.

Pha dung d ch cht ch th : Ha tan 8g bromthymol blue vo hn hp 250 ml ethanol 90% v 250 ml nc ct Tit trng mi trng 121 oC trong thi gian 15 pht 8. Mi trng DRBC _ Dichoran Rose Bengal Chloramphenicol Thnh phn Glucose Peptone K2HPO4 MgSO4 Rose bengal (5%w/v) Dichloran (0,2% w/v trong ethanol) Chloramphenicol* Agar Nc ct pH cui (25oC) S lng 10 5 1 0,5 0,5 1 0,1 15 1000 7,0 0,2 n v g g g g ml ml g g ml

* Chloramphenicol ngui n 50 oC mi b sung vo Tit trng mi trng 121 oC trong thi gian 15 pht Ch : Mi trng c cha dichloran v rose bengal nn c ch s ko di khun ty n m mc mc nhanh , v th cho php pht hin nhng nm mc mc chm. Rose bengal l cht d b phn hy trong nh sng, n n cn gi mi trng trong ti.

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Tit trng mi trng 121oC trong thi gian 15 pht 5. Mi trng thch nm men nm si (Yeast Mould Agar) Thnh phn Bt chit nm men Bt chit malt Peptone Dextrose Agar Nc ct pH cui (25oC) S lng 3 3 5 10 20 1000 6,2 0,2 n v g g g g ml ml

* ng c th l glucose, saccharose hoc lactose

Tit trng mi trng 121 oC trong thi gian 15 pht 6. Mi trng MRS (Man, Rogosa, Sharpe) Thnh phn Peptone Bt Lab-Lemco Bt chit nm men Glucose Sorbitan mono-oleate K2HPO4 Sodium acetate 3H20 Triammonium citrate MgSO4.7H20 MgSO4.4H20 Agar Nc ct pH cui (25oC) S lng 10 8 4 20 1 2 5 2 0,2 0,05 10 1000 6,2 0,2 n v g g g g ml g g g g g g ml

Tit trng mi trng 121 oC trong thi gian 15 pht 7. Mi trng ln men Thnh phn ng* Peptone Bt chit nm men Dd ch t ch th Nc ct pH cui (25oC) S lng 5 10 5 2 1000 7,0 0,2 n v g g g ml ml

* ng c th l glucose, saccharose hoc lactose

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PH LC
1. Mi trng Cao tht Pepton Thnh phn Cao tht Pepton NaCl Agar Nc ct pH cui S lng 5 10 5 15 1000 7,0 0,2 n v g g g g ml

Tit trng mi trng 121oC trong thi gian 15 pht 2. Mi trng Czapek Dox Thnh phn
NaNO3

S lng 2,0 1,0 0,5 0,5 0,1 30 15 1000 7,3 0,2

n v g g g g g g g ml

K2HPO4 MgSO4 KCl FeSO4 Saccharose Agar Nc ct pH cui (25oC)

Tit trng mi trng 121oC trong thi gian 15 pht 3. Mi trng PDA _Potato Dextrose Agar Thnh phn Khoai ty* Dextrose Agar Nc ct pH cui (25oC) S lng 200 20 20 1000 5,6 0,2 n v g g g ml

* Dch chit t 200 g khoai ty c th thay th bng 4 g bt chit khoai ty

Tit trng mi trng 121oC trong thi gian 15 pht 4. Dung d ch pha long SPW _ Salt Pepton Water Thnh phn NaCl Pepton Nc ct S lng 85 10 1000
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n v g g ml

TI LIU THAM KHO

1. Trn Linh Thc, Phng php phn tch vi sinh vt trong thc phm, nc v m phm, NXB Gio dc, 2005 2. L Vn Vit Mn - Li Mai Hng, Th nghim vi sinh vt hc thc phm, NXB i hc Quc gia Tp. HCM, 2006 3. Nguyn Thnh t Mai Th Hng, Sinh hc vi sinh vt, NXB Gio dc, 2000 4. Gio trnh thc tp Vi sinh nm IV, Khoa Sinh hc Trng H Khoa hc t nhin Tp. HCM, nm hc 2000 2001 5. Adams M.R., Moss M.O. Food Microbiology 3rd ed. Chapter 10 Method for the microbiological examination of foods, RSC, 2008

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6.5. 6.6.

Yu cu vit bo co Nu c ch ln men ethanol Quy trnh cc b c tin hnh ln men sa chua Gii thch cc thng s trong tng qu trnh. Tiu ch nh gi Bng 6.2. Tiu ch nh gi bi 6

STT 1 2 3 4 5 An ton, v sinh Th i gian Chun b Thao tc : 6

Tiu ch nh gi thc t chc, k lut

im 1 1 1 1 1,0 1,0

Pha ch mi trng ln men ng Kim tra cc thng s bin i trong qu trnh ln men (VD : pH)

Kt qu : - Xc nh hm lng acid lactic to ra trong qu trnh ln men hoc gin tip qu s gim gi tr pH. - Th o lun kt qu Bo co - Hnh thc trnh by bo co - Ni dung bi bo co y yu cu ra TNG

1,0

1,0

0,5 1,5 10

6.7.

Cu hi tho lun

1. Mc tiu ca vic o hm lng cht kh trong dch sa nguyn liu? 2. Ti sao phi dch ln men 40 43 oC? 3. Nu thay i nhit ln men th cht lng sn phm c thay i khng v thng s no s thay i theo? 4. Ngoi nh tnh acid lactic sinh ra trong qu trnh ln men sa chua bng pH k, ngi ta c th s dng phng php no chng minh acid lactic sinh ra mt cch chnh xc ?

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6.3.

Cc bc tin hnh Sa ti Lm m Ph gia Phi ch Cy men Rt hp Bo qun Sn phm Sa bt Men ging H nha

Quy trnh cng ngh ln men sa chua:

Bc 1. Chun b nguyn liu v cc ph liu. Bc 2. Xc nh hm lng cht kh ca sa bng Brix k. Bc 3. Tnh ton hm lng cc cht ph gia v sa bt cn phi ch (iu chnh Brix > 16% v thm cc ph gia khc ...) Bc 3. Duy tr nhit canh trng 40 45 oC. Bc 4. Cy men vo canh trng theo t l 10% men ging. Bc 5. Sau khi khuy u men vo sa, xc nh pH im u v rt dch sa vo h nha Bc 5. 40o C trong 5 6 gi. Bc 6. Sau 6 gi ln men, xc nh pH im cui v bo qun sa chua trong t lnh 2 4 oC 6.4. Kt qu nh gi cc thng s kim sot qu trnh ln men (pH, nhit (oC)) Xc nh lng acid lactic sinh ra trong qu trnh ln men

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cc sn phm ph diacetin, ester v cc acid hu c bay hi nn sa chua b dng v c hng v thm ngon. C th sn xut bng phng php ln men t nhin (nh h VSV nhim t nhin) v phng php cy ging thun khit. 6.2. Dng c, thit b v ha cht Bng 6.1. Dng c, thit b v ha cht bi 6
A. HA CHT: STT TN HA CHT QUY CCH SL/VT GHI CH

1 2 3 4 5
STT

Bng thm nc C n Sa c /sa ti Sa chua Gelatin

TN DNG C

1,0 kg 95 0+ 700 2 + 3 lt 1 hp 1 hp Tinh khit

QUY CCH

Lu t u 2 t 1 t 1 lp
GHI CH

60 g
SL/VT

B. DNG C :

1 2 3 4 5 6 7 8
STT

C c th y tinh C c th y tinh a th y tinh N i va C c th y tinh Nhit k khui sa hp Ti nylon nh

TN THIT B

100ml 250ml

1000ml 0 0-1000C
8cm x 15cm
QUY CCH

2 ci 1 ci 1 ci 1 ci 10 ci 5 ci 1 ci 150 g
SL/VT

1 t 1 t 1 t
1 t 1 lp 1 lp 1 lp SV chu n b
GHI CH

C. THIT B :

1 2 3 4 5 6

N i hp tit trng T m T lnh Bp in pH k Brix k

10-30%

1 ci 1 ci 1 ci 10 ci 2 ci 2 ci

1 lp 1 lp 1 lp 1 lp 1 lp 1 lp

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BI 6.

LN MEN LACTIC

Mc tiu: Sau khi h c xong bi ny, sinh vin c kh nng: Thc hin qu trnh ln men lactic Nh n bit du hiu ln men lactic

6.1. C s l thuyt Ln men lactic l qu trnh chuyn ha k kh ng vi s tch ly acid lactic trong mi trng. Con ngi bit ng dng hin tng ny t lu ch bin cc loi thc n chua (sa chua, mui da, mui c), chua thc n cho gia sc hay sn xut acid lactic phc v cho ngnh cng nghip thuc da, dt nhum, cng nghip thc phm Cc vi khun lactic c xp chung vo h Lactobacteriaceae. Mc d cc loi vi khun ny khng ng nht vi nhau v mt hnh thi (hnh que ngn, d i, hnh cu), song v mt sinh l chng li tng i ng nht. Tt c u l nhng vi khun Gram dng, khng to bo t v hu ht khng di ng. Thu nhn nng lng t vic phn gii hydrate carbon v tit ra acid lactic. 6.1.1. C ch ln men lactic Khi thy phn ng trong iu kin k kh, VSV lactic chuyn hydro t NADH sang pyruvate to thnh acid lactic ti to NAD+
NADH NAD+

C6H12O6

CH3COCOOH

CH3 - CHOH - COOH + Q

Cn c vo sn phm sinh ra, qu trnh ln men lactic c chia lm 2 kiu: Ln men lactic ng hnh Cc vi khu n lactic ng hnh chuyn ha ng to th nh sn phm ch yu l acid lactic (95%). VSV ln men lactic ng hnh: Ging Streptococcus: S. cremoris, S. lactis, S. thermophillus Ging Lactobacillus: L. bulgaricus, L. acidophillus, L. platarum

Ln men lactic d hnh Khi ln men cc vi khun ch to ra 60% acid lactic phn cn li l acid acetic, ru ethanol, glycerin v mt s sn phm khc. VSV ln men lactic d hnh: Lactobacillus brasicar fermentatae, Leuconostoc mesenteroides, Escherichia coli 6.1.2. Ln men sa chua Sa chua l loi sn phm ln men t sa ti. Sa chua c gi tr dinh dng cao v c hng v thm ngon nn c nhiu ngi a thch. nhiu quc gia, sa chua l mn n hng ngy ca nhn dn. V sa chua c hm lng acid lactic cao n n protein sa khng tip tc b phn gii na, m b ng t. Bn cnh , qu trnh ln men cn to ra
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5.4. 5.5.

Yu cu vit bo co Nu nguyn tc cc phng php nh lng trc tip v gin tip VSV Trnh by c ch ln men ethanol. Nu cc bc tin hnh qu trnh ln men ethanol. Phn tch cc yu t lm nh hng n qu trnh ln men ethanol Tiu ch nh gi Bng 5.4. Tiu ch nh gi bi 5 STT 1 2 3 4 5 An ton, v sinh Thi gian Chun b Thao tc : 6 7 K thu t m khun lc chnh xc trn my m khun lc. Ho t ha v nh lng trc tip t bo nm men trn knh hin vi Nu cm np khng kh, chy. Sn ph m cm ru ln men khng b h Gii thch v bin lun cc hin tng ln men ethanol 0,5 1,0 0,5 Tiu ch nh gi thc t ch c, k lut im 1 1 1 1

Kt qu: 1,0 1,0

Bo co - Hnh thc trnh by bo co - Ni dung bi bo co y yu cu ra T NG

0,5 1,5 10

5.6.

Cu hi tho lun

1. Nu nhc im v u im ca phng php m VSV bng bung m hng cu. 2. Trong cng nghip ln men, ngi ta thng s dng phng php nh lng VSV no nht, ti sao? 3. Hy phn tch s khc bit ca phng php nh lng VSV trc tip v gin tip? 4. Trong k thut ln men cm ru, ti sao ta phi 1/4 - 1/3 th tch trng trong vt cha c cht ln men? 5. Ngoi phng php cm quan, ngi ta cn dng phng php ha hc nh tnh ethanol c sinh ra trong qu trnh ln men cm ru. Nu phng php .
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Bc 5. cm np gieo men vo trong vt cha t 2/3 3/4 th tch (tu theo hnh th ca vt cha) v bt li bng mng bao PVC (mng mng dng bao a thc n). Bc 6. 30 oC, 3 - 4 ngy s cho sn phm cm ru.

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10-1 s m nh hn 25 th kt qu ghi: < 2,5 x 10 -2 CFU/g. Hnh 5.6. S lng v dng khun lc trn Petri sau nui cy 5.3.1.2. Phng php m trc tip Phng php m bng bung m hng cu Bc 1. Nghin vin nm men v cn 1 g bt nm men Bc 2. Cho 1 g bt nm men vo 49 ml nc ct (37 oC), lc u v a vo t m (37 o C) hot ha 20-30 pht. Bc 3. Dch bt nm men c ly ra t t m c pha long gp 4, 8, 16, 32 ln. Bc 4. Chn pha long sao cho trn mt bung m c 5 10 t b o/1 ln. Bc 5. Chn 5 ln 4 gc v 1 ln trung tm v m 5 n y. Bc 6 . Trong mt ln, m t bo nm men quy nh theo ng dzch dzc t tri sang phi ca di cc nh v n u t bo nm men nm trn cnh bn phi v cnh y ca vung nh th thuc v . Bc 7 . Ly s trung bnh cng ca s t b o nm men trong mt ln (A) ri tnh theo cng thc trn ta c s lng t bo nm men c trong 1 g bt nm men.

