BI 1: CHUN B MI TRNG V THAO TC V TRNG

CC THIT B PHNG TH NGHIM VI SINH V PHNG PHP KH TRNG 1) Mt s dng c v thit b phng th nghim vi sinh: Cc dng c thy tinh: ng nghim: c s dng cha mi trng nui cy vi sinh vt, c nt bng gn khng thm nc hay nt bng nha chu nhit.

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

ng ht (pipette) ng ht c chia . Micropipette (Pipetman).

 Cc dng c bng thy tinh khc: Becher. Bnh cu y bng v y trn. Bnh tam gic (erlen). Cc dng c, thit b khc: 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 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 hkng b xy ha nhit cao. T m: dng vi sinh vt hoc theo di s tng trng ca vi sinh vt.

 T cy v trng:

 Autoclave:

2) Cc phng php kh trng dng c: a) 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 t cho mu vt. b) Cc phng php kh trng: Khi kh trng bng nhit, cc t bo sinh dng ca vi sinh vt 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. S lng t bo. pH 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 vi sinh vt v bo t ca chng c trong dng c cn kh trng. C th kh trng bng nhit kh hay nhit t. 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 (110oC trong 2 -3 ngy). Tt t sy, ngui ti 60oC 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 hi mu vng l t yu cu. 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. 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 Cch tin hnh: Dng nc sch ngp dng c. un si t 10 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 70oC trong 15 30. Phng php ny ch c tc dng c ch vi sinh vt khng c bo t. Hp cch qung 100oC (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 100oC t 30 40. Ly ra t m 24h cho bo t vi khun pht trin.

 Hp mi trng ln th hai 100oC trong 30 40 tiu dit cc bo t va ny mm. Lp li qu trnh ny 3 -4 ln. 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. Cch s dng: Chun b ccdng c cha mi trng cn kh trng. Cho 3 lt 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, ni s bo hiu bng ci. Gt cng tc kh trng v v tr dry. Ch kim ch p sut 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. Kh trng bng s lc: S dng cho mi trng lng, trong, c nhy yu, khng chu c nhit cao hn 60oC. Cho mi trng i qua mt mng lc xp c ng knh l nh hn ng knh 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. Dit trng bng bc x: Tia t ngoi: bc x thng dng nht l tia UV. Dng tia UV tit 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. PHNG PHP LM MI TRNG

Lm mi trng thc hin vic phn lp, nhn ging, gi ging vi sinh vt, ng thi nui cy v nghin cu cc c im sinh hc ca chng.

1) Nguyn tc ca vic ch to 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 v s cn bng p sut thm thu gia mi trng v t bo vi sinh vt nn cn iu chnh t l v nng cc cht trong thnh phn mi trng. m bo cc iu kin ha l cn thit cho cc hot ng trao i cht ca vinh vt. pH thch hp. Khng c cht c hi. V trng. Phn loi: Ngi ta da trn cc c s khc nhau phn loi mi trng. 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, chnh xc. Mi trng bn tng hp: cha c cht ha hc ln sn phm t nhin. 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 ch 1,5 2% agar hoc 10 20% gelatin. Mi trng ny c s dng nghin cu cc c im hnh thi, sinh l vi sinh vt. Mi trng bn lng: ch cha 0,3 0,7% agar. 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 . 2) Cc bc ch to mi trng dinh dng: Mi trng Hasen:

 Glucose: 50g. Pepton: 10g. K2HPO4: 3g/l. MgSO4: 3g/l. Agar: 20g/l (+/-). Nc ct 1 lt. pH = 6. Mi trng GPA: Glucose: 50g. Agar: 20g/l (+/-). Dch chit khoai ty/ gi 1 lt (500g khoai ty gt v/ ct thi/ nu k vi 1 lt nc trong 2 gi. Lc/ thm nc cho va 1 lt). Trong bi thc hnh, chng ta s dng mi trng GPA. Pha ch: Cn, ong tht chnh xc tng 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 ha tan vo nc ct. Mi trng c: Cn agar ri ha tan vo nc. un trn bp, khuy u tay cho n khi agar tan ht vo trong dung dch dung dch tr nn trong. iu chnh pH ca mi trng (nu cn): Trong bi, ta s dng mi trng t nhin t nc chit khoai ty nn khng cn iu chnh pH. Mun iu chnh pH ca mi trng ngi ta dng HCl 10% hay NaCl 10%. Ngai ra, c th dng mt s ha cht khc nh: H3PO4, H2SO4, KOH, NaHCO3, Na2CO3 Mun kim tra pH ca mi trng ta nn dng my o pH (pH metre). Phng php ny nhanh nhy v cho chnh xc cao. Trong phng th nghim, c th dng ch th mu xanh bromotomol hay giy qu o pH. Phng php ny tin li, nhanh nhng khng chnh xc cao. Phn phi mi trng vo dng c: Ngi ta thng phn phi mi trng vo ng nghim, a petri, bnh tam gic. Trnh t phn phi gm cc bc sau:

 Mi trng cn c un cho ha cht lng ri vo cc dng c. Tay tri gi dng c cha mi trng. Tay phi kp nt bng ko ra. Nhanh tay rt mi trng vo dng c v y nt bng li. Ch : i vi ng nghim: Nu dng mi trng lm thch nghing th lng mi trng cn c phn phi chim 1/4 th tch ca ng nghim. Nu lm thch lng th lng mi trng cn c phn phi 1/3 th tch ng nghim. i vi bnh cu hay bnh tam gic: lng mi trng c phn phi chim 1/2 n 1/3 th tch bnh. Cc thao tc phn phi phi nhanh, gn, kho lo mi trng khng dnh ln ming dng c hoc nt bng v vic phn phi cn thc hin xong trc khi mi trng b ng c. Lm thch nghing, thch v a petri:

Lm thch nghing: cn tin hnh ngay sau khi kh trng mi trng va kt thc v mi trng cha ng c. thch vo a petri: Ton b quy trnh thch vo a petri u thc hin trong t cy v trng. Ch :

 Thao tc thch phi ht sc khn trng v kho lo hn ch s nhim khun. Mt thch phi phng, nhn c dy khong 2mm. Thng thng c 1/4 lt mi trng c th phn phi c 22 25 a petri. Kh trng mi trng: Sau khi phn phi mi trng vo dng c, bao gi cn thn, vit nhn (tn mi trng; ngy, thng kh trng). Kh trng bng autoclave nhit 171oC. Trong thi gian 2 3 gi, nu nhit thp hn th kh trng nhiu ln hoc thi gian nhiu hn. Bo qun v kim tra mi trng: Mi trng cha dng cn c bo qun ch mt, hn ch tc dng ca nh sng, nhit t 0 5oC v khng mi trng bi kh. Trc khi s dng, kim tra v khun ca mi trng, ngi ta thng t chng vo t m 37oC, trong 2 3 ngy. Sau ly ra quan st, loi b cc ng c vi sinh vt pht trin v ch s dng nhng ng nghim. nhng a petri c mi trng t yu cu.

BI 2: CC PHNG PHP CY CHUYN V PHN LP VI SINH VT

PHNG PHP NUI CY VI SINH VT 1) Khi nim: Qu trnh nui cy vi sinh vt gm hai khu: nui v cy. Nui: l qu trnh m bo v duy tr nhng iu kin thun li nht cho s hat 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) 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. 3) Phn loi: Cy ging t mi trng lng sang mi trng lng. Cy ging t ng thch nghing sang ng thch nghing. Cy ging t ng thch nghing sang mi trng lng. 4) Cc phng php cy: Cy ging t ng thch nghing sang mi trng lng:

Phng php cy chuyn mi trng lng sang mi trng lng

Dn nhn ghi: tn ging vi sinh vt + ngy cy. Mt tay cm hai ng nghim: mt ng ging v mt ng mi trng. Tay cn li cm que cy v t dy cy trn ngn la n cn kh trng. Dng ngn t v ngn p t rt nt bng ca ng ging ra. H nng kh trng ming ng nghim. i que cy ngui, a que cy vo ng nghim ly khun lc trong ng ging. Rt que cy ra v tin hnh cy chuyn trong ng mi trng. Kh trng li phn khng kh ni ming ng nghim ri y nt bng. Kh trng li que cy sau khi s dng. Cy ging t ng thch nghing sang ng thch nghing: Phng php ny cy chuyn cc vi sinh vt hiu kh: S dng que cy vng tin hnh cc thao tc theo ng trnh t nu trn.

 Thc hin vic cy ging vo ng thch nghing bng cc thao tc tip theo: Ha ging u que cy vo git nc y ng nghim. Nh nhng lt que cy trn mt thch theo cc kiu: hnh ch chi, vng xon, vch ngang song song.

