-
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
21/37
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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
22/37
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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
23/37
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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
24/37
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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
25/37
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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
26/37
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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
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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
28/37
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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
29/37
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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
30/37
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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
31/37
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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
32/37
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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
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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
34/37
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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
35/37
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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
36/37
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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)