B GIO DC & O TO TRNG I HC K THUT CNG NGH TP.

HCM KHOA MI TRNG & CNG NGH SINH HC

BI GING

THC TP VI SINH I CNG

Bin son: ThS. L TH VU LAN KS. PHM MINH NHT

- 2008 -

NI DUNG THC HNH

-------------------Bi s 1: Cc quy tc an ton trong phng th nghim vi sinh vt Bi s 2: Cc thit b phng th nghim vi sinh v cc phng php kh trng Bi s 3: Thc hnh pha mi trng dinh dng Bi s 4: Phn lp Nui cy Bo qun vi sinh vt Bi s 5: Nghin cu mt s c im sinh hc ca vi sinh vt trn knh hin vi

BI S 1: CC QUY TC AN TON TRONG PHNG TH NGHIM VI SINH VT -------------------------------------Thao tc an ton l yu cu cc k quan trng trong kim nghim vi sinh vt. Khi lm vic vi vi sinh vt, chng ta thng thao tc vi s lng rt ln v m c t bo vi sinh vt ( mc 109 t bo/ml). Nhiu chng vi sinh vt l tc nhn gy bnh nn cn lun lun cn thn vi tt c cc chng ang thao tc. Mt khc, nhn vin kim nghim cng phi s dng nhiu loi ha cht, trong c cc acid hoc nhng ha cht c c tnh. Do vy, cn tun th mt s quy tc an ton m bo an ton cho bn thn v cho nhng ngi khc trong phng th nghim nh sau: Nm vng nguyn tc, phng php lm vic vi vi sinh vt. Khng n ung, ht thuc trong phng kim nghim. Mang khu trang khi thao tc vi vi sinh vt. Mc o blouse trong thi gian lm vic. Trc khi bt u lm cn st trng mt bn bng giy lau tm cn 700 hoc dung dch cht dit khun khc (lysol 5%, amphyl 10%, chlorox 10%), kh. Thc hin tng t cho hai tay. Ch cha t n cn hoc n Bunsen khi tay cha kh cn. Lp li vic st trng ny sau khi hon thnh cng vic. Cn ghi ch tn chng, ngy thng th nghim ln tt c cc hp petri, ng nghim mi trng, bnh nui cy. Khi l tay lm , nhim vi sinh vt ra ni lm vic, dng khn giy tm cht dit khun lau k, sau thc hin kh trng li bn lm vic. Cn thn khi thao tc vi n cn hoc n Bunsen. Tt ngn la khi cha c nhu cu s dng hoc ngay sau khi thc hin xong mi thao tc. Lu trnh a tay, tc qua ngn la. Cn c cch bo v tc thch hp trng hp tc di. S dng qu bp cao su khi thao tc ng ht nh lng (pipette), khng ht bng ming. Khi lm v dng c thy tinh, cn thn mang gng tay thu gom tt c mnh v vo mt ti rc ring. Tch ring cht thi rn v cht thi lng. Tt c cht thi rn, mi trng cha hoc nhim vi sinh vt cn c hp kh trng trc khi thi b vo cc bi rc. Cc dng c, bnh cha nhim vi sinh
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vt cn c ngm vo dung dch cht dit khun (nc javel) trc khi ra v ti s dng. Cn gi hoc rng bng bng keo khi t chng cc a petri ln nhau. Khng m hp petri v dng mi ngi trnh nhim vi sinh vt vo ng h hp. Khi t que cy c dnh sinh khi vi sinh vt, cn t vng hoc u que cy vo chn ngn la trnh s vng nhim vi sinh vt vo khng kh. St trng v ra tay sch s trc khi ri phng th nghim.

BI S 2: CC THIT B PHNG TH NGHIM VI SINH V CC PHNG PHP KH TRNG ------------------------I. MC CH YU CU 1. Kin thc l thuyt: Cng c cc kin thc sau: nh hng ca cc nhn t vt l, ha hc i vi s tn ti v pht trin ca vi

sinh vt + Nhn t vt l bao gm: nhit , m, nh sng, pH ... + Nhn t ha hc bao gm: acid, base, mui kim loi, cn ... Nguyn nhn gy nhim cc dng c l do s tip xc vi khng kh, cc dng

c hay vt phm c vi sinh vt 2. K nng thc hnh: Hnh thnh v rn luyn cc k nng: Bao gi dng c v lm nt bng cho ng nghim Kh trng dng c v mi trng bng ni hp p sut cao v t sy

II. MT S DNG C V THIT B PHNG TH NGHIM VI SINH 1. Cc dng c thy tinh a. ng nghim: c s dng cha mi trng nui cy vi sinh vt, c nt bng gn khng thm nc hay bng nha chu nhit Nt gn

ng nghim

Hnh 1: ng nghim b. a petri: gm mt np ln v mt y nh p lng vo nhau, ng knh 8cm, 10cm, 12cm ......

Hnh 2: a petri
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c. ng ht (pipette) ng ht c chia ng ht Pasteur

Nu khng c sn pipette Pasteur ta c th ch to t ng thy tinh ng knh 7mm, di khong 25cm vi 2 u c t trn cnh v nht gn khng thm nc. khong gia ng thy tinh trn ngn n cn, xoay u cho n khi thy tinh chy ra, mang khi ngn la v ko u tay nh th ta c c 2 pipette Pasteur
Gn khng thm nc

n cn

ng ht Pasteur

Hnh 3: Cch lm mt pipette Pasteur d. Micropipettes (Pipetman) y l pipet chnh xc, cho php ta ht c mt lng cht rt chnh xc.