Mt s kiu cy trn ng thch nghing

5) Kt qu ca vic nui cy: Sau khi nui thi gian v nhit thch hp cho mi loi vi sinh vt, ta thy: Trong mi trng lng: vi khun pht trin s lm c mi trng. Trong mi trng c thch nghing: Nm men, vi khun pht trin s to thnh vt ni trn mt thch trong hay trng c. Nm mc s to nn nhng si mnh t vt cy.

PHN LP GING VI SINH VT 1) Khi nim: Phn lp vi sinh vt l qu trnh tch ring cc loi vi sinh vt t qun th ban u v a v dng thun khit. y l mt khu c ngha rt quan trng trong vic nghin cu v ng dng vi sinh vt. Vi sinh vt dng thun khit l ging vi sinh vt c to ra t mt t bo ban u.

 Trong thin nhin hoc trong cc vt phm nghin cu, vi sinh vt thng tn ti dng hn hp gm nhiu loi khc nhau. Mun nghin cu v hnh thi, sinh l, l ha hoc s dng vo thc tin mt loi no th cn phi a chng v dng thun khit. 2) Nguyn tc: Tch ri cc t bo vi sinh vt Nui cy cc t bo trn trong mi trng dinh dng c trng cho khun lc ring r, cch bit nhau. 3) Phng php phn lp vi sinh vt: Trong bi thc hnh, ta s dng k thut hp ria (ria bn gc) to khun lc n. C th dng thao tc cy tri, a. K thut hp ria: Dng que cy vng thao tc v trng thu ging. Ria cc ng trn a petri cha mi trng thch hp (ria ch T v tia bn gc). Sau mi ng ria, t kh trng u que cy (nhm loi b cc vi sinh vt cn d, dnh trn que cy) v lm ngui trc khi thc hin ng ria tip theo.

Cc kiu ria trn mt thch

kt qu th nghim:
- Cc t bo VSV b tch ring r,sau thi gian nui cy to thnh cc khun lc trn nn thch - Phng php ny gip phn lp v to thnh dng thun,c ngha c bit trong cng vic to ging trong sinh hoc.

BI 3: QUAN ST V PHT HIN VI SINH VT BNG PHNG PHP NHUM

1) Nguyn tc: S dng thuc nhum c kh nng thm thu qua mng t bo v kt hp vi cc thnh phn khc nhau ca t bo to thnh nhng hp cht mu c trng bn vng. Ty theo mc ch nghin cu v kh nng bt mu khc nhau ca cc thnh phn t bo m chn loi thuc nhum v cch nhum cho ph hp. C hai cch nhum chnh: Nhum n: ch dng mt loi thuc nhum trn mt tiu bn. Nhum kp: dng ng thi hai hay nhiu loi thuc nhum trn mt tiu bn. 2) Thao tc thc hnh: a) Cch lm tiu bn c nh: Cc c im ca tiu bn c nh: Quan st tiu bn c nh l mt phng php ph bin trong nghin cu vi sinh vt v n c nhng u im sau: Thao tc tuy phc tp nhng tiu bn c mu sc p, gi c lu. Khng s ly nhim khi lm vic vi vi sinh vt gy bnh. Cho php ta quan st r hnh dng, cu to ca t bo cng nh d dng m s lng t bo. Cch lm tiu bn: Lm vt bi: t mt git nc sch vo gia phin knh, sau dng que cy kh trng ly mt vng que cy vi sinh vt cho vo git nc v dn u ra (1 2 cm). Trng hp nui cy trn mi trng lng, th dng pipet ly dung dch nghin cu. H kh phin knh c vi sinh vt trn ngn la n cn hoc kh t nhin ta c mt vt m trn phin knh gi l vt bi. Ch : Lng vi sinh vt ly va phi. Vt bi trn, gn, tht mng. C nh vt bi:

Vic c nh vt bi nhm cc mc ch sau: Git cht vi sinh vt chng bt mu v an ton khi tip xc (nu l vi sinh vt gy bnh). Gn cht vi sinh vt vo phin knh lc nhum khng b ra tri. C nh bng nhit: (trong bi ta s dng cch c nh bng nhit, c th dng cch c nh bng ha cht). Dng kp g kp phin knh, h qua li trn ngn la n cn 2 3 ln (chng vi ba giy), trnh khng tiu bn qu nng. b) Phng php nhum n: Cc loi thuc nhum thng dng nhum n: Fuchsin. Xanh metylen. Tm gentian. trung tnh. Trong bi s dng xanh metylen hay Fuchsin. Nh thuc nhum ln tiu bn c nh. yn trong 1. Ra nh bng nc ct ti khi no thy nc ra chy qua vt bi khng cn mu na (3 ln). tiu bn kh dn. Khi tiu bn kh hn, nh mt git du soi knh, v em i quan st di knh hin vi vt knh 100.