Hnh 5.7. Cch cho dch vi sinh vt vo bung m

Lu : Mu cn m l bt nm men d c sai s do cht mang l tinh bt d nhm ln vi t bo nm men

5.3.2. Ln men cm ru Bc1. Vo go np trng hay n p lt ( ngm c 30 60 pht), thm nc v a ln b p nu. Bc 2. Khi cm si, vn bp in nh li v lt ln bp 1 ming amiante m cho ti khi cm chn. Bc 3. Xi cm ra mt ci thau nha sch, ngui ni lng gi. Bc 4. Khi cm np ngui n 35 40 oC (m m), ta rc bt nm men vo vi t l 35 40 t t bo/1kg cm np v o u bng bao tay nylon hay a tre. Lu : khng nn o qu nhiu lm nho cm th ln men s khng t.
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Bc 2 . Dng vit lng phn mt y Petri thnh 4 phn bng nhau hay dng my m khun lc (nu c). Bc 3. Mi mt khun lc m c nh du mt chm mc b ng bt m khun lc Bc 4. Khi c tng s s khun lc cc a Petri th p dng cng thc (*) suy ra s lng VSV c trong mt n v trng lng hay th tch thc phm. Mt tng vi khun hiu kh trn 1 g m u c tnh nh sau:
{EMBED Equation.3} (*)

Trong :

A l s t bo vi khun /1g m u N l tng s khun lc trn cc a chn n i l s lng a cy ti m t pha long V l th tch d ch cy vo Petri fi l pha long tng ng

V d : Phn tch tng s vi khu n hiu kh trn 1 gram th t heo sng qua bo qun 3 ngy 10 oC. Kt qu: pha long (fi) a s 1 (n1) a s 2 (n2) p dng cng thc, ta c kt qu: {EMBED Equation.3} 10-3 224 235 10-4 20 26

Hnh 5.5. m khu n l c TS VKHK (Mi trng cao tht-pepton) Cc kt qu ca t ng s vi khu n hiu kh thng c biu din di dng s m ca c s thp phn. Trng hp khun lc vi sinh vt mc loang, mi vt loang c tnh l mt khu n lc. Nu pha long cao nht, s khu n lc m c trn 1 a > 250, v d nng 10-4 s m > 250 th kt qu ghi: > 2,5 x 10-6 CFU/g. Nu pha long th p nht, s khun lc m c trn 1 a < 25, v d nng

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Petri c > 250 khun lc Petri c 25 250 khun lc Petri < 25 khun lc Petri c khun lc mc loang

4 5 6 7 8 9
STT

ng Saccharose Cn Go np lc vin nm men Mng bao PVC (wrap PVC) Ti nilon PP/PE
TN DNG C

95 0+ 700

Bao thc n Lo i 1 kg
QUY CCH

60 g 1+2 lt 1 kg 10 vin 1 cun 100 g

SL/VT

SV chun b, 2 t SV chun b, 1 t SV chun b, 1 lp

GHI CH

B. DNG C :

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
STT

ng nghim Hp petri Bnh tam gic ng ht ng ht Qu bp cao su Bnh tia n cn Cc th y tinh Cc th y tinh Cc th y tinh Gi ng nghim Gi ng ht a th y tinh Giy lau knh Bung m hng cu Lame + lamelle Cn ng h a tre Chu Ci v chy N i ng ht nh git Pipetman + u tp
TN THIT B

=18mm =100mm 250 ml 1ml 10ml 500ml 100ml 250ml 1000ml

2kg Di va va 1 ml
QUY CCH

80 ci 80 ci 22 ci 30 ci 1 ci 1 ci 1 ci 1 ci 2 ci 1 ci 1 ci 1 ci 1 ci 1 ci 2 t 1 ci 2 hp 2 ci 1 i 10 ci 1 b 1 ci 5 ci 1 + 5 ci
SL/VT

lu nhn t bu i 1 1t 1t 1t 1t 1t 1t 1lp 1t 1t 1t 1t 1t 1 lp 1t 1t Lu bu i 5, tr bu i cu i 1 t 1t 1lp 1t

GHI CH

C. THIT B :

1 2 3 4 5 6 7 8

Ni hp tit trng T m T lnh Cn in t Bp in Cn ng h Knh hin vi My m khun lc

1 ci 1 ci 1 ci 1 ci 1 ci 1 ci 1 ci 2 ci

1lp 1lp 1lp 1lp

1 t 1 t 1 t
1lp

5.3. Cc bc tin hnh 5.3.1. nh lng VSV 5.3.1.1. Phng php m gin tip Phng php m khun lc Bc 1. Sinh vin ly kt qu t cc a Petri cy tri (kt qu bi 4), chn m a Petri c s lng 25 250 khun lc.

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Qu trnh ln men ethanol nh nm men l c s ca vic sn xut cc loi ru, bia, cn v glycerin. Qu trnh ny cn c ng dng trong sn xut bnh m (lm n bt m ) v mt s loi nc gii kht. 5.1.3.1. C ch qu trnh ln men ethanol Glucose
EMP

Tinh b t { EMBED Equation.3 } Dextran { EMBED Equation.3 } Pyruvate Acetaldehyde

pH 4 - 5

Ethanol

Khi phn hu ng trong iu kin k kh, VSV chuyn hydro t NADH n acetaldehyde to thnh ru ethanol ti to NAD+. Sn phm to thnh l ethanol v CO2 Phng trnh phn ng :
NADH NAD+

C6H12O6

CH3COCOOH

CH3CHO

C2H5OH + CO2 + 113,4 Kj

Cc VSV tham gia trong qu trnh ln men ru ngoi ging Saccharomyces (S. cerevisiae, S. ellipsoideus), Schizosaccharomyces cn c nm mc Mucor (M. rouxii) hay vi khu n Sarcina (S. ventriculi)... 5.1.3.2. Quy trnh ln men cm ru Np trng Vo go Nu ngui (35 40 oC) Phi trn Nghin mn Men ngt Np lc (Ngm 30 60 pht) Vo go

(30 oC, 3 4 ngy) Sn phm cm ru 5.2. Dng c, ha cht v thit b Bng 5.3. Dng c, ha cht v thit b bi 5
A. HA CHT: STT TN HA CHT QUY CCH SL/VT GHI CH

1 2 3

Nc ct Bng khng thm nc Bng thm nc

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6 lt 1,0 kg 0,5 kg

Lu t u

Phng php m khun lc c thc hin bng k thut hp hay hp tri vi cc thit b h tr (my m khun lc, bt m khun lc) c kt qu. Trong phng php ny cn thc hin nhiu pha long theo b c 10 vi mt t bo thch hp c c s lng t bo ring r trn mi trng thch vi s lng ln hn ch sai s khi m v tnh ton. Mt t bo trn 1 a qu ln, cc khun lc s mc chng cho ln nhau khng th m c. Nu mt t bo trn 1 a qu nh, s khng c gi tr thng k. S lng khun lc ti u trn 1 a c ngh bi cc c quan c uy tn (FDA, AOAC) l 25 250 khun lc/a. 5.1.2.1. Phng php o c Mt VSV c th c xc nh mt cch gin tip thng qua cn quang ( c) ca t b o. T bo VSV cn cc chm sng (, 550 610 nm) chiu qua v mn chn nh sng pha sau s nhn c nh sng t hn so vi cc chm sng chiu n. T l nh sng nh n trn nh sng chiu i cho ta c gi tr mt quang (OD_Optical density) tuyn tnh vi mt t bo VSV c trong mu. Dng ng tng quan tuyn tnh theo v d sau : Ly dch huyn ph t b o pha long cc cp khc nhau c c cc gi tr theo b ng sau: Bng 5.2. Cc gi tr tng quan gia mt quang v s lng t bo vi sinh vt pha lo ng (ln) OD610nm Mt t bo m trc tip (Triu t b o/ml) Nc ct 0 0 5 0,16 5 4 0,28 10 3 0,44 15 2 0,61 20 1 0,71 25

Mt t bo ca ng tuyn tnh c th c m trc tip bng bung m hay knh hin vi hunh quang.

Hnh 5.4. th ng tng quan tuyn tnh gia mt quang v s lng t bo VSV

ng tuyn tnh c th hin bng h m s y = ax + b (x l gi tr OD ca mu cn xc nh, y l kt qu mt t bo c trong mu) 5.1.3. Ln men ethanol Ru ethanol l mt loi sn phm ln men ng ph bin ca nhiu nhm VSV. Song tc nhn ln men ch yu l n m men, c bit l loi Saccharomyces cerevisiae. Nhiu loi vi khu n k kh v hiu kh ty tin cng c kh nng to thnh ru nh l mt sn phm ch yu hay m t sn phm ph ca qu trnh ln men hexose v pentose.

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Loi li m (Grid type) Thoma

Chiu cao (h) 0,1

Kch thc vung nh (mm) 0,05

Bung m (Chamber)

Li m (Grid)

Semen

0,01

0,10

Neubauer ci tin

0,1

0,05

Brker

0,1

0,05

{ INCLUDEPICTURE { INCLUDEPICTURE "http://www.evolution. "http://www.evolution.unibas.c unibas.ch/ebert/lab/img h/ebert/lab/img/thoma.jpg" \* /thoma.gif" \* MERGEFORMATINET } MERGEFORMATINE T} { INCLUDEPICTURE { INCLUDEPICTURE "http://www.evolution. "http://www.evolution.unibas.c unibas.ch/ebert/lab/img h/ebert/lab/img/semen.jpg" \* /semen.gif" \* MERGEFORMATINET } MERGEFORMATINE T} { INCLUDEPICTURE { INCLUDEPICTURE "http://www.evolution. "http://www.evolution.unibas.c unibas.ch/ebert/lab/img h/ebert/lab/img/neubauer2.gif" /neubauer.gif" \* \* MERGEFORMATINET } MERGEFORMATINE T} { INCLUDEPICTURE { INCLUDEPICTURE "http://www.evolution. "http://www.evolution.unibas.c unibas.ch/ebert/lab/img h/ebert/lab/img/semen.jpg" \* /buerker.gif" \* MERGEFORMATINET } MERGEFORMATINE T}

m trc tip bng knh hin vi hunh quang quan st v m trc tip trn knh hin vi hunh quang, ngi ta phi nhum VSV bng cc cht pht hunh quang nh: acridin cam (AODC), 4,6-dianidino-2-phenyl indol (DAPI), flourescent isothiocyanate (FITC). S m nhn c t phng php ny lun cho kt qu cao khong gp i so vi phng php m gin tip bng khun lc. S khc bit ny phn nh s chn lc ca mi trng, iu kin nui cy VSV, mt phn t bo b cht hay b tn thng khng th mc th nh khun lc c. S lng VSV m trc tip bng knh hin vi tng ng vi s lng t bo thc t c trong mu.

5.1.2. Phng php nh lng gin tip 5.1.2.1. Phng php m khun lc Khc vi phng php m trc tip, phng php m khun lc cho php xc nh s lng t bo VSV cn sng hin din trong mu. T b o sng l t b o c kh nng phn chia v to th nh khun lc trn mi trng chn lc. Do vy, phng php ny cn c tn l phng php m khun lc (colony count) hay m a (plate count). Phng php ny c c im l cho php nh lng chn lc VSV tu thuc vo mi trng v iu kin nui cy.
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Bung m hng cu l mt phin knh dy 2 - 3 mm c mt vng a m nm gia phin knh v c bo quanh bi hai ng rnh hai bn. a m c hnh trn, thp hn b mt ca phin knh khong 1/10 mm v th khi ph ln 1 l knh th su ca a m ng u nhau. Bung m Neubauer ci tin c 16 hoc 25 ln h nh vung, mi ln gm 16 nh hnh vung .

Hnh 5.3. Bung m Neubauer ci tin Chiu cao bung m (h): Cnh 1 vung nh: Din tch ca 1 vung nh: Din tch 1 vung ln (S): Th tch 1 vung ln (V): h = 0,1 mm 50 m = 0,05 mm = 1/20 mm (1/20) mm x (1/20) mm = (1/400) mm 2 (1/400) mm 2 x 16 = (1/25) mm 2 (1/25) mm 2 x 0,1 mm = (1/250) mm3

Khi thc hin quan st VSV cn pha long m u sao cho mi ln trong bung m khong t 5 10 t bo VSV. Sau nh 1 git mu ln bung m v y li bng l knh, quan st vt knh 10X tm ng li m ri chuyn sang vt knh 40X. Chn 5 ln, gm 4 4 gc v 1 gia. Cc c chn phi c s lng mang tnh i din cho c bung m. Kt qu c bao nhiu ly s trung bnh cng ca 5 (A). S t b o c trong 1 ml mu ban u c tnh theo cng thc: { EMBED Equation.3 } (T bo/ml) Trong : A. - s t bo trung bnh trong 1 vung ln. 1000 - h s chuyn i t mm3 sang ml (1 ml = 1 cm 3 = 103 mm3) F h s pha long ca mu trc khi m V Th tch ca 1 vung ln

Bng 5.1. Cc loi bung m

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Hnh 5.2. Cu to bung m Neubauer ci tin

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BI 5.

