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THNH PH H CH MINH
MC LC
Bi 1: Thit b, dng c phng th nghim v cc phng php
tit trng vi sinh vt..............................................................................................3
Bi 2: Phng php pha ch mi trng nui cy vi sinh vt..............................6
Bi 3: Phng php nui cy vi sinh vt............................................................10
Bi 4: phng php quan st vi sinh vt bng knh hin vi quang hc...............16
Bi 5: Phng php nhum mu vi sinh vt.......................................................19
Bi 6: Phng php phn lp vi sinh vt............................................................20
Bi 10: Kho st c tnh sinh ha ca vi sinh vt.............................................23
Bi 13: nh lng vi sinh vt bng phng php m trc tip.......................35
Bi 14: nh lng tng vi khun hiu kh bng
phng php m khun lc...............................................................................37
Bi 15: nh lng Coliform bng phng php MPN.....................................39
Bi 19: Phng php nh lng E.coli trong thc phm..................................41
Bi 20: Phng php phn tch Salmonella spp. trong thc phm.....................43
Bi 21: Phng php phn tch nh lng Bacillus cereus trong thc phm....46
Bi 22: Phng php phn tch nh lng Staphilococcus aureus
trong thc phm.................................................................................................48
Bi 24: Phng php phn tch Clostridium perfringens trong thc phm........50
Vi cc dng c nh pipet, que trang phi dng giy bao kn ton b. C th dng hp
nhm ng cc dng c trn kh trng.
2. Cng dng v cch s dng cc dng c, thit b phng th nghim vi sinh
vt?
Cng dng v cch s dng cc dng c, thit b phng th nghim vi sinh
vt:
a. Ni hp t (autoclave):
Thit b ny cp nhit bng hi nc p sut cao, c s dng hp kh trng
m trng, mt s nguyn liu v dng c th nghim.
b. Knh hin vi:
Cng dng: dng nghin cu, quan st t bo vi sinh vt v c im hnh thi,
sinh l nh vo kh nng phng i ca knh.
c. Cc dng c th nghim
Dng c thy tinh c nhiu loi vi nhiu kch c khc nhau nh bnh tam gic,
ng nghim, a petri, lam knh, a thy tinh, que trang, ng ong, cc ong, bnh
nh mc
-
Phin knh (lame): dng lm tiu bn quan st hnh thi, sinh l t bo vi sinh vt.
Que cy:
i vi dng c cy kim loi, i khi c thy tinh, phng php thng dng l t
trc tip trn ngn la n cn.
Phng php Pasteur: ch dit vi khun gy bnh, khng dit bo t v vi khun hoi
sinh. Phng php ny thng dng nhit 70-750C trong thi gian 10-15 pht.
Phng php Tyndall: un cch thy nhiu ln nhit 70-80 0C, mi ln 30-60 pht
v lin tip trong ba ngy lin.
Tia m cc dng trong tit trng dng c gii phu, thuc, thc phm. Vt kh trng
phi bao gi knh.
Dit trung bng cch lc:
4
Mi trng phn lp
Mi trng tng sinh
Mi trng nui gi ging: ngho dinh dng
Mi trng th nghim sinh ha
2. Trnh by qui trnh pha ch mi trng nui cy vi sinh vt
Bao gm cc bc sau:
3.
Chun b dng c
Cn ha cht
Phi ch to mi trng nui cy
iu chnh pH ca mi trng
Phn phi mi trng vo dng c
Kh trng mi trng
Lm thch nghin, thch ng, thch vo a petri
Kin tra v trng v bo qun
Yu cu ca mi trng trong a petri, ng nghim thch nghing v thch
ng
Lm thch nghing:
Cn tin hnh ngay sau khi kh trng mi trng v mi trng cha ng
c.
-
nhn v lin tc
Lm thch ng:
- t cc ng nghim c mi trng l thch ng vo gi, yn cho mi
trng ng c
mi trng vo a petri:
- Ton b qu trnh thch vo a petri c thc hin trong t cy v
trng v gm cc thao tc sau:
o M bao giy gi cc a petri
7
o
o
o
o
o
o
4.
Nguyn tc
1. Mc ch ca vic nui cy
- Pht hin s c mt ca vi sinh vt trong cc nguyn liu vt phm cn nghin cu.
- Tin hnh vic phn ging cc vi sinh vt mt cch nhanh chng.
- Bo tn cc ging vi sinh vt thun khit.
- Nghin cu cc c tnh sinh hc v s pht trin tng loi ca vi sinh vt.
2. Nguyn tc nui cy vi sinh vt
Mi thao tc nui cy u phi thc hin trong iu kin v trng trnh tp
nhim cc vi sinh vt khng mong mun t mi trng ngoi.
