Professional Documents
Culture Documents
Phn loi
Gii: Bacteria
Ngnh : Firmicutes
Lp: Bacilli
B: Bacillales
H: Listeriaceae
Chi: Listeria
Loi : Listeria monocytogenes
2.1.2.3
c im
- Cc loi Listeria l trc khun Gram dng, c kch thc ngn (0,4
0,5 0,5 2,0 m), chng mc trn cc mi trng nui cy khng acid, khng sinh
GVHD: KS. Hunh Vn Thnh
SVTH: Nguyn Hu Lim
Hnh 2.2:
Vi
khun Listeria
monocytomonogenes
2.1.2.4
Yu t c lc
- Listeriolysin O (LLO) l yu t c lc chnh ca Listeria
monocytigenes cho php n thot khi s thc bo. LLO c khi lng phn t 58,6
kDa, hnh thnh bi mt chui polypeptide n gm 529 amino acids. Trnh t u N
i din cho c im ca trnh t du hiu ca vi khun Gram dng in hnh: phn
u tin c tnh i nc v mang in tch dng c theo sau bi 20 u k nc.
Trnh t ny m ha trc tip cho protein v c phn ct sau v tr lysine 25. Kt qu
l LLO c bt u t amino acid 26, l mt phn t protein di 504 aa v c trng
lng phn t 55,8 kDa.
- LLO thuc v h c t gn cholesterol c hot bi nhm thiol c
to bi hu ht cc vi khun Gram dng. Hot tnh ly gii ca cc c t ny bi
gim bt mt s tc nhn v b km hm bi s oxy ha v biu hin khi c cholesterol
hoc khng th khng streptolysin O. Cytolysin c hot ha bi thiol c mt cystein
n vng u C lm cho chng d b bt hot bi s oxy ha mc d cystein ny c
th c thay th bng alanine m khng lm mt chc nng ly gii. Chng to nn
nhng l khng lin tc vi ng knh bn trong ln n 30 nm trong nhng mng
cha cholesterol. C ch hot ng ca chng da vo s tng tc vi cholesterol c
GVHD: KS. Hunh Vn Thnh
SVTH: Nguyn Hu Lim
10
11
12
13
Hnh 2.3 : Vi
khn
2.2.3.2
Salmonella
Tnh cht
ha
sinh
Salmonella khng
ln men lactose, ln
men
ng
glucose v sinh hi. Thng khng ln men sucrose, salicin v inositol, 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 H2S, khong 5% cc chng Salmonella sinh c t sinh
bacteriocin chng li E.Coli, Shigella v ngay c 1 s chng Salmonella khc.
2.2.4 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 lng H v
khng nguyn v K. Vi khun thng hn (S.typhi) c khng nguyn V (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 t p gm 2
lp. Trong cng l mt lp peptidoglycan mng, cch mt lp khng gian chu cht v
GVHD: KS. Hunh Vn Thnh
SVTH: Nguyn Hu Lim
14
15
Ni c t - Endotoxin
16
c t ng rut
17
c t t bo
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19
20
21
22
23
24
c trng ca
Salmonella trn mi
trng XLD
+
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
25
Hnh
ca
2.6:
Khn lc c trng
Salmonella
d)
trn mi trng BS
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. 370C trong 18 24 gi. Cc khun lc c trng trn mi trng
ny c s dng cho cc test sinh ha v huyt thanh
+ Cc th nghim sinh ha chnh cn thc hin cho Salmonella
l: ln men glucose, urea, manitol, indol, H2S, VP, ODC, LDC, saccharose, sorbitol.
Cc chng Salmonella cho kt qu th nghim nh sau
+ Th nghim H2S (KIA hay TSI): Salmonella ch ln men c
ng glucose trong mi trng, v vy phn nghing ca mi trng c mu ,
phn su c mu vng. a s cc chng Salmonella u c kh nng sinh H 2S nn c
cc vch mu en trong mi trng. C th quan st thy hin tng sinh hi qua hin
tng lm v thch hoc mi trng b y ln li mt khong h di y ng
nghim
+ Th nghim LDC (+): sau khi nui cy, mi trng b kim
ha, mu khng i ( mu tm hay xanh )
+ Th nghim urea (-): Salmonella khng phn gii urea nn
khng lm thay i pH ca mi trng, sau khi nui cy mi trng vn gi nguyn
mu
+ Ln men manitol, sorbitol (+): mi trng sau khi nui cy b
acid ha chuyn sang mu vng.
GVHD: KS. Hunh Vn Thnh
SVTH: Nguyn Hu Lim
26
Mi trng
TSI/KIA
LDC
Urea
Manitol, sorbitol
Trypton
hin vch en
Khng i mu
Khng mu
B acid ha,mi trng chuyn sang mu vng
Khng xut hin vng khi nh thuc th
VP
Kovacs
Khng i mu khi nh napthol 5% v
KOH 40%
Bng 2.1: Biu hin c trng ca Salmonella trong test sinh ha
- 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
2.3.2 Phng php hin 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 d ng. Phng php PCR gip pht hin nhanh; tuy nhin d xy ra hin
tng m tnh gi, i hi cc 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 c n kt hp nhiu phng php vi nhau, v d: IMS-PCR
27
28
29
30
31
32
Thu mu ti cc a im kho st
GVHD: KS. Hunh Vn Thnh
SVTH: Nguyn Hu Lim
33
nh gi cc ch tiu
Cm quan
Salmonella
25 g mu + 225 ml BPW,
ng
30 giy
Nhn
xt,nht
Ktmu
luntrong
-1
Hnh 3.1: Ni dungthc
hin
th
nghim
pha long 10
3.5 Phng php nghin cu
3.5.1 Quy trnh phn tch
em 37oC trong 24 gi
37oC trong 24 gi
Thuyt minh quy trnh
35
Negati
ve
Negati
ve
Positi
ve
36
Negati
ve
Negativ
e
Negati
ve
37
Negativ
e
Positiv
e
Negativ
e
38
Negati
ve
Hnh 3.7: Th nghim LDC
Th nghim
kh nng sinh Indol:
- Cy VSV th nghi m qua mi trng canh trypton khong 24 gi
37oC.
- Nh vi git ether ko indol ln b m t mi trng, thm vi git
thuc th Kovacs
- Quan st sau vi pht.
- Th nghi m (+): trn b m t mi trng xut hi n vng mu cnh
sen.
- Th nghi m (-): khng xut hi n vng .
39
Positiv
e
Negativ Negativ
e
e
40
Negativ
e
Hnh 3.9: Th nghim Voges Proskauer
3.4.5.
41
Hnh
3.10 :
T l
nhim
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