Cc c im sinh haBi:

Nguyn Ln Dng

CC C IM SINH HA:

Xt nghim oxidaza

Mc ch: phn bit cc nhm vi khun da trn hot tnh cytochrom oxidaza

? Pha dung dch Tetramethyl-p-phenylen diamin dihydrochlorid (TPPDD) 1% trongnc, bo qun trong l mu ti 4 C, s dng trong 2 tun.

? t mt ming giy lc trong np hp Petri sch, nh dung dch TPPDD 1% lntrn ming giy lc sao cho va m, khng qu t.

? Dng que cy c u que lm bng si platin hay dng a thy tinh (khng dngu que cy bng si kim loi st, niken...) ly mt t vi khun hot ho (18-24 gi)bi ln ming giy lc.

? Sau 10 giy nu vi khun chuyn sang mu hng tc l c oxydaza dng tnh;nu sau 60 giy mi chuyn mu l oxydaza m tnh.

? Ch : nu dung dch t chuyn sang mu hng ri th khng c s dng.Nu giy lc qu t s cn tr khun lc tip xc vi khng kh nn chuyn mu chm,to nn m tnh gi.

Xt nghim catalaza

Mc ch: kim tra kh nng phn hu H2O2 ca vi sinh vt nh sn sinh ra enzymecatalaza.

? Chun b dung dch H2O2 nng 3-10%, nh mt git ln phin knh.

? Dng u que cy platin ly mt t vi khun mi hot ho (24 gi) trn vo gitH2O2 trn phin knh.

? Nu thy si bt l dng tnh, khng si bt l m tnh.

Cc c im sinh ha

1/37

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? C th nh trc tip dung dch H2O2 ln khun lc trn thch a cng cho kt qutng t.

Hnh 3.1. Phn ng si bt khi tip xc vi dung dch H2O2 ca vi khun ccatalaza dng tnh.

Kh nng ln men/xy ha glucoza

C th dng mt trong hai mi trng sau lm th nghim

? Mi trng I:

Pepton 2 g

NaCl 5 g

K2HPO4 0,2 g

Glucoza 10 g

Thch 6 g

Dung dch BTB 1% 3 ml (pha trong mt t cn 95%, sau mi thm nc thnh dung dch 1%)

Nc ct 1000 ml.

pH = 7,0 - 7,2.

Phn mi trng vo cc ng nghim (4-5 ml), kh trng 115 0C trong 20 pht.

? Mi trng II:

NH4H2PO4 0,5 g

Cc c im sinh ha

2/37

K2HPO4 0,5 g

Cao men 0,5 g

Glucoza 10 g

Thch 5-6 g

Dung dch BTB 1% 3 ml (pha nh trn)

Nc ct 1000 ml

pH = 7,0 - 7,2.

Phn mi trng vo ng nghim v kh trng nh trn.

? Ly vi khun mi hot ho (18-24 h) cy chch su vo mi trng bng que cythng (mi chng vi khun cy vo 4 ng). Bt kn 2 ng nt bng bng vaselin-paraffin(ly vaselin lm chy ra, thm 1/3 du paraffin) cch ly vi khng kh. Ngoi ra lythm 2 ng khng cy vi khun lm i chng. Theo di kt qu sau 1,2,3,7 v 14 ngy.

? Kt qu: - Nu ch c ng khng bt kn sinh axit (chuyn mu vng)

tc l vi khun thuc dng xy ha.

- Nu c ng khng bt v ng bt kn u sinh axit (chuyn mu vng) tc l vi khunthuc dng ln men.

Kh nng ln men ng, ru

? Mi trng:

Cao tht 3 g

Pepton 10 g

NaCl 5 g

Cht ch th mu Andrade* 10 ml

(hay dung dch Xanh bromophenol 0,2%)

Cc c im sinh ha

3/37

Nc ct thm n 1000 ml

? B sung ng vi nng 0,5%. Phn mi trng vo cc ng nghim, mi ng1ml.

? t vo mi ng nghim 1 ng nh (ng Durham) ln ngc u hng khCO2 sinh ra nu vi khun c kh nng ln men ng. Kh trng trong 15 pht 1210C. ng arabinoza, xyloza, v cc ng kp cn kh trng ring bng mng lc rimi b sung vo mi trng.

* Cch pha cht ch th mu Andrade:

Fuchsin axit 0,5 g

NaOH 1M 16 ml

Nc ct 100 ml

Nu dung dch c mu hng, dng NaOH 0,1 M (1-2 ml) trung ha cho n khi mtmu.

Xa nh bromophenol (BPB):

2 g BPB, b sung dn 5 ml NaOH 0,1M, nghin trong ci s, ha vi nc ct cho 100 ml.

? Cy vi khun mi hot ho vo cc ng nghim, t 36 0C, theo di hin tngsinh axit sau 1-3 ngy. C trng hp cn dng paraffin bt kn nt bng v theo ditrong 14-30 ngy

? Nu vi khun c kh nng ln men ng (sinh axit) th cht ch th Andrade schuyn mu , cht ch th BPB s chuyn mu vng lc.

C th lm cch khc nh sau:

? Vi vi khun ni chung dng mi trng I (xem mc 3.3), thay glucoza cc ngkhc hay ru (nng 1).

? Vi vi khun sinh bo t dng mi trng sau:

(NH4)2HPO4 1 g

KCl 0,2 g

Cc c im sinh ha

4/37

MgSO4 0,2 g

Cao men 0,2 g

Thch 5-6 g

ng hay ru 10 g

Nc ct 1000 ml

Dung dch BTB 0,04% 15 ml

pH = 7,0-7,2.

Phn mi trng vo cc ng nghim (4-5 ml), kh trng 112 0C trong 30 pht.

? Vi vi khun lactic dng mi trng sau:

Pepton 5 g

Cao tht 5 g

Cao men 5 g

Tween 80 0,5 ml

Thch 5-6 g

Nc ct (hay nc my) 1000 ml

Dung dch BTB 1,6% 1,4 ml

pH = 6,8-7,0.

Phn mi trng vo ng nghim, kh trng ti 112 0C trong 30 pht.

