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

Trypticase NaCl Cao tht

10g 5g 1g 0,018g 1g

Phenol red (7,2ml ca dung dch phenol red 0,25%)

Carbohydrate*

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 Bromothymol blue Agar 1.0g 1.0g 5g 2g 0,2g 0,08g 13g

Th nghim Citrate

Ch - Cy lng sinh khi va - C i chng trng i km

i chng trng

P m tnh

P dng tnh

Th nghim Urease

Mc ch: pht hin VSV c mang enzym urease

C 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

Urea
Cao nm mem Na2HPO4 K2HPO4 Phenol red

20g
0,1g 9,5g 9,1g 0,01g

Nc ct

1 lt

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 Thiosulfate

desulfohydrase

H2S H2S

thiosulfate reductase

H2S sinh ra c nhn bit bi ion st, ch to kt ta mu en (FeS, PbS)

Th nghim kh nng sinh H2S

phn bit cc loi thuc h Enterobacteriaceae v ging Proteus Mi trng s dng:

KIA, TSI SIM, PIA BSA

(thch nghing) (thch su) (thch a) (37oC, 24 48h)

Cy vsv ln mi trng

Th nghim kh nng sinh H2S

c kt qu:
Xut hin mu en trong mi trng Khng xut hin mu en trong mi trng

(+)

(-)

(+)

Th nghim kh nng sinh H2S

Pancreatic digest of casein (casitone)
Peptic digest of animal tissue (beef extract) Ferrous ammonium sulfate

20.0 g
6.1 g 0.2 g

Sodium thiosulfate
Agar

0.2 g
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 (-)

i 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 / H2S

Th nghim KIA/TSI

10

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 C 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

c 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 Pepton Esculin Mt b (Oxgall) Ferric citrate Agar Nc ct 3g 5g 1g 40g 0,5g 15g 1 lt

Esculetine

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)

6,0 7,6 pH >7,6

pH <6,0

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 H2O2 (hydrogen peroxide)
catalase

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

3 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 cy

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) F (fermentation) Pseudomonas aeruginosa 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:

nitratase NO3 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

i 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

C 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)