Hnh 4: Micropipette e. Cc dng c bng thy tinh khc Becher Bnh cu y bng v y trn Bnh tam gic (Erlen) Bnh Roux

2. Cc dng c thit b khc

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a. Dy cy Dy cy thng: s dng cy su hay ly trch vi sinh vt trn mi trng c Dy cy vng: dng cy ria vi sinh vt trn trn mt thch hay phn lp vi sinh vt trong mi trng lng hoc mi trng c Dy cy thc th: dng cy cc loi nm men, nm mc, x khun

Nhng loi dy cy ny thng lm bng kim loi khng b oxy ha nhit cao b. T m: dng vi sinh vt hoc theo di s tng trng ca vi sinh vt

Hnh 5: T m c. L Pasteur (xem phn sau) d. Autoclave (xem phn sau) e. Ni chng cch thy III. BAO GI DNG C

1. Nguyn tc Dng c c bao gi phi m bo sch v kh. Bao gi phi kn v cn thn sau khi kh trng vn m bo s v trng ca

dng c trong lp giy gi v ly ra s dng d dng. 2. Phng php bao gi dng c Vic bao gi dng c gm 2 khu: Lm nt bng: cho cc ng nghim, bnh tam gic, pipet, que trang Bao gi: cho hu ht cc dng c khc

a. Cch lm nt bng Vi cc ng nghim: Ly mt t bng khng thm nc cun li Dng que tre n vo gia cun bng y cun bng ny gp i v t t vo ming ng nghim Yu cu:
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 Nt c kch thc v cht va phi. u nt trn, gn, phn ngoi ln hn phn trong. Ly nt ra hay ng vo d dng Vi cc chai, l, bnh tam gic c kch thc ln: cch lm tng t nhng s dng lng bng nhiu hn Vi cc pipet: dng mt si dy thp nh nht mt t bng vo u ln ca

pipet hn ch khng kh t ming ngi ht vo pipet b. Cch bao gi dng c Vi cc dng c sau khi lm nt bng cn bao gi phn c nt bng bng giy bo khi kh trng nt bng khng b t v m bo iu kin v trng tt hn. Cch lm nh sau: Ct cc on bng giy hnh ch nht vi kch thc ty theo dng c cn bao gi. Qun quanh phn u c nt bng. Ct li tht cht

Yu cu: Phn giy bao bn ngoi phi cht v kn Bao bng giy du vi dng c hp t Bao bng giy bo vi dng c sy kh khi kh trng t.

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. IV. CC PHNG PHP KH TRNG DNG C 1. Nguyn tc Sau khi kh trng cn m bo: S v trng tuyt i cho dng c v vt phm Khng lm thay i cht lng mu vt Bo m an ton tuyt i cho con ngi

2. Cc phng php kh trng Khi kh trng bng nhit, cc t bo sinh dng ca VSV b tiu dit d dng trong khi cc bo t vn cn tn ti ngay nhit Kh nng chu nhit ca vi sinh vt ph thuc vo: Tnh cht mi trng
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S lng t bo pH ca vt cn kh trng

Do vy kh trng bng nhit hiu qu cn xc nh ngng nhit thp nht v khong thi gian ngn nht cn thit tiu dit ton b VSV v bo t ca chng c trong dng c cn kh trng C th kh trng bng phng php nhit kh hay nhit t a. Phng php nhit kh Kh trng bng t sy c thc hin trong t sy Cch tin hnh: t cc dng c c bao gi vo t sy Bt cng tc t hot ng iu chnh thi gian v nhit thch hp (1600C trong 2h hoc 1800C trong 30 pht) Tt t sy, ngui ti 600C ri m t ly dng c ra. Trnh m t ly dng c khi nhit t cn cao s lm dng c thy tinh d v Cc dng c sau khi sy m giy bao c mu hi vng l t yu cu. Nu giy bao c mu nu chng t nhit kh trng cao lm bng v giy bin thnh gondron (hp cht c tnh st trng) th khng th s dng dng ny nui cy VSV c. Kh trng bng cch t que la nng : Phng php ny dng kh trng que cy, ng ht, u ng nghim, ming

bnh tam gic sau khi ly nt bng ra. Cch kh trng: H dng c trn ngn la n cn, a qua a li n 3 4 ln. Vi cc dy mayxo u que cy phi nung cho tht ht chiu di dy cy. i dng c ngui mi c s dng trnh v v vi khun khng b tiu dit khi ly ging. b. Kh trng bng sc nng t un si trong nc Phng php ny c s dng khi cn kh trng nhanh cc dng c: kim tim, dao, ko, kp, cc ....
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Cch tin hnh: Dng nc sch ngp dng c un si t 10 pht n 1h un cch thy nhit thp (phng php kh trng Pasteur) Phng php ny c dng kh trng nhanh cc thc phm d bin tnh nhit cao Cch tin hnh: un nng mi trng ln 65 700C trong 15 30 pht Hp cch qung 1000C (phng php Tyndal) Phng php ny dng kh trng mt s loi mi trng nui cy men bnh m, men gia sc, mc lm nc chm.... Cch kh trng: Hp trong trng 1000C t 30 40 pht. Ly ra t m 24 gi cho bo t vi khun pht trin Hp mi trng ln th hai 1000C trong 30 40 pht tiu dit cc bo t va ny mm. Lp li qu trnh ny 3 4 ln