? Ly vi khun mi hot ho (18-24 gi) cy trch su vo mi trng thch, t nhit thch hp v quan st sau 1,3,5 ngy.

? Nu ch th mu bin vng l vi khun c kh nng ln men sinh axit (phn ngdng tnh); nu vn gi mu lam l phn ng m tnh.

Cc c im sinh ha

5/37

Phn ng M.R. ( Methyl - Methyl Red)

? Mi trng:

Pepton 5 g

Glucoza 5 g

K2HPO4 hoc NaCl 5 g

Nc ct 1000 ml.

pH = 7,0 - 7,2.

Phn mi trng vo ng nghim (4-5 ml), kh trng 115 0C trong 30 pht.

? Thuc th:

Methyl 0,1 g

Etanol 95% 300 ml

Nc ct 200 ml.

? Cy vi khun vo mi trng (lp li 2 ln), t nhit thch hp trong 2-6ngy (nu kt qu m tnh cn ko di thm thi gian). Vi Vi khun ng rut thuch Enterobacteriaceae, t ng nui cy 37 0C v kim tra sau 4 ngy.

? Nh 1 git thuc th vo dch nui cy, nu chuyn mu l phn ng dngtnh, mu vng l m tnh (mu pH 4,4; mu vng pH 6,0).

Cc c im sinh ha

6/37

Hnh 3.2. Cht ch th methyl chuyn mu khi tip xc

vi dch nui cy vi khun ln men ng.

Phn ng V.P. (Voges-Proskauer)

? Mi trng: xem phn 3.5.

? Thuc th:

Creatin 0,3% (hoc nguyn dng tinh th)

NaOH 40%

? Cy vi khun mi hot ho, t nhit thch hp trong 2-6 ngy.

? B sung NaOH 40 (bng th tch dch nui cy), sau nh mt t dung dchcreatin (hoc thm mt t tinh th), i khong 10 pht (c khi lu hn). Nu dch thchuyn mu l phn ng dng tnh.

Cch khc:

? Mi trng Clark-Lubs:

Pepton 3 g

K2HPO4 5 g

Cc c im sinh ha

7/37

Glucoza 5 g

pH = 7,5.

? Phn ng V.P: nh 5 git dung dch alpha naphtol 6% (trong cn 90%, gi 4 Ctrc khi dng) v 5 git NaOH 16% (trong nc), lc nh. Nu dch th chuyn sangmu nu l phn ng dng tnh, mu vng nht l m tnh.

? Phn ng M.R: nh 2-3 git dung dch methyl 0,5% (trong cn 60%), lc nh.Nu dch th chuyn mu l phn ng dng tnh, mu vng nht hay khng mu lm tnh.

Hnh 3.3. V d minh ho kt qu phn ng V.P. v M.R.

Phn ng ONPG-aza (O-nitrophenyl--D-galactopyranosidase)

? Mi trng:

ONPG 0,6 g

m phosphat 0,01M pH 7,5 100 ml

Dung dch pepton 1% (pH 7,5) 300 ml.

(Ho 0,6 g ONPG trong 100 ml dung dch m, kh trng bng mng lc, sau trnvi 300 ml dung dch pepton kh trng).

Cc c im sinh ha

8/37

Bng thao tc v khun phn mi trng vo cc ng nghim nh, bo qun 4 Ctrong vng 1 nm.

? Ct nhng khoanh giy lc, kh trng 112 C, 30 pht.

? Nh vo mi khoanh giy lc mt git dung dch sau:

ONPG 0,06 g

Na2HPO4.2H2O 0,017 g

Nc ct 10 ml

Lm kh 37 0C trong 24 gi, bo qun trong cc ng nghim c nt xoy nhit phng.

? Cy vi khun mi hot ho (1 vng que cy) vo mi trng chun b nh trn,t nhit thch hp trong 24 gi.

? Ly tm dch nui cy thu t bo, lm dch huyn ph m c trong 0,5 ml ncmui sinh l.

? a khoanh giy ONPG vo dch huyn ph, gi 35-37 0C trong 24 gi.

Quan st kt qu: mu vng l phn ng dng tnh (Escherichia coli); khng mu lm tnh (Salmonella paratyphi B).

Proteus mirabilis- ONPG (ng th 8) m tnh

Cc c im sinh ha

9/37

Hnh 3.4. V d minh ho kt qu phn ng OPNG-aza

Kh nng thy phn tinh bt

? Mi trng: B sung tinh bt tan (0,2%) vo mi trng nc tht pepton, khtrng 121 0C trong 20 pht, a Petri.

? Ly vi khun mi hot ho cy vch hay cy chm ln a thch. Sau 2-5 ngynh thuc th Lugol (xem phn nhum Gram) ln vt cy quan st kh nng phngii tinh bt. Nu thuc th Lugol khng bt mu quanh vt cy tc l vi khun c khnng phn gii tinh bt.

Hnh 3.5. Phn ng vi dch Lugol kim tra kh nng phn gii tinh bt ca vikhun.

Kh nng to tinh th Dextrin

? Mi trng: ho 50 g bt go vo 200 ml nc, quy k, thm 20 g CaCO3, sau b sung dn dn 750 ml nc si, ng thi quy u ri un si 10 pht. Phn mitrng vo cc ng nghim (15 ml/ng), kh trng 121 0C trong 30 pht.

? Cy vi khun mi hot ho (18-24 gi) vo mi trng trn, t 300C trong 5-10ngy.

? B sung 1ml dung dch tinh bt 3%, gi 40 0C trong 15 pht.

? Ly 3 git dch trong pha trn ho vi 1 git dung dch Lugol v dn ln phinknh, lm kh trong khng kh v quan st di knh hin vi.

? Nu c sn sinh ra, nhng tinh th dextrin hnh lc gic bt mu lam c thquan st c st mp vt bi.

Kh nng phn gii celluloza

? Mi trng khong:

NH4NO3 1 g

K2HPO4 0,5 g

Cc c im sinh ha

10/37

KH2PO4 0,5 g

MgSO4. 7H2O 0,5 g

NaCl 1 g

CaCl2 0,1 g

FeCl3 0,02 g

Cao men 0,05 g

Nc ct 1000 ml

pH = 7,0- 7,2.