c) Phng php nhum kp: Nguyn tc ca vic nhum kp: Cc cu trc khc nhau, thm ch cc phn khc nhau ca mt cu trc thng c tnh cht ha hc, l hc cng nh kh nng bt mu khc nhau. Vic s dng ng thi cc loi thuc nhum trn cng mt tiu bn cho php ta c th quan st v phn bit cc cu trc d dng hn. Phng php nhum Gram: Nguyn tc:

 Da trn kh nng bt mu ca t bo cht v mng t bo vi thuc nhum tm tinh th v iot m hnh thnh nn hai loi phc cht khc nhau. Loi phc cht th nht vn gi nguyn mu ca thuc nhum nn khng b ra tri khi x l bng cn. Vi sinh vt c phc cht ny thuc loi Gram dng. Loi phc cht th hai khng cn gi c mu ca thuc nhum nn mt mu khi x l bng cn v bt mu ca thuc nhum b sung. Vi sinh vt c loi phc cht ny thuc Gram m. Cch tin hnh: Sau khi c nh tiu bn, nh tm tinh th ln trn phin knh. yn trong 1. Nh lugol ( yn trong 30, gi mu ca tm tinh th). Ra s bng nc ct. Ty cn (nhng qua cn 3 ln, dung dch cn s ra sch thuc nhum khng gn kt, vi khun gram dng gi li mu tm, vi khun gram m mt mu). kh t nhin hoc sy. B sung Fuchsin (trong 1). Ra sch bng nc ct. i tiu bn kh, nh mt git du soi knh v em quan st di knh hin vi vt knh 100. Quan st di knh hin vi: Vi khun gram dng: tm. Vi khun gram m: hng.

Nhn xt:
- Phng php nhum n:dng kho st hnh thi v cch sp xp ca VSV bit c ging VSV o u im : d thc hin trong phng th nghim, o nhc im: khng phn bit c t bo sng v cht. - phng php nhum kp:cho ta bit c cu trc mng t bo ca VSV

BI 4: CC PHNG PHP NH LNG VI SINH VT

1) Phng php nh lng trc tip: Mt vi sinh vt n bo c k ch thc ln nh nm men, to c th xc nh bng cch m trc tip bng bung m trn knh hin vi. Quy trnh m trc tip cho php xc nh nhanh chng mt vi sinh vt trong mu nhng phng php ny c mt s nhc im l khng phn bit gia t bo sng v t bo cht, d nhm ln t bo vi sinh vt vi cc ht vt th khc trong mu, kh t chnh xc cao, khng thch hp cho huyn ph vi sinh vt c mt thp. m trc tip bng bung m hng cu: Cu to bung m hng cu: Bung m thng l mt phin knh dy 2- 3mm c mt vng a m nm gia phin knh v c bao quanh bi mt rnh. a m thp hn b mt ca phin knh khong 1/10 m, c hnh trn v th khi c ph ln bng mt l knh th su ca a m s ng u nhau. Vng a

m c din tch 1mm2 v c chia thnh 25 vung ln c din tch mi l 1/25 mm2 v 400 vung nh hn, mi c din tch 1/400 mm2.

Cu to bung m hng cu

Khi thc hin quan st v m vi sinh vt, phi pha long mu cn m sao cho mi ln ca bung m c khong 50 200 t bo vi sinh vt. t c pha long nh vy cn phi c lng c s t bo vi sinh vt trong mu, ng thi phi th vi ln trong qu trnh pha long. t mt git mu c pha long vo vng m trn bung m. Chnh th trng sao cho mt th trng cha trn mt ln. m s t bo hin din trong mt ln. Sau , chnh th trng tm mt ln khc. m s t bo ca t nht nm ln. Ly tr s trung bnh. Cch tnh mt t bo nh sau: Mt t bo = (TB/ml).

a: tng s t bo trong nm ln i din. H s pha long ca mi trng bng nghch o nng 10-n. Cch tin hnh: Dch mu em i pha long thch hp. Ht mt t dch mu nh ln hai rnh ca bung m. y lamelle.

