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THC TP VI SINH
Chng trnh i hc
NI DUNG CHNH
Phn 1. CHUN B MI TRNG, THAO TC V
TRNG V BO QUN GING VI SINH VT
Phn 2. QUAN ST, PHT HIN VI SINH VT
Phn 3. NH LNG VI SINH VT
NH GI MN HC
1.
2.
3.
4.
Bi 2. K thut v trng
Nguyn tc: Loi tr vi sinh vt tp nhim
Dng c
Thit b h tr
Thao tc
Bi 3. Pha ch mi trng
Nguyn tc:
- Dinh dng ph hp
- pH thch hp
- Khng c cht c hi
- V trng
Phn loi:
- Mi trng t nhin, tng hp, bn tng hp
- Mi trng lng, rn, rn mm
- Mi trng chn lc, kim nh
Thc hnh
Bi 3. Pha ch mi trng
Mi trng Hansen:
- Glucose: 50g
- Pepton : 10g
- K2HPO4 : 3g/l
- MgSO4 : 3g/l
- Agar
: 20 g/l (+/-)
- Nc ct 1 lt
pH= 6
Mi trng PGA
- Glucose: 50g
- Agar
: 20 g/l (+/-)
- Dch chit khoai ty/gi
1 lt
( 500 g khoai ty gt v/ ct
thi/ nu k vi 1 lt nc
trong 2h. Lc/ thm nc
cho va 1 lt)
13
14
Thao tc cy chuyn
(thch nghing thch nghing)
15
Thao tc cy chuyn
(thch a thch nghing)
16
PHN LP GING
Tch ring vi sinh vt thu ging thun
Nguyn tc: dn mng (pha long) hn hp vi
sinh vt sao cho c th thu c khun lc
thun
Thng dng thao tc cy ria
C th dng thao tc cy tri, a
17
Thao tc cy ria
(ng nghim-thch a)
18
Thao tc cy ria
(thch a-thch a)
19
ng di chuyn u que cy
20
t nng que cy
ngui
26
Cy chuyn
Bo qun ging bng glyceron
Bo qun ging bng phng php lm kh
ng kh
LOGO
Bi 8. CC PHNG PHP
NH LNG VI SINH VT
2
4
Phng
php m Phng php
t bo qua
MPN
knh hin vi
Phng
php mng Phng php
Phng php
lc
m khun lc
o ATP
6
7
Phng
php
o
c
Phng
php xc
nh sinh
khi
Phng
php
PCR
S tin hnh
Nc
Nh ln hai khoang
Nh ln cnh bung m
Thm nc d
m s t bo trn 5 ln
Mu
PhaA long
Nc
Phng php m
x1
x4
L knh
x3
Vng cha
t bo VSV
Cu to bung m
hng cu
x2
x5
xi [50- 200]
Kt qu
a * 4000*b
x=
*1000
c
x : lng t bo trong mt ml
a : s lng t bo trong 5 ln.
b : t l pha long canh trng (nghch o nng pha
long)
c : s nh trong 5 ln (c=16x5=80)
Nhn xt
u im ca phng php m bng bung m
Nhanh chng
n gin
Nhn xt
Nhc im:
1.Khng phn bit c t bo sng v t bo
cht.
2.Ch thch hp vi t bo c kch thc ln (
nm men, nm mc.)
3.Kh t chnh xc cao
4.Cn c knh hin vi phn pha khi mu khng
c nhum.
5.Phng php ny khng thch hp trong
trng hp huyn ph mt thp
Cc mu pha long
S ng dng tnh
S ng dng tnh
Khong tin cy
0.1
0.01
0.001
MPN/g
< 3.0
Khong tin cy
thp
cao
0.1
0.01
0.001
MPN/g
thp
cao
9.5
21
4.5
42
0.15
9.6
28
8.7
94
0.15
11
35
8.7
94
6.1
1.2
18
29
8.7
94
6.2
1.2
18
36
8.7
94
9.4
3.6
38
23
4.6
94
3.6
0.17
18
38
8.7
110
7.2
1.3
18
64
17
180
11
3.6
38
43
180
7.4
1.3
20
75
17
200
11
3.6
38
120
37
420
11
3.6
42
160
40
420
15
4.5
42
93
18
420
16
4.5
42
150
37
420
9.2
1.4
38
210
40
430
14
3.6
42
290
90
1000
20
4.5
42
240
42
1000
15
3.7
42
460
90
2000
20
4.5
42
1100
180
4200
27
8.7
94
>1100
420
3.nh gi :
Vi phng php ba nng pha long ta
ch cn lp dy s cc ng dng tnh v tra
theo bng bit gi tr MPN tng ng ca
mu th.
Phng php MPN c th dung nh
lng bt k loi vi sinh vt no vi iu kin
phi dng mi trng tng sinh chn lc
(mi trng lng) cho kt qu kh ph hp,
chng hn BGBL kt hp vi ch nhit
37oC cho Coliform, 45oC cho Coliforms phn,
Manitol Salt Broth cho Staphylococus
Xc nh s t bo sng
bng phng php m khun lc
a, cy tri
Nhn xt
u im:
_ Ta nh lng nhng t bo sng c trong mu kho st ban
u.
_ c ng dng rng ri hn cc phng php khc, c bit
l kim tra thc phm,bnh phm, cht lng v sinh ca
nc.