Phn mi trng vo ng nghim, kh trng 121 0C trong 20 pht.

? Mi trng Pepton:

Pepton 5 g

NaCl 5 g

Nc my 1000 ml

pH = 7,0-7,2

Phn mi trng vo ng nghim, kh trng 121 0C trong 20 pht.

? Cho vo ng nghim mt bng giy lc di 5-7 cm (vi vi khun hiu kh mtphn bng giy lc nh ln khi mi trng; vi vi khun k kh th bng giy lcngp trong mi trng).

? Cy vi khun mi hot ho, t nhit thch hp, quan st nh hng ca vikhun ti bng giy lc sau 1-4 tun. Nu vi khun pht trin v lm nt giy lc tcl chng c kh nng phn gii celluloza (phn ng dng tnh); m tnh l khng lmbin i giy lc.

Cch khc:

? vo a Petri mt lp thch 2% (15 ml thch cho mt a 9 cm).

Cc c im sinh ha

11/37

? B sung bt celluloza (0,8%) v thch (1,5%) vo mi trng ghi trn, 5 mlln trn lp thch 2% chun b trong a Petri.

? Cy vi khun mi hot ho thnh im trn mi trng, t nhit thch hptrong 1-4 tun v quan st vng phn gii celluloza c to ra quanh vt cy.

Kh nng thy phn pectin

? Mi trng:

Cao men 5 g

CaCl2.2H2O 0,5 g

Thch 8 g

Na-polypectat 10 g

Nc ct 1000 ml

NaOH 1N 9 ml

Dung dch BTB 0,2% 12,5 ml.

ho tan Na-polypectat v cc thnh phn khc cn khuy mnh v lm nng mitrng trong ni cch thy. Kh trng 121 0C khng qu 5 pht ri a Petri.

? Cy vi khun mi hot ho thnh 8 chm trn thch a, t nhit thchhp 3 ngy ri quan st. Nu quanh vt cy c vt lm xung l dng tnh (Erwiniacarotova); khng c vt lm xung l m tnh (Erwinia herbicola).

Kh nng thy phn Esculin

? Mi trng:

B sung Esculin (0,1%) v citrat st (0,05%) vo mi trng nc tht pepton. Phn mitrng vo cc ng nghim lm thch nghing. Kh trng 121 0C trong 20 pht.

? Cy vi khun mi hot ho (18-24 gi), t nhit thch hp sau 3,7 v 14ngy ri ly ra quan st.

Cc c im sinh ha

12/37

? Kt qu: xut hin sc t mu en nu l phn ng dng tnh, khng c l mtnh

Kh nng to Dextran v Levan

? Mi trng

Casein thy phn 15 g

Pepton 5 g

ng knh 50 g

K2HPO4 4 g

Thch 10 g

Nc ct 1000 ml

Dung dch Xanh Trypan (Tripan blue) 1% trong nc 7,5 ml

Dung dch Tm kt tinh 1% trong nc 0,1 ml

pH 7,0

Kh trng 115 0C trong 20 pht. ngui n 50 0C, thm 1ml dung dch Kali-tellurit1% ( kh trng bng mng lc) ri a Petri.

? Cy ria to khun lc n. t 37 0C trong 24 gi, sau gi thm nhit phng trong 24 gi.

? Vi khun sinh dextran s c khun lc nh, mu lam ti, b mt nhy v mc lmvo thch (loi Streptococcus sanguis).

Vi khun sinh levutan c khun lc nhy mu phn hng (Streptococcus salivarius).

Nu khng sinh dextran v levan th vi khun c mu lam nht hoc ti, kch thc nh,d ha sa (Streptococcus mitis).

Cc c im sinh ha

13/37

Xc nh 3-Ketolactoza

? Mi trng:

Lactoza 10 g

Cao men 1 g

Thch 20 g

Nc ct 1000 ml

pH = 7,0-7,2

Kh trng 115 0C trong 20-30 pht, a Petri.

? Ly vi khun mi hot ho (18-24 gi) cy im ln thch a, t nhit thchhp trong 2 ngy to khun lc r rt.

? Pha thuc th Benedict:

CuSO4.5H2O 17,3 g

Na2CO3 (khan) 100 g

Na-Citrat 173 g

Nc ct thm ti 1000 ml

Cch pha: ho Na2CO3 v Na-Citrat trong 600 ml nc ct, lc trong, sau thm ncti 850ml. Ho tan CuSO4 trong 100 ml nc, b sung nc cho ti 150 ml. Cui cngtrn dung dch CuSO4 vo dung dch u, va va khuy.

? Nh thuc th Benedict ln khun lc trn mt a thch, t 30 pht tr ln nhit phng.

? Kt qu: nu quanh khun lc xut hin nhng kt ta mu nu th l phn ngdng tnh, nu khng th l m tnh.

Kh nng kh Nitrat

? Mi trng:

Cc c im sinh ha

14/37

Nc tht pepton 1000 ml

KNO3 1 g

pH = 7,0-7,6

Phn mi trng vo cc ng nghim (4-5 ml/ng), kh trng 121 0C trong 15-20pht.

? Chun b thuc th Griess:

Dung dch A: Acid sulfanilic 0,5 g

Acid acetic long (khong 10%) 150 ml.

Dung dch B: Alpha Naphtylamin 0,1 g

Nc ct 20 ml

Acid acetic long (khong 10%) 150 ml.

? Chun b thuc th Diphenylamin: 0,5 g Diphenylamin ha vo 100 ml H2SO4c, thm 20ml nc ct.

? Cy vi khun mi hot ho vo mi trng (mi chng cy 2 ng), t nhit thch hp trong 1,3,5 ngy. Chn 2 ng khng cy vi khun lm i chng.

? Ly ng nghim sch v b sung ln lt cc dung dch nh sau:

Dch nui cy vi khun (hoc mi trng ng i chng)

1 git dung dch A

1 git dung dch B

? Kt qu:

- Nu dch nui cy chuyn mu (, hng, da cam hay nu) l biu th c nitrit, tc lvi khun c kh nng kh Nitrat.

