S2O5 0,04 g/l, bromocresol purple 0,016g/l, pH 6,7.
Mi trng Urea broth: c dng th nghim kh nng thy phn urea ca
Salmonella. Thnh phn mi trng nh sau: cao nm men 0,1 g/l, KH2PO4 9,1 g/l,
Na2HPO4 9,5 g/l, urea 20 g/l, phenol red 0,08 g/l, pH 6,8.
Mi trng Sorbitol phenol red broth: c s dng th nghim kh nng ln
men ng sorbitol ca Salmonella. Thnh phn mi trng gm c: peptone tht 5 g/l,
peptone u nnh 5 g/l, sorbitol 10 g/l, NaCl 5 g/l, phenol red 0,018 g/l, pH sau khi thanh
trng l 7,4 0,2.
Mi trng Dulcitol phenol red broth: c s dng th nghim kh nng ln
men ng dulcitol ca Salmonella. Thnh phn mi trng gm c: peptone tht 5 g/l,
32

peptone u nnh 5 g/l, dulcitol 10 g/l, NaCl 5 g/l, phenol red 0,018 g/l, pH sau khi thanh
trng l 7,4 0,2.
Mi trng Malonate broth: c s dng th nghim kh nng s dng
malonate ca Salmonella. Thnh phn mi trng bao gm: ammonium sulphate 2 g/l,
dipotassium phosphate 0,6 g/l, monopotassium phosphate 0,4g/l, sodium chloride 2 g/l,
sodium malonate 3 g/l, bromthymol blue 0,025 g/l. Phn phi 3 ml vo cc ng nghim.
Hp 115oC trong 10 pht. pH cui 6,7 0,2.
Mi trng Lactose agar 1%: c s dng cm ng to ra enzyme -
galactosidase dng trong th nghim ONPG. Thnh phn mi trng nh sau: lactose 10
g/l, peptone 5 g/l, cao tht 3 g/l, NaCl 10 g/l, agar 15 g/l. a giy ONPG: c s dng
th nghim ONPG v kh nng tng hp enzyme - galactosidase. Mi a c phn
phi vo mi ng nghim cha 0,5ml nc ct v trng. a giy ONPG. c sn
xut bi hng Biorad dng thng phm.
3.1.3. So snh mi trng SPW v BPW
Tiu ch so snh BPW SPW
Thnh phn mi trng
NaCl 5g
Pepton 10g
Nc ct 1 lt
Na
2
HPO
4
3,5g
KH
2
PO
4
1,5g
NaCl 1g
Pepton 8,5g
Nc ct 1 lt
Vai tr
L mi trng tim tng
sinh khng chn lc
phn lp samollena t thc
phm
L mi trng dng
pha long mu.
C ch
Cung cp dinh dng cho
qu trnh hi phc cc t
bo b tn thng trong qu
trnh bo qun thc phm.

pH sau kh trng pH = 7.2 0.2 pH = 7 0.2
Nhit v thi gian kh
trng
Hp p lc 15 pht
nhit 121
o
C
Hp p lc 20 pht
nhit 121
o
C
Phm vi s dng
S dng trong mi trng
kim tra nhiu loi vi sinh
vt gy bnh thuc h
S dng trong cc mi
trng kim tra vi sinh vt
hiu kh, coliforms,
33

3.2. Pg pp thc hin

Enterobacteriaceae E.coli, Clostridium
perfringens, Staphylococcus
aureus.
iu kin bo qun sau khi
hp kh trng
Mi trng c bo
qun mt 45
o
C trong b
iu nhit
Mi trng c bo
qun mt 45
o
C trong b
iu nhit
34

3.2.1. Tng sinh
i vi cc loi mu thng thng, tin hnh cn 25g mu vo ti PE v trng, b
sung 225ml dung dch BPW v ng nht mu trong 15 hoc 30 giy. 37
0
C trong 18
24 gi. i vi mt s loi mu c cha cc cht gy c hoc c ch s pht trin ca
Salmonella cn tin hnh quy trnhc bit :
- i vi tht gia cm ti sng : t gia cm vo mt ti PE ln, cho thm 1 lt
BPW, lc bng my lc 30 giy mi trng thm vo ton b mu
- i vi mu sa kh : Cn 25g mu vo ti PE v trng, cho thm 225ml BPW,
yn nhit phng khong 60 pht. Sau lc cho tan hon ton
- i vi cc mu gia v hay cc loi thc phm cha nhiu gia v : ng nht mu
vi t l 1/100 trong BPW ( thay v 1/10 nh bnh thng ) trc khi tng sinh. Bin
php ny nhm gim nng cc cht c ch s tng trng ca Salmonella trong bc
tng sinh
- i vi cc loi mu nh casein, phomat, b v cc sn phm tng t khc : thc
hin dng nht mu trong mi trng tng sinh c lm m n 40 0 C
- i vi cc sn phm cha coca: ng nht mu trong mi
- trng Skim Milk Broth c lm m n 40 0 C
- i vi da, cc sn phm t da v cc mu c hm lng cht bo cao: ng
nht mu trong mi trng BPW, sau cho thm 2 3 git Triton X 100 trc khi
tng sinh