_ nhy kh cao, cho php pht hin tng i chnh xc mc
nhim khun i vi sn phm.
Nhc im:
_ Thi gian di v tn km chi ph ho cht
_ Nhn cng nhiu hn so vi phng php khc
Phng php cy gt
_
Chun b mi trng thch trong erlen v i hp tit trng.
Sau khi lm ngui mi trng thch v nhit 50oC, mi
trng thch vo hp petri v khun. Cc hp c xp trn mt
phng nm ngang cho n khi thch ngui. Ngi ta bao gi v
lt p cc hp petri li ri em i bo qun trong t nhit
phng trong thi gian 2-3 ngy kim tra v trng ca mi
trng.
_
ng vi pha long, lc u mu v dng pipet v trng
ly chnh xc mt lng th tch mu cho ln b mt thch hp
petri. Th tch ly mu khng qu 0,1ml.
Phng php cy gt
_ Sau dng que cy gt v trng dn u mu trn b mt
thch. v trng que cy gt. Ta nhng que cy vo dung
dch cn 70o. Ta vy ro v a que cy ln ngn la n cn
lm chy i phn cn cn ng li trn qua. Ngay sau , ta
c th s dng que cy gt dn u mu trn b mt thch.
_ Ch cho b mt thch kh, tin hnh bao hp petri, lp p hp
v t chng vo t m. Nui cy trong 2-3 ngy nhit
thch hp.
_ i vi mi pha long, cn tnh hnh gieo cy t nht l
trong 2 hp petri. S dng 1 pipet v trng v 1 que cy gt
cho mi pha long.
Phng php hp
_ Chun b mi trng thch v hp tit trng. Mi ng nghim
cha 25ml mi trng thch. Trc khi em s dng, t cc
ng nghim vo b iu nhit nhit 50oC thch bn
trong ng tan chy tr li.
_ S dng cc hp petri c v khun trc. ng vi mi
pha long, dng pipet v trng ly chnh xc mt th tch
mu cho vo hp petri . Th tch mu c th dao ng t 0,11,0ml.
_ Lc u phn mi trng trong ng nghim v tip vo hp
petri. Xoay hp mu huyn ph vi sinh vt v mi trng
thch c trn u vi nhau.
_ Ch cho thch ng li ri tin hnh bao gi cc hp petri, lt
p hp ri t vo t m.
Hot ha: phn t ATP v luciferin s cng to phc vi phn t enzyme, sau ATP s chuyn thnh AMP v gii phng ra
pyrophosphate.
Decarboxy ha: giai on ny, s gii phng ra mt phn t CO2, AMP v mt photon.
Cng nh sng pht ra t phn ng trn t l thun vi hm lng ATP tham gia phn ng. Bng cch dng th ng
chun, ta c th nh lng hm lng ATP trong mu phn tch.
Nhn xt:
u im:
_ n gin
_ Nhanh gn
Nhc im:
_ Tn ho cht
_ Ch nh gi tnh trng v sinh b mt
Pha long dung dch ATP chun thu c cc dung dch ATP c nng thay i t 10-8
n 10-4 mol/l.
_ ng vi mi pha long, ly 50l mu cho vo ng nghim cha 2.5ml dung dch biofax
A2. Khuy trn trn my vortex trong thi gian 30 giy.
_ Ly 200l hn hp trn cho vo mt ng nghim khc. Thm vo 10l dung dch luciferin v
khuy trn trn my vortex trong thi gian 30 giy.
_ c kt qu cng nh sng ca mu o. Kt qu o c biu din di dng n v nh
sng tng i. Tin hnh c 3 ln, mi ln cch nhau 10 giy. Ly gi tr cao nht trong ba
gi tr o c tnh kt qu.
_ Thc hin mt mu i chng thay dung dch ATP bng nc ct. Cng nh sng ca
cc mu ATP chun RLUATP s c tnh theo cng thc:
RLUATP= RLUATP* - RLUH2O
Trong :
RLUATP*-gi tr o c ca mu cha ATP trn thit b o cng nh sng.
RLUH2O -gi tr o c ca mu nc ct trn thit b o cng nh sng.
PHNG PHP O C
Khi mt pha lng c cha nhiu phn t khng
tan => to thnh mt h huyn ph v c c
bi cc phn t hin din trong mi trng lng
cn nh sng, lm phn tn chm nh sng ti
T bo vi sinh vt l mt thc th nn khi hin
din trong mi trng cng lm mi trng c
Xc lp quan h t l tuyn tnh gia mt t
bo vi sinh vt v c
nh lng gin tip thng qua c
Da vo ng chun
o mt quang (OD)
B phn x lxut kt qu
Ngun sng
Dch lng
cha VSV
T bo quang in
Gi s c bit mt t bo nhn
ging nm men S. cerevisiae nm
trong khong 2.109 -3.109 t bo/ml.
Xc nh mc pha long thch hp
p dng phng php nh lng
bng bung m
Xc nh mt t bo nm men S.
cerevisiae trong giai on nhn ging bng
phng php m trc tip ghi nhn c
5 ln i din nh sau:
[1: 74 _ 2: 86 _ 3: 88_ 4: 75 _ 5:81].
Bit nng pha long huyn ph nm men
bia l 10-2.
Hy trnh by cc bc cn tin hnh xc
nh t l t bo sng trong huyn ph nu
trn.