- Nu dch nui cy khng chuyn mu, thm 1-2 git thuc th Diphenylamin kimtra s c mt ca Nitrat (chuyn mu xanh lam l c Nitrat chng t vi khun khngkh Nitrat; khng chuyn mu tc l Nitrat c kh ht v nitrit c kh tip tcthnh cc cht khc nh N2).

Cc c im sinh ha

15/37

? Ch : phn ng kh Nitrat thc hin trong iu kin k kh, v vy khng cphn vo ng nghim qu t mi trng.

i vi cc vi khun khc nhau nitrit c th l sn phm cui cng ca qu trnh khNitrat, nhng cngc th ch l sn phm trung gian. Ngoi ra, tc kh ca cc loicng khc nhau, v th cn theo di thng xuyn mu sc ca mi trng. Trong mitrng hp cn phi lm thm phn ng vi cht ch th diphenylamin.

Kh nng kh Nitrit

? Mi trng:

Peptone 5 g

NaNO2 1 g

Nc ct 1000 ml

pH = 7,3-7,4

Phn mi trng vo cc ng nghim, kh trng 121 0C trong 15 pht.

? Chun b thuc th Griess: ging nh phn kh Nitrat.

? Cy vi khun, t 30 0C trong 1,3,7 ngy ri lm phn ng xc nh.

? Nh vo dch nui cy 1 git dung dch A v 1 git dung dch B (xem phn khNitrat), lc nh. Nu mt mu v sinh ra NH3 l kt qu dng tnh (Alcaligenesodorans); nu vn gi mu l phn ng m tnh, khng kh nitrit (Acinetobactercalcoaceticus).

Kh nng phn nitrat ha (Denitrification)

? Mi trng:

Nc tht pepton 100 ml

KNO3 1 g

Cc c im sinh ha

16/37

pH = 7,2-7,4

Phn mi trng vo cc ng nghim (4-5 ml), kh trng 121 0C trong 30 pht.

? Cy vi khun mi hot ho. Dng vaselin bt kn nt ngn xy, t nhit thch hp trong 1-7 ngy v quan st s pht trin ca vi khun (tng c ca dchnui cy, sinh kh NH3). Vi khun c pht trin l phn ng dng tnh, khng phttrin l m tnh.

Kh nng sinh amonia

? Mi trng:

Pepton 5 g

Nc ct 1000 ml

pH= 7,2

Phn mi trng vo cc ng nghim, kh trng 121 0C trong 15-20 pht.

? Chun b thuc th Nessler:

Ho tan 20 g IK trong 50 ml nc; b sung I2Hg cho n khi bo ha (khong 32g), sau thm 460 ml nc. Cui cng b sung 134 g KOH. Bo qun trong l ti nhit phng.

? Cy vi khun mi hot ho (18-24 gi), t nhit thch hp trong 1,3,5 ngy.

? Ly vo ng nghim sch mt t dch nui cy, nh vi git thuc th Nessler.Nu xut hin kt ta mu vng nu l phn ng dng tnh.

Xt nghim Ureaza

Mc ch: kim tra kh nng phn hu ur nh enzyme ureaza

? Mi trng:

Pepton 1 g

Cc c im sinh ha

17/37

NaCl 5 g

Glucoza 1 g

KH2PO4 2 g

Dung dch phenol 0,2% trong nc 6 ml

Thch 20 g

Nc ct 1000 ml

Kh trng xong chnh pH n 6,8-6,9, mi trng c mu vng hi nh l c. Phnmi trng vo cc ng nghim lm thch nghing. Kh trng li 115 0C trong 30pht.

? Chun b dung dch Ur 20%, kh trng bng mng lc, b sung vo cc ngnghim khi ngui n 50-55 0C (t nng Ur 2%), t thch nghing.

? Cy vi khun mi hot ho, t nhit thch hp, sau 2-4 gi ly ra quan st.Kt qu m tnh cn tip tc quan st sau 4 ngy.

? Kt qu: mi trng chuyn mu cnh o l phn ng dng tnh, mu sckhng thay i l m tnh.

? Ch : cn lm i chng m tnh (khng b sung Ur), nht l khi xc nh ccloi Pseudomonas, v i chng dng tnh (so snh vi 1 chng bit c hot tnhureaza).

Lm cch khc:

? Cy vi khun vo mi trng thch nghing ni trn v xc nh hot tnh ureazasau 3 ngy v 7 ngy.

? Ly vi khun t thch nghing lm dch huyn ph m c trong ng nghimsch.

? Nh 1 git phenol vo dch huyn ph, chnh pH n 7 ( phenol chuyn tvng sang da cam).

? Chia dch huyn ph vo 2 ng nghim sch. Trong ng 1 thm vi tinh th Ur(khong 0,05-0,1 g), ng th 2 gi nguyn lm i chng. Sau vi pht nu dch

Cc c im sinh ha

18/37

trong ng 1 (c Ur) chuyn sang kim ( phenol chuyn mu ), biu th vi khunc hot tnh Ureaza; nu khng th l m tnh.

Xt nghim sinh Indol

? Mi trng:

Dung dch Pepton 1% trong nc

pH n 7,2- 7,6.

Phn mi trng vo cc ng nghim (1/3-1/4 th tch ng), kh trng 115 0C trong30 pht.

? Chun b thuc th:

Para-dimethyl-amino-benzaldehyde 8g

Etanol 95% 760 ml

HCl c 160 ml

? Cy vi khun mi hot ho (18-24 gi), t nhit thch hp v lm php thti cc thi im 1, 2, 4, 7 ngy.

? Nh thuc th theo mp ng nghim (to thnh lp dy 3-5 mm). Gia hai lpthuc th v dch nui cy nu c mu l phn ng dng tnh. Nu mu sc khngr rt th thm 4-5 git eter vo dch nui cy, lc nh lm cho eter khuch tn vo lpdch, yn mt lt khi eter ni ln b mt th li thm thuc th ni trn. Nu trongmi trng c indol th s xut hin mu trong lp eter.

Xt nghim Phenylalanin desaminaza

(kim tra kh nng chuyn ho nhm amin (?NH2) trong acid amin)

? Mi trng:

Cao men 3 g

Na2HPO4 1 g

Cc c im sinh ha

19/37

DL-Phenylalanin (hoc L-Phenylalanin) 1 g

NaCl 5 g

Thch 12 g

Nc ct 1000 ml

pH = 7,0

Phn mi trng vo cc ng nghim, kh trng 121 0C trong 10 pht, t thchnghing.