Phng php ny ch c tc dng c ch VSV khng c bo t

Kt qu: mi trng va c kh trng va c m bo khng thay i cht lng. Kh trng bng hi nc bo ha p sut cao (Autoclave) Phng php ny c thc hin trong ni hp v trng p sut cao. l thit b lm bng kim loi c tnh chu nhit cao c kh nng t ng iu chnh nhit v thi gian Nguyn tc hot ng Lm tng nhit kh trng cc vt bng hi nc

di p sut ln hn p sut kh quyn. Khi p sut tng lm nhit tng nh h thng van rt cht ch Hnh 6: Autoclave

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Mi quan h gia p sut v nhit ca ni c biu hin qua bng sau: p sut (atm) 0 0,5 1,0 1,5 2,0 Nhit (0C) 100 112 121 128 134

Cch s dng: Chun b cc dng c cha mi trng cn kh trng Cho 3l nc ct vo ni hp ri kim tra mc nc cho ph hp. Ln lt a cc dng c, mi trng cn hp vo bn trong ni. ng cht ni hp. iu chnh kim ng h thi gian v p sut v v tr mong mun. Cm phch in Bt cng tc kh trng ni hp tin hnh hp. Khi kt thc qu trnh hp, ni s bo hiu bng ci. Gt cng tc kh trng v v tr dry Ch kim ch p sut tr v 0 mi m np ni ly dng c ra. Rt phch in Ghi nhn ngy - thng nm vo dng c sau khi kh trng.

CH : m bo an ton v hiu qu ca vic kh trng, ngi thc hin cn phi: Kim tra li ni hp trc khi s dng Thc hin ng quy trnh hng dn Trnh cung cp in t ngt khng gy v dng c, nguyn liu hoc gy n nguy him. Trc tip theo di qu trnh kh trng cho n khi kt thc v ngt in. nh k kim tra cht lng ng h p k v van an ton.

Tm li, phng php kh trng bng hi nc bo ha p sut cao l phng php ph bin v hiu qu nht trong cc phng php kh trng nh kh nng tiu dit cc t bo sinh dng ln bo t ca vi sinh vt.
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c. Kh trng bng s lc S dng cho mi trng lng, trong, c nhy yu, khng chu c nhit cao hn 600C. Cho mi trng i qua mt mng lc xp c ng knh l nh hn ng knh ca vi khun. Khi , vi khun s b gi li trn mng lc cn dung dch i qua s v trng. Mng lc thng l mng cellulose d. Dit trng bng bc x Tia t ngoi: Bc x thng dng nht l tia UV. Dng tia UV dit trng khng kh phng bnh vin, phng v trng. Tia UV ch kh trng b mt m khng thm su vo bn trong mu vt. Tia m cc: Dit trng cc dng c gii phu, thuc, thc phm, tia m cc c th tiu dit cc vt cho vo bao gi kn.

CU HI V BI TP 1. Thc hnh bao gi cc loi dng c? 2. Phn tch c s vi sinh vt hc ca cc phng php kh trng Pasteur, Tyndal? 3. Tm tt cch s dng ni hp p sut cao (autoclave)?

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BI S 3: CHUN B MI TRNG NUI CY VI SINH VT -----------------------I. MC CH V YU CU 1. Kin thc l thuyt: Cng c v hnh thnh mi cc kin thc sau: Khi nim mi trng dinh dng. Yu cu c bn ca mi trng dinh dng. C s phn loi mi trng dinh dng. Nguyn tc c bn ca vic ch to mi trng dinh dng

2. K nng thc hnh K nng s dng cc dng c: cn phn tch, ng ong, pipet, autoclave, t m. Cn ong, pha ch ha cht. Lm trong mi trng. iu chnh pH v kh trng mi trng. Kim tra kt qu kh trng. Bo qun mi trng

II. HA CHT NGUYN LIU DNG C 1. Ha cht nguyn liu Ty theo yu cu v iu kin thc t ca PTN m ta chun b cc nguyn liu, ha cht ca mt s loi mi trng SV thc hnh. 2. Dng c III. ng nghim Gi ng nghim a petri Bnh tam gic 250 ml Cc thy tinh 250 ml Pipet cc loi ng ong Phu thy tinh Giy lc, giy bao gi Bng khng thm nc, bng y t n cn MI TRNG DINH DNG
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1. Khi nim mi trng dinh dng bo. Mi trng dinh dng l hn hp cc cht dinh dng v cc cht ny c Cc cht dinh dng l nhng hp cht tham gia vo qu trnh trao i cht ni

nhim v duy tr th oxy ha kh, p sut thm thu ca t bo v s n nh ca pH trong mi trng. 2. Cc yu cu c bn ca mi trng dinh dng C y cc cht dinh dng cn thit C pH thch hp C nht nht nh Khng cha cc yu t c hi Tuyt i v trng

3. Phn loi mi trng dinh dng Ngi ta da trn cc c s khc nhau phn loi mi trng a. Cn c theo thnh phn v ngun gc: gm 3 loi Mi trng t nhin: c thnh phn l cc sn phm t nhin nh: trng, sa,

khoai ty, dch chit nm men, ng, cm. Thnh phn ha hc ca loi mi trng khng c xc nh chnh xc do s khng n nh ca sn phm t nhin. Mi trng tng hp: cha cc cht ha hc m thnh phn ca chng c xc

nh v nh lng mt cch c th v chnh xc. Mi trng bn tng hp: cha c cc cht ha hc ln cc sn phm t nhin.

b. Cn c theo tnh cht l hc: gm 3 loi Mi trng lng: thnh phn mi trng ny khng cha agar v thng c

s dng nghin cu qu trnh tng hp ca vi sinh vt. Mi trng c: mi trng ny cha 1,5 2% agar hoc 10 20% gelatin.