Mi trng v dng c nui cy u phi kh trng trit . Duy tr tt cc iu
kin nui cy vi sinh vt pht trin thun li.
II.
III.
1.
1.1.
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Hnh 3.2:
Cch
dn vi sinh vt
ln b
mt mi trng
Hnh ch chi
Hnh vng xon
Hnh vch ngang song song
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thnh ng tu yu cu.
ng cy phi thng, nh nhng khng gy nt, v mi trng
IV. Tr li cu hi.
1. Cc phng php gieo cy vi sinh vt
- Cy truyn vi sinh vt trn mi trng lng: cy truyn bng pipette Pasteur, cy
-
nhm chn la ging vi sinh vt thun khit cho nhng mc ch khc nhau.
4. Mun phn lp nm men, nm mc, vi khun th ngun mu cn chn l g?
Tr li:
- Phn lp vi khun: ngun mu l c kh ct nh.
- Nm mc: ngun mu l cm ngui, xi lm mc tng, bnh m kh
- Nm men: ngun mu l b mt tri cy, dch p nh to, l hoc trong ru np, bia,
nc ma.
5. So snh s ging v khc nhau ca cch phn lp vi sinh vt hiu kh v k kh.
Tr li:
- Ging nhau: nhm phn tch cc chng vi sinh vt trong mi trng t nhin v c
lp chng nhm chn la nhng ging vi sinh vt thun khit.Khc nhau:
Phn lp vi sinh vt hiu kh
- Ht dch mu pha long cho
vao da petri c mi trng thch
hp.
- Dng que gt thy tinh phn phi
dch mu tri u khp mt thch.
- Tip tc dng que gt mu cho
Phn lp vi sinh vt k kh
- Dng mi trng c trong ng nghim em
chng cch thy loi b khng kh.
- ngui mi trng cn 45-50oC
- Ht dch nghin cu cho vo ng mi trng, y
nt li, lc trn quanh trc ng nghim.
- Rt nhanh, y nhanh trnh cho ng nghim c
khng kh.
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u khp mt thch a th 2 ri
a th 3.
- m nhit thch hp sau
c cc khun lc ring r t cc
a th 2, 3
a. Cu to
Knh hin vi quang hc gm c hai phn:
- H thng c hc: gi knh gm c chn knh, thn knh, tr v xoay th knh, gn
v xoay vt knh, bn knh, thanh trt di chuyn tiu bn, c di chuyn thanh trt,
kp gi tiu bn, c di chuyn t quang knh, c iu chnh th s cp, c iu chnh
-
th cp (vi cp).
H thng quang hc gm: th knh, vt knh, t quang knh, mng chn sng, ngun
chiu sng (n in chiu sng hoc knh chiu sng). Ngoi ra, knh dng ngun
chiu sng l n in th c thm b phn cung cp in nh phch cm, dy in,
Knh hin vi in t
Dng bc sng 500 nm 560 nm trong chm nh sng thng to nng sut
phn li ln gip phn bit hai im cch nhau khong 0.2 m tr ln. H thng phng
to ca knh hin vi quang hc gm hai b phn: vt knh v th knh. Mi b phn l
mt h thng thu knh hi t phc tp. Chc nng ca knh hin vi quang hc l dng
quan st t bo vi sinh vt, k sinh trng, t bo ng vt, thc vt.
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3. Ti sao khi quan st vt knh 100 phi cho 1 git du soi knh?
Do chit sut nh sng ca khng kh nh hn thu tinh, nn tia sng khi i qua
tiu bn thu tinh s b khc x mt phn. Phn pha ngoi cu tia sng do b khc x
nn khng i vo c vt knh. Vt knh c phng i cng ln th ng knh
ca thu knh cng nh, lng tia sng i vo vt knh rt t nn khng nhn r nh.
hn ch nhc im ny ta dng du soi knh c chit sut nh sng gn bng thu
tinh. Thu tinh v du soi c xem l mt mi trng ng nht. nh sng i qua
khng b khc x nn tp trung y vo vt knh, gip xem r nh.
Gieo cy vi khun pha long trn mi trng dinh dng c trng (2% thch hay
cn gi l agar).
Nui dng trong iu kin thch hp cho mc cc khun lc tch bit nhau.
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Cy tch t khun lc mc tch bit sang ng mi trng dinh dng thch nghing
thu nhn chng vi khun thun khit.
Phng php phn lp vi khun thun khit:
1.1.