PHNG PHP NH LNG VI SINH VT LN MEN ETHANOL

Mc tiu: Sau khi h c xong bi ny, sinh vin c kh nng: nh lng vi sinh vt bng mt s phng php phn tch gin tip v trc tip Thc hin qu trnh ln men ethanol Nh n bit cc du hiu ln men ethanol

5.1. C s l thuyt 5.1.1. Phng php nh lng vi sinh vt S hin din ca VSV c th c nh lng bng nhiu phng php khc nhau, nh: m s lng t bo trc tip trn bung m hng cu bng knh hin vi quang hc ho c bng knh hin vi hunh quang. m gin tip bng s lng khun lc mc trn mi trng xc nh hoc o c (mt quang) ca t bo

Sau y l mt s phng php thng dng nh lng VSV: 5.1.1.1. Phng php nh lng trc tip Vi sinh vt n bo c kch thc ln nh: nm men, to c th c nh lng bng cch m trc tip bng bung m trn knh hin vi. Phng php ny c u im l c th xc nh c ngay lng t b o trong mu, nhng li c nhc im l khng xc nh c t bo cn sng hay cht, d lm t b o VSV vi cc ht vt th khc c trong mi trng v dch huyn ph VSV phi c mt t bo ph i ln. m trc tip bng bung m hng cu

Hnh 5.1. Bung m hng cu

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4.4. 4.5.

Yu cu vit bo co Nu nguyn tc ca k thut ly m u dng rn, lng, v bn rn Trnh by cc quy trnh cc bc t ly m u, ng nht m u, pha long v cy mu. Phn bit s khc nhau c a k thut h p v hp tri. Tiu ch nh gi Bng 4.3. Tiu ch nh gi bi 4

STT 1 2 3 4 5 An ton, v sinh Th i gian Chun b Thao tc : 6

Tiu ch nh gi thc t chc, k lut

im 1 1 1 1 0,5 0,5 0,5 0,5

Pha ch mi trng ng Ly mu ng k thu t Pha long ng k thut, chun xc. Cy m u ng k thut

Kt qu : - m khun lc cc a cy Tnh kt qu ng Bo co - Hnh thc trnh by bo co - Ni dung bi bo co y yu cu ra TNG

1,0 1,0 0,5 1,5 10

4.6.

Cu hi n tp

1. Hy nu cc nguyn nhn lm sai lch kt qu thng gp khi phn tch VSV ? 2. Trong k thut phn tch tng s VKHK, ti sao mi trng thch c 45 oC. 3. Trong ch tiu phn tch tng s NM-NM, ti sao ta khng s dng k thut hp m li s dng k thut hp tri ? 4. Khi s dng mi trng Czapek-Dox thay th mi trng DRBC trong phn tch tng s NM-NM c nh hng n kt qu phn tch khng ? Gii thch ?

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Bc 3. t a Petri trn mt phng ngang, xoay nh a Petri theo hai chiu ngc nhau mi chiu t 3 5 ln dch VSV dn u mt y ca a Petri v trn u trong mi trng cy. Bc 4. y a Petri, ng t nhin. Bc 5. Gi giy v nhit thch hp.

Hnh 4.5. Phng php phn tch vi sinh bng k thut hp

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Bc 5 Bc 6

Dng pipetteman vi u tip v trng (hay pipette 1ml v trng) ht 1ml d ch mu vo ng nghim (theo hnh) c cc pha long tip theo. Trn mu trong ng nghim cho u b ng my vortex mixer hay lc m nh bng tay ta c cc pha long.

4.3.2. K thut hp tri S dng cc a Petri cha mi trng Cao tht-pepton v Czapek-Dox hp kh trng. Cc Petri cha mi trng Cao tht pepton dng phn lp vi khun. Cc Petri cha mi trng Czapek-Dox dng phn lp nm men hay nm mc. Cc bc thc hin: Bc 1 . Dng micropipette v u tip v trng ht 0,1 ml d ch VSV ln b mt mi trng thch a trong khng gian v trng. Bc 2. Nhng u que trang trong cc thu tinh cha cn 70 o, t trn ngn la n cn kh trng. u que tri ngui trong khng gian v trng (gn ngn la n cn). Bc 3. M a Petri, dng que trang gt u git VSV trn b mt thch. Dng que trang gt xoay u trn b mt thch. Trong khi gt, xoay a Petri ti lui 3 4 ln, mi ln chu vi cho dch VSV c tri u khp trn b mt mi trng. Bc 4 . Rt que trang khi a, y a, gi giy v nhit v thi gian thch hp theo tng i tng VSV mc tiu.

Hnh 4.4. M hnh k thut hp tri 4.3.3. K thut hp - Pha ch mi trng cao tht-pepton nh bi 2. - Chu n b Petri, gi giy khng thm nc. - Chu n b ng nghim th ch, gi giy khng thm nc. Hp kh trng Sau khi hp kh trng, thc hin cc bc sau: Bc 1. Dng micropipette v u tp v trng hoc pipette v trng chuyn 1 ml dch VSV ln a Petri v trng cha c mi trng. Bc 2. khong 15 20 ml mi trng nng chy 45 oC vo a Petri cy ging VSV.

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9 10 11
STT

Cn Nc ma Giy nhm
TN DNG C

95 0+ 700

1+2 lt 100 ml 1 cu n

1 lp, SV chu n b

B. DNG C : QUY CCH SL/VT GHI CH

1 2 3 4 5 8 7 10 13 15 16 17 18 19 20 21 22
STT

ng nghim Hp petri Bnh tam gic ng ht ng ht u tp Micropipette Qu bp cao su Que tri n cn Cc th y tinh Cc th y tinh Cc th y tinh Gi ng nghim Gi ng ht a th y tinh Bnh tia
TN THIT B

=18mm =100mm 250 ml 1ml 10ml 1000 l 1000l

100ml 250ml 1000ml

80 ci 80 ci 22 ci 30 ci 2 ci 80 ci 1 ci 1 ci 1 ci 1 ci 3 ci 2 ci 2 ci 1 ci 1 ci 1 ci 1 ci
SL/VT

lu nhn t bui 1

lu t bui 3 1t 1t 1t 1t 1t 1t 1lp 1t 1t 1t 1t
GHI CH

C. THIT B : QUY CCH

2 3 4 5 6 7 9 10 11

Ni hp tit trng T sy T cy T m T lnh B iu nhit Memmert Cn in t Bp in My m khun lc

C gi

1 ci 1 ci 1 ci 1 ci 1 ci 1 ci 2 ci 10 ci 1 ci

1lp 1lp 1lp 1lp 1lp 1lp 1lp 1lp 1lp

4.3.

Cc bc tin hnh

4.3.1. K thut pha long m u Mu th c phm c th l dng rn hoc dng lng Bc 1 Bc 2 Bc 3 Cn 10,0 g (hoc 25,0 g) ho c ht 10,0 ml (ho c 25,0 ml) mu vo bnh tam gic c 90 ml (hay 225 ml) SPW. Nu l m u rn th ng nht mu b ng my dp m u (stomacher) hay xay m u trong ci v trng ti a 2,5 pht. Lc u mu trong bnh tam gic t nht 2 pht ta c mu c pha long 10-1 { EMBED ACD.ChemSketch.20 } Hnh 4.3. K thut pha long
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thnh nhng khun lc ring bit. T chng ta c th gin tip xc nh c mt t bo hay bo t g i chung l CFU (Coloning Form Unit) c trong th c phm cng nh phn lp to dng thu n vi sinh vt. 4.1.4. K thut hp tri (spread plate) L k thut pha long theo bc 10 dch cha VSV thnh cc mc pha lo ng khc nhau v chuyn 0,1 ml dch cc mc pha lo ng ln b mt mi trng thch. Sau thi gian , mi mt t bo s pht trin thnh mt khun lc.

Hnh 4.1. K thut hp tri Ch : Ngi ta cng thng dng k thut ny nui cy, phn lp hay phn tch tng s nm men-nm mc 4.1.5. K thut hp (pour plate) L k thut pha lo ng ging VSV nh k thut hp tri, nhng k thut ny chuyn 1ml dch pha long ln hp Petri v trng, sau mi trng nng chy 45 oC (trong b n nhit) ln a v xoay vng theo hai chiu ngc nhau vi ln cho cc t bo phn tn vo mi trng. Sau thi gian , mi t bo s pht trin thnh mt khun lc mc di v trn mi trng thch.
{ INCLUDEPICTURE "http://www.practicalbiology.org/data/images/originals/making-apour-plate-adding-agar-600-329.jpg" \* MERGEFORMATINET }

Hnh 4.2. K thut hp Ch : K thut hp thng c p dng trn i tng vi khun, v d nh phn tch ch tiu tng s vi khun hiu kh 4.2. Ha cht, dng c v thit b Bng 4.2. Dng c, thit b v ha cht bi 4
A. HA CHT: STT TN HA CHT QUY CCH SL/VT GHI CH

1 2 3 4 5 6 7 8

DRBC Cao tht Pepton Agar NaCl Nc ct Bng khng thm nc Bng thm nc
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1500 ml 10 g 40 g 60 g 200 g 6 lt 1,0 kg 0,5 kg

Lu t u

Tin hnh kim nghim. Trng hp c nhiu mu hng cha kim nghim ngay mt lc th phi m bo iu kin bo qun sao cho vi sinh vt trong thc phm khng b thay i cho n khi kim nghim. Trong trng h p m u c khi lng ln hoc th tch ln, ta cn tin hnh ly m u trong iu kin an ton vi sinh vt trnh cho vi sinh vt t mi trng xung quanh nhim thm vo m u va ly. Ly m u thc ph m d ng lng

4.1.1.4.

Cc loi thc phm d ng lng, nh nc ch m, nc mm, nc tng, du n thng c cha ng trong cc thng to hay bn th dng ng cao su sch, kh em cm vo nhng v tr trn, di, gia, bn cnh ht . Sau trn u cc m u , r i mi ly lng m u chnh xc phn tch. i vi cc loi th c phm lng ng gi, chai, hp mu ly s gi nguyn bao b. Sau khu y ho c lc u cho k trc khi ht. 4.1.1.5. Ly m u thc ph m d ng rn Thc phm l mt khi rn to, ng nht, ly mu cc gc, trong gia. Sau , mu c em nghin, trn u, ri mi em cn v cho vo ti dp m u. Thc phm dng bt em trn u, cn v cho vo ti dp m u. i vi cc nguyn liu v sn phm thc phm d ng rn, nh go, c ph, ht tiu, tr, c th dung cy xm hay d ng c chuyn d ng khc ly mu trn, di, gia, cnh cc bao. Ri c chia m u trung bnh trong cc my chia m u. Trng hp thc phm c ln lng khng ng nht Xay nt mu thc phm c ln lng thnh mt khi ng nht. Sau cn mt lng xc nh theo yu cu ca ch tiu phn tch. Thng m u c nh lng l 10 ho c 25 gram. 4.1.2. Ghi kt qu kim nghim Kt qu kim nghim c ghi trong mt phiu kim nghim bao gm: Tn v a ch c quan kim nghim. S th t mu hay m s mu ghi trong s kim nghim . Tn mu thc phm th v tn nh sn xut. Tn c quan ly mu v gi mu. Ngy gi tip nhn mu ti PTN. Trng thi bao b. Yu cu kim nghim. Kt qu phn tch

Ch : Kt qu kim nghim cn ghi theo yu cu kim nghim. Phiu kim nghim do ngi ph trch
k v c ch k duyt ca th trng.

4.1.3. K thut pha long Mu th c phm dng rn c ng nht ho c dng lng s c pha long theo b c 10 trong dung dch pha long SPW hoc BPW. Sau khi pha long d ch pha long s c mt vi sinh vt ph hp cho k thu t pht hin, nh lng hoc phn lp vi sinh vt
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4.1.1.2.

Nguyn tc gi mu v bo qun mu

- Mu thc phm gi kim nghim c gi trong bao b ban u ca n, hoc c cha trong nhng dng c khng lm nh hng n thc phm , tt nht l trong cc bao b kh trng v trong iu kin lnh. - Trng hp thc phm phi gi i xa kim nghim, hoc c nghi vn, tranh chp phi ng gi k, pha ngoi dn giy c ng du ln nt buc hoc kp seal ch cn thn, trnh mu thc phm b nh tro. - Thc phm b h hng phi m bo gi gp n ni kim nghim trong thi gian ngn nht cho kt qu chnh xc tnh trng ca thc phm . - Thc phm gi n PTN phi c phiu yu cu kim nghim km theo nhn dn bao gm: o C quan hoc nh my sn xut. o Ngy, gi ly mu, ni ly mu. o C quan ly mu , l do ly mu. o Ni gi kim nghim. o Yu cu kim nghim. o Bin bn ly mu : Tn kim nghim vin ly mu , c quan ly mu. Nh my sn xut v a ch. L do ly mu. Loi hng v lng hng ly mu. Lng mu hng. Nhng li ch dn cn thit. Ch k ca i din hai bn hu quan. Ch :
Trng hp mu hng hng khi xc nh r bng trng thi cm quan, ch ly mu hng khi ch hng khng xc nhn s h hng ca hng. Trng hp c s khiu ni v kt qu kim nghim, mu ly li phi tin hnh k hn, t l ly mu phi cao hn. Mu ly kim nghim phi gi li 40% lm i chng khi c khiu ni thi hn lu mu t 1 n 3 thng, ty theo mc d h hng ca mu hng.

4.1.1.3. -

Chun b th mu

Thc phm khi a n PTN cn tin hnh theo trnh t sau: Kim tra bao b, xem c hp l hay khng. Kim tra li phiu gi kim nghim , bin bn ly mu , dn nhn hoc seal ch, xc nh loi thc phm Xc nh yu cu kim nghim. Vo s nhn mu hng kim nghim vi cc li ch dn ghi ch cn thit.

