TRNG H CNG NGHIP THC PHM

TP.HCM
KHOA CNG NGH THC PHM



MN: PHN TCH VI SINH THC PHM

TI:
QUY TRNH NH TNH SALMONELLA
GVHD: PHAN TH KIM LIN
NHM: 05

TP. HCM, THNG 4 NM 2014
DANH SCH THNH VIN NHM
ON NGC TM THIN
NGUYN TH I LY
PHAN TH KIM NGN
NGUYN TIN PHC
NI DUNG
TNH
HNH
NG
C
THC
PHM
C
IM
VI
KHUN
QUY
TRNH
NH
TNH
TNH HNH NG C DO SALMONELLA
Trn th gii, mi nm nhim khun salmonella t thc phm xy
ra 1.4 triu ngi vi 95% cc trng hp c triu chng ng
c.
Cc ngun gc chung ca ng c thc phm salmonella l cc
thc phm sng hoc cha nu chn nh trng sng, trng hp,
sa cha c tit trng, nc cha c un si k, cc sn
phm t tht v t gia cm cha chn k.
Salmonella
xm nhp vo
c th????
Bt c thc phm sng hay cha nu chn.
Sn phm t gia cm( k c cc sn phm b sa
bo qun khng tt)
Tht g v trng: thc phm c nguy c nhim
cao.
Khi tip xc vi ngi b nhim salmonella hoc
phn ngi v sc vt c nhim salmonella.
C th b nhim khun cho

TRIU
CHNG
Tiu chy, au bng, bun i v i, st, au
u,
Thng xy ra khong 12-36h sau khi n.
Cc triu chng c th ko di t 2 ngy n 1
tun
TNH HNH NG C DO SALMONELLA
Bin php ngn chn nhim khun do Salmonella:
Hn ch n trng sng v cha nu chn. Hn ch hoc
trnh mn n c cha trng sng.
Luc chn k tht g (ti khi tht khng cn hoc
mu hng).Tch bit gia thc phm nu chn v cha nu.
Sau khi ch bin nhng mn sng lm sch cc dng
c ch bin, bp bng x bng v nc hoc dung dch ty ra.
NI DUNG
TNH
HNH
NG
C
THC
PHM
C
IM
VI
KHUN
QUY
TRNH
NH
TNH
C IM SALMONELLA
c im chung v c im nui cy:
Salmonella l trc
khun Gram(-), hiu
kh v k kh ty , c
kh nng di ng(tr
S.gallimarum v
S.pullorum), khng to
bo t.
Pht trin tt 5-45
C, thch hp nht t
37 C. pH thch hp
6.8-7,2.
Pht trin c trong cc
mi trng nui cy thng
thng. Trn mi trng
thch hp s pht trin sau
24h.
C th mc trn cc mi
trng c cht c ch chn
lc nh DCA v XLD.
Khun lc c trng ca
Salmonella trn mi trng
l trn, li, trong sut, c
tm en,..
1
Ln men
glucose v
mannitol sinh
acid
2
khng ln men
saccharose v
lactose, khng
sinh indol,
khng phn
gii ure,
3
C th tit ra 2
loi c t:
Ngoi c t.
Ni c t.
C IM SALMONELLA
NI DUNG
TNH
HNH
NG
C
THC
PHM
C
IM
VI
KHUN
QUY
TRNH
NH
TNH
QUY TRNH NH TNH SALMONELLA
Salmonella c
th c pht
hin bng quy
trnh gm 4
bc
Tng sinh
Tng sinh
chn lc
Phn lp
Khng
nh
QUY TRNH NH TNH SALMONELLA
MI
TRNG
V