Mi trng ny c s dng nghin cu cc c im hnh thi, sinh l ca vi sinh vt. Mi trng bn lng: ch cha 0,3 0,7% agar

c. Cn c vo cng dng: gm cc loi sau: Mi trng c bn: thch hp cho nhiu loi vi sinh vt khc nhau. Mi trng chn lc: l mi trng m bo cho s pht trin u th ca mt

loi hay mt nhm loi vi sinh vt xc nh no .

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Mi trng kim nh: l mi trng cho php phn bit c mt s c im

ca mt s loi vi khun xc nh. Thng thng, ngi ta cho vo mi trng cht ch th mu to ra nhng mu c trng. 4. Phng php pha mi trng Pha mi trng thc hin vic phn lp, nhn ging, ging vi sinh vt, ng

thi nui cy v nghin cu cc c im sinh hc ca chng a. Nguyn tc pha mi trng Da trn c s nhu cu v cc cht dinh dng v kh nng ng ha cc cht

dinh dng ca tng loi vi sinh vt. m bo s cn bng v p sut thm thu gia mi trng v vi sinh vt

nn cn iu chnh t l v nng cc cht trong thnh phn mi trng dinh dng. m bo cc iu kin ha, l cn thit cho cc hot ng trao i cht ca vi

sinh vt. b. Cc bc pha mi trng dinh dng Pha ch Cn, ong tht chnh xc cc thnh phn mi trng v pha ch theo ng trnh t hng dn trong ti liu. Mi trng lng: cn, ong cc cht ri cho vo nc Mi trng c: Cn cc thnh phn ca mi trng cho vo nc ct. Cn agar ri cho vo dung dch trn Lm trong mi trng Vic lm trong mi trng gip ta d dng quan st s pht trin ca vi sinh vt. Vi mi trng lng: tin hnh lc bng vi tha hoc giy lc. Vi mi trng c: thng lc qua vi mn 2 lp hoc giy lc trong iu kin c phu lc nng. iu chnh pH ca mi trng Dng HCl 10% hoc NaOH 10% iu chnh pH. Mun kim tra pH, ta s dng my o pH v n nhy v cho chnh xc

cao. Nu khng c th s dng giy qu o pH nhng khng c chnh xc cao. Phn phi mi trng vo dng c
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Ngi ta thng phn phi mi trng vo a petri, ng nghim, bnh tam gic. Trnh t phn phi nh sau: un cho mi trng ha lng. Mt tay gi dng c cha mi trng Tay cn li kp nt bng v ko ra. Nhanh tay mi trng vo dng c v y nt bng li. Ch : i vi ng nghim: Nu mi trng lm thch nghing th lng mi trng cn c phn phi chim 1/4 th tch ng nghim. Nu lm thch ng th lng mi trng c phn phi chim 1/2 - 1/3 th tch ng nghim. i vi bnh cu hay bnh tam gic : lng mi trng c phn phi chim 1/2 - 2/3 th tch bnh. Cc thao tc phn phi phi nhanh gn, kho lo mi trng khng dnh vo ming dng c hoc nt bng v vic phn phi cn c thc hin xong trc khi mi trng b ng c. Kh trng mi trng Ty theo tnh cht v iu kin c th ca tng loi mi trng m c ch v phng php kh trng khc nhau. Cc phng php kh trng thng c s dng l: Phng php Pasteur Phng php Tyndal Phng php lc bng dng c lc vi khun Phng php hp bng hi nc bo ha p sut cao i vi cc dng c chu nhit (s, vi, thy tinh) kh trng 1,5 atm / 20 30 pht i vi dng c cha 1l mi trng tr ln th hp 1 atm / 30 pht Vi cc loi mi trng cha ng d bin tnh nhit cao th hp kh trng 0,5 0,6 atm / 15 pht Lm thch nghing, thch ng v thch vo a petri Lm thch nghing: Cn tin hnh ngay sau khi kh trng mi trng v mi trng cha ng c.

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t ng nghim c mi trng ln gi t nghing v khng c

mi trng chm vo nt bng. yn cho n khi mi trng ng c. Yu cu mt thch phi thng,

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: - M bao giy gi cc a petri - Mt tay cm dng c cha mi trng - Tay cn li m nt bng v h ming bnh trn ngn la n cn. - M h np a petri. Nghing bnh v rt mi trng vo a petri. - y np a li, xoay trn a mi trng c phn phi u bn trong a. - yn cho mi trng ng c. - Lt ngc a li v bo qun. CH : Thao tc mi trng phi nhanh v kho lo hn ch s nhim khun. Mt thch phi phng, nhn, c dy khong 2mm. Thng thng c khong

200 ml mi trng s phn phi c t 22 25 a. Sau khi phn phi mi trng vo a petri, kim tra li xem mi trng c b

nhim khun sau 1 2 ngy. Ghi ch mi trng (tn mi trng, ngy kh trng, hn s dng) Bo qun v kim tra mi trng Mi trng cha s dng c bo qun ch mt (0 50C) v khng mi

trng b kh. Trc khi s dng, kim tra v khun ca mi trng, ngi ta thng t vo t m 37oC trong mt khong thi gian nht nh. Loi b mi trng b nhim khun v ch s dng mi trng t yu cu. IV. CNG THC V CCH PHA MT S LOI MI TRNG THNG DNG 1. Mi trng nui cy vi khun (mi trng cao tht pepton)
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Cng thc: Cao tht: Pepton: Nc ct: 5g 10g 1000ml

Cch pha mi trng nui cy: Cn chnh xc v cho cc thnh phn ca mi trng vo trong cc cha sn