3.2.2. Tng sinh chn lc
Lc trn u dch tng sinh, sau chuyn 0,1ml sang 10ml mi trng tng
sinh chn lc RV c m n 42
0
C. 42
0
C trong 18 24 gi, khi cn thit c
th ko di thm 24 gi
Mt s mi trng tng sinh chn lc khc cng c s dng, mi loi mi
trng ch c tc dng chn lc da trn mt c im pht trin ca Salmonella, mt s
dng ch c th tng trng trong mi trng ny m khng th tng trng trong mi
trng khc. Do vy, tng kh nng pht hin tt c cc dng Salmonella hin din
35

trong thc phm cn phi dng t nht hai loi tng sinh chn lc khc nhau cho cng
mt mu. Ngoi ra mi mi trng cn c cc nhit khc nhau
3.2.3. Phn lp v nhn din
Dng que cy vng thc hin k thut cy phn lp khun lc n vi ging t
dch tng sinh chn lc trn a mi trng phn lp c trng cho Salmonella nh XLD,
HE, BS Cng chn lc v mc phn bit th hin hnh thi ca khun lc
Salmonella trn tng mi trng cng khc nhau. chn lc v nhn dng tt c cc
dng Salmonella cn s dng t nht hai loi mi trng chn lc phn bit khc nhau
cho cng mt mu. Cc biu hin ca Salmonella trn tng mi trng khc nhau nh
sau
- Mi trng XLD: khun lc trn, li, trong sut, c hay khng c tm en, i khi
tm en qu ln bao trm khun lc, mi trng xung quanh chuyn sang mu hng.

- Mi trng HE: khun lc c mu thay i t xanh dng n xanh lc, c hay
khng c tm en, i khi tm n qu ln bao trm khun lc.
36

- Mi trng BS: khun lc c mu nu xm hay mu en, thnh thong c nh kim
tm, mi trng chuyn thnh mu nu v sau chuyn sang mu en nu ko di thi
gian .

3.2.4. Khng nh
- T mi mi trng phn lp cy chuyn t nht 5 khun lc c trng qua mi
trng khng chn lc nh TSA. 37
0
C trong 18 24 gi. Cc khun lc c trng
trn mi trng ny c s dng cho cc test sinh ha v huyt thanh
37

- Cc th nghim sinh ha chnh cn thc hin cho Salmonella l: ln men glucose,
urea, manitol, indol, H
2
S, VP, ODC, LDC, saccharose, sorbitol.
- Cc chng Salmonella cho kt qu th nghim nh sau :
Th nghim H
2
S (KIA hay TSI): Th nghim kh nng ln men lactose, glucose
v sinh kh H
2
S.
V ngun carbon, mi trng KIA cha 2 loi ng l 1% lactose v 0.1%
glucose. Khi cy chng vi sinh vt ln mi trng ny c 3 trng hp xy ra i vi s
tng trng ca VSV: ch s dng glucose, s dng glucose v lactose, khng s dng c
hai ng ny. Salmonella ch ln men c ng glucose v th phn nghing ca mi
trng c mu , phn su c mu vng. Thi gian t 18-24 gi.
V sinh hi: a s cc dng Salmonella u c kh nng sinh H
2
S nn c xut hin
cc vt mu en trong mi trng ny. C th thy hin tng sinh hi qua hin tng
lm v thch mi trng hoc mi trng b y ln trn to mt khong khng bn di
y ng nghim.

Th nghim KIA.

ng 1: mi trng KIA khng c VSV.
ng 2: xut hin mu vng chng t VSV ln men lactose tt (ln men glucose cngtt).
ng 3: c sinh kh, chng t ln men c sinh kh.
ng 4: mu trn phn thch nghing, mu vng di y chng t VSV ch ln men
glucose.
ng 5: mu en xut hin chng t c qu trnh kh lu hunh v sinh H
2
S.
38

Th nghim kh nng tng hp enzyme lysine decarboxylase (th nghim LDC):
dng que cy vng chuyn sinh khi t mi trng TSA sang mi trng LDC broth.
37oC trong 24 gi, Salmonella cho phn ng kim c biu hin LDC dng tnh khi
mi trng c sinh khi v chuyn sang mu tm hoc mu xanh.

