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