Ria cc ng trn a ptri cha mi trng thch hp (ria ch T v ria bn gc). Sau
mi ng ria, t kh trng u que cy v lm ngui trc khithc hin ng ria
tip theo.
b. K thut hp tri:
-
Dng pipetman v u tp v trng, thao tc v trng chuyn 0,1 ml dch cha ging
vi sinh vt ln b mt mi trng trong a ptri.
Dng u thanh gt xoay, tri u dch ging ln b mt thch. Trong khi tri, thc
hin xoay a mt vi ln, mi ln khong 1/2 chu vi a to iu kin cho thanh gt
tri dch ging u khp b mt mi trng.
c. K thut hp :
-
Xoay nh ia petri cng chiu v ngc chiu kim ng h vi ln dung dch ging
c trn u trong mi trng cy.
y np a ptri, ng t nhin.
1.2.
Sau khi vi sinh vt pht trin, chn cc khun lc ring r trong khi mi trng, dng
que cy ct c khi mi trng ri cy vo mi trng lng thch hp.
2. Nguyn tc ca vic nui tch ly?
Nguyn tc ca vic nui tch ly: trng hp s vi khun c trong mu t th
phi nui tch ly bng cch mu, b sung cht dinh dng v cc iu kin l ha
cn thit hoc b sung cc cht c ch sinh trng ca cc vi sinh vt i km.
3. Th no l chun vi khun thun khit (chng sch)?
Chun vi khun thun khit (hay cn gi l chng sch) c hiu l th h con,
chu, dng c ngun gc t mt t bo ring l. Cc chng vi sinh vt c thu nhn
t khun lc phn lp ln u cha chc thun khit v mt khun lc c th c
hnh thnh bi mt hoc nhiu t bo, bo t, v th phi lm sch nhiu ln mi thu
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Chng
Vi Sinh Vt
Mi Trng: NB v Tripton
Thuc
th
Kovacs
37oC
24h
37oC / 24h
m tnh
Chng
Vi Sinh Vt
Mi Trng
Dng tnh
m tnh
i chng
Mi trng MR-VP
Methyl
Red
24h,
37oC
2-4 ngy
Chng
Vi Sinh Vt
Mi Trng
Dng tnh
m tnh
C ch:
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C s sinh ha:
5,0 5,8
Trn 6,0
Mi trng MR-VP
24h, 37oC
KOH 10% (NaOH 40%)
-naphthol
Kt qu,mu hng (Dng tnh), khng i mu (m tnh)
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Dng tnh
4. Th nghim Citrate
Mc ch: Xc nh kh nng vi sinh vt s dng ngun citrat nh l ngun
cacbon duy nht.
C s sinh ha:
1.0g
1.0g
NaCl
5g
Sodium citrate
2g
MgSO4
0,2g
Bromothymol blue
0,08g
Agar
13g
Chng vi sinh vt
24h, 37oC
Kt qu, mu xanh dng (Dng tnh), mu xanh lc (m tnh)
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P m tnh
i chng trng
P dng tnh
Sinh H2S
To hi (gas)
Chng vi sinh vt
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Kt qu
370C/24h
peptone.
Khng s dng glucose, lactose: vi sinh vt s bin dng peptone thu ly nng
lng v vt cht cho s tng trng. tuy nhin do peptone ch c bin dng trong
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mi trng.
Kh nng sinh H2S do trong mi trng sodium thiosunfat, vi sinh vt kh sunface c
th kh cht ny, nh c enzyme thiosunfat reductase gii phng H2S, H2S s phn
ng vi Ion Fe2+ ca ch th amonium citrate to kt ta mu en F2S.
Mt s trng hp c th xy ra khi dng mi trng KIA/TSI
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mu tm khi pH>6.8.
Cresol red c mu vng tm khi pH<7.2, c mu tm khi 7.2<pH<8.3 v mu tm
khi pH>8.3
V sau khi vi khn phn hu NH2-CH2-(CH2)3-CH2-NH2-COOH v Glucozo mi
Mi trng
37oC, trong 24-48h
c kt qu
Cch c kt qu: vi sinh vt c tim mao c kh nng mc lan ra khi ng cy,
nu khng c kh nng mc lan th ch c th pht trin trn ng cy
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Cress A c A.sulfanilic
Cress B c -naphthilamine
Cress A v Cress B c kh nng kt hp vi NO2- to thnh phc cht c mu nu
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BI 13:
NH LNG VI SINH VT BNG PHNG PHP M TRC TIP
Bo co thc tp
1. Nguyn tc v cch tin hnh ca phng php nh lng vi sinh vt bng
phng php m trc tip
a. Nguyn tc
Da trn s quan st v m trc tip s lng t bo VSV bng knh hin vi
v bung m. Phng php ny dng xc nh s lng cc loi VSV n bo c
kch thc ln nh: nm men, to n bo, bo t mc d quan st trn knh hin vi.