HA CHT
Nc peptone m (Buffered Peptone Water, BPW).
Canh Rappaport-Vassiliadis Soya Peptone (RV).
Canh Malachite Green Magnesium Chloride (canh RV ci
tin).
Tetrethionate Muller-Kauffmann( TT).
Thch Xylose Lysine Desoxycholate ( XLD ).
Thch Hektoen Entric Agar ( HE ).
Thch Bismuth Sulphite Agar ( BS ).
Thch Brilliant Green Phenol Red Lactose Sucrose ( BPLS ).
Thch Triple Sugar Iron ( TSI ).
Canh Urea.
Canh Lysine Decarboxylase.
Canh Ornithine Decarboxylase.
Canh Mannitol ( Phenol Red Broth Base).
Canh Sucrose (Phenol Red Broth Base).
Canh Sorbitol (Phenol Red Broth Base).
Canh Tryptone.
Thuc th Kovacs.
Khng huyt thanh Salmonella a gi.
QUY TRNH NH TNH SALMONELLA
MI TRNG-HA
CHT
VAI TR
BPW Tng sinh
Canh RV, RV ci tin, TT Tng sinh chn lc
XLD, HE, BS, BPLS Phn lp, nhn din Sal
TSI, canh urea, canh
tryptone,
Khng nh li s hin din
ca Sal trong mu
Thuc th:
Thuc th kovac
Thuc th methyl red
Phenol red
QUY TRNH NH TNH SALMONELLA
ng nht 25g mu trong
225ml mi trng tng sinh
BPW, 37
0
C, 18-24 gi
Cy 0,1ml dch tng sinh sang
mi trng tng sinh chn lc
RV, 42
0
C, 18-24 gi
Phn lp khun lc n trn t
nht 2 mi trng chn lc
phn bit, 37
0
C, 24 gi
Chn cc khun lc c trng
cho Sal, cy sang BHI hay
TSA, qua m
Th nghim sinh ha
Th nghim ngng kt khng
huyt thanh
Kt lun
QUY TRNH NH TNH SALMONELLA
BC 1: TNG SINH
y l khu quan trng khng th thiu trong
quy trnh kim nghim.
L mi trng tng sinh khng chn lc, giu
dinh dng, khng cha cc cht c ch.
Ty theo c tnh thnh phn ha hc ca
mu, chn quy trnh tng sinh ph hp.
T l thng thng gia mu v mi trng l
1:9, trong nhiu trng hp c th thay i
QUY TRNH NH TNH SALMONELLA
BC 1: TNG SINH
Tn mi trng Thnh phn
BPW
Pepton 10g
NaCl 5g
Na
2
HPO
4
3,5g
KH
2
PO
4
1,5g
Ngoi ra cn c th s dng mt s mi trng:
- Mi trng dch th Tryptic soy broth(TSB)
- Mi trng dch th lactose
QUY TRNH NH TNH SALMONELLA
BC 1: TNG SINH
25g mu
ng nht mu bng
my Stomacher
15 hoc 30 giy
25g mu
+
225ml BPW
37 C
trong 18-24h
QUY TRNH NH TNH SALMONELLA
25g mu
+
225ml BPW
Ly 0,1ml
10ml mi
trng
tng sinh
RV
c m
n 42
42 0,2
0
C
trong 18-24h
BC 2: TNG SINH
CHN LC
QUY TRNH NH TNH SALMONELLA
BC 2: TNG SINH
CHN LC
Tn mi trng Thnh phn
RV
- Mi trng c bn(
tryptone, NaCl, K
2
PO
4
,
nc ct)
- Dung dch MgCl
2