1 t nc Khuy cho tan ht v thm lng nc theo yu cu un ln cho tan hon ton ngui v lng dung dch va un Lc bng vi hay giy lc Phn phi mi trng vo cc dng c kh trng

2. Mi trng nui cy nm men (Hansen) Cng thc: Saccharose hoc Maltose: Pepton: KH2PO4: MgSO4: Agar: Nc ct: 50g 10g 3g 35g 20g 1000ml

Cch pha ch mi trng nui cy: Cn chnh xc v cho cc thnh phn ca mi trng vo trong cc cha sn

1 t nc Khuy cho tan ht v thm lng nc theo yu cu un ln cho tan hon ton ngui v lng dung dch va un Lc bng vi hay giy lc Phn phi mi trng vo cc dng c kh trng

3. Mi trng nui cy nm mc (mi trng Czapek) Saccharose: NaNO3: K2HPO4: 30g 30g 1g
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MgSO4: FeSO4: Agar: Nc ct:

0,5g 0,01g 20g 1000ml

pH = 6, kh trng 1atm/30 pht 4. Mi trng nui cy x khun (mi trng Gauze 1) Tinh bt tan: K2HPO4: MgSO4.7H2O: KNO3: NaCl: FeSO4: Agar: Nc ct: 20g 0,5g 0,5g 1,0g 0,5g 0,01g 20g 1000ml

pH = 7,2 7,4 kh trng 0,5 atm/30 pht V. CC PHNG PHP KH TRNG MI TRNG DINH DNG 1. Nguyn tc kh trng Kh trng mi trng dinh dng nhm mc ch: Tiu dit ton b vi sinh vt ngoi lai hin din trong mi trng To iu kin v trng cho mi trng kt qu phn lp, nui cy chnh xc.

Do , khi kh trng mi trng phi m bo mt s iu kin nh sau: Khng lm bin tnh mi trng, khng lm bin tnh mt s cht trong mi trng Sau khi kh trng, trong mi trng khng sn sinh ra cc cht c gy cht vi sinh vt nui cy Phi m bo iu kin v trng tuyt i sau khi kh trng Bo m an ton i vi ngi s dng

2. Mt s phng php kh trng mi trng dinh dng Phng php kh trng c s dng ch yu kh trng mi trng l autoclave. Tuy nhin, ty tng loi mi trng khc nhau s c nhng iu kin kh trng khc nhau a. i vi mi trng cha cc cht d bin tnh (sa, cc ng)
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i vi mi trng cha sa: khng c kh trng trn 1000C v nhit

cao sa d b bin tnh. Do , i vi loi mi trng ny, ta tch ra lm 2 phn: sa tin hnh thanh trng Pasteur, cn cc thnh phn khc c kh trng trong autoclave. Sau , ha trn 2 thnh phn vi nhau. i vi mt s mi trng cha mt s cht d b phn hy nhit cao nh

urea th nu chng ta t pha ch th chia ra 2 phn kh trng cn nu mi trng tng hp th ta kh trng bng cch s dng mng lc c kch thc l nh (0,45 m) lc kh trng. b. Mi trng thng thng Tin hnh kh trng bnh thng vi cc iu kin thi gian v nhit c khuyn co cho tng loi mi trng

CU HI V BI TP
1. Trnh by cc nguyn tc c bn ca vic pha mi trng dinh dng? 2. Cc bc pha mi trng dinh dng? 3. Mi nhm sinh vin thc hnh 3 loi mi trng. 4. Mi nhm SV thc hnh kh trng 1 trong 3 loi va pha bng autoclave v phn phi vo ng nghim, a Petri. 5. Kim tra kt qu kh trng bng cch mi trng vo t m 37oC t 2 3 ngy xc nh c vi sinh vt khng? Nu c, hy gii thch nguyn nhn ca hin tng ny.

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BI S 4: PHN LP NUI CY BO QUN VI SINH VT ---------------------------------1. MC CH V YU CU CA BI 1. Kin thc l thuyt ngha ca vic phn lp, nui cy v bo qun vi sinh vt trong cng tc

nghin cu vi sinh vt hc Cc nguyn tc c bn ca qu trnh phn lp nui cy bo qun vi sinh vt

2. K nng thc hnh K nng phn lp vi sinh vt hiu kh To ra cc khun lc ring r t qun th vi sinh vt To ra cc khun lc ring r mi trng trn a petri cy chuyn Kim tra tinh khit ca ging mi phn lp K nng nui cy vi sinh vt hiu kh Cc thao tc cy chuyn t ng ging sang cc loi mi trng: thch nghing, thch ng, thch a Cc kiu cy khc nhau trn cc kiu mi trng trn K nng bo qun cc chng vi sinh vt thun khit Bo qun trn mi trng thch Bo qun trn ct, trn ht Bo qun bng phng php ng kh 2. HA CHT NGUYN LIU DNG C 1. Ha cht, nguyn liu Mi trng thch ng, thch a, thch nghing nui cy vi sinh vt hiu kh Cc ng ging nm men, nm mc t vn, ct, la

2. Dng c 3. Que cy u trn, u nhn, hnh thc th Que trang, ng ht chia 1ml ng nghim, gi ng nghim, a petri n cn, dim qut CCH THC PHA LONG MU
21