Quy trnh m n gin, cho php xc nh nhanh chng s lng VSV. Tuy nhin, do
quan st v m bng mt trn knh hin vi nn c mt s tr ngi
- Khng phn bit cc t bo VSV sng v cht trong mu.
- Khng phn bit cc t bo VSV v ht vt th.
- Hn ch i vi huyn ph c mt thp do lng dd em m nh
Ty mc c mt ca VSV trong mu ban u, dd em m cn c pha
long n mc ph hp ( s tb trong mt nh khng qu 10 tb). Nguyn tc pha
long:
-
c lng pha long 1/5, 1/10, 1/20 hay 1/10, 1/100, 1/1000
Dung dch pha long mu thng l NaCl 9% hay phosphat m c v
trng.
Cch pha:
+ Mu 1/5: 1ml mu+4ml NaCl 9%
+ Mu 1/10: 1ml mu+9ml NaCl 9%\
T mu 1/10 ly 1ml +9ml NaCl 9% ta c pha long 10 -2, c nh vy ta
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Thao tc knh hin vi, dng vt knh x10 iu chnh s b trc, sau
ln.
- Trng hp 1 ln c 16 nh: m tt c 80 nh trong 5 ln.
- Trng hp 1 ln c 25 nh: m tt c 125 nh trong 5 ln.
2. Phng php nh lng vi sinh vt bng cch m trc tip thng p dng cho
nhng i tng vi sinh vt no? Gii thch
i tng: nhng vi sinh vt c kch thc ln nm men, to n bo, bo t mc
d quan st trn knh hin vi.
Do quan st bng knh hin vi vt knh x40, ngha l phng i ln 400 ln
nn ch c th m c nhng vi sinh vt c kch thc ln.
Mu
ng nht mu ( ht 1ml mu trong 9 ml nc mui sinh l)
Tng sinh ( chn 3 nng lin tip thch hp cy 1 ml mu vo 10ml BGBL, mi nng cy 3 ng, 370C/48h)
Phng php to hp :
u im: th tch mu cy ln, mt vi sinh vt cao
Nhc im: D lm vi sinh vt cht nu mi trng cha h xung
nhit 450C. Khng xc nh c hnh dng khun lc nht nh
36
38
Xc nh ng (+) mi nng , cy sinh khi VSV t cc ng (+) sang a mi trng EMB phn lp E.c
a. C ch ca cc th nghim sinh ha
Phn ng Indol
Trong mi trng NB/ tryptone s c Tryptophan
Nu trong mi trng c vi khun E.Coli th s sn sinh ra Emzyme
Tryptophanase
Tryptophanase s kh Tryptophan th Indol
nhn bit c Indol trong mi trng hay khng th chng ta s nh
thuc th kowacs vo. Nu sinh ra hp cht c mu hng cnh sen th
c mt ca Indol
Suy ra phn ng Indol (+)
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Th nghim MR
Nguyn tc: pht hin cc vi khun ln men glucose to sn phm acid
hu c
Trong mi trng c h m phosphat ( K2HPO4) nhm n nh pH
(6,9)mi trng. E.coli c kh nng ln men glucose to ra acid hu c
bn th n s ph v h m lm pH gim mnh xung khong 4 4,3.
Nh MR nu pH <4,3 th mi trng c mu th (+) cn mu vng th
(-)
Kt qu MR(+)
- Th nghim VP
Nguyn tc: cng ging nh vi MR nhng n s khng to ra acid hu c m to
thnh acetoin
Sau nh thm KOH 10% v
pht
Phn ng (+): c mu hng
Phn ng (-): mi trng c mu vng hoc mu nu t
=>E.coli khng c kh nng sinh acetoin nn phn ng cho kt qu m tnh.
40
41
Mu
Tin tng sinh( ng nht 25ml mu trong 225ml canh BPW 370C trong 16-24h)
3h
3h
Phn lp
3h
42
43
Mu
ng nht mu
m s khun lc in hnh
Khng nh
Tnh kt qu
45
rut
Pha long
m s khun lc in hnh
ng huyt tng. Mc ng tr ln c
Khng
xem
hnh
l thnh
dngkhi
tnh ng huyt tng( hn46
hp ng nht
47
48
Mu
ng nht mu
Pha long mu
n nng thch hp cy 10ml mu vo 2 ng mi trng thch Wilson Blair b sung thm 0,5 ml Na2SO3 5% v 0,25ml Fe
Lm ng thch nhanh
Motility
Nitrate
Lactose
Gelatine
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