- Dung dch Malachite
green oxalate
Canh RV c ch vi khun khc nh MgCl
2
bng cch tc ng ln
t bo vi khun v nh mu xanh Malachite c ch cc vi khun
Gr(+)
QUY TRNH NH TNH SALMONELLA
BC 2: TNG SINH
CHN LC
Mt s mi trng
tng sinh chn lc
khc cng c
dng
Selenite Cysteine Broth
Mi trng dch th TGB


Mi loi mi trng ch c tc dng chn lc trn mt c im
pht trin ca Salmonella.
Mi mi trng chn lc c mt nhit khc nhau
tng kh nng pht hin tt c cc dng Salmonella cn
dng t nht hai loi mi trng tng sinh chn lc khc nhau cho
cng mt mu.


QUY TRNH NH TNH SALMONELLA
BC 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 ln a mi
trng chn lc phn bit c trng ca
Salmonella.
Mi trng XLD: khun lc c
mu hng trong sut, c hay khng
c tm en. Mt s dng
Salmonella c th c tm en bng
rt ln c th chim gn ht khun
lc
QUY TRNH NH TNH SALMONELLA
Mi trng Thnh phn mi trng
XLD
(Xylose Lysine Desoxycholate)
Cao nm men: 3g
L-lysine: 5g
Xylose: 3.75g
Lactose: 7.5g
Sucrose: 7.5g
Sodium deoxycholate: 2.5g
Ferric ammonium citrate: 0.8g
Sodium thiosulfate: 6.8g
NaCl: 5g
Agar:15g
Phenol red: 0.08g
QUY TRNH NH TNH SALMONELLA
BC 3: PHN LP
V NHN DIN
Mi trng HE: khun lc c mu
thay i t xanh dng n mu
xanh lc, c hay khng c tm en.
QUY TRNH NH TNH SALMONELLA
Mi trng Thnh phn
HE
(Hektoen Entric Agar)
Proteose Peptone: 12.0g
Yeast Extract: 3.0g
Bile Salts No. 3: 9.0g
Lactose: 12.0g
Saccharose: 12.0g
Salicin: 2.0g
Sodium Chloride: 5.0g
Sodium Thiosulfate: 5.0g
Ferric Ammonium Citrate : 1.5g
Agar: 14.0g
Bromthymol Blue: 65.0mg
Acid Fuchsin : 0.1g
QUY TRNH NH TNH SALMONELLA
BC 3: PHN LP
V NHN DIN
Mi trng BS:
- Bismuth sulfite v brilliant
green: c ch Gr(+).
- Disodium phosphate: to
tnh m cho mi trng.
- Dextrose: cung cp nng
lng cho VSV.
-
Khun lc c mu nu xm hay
mu en, thnh thong c xut
hin nh kim tm.
QUY TRNH NH TNH SALMONELLA
BC 4:
KHNG NH
T mi mi trng chn lc phn bit chn v cy
chuyn t nht 5 khun lc nghi ng sang mi
trng khng chn lc TSA. 37
0
C, 18-24 gi.
Cc khun lc xut hin trn mi trng ny c
s dng cho cc th nghim sinh ha nh sau:
Th nghim sinh H
2
S trn mi
trng KIA
Hnh 15: 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 cng
tt). 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 H2S.
QUY TRNH NH TNH SALMONELLA
Th nghim urea
c thc hin trn mi trng urea lng
RSU (Rustigian Stuarts Urea Broth), cha
ch th phenol, chuyn mu t vng sang
(vng chuyn mu l pH 6.8-8.4). Dng
que cy vng cy lng sinh khi ln t
khun lc ca chng thun vo ng cha 3ml
mi trng v trng, lc nh ng trn u
vi khun v 37
0
C trong 48 gi.
Salmonella khng phn gii urea thnh NH
3

nn khng lm thay i pH mi trng; sau
khi nui cy mi trng vn gi nguyn mu
vng cam.
QUY TRNH NH TNH SALMONELLA
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. 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.
QUY TRNH NH TNH SALMONELLA
Ln men mannitol
(+)
Salmonella c kh nng ln men
ng mannitol sinh acid. Khi
c mt thuc th Phenol Red s
lm mi trng chuyn t
sang vng
QUY TRNH NH TNH SALMONELLA
Ln men LDC (+)
Sau khi nui cy mi
trng chuyn thnh kim,
mu mi trng c
chuyn thnh mu nh ban
u.
QUY TRNH NH TNH SALMONELLA
Th nghim kh
nng sinh indol (-)
Mi trng th nghim l
nc tryptone, trong thnh
phn c cha tryptophan.
Nh vi git ether ko
indol ln b mt mi trng,
thm vi git thuc th
Kovacs. Quan st sau vi
pht.
- Th nghim (+): trn b mt
mi trng xut hin vng
mu o cnh sen.
QUY TRNH NH TNH SALMONELLA
Th nghim 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
Mannitol,
sorbitol(+)
Mannitol,
sorbitol
B acid ha, mi trng
chuyn sang mu vng
Indol(-) Tryptone Khng xut hin vng
khi nh thuc th Kovacs
VP(-) VP Khng i mu khi nh -
napthol 5% v KOH 40%
QUY TRNH NH TNH SALMONELLA
KT QU
Quy trnh kim nghim ny ch cho
php phn tch nh tnh Salmonella,
gip kt lun c hay khng c s hin
din ca Salmonella trong mu.
khng nh c tn chng phn
lp c cn gi chng n cc phng
th nghim chuyn nh danh vi sinh
vt
Salmonella cha dng bnh rt nguy
him nn cn cc bin php an ton khi
lm vic
QUY TRNH NH TNH SALMONELLA
Kim tra cc phn
ng khng huyt
thanh
Mc ch: khng nh tn chng Sal
phn lp c.
Sal c 3 loi khng nguyn:
- Khng nguyn O(khng nguyn thn): bn vi nhit, c
dng chia thnh nhm.
- Khng nguyn H(khng nguyn tin mao): khng bn
vi nhit dng nh loi, c bn cht l protein
- khng nguyn Vi: khng bn vi nhit thng che lp
ngn chn khng nguyn O.