1. Nguyn tc Pha long mu l mt trong nhng cng on c bn nhng rt quan trng trong qu trnh phn tch vi sinh vt. Vic pha long mu cc nng thch hp s gip ch rt nhiu trong qu trnh nh lng cng nh phn tch vi sinh vt 2. Phng php i vi mu cht lng: dng pipet ht 1 ml mu cho vo ng nghim cha 9 ml dung dch pha long, khi ta s c nng pha long l 10-1. Tip tc t ng nghim 10-1 ht tip 1ml v cho vo ng nghim cha 9 ml dung dch pha long pha long 10-2. Tip tc nh vy n nng cn thit long i vi mu rn: cn chnh xc 10 g mu, sau cho vo 90 ml dung dch pha nng pha long 10-1. V tip tc pha long tng t nh mu nc

Hnh 7: Phng php pha long 4. PHN LP VI SINH VT 1. Nguyn tc Tch ri cc t bo vi sinh vt Nui cy cc t bo trn trong mi trng dinh dng to khun lc ring r

2. Qu trnh phn lp vi sinh vt dng thun khit: gm cc bc: To ra cc khun lc ring r t qun th vi sinh vt ban u Phn lp cc vi sinh vt thun khit Kim tra tinh khit ca vi sinh vt

a. To ra cc khun lc ring r trn mi trng phn lp Nu mu ban u dng rn th phi chuyn v dng lng bng cch: Nghin mu Ha tan mu vo nc ct v trng ri pha long
22

Sau thc hin nh l mu dng lng: Tip tc pha long cc nng cn thit Cy mu trn mi trng c trng Ch : nu khng c mi trng c trng c th s dng mi trng m vi sinh vt pht trin bnh thng nhng c b sung cc cht c ch cc vi sinh vt khc pht trin. b. Phn lp vi sinh vt trn mi trng thch trn a petri i vi vi sinh vt hiu kh Ht 0,1 ml dch mu pha long cho vo a petri c mi trng thch hp. Dng que trang tri u mu khp mt thch Tip tc s dng que trang ny tri u khp mt thch a th 2, th 3.. cc a trn nhit thch hp trong 1 khong thi gian nht nh ta s thu

c cc khun lc ring r c. Kim tra tinh khit ca ging mi phn lp Kim tra vt cy Quan st s sinh trng ca vi sinh vt trn mi trng thch Nu vt cy c b mt v mu sc ng u, thun nht chng t ging mi

phn lp tinh khit th gi li Nu vt cy khng thun nht th loi b Kim tra thun ca cc khun lc Chn cc khun lc ring r trn mi trng thch nghing Tch cc khun lc ny ra, ha tan v pha long nng cn thit trong nc

ct v trng. Nh 1 git dch trn vo a petri c mi trng. Dng que trang tri u dch khp mt a petri th nht, ri a th 2, th 3. a petri nhit v thi gian thch hp. Sau quan st cc khun lc ring r. S thun khit ca khun lc l biu

hin s thun khit ca ging 3. Phn lp mt s vi sinh vt trong thc t a. Phn lp vi khun Bacillus subtilis C kh ct nh cho vo bnh tam gic B sung thm: mt t phn v nc sch cho ngp c
23

un si 15 pht dit cc t bo sinh dng v cc t bo khng sinh bo t. y nt bng v t m 25 260C trong vng 48 72h Kt qu: Xut hin lp vng xm c nhiu vi khun B.subtilis v c kh bao gi cng xut hin bo t ca vi khun ny Trn knh hin vi: T bo vi khun B.subtilis c hnh que, di, bo t hnh ovan nm xa tm hay gn tm khun lc. T bo c kch thc 3 5 x 0,6 m.

Hnh 8: T bo vi khun Bacillus subtilis quan st di knh hin vi b. Phn lp nm mc Aspergillus oryzae, Aspergillus niger v Mucor C th phn lp nm mc ny trn cm ngui, xi lm mc tng, bnh m

kh t ngy. Thng qua mu sc ca mc nhn din: Mc c mu trng: Mucor hoc Rhizopus Mc c mu en: Aspergillus niger Mc c mu xanh lc: Penicillum italicum Dng que cy hnh thc th ly 1 t si nm cy vo mi trng thch nghing thch hp (Czapek)

Penicillum

Aspergillus niger Hnh 9: Hnh dng mt s nm mc

24

Rhizopus

c. Thu nhn nm men C th phn lp nm men d dng t cc mi trng nh: B mt tri cy hoc dch p mt s tri cy nh to, l, nho, du.... Trong ru np, trong cc bnh men ru, bia, nc ma vi hm, ht kefir.... Di knh hin vi, nm men c dng hnh cu hay hnh trng. T bo c kch

thc ln, c kh nng ny chi. Khun lc c mu trng sa Chn khun lc nm men ring r v cy vo mi trng thch hp (mi trng

Hansen).

(a)

(b)

Hnh 10: Nm men Saccharomyces cerevisiae di knh hin vi quang hc (a) v knh hin vi in t (b) d. Thu nhn x khun C th thu nhn x khun d dng t t, nc, trn cc c cht ng vt, thc

vt v s dng mi trng Gauze 1 phn lp Phn bit x khun trn mi trng Gauze 1: Khun lc vi khun nhy, t Khun lc vi khun bng xp, c dng si Sau khi cy 0,1 ml dch mu t vo mi trng, cn ch : nui trong t m (280C) t 7 15 ngy mi ly ra chn khun lc ring r cy chuyn

Hnh 11: X khun

25

5. PHNG PHP NUI CY VI SINH VT 1. Khi nim Qu trnh nui cy vi sinh vt gm 2 khu: nui v cy Nui: l qu trnh m bo v duy tr nhng iu kin thun li nht cho s

hot ng v pht trin ca vi sinh vt Cy: l nhng thao tc chuyn vi sinh vt t mi trng ang sng sang mi

trng thun li hn cho s pht trin ca chng Hai khu ny gn b vi nhau trong qu trnh nghin cu v ng dng vi sinh vt 2. Mc ch Pht hin s c mt ca vi sinh vt trong cc nguyn liu vt phm cn nghin cu. Tin hnh nhn ging vi sinh vt mt cch nhanh chng Bo tn cc ging thun khit. Nghin cu cc c tnh sinh hc v h thng sinh hc ca chng.

