Bacterial Characterisation

Question 1

List the five most