PHT 381 Lab# 6. 1- Catalase Test H2O2H2O2 Catalase enzyme H 2 o + O 2 Air bubbles Principle: Procedure: 1 3 H2O2H2O2 2.

PHT 381PHT 381Lab# 6Lab# 6

1- Catalase Test1- Catalase Test

H2O2

Catalase enzymeH2o + O2

Air bubbles

Principle:

Procedure:

1

3 H2O2

2

Results:

Positive test: rapid appearance of gas bubbles.

Catalase –veCatalase +ve

2-. Coagulase Test:2-. Coagulase Test:

Principle:

FibrinogenPlasma

Coagulase enzymeFibrin

Visible Clot

Procedure:2

3 Place at water bath at 37oC, observe for formation of visible clot for up to 4 hrs.

1 ml rabbit plasma

1

Results:Positive test: formation of visible clot.

Coagulase +ve

S.aureus

Coagulase -ve

S.epidermidis

Principle:

Tetramethyl p-phenylene diamine(oxidase reagent)

colourless

Cytochrome oxidase enzyme

Indophenol(Purple colour)

3- Oxidase Test:3- Oxidase Test:

• Oxidase test:Oxidase test:

Results:

+ve Test: Appearance of purple colour within 1-2 min.

purple colour

+ve test

No colour

-ve test

• Oxidase test:Oxidase test:

Results:+ve Test: Appearance of purple colour within 1-2 min.

purple colour

+ve test

No colour

-ve test

Principle:

UreaUrease enzyme

NH3 ( alkaline)

Phenol red indicator

Pink colour

4- Urease Test:4- Urease Test:

Results:

+ve

-ve

5- Deoxyribonuclease (DNase) Test:5- Deoxyribonuclease (DNase) Test:

Principle:

DNADNase enzyme

NucleotidesInsoluble

In acidsoluble In acid

Procedure:

1. Inoculate DNase agar plate with the test organism.

2. Incubate the plate at 35oC for 24 hrs.

3. Flood the plate with 1M HCl.

Results:DNase activity is indicated by a clear zone around the growth after addition of Hcl

Clear zone around the growth while the rest of the plate appears cloudy

S.aureus

Cloudiness in all the plate

S.epidermidis

Glucose Lactose maltose mannite Maltose Sucrose

Sugar fermentation can be indicated by change colour of the medium from red to yellow.

+ve - ve+ve +ve +ve +ve

1. Sugar fermentation:1. Sugar fermentation:

Principle:(glucose 0.1%, lactose 1%, sucrose 1%).

butt slant

2- 2- Triple Sugar Iron (TSI) agar:Triple Sugar Iron (TSI) agar:

1. No Fermentation:1. No Fermentation:

Butt: alkaline (red) Slant: alkaline (red)

2.Glucose Fermentation:2.Glucose Fermentation:

a) Initial reaction:

Dextrose

acid

(after 10 – 12 hrs)

Butt: acidic (yellow) Slant: acidic (yellow)

2. glucose Fermentation:2. glucose Fermentation:

b) Delayed reaction:(after 24 hrs)

Peptone O

2

Alkaline amines

Butt: acidic (yellow) Slant: alkaline (red)

Small amount of acid

3. Lactose/ sucrose Fermentation:3. Lactose/ sucrose Fermentation:

Lactose

Large amount of acid

Peptone O2

Alkaline amines

Butt: acidic (yellow) Slant: acidic (yellow)

This medium contains also ferrous sulfate as an indicator for H2S production.

H2S + FeSo4 FeS

Black ppt. of ferrous sulfid

Results:

Butt:

Slant:

H2S Production:

acidic (yellow)

acidic (yellow)

-ve

acidic (yellow)

alkaline (red)

-ve

acidic (yellow)

alkaline (red)

+ve

3-O/F Test (Oxidation Fermentation Test)3-O/F Test (Oxidation Fermentation Test)

Principle:

Saccharolytic bacteria attack carbohydrates either:

• fermentatively ( in absence of oxygen) to yield relatively strong acids, or

• oxidatively ( in presence of oxygen) to yield weak acids.

Oxidation process is much more easier than Fermentation process.

Procedure:

O F

1 ml liquid paraffin

O-/F- O+/F+ O+/F- O-/F+

Results:

Fermentative OxidativeNon Saccharolytic

Positive Test:

Principle:

sensitive O/F medium is a specifically formulated medium to detect weak acids produced from saccharolytic Gram’s –ve bacteria.

sensitive O/F Test (Oxidation O/F Test (Oxidation Fermentation Test)Fermentation Test)

To be more sensitive this medium contains:

Higher conc. Of sugar to increase amount of acid produced. Lower amount of peptone to reduce formation of alkaline amines which neutralize weak acids formed. Lower conc. Of agar making the medium semisold to facilitate diffusion of acid throughout the medium.

• Oxidation Fermentation (O/F) Test:Oxidation Fermentation (O/F) Test:

Procedure:

O F

1 ml liquid paraffin

Positive Test:

• Oxidation Fermentation (O/F) Test:Oxidation Fermentation (O/F) Test:

O-/F- O+/F+ O+/F- O-/F+

Results:

Fermentative OxidativeNon Saccharolytic

Principle:

Dextrose

Acidic pathway OROR Neutral pathway

Mixed acids pH less than

4.4MR indicator

Red colour

Acetyl methyl carbinol

α-naphtholKOH

Brick red colour

MR +ve VP +ve

4- 4- MR-VP Test:MR-VP Test:

Principle:

TryptophaneTryptophanase enzyme

Indole

Kovac’s reagent

Purple red color in the

upper organic layer

4- 4- Indole testIndole test

Results:

+ve-ve