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BI 4.
Mc tiu:

K THUT PHN TCH VI SINH VT

Sau khi h c xong bi ny, sinh vin c kh nng: Th c hin c th nghim ly m u phn tch Th c hin c k thu t pha long bc 10 Th c hin c k thu t hp tri v hp

4.1. C s l thuyt 4.1.1. Phng php l y m u thc ph m Ly mu nguyn liu hoc sn ph m thc phm xc nh cht lng bng cm quan v phn tch trong phng th nghim (PTN) l khu u tin rt quan trng trong cng tc phn tch, gp ph n vo tnh chnh xc c a kt qu kim nghim v x l th c phm sau ny. 4.1.1.1. Nguyn tc ly mu Mu thc phm phi c tnh cht i din cho c m t l hng thc ph m ng nht.

L hng ng nht: l l hng bao gm nhng sn phm c cng mt tn gi, cng m t loi phm cht v cng mt khi lng, ng trong bao b cng 1 kiu, cng mt kch thc, sn xut trong cng mt thi im nht nh theo cng mt quy trnh cng ngh. - Trc khi ly mu trung bnh, ph i xem xt l hng c ng nht khng v kim tra tnh trng bao b ca l hng . - T l ly mu t 0,5 1,0% ty theo s lng, nhng mi ln khng t hn lng cn thit phn tch. - Mu hng ly a i kim phi l mu trung bnh. Ngha l sau khi chia thnh l hng ng nht, m u s ly u cc gc, cc pha trn di, gia l hng v trn u. Bng 4.1. Lng m u ti thiu c n thit phn tch STT 1 2 3 4 5 6 7 8 9 10 11 12 13 Tn thc phm Tht v cc sn phm ca tht C, tm, cua (nguyn con) Trng Nc mm, nc chm Dm Sa ti Du, m Ru cc loi Bt ng cc, sn phm ca bt Bnh, mt, ko Gia v, mui Phm m u hp, nc gii kht Lng mu 200 500 200 500 5 10 500 750 500 750 500 750 500 750 750 1000 250 500 250 500 50 100 20 50 5 10 n v gram gram Qu ml ml ml ml ml gram gram gram gram hp, chai

Ty theo l hng v yu cu kim nghim, thc phm c th ly nhiu hn mc trn.

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3.4.

Tiu ch nh gi Bng 3.2. Tiu ch nh gi bi 3

STT 1 2 3 4 5 An ton, v sinh Th i gian Chun b Thao tc : 6

Tiu ch nh gi thc t chc, k lut

im 1 1 1 1 1,0 1,0 1,0 1,0 0,5 1,5 10

K thut cy gi ng K thut phn lp vi sinh vt

Kt qu : - Phn lp c khun lc ring bit - ng ria ng k thu t Bo co - Hnh thc trnh by bo co - Ni dung bi bo co y yu cu ra TNG

3.5.

Cu hi n tp

1. Ngi ta phn lp VSV t mi trng t nhin da trn nhng nguyn tc g ? 2. Trong mt nh my ln men bia, do s v k thut, ngi ta lm mt mt ging nm men c kh nng chu c nng ethanol cao. Em hy xut cc bc tin hnh phn lp ging nm men c c tnh trn thu hi v tng tr n . 3. Trnh by phng php phn lp vi sinh vt bng k thut cy ria v k thut pha long kt hp vi cy tri?

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Phn lp vi sinh vt - K thut cy ria trn a thch S dng cc a Petri c chun b t bi 2: Petri cha mi trng Cao tht-pepton v Czapek-Dox hoc DRBC h p kh trng. Cc Petri cha mi trng Cao tht pepton dng phn lp vi khun. Cc Petri cha mi trng Czapek-Dox ho c DRBC dng phn lp nm men hay nm mc.

1. Lc u ng nghim nui cy VSV

2. Kh trng u que cy trn ngn la cho n khi nng

3. M nt bng v kh trng ming ng nghim

4. Nhng que cy vo canh khun ly ging

5. Tit trng ming ng nghim v que cy c VSV trong khng gian v trng

6. Tit trng nt bng v ng nt bng ng nghim

7. Tay tri m np Petri, tay ph i cm que cy ria trn b mt thch

8. Kh trng que cy sau khi cy ria xong

Hnh 3.2. K thut cy ria trn a thch 3.3. Yu cu vit bo co Trnh by nguyn tc phn lp vi sinh vt Trnh by cc k thut gieo cy vi sinh vt

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Cc bc tin hnh 3.2.1. K thut cy ria 3.2.1.1. K thut cy ria trn ng nghim thch nghing S dng cc ng n ghim c chun b t bi 2: ng thch nghing mi trng Cao tht-pepton v Czapek-Dox. ng nghim thch nghing mi trng Cao tht pepton dng cy ria cc ging vi khu n. ng ngim thch nghing mi trng Czapek-Dox dng cy ria ging nm men hay nm mc. Bc 1. 2 ng nghim c cm tay tri, tay ph i cm que cy, ngn t v lng bn tay ca tay phi dng m v gi nt bng. Sau khi kh trng que cy, m nt bng c a ng cha VSV, kh trng ming ng nghim bng cch h qua ngn la kh trng. Bc 2. a que cy kh trng vo bn trong ng nghim hay bnh tam gic v nhng vo canh trng ly m t t sinh khi vi sinh vt m khng u que cy chm vo thnh v ming ng nghim. Bc 3. M nt bng ng th ch cn cy, kh trng ming ng nghim, ri a que cy vo y ng nghim ria theo ng dzch dzc t di ko v hng ming ng nghim. Bc 5. Rt que cy ra, t ming ng nghim v nt bng ri y li. Bc 6. cc ng nghim vo gi, t que cy trn n cn ngay trc khi tr v gi que cy. Bc 7. Dn nhn ln ng mi trng m i c gieo c y, ghi tn vi sinh vt v ngy gieo c y.

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3.2.

Dng c, ha cht v thit b Bng 3.1. Dng c, thit b v ha cht bi 3

A. HA CHT:
STT TN HA CHT QUY CCH SL/VT GHI CH

1 2 3 4 5 6 7 8 9 10

DRBC Cao tht Pepton Agar NaCl Nc ct Bng khng thm nc Bng thm nc Ging vi sinh Giy sticker
A4 c nhiu ming nh QUY CCH

1500 ml 10 g 40 g 60 g 200 g 6 lt 1,0 kg 0,5 kg Gio vin chu n b 8 ming

SL/VT

Lu t u

Lp
GHI CH

B. DNG C : STT TN DNG C

1 2 3 4 5 8 9 10 11 12 13 14 15 16 17 18 19 20

ng nghim H p petri Bnh tam gic ng ht ng ht u tp Pipetman Bnh tia Qu bp cao su Que cy vng Que cy thng Que trang n cn Cc th y tinh Cc th y tinh Cc th y tinh Gi ng nghim Gi ng ht
TN THIT B

=18mm =100mm 250 ml 1ml 10ml 1000 l 500ml

100ml 250ml 1000ml

80 ci 80 ci 22 ci 30 ci 1 ci 80 ci 1 ci 1 ci 1 ci 1 ci 1 ci 1 ci 1 ci 2 ci 1 ci 2 ci 1 ci 1 ci
SL/VT

lu 1t Lu bui 3 1t 1t 1t 1t 1t 1t 1t 1t 1t 1lp 1t 1t
GHI CH

C. THIT B : STT

QUY CCH

1 2 3 4 5 6 7 8 9

N i hp tit trng T sy T c y T m T lnh B iu nhit Cn in t Bp in L vi ba

1 ci 1 ci 1 ci 1 ci 1 ci 1 ci 2 ci 10 ci 1 ci

1 lp

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Quay ngc ng mi trng cho ming ng nghim xung di trnh vic nhim khun lc gieo cy. a ng nghim c vi sinh vt m su vo d c theo ng th ch cho n gn y ng nghim.

Lu : Khi cm que cy vo cng nh khi rt que cy ra, ch phi gi que cy thng, nh nhng v khng lm cho mi trng b nt n. 3.1.2. Phn lp vi sinh vt VSV tn ti trong t nhin dng qun x gm nhiu qun th cc lo i VSV. Vic phn lp 1 lo i nht nh trong qun x VSV d a vo kh nng phn tch cc t b o ra thnh ring l trn mi trng chn lc. Hu ht cc phng php phn lp v thu n chng VSV u da trn mt s k thut pha long phn tch t bo VSV kt hp iu kin nui cy chn lc to u th tng trng cho chng quan tm. Hin nay, c cc k thut ph bin sau: 3.1.2.1. K thut hp ria (streak plate) L k thut phn tch hn hp VSV bng cch ria trn b mt a thch sao cho cc t bo ring r nhau ra. Sau khi c trong mt thi gian, mi t bo s tng trng thnh mt khun lc ring bit. Khu n lc ny hnh thnh do s sinh sn, phn chia t mt t bo ban u. Trong k thut ria, c nhiu cch ria khc nhau v khng c k thut no hon ho tuyt i. Hiu qu ca k thut phn ln ph thuc vo ngi cy. Sau y l mt s k thut ria thng dng:

K thut ria ch T (T streak)

K thut ria lin tc (continuous streak)

K thut ria 4 gc (quadrant streak)

K thut ria tia (radiant streak)

Hnh 3.1 Cc kiu ria trn mi trng thch Ch : Sau mi vng ria, ngi ta phi tit trng que cy trn ngn la n cn. K thut hp tri (hc bi 4)
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BI 3.
Mc tiu:

K THUT GIEO CY V PHN LP VI SINH VT

Sau khi h c xong, sinh vin c kh nng: 3.1. Thc hin cc k thut gieo cy vi sinh vt Thc hin cc k thut phn lp vi sinh vt C s l thuyt

3.1.1. K thut gieo cy vi sinh vt Mc ch ca vic gieo cy VSV nhn ging vi sinh vt; pht hin s c mt c a vi sinh vt trong cc mu (th c phm, bnh phm, dc phm, m phm, nc, t...) cn phn tch; nghin cu cc c tnh hnh thi, sinh l, sinh ha ca i tng VSV mc tiu. Cy ging t mi trng lng sang ng nghim cha mi trng lng Dng que cy vng v lm theo trnh t cc bc nh sau: ng nghim c cm tay tri, tay ph i cm que cy, ngn t v lng bn tay ca tay phi dng m v gi nt bng. Sau khi kh trng que cy, m nt bng, kh trng ming ng nghim b ng cch h qua ngn la. a que cy kh trng vo bn trong ng nghim hay bnh tam gic v nhng vo canh trng ly m t t canh trng ch a vi sinh vt m khng u que cy ch m vo thnh v ming ng nghim. u que cy c dnh VSV c gi khng gian v trng gn ngn la n cn. Dng tay tri l y ng nghim m i, m nt bng, kh trng ming ng nghim, r i a que cy vo bn trong ng nghim nhng vo canh trng v khuy nh que cy. Rt que cy ra, t ming ng nghim v nt bng ri y li. cc ng nghim vo gi, t que cy trn n cn ngay trc khi tr v gi que cy. Dn nhn ln ng mi trng mi c gieo cy: ghi tn vi sinh vt v ngy gieo cy.

Ch : Khi cy chuyn t ng nghim ny sang ng nghim kia c th c m hai ng nghim cng mt lc trn cng bn tay tri. Cy ging t ng nghim thch nghing hay mi trng lng sang ng nghim thch nghing Thng s dng que cy vng. Cc bc tin hnh ging k thut cy ging t mi trng lng sang mi trng lng, nhng ch khc bc 4. Sau khi ly c sinh kh i vi sinh vt vo u que cy ri, ta tin hnh a u que cy vo trong ng nghim v cy ria theo ng dzch dzc t y ng ln. Cy m su trn ng nghim thch ng Ngi ta s dng que cy th ng m su vo trong mi trng thch ng. Cc bc tin hnh cng tng t nh mc 1.1 v 1.2, nhng c im khc nh sau:

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2.5.

Tiu ch nh gi Bng 2.5. Tiu ch nh gi bi 2

STT 1 2 3 4 5 An ton, v sinh Th i gian Chun b Thao tc : 6

Tiu ch nh gi thc t chc, k lut

im 1 1 1 1 1,0 1,0 1,0 1,0 0,5 1,5 10

Pha mi trng khng b chy kht, ln cn hoc kt ta. Lm tiu bn cho hnh nh quan st m u p

Kt qu : - Mi ng trn a u, phng, khng vn c. Mi trng sau khng b nhim VSV Bo co - Hnh thc trnh by bo co - Ni dung bi bo co y yu cu ra TNG

2.6.

Cu h i tho lu n

(1). Trong k thut nui c y VSV, mi loi hoc mi nhm VSV khc nhau, t i sao cn ph i pha ch cc lo i cht khc nhau v hm lng cng khc nhau? (2). Khi pha ch mi trng Czapek-Dox, ti sao phi cn ring thnh phn K2HPO4 v cho vo sau khi trn chung th tch agar nng ch y v th tch hn hp cc cht ha? (3). Hy cho bit ti sao mi trng thch a cn phi c p ngc sau khi lm ngui? (4). Gi i thch ti sao mi trng nui cy cn nn c lm ngui khong 45-55oC trc khi chng c rt vo a Petri?

