biochemical reaction - prac. microbiology

Bacteria vary in their metabolic & enzymatic activities.

Used in identification of different genera & species of bacteria.

Biochemical reactions are done on bacteria grown in pure culture.

Biochemical Reactions

1. Sugar fermentation2. Oxidase test3. Catalase test4. Coagulase test5. API Test

1. Sugar fermentationSugar media are composed of: Peptone water + 1% test sugar + Andrade’s indicator + A small inverted tube (Durham’s

tube) .

Sugar fermentation test

uninoculated

fermented with A only

Not fermented fermented with A & G

Glucose Lactose maltose mannite Sucrose

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

+ve - ve+ve +ve +ve

1. Sugar fermentation1. Sugar fermentation

Catalase TestCatalase Test

H2O2 Catalase enzyme H2 O + O2

Air bubbles

Principle:

Procedure:

1

3 H2O2

2

Results:

Positive test: rapid appearance of gas bubbles.

Catalase –veCatalase +ve

Catalase TestCatalase Test

Catalase TestCatalase Test

Coagulase TestCoagulase TestPrinciple:

FibrinogenPlasma

Coagulase enzyme FibrinVisible Clot

Procedure:2

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

1 ml rabbit plasma

1

Coagulase Test

Coagulase -ve

Coagulase +ve

Principle:

Tetramethyl p-phenylene diamine hydrochloride(oxidase reagent)

colourless

Cytochrome oxidase enzyme

Indophenol(Purple colour)

within 1-2 min

Oxidase TestOxidase Test

Oxidase Test

API (Analytical Profile Index)

Incubation Interpretation acc. to given charts

Bacterial Typing

Classification of a certain species of bacteria into Types.Importance: Epidemiological investigation (e.g. food

poisoning)

Examples of Typing methods: bacteriophage typing bio-typing serotyping plasmid analysis + others

Bacteriophage TypingBacterial strain on surface of media + drops of different phages

homogenous bacterial growth + areas of lysis (= susceptibility)

Questions

1- In sugar fermentation, Andrade's indicator is added to the sugar media to test for gas production:

( ) True ( ) False2- In the coagulase test, coagulation of plasma

occurs in positive cases: ( ) True ( ) False3- Biochemical reactions must be done on a pure

culture of the organism to be identified: ( ) True ( ) False 

M C Q4-Catalase test is: a) negative if oxygen bubbles are released b) positive if a visible clot is formed c) done by adding a drop of H2O2

d) done to test for the presence of urease enzyme5- Oxidase test is: a) done using catalase reagent b) positive when a deep purple color is produced c) positive after a few hours d) negative when a yellow color is produced  

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