 m di knh hin vi ( vt knh 10 hay 40). Ch : Khng c m bn cnh ca mt gy sai s rt ln. Phi vortex k trc khi tin hnh pha long hoc m do cc t bo vi sinh vt c th b lng ng. - kt qu th nghim: m tng trng 5 theo hang cho thu c s VSV 2) Phng php nh lng gin tip: Phng php o c: L phng php gin tip xc nh mt vi sinh vt. nh lng mt t bo thng qua o c bng my o quang cc bc sng 550 610nm. Phng php xc nh mt t bo theo c c th c dng so snh mc tng trng ca hai hay nhiu chng vi sinh vt trong mi trng lng. Phng php ny cho kt qu nhanh chng v thng c ng dng trong theo di hoc nghin cu c trng tng trng ca cc chng vi sinh vt trong phng th nghim hoc trong sn xut. Tuy nhin, khng thch hp dng trong kim nghim vi sinh vt. Nguyn tc: Khi mt pha lng c cha nhiu phn t khng tan to thnh mt h huyn ph c c bi cc phn t hin din trong mi trng lng cn nh sng, lm phn tn chm nh sng ti. T bo vi sinh vt l mt thc th, khi hin din trong mi trng cng lm cho mi trng c. nh lng gin tip thng qua c. Da vo ng chun. Cch tin hnh: Xy dng ng chun: Ly dch mu, pha long cc nng khc nhau o c bc sng 610nm. Ghi nhn kt qu. m trc tip bng bung m. Dng ng chun mt t bo theo quang.

 tin hnh o mu: ta lm tng t nh trn, pha long dch mu sao cho OD lt vo ng chun ri t suy ra c mt t bo. Kt qu th nghim: Mu ban u: OD = 1,94. Tin hnh pha long:

Mu 1 2 3 4 5

Pha long 10 ln 5 ln 3 ln 2,5 ln 2 ln

OD 0,62 1,00 1,12 1,44 1,55

m trc tip bng bung m: Mu 1 2 3 4 5 OD 0,62 1,00 1,12 1,44 1,55 S t bo vi sinh vt 208 495 573 1293 1501 Log (t bo vi sinh vt) 2,318 2,695 2,758 3,112 3,176

Dng ng chun:

Log ( TB)

3.5 3 2.5 2 1.5 1 0.5 0 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 OD

y = 0.9368x + 1.7382 R = 0.9958

th Log(t bo vi sinh vt) theo c OD

Nhn xt: Phng php ny cho kt qu nhanh chng, chnh xc. Tuy nhin, cn quan tm n mt s thao tc c th gy sai s cho phng php: Thao tc pha long mu (thao tc chm, pha long khng chnh xc, vortex khng k). Thao tc o c (thao tc cm cuvette, thao tc chm lm t bo vi sinh vt b lng, vortex khng k). Sai s do my o. Sai s khi m trc tip trn bung m. Phng php m s khun lc:

 Nguyn tc: Mt t bo c th phn chia theo cp s nhn cho n khi hnh thnh khun lc c th trng thy c. y l c s ca vic nh lng t bo trn thch a, bi v s lng khun lc sinh ra t mt th tch ging vi sinh vt nht nh ch s lng t bo sng trong th tch ging . Thng thng tt c t bo pha tng trng hay giai on sm ca phage n nh u c kh nng hnh thnh khun lc. V vy, s lng khun lc m c gn nh tng ng vi s lng t bo sng. Cch tin hnh: Dch mu pha long theo nng thch hp. Trnh t pha long nh sau:

 Nh vo a petri chun b mi trng thch a (0,1 1ml dung dch mu). Tri n bng que tri ( kh trc khi em ). mu (2 3 ngy). m s khun lc mc trn mt thch.

Cng thc tnh s t bo: S t bo sng = X . k . 10-n X: s khun lc trn a petri. K = 1/m : (m l s ml dung dch trn a petri 0,1 1ml). H s pha long bng nghch o nng 10-n. T l t bo sng: (% t bo sng) = (t bo sng/t bo) . 100%. Nhn xt: u im: Cho php xc nh t bo sng. nh lng chn lc vi sinh vt. C th nhn dng c khun lc c trng. Nhc im: Mt nhiu thi gian. Khng xc nh c t bo ym kh hoc hiu ym kh. Mt mt lng t bo dnh trn que tri. D b nhim khun, gy kt qu khng chnh xc. Khun lc mc cnh tranh hoc trong mi trng gy kh m.