? Thuc th: dung dch FeCl3 10% (W/V)

? Cy vi khun mi hot ho, t 37 0C, lm php th sau 4 gi hoc 8-24 gi.

? Nh 4-5 git thuc th ln b mt thch nghing c vi khun pht trin, nu xuthin mu lc l phn ng dng tnh (do sn sinh ra acid Phenylpyruvic), nu khng imu th l phn ng m tnh.

Tryptophan desaminaza

C hai cch kim tra:

Cch th nht:

xc nh ng thi Tryptophan desaminaza, Ureaza v kh nng sinh Indol.

? Mi trng:

L-Tryptophan 3 g

KH2PO4 1 g

K2HPO4 1 g

NaCl 5 g

Etanol 95% 10 ml

Nc ct 900 ml.

Cc c im sinh ha

20/37

Thm phenol (khong 25- 30mg)

pH = 6,8-6,9

Phn mi trng vo cc bnh tam gic, kh trng 121 0C trong 20 pht.

? Ho 20 g Ur vo 100ml nc, kh trng bng mng lc, b sung vo mi trng chun b trn (thao tc v trng). Phn mi trng vo cc ng nghim v khun(3-4 ml).

? Cy vi khun mi hot ho (18-24 gi), nui nhit thch hp trong 24 gi.

? Ly ra 2-4 git dch nui cy, thm 1 git dung dch FeCl3 (khong 33%). Nuhin mu nu l phn ng Tryptophan desaminaza dng tnh, khng hin mu lphn ng m tnh. Dng vi khun Proteus lm i chng dng tnh.

? Nu mi trng sau khi nui cy vi khun chuyn t mu vng sang l biuhin c hot tnh Ureaza. Dng thuc th para-dimethylaminobenzaldehyd kim tras hnh thnh indol. Nu khng nh kim tra ureaza th khng cn b sung phenolv Ur vo mi trng.

Cch 2 th hai:

? Chun b ho cht:

L-Tryptophan 0,2-0,5%

Nc mui sinh l hoc dung dch m phosphat pH 6,8

Dch A: 50 ml KH2PO4 0,2 M (27,2g/L)

Dch B: 23,6 ml Na2CO3 0,2 M (8g/L)

Trn dch A v B vi nhau

FeCl3 33%

? Cy vi khun vo mi trng thch nghing nc tht pepton, t nhit thchhp trong 24 gi.

? Ly 4 ng nghim sch, thm vo mi ng 4 git dung dch L- Tryptophan0,2-0,5% v 4 git nc mui sinh l (hay dung dch m phosphat pH 6,8).

Cc c im sinh ha

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? Ly vi khun t thch nghing lm thnh dch huyn ph m c trong 2 ng, li 2 ng lm i chng, gi nhit phng trong 15-20 pht.

? Thm vo mi ng 1 git dung dch FeCl3 (33%). Nu hin mu nu l phnng dng tnh, khng i mu l m tnh. C th dng vi khun Proteus lm i chngdng tnh.

Carboxylaza i vi Ornithin, Lysin, v Arginin

? Mi trng:

Pepton 5 g

Cao tht 5 g

D-Glucoza 0,5 g

Bromocresol purple (BCP) 1,6% 0,625 ml

Cresol 0,2% 2,5 ml

Thch 3-6 g

Nc ct 1000 ml

Ha tan cc thnh phn trn trong ni cch thy, chnh n pH 6, thm ch th mu.

? Chia mi trng thnh 4 phn u nhau, b sung tng cht L-Ornithin, L-Lysin,L-Arginin vi nng 1% (nu dng DL-acid amin th ly nng 2%), sau chnhpH n 6,0-6,3. Mt phn khng thm acid amin dng lm i chng. Phn vo ccng nghim nh (mi ng 3-4 ml), kh trng 121 0C trong 10 pht. Mi trng chaOrnithin c th to mt t kt ta nhng khng nh hng n kt qu th nghim.

? Cy vi khun mi hot ho (18-24 gi), sau vaselin bt kn nt, nui iukin thch hp. Vi khun ng rut thuc h Enterobacterobacteriaceae cn nui 370C trong 4 ngy v theo di kt qu hng ngy. Cc vi khun phi lm sng nui 30 0C,quan st trong 7 ngy. Nu ch th mu chuyn sang mu ta hay mu ta c nh ldng tnh, nu mu vng (nh ng i chng) l m tnh. Vi khun ng rut thngbiu hin phn ng dng tnh sau 1-2 ngy, nhng cng c khi chm hn, cn theo diqua 3-4 ngy.

Cc c im sinh ha

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Arginin dihydrolaza

? Mi trng Thornley:

Pepton 1 g

NaCl 5 g

K2HPO4 0,3 g

Thch 6 g

Phenol 0,01 g

L-Arginat 10 g

Nc ct 1000 ml

pH = 7,0-7,2.

Phn mi trng vo cc ng nghim (4-5 ml), kh trng 121 0C trong 15 pht. Ch lm ng i chng khng c Arginat.

? Cy vi khun mi hot ho, dng vaselin bt kn nt ng nghim, nui nhit thch hp trong 3, 7, 14 ngy quan st. Mi trng chuyn sang mu l dngtnh, khng chuyn mu l m tnh.

Acetylamin

? Dung dch Acetylamin:

Acetylamin 2 g

Nc ct 20 ml

(khng cn kh trng)

? Dung dch m:

K2HPO4 0,4 g

Cc c im sinh ha

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KH2PO4 0,1 g

KCl 8 g

Nc ct 1000 ml

Kh trng 115 0C trong 20 pht.

? Dung dch lm th nghim: Pha long dung dch Acetylamin trong dung dch mtheo t l 1:99 vol/vol.

? Thuc th Nessler:

KI 5 g

Nc ct 5 ml

Thm dch HgCl2 bo ho, lnh cho n khi lc mnh m vn cn mt t kt ta thdng. Thm 40ml NaOH 9N ri b sung nc n 100 ml.