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Sau khi pha ch mi trng Cao tht-pepton v Czapek-Dox cn ang nng chy, phn phi vo cc ng nghim, mi ng nghim c 7 8 ml mi trng. Sau , lm nt bng bng bng khng th m nc v bt np ng nghim. Sau khi hp tit trng, Cc ng nghim thch nghing s c nghing < 25 o sao cho phn thch nghing khong 1/2 - 2/3 ng nghim. Khng c mi trng chy gn ming v nt bng. yn, ch cho mi trng ng li. em bo qun nhit 4oC. 2.3.7. a Bc 1. a Petri sau khi hp tit trng phi c sy kh Bc 2 . Tt qut, lau khu vc lm vic, lau tay b ng cn 70o C v khi mt bn kh cn ri t n cn . Bc 3. Ch cho mi trng ngui n 45 55o C, dng khn trng h p tit trng cm bnh tam gic mi trng vo cc a Petri vi dy 3 4 mm tnh t y ln trong khng gian v trng gn ngay trn ngn la n cn. Trong qu trnh a, sinh vin khng c ni chuyn v lm cc ng tc m nh gy xo trn khng kh. Bc 4. t a Petri trn mt phng ngang, ch cho ng thch, gi li v b o qu n. Ch : Bnh th ng chng ta phi lm cc thao tc ny trong t cy v trng. 2.4. Yu cu vit bo co Nu vn tt c s l luyt v dinh dng VSV Trnh by d ng c, ha ch t v thit b cn thit trong bi. Nu cc bc tin hnh pha ch mi trng dinh dng c bn, h p kh trng v mi trng vo a Petri. ngh a ca vic bao gi v hp kh trng dng c v mi trng nui cy VSV Nh n xt v th o lun cc nguyn nhn b nhim VSV trong qu trnh a.

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khi h p, mi trng s b y ht ra ngoi do p lc tng khi gia nhit trong qu trnh h p . Cun giy bnh tam gic : Bnh tam gic cng s c cun giy nh phn ca ming ng nghim. Cun giy dng c khc : Cc dng c cng c cun giy nh: cc loi que cy, pipette, cc thy tinh , khn lau

2.3.5. Hp tit trng mi trng v dng c Mi trng hp tit trng: Cc bnh tam gic v cc ng nghim cha mi trng cao tht-pepton, Czapek-Dox Dng c hp tit trng : a Petri, ng nghim, que cy vng, que trang, khn trng... Tt c u c bao bng giy trc khi cho vo ni hp. Cch vn hnh ni hp c (ti PTN): Bc 1. Kim tra mc nc trong khong an ton Bc 2. Bt cng tc gia nhit trc (10 15 pht) Bc 3. t cc dng c v dng c cha mi trng vo bung h p Bc 4. Kim tra v ng cc valve x v valve an ton Bc 5. y np v vn c i xng nhau tng i mt Bc 6. Khi ng h p lc ch 0,4 0,5 kg/cm2 th x kh ln mt cho ti khi ng h p lc ch 0,1 kg/cm2 hay khi thy lung kh x xut hin dng kh trng lin tc th ng valve x Bc 7. Ch cho ng h p lc ch 1 atm hay nhit ch 121 oC, tnh gi v canh 15 pht Bc 8. Ht gi, m valve x kh t t, khi p su t cng gim th m valve cng ln Bc 9. Khi ng h p lc ch 0 kg/cm2 v valve x khng cn thy dng kh x mu trng, m np ni hp v ly dng c v dng c cha mi trng trong ni h p ra. Cch vn hnh ni hp t ng : Bc 1. M np ni hp ra, ly hai ci gi inox nh v 1 ci gi inox ln ra. Bc 2. Cho nc ct vo ni hp sao cho ngp con c cm ng mc nc khong 1 2 cm (bn cnh vng in tr). Sau cho gi inox ln vo ni. Bc 3. Cho cc dng c v mi trng vo cc gi nh, ri a vo ni hp. Bc 4. ng np ni hp v kha valve exhaust. Bc 5. Bt CP bn hng, ci t thng s 121 oC, 15 pht, bm nt set v b m nt start. Bc 6. Khi ni hp c bo hiu tittit ln 2, m t hin th bo nhit 97 oC v c n bo complete chp chp mu , th m ni hp ra, ch 1 pht cho bt nng v ly dng c v mi trng ra. 2.3.6. Lm ng nghim thch nghing ng nghim thch nghing dng nui cy v gi ging theo phng php cy chuyn v bo qun lnh.
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Bc 4. th tch dch cha thnh phn cc mui khong vo th tch dch agar ang si, khuy u v tt bp. Bc 5. Khi nhit mi trng h xung 50 oC, cho K2HPO4 vo hn hp bc 4 v khuy u. Bc 6. Phn phi vo cc dng c cha, lm nt bng khng thm nc, bt np giy , dn nhn tn nhm v tn mi trng, v em i hp tit trng. 2.3.3. Lm nt bng Cch lm ny dng ngn nhim vi sinh vt t ngoi vo mi trng trc vo sau khi cy vi sinh vt. Bc 1 . Dng tay x mt ming lp bng hnh ch nht, gp cc ra ming bng vo trong cho gn khng b x. Bc 2 . Cun cht li thnh nt sao cho va kht ng nghim (khng qu cht, nhng cng khng qu lng). Ngi ta cng c th lm nt bng bng cch cun vi mng bn ngoi v ct li bng mt si ch sao cho c th a nt bng vo v ly ra mt cch d dng (cch lm ny tt nhng phi mt nhiu cng sc v th i gian). 2.3.4. Bao giy dng c : Mc ch l ngn nga vi sinh vt nhim vo mi trng trc v sau khi cy; trnh v dng c bng thy tinh trong qu trnh thao tc. Tt nht l dng giy khng thm nc. V giy thm nc d b rch trong qu trnh thao tc sau khi hp kh trng. Trong phng th nghim vi sinh vt, ngi ta thng cun giy trn nhiu dng c : Cun giy ng nghim : ty theo mc ch ca vic nui cy m nn cun theo tng kiu cho ph hp : o Mc ch l gi ging : Ngi ta cun giy to thnh nh mt np chp c th ly ra v chp vo d d ng. V np chp ny s c dng i dng li nhiu ln sau mi ln ly ging trong ng nghim ra. k thut n y, mp cun sau cng s gp nht hoc c dn bng bng keo, nhng khng dng thun ct. o Mc ch l phn tch : np giy ng nghim s dng mt ln ri b. Nn ta c th cun n gin hn v gi mp giy bng mt si thun. Gi giy a Petri : Bc 1. t 1 hoc 2, 3 ci a trn mt t giy hnh ch nht c ct ph hp. Bc 2. Tm ly hai cnh nh ca hnh ch nht gp cun mp 2 3 ln cho cht va cc a. Bc 3. Sau n hai u mp cn li xung v gp mp h nh tam gic nhn gia nh gi qu. Bc 4. Gp ngc hai hnh tam gic xung sao cho chng a ln n p gp. Cun giy b ng nghim c cha mi trng: B ng nghim thng t 4 6 ng s c cun xung quanh y ng nghim. Chiu cao giy t y ln 1/3 ng nghim v c ct li bng mt si thun. Ph n trn ming ng nghim cng cun tng t nh phn y v h phn gia ca b ng nghim. Cn phn bit c phn u v y ca b ng nghim trnh cho vo ni hp ln ngc v kt qu sau
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3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
STT

Bnh tam gic ng ht ng ht Bnh tia Cc th y tinh Cc th y tinh Gi ng nghim Gi ng ht a th y tinh Qu bp cao su Cc th y tinh Giy bo Thun buc Ko Hp qu t Tm amiang
TN THIT B

250 ml 1ml 10ml 500ml 100ml 250ml

500ml

22 ci 30 ci 1 ci 1 ci 2 ci 1 ci 1 ci 1 ci 1 ci 1 ci 2 ci 2 kg 1g 1 ci 1 ci 1 ci
SL/VT

1t 1t 1t 1t 1t 1t 1t 1t 1 lp SV chun b SV chun b

1 t
GHI CH

C. THIT B QUY CCH

1 2 3 4 5 6 7 8 9

Ni hp tit trng T sy T m T lnh B iu nhit Cn in t Bp in L vi ba pH k

bn

1 ci 1 ci 1 ci 1 ci 1 ci 2 ci 1 ci 1 ci 1 ci

L p L p L p L p L p L p 1 t L p L p

2.3.

Cc bc tin hnh

2.3.1. Pha ch mi trng Cao tht pepton : Bc 1 . Cn agar cho vo th tch nc nh sn cho cc nhm. Sau , nu si trn b p in (c ming amiante) cho agar n ht. Bc 2. Cn thnh phn cao tht, pepton v NaCl ca mi trng theo cng thc cho trc. Sau , ha tan chng vo th tch nc cn li. Bc 3. th tch dch cha cao tht, pepton v NaCl vo th tch dch agar ang si, khuy u v tt bp. Bc 4 . Phn phi vo cc dng c cha, lm nt bng khng th m nc, bt np giy, dn nhn c tn nhm v tn mi trng, v em i hp tit trng. 2.3.2. Pha ch mi trng Czapek Dox: Bc 1. Cn agar cho vo th tch nc nh sn cho cc nhm. Sau , nu s i trn bp in (c ming amiante) cho agar n ht. Bc 2 . Cn thnh ph n khong c kh nng gy ta (K2HPO4) vi cc mui khong khc ring. Bc 3. Cn cc mu i khong cn li ca mi trng theo cng thc cho trc. Sau , ha tan chng vo th tch nc cn li.

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Hp bng nhit m 121 oC, 1 kg/cm2 (1 atm 1 bar 1 kg/cm2) trong 15 30 pht tiu dit hon ton cc dng sng ca VSV. Bng 2.2. Mi quan h gia nhit v p sut p sut (kg/cm2) 0,00 0,25 0,50 1,00 1,50 2,00 Nhit (oC) 100 107 112 121 129 135

2.2.

Dng c, ha cht v thit b

2.2.1. Mi trng v ha cht B ng 2.3. Thnh phn ca m t s loi mi trng nui cy vi sinh vt Mi trng cao tht pepton Mi trng Czapek - Dox
Thnh phn Cao tht Pepton NaCl Agar Nc ct Hm lng 3g 10 g 5g 20 g 1000ml Thnh phn NaNO3 K2HPO4 MgSO4 KCl pH cui Hm lng 2,0 g 1,0 g 0,5 g 0,5 g 7,3 0,2 Thnh phn FeSO4 Sucrose Agar Nc ct Hm lng 0,1 g 30 g 20 g 1000 ml

2.2.2. Dng c v thit b Bng 2.4. Dng c v thit b bi 2

A. HA CHT: STT TN HA CHT QUY CCH SL/VT GHI CH

1 2 3 4 5 6 7 8 9 10 11
STT

DRBC Cao tht Pepton Agar NaCl HCl NaOH Nc ct Bng khng thm nc Bng thm nc Cn
TN DNG C

1500 ml 10 g 40 g 60 g 200 g Dd 20% Dd 20% 20 ml 20 ml 6 lt 1,0 kg 0,5 g 95 0+ 700

QUY CCH

Lu t u

1+2 lt
SL/VT GHI CH

B. DNG C :

1 2

ng nghim Hp petri

=18mm =100mm
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80 ci 80 ci

Lu

L loi mi trng c pha ch sn (c th l tng hp hay bn tng hp) dng kh vi cc thnh phn c nh sn bi nh sn xut. Cho nn, cc loi mi trng ng kh ny h n ch s bin ng cc thnh phn ca mi trng . Ngi ta thng dng nhng loi mi trng ny phn tch VSV. V d : Mi trng nh lng tng s nm men nm mc DRBC (Dichloran Rose Bengal Chloramphenicol, sn phm ca hng Merck). Mi trng hp PCA _Plate count agar (hng Merck cung cp ). 2.1.3. Pha ch mi trng 2.1.3.1. Chun b mi trng lng Cn tng thnh ph n ca mi trng. Ha tan tng thnh phn mi trng trong mt lng nc nh. Sau trn ln tt c tt c cc thnh phn dinh dng vi nhau v thm nc cho th tch xc nh. Kim tra pH mi trng bng pH k v ch nh pH bng dung dch HCl v NaOH. Phn phi mi trng vo cc dng c cha, ng nt bng (bng khng thm nc), bt np giy hay vn np i vi ng nghim c np. Hp tit trng (121 oC, 15 pht) trong ni hp v lm ngui nhit phng, gieo cy VSV ngay ho c em i bo qun trong t lnh ( 4 oC). Chun b mi trng rn v bn rn i vi mi trng c cha cht lm c nh : agar, gelatin cn phi nu chy agar trong th tch nc dng pha mi trng. Cn v khu y tan cc thnh ph n khc ca mi trng trong lng nc cn li. Khi agar tan chy hon ton, trn ln dung dch cc cht dinh dng vi dung dch c cha agar vi nhau, khuy u . Phn phi mi trng ang nng chy vo cc dng c cha. Cc bc tip theo tng t nh mi trng lng

2.1.3.2.

2.1.4. Kh trng dng c v mi trng VSV c th b tiu dit bi cc tc nhn nh : nhit , tia bc x, lc v ha ch t. i PTN vi sinh thng ngi ta kh trng dng c v mi trng bng nhit, trong mt s trng hp khi mi trng c thnh phn d b phn hu bi nhit th ngi ta dng phng php lc. Kh trng bng nhit kh : Cc dng c thu tinh c th c kh trng bng cch sy 170 180 oC trong 2 gi. Cc dng c kh trng dng n y cn c gi v cun giy trc khi cho vo t sy. Cc que cy c th c kh trng b ng cch t trn ngn la n cn. un 63 oC trong 30 pht ho c 72 oC trong 15 pht c th tiu dit c VSV gy bnh hay VSV hoi sinh trong thc phm. un 100 oC c th tiu dit c t bo sinh dng VSV.

