Chng 4

Cc th nghim sinh haGV: 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 menMc 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 BrothTrang 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 CitrateMc 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 CitrateMi 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 BrothUreaCao nm mem Na2HPO4 K2HPO4 Phenol red

20g0,1g 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 H2SMc 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

(+)

(-)

C

(+)

Th nghim kh nng sinh H2SPancreatic digest of casein (casitone)Peptic digest of animal tissue (beef extract) Ferrous ammonium sulfate

20.0 g6.1 g 0.2 g

Sodium thiosulfateAgar

0.2 g3.5 g

(-)

(+)

(+)

Th nghim kh nng sinh Indol

Mc chPht 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

C

1

2

3

4

5

6

7

8

9

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

C

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

Esculetine

Phn t EsculineGlucose

S phn gii Esculine thnh Glucose v Esculetine

Th nghim Bile Esculin

Khun lc Enterococcus faecalis cho kt qu BEA (+)

Th nghim MalonateMc 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