Th nghim urea : Dng que cy vng ly sinh khi t mi trng TSA cy
chuyn sang mi trng Urea phenol red. 37
0
C trong 24 gi, Salmonella khng thy
gii urea nn khng lm chuyn mu mi trng. Nhng vi sinh vt c phn ng urea
dng tnh lm tng pH mi trng nn sau khi nui cy mi trng chuyn sang mu
hng.

Kt qu th nghim urea
39

Th nghim VP (-):mi trng c s dng l mi trng lng Clark-Lubs (mi
trng MR-VP), pH 6.9. Dng que cy vng cy vo cc ng mi trng MR-VP mt t
sinh khi t khun lc ca chng thun 18-24 gi trn mi trng KIA. Sau thi
gian , b sung thuc th trc tip vo ng mi trng. Thuc th Barritt gm dung dch
A l 5% -naphthol trong cn tuyt i, dung dch B l 40% NaOH hay KOH. Trc tin
nh 6 git dung dch A, sau nh 2 git dung dch B. c kt qu sau 20 pht hoc
chm nht 4 gi. Salmonella cho phn ng m tnh vi th nghim VP c hin tng
khng i mu trn b mt mi trng. Th nghim VP (+) khi c mu trn b mt
mi trng.

Kt qu th nghim VP(-)

Bng biu hin c trng caSalmonella trong test sinh ha
Thnghim Mi trng Biu hin c trng

H
2
S TSI/KIA Phn nghing , su vng,
nt thch v xut hin vch
en
LDC LDC Khng i mu

Urea Urea Khng i mu

Manitol,sorbitol Manitol, sorbitol B acid ha,mi trng
40

chuyn sang mu vng

Indol Trypton Khng xut hin vng
khi nh thuc th Kovacs

VP VP Khng i mu khi nh
napthol 5% v KOH 40%

3.2.5. Khng nh Salmonella bng khng huyt thanh
Cc th nghim khng nguyn cn c th nghim song song vi mu i chng
bng nc mui sinh l trnh hin tng ngng kt gi. Hai loi huyt thanh a gi
cn dng l Salmonella Polyvalent O v Salmonella Polyvalent H. Phn ng (+) khi
chng th nghim ngng kt khng huyt thanh nhng khng ngng kt vi nc mui
sinh l
Thng thng tin hnh th phn ng ngng kt vi khng huyt thanh O a gi
v n gi xc nh chng phn lp l Salmonella. Chia lame knh sch lm hai phn:
mt phn nh mt gii nc mui sinh l lm i chng m v nh mt git huyt
thanh. Ly mt t sinh khi vi sinh vt t mi trng TSA khuych tn vo git huyt
thanh, lm tng t vi git nc mui sinh l, tn u. Ch 30 60 giy quan st trn
nn en, phn ng ngng kt khng huyt thanh xy ra khi khng c hin tng ngng
kt git nc mui sinh l hn hp vn c u nh sa, cn git huyt thanh th c
hin tng ngng kt, xut hin cc kt ta dng ht hoc dng si, dch huyt thanh th
li trong sut . Sau khi qu trnh th nghim phn ng sinh ha ph hp v c s ngng
kt huyt thanh O a gi v n gi th kt lun Salmonella dng tnh trong 25 g mu.
Ch lm phn ng vi khng huyt thanh sau khi xc nh sinh ha. Thc hin phn
ng ngng kt vi khng huyt thanh O a gi ca vi khun Salmonella trc, sau
mi lm khng huyt thanh n gi sau

41

TI LIU THAM KHO
1. Phng php phn tch vi sinh vt trong nc, thc phm v m phm- Trn Linh
Phc.
2. Cng ngh vi sinh vt ( tp1-2) Nguyn c Lng.
3. Cng ngh vi sinh vt-Lng c Phm
4. Ng c v an ton thc phm-L Ngc T.
5. Vi sinh vt hc-Nguyn Ln Dng
6. http://doc.edu.vn/tai-lieu/khoa-luan-tim-hieu-ve-vi-khuan-salmonella-11369/
7. http://www.about-salmonella.com/
8. http://www.textbookofbacteriology.net/salmonella.html
9. http://en.wikipedia.org/wiki/Salmonella
10. http://www.cdc.gov/salmonella/general/

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TRNG I HC CNG NGHIP THC PHM THNH PH H CH MINH

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