Kh trng bng nhit m :

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L mi trng lng dng tng sinh khng c tnh chn lc, lm giu mt ca cc VSV hin din trong mu. Loi mi trng ny giu dinh dng khng c cc cht c ch tng trng chn lc, c tc dng phc hi v gip s tng trng ng thi ca nhiu loi VSV hin din trong thc phm nhng t nhiu b tn thng trong qu trnh ch bin v b o qun thc phm. V d : Nc mui pepton (SPW_ Saline Pepton Water) kch thch s tng trng ca cc loi vi khun ng rut thuc h Enterobacteriaceace trong kim nghim mt s ch tiu an ton v sinh thc phm. Mi trng tng sinh (enrichment media) L mi trng lng dng tng sinh chn lc i tng VSV cn kim nghim v c ch s tng trng ca cc loi VSV khc do c cha cc cht c ch tng trng c hiu. V d: Mi trng Tetrathionate l mi trng tng sinh chn lc cho Salmonella . Mi trng Listeria enrichment Broth I & II (LB I & II) l mi trng tng sinh chn lc cp I v II cho Listeria. Mi trng chn lc (selective media) L mi trng rn dng phn lp cc khun lc n ca i tng cho VSV mc tiu. Cc mi trng phn lp khc nhau c mc chn lc khc nhau. Thng thng phn lp v xc nh VSV, ngi ta s dng vi mi trng c mc chn lc khc nhau. Mc chn lc c th kim sot bng cc cht c ch tng trng, thnh phn mi trng, nhit , oxi V d : Mi trng DRBC l mi trng chn lc nm men n m mc nhng c ch s tng trng ca vi khun. Mi trng phn bit (differential media) L mi trng rn c ch th gip cho s pht hin d dng i tng VSV mc tiu. V d: Mi trng thch mu Trypticase Soy Sheep l mi trng phn bit mt s loi vi khun c kh nng lm tan huyt xung quanh khu n lc, nh Bacillus cereus, B. thuringiensis v B. mycoides vi loi vi khu n khng c kh nng lm tan huyt B. anthracis sau 24 gi. Mi trng chn lc phn bit (selective different media) L mi trng kt hp gia cng dng chn lc v phn bit nh s s dng kt hp cc ha cht khc nhau. V d : Mi trng Manitol Salt Agar cha mui c nng cao (7,5%) gip s tng trng chn lc ca Staphylococcus trong khi c ch s tng trng ca a s cc VSV khc. Mt khc manitol v ch th pH trong mi trng gip nh n din cc khun lc Staphylococcus da vo kh nng ln men manitol sinh acid lm i mu ca ch th phenol. Mi trng th nghim sinh ha L mi trng dng xc nh mt hoc mt vi c im sinh ha ca ca chng VSV c phn lp v lm thun, to c s nh danh chng phn lp. V d : Mi trng MIR (Motility Indol Red) dng xc nh tnh di ng, kh nng to indol v kh nng phn gii urea ca mt chng phn lp. Mi trng ng kh thng phm
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Nng thch hp c a cc g c kim purin, pirimidin cho vi sinh vt vo khong 5 20 g /ml Nng thch h p ca cc vitamin vo khong 0,1 0,5 g/ml (1 g = 10-6 g ) 2.1.2. Cc loi mi trng nui cy vi sinh vt Mi trng ni cung cp y cc thnh ph n v dinh dng (ngun nit, ngun carbon, khong a-vi lng, cht sinh trng...) cho s pht trin ca VSV. Tu theo mc ch m thnh phn, hm lng cc cht trong mi trng l khc nhau. Mi trng c th c phn loi theo cu trc, th nh phn v cng dng. 2.1.2.1. Phn loi mi trng nui cy VSV theo cu trc Mi trng lng Thng dng tng sinh, th nghim sinh ha, nhn ging th cp, ln men ca VSV. Mi trng lng khng cha cht lm c mi trng , nh : agar, gelatin... Vd : Mi trng cao malt ln men bia (ln men chm) Mi trng rn Ngoi cc thnh phn dinh dng, mi trng rn l mi trng c th m thnh ph n lm c (agar, gelatin...). Ngi ta thng dng loi mi trng ny phn lp VSV, quan st khun lc, nh lng VSV (trong phn tch cc ch tiu tng s vi khun hiu kh, tng s nm men-n m mc...) hay nghin cu nhng c tnh khc ca VSV. V d : Mi trng thch Hansen nui cy nm men Mi trng bn rn Ging nh mi trng rn, nhng mi trng bn rn c hm lng cht lm c t hn nn cu trc ca mi trng mm v xp hn. Ngi ta thng dng n trong vic xc nh kh nng chuyn ng ca VSV. V d : Mi trng thch mm Oxford xc nh kh nng di ng ca Listeria monocytogenes 2.1.2.2. Phn loi mi trung nui cy VSV theo thnh phn Mi trng tng hp L loi mi trng c th nh ph n ha hc xc nh, c pha ch t nhng ha cht tinh khit. V d : Mi trng nui cy nm men-nm mc Czapek-Dox Mi trng t nhin L lo i mi trng cha cc th nh ph n c ngun gc t t nhin, khng c thnh ph n ha hc r rng. V d : Mi trng nui cy n m mc cm tru Mi trng bn tng hp L loi mi trng va c mt s thnh ph n ha hc xc nh va c mt s th nh ph n c ngun gc t t nhin gm nhng hn hp cht cha xc nh r. V d : Mi trng nui cy vi khun thch mu c bn, nui cy nm men n m mc PGA (Potato, Glucose, Agar) 2.1.2.3. Phn loi mi trung nui cy VSV theo cng dng
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Mi trng tin tng sinh (preenrichment media)

BI 2.
Mc tiu:

K THUT PHA CH MI TRNG

Sau khi h c xong, sinh vin c kh n ng: - Phn bit cc loi mi trng nui cy v phn tch. - Pha ch c cc lo i mi trng c bn. - p dng c cc phng php tit trng mi trng, dng c - Bao gi dng c - mi trng vo a Petri, lm ng nghim thch nghing 2.1. C s l thuyt 2.1.1. Dinh dng vi sinh vt Mi trng dinh dng ca vi sinh vt bao gm nhiu thnh phn dinh dng cn thit khc nhau. Thnh phn dinh dng ny tu thu c vo nhu cu c a tng loi vi sinh vt. V d : C loi vi sinh vt cn nhiu protein, nhng cn t glucide; C loi cn nhiu nguyn t vi lng, nhng c loi cn t; C loi trong qu trnh pht trin khng th thiu cc cht tng trng. Mi c th sng u c cu to t nhng hp cht hu c. Cc hp cht hu c c cu to t cc nguyn t C, H, O v N gi l cc nguyn t thit yu ca s sng. Nhn chung cc nguyn t thit yu khng th thiu trong qu trnh sinh trng v pht trin c a vi sinh vt. Ngoi ra cc nguyn t thit yu, trong cc c th vi sinh vt cn c cc nguyn t a lng nh: S v P, cc nguyn t vi lng nh: Fe, Cu, Mg, Mn, Zn, K, Ca, Cl, Bo, IV th trong thnh phn dinh dng khng th cung cp n thun mt s ch t no hoc m t nhm nguyn t no m trong t nhin, VSV s dng cc ngun dng cht t c th thc vt, ng vt hoc VSV. Nng ng ph hp nui cy vi khun v x khun khong 0,05 0,20%, n m m c v n m men kho ng 3 15%. Bng 2.1. Nhu cu khong i vi mt s vi sinh vt
Loi mui khong K2HPO4 KH2PO4 MgSO4.7H2O MnSO4.7H2O FeSO4.7H2O Na2MoO4 ZnSO4.7H2O CoCl2 CaCl2 CaSO4.5H2O Nng cn thit (%) Vi khun Vi n m v x khun 0,2 0,5 12 0,2 0,5 12 0,1 0,2 0,2 0,5 0,005 0,01 0,02 0,1 0,005 0,01 0,05 -0,2 0,001 0,005 0,01 0,02 0,02 0,03 0,03 0,06 0,01 0,03 0,02 0,1 0,001 0,005 0,001 0,05

Cc ch t tng trng ch yu l cc vitamin v cc gc kim purin, pirimidin, cc acid bo, cc thnh phn ca mng t bo...
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- Hnh thc trnh by bo co - Ni dung bi bo co y yu cu ra TNG 1.6. Cu hi tho lun

0,5 1,5 10

1. Nu k thut chnh knh hin vi khi quan st trng thi sng v trng thi cht. 2. Sau khi quan st hnh thi ca VSV, em hy nu s khc bit c bn v hnh dng ca vi khun, nm men v nm m c ? 3. Sau khi thao tc vi VSV, ngi ta thng dng cn 70o tit khu n tay v ni lm vic m khng dng c 90o hay 60o, gi i thch ?

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Hnh 1.26. Vi khun Gram dng b t mu tm { INCLUDEPICTURE "http://3.bp.blogspot.com/_X5UZqPW3nj4/RXhFxtYitkI/AAAAAAAAABM/i Abqr0vIV3I/s320/gram+neg.jpg" \* MERGEFORMATINET } Hnh 1.27. Vi khun Gram m (c mu hng ) 1.4. 1.5. Yu cu vit bo co Trnh by cc quy tc an ton trong PTN vi sinh vt Trnh by cc k thut lm tiu b n nhum n v nhum kp (nhum Gram). V kt qu quan st hnh thi ca nm men, nm mc v vi khun Phn bit hnh thi ca ba nhm vi khun, nm men v n m mc Tiu ch nh gi Bng 1.2. Tiu ch nh gi bi 1 STT 1 2 3 4 5 An ton, v sinh Th i gian Chun b Thao tc : 6 iu chnh knh quan st th y mu vt Lm tiu bn cho hnh nh quan st m u p 1,0 1,0 1,0 1,0 Tiu ch nh gi thc t chc, k lut im 1 1 1 1

Kt qu : - V hnh 3 mu n m men, nm mc v vi khun Phn bit c hnh thi c a 3 nhm vi sinh vt

Bo co
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Bc 6. Nhn vo th knh, iu ch nh c ch nh th t t cho n khi thong thy vt th iu chnh c chnh tinh trng r vt. 1.3.2. To tiu bn quan st khu n ty nm mc (hay khun ty x khu n) quan st nm mc hay x khun ngi ta cn to m t trong hai dng tiu bn l tiu bn nhum sng v tiu b n phng m. Nhng vi thi lng th c hnh c gii hn nn chng ta ch th c hin tiu b n nhum sng. Cc bc to tiu bn nhum sng khun ty nm mc: Bc 1. S dng que cy mc ly mt t khun ty nm mc, ra bt bo t bng cch t sinh khi khun ty vo dung dch x phng (nc ra chn) pha long v khuy nh. Bc 2. t sinh khi khun vo git carbofuchsin trn phin knh. Dng hai que cy mc hoc 1 que cy mc v 1 que cy thng tch ri cc si khun ty trong git carbofuchsin trn phin knh v y l knh. Bc 3. t ln bn mu, quan st b ng vt knh 10X tm nh v chuyn qua vt knh 40X quan st chi tit.

1.3.3. Nhum Gram (tiu bn nhum mu kp) Nhum Gram l phng php nhum kp phn bit hai nhm vi khu n Gram dng v Gram m do Han Christian Gram ngi an Mch ln u tin m t vo nm 1844. Khi cng nhum mu t bo theo cng phng php, t bo vi khun G(+) vn gi c mu tm, cn vi khu n G(-) b mt mu tm. Tin hnh: Bc 1 . Cho 1 gi t sinh khi vi khun ln phin knh, c nh vi khun trn phin knh b ng cch h kh vt bi trn ngn la n cn. Ch khng qu gn ngn la lm chy vt bi. Bc 2. Nhum vt bi bng dung dch tm kt tinh, nh 2 git methyl violet ln vt bi, gi 30 giy. Tip theo, dng bnh tia ra sch phm nhum, ri h kh. Bc 3 . Nhum vt bi bng dung d ch Lugol, nh 2 git I2/KI ln vt bi, 1 pht. Sau , dng bnh tia cha cn 95% ra th t sch phm nhum n khi mt ht mu, r i ra li bng nc v h kh. Bc 4. Nhum vt bi bng safranin (hoc carbofuchin), nh 2 git safranin ln vt bi, yn 30 giy. Sau , ra vt bi bng nc, h kh, nh 2 git du soi knh ln vt bi v quan st vt knh 100X.

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2 3 4 5 6 7 8 9 10 11

T sy T cy T m T lnh B iu nhit Cn in t My dp mu My lc (votex) Knh hin vi quang hc L vi ba

1 ci 1 ci 1 ci 1 ci 1 ci 1 ci 1 ci 1 ci 1 ci 1 ci

gii thiu cho hc sinh, khng dng

1t

L p

1.3.

Cc bc tin hnh

1.3.1. To tiu bn quan st vi khun v nm men 1.3.1.1. To tiu bn quan st VSV tr ng thi sng hay cn gi l tiu b n gi t p (quan st VSV v t knh 40X) Bc 1 . Ly mt phin knh sch cho m t git nc hay 1 gi t phm mu methylene blue (0,1%). Sau cho VSV ha tan trong git nc hay git mu long . Bc 2. y l knh sao cho chn ca l knh ch m vo git nc hay git mu, nghing mt gc 45o v h l knh xung khng c b t kh.

