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Ghi nhn s luong ng nghim
duong tinh
o tung d pha long. Tra bang Mac
Crady
U o nhit d va thoi gian thich
hop
Kt qua biu kin chung minh su
tng
truong cua vi sinh vt cn kim
Pha long mu phn tich o 3 nng
d pha long bc 10 lin tip.
ColiIorms
E.Coli
. TIEN HNH PHAN TICH
Mu phn tich: Nuoc mia nguyn cht
.. Pha mi trung v dung cu
Tn mi truong Thanh phn Tng th tich
SPW (Saline
Pepton
Water)
NaCl : 42.5g
Pepton: 5g
500 ml nuoc
ct
LSB
(LaurylSulphateBr
oth)
Tryptose: 20g
Lactose: 5g
Sodiumchloride: 5g
KH
2
PO
4
: 2.75g
K
2
HPO
4
: 2.75g
Lauryl sulphate: 0.1g
1000 ml nuoc
ct pH 6.8
0.2
BGBL(Brilliant
Green Bile
Lactose Broth)
Peptone: 10g
Lactose: 10g
Mt bo: 20g
Brilliant green: 0.0133g
1000 ml
nuocct pH
7.4
EC
(E.coli medium)
Trypticase or tryptose: 20g
Mui mt No.3: 1.5g
Lactose: 5g
KH
2
PO
4
: 1.5g
K
2
HPO4: 4g
NaCl: 5g
1000 ml nuoc
ct pH 6.9
EMB
(Eosin Methylene
Blue Agar)
Pancreatic digest oI gelatin:
10g
Lactose: 10g
K
2
HPO
4
: 2g
Eosin Y: 0.4g
Methylene blue: 65mg
Agar: 15g
1000 ml nuoc
ct. pH 7.1
0.2
Tryptone water Tryptone hoc
trypticase:10g
1000ml nuoc
ct pH 7.2
0.2
MR-VP
Both
(Glucose
Phosphate)
trung 1:
BuIIered peptone-water
powder: 7g
Glucose: 5g
K
2
HPO
4
: 5g
1000ml nuoc
ct. pH 6.9
0.2
trung 2:
Casein Pancreatic
Digest: 3.5g
Peptic digest oI animal
tissue:3.5g
Dextrose: 5g
Potassium phosphate: 5g
pH 6.9 0.2
trung 3:
Peptone: 5g
Glucose: 5g
Phosphate buIIer: 5g
pH 7.5 0.2
SCA
(Simmons Citrate
Agar)
Sodium citrate: 2g
NaCl: 5g
K
2
HPO
4
: 1g
NH
4
H
2
PO
4
: 1g
MgSO
4
: 0.2g
Bromothymol blue: 0.08g
Agar: 15g
1000ml nuoc
ct. Dun nong
nhe va thinh
thoang lc.
Dun si 1-2
phut cho dn
khi hoa tan
ht. pH 6.8
0.2
.. Khu trng dung cu v mi trung d pha
Bao goi dung cu va phn phi mi truong vao dungcu, gn nut va
bao goi. Hp tit trung o 121
0
C trong 15 phut.
.. Tin hnh phn tch.
f !ha long mu.
/. Th nghim IMJi
Kt qua IMViC cua Escherichia coli sau 24gio u o 37C. (Anne Hanson,
University oI Maine, Orono%
Ong A: duong tinh thu nghim indole trn mi truong tryptone. Xut hin
lop mau do trn b mt mi truong sau khi nho thuc thu Kovacs.
Ong B: duong tinh thu nghim methyl red xut hin mau do cua methyl red.
Ong C: m tinh thu nghim Voges-Proskauer biu hin qua su khng thay
di mau sau khi cho thuc thu Barritt`s A va Barritt`s B.
Ong D: m tinh thu nghim citrate biu hin qua su thay di mau sc va
khng co khun lac trong ng nghim.