3. Nguyn tc Mi thao tc nui cy u phi thc hin trong iu kin v trng trnh nhim khun. Mi trng v dng c nui cy phi c kh trng Duy tr cc iu kin thun li vi sinh vt pht trin

4. Cc phng php cy a. Phng php chung ca vic cy chuyn

Hnh 11: Phng php cy chuyn mi trng lng sang mi trng lng
26

Dn nhn ghi: tn ging vi sinh vt + ngy c M Tay cn l m: m 1 n ng n c ng

Dng ngn t v ngn p t rt nt bng c ng mi o Rt que c Kh Kh ng l ng l ti nh c khu

CH : N Thao tc kh gi. Sau khi s ng, c o c ng ng dng pipet ht ca tho gi

b/ Phng php cy trn thch nghing vi sinh v S Th Ha gi Nh xo c. Phng php cy trn thch ng S Sau khi l nh kim. dng que c y su vo kh nh d. Phng php cy trn a petri Dng que cy vng ng ti nh cc thao tc vo o gi n m cc ki nh ch ng ng b : nh t nu trn

thao tc ti

27

Hnh 12: Phng php cy chuyn t mi trng lng sang mi trng thch i ln bn Dng que c M Nh Theo hnh ch Theo nh Theo 4 hnh ch 4 gc n ton b o n m theo 1 trong cc ki

Hnh 13: Kt qu sau khi cy Dng pipet

28

Cch 1: Tr Xoay trn kh ng d o th b

0

n c C

Cch 2: Ht 0,1 ml d Dng que trang tr Sau khi nui c th n c

e. Kt qu ca vic nui cy th cho m

th N c N n nh ng:

6. BO QUN CHNG VI SINH VT THUN KHIT 1. Nguyn tc ph Lm ch nh trao h h nh b

qu trnh sinh s 2. Cc phng php bo qun a. Phng php cy chuyn nh k trn mi trng mi

Th nhi

V V c c th 50C)

n sau 1 6 thng

2 thng

29

gi chuy

m, th . Ph n nhn kh nh c

b. Phng php gi ging trn t, ct, ht Trn t, ct: b qu m nhi o t sinh (bo t th gian b

Cch bo qun: 12 l Rt ton b Hn kn mi Trn ht: b Th Cch b H N Cho cc h Ph C Gi n m o cc y m n l 200C. qu vi sinh v vo m y s o t R S gi o ghi ch i n ng. ngm trong HCl hay trong H2SO4 8

c. Phng php ng kh gi ch ng nhi n t i ch o m nh phn chia c m

30

CU HI V BI TP
1. Nu tm t thu 2. 3. Th nh vi t gi nh nui c n t

31

BI S 5: NGHIN CU MT S C IM SINH HC CA VI SINH VT TRN KNH HIN VI

----------------------I. MC CH YU CU 1. Kin thc l thuyt 2. K nng thc hnh K K K K h II. HA CHT NGUYN LIU DNG C 1. Khi nim thuc nhum Thu Thu nhu u, chng k V d Thu ch nh ph Trong nghin c bo c u r y thu o ch thu t g h h o tnh ch m 2 lo thu o. u m tiu b u cc tiu b n knh hi u b ngh n c m

2. Mt s loi thuc thum c s dng Thuc nhum Fuchsin: 1/ Fuchsin ki 2/ Phenol: c 960 : 10 ml 0,3g 5g 95 ml Tr dung d
32

ng 5 l

Xanh Methylen 0,001% Xanh methylen: 0,1g 1000ml Xanh Methylen Loeffler 300ml Xanh methylen: Ha tan h D KOH 1%: n r 30ml 1ml 1000ml Dung dch lactophenol Phenol k Acid lactic: Glycerin: 10g 10g 20g 10ml Tm gentian Cng th 1/ Gentian violet:
0

20g

1g : 10ml 5g 100ml

2/

Phenol tinh khi

Cch pha ch Tr (2) r u id vo, l n m

Ghi ch: Ha m m c Dung dch lugol

Cng th

Iod tinh th KI:

1g 2g 300 ml

Cch pha ch
33

Ha KI vo trong 5ml n B Cn 95% Aceton 3. Nguyn liu ng c i vi sinh v Bacillus subtilis, Sarcina lutea Penicillum italicum, Aspergillus niger Streptomyces grisea Saccharomyces cerevisiae 4. Dng c III. Knh hi LM TIU BN TM THI Lame, lamelle Que c Gi Bocan, c Bnh tia n c M Hansen nui c th ng pepton d Saccharomyces cerevisiae E.Coli. t

1. Cc c im ca tiu bn tm thi s Ch nh thnh bo t Thao tc lm tiu b nh nhanh. n mao,

2. Cch ly ging vi sinh vt lm tiu bn M Tay cn l Ko nt bng ra kh ng kh

34

n c

ng mi

Rt que c a gi m v Kh ng l

o l ng mi kh o gi

3. Cch lm tiu bn git p Dng que c n gi Quan st trn knh hi Ch : N N b 4. Cch lm tiu bn tm thi c nhum mu a. Nguyn tc v ng nhu u b ho n ra ngoi lamelle th dng gi dng vaselin bi quanh mp lamelle m v

b. Cch nhum: C 2 cch nhu Cch 1: Quan st tiu b Cch 2 Nh Dng que c Nh Quan st tiu b iv nh 1 vng nh kh nh X10 v X40 n lame n. X40 Nh Nh v n lame