Hnh 1.25. Cch lm tiu bn quan st vi sinh v t tr ng thi sng Bc 3. t phin knh ln bn mu, h t quang, ng bt chn sng, quan st vt knh 10X, dng c chnh th nng bn vt mu ch m ti vt knh, t m t vo quan st, dng c chnh th h bn mu xung cho n khi thy nh VSV r i dng nt ch nh tinh vn xem r nh VSV. Trn bn mu, dng c di chuyn m u vt di chuyn phin knh n vng mun quan st trong th trng. Bc 4. quan st nh c phng i ln hn, chuyn sang vt knh 40X v iu chnh c chnh tinh tm nh. Ch : Ngoi nm men v vi khun, i tng quan st cng c th l bo t nm mc... 1.3.1.2. To tiu bn quan st VSV trng thi cht (tiu bn nhum mu n) Bc 1. C nh VSV bng cch cho mt git sinh kh i VSV tri u ln phin knh sch, h trn ngn la n cn cho n khi kh (khng cn t, nhng khng c chy). Bc 2. Cho 1 gi t (ln) phm nhum n (methylene blue 0,1% hoc Carbofuchsin 0,1% ) ln vt VSV c nh trong 5 pht. Bc 3. Ra nc bng u xt ca bnh tia cho n khi sch mu v lm kh b ng ngn la n cn (c th dng giy mm thm nh nc, nhng khng dng giy m m lau). Bc 4. Nh m t git d u cdre ln vt nhum, a ln bn mu. Bc 5. Nng t quang, m chn sng. Nhn t ngoi, h vt knh 100X xung chm vo git d u cdre.
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
STT

Du soi knh Dd lau knh Xylol Dd xanh methylen Lugol(I2/KI) Safranin Methylviolet DRBC Cao tht Pepton Agar NaCl Nc ct Bng khng thm nc Bng thm nc ng Saccharose Cn Ging vi sinh Gi y bo Thun buc
TN DNG C

40 ml 30 ml 1% B nhum Gram 30 ml 20 ml 20 ml 20 ml 1500 ml 10 g 40 g 60 g 200 g 6 lt 0,8 g 500 g 250 g 95 0+ 700 2 + 3 lt

1lp 1lp 1lp 1lp 1lp 1lp

Lu t u

Gio vin chun b 3 kg Vng si

QUY CCH

SV chun b SV chun b
GHI CH

100 g
SL/VT

B. DNG C :

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
STT

ng nghim Hp petri Bnh tam gic ng ht ng ht ng ht nh git Micropipette u tp Que tri Lame + lamelle Gi y lau knh Que cy vng Que cy thng Bnh tia n cn Cc th y tinh Kp vi sinh Gi ng nghim R ng d ng c
TN THIT B

=18mm =100mm 250 ml 1ml 10ml 1000l 1000 l

500ml 100ml

80 ci 80 ci 22 ci 30 ci 2 ci 5 ci 1 ci 1 ci 1 ci 2 hp 20 t 1 ci 1 ci 1 ci 1 ci 2 ci 1 ci 1 ci 2 ci
SL/VT

1lp 1lp 1lp 1lp 1lp 1 lp

lu lu lu lu lu 1 t 1 t

1 lp 1 lp 1lp 1t 1t 1t 1t 1t 1t 1 t 1lp, lu
GHI CH

C. THIT B : QUY CCH

Ni hp tit trng
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1 ci

1lp

Hnh 1.23. Knh hin vi hunh quang Knh hin vi in t: Knh hin vi in t (Transmission electron microscope_TEM) l loi knh khng s dng cc chm tia sng khuych i hnh nh vt quan st qua thu knh nh knh hin vi quang hc. Knh hin vi in t s dng cc chm in t v khuch i hnh nh nh cc cm in t trng, hnh nh c chiu ln m t mn hu nh quang.

Hnh 1.24. Knh hin vi in t Ngi ta dng knh hin vi in t quan st v nghin cu cc bo quan ca vi khu n, nm men, nm si v hnh thi-cu trc ca virus hay cc i phn t sinh hc. 1.2. Dng c, thit b v ha cht Bng 1.1. Dng c v ha cht bi 1
A. HA CHT: STT TN HA CHT QUY CCH SL/VT GHI CH

1.2.1. Dng c v ha cht

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Hnh 1.21. Knh hin vi soi ni Knh hin vi i pha: Knh hin vi i pha (Phase contrast microscope_PCM) c s dng quan st VSV sng khng nhum mu. Khi xuyn qua vt th c dy mng khc nhau, nh sng c cng khng thay i nhng pha b thay i. C th lm tng r c a nh bng cch bin s thay i pha thnh thay i cng nh b phn c bit ca knh.

Hnh 1.22. Knh hin vi i pha Knh hin vi hunh quang: Knh hin vi hu nh quang (Flourescent microscope_FM) l lo i knh c lp b ngun cc nh sng kch thch c di sng khc nhau kch thch to hu nh quang. Ph n ln VSV khng c kh nng pht hunh quang hoc c nhng rt yu. Tuy nhin c th nhum t bo VSV bng cc ph m nhum hunh quang khc nhau. Nh vy c th quan st, theo di chuyn bit bo quan hoc i phn t ang quan tm, trong khi t bo vn sng, tng trng.

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Vi khun c kch thc rt nh m t thng khng th th y c. Cho n nm 1676 Antony Leuwenhook hon thin knh hin vi u tin ca nhn loi v ngi ta c th thy c hnh nh ca cc loi vi khu n. Ngy nay chng ta ch to ra rt nhiu loi knh hin vi khc nhau c th th y hnh nh ca vi sinh vt r nt t ngoi vo trong t bo, thm ch c cc bo quan v i phn t sinh hc (protein, lipid). Knh hin vi quang h c: Knh hin vi quang hc (optical microscope) l h thng dng phng i VSV c kch thc nh. phng i c th t vi ch c ln n 2000 ln. C u to: Th knh: ni t m t nhn. V t knh: ni k st vi m u quan st, tip nhn nh sng t mu vt i qua. Bn mu: ni phin knh c cha mu cn quan st. T quang: Ni tip nh n nh sng t ngun sng (n) hi t li thnh chm sng hp c t quang: iu chnh t quang ln hay xung khi quan st m u nhum mu hay mu sng. c chnh th ( c th cp): nng ln hay h bn mu xung thay i tiu c tc ln. c chnh tinh ( c vi c p): ging nh c chnh th nhng thay i tiu c tc vi cp cho php nhn c nh nt hn.

Hnh 1.20. Knh hin vi quang hc Knh hin vi soi n i: Knh hin vi soi n i (Stereoscopic microscope) l d ng knh lp c phng i thay i t 10 60 ln thng dng v quan st trn nhng vt c kch thc tng i ln. Ngun sng thng c chiu t pha trn xung vt v phn x vo knh, nh vy c th quan st vt th trng thi khng gian ba chiu.
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c dng m bo tnh v trng khi thao tc vi VSV. Khng kh trong t cy v trng c lc bng mng lc v trng. Trong t c n UV (Ultra Violet) kh trng mi trng trong t cy. Trc khi th c hin, bt n UV t 30 - 60 pht, bn ngoi t phi c che li bng mn ti mu.

Hnh 1.17. T c y v trng (8). Ni hp (autoclave) L thit b b t bu c phi c trong PTN vi sinh cho php tiu dit tt c cc d ng sng ca VSV (t bo sinh dng, bo t). Ni h p c cu to 2 lp v nn c kh nng chu c p sut cao. Bung kh trng c gn valve thot kh, p k, nhit k.

Hnh 1.18. Ni hp kh trng Khi s dng nn dng nc ct hay nc v khong chm vo ni, trnh b ng cn. Nc phi c chm n mc quy nh. (9). pH k (pH meter) S dng my o pH bn hay pH k di ng xc nh pH ca mi trng nui cy VSV, d ch ln men hay dung dch ha ch t. Gi tr pH cui ca mi trng nui cy VSV thng c xc nh 25 oC. { INCLUDEPICTURE "http://www.thanglonginst.com/admin/anhnho/mV.jpg" \* MERGEFORMATINET } Hnh 1.19. pH k (9). Knh hin vi (Microscope)
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Hnh 1.12. T s y (3). T m (lab incubator) T m c dng cho mc ch nui cy VSV. T m duy tr c nhit n nh. Di nhit trong t m thng t 0 50 oC. Vi khun thng c nui cy 37 oC. i vi mi i tng VSV cn c iu chnh nhit cho ph h p.

Hnh 1.13. T m (4). My l c (orbital shaker) My lc nh m o trn mi trng nui cy VSV, tng cng oxi tan trong mi trung. c dng nui VSV hiu kh trong mi trng lng. { INCLUDEPICTURE "http://photos.labwrench.com/equipmentPhotos/2000/25714614.jpg" \* MERGEFORMATINET } Hnh. 1.14. My lc (5). B n nhit (water bath) Trong PTN VSV, b n nhit c s d ng duy tr nhit nng ch y ca mi trng thch (45 oC) trc khi a Petri. Hin nay, cn c loi b iu nhit lc (shaking water bath), loi ny cn c tc dng o u mi trng trc khi s dng. { INCLUDEPICTURE "http://img.directindustry.com/images_di/photo-g/shakingwater-bath-375366.jpg" \* MERGEFORMATINET } Hnh 1.15. B n nhit lc (6). My l c ng nghim (vortex mixer) Dng o trn d ch VSV cn phn tch hay dung d ch ha cht cha trong ng nghim cho u.

Hnh 1.16. My l c ng nghim (7). T cy v trng (flow cabinet)

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(1)

(2)

(3)

(4)

(5)

(6)

Hnh 1.10. Pipetteman

(1). 2 20 l, (2). 20 100 l, (3). 20 200 l, (4). 200 1000 l, (5). 1000 5000 l, (6) 1000- 10000 l

Ch : Khi s dng cn lu , cn bm micropipette c hai nc: nc 1 tng ng vi dung tch c chn khi ht dch; nc hai n vt qu nc 1 u i ht dch cn mao qun u tip. Khng dng micropipette ht cc loi ha cht d bay hi, nh: acetic, HCl

1.1.2.2.

Thit b

(1). Cn (lab scale) Thng s dng loi cn k thu t (lo i 2 s l) c chnh xc 0,01 g. Khi cn xc nh m t lng chnh xc, ngi ta dng cn phn tch (loi 4 s l) c chnh xc n 0,0001 g (0,1 mg).

{ INCLUDEPICTURE "http://atlanticsupply.com/pictures/%7B23A465F0-AE63-46A2-9D08C02567CD7EC7%7D_PIONEERANALYTICALBALANCE.jpg" \* MERGEFORMATINET } Hnh 1.11. Cn k thu t (tri) v cn phn tch (phi) Ch : cn phi c t trn b m t phng, trnh gi la, khng b rung. (2). T sy (drying oven) Dng sy kh cc dng c ho c kh trng theo phng php nhit kh. T sy thng c t nng b ng in tr, nhit c th c ln n 200 oC. Khi nng, nhit c o u b ng qut trnh sai lch nhit cc b.

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thi sng, l knh cn c r a li bng nc ra chn hay x phng, lm kh v ngm trong cn 95o. Cc loi dng c thu tinh khc: cc loi c c thu tinh (becher), ng ong, ng ht Que cy thng : dng cy su. Hnh 1.6. Que cy thng Que cy vng: dng cy ria trn m t th ch hay phn lp VSV trn b mt thch hoc cy chuyn trn mi trng lng.

(2). Cc loi que cy -

Hnh 1.7. Que cy vng Que cy mc: dng cy cc loi nm mc hay x khu n. Hnh 1.8. Que c y mc Que trang (tri): dng tri git sinh khi VSV trn b mt thch.

Hnh 1.9. Que trang (3). Micropipette L lo i ng ht my, c chnh xc n l (tu theo loi) dng nh lng m t th tch chnh xc dung d ch VSV, mi trng nui cy (lng) hay dung d ch ha cht. Micropipette (pipetteman) c nhiu lo i, m i loi u c d i gii h n dung tch, nh: 0,1 10,0 l; 1 20 l; 20 200 l; 100 1000 l; 1000 5000 l v 1000 10.000 l (1000 l = 1 ml). Ty theo hng sn xut m quy cch dung tch c thay i. Trong di dung tch cho php, ngi ta c th iu chnh dung tch chnh xc nh mong mun. M i loi micropipette u c loi u tip tng ng.

