Chng 4

Cc th nghim sinh
ha
GV: Nguyn Vn Hnh

Phn

lp khun lc thun khit l

cn thit cho nh danh VSV
Vic nh danh da ch yu vo
c im kiu hnh c bit l cc
phn ng sinh ha.
C 3 cch s dng cc th nghim
sinh ha nh danh VSV:
Cch truyn thng
S dng cc b KIT
S dng cc thit b t ng

Th nghim kh nng ln
men
Mc

ch: th nghim kh nng s dng cc

ngun CH ca cc VSV
Nguyn tc: VSV s dng CH tao acid
gim pH mi trng
Cc loi carbonhydrate

Monocarbonhydrate: glucose, xylose, rhamnose

Dicarbonhydrate: sucrose, lactose
Polycarbonhydrate: tinh bt, cellulose
Cc loi ng kh: ng mono cha chc CHO
Cc loi ng ru: cha chc -OH

Phenol Red Carbohydrate

Broth
Trang
104

Hp 115oC trong 15 pht

Th nghim kh nng ln
men
Mi

trng: Phenolred broth

base b sung 0,5-1% ng
cn th nghim

VSV s

dng c ngun
ng trong mi trng s
lm gim pH thay i
mu cht ch th phenolred

Phn

ng (+): mi trng
chuyn vng

Phn

ng (-): mi trng c
mu

Th nghim Citrate
Mc

ch: Xc nh kh nng vi sinh vt s

dng ngun citrat nh l ngun cacbon duy
nht.
C s sinh ha:
VSV s dng citrate, sinh ra CO2 lm kim
ha MT
VSV s dng mui ammonium l ngun m
duy nht to ra NH3 lm kim ha MT

Th nghim Citrate
Mi trng Simmon citrate agar (tr. 105)
Ammonium dihydrogen phosphate
Dipotassium hydrogen phosphate
NaCl
Sodium citrate
MgSO4

1.0g
1.0g
5g
2g
0,2g

Bromothymol blue
Agar

0,08g
13g

Th nghim Citrate
Ch

- Cy lng sinh khi va

- C i chng trng i km

i chng

P m tnh

P dng tnh

Th nghim Urease
Mc

ch: pht hin VSV c mang enzym urease

s sinh ho:
(NH2)2CO + H2O 2 NH3 + CO2
tng pH mi trng phenol (vng )

Mi

trng s dng:
Urea Broth (Rustigian Stuart)
Christensen Urea (mi trng thch nghing)

Mi trng Urea Broth

Th nghim Urease
Thc

hin

Chun b mi trng
Cy VSV vo 5ml mi
trng
37oC/24 gi
Quan st

Th nghim kh nng sinh

H2S
Mc

ch: pht hin kh nng sinh H2S

C s sinh ha:
Acid amin cha S

desulfohydras
e
2

HS

thiosulfate
2reductase

Thiosulfate H S

H2S sinh ra c nhn bit bi ion st, ch

to kt ta mu en (FeS, PbS)

Th nghim kh nng sinh

H2 S

phn bit cc loi thuc h

Enterobacteriaceae v ging Proteus
Mi trng s dng:
KIA, TSI

(thch nghing)

SIM, PIA

(thch su)

BSA

(thch a)

Cy vsv ln mi trng

(37oC, 24 48h)

Th nghim kh nng sinh

H2 S
c
(+)
(-)

kt qu:

Xut hin mu en
trong mi trng
Khng xut hin mu
en trong mi trng

(+)

Th nghim kh nng sinh

H2 S

Pancreatic digest of
casein (casitone)

20.0 g

Peptic digest of
animal tissue (beef
extract)

6.1 g

Ferrous ammonium
sulfate

0.2 g

Sodium thiosulfate

0.2 g

Agar

3.5 g

(-)

(+)

(+)

Th nghim kh nng sinh

Indol

Mc

ch

Pht hin cc VSV c kh nng sinh indol

cc VSV c h emzym tryptophanase

37oC / 24h
Thuc th
Kovacs
Chng VSV

MT canh trypton

P dng tnh P m tnh

Th nghim kh nng sinh

Indol
L

phn ng gip phn bit

E. coli (+) vi Klebsiella (-)

Proteus mirabilis (-) vi Proteus khc
(+)
Bacillus alvei (+) vi Bacillus khc (-)

chng (+) Proteus rettgeri

(-) Serratia marcescens

Th nghim KIA/TSI
KIA:

Kligler iron agar (trang 99)

Pepton
Lactose
Glucose
NaCl
Feric ammonium citrate
Sodium thiosulphate
Agar
Phenol red
Nc ct
pH 7,40,2

20g
20g
1g
5g
0,5g
0,5g
15g
0,025g
1 lt

Th nghim KIA/TSI
TSI:

Triple sugar iron agar (trang 106)

Pepton
Lactose
Sucrose
Glucose
NaCl
Feric ammonium sulphate
Sodium thiosulphate
Agar
Phenol red
Nc ct
pH 7,40,2

20g
10g
10g
1g
5g
o,2g
0,2g
13g
0,025g
1 lt

Th nghim KIA/TSI
Mc

ch: pht hin kh nng

s dng cc ngun cacbonhydrate

sinh H2S
to hi (gas)

37oC/24 gi

Quan st:
Phn nghing / phn su / hi /
H2 S

Th nghim KIA/TSI

11

Th nghim MR (Methyl red)

Mc

ch: xc nh vi sinh vt sn xut v duy

tr cc acid bn trong qu trnh ln men
glucose.
C s sinh ha:
Cht ch th pH: methyl red

di 4,4

5,0 5,8

trn 6,0

MR (+) cng ko di thi gian nui cy mi trng

cng acid
MR (-) cng ko di thi gian nui cy cc cht c
tnh acid b chuyn ha mi trng dn trung tnh
Thi gian 2 5 ngy 37oC

Th nghim MR (Methyl
red)
Mi

trng: Glucose Phosphate (MR-VP broth)

2 5 ngy
37oC

Chng VSV

MR-VP broth

P m tnh P dng tnh

Th nghim VP (Voges
Proskauer)
Mc

ch: Pht hin vsv to sn

phm trung tnh (acetoin) trong qu
trnh ln men glucose

s sinh ha: Acetoin c to ra

trong iu kin ym kh hon ton.
2 pyruvate
acetoin + 2 CO2

Th nghim VP (Voges
Proskauer)
Mi

trng s dng: MR-VP

Phng php tin hnh:

Cy vi sinh vt trong mi trng MR-VP

24 48 gi, nhit 37oC
B sung thuc th vo mi trng, lc nh
c kt qu sau 20 pht v chm nht l 4
gi.

Th nghim VP (Voges
Proskauer)
Kim

tra thuc th
bng i chng
(+) : Enterobacter cloacea
(-) : E. coli

kt qu:

(+): mu trn mi
trng
(-): mt mi trng khng
i mu

(-)

(+)

Th nghim Bile Esculin

Mc

ch: xc nh kh nng thy gii

glucoside esculin thnh esculetin v
glucose khi c s hin din ca mui
mt.
C s sinh ho:
Esculin l hp cht nhn to
Esculetine c phng thch phn ng
vi Fe2+ to thnh phc hp mu en

Mi trng Bile Esculine

Agar
Beef extract
3g
Pepton
Esculin
Mt b (Oxgall)
Ferric citrate
Agar
Nc ct

5g
1g
40g
0,5g
15g
1 lt

Esculetin
e

Phn t
Esculine
Glucose

S phn gii Esculine thnh Glucose v

Esculetine

Th nghim Bile Esculin

Khun lc Enterococcus faecalis cho kt qu BEA

(+)

Th nghim Malonate
Mc

ch
Pht hin cc VSV c kh nng s dng
malonate nh ngun carbon duy nht
C s sinh ho
Malonate l cht cnh tranh vi succinate
Khi VSV phn hy c malonate th cng
phn hu c cc ngun m v c khc
to thnh sp kim lm tng pH mi trng

Th nghim Malonate
Mi

trng s dng:
Malonate broth
(bromothymol blue)

pH <6,0

6,0 7,6

pH >7,6

Dng

tnh: MT
chuyn mu xanh da
tri
m tnh: MT khng i
mu v khng sinh khi

(+
)