Thu nghim Indol (I):
Nguyn tc: Tryptophan la mt acid amin co th bi oxi hoa boi mt s vi
sinh vt co h enzyme tryptophanase tao nn cac san phm chua gc indol. Vic
phat hin indol duoc thuc hin bng phan ung cua phn tu nay voi cac thuc
thu chua p-Dimethylaminbenzaldehyde (p-DMABA) tao nn mt phuc cht
dang quinone co mau do.
Phuong phap tin hanh: Mi truong su dung cho thu nghim nay la
nuoc tryptone. Sinh khi chung thun duoc cy vao mi truong long tryptone,
u o nhit d 37
0
C trong 24-48 gio. B sung 1ml ether hoc xylene vao ng
nghim, lc du d chit tach indol ln lop dung mi huu co. Nho 5 giot thuc
thu vao ng dich nui cy, d yn vai phut, theo di su tao mau do trong lop dung
mi huu co.
Thu nghim Indol.
Doc kt qua: () xut hin cua lop mau do trn b mt mi truong. (-) lop
mau vang cua thuc thu. Di khi co su xut hin cua mau cam do skatol, la tin
cht cua methyl hoa cua indol, tao ra.
Thu nghim Methyl Red (MR):
Nguyn tc: chi thi do methyl red giup phn bit nng d H hin din
trong mi truong sau khi vi sinh vt ln men glucose. Chi thi nay thay di mau nhu
sau: pH4.4 do; pH 5.0-5.8 mau cam; pH~6.0 mau vang.
Phuong phap tin hanh: Mi truong su dung cho thu nghim nay la mi
truong long Glucose Phosphate (MR-VP Broth). Dung que vong cy mt luong
nho khun lac trn mi truong MR-VP, u o 37
0
C trong khoang 2-5 ngay. Thm
vao vai giot thuc thu do methyl red (0.02 trong hn hop cn nuoc co ty l 3:2,
bao quan o 4
0
C) vao trong ng nghim dich nui cy, doc kt qua ngay.
Thu nghim MR.
Doc kt qua: () mi truong co mau do sau khi b sung thuc thu. (-)
khi co mau vang. Truong hop mau cam, cn tip tuc u ng mi truong co ging
trong 4 ngay va thuc hin lai thu nghim.
Thu nghim Citrate (iC)
Nguyn tc: su bin dung citrate boi vi sinh vt s tao ra CO2 lam km hoa
mi truong. Mt khac moi VSV co kha nng su dung citrate lam ngun carbon duy
nht du co kha nng dung mui ammonium lam ngun dam duy nht. Su
phn giai cua mui ammonium dung lam ngun dam trong mi truong s
sinh NH3 lam kim hoa mi truong. Su gia tng gia tri pH nay duoc chi thi
bng su di mau cua chi thi pH trong mi truong.
Phuong phap tin hanh: Mi truong su dung cho thu nghim nay la mi
truong long SCA. Dung que vong cy ria mt luong nho khun lac ln mi
truong SCA rn trong ng nghim thach nghing, u o 35
0
C trong khoang 24-48
gio.
Thu nghim Citrate.
Doc kt qua: () khi xut hin khun lac va mi truong chuyn sang
mau xanh duong. (-) khi khng co khun lac va mi truong giu nguyn mau xanh
luc.
.4. Cch Tnh Kt Qua.
Tu s luong cac ng nghim co E. coli () o mi d pha long cua mu,
dung bang MPN loat 3 ng nghim o 3 nng d pha long lin tip d tinh ra
mt d vi sinh vt trong mu va biu din duoi dang tri s MPN/ml mu ban du
chua pha long.
Bang Tra MPN dung cho loat 3 ng nghim o 3 nng d pha long lin tip.
KET LUAN
1. Giao trinh phn tich Vi Sinh Thuc Phm, Ths. L Thuy Linh , Truong Dai
Hoc Cng Nghip Thuc Phm Tp.HCM
2. TCVN 6505-2:1999 Sua va cac san phm tu sua dinh luong E.Coli gia
dinh bng phuong phap MPN.
3. Google.com.vn
4. Giao trinh Vi Sinh Thuc Phm, truong Dai Hoc Cng Nghip Tp.HCM.