IV. LM TIU BN C NH 1. Cc c im ca tiu bn c nh

35

Quan st tiu b sinh v n c Tuy thao tc ph

n c

Cho php ta quan st r rng hnh d

oc

o, d

ng vi sinh v Khng s m vi v

2. Cc bc tin hnh tiu bn c nh a. Lm vt bi Ch : V Cc t Vi Gi G Cc cch c C Dng k C Cch ny tuy ph bi Dng cc ha ch Cch c Nhng v v Ngm v 5 pht. o dung d
36

Ch L

kh o gi n trong khng kh

n g o vi sinh v

b. C nh vt bi

u v an ton khi ti

ph bi k n ng n c

o, khng gy n mao.

u. V

15 pht, cn

Nh i l

i gi

n v

3. Nhum mu tiu bn c nh a. Nguyn tc S cc thnh ph v Ty theo m ph o m ch Nhu Nhu b b. Cch nhum Nh R v Dng gi Quan st tiu b u b u b o v n c i gi n 1 2 pht. n c kh u khc nhau c cch nhu h nh uc o thnh nh ng t o v k

C 2 cch nhu ng m n 1 tiu b n 1 tiu

ng lame, dng bnh tia x n mu n u b X100 dng d i

V. QUAN ST C IM SINH HC CA CC NHM VI SINH VT 1. Quan st cc c im sinh hc ca vi khun (Bacteria) a. Cc c trng sinh hc cn quan st vi khun Cc ki Cc ki Kh Kh Nhu nh thnh bo t nh d n k o o

b. Cch quan st Quan st cu khun Lm tiu b Quan st cc tiu b n knh hi

37

ng c

Sarcina lutea.

Yu cu: V nh d o, cc ki n k o o Nh n xt v Quan st trc khun

Lm tiu b Lm tiu b

E.Coli Bacillus subtilis nui c ng th

nghing (2) Yu cu: Tiu b c V c o. X100 b nh d o o; hnh d v Tiu b nh v nh o t hnh, nh

Quan st xon khun Lm tiu b Spirochae, Spirillum. Ch L Quan st V nh cc d X100 b ng nhi m v u v Vibrio,

vng)

a)

b) Hnh 13: Cc hnh dng chnh ca vi khun a) c b) tr c) ph khu hay xo

c)

2. Quan st c im sinh hc ca x khun (Actinomycetes)

38

a. Cc c im sinh hc c trng cn quan st Hnh d o t o t nh thnh chu o t

b. Cch quan st Lm tiu b b y ph cc s n u b X40. V nh v nh nh ng que c Streptomyces grisea trn th m khu ng (khi lm tiu

tch r -

Lm tiu b Yu c d

Lm tiu b C Dng lamelle c o b

o t ng m
0

n lame v quan st o t n m c x

Lm tiu b Dng lame V

Quan st trn knh hi nh v nh o t bo t

3. Quan st c im sinh hc ca nm mc (Molds) a. Cc c im sinh hc c trng cn quan st quan sinh s Hnh d , cch s o t u s quan sinh s nh d

b. Cch quan st Lm tiu b Nh Dng que c dn m quan st

39

u n lame Penicillum ho Aspergillus niger v

X40

V chung c -

nh v nh o t Nh L V nh c nh, cu

nh d nh, cu nhu u: n lame n lame quan st o t

nh d

x n

Lm tiu b

4. Quan st c im sinh hc ca nm men (Yeasts) a. Nhng c im sinh hc cn quan st Hnh thi, kch th S Hnh d o t o t o n

b. Cch quan st Lm tiu b Yu cu: Quan st knh hi V nh ch thch mng t o ch X100 o c o

VI. PHNG PHP NHUM GRAM 1. Ha cht nguyn liu - dng c a. Ha cht Nguyn liu Ha cht Nguyn liu Cc ng nui c i B.subtilis, E.Coli. Cc lo

b. Dng c Lame, lamelle Que c Bnh Ch C h

40

ng

n c

m qu u b

Knh hi Gi

2. Nguyn tc nhum gram D n kh u c o ch mng t o v

tinh v iod m hnh thnh nn hai lo r x thu 3. Cc bc tin hnh Lm tiu b Dng que c lame). Dng que c Bn tri lame l B.subtilis Bn ph Nhu Nhu Nhu R T khi tan h R Nhu R Lm kh v soi tiu b K V B.subtilis mu tm
41

Lo

n mu c y l vi khu

n khng b

Lo b u c

u c

n m

u khi y

m 3 v

gi

m v

E.Coli B.subtilis tr ong khng kh ho E.Coli

u b n 3 v u b

ng tiu b u

30 giy

 V V

B.subtilis + E.Coli c mu h E.Coli mu h - gram m

u tm

Hnh 14: Cc bc nhum gram

Hnh 15: Kt qu sau khi nhum gram

CU HI V BI TP
1) Th hnh lm tiu b E.Coli. V nh v ch thch cc

2) Th

nh lm tiu b

B.subtilis, vi khu

hnh v ch thch cc

42

3) Lm tiu b c 4) Trnh by nguyn t 5) Nu tn cc lo 6) Trnh by c 7) Tm t n t

u A.niger. V

nh v ch thch cc c

ngh

nh, ch thch k

nhu

43