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VSV, s dng nt bng thay cho n p vn. Khi phn tch VSV, s dng giy nhm hay nt silicon h n ch s nhim VSV. { INCLUDEPICTURE "http://www.indigo.com/images/product/60.jpg" \* MERGEFORMATINET } Hnh 1.1. ng nghim v gi ng nghim - a Petri (Petri dishes): Gm np ln y ln np nh c hnh trn, ng knh 6, 8, 10, 12 cm. a Petri c gi li b ng giy trc khi em hp tit trng bng nhit m hay sy bng nhit kh. Hin nay c cc loi a Petri bng nha v trng bn sn, rt tin li v thng c s dng trong phng phn tch VSV. Khc vi a thu tinh c th ti s dng nhiu ln, a nha c ch to s dng 1 ln. { INCLUDEPICTURE "http://sinhviet.com/sanpham/barloworld_files/0.jpg" \* MERGEFORMATINET } Hnh 1.2. a Petri - Bnh tam gic (erlenmeyers): cn c gi l bnh nn dng cha mi trng nui cy (lng hay rn) hay dung mi ha cht trong pha ch mi trng. { INCLUDEPICTURE "http://hoahocdoisong.com/image/Articles/erlenmeyer%20flask%20three%20glassware.j pg" \* MERGEFORMATINET } Hnh 1.3. Bnh tam gic - n cn (Alcohol bunners): l lo i n c c t bng cn 90o, to nhit to ra mt vng khng gian xung quanh v trng. n cn c th c dng kh trng cc loi que cy b ng cch h hay t trn ngn la n cn. { INCLUDEPICTURE "http://hoahocdoisong.com/image/Articles/alcoholburner%2001.jpg" \* MERGEFORMATINET } Hnh 1.4. n c n Phin knh (lame) v l knh (lamelle): Phin knh l ming thu tinh hnh ch nht, c trong sut cao, dy 2 mm dng ch a gi t VSV trng thi sng hay nhum mu khi quan st chng b ng knh hin vi. L knh l ming thu tinh hnh vung, c trong sut cao dy 0,1 - 0,2 mm dng y ln git VSV trn phin knh quan st trng thi sng ca t bo VSV.

Hnh 1.5. Phin knh v l knh Phin knh v l knh khi mi mua v cn c lm sch bng cch un si trong NaOH 10% trong 10 pht, r a b ng nc ct, ra bng HCl long v sau cng ra sch li bng nc ct. Phin knh s dng quan st nhu m mu cn c s l bng cch ngm trong dung dch sulfocromate (100 g H2SO4 , 50 g K2Cr2O7 v 1000 ml nc ct) trc khi ra sch bng nc v trng li b ng nc ct. Sau khi quan st VSV trng
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BI 1.

HNG DN S DNG THIT B, DNG C VI SINH VT DI KNH HIN VI QUANG HC

PHNG TH NGHIM VI SINH V QUAN ST TIU BN

Mc tiu: Sau khi h c xong bi ny, sinh vin c kh nng: Chu n b trang thit b cn thit ca m t phng th nghim vi sinh vt. Vn dng c cc quy tc an ton trong phng th nghim vi sinh vt. Lm c cc tiu b n quan st vi sinh vt. C s l thuyt Hiu ng nguyn tc, phng php lm vic vi vi sinh vt (VSV). Thao tc an ton trn i tng VSV m t cao. Khng n ung, ht thu c trong phng th nghim (PTN) VSV. Mc o blouse trong th i gian th c hnh. Khng dng ming ht bng ng ht (pipette) nh lng VSV hay ha cht, m ph i dng qu bp cao su khi thao tc ht bng ng ht. Hp Petri mi trng ho c sau nui cy VSV, khng m np, dng tay s v dng mi ng i. Khi l tay lm VSV ra ni lm vic, dng khn hay giy tm cn 70 o lau v lau sch li b ng khn hay giy tm cn 70 o khc. Khi s dng que cy gieo cy VSV cn phi c kh trng bng cch t trn ngn la n cn trnh vng vi ra xung quanh. Trc v sau khi thao tc trn VSV cn lau tay k bng cn 70 o. Khi th c hin thao tc ny cn phi trnh xa ngn la n cn khi tay cn t. Cn ghi ch tn ch ng VSV v ngy thng th nghim ln cc d ng c cha mi trng nui cy VSV. Tt c cc cht thi v mi trng cha ho c nhim VSV cn c h p kh trng trc khi em vo thng rc. Cc dng c nhim VSV cn c ngm trong dung d ch st khu n trc khi ra. Khng thch vo bn ra v ng thot nc. Dng c

1.1. -

1.1.1. Cc quy t c an ton trong phng th nghim

1.1.2. Dng c v thit b th nghim vi sinh v t 1.1.2.1. (1). Dng c bng thu tinh: - ng nghim (test tubes): c dng cha mi trng nui cy v nui cy VSV. Mi trng c th dng l ng hay rn. Ming ng nghim c th c y b ng nt bng khng thm nc, bng silicon, giy nhm hay np vn. Trong trng hp nui cy

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4. nh gi bi thc hnh Bng. Tiu ch nh gi chung STT 1 2 3 4 5 6 7 Tiu ch nh gi th c t chc, k lu t An ton, v sinh Thi gian Chu n b Thao tc Kt qu Bo co TNG im 1 1 1 1 2 2 2 10

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GII THIU HC PHN

1. Mc tiu h c phn Sau khi hc xong hc phn ny, sinh vin c kh nng v: Kin thc: Phn bit chnh xc nm men, nm mc, vi khun; Cng c kin thc v sinh l, sinh ho vi sinh vt m sinh vin hc hc phn l thuyt. K nng : Thc hin c cc thao tc s dng knh hin vi, lm tiu bn v quan st vi sinh vt; Pha ch mt s mi trng thng dng v mi trng c hiu nui vi sinh vt; Thc hin k thut gieo cy, nui, phn lp vi sinh vt; Thc hin cc k thut pht hin v nh lng vi sinh vt trong cc ch tiu v sinh an ton th c phm. Thi : 1. Rn luyn tnh cn thn, lm vic c khoa hc; 2. C quan im ng v vi sinh vt trong ln men, bo qun v v sinh an ton thc ph m. 2. Phn b chng trnh thc hnh Th i gian l thuyt: 0 tit Th i gian thc hnh: 30 tit, chia thnh 6 bi, mi bi 5 tit. Ni dung cc bi th c hnh v vi sinh vt bao gm: - Chun b trang thit b v dng c phng th nghim, quan st vi sinh (tiu b n sng, nhum mu); - K thu t pha ch mi trng v nui cy vi sinh vt; - K thu t c bn phn tch vi sinh vt; - Mt s qu trnh ln men thc phm. 3. nh gi hc phn Mi bu i thc hnh, sinh vin ph i so n bi bo co trc (kt qu ghi sau) khi n phng th nghim v c k xc nhn ca th y c. Sinh vin no khng son bi bo co trc xem nh khng i thc hnh bu i th nghim . Sinh vin lm 6 bo co v ng thnh 1 cun, np vo bui cui. B cc bi bo co v thang im: vit ngn gn cc ni dung sau trn kh giy A4: 1. C s l thuyt 2. Chun b dng c, ha cht v thit b 3. Cc bc thc hin 4. Kt qu 5. Nh n xt v th o lun

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DANH MC CH VIT TT & THUT NG

Ch vit tt: VSV_ Vi sinh vt PTN _ Phng th nghim OD _ Optical density EMP _ Embden Meyerhof Parnas CFU _ Coloning Form Unit VKHK _ Vi khun hiu kh NM-NM _ Nm men n m mc NADH _ Nicotinamide Adenine Dinucleotide Thut ng: Tiu bn: Lame: Lamelle: l phin knh c cha vi sinh vt trng thi sng hoc cht, dng quan st hnh thi vi sinh vt Hay cn gi l phin knh, dng tiu bn vi sinh vt Hay cn g i l l knh, dng lm tiu bn git p quan st vi sinh vt Mi trng dinh dng lng nui cy hoc nh danh vi sinh vt Mi trng lng ang c cc vi sinh vt sinh trng

Mi trng thch: Mi trng dinh dng rn c ng khi bi thnh phn agarose Canh trng: Canh khun:

Mi trng ng kh thng phm: Mi trng dinh dng thng phm nui cy vi sinh vt c ph i ch sn Sinh khi: Khun lc: Khi lng ca cc t bo vi sinh vt trong mt khng gian xc nh Mt khi t bo vi sinh vt c pht trin t mt t bo hoc bo t

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Hnh 4.4. Hnh 4.5. Hnh 5.1. Hnh 5.2. Hnh 5.3. Hnh 5.4. Hnh 5.5. Hnh 5.6. Hnh 5.7.

M hnh k thut hp tri ............................................................................ 44 Phng php phn tch vi sinh bng k thut hp ................................... 45 Bung m hng cu .................................................................................. 47 Cu to bung m Neubauer ci tin ......................................................... 48 Bung m Neubauer ci tin ..................................................................... 49 th ng tng quan tuyn tnh gia mt quang v s lng t bo VSV............................................................................................................ 51 m khun lc TS VKHK (Mi trng cao tht-pepton) ............................. 54 S lng v d ng khun lc trn Petri sau nui cy ..................................... 55 Cch cho dch vi sinh vt vo bung m ................................................... 55

DANH MC BNG
Trang Bng 1.1. Dng c v ha ch t bi 1 ........................................................................... 18 Bng 1.2. Tiu ch nh gi bi 1 ................................................................................ 22 Bng 2.1. Nhu cu khong i vi m t s vi sinh vt.................................................. 23 Bng 2.2. Mi quan h gia nhit v p sut .......................................................... 27 Bng 2.3. Thnh ph n c a mt s loi mi trng nui cy vi sinh vt ....................... 27 Bng 2.4. Dng c v thit b b i 2 ............................................................................. 27 Bng 2.5. Tiu ch nh gi bi 2 ................................................................................ 31 Bng 3.1. Dng c, thit b v ha cht bi 3 .............................................................. 34 Bng 3.2. Tiu ch nh gi bi 3 ................................................................................ 37 Bng 4.1. Lng m u ti thiu cn thit phn tch .................................................. 39 Bng 4.2. Dng c, thit b v ha cht bi 4 .............................................................. 43 Bng 4.3. Tiu ch nh gi bi 4 ................................................................................ 47 Bng 5.1. Cc lo i bung m .................................................................................... 51 Bng 5.2. Cc gi tr tng quan gia mt quang v s lng t bo vi sinh vt .... 52 Bng 5.3. Dng c, ha cht v thit b bi 5 .............................................................. 54 Bng 5.4. Tiu ch nh gi bi 5 ................................................................................ 58 Bng 6.1. Dng c, thit b v ha cht bi 6 ............................................................. 59 Bng 6.2. Tiu ch nh gi bi 6 ................................................................................ 61

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DANH MC HNH
Trang Hnh 1.1. Hnh 1.2. Hnh 1.3. Hnh 1.4. Hnh 1.5. Hnh 1.6. Hnh 1.7. Hnh 1.8. Hnh 1.9. ng nghim v gi ng nghim................................................................ 8 a Petri........................................................................................................ 8 Bnh tam gic ............................................................................................... 8 n cn ....................................................................................................... 9 Phin knh v l knh..................................................................................... 9 Que cy thng ............................................................................................... 9 Que cy vng .............................................................................................. 10 Que cy mc ............................................................................................... 10 Que trang .................................................................................................... 10

Hnh 1.10. Pipetteman .................................................................................................. 10 Hnh 1.11. Cn k thu t (tri) v cn phn tch (phi)................................................... 11 Hnh 1.12. T sy ......................................................................................................... 11 Hnh 1.13. T m ......................................................................................................... 12 Hnh. 1.14. My lc ....................................................................................................... 12 Hnh 1.15. B n nhit lc ............................................................................................ 12 Hnh 1.16. My lc ng nghim ................................................................................... 13 Hnh 1.17. T cy v trng ........................................................................................... 13 Hnh 1.18. Ni h p kh trng ....................................................................................... 13 Hnh 1.19. pH k .......................................................................................................... 14 Hnh 1.20. Knh hin vi quang hc .........................................................................................15 Hnh 1.21. Knh hin vi soi ni..................................................................................... 15 Hnh 1.22. Knh hin vi i pha.................................................................................... 16 Hnh 1.23. Knh hin vi hu nh quang ........................................................................... 16 Hnh 1.24. Knh hin vi in t .................................................................................... 17 Hnh 1.25. Cch lm tiu bn quan st vi sinh vt trng thi sng.............................. 19 Hnh 1.26. Vi khun Gram dng bt mu tm ............................................................. 20 Hnh 1.27. Vi khun Gram m (c mu hng ) ......................................................... 20 Hnh 3.1 Hnh 3.2. Hnh 4.1. Hnh 4.2. Hnh 4.3. Cc kiu ria trn mi trng thch ........................................................... 33 K thut cy ria trn a thch..................................................................... 36 K thut hp tri ......................................................................................... 41 K thut hp .......................................................................................... 41 K thut pha long ...................................................................................... 43
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MC LC
Trang Danh m c hnh ................................................................................................................ 2 Danh m c b ng................................................................................................................ 3 Danh m c ch vit tt v thut ng.................................................................................. 4 Gii thiu hc phn ........................................................................................................ 5 Bi 1. HNG DN S DNG THIT B, DNG C PHNG TH NGHIM VI SINH V QUAN ST TIU BN VI SINH VT DI KNH HIN VI QUANG HC ...................................................................................................... 7 Bi 2. K THUT PHA CH MI TRNG ............................................................ 22 Bi 3. K THUT GIEO CY V PHN LP VI SINH VT .................................. 32 Bi 4. K THUT PHN TCH VI SINH VT .......................................................... 38 Bi 5. PHNG PHP NH LNG VI SINH VT - LN MEN ETHANOL ....... 47 Bi 6. LN MEN LACTIC ........................................................................................... 58 Ph lc .......................................................................................................................... 62

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TR NG I HC CNG NGHIP THC PH M THNH PH H CH MINH

KHOA CNG NGH THC PHM

Th nghim

VI SINH VT HC THC PHM

(Dnh cho h i hc, Cao ng v Trung cp)

Bin son: Tr n Quc Huy

TP. H Ch Minh, thng 09 n m 2011