Ly 1 vng que cy vi khun trn vi dch th nghim ni trn, t nhit thch hptrong 24 gi. Thm 1 git thuc th Nessler. Phn ng l dng tnh khi to kt ta mu nu hay nu (vi khun Comamonas acidovorans), phn ng m tnh khi thy muvng (Pseudomonas stutzeri).

Thy phn Hippurat ; Phng php Yong & Thompson

(dng khi nh tn Streptococcus, Campylobacter v Gardnerella vaginalis):

? Dung dch c cht:

Na-Hippurat 0,25 g

Nc ct 25 ml

Kh trng bng mng lc

? Thuc th :

Ninhydrin 3,5 g

Aceton-butanol (1:1 vol/vol) 100 ml

Cc c im sinh ha

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Bo qun trong l ti

? Cy 2 git huyn ph vi khun vo dung dich c cht, gi 37 0C trong 1 gi. Thm2 git thuc th, gi 15 pht.

? Phn ng l dng tnh nu xut hin mu ta (Campylobacter jejuni, Gardnerellavaginlis, Streptococcus agalactiae), phn ng l m tnh nu sau 15 pht cha i mu(Campylobacter coli, Streptococcus agalactiae).

? Ch : lng vi khun cy phi tha ng, thi gian trc v sau khi thm thucth phi chun xc. Trnh nh sng khi gi thuc th.

Phng php Baird-Parker:

? Mi trng:

Pepton ty tng 10 g

Cao tht 1 g

Glucoza 1 g

NaH2PO4 5 g

Na-Hyppurat 10 g

Nc ct 1000 ml

Phn mi trng vo cc ng nghim, kh trng 121 0C trong 30 pht.

? Thucth:

H2SO4 c 50 ml

Nc ct 50 ml

t t H2SO4 c vo nc ct

? Cy vi khun mi hot ho (18-24 gi) vo mi trng trn, nui nhit thch hptrong thi gian 4-6 tun.

Cc c im sinh ha

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? Phn ng xt nghim: trn 1 ml dch nui cy vi 1,5 ml thuc th. Phn ng ldng tnh khi xut hin tinh th (do Hyppyrat c chuyn ho thnh Benzoin); khngxut hin tinh th l m tnh.

Hot tnh ADN-aza

Mi trng: Pepton casein Pepton utngNaCl ADNToluid-BlueThch Nc ct

10 g5 g5 g2 g0,1 g (c thpha thnh dung dch ri chovo)15 g1000 ml

Ho tan cc thnh phn ca mi trng bng nhit, sau b sung ADN v Toluid-blue,trn u ri phn vo bnh. Kh trng 121 0C trong 30 pht, thch a.

? Cy vi khun mi hot ho (18-24 gi) thnh im trn a thch, nui iu kinthch hp trong 2 ngy.

? Phn ng l dng tnh trong trng hp quanh cm cy c vng mu (dngchng Salmonella lm i chng dng tnh).

Hot tnh Phosphataza

? Mi trng:

Lm nng chy mi trng thch-nc tht-pepton ( kh trng)

Thm 1% Phenolphthalein diphosphat (kh trng bng mng lc).

thch a.

? Cy vi khun mi hot ho thnh im trn thch a, nui iu kin thch hptrong 2 ngy.

? Phn ng xt nghim: ly 0,1ml nc amonia ph ln mt thch, sau 20 pht xemkt qu. Phn ng l dng tnh nu khun lc chuyn mu phn (Staphylococcusaureus); khng chuyn mu l m tnh.

Cc c im sinh ha

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Kh nng lm dch ha Gelatin

? Mi trng:

Pepton 5 g

Gelatin 100-150 g

Nc ct 1000 ml

pH = 7,2-7,4

Phn mi trng vo cc ng nghim (4-5 ml), kh trng 115 0C trong 20 pht.

? Cy vi khun mi hot ho (18-24 gi) chch su vo mi trng gelatin, gi 2ng khng cy lm i chng, nui 20 0C trong thi gian 2,7,10,14,30 ngy.

? Quan st kh nng lm dch ho gelatin ti nhit phng t 20 0C tr xung.Nu b mt mi trng gelatin khng lm xung, gelatin vn trng thi n nh lphn ng m tnh (khng sinh gelatinaza); nu mt phn hay ton b gelatin ha lngth l phn ng dng tnh. Nu so vi i chng m tnh thy vi khun mc, gelatincha ha lng nhng b mt lm xung th cng vn coi l dng tnh (mc dchha thp). Nu vi khun hon ton khng sinh trng th c th l khng mc c trngelatin hoc mi trng c s cha thch hp.

? Ch :

- Nu vi khun ch sinh trng nhit trn 20 0C, lc quan st gelatin ho lng cnt ng nui cy mt lc vo nc lnh ri so snh vi i chng m tnh.

- Kh trng nhit qu cao hay qu thp u nh hng n kt qu, nn kh trng 115 0C trong 15 pht.

- Gelatin cht lng khng u nhau, lng dng kh thng nht, nn chn nng tong tt 20 0C l c. Nn dng thng nht mt loi gelatin cho ton b th nghim.

Cc c im sinh ha

27/37

Hnh 3.7. V d minh ho kt qu kim tra kh nng lm dch ho gelatin (sinhgelatinaza), Escherichia coli- m tnh, Pseudomonas aeruginosa- dng tnh.

Hot tnh Lipaza (vi Tween 80)

? Mi trng:

Pepton 10 g

NaCl 5 g

CaCl2. 2H2O 0,1 g

Thch 9 g

Nc ct 1000 ml

pH = 7,4.

Kh trng 121 0C trong 20 pht, ngui n 50 0C ri thm Tween 80 n nng 1%, thch a (c th thay Tween 80 bng du tributyrin).

? Cy vi khun mi hot ho (18-24 gi) thnh vch, nui trong 7 ngy, hng ngyly ra quan st.

? Phn ng l dng tnh nu quanh vt cy c vch trong, nu khng c th l mtnh.

Hnh 3.8. V d minh ho kt qu phn ng th hot tnh Lipaza: m tnh - Salmonellatyphimurium (bn tri), dng tnh - P. aeruginossa (bn phi).