(-)

Th nghim catalase
Mc

ch: pht hin cc vi sinh vt c h

enzym catalase.
C s sinh ho:
Catalase hin din cc VSV hiu kh v k kh
ty
catalase

H2O2

(hydrogen peroxide)

H2O + O2 (bt kh)

Th nghim catalase
Thc

hin
VSV ly t mi trng nui cy (lng, rn)
t VSV ln lam knh sch
Nh H2O2 30%
Quan st sau 1-2 giy
Phn ng (+): c bt kh xut hin
Phn ng (-): khng c bt kh xut hin

Th nghim catalase

Th nghim catalase

Th nghim catalase trn a petri: s dng

H2O2 30%

Th nghim
decarboxylase
Mc

ch: xc nh kh nng to enzyme

decarboxylase xc tc phn ct nhm carboxyl
mt s acid amin

Cu trc ca phn t
Amino acid

th nghim quan trng

Lysine decarboxylase (LDC)
Ornithine decarboxylase (ODC)
Arginine decarboxylase (ADC) /
Arginine dehydrolase (ADH)
Cc enzyme trn l cc enzyme cm ng,
ch c to ra khi trong mi trng
nui cy c c cht tng ng

Th nghim decarboxylase
C

s sinh ho
Cc sp to ra lm tng pH mi trng
i mu cht ch th
Mi trng s dng:
Decacboxylase Basal Medium
ch th bromocresol purple (5,2 6,8)
pH <5,2

5,2
6,8

pH >6,8

Th nghim decarboxylase
Biu

hin sinh ho
Dng tnh: pH mi trng tng
m tnh: pH gim

MT trc khi

P dng tnh

P m tnh

TH NGHIM COAGULASE
Mc

ch: Th nghim kh nng lm ng

t huyt tng bi enzyme coagulase
L bc cui trong nh danh cc ging
Staphylococcus
Chng i chng (+): S. aureus
(-): S. epidermidis
Th nghim tin hnh vi huyt tng v
fibrinogen.

TH NGHIM COAGULASE
Th nghim bng 2 cch

Th trn phin knh

c
kt
qu
Git nc

Sinh khi vsv + Huyt tng ngi

Th nghim trong ng nghim:

0,5ml huyt tng
v c kt qu mi
0,5ml huyt dch sinh
khi

30 pht

TH NGHIM COAGULASE

Kt qu th nghim Coagulase
(+) khi xut hin khi ng t huyt tng
(-) khng xut hin khi ng t, dung dch
ng nht

Th nghim gelatinase
Mc

ch: th nghim kh nng phn gii

gelatine bi gelatinase.
C s sinh ha:
Gelatine

gelatinase

polypeptide + acid amin

Gelatine trong mi trng dinh dng

mi
trng ng c
VSV phn hy gelatine
mi trng lng

Th nghim gelatinase
i

chng dng: Aeromonas

hydrophila
m: E. coli
Mi trng s dng Nutrient Gelatine
Dng ng nghim thch su
Cy vi sinh vt v nhit phng.

Th nghim gelatinase
c

kt qu

(+) mi trng tan chy

(-) mi trng khng tan chy

TN KH NNG LN MEN XI HA
Mc

ch: th nghim kh nng chuyn

ho glucose theo cc con ng khc
nhau
C s sinh ha:
Ln men: l qu trnh k kh, to mi trng
acid cao
xi ha: l qu trnh hiu kh

TN KH NNG LN MEN XI HA
Mi

trng s dng:
Oxidation/Fermentation media (Hugh &
Leifson media)
Ch th pH: bromocresol purple
pH <5,2

5,2
6,8

pH >6,8

TN KH NNG LN MEN XI HA
Trc khun gram m
O (oxidation)

Pseudomonas aeruginosa

F (fermentation)

Serratia marcescens

Cu khun gram dng

O (oxidation)

Micrococcus luteus

F (fermentation)

Staphylococcus aureus

TN KH NNG LN MEN XI
HA
c

kt qu:

A: i chng
B: phn ng xi ha
C: phn ng ln men

Th nghim nitratase (kh

nitrate)
Mc

ch: Th nghim kh nng kh

nitrate
C s sinh ha:

NO
3

nitratase

NO2 , NH 3 , N 2 , NH 2OH , NO

NO2 + sulphanilamine/N-napthylethylenediamine
hydrochloride cht mu hng
NO3 + bi km mu hng

Th nghim nitratase (kh

nitrate)
Phng

php tin hnh:

Nui cy chng vi sinh vt trong mi

trng cha nitrate
B sung cht th kim tra s hin
din ca nitrite
Phn ng nh tnh nitrite (-) b sung
lng km nh nh tnh nitrate

Th nghim nitratase (kh

nitrate)

Th nghim oxydase
Mc

tiu: pht hin VSV c h

enzym oxydase (h cytochrom C)
C s sinh ho
Cytochrom C kh + H+ + O2 Cytochrom C xi ho +
H2O
Cytochrom C xi ho + TMPD kh TMPD xi ho
(mu xanh)

Th nghim oxydase
Thuc

th
TMPD (0,1%): N,N,N,N-tetramethyl-p-phenylenediamine
Bo qun lnh trong ti
Thi hn bo qun: 2 tun

chng (+): Serratia marcescens

(-) : Proteus rettgeri

Th nghim oxydase
Thc

hin:

Ly VSV t Nitrient Agar t ln giy thm

Nh thuc th TMPD
Quan st sau 30 giy
Phn ng (+): sinh khi chuyn mu xanh
Phn ng (-): sinh khi vn mu trng

Th nghim oxydase

Th nghim ONPG
Mc

ch: pht hin cc vi sinh

vt c h enzyme -galactosidase
enzyme cm ng.
C s sinh ha:
ONPG
o-nitrophenol
Khng mu

Mu vng

Th nghim ONPG

qua m

Lactose agar

37o
C
Mu vng

Chng VSV

2ml ONPG broth

P (+)

P (-)

Th nghim kh nng tan

huyt
Mc

tiu: pht hin cc vi sinh

vt c kh nng lm tan hng
cu
Mu s dng: cu, b non, th

s sinh ho:

Cc heamolysine l cc tc nhn lm
tan hng cu ng vt
Vi sinh vt khc nhau heamolysine
khc nhau cng v biu hin
tan hng cu khc nhau

Th nghim kh nng tan

huyt
Phn

loi: 3 kiu tan huyt

Tan huyt hon ton (): vng tan

huyt trong, r
Tan huyt khng hon ton ():xung
quanhv di khun lc chuyn c
v c mu khc
Khng tan huyt (): hon ton
khng tan huyt

Th nghim CAMP
Mc

tiu: th nghim kh nng cng

hng tan huyt gia cc VSV
C s sinh ha:
S. aureus tit -lysin gay tan hng cu
VSV tit CAMP gy tan huyt
-lysin + CAMP gy tan huyt mnh,
hon ton
ngha: dng phn bit Streptococcus
nhm B (+) vi Streptococcus nhm khc
(-)

Staphylococcus
aureus
Streptococcus
agalactiae
(+)

Streptococcus pyogenes
(-)

Th nghim tnh di ng
Mc

ch: Xc nh kh nng di ng ca
vi sinh vt.
C s: Vi sinh vt di ng nh tim mao
Cc tin hnh: Cy m su vi sinh vt
vo mi trng thch mm (0,5% agar).
Vi sinh vt di ng s lm mi trng c,
pht trin lan ra khi vt cy.
Vi sinh vt khng di ng s pht trin quanh
ng cy, mi trng khng b c.

Th nghim tnh di ng

(-)

(+)

(+)

ng dng th nghim sinh

ha nh danh VSV
Mi

loi vsv c nhng c tnh sinh

ha khc nhau
Thc hin kim tra cc th nghim
sinh ha c th gip xc nh tn
loi (nh danh) vi sinh vt .
Bng sinh ha dng nh danh cc
loi vi sinh vt ng rut (trang 23)

H thng xc nh vi sinh vt API-20E

(bioMerieux, Inc)