Transcript LAB 6A

PHT 381
Lab# 6
1- Catalase Test
Principle:
Catalase enzyme
H 2 O2
Procedure:
3H2O2
1
2
H 2o
+ O2
Air bubbles
Results:
Positive test: rapid appearance of gas bubbles.
Catalase +ve
Catalase –ve
2-. Coagulase Test:
Principle:
Fibrinogen
Plasma
Coagulase enzyme
Fibrin
Visible Clot
Procedure:
2
1
1 ml rabbit plasma
3
Place at water bath at 37oC, observe for formation of visible clot for up to 4 hrs.
Results:
Positive test: formation of visible clot.
Coagulase -ve
S.epidermidis
Coagulase +ve
S.aureus
3- Oxidase Test:
Principle:
Tetramethyl p-phenylene diamine
(oxidase reagent)
colourless
Cytochrome oxidase enzyme
Indophenol
(Purple colour)

Oxidase test:
Results:
+ve Test: Appearance of purple colour within 1-2 min.
No colour
purple colour
+ve test
-ve test

Oxidase test:
Results:
+ve Test: Appearance of purple colour within 1-2 min.
No colour
purple colour
+ve test
-ve test
4- Urease Test:
Principle:
Urease enzyme
Urea
NH3 ( alkaline)
Phenol red indicator
Pink colour
Results:
+ve
-ve
5- Deoxyribonuclease (DNase) Test:
Principle:
DNA
Insoluble
In acid
DNase enzyme
Nucleotides
soluble
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
Cloudiness in all
the plate
S.epidermidis
Clear zone around the
growth while the rest of
the plate appears
cloudy
S.aureus
1. Sugar fermentation:
Sugar fermentation can be indicated by change of color of the
medium from red to yellow.
Glucose
+ve
Maltose
+ve
Sucrose
+ve
Glucose
+ve
Maltose
+ve
Sucrose
- ve
2- Triple Sugar Iron (TSI) agar:
Principle:
(glucose 0.1%, lactose 1%, sucrose 1%).
butt
slant
1. No Fermentation:
Butt: alkaline (red)
Slant: alkaline (red)
2.Glucose Fermentation:
a) Initial reaction:
(after 10 – 12 hrs)
Dextrose
acid
Butt: acidic (yellow)
Slant: acidic (yellow)
2. glucose Fermentation:
b) Delayed reaction:
(after 24 hrs)
Small amount of acid
Butt: acidic (yellow)
Slant: alkaline (red)
3. Lactose/ sucrose Fermentation:
Lactose
Large amount of acid
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:
acidic (yellow)
acidic (yellow)
acidic (yellow)
Slant:
acidic (yellow)
alkaline (red)
alkaline (red)
H2S Production:
-ve
-ve
+ve
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:
1 ml liquid paraffin
O
F
Results:
Positive Test:
O-/FNon Saccharolytic
O+/F+
Fermentative
O+/FOxidative
O-/F+
sensitive O/F Test (Oxidation
Fermentation Test)
Principle:
sensitive O/F medium is a specifically
formulated medium to detect weak acids
produced from saccharolytic Gram’s –ve
bacteria.
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:
Procedure:
1 ml liquid paraffin
O
F

Oxidation Fermentation (O/F) Test:
Results:
Positive Test:
O-/FNon Saccharolytic
O+/F+
Fermentative
O+/FOxidative
O-/F+
4- MR-VP
Test:
Principle:
Dextrose
Acidic pathway
Mixed acids
 pH less than
4.4
MR indicator
Red colour
MR +ve
OR
Neutral pathway
Acetyl methyl carbinol
α-naphthol
KOH
Brick red colour
VP +ve
4- Indole
test
Principle:
Tryptophane Tryptophanase
enzyme
Indole
Kovac’s reagent
Purple red color
in the upper
organic layer
Results:
+ve
-ve