Cc c im sinh ha

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Hot tnh Lipaza (vi du ng)

? Mi trng:

Pepton 10 g

Cao 3 g

NaCl 3 g

Thch 20 g

Xanh Victoria (Victoria Blue)

dung dch 1:5000 trong nc 100 ml

Du ng 50 ml

Nc ct 900 ml.

Ho tan cc thnh phn ca mi trng (tr du ng) bng un nng, sau b sungdu ng, khuy u bng my khuy t, chnh n pH 7,8. Phn mi trng vo cc ngnghim, kh trng 115 0C trong 30 pht.t thch nghing hoc thch a, mitrng c mu nht.

? Cy vi khun mi hot ho (18-24 gi), nui nhit thch hp trong 24 gi.

? Quan st: phn ng l dng tnh khi mi trng chuyn sang mu lam, khngchuyn mu l m tnh.

Cc c im sinh ha

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Hnh 3.9. V d minh ho kt qu phn ng th hot tnh lipaza vi du ng.

Hot tnh Lecithinaza

? Trn lng trng vi cng trng lng nc mui sinh l (thao tc v trng) tothnh dch huyn ph.

? Ly ra 10 ml dch huyn ph trn ha tan vo mi trng thch-nc tht-peptonva kh trng, ngui n 50-55 0C ri a Petri.

? Cy vi khun mi hot ho (18-24 gi) thnh im trn a thch, mi im ngknh khong 2-3mm. Cy 5-7 chng trn mt a. Vi vi khun k kh c th y l knhmng (lamelle) ln vt cy, tuy nhin tt nht l a vo t nui k kh.

? t nhit thch hp trong 18-24 gi, mt s chng (nh chi Bacillus) cn thigian lu hn (48 gi) v quan st bin i ca mi trng thch.

? Nu xung quang v di vt cy c vch trong l phn ng dng tnh (Lecithinc chuyn ho thnh lipid do vi khun sinh men lecithinaza).

? Ch : khi trn dch huyn ph lng trng vo mi trng thch khng nntin hnh nhit qu cao v s lm ngng kt lecithin c trong lng trng.

Cc c im sinh ha

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Hnh 3.10. Kh nng phn hy Lecithin ca Clostridium

Kh nng sn sinh H2S

Phng php gii giy

? Mi trng:

Pepton 10 g

NaCl 5 g

Cao tht 10 g

Cystein 0,5 g

Nc ct 1000 ml

pH = 7,0-7,4

Phn mi trng vo cc ng nghim (4-5 ml), kh trng 112 0C trong 20-30 pht.

? Ct giy lc thnh di rng 0,5-1cm, di ty thuc vo ng nghim v caoca mi trng. Tm vo giy dung dch Ch-acetat, sy kh giy trong t sy t tronghp Petri v kh trng.

? Cy vi khun mi hot ho (18-24 gi). Dng panh v khun gp giy tm ch-acetat a vo tng ng nghim, di n nt bng nhng khng chm vo mi trng.Nui vi khun nhit thch hp, sau 3,7,14 ngy th ly ra quan st. Nu giy binen l phn ng dng tnh, nu khng i mu th l m tnh.

Cc c im sinh ha

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? Ch : phng php ny rt mn cm, khng thch hp i vi trc khun ngrut. Khng t giy lc tm ch-acetat gn mt mi trng qu trnh b ht m,nhng cng khng nn t cch xa qu. Ngoi ng i chng khng cy vi khun nnly chng vi khun bit l m tnh lm i chng.

Phng php i vi Trc khun ng rut

? Mi trng:

Cao tht 7,5 g

Pepton 10 g

NaCl 5 g

Gelatin 100-120 g (hay thch 15 g)

Dung dch FeCl2 10% 5 ml (kh trng ring bng mng lc)

Nc ct 1000 ml

pH = 7,0

Kh trng 112 0C trong 20 pht, b sung dung dch FeCl2 ( kh trng) vo khithch hay gelatin cha ng. Phn vo cc ng nghim v khun (4-5 ml), ngay lp tcnhng vo nc lnh cho ng li.

? Cy chch su vi khun vo cc ng, nui 30 0C trong 1,3,7 ngy. Nu mitrng chuyn thnh mu en l phn ng dng tnh, nu khng i mu l m tnh.

? Ch : phng php ny dng khi cn nh tn vi khun thuc hEnterobacteriaceae. C th dng FeSO4 thay th cho FeCl2. Nu nui cy 20 0C cth dng kt hp xc nh gelatinaza.

Kh nng phn gii sa (Litmus Milk Reaction)

? Mi trng: sa ti un si, lnh qua m, ly tm v ht b b lp trn,phn di l sa khng cha lipid. C th dng sa bt loi cht bo (ho tan 100 gsa bt vi 1000 ml nc).

? Thuc th Litmus:

Cc c im sinh ha

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ho tan 2,5 g Litmus trong 100 ml nc ct, lc bng giy lc, qua m mi s dng.C th bo qun lu.

? Mi trng sa Litmus:

Dung dch Litmus 2,5% 4 ml

Sa loi b 1000 ml

Mi trng c mu ta.

Phn mi trng vo cc ng nghim (4 ml), kh trng gin on hay kh trng 1120C trong 20-30 pht.

? Cy vi khun mi hot ho (18-24 gi), nui nhit thch hp, sau 1,3,5,7,14ngy ly ra quan st.

? Da vo bin i ca mi trng m c nhng kt lun nh sau:

Mi trng chuyn thnh mu trng phn ng kh Litmus

Mi trng tr nn trong phn ng peptn ho

Mi trng chuyn mu phn ng sinh acid

Mi trng chuyn mu xanh lam phn ng sinh kim

Mi trng chuyn mu , sa ngng kt sinh acid v ngng kt

Ngng kt do men: khng chuyn mu hoc c mu lam, sa vn cc v ngng kt

? Ch : tt nht nn dng sa ti, khng cn iu chnh pH.

Kh nng oxy ha Gluconat

? m Kali phosphat 1/15 mol/L, pH 7,2:

A) KH2PO4 9,078 g/L

B) K2HPO4.12H2O 23,876g/L

Cc c im sinh ha

33/37

Trn 3 ml dung dch A vi 7 ml dung dch B c dung dch m phosphat 1/15 mol/L, pH 7,2

? Thuc th Feling:

Dung dch A: CuSO4 tinh th 34,64 g

Nc ct thm ti 500 ml

Dung dch B: Na-Tartrat 173 g

KOH 125 g

Nc ct thm ti 500 ml

Trc khi dng trn hai dung dch A v B theo t l 1:1 (vol/vol), s dng trong ngy.

? Chun b dung dch gluconat 1% trong m phosphat pH 7,2, phn vo cc ngnghim, mi ng 2ml. Kh trng 112 0C trong 30 pht.

? Ly vi khun mi hot ho (18-24 gi) to dch huyn ph m c trong mphosphat, gi 30 0C qua m.

? Thm vo mi ng 0,5 ml thuc th Feling, t trong bnh cch thy si 10 pht.

? Kt qu: nu dch huyn ph chuyn t mu lam sang mu vng lc, lc da camhoc c kt ta l phn ng dng tnh; nu khng i mu l m tnh.

? Ch : nu dng gluconat canxi th d to kt ta vi gc phosphat, tuy nhinkhng nh hng n kt qu th nghim.

Kh nng oxy ha Etanol

? Vi vi khun Acetobacter dng mi trng sau:

Cao men 10 g

Nc my 1000 ml

Xanh Bromophenol (BPB) 0,04% 20 ml.

pH = 6,8-7,0

Cc c im sinh ha

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Phn mi trng vo cc ng nghim (4-5 ml), kh trng 121 0C trong 20 pht. Lcs dng thm ethanol vo mi ng nng khong 2-10% (vol/vol)

? Vi cc vi khun khc: dng mi trng nh trong th nghim oxy ha/ln men,thay ng bng etanol vi nng 1%. C th khng cn thch.

? Cy vi khun mi hot ho, nui 1,3,7,14 ngy trong iu kin thch hp

? Kt qu: nu mi trng chuyn mu vng (do sinh acid) th l dng tnh, khngi mu l m tnh.

Phng php khc (dng phn lp v kim nh Acetobacter):

? Thm vo cc mi trng ni trn 2% thch v 1% CaCO3; nng etanol cuicng l 2%; khng thm ch th mu. thch a.

? Cy vi khun trn thch a, nui 3, 7, 14 ngy iu kin thch hp.

? Kt qu: nu xung quanh khun lc c vng phn gii trong l kt qu dng tnh,nu khng l m tnh (Acetobacter lc u phn gii CaCO3 nn to vng trong, nhngsau acetat canxi to ra b oxy ha tip chuyn thnh CaCO3 vng trong chuyn mutrng sa sng do acid acetic b oxy ha).

Kh nng oxy ha acid acetic

? Mi trng:

Cao men 10 g

Ca-Acetat 10 g

Thch 20 g

Nc my 1000 ml

pH 7,0-7,2

Phn mi trng vo bnh tam gic hoc cc ng nghim ln kh trng, thch ahoc lm ng thch nghing.

? Cy vi khun mi hot ho (18-24 gi), nui 3-5 ngy iu kin thch hp

Cc c im sinh ha

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? Kt qu: nu xung quanh khun lc c vng trng sa l phn ng dng tnh(acetat b oxy ha, canxi gii phng ra to mu trng sa), nu khng th l m tnh.

Xc nh Indol-Pyruvic acid (IPA)

? Mi trng SIM:

Cao tht 3 g

Pepton 30 g

Na2S2O3.5H2O 0,05 g

Cystin hydrochlorid 0,2 g

Ammonium-ferric-citrat 0,5 g

Thch 4 g

Nc ct 1000 ml

Ho tan cc thnh phn mi trng trong ni cch thy, chnh pH n 7,4, phn vo ccng nghim nh, kh trng 121 0C trong 15 pht, t ng thch ng.

? Thuc th Kovac (c th mua sn hoc t pha):

p-dimethyl aminobenzaldehyd 8 g

Etanol 760 ml

HCl c 160 ml

? Cy vi khun mi hot ho (18-24 gi) vo mi trng SIM, nui 300C trong24 gi.

? Kt qu: nu phn trn ca mi trng chuyn mu nu l phn ng IPA dngtnh, nu khng l phn ng m tnh.

? Sau nh thm thuc th Kovac vo v quan st. Nu trn mt thch xut hinmu o l phn ng Indol dng tnh.

Cc c im sinh ha

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Hnh 3.11. V d minh ho phn ng Indol dng tnh.

Cc c im sinh ha

37/37

Cc c im sinh haCC C IM SINH HA:Xt nghim oxidazaXt nghim catalazaKh nng ln men/xy ha glucozaKh nng ln men ng, ruPhn ng M.R. ( Methyl - Methyl Red)Phn ng V.P. (Voges-Proskauer)Phn ng ONPG-aza (O-nitrophenyl--D-galactopyranosidase)Kh nng thy phn tinh btKh nng to tinh th DextrinKh nng phn gii cellulozaKh nng thy phn pectinKh nng thy phn EsculinKh nng to Dextran v LevanXc nh 3-KetolactozaKh nng kh NitratKh nng kh NitritKh nng phn nitrat ha (Denitrification)Kh nng sinh amoniaXt nghim UreazaXt nghim sinh IndolXt nghim Phenylalanin desaminazaTryptophan desaminazaCch th nht:Cch 2 th hai:

Carboxylaza i vi Ornithin, Lysin, v ArgininArginin dihydrolazaAcetylaminThy phn Hippurat ; Phng php Yong & ThompsonHot tnh ADN-azaHot tnh PhosphatazaKh nng lm dch ha GelatinHot tnh Lipaza (vi Tween 80)Hot tnh Lipaza (vi du ng)Hot tnh LecithinazaKh nng sn sinh H2SPhng php gii giyPhng php i vi Trc khun ng rut

Kh nng phn gii sa (Litmus Milk Reaction)Kh nng oxy ha GluconatKh nng oxy ha EtanolKh nng oxy ha acid aceticXc nh Indol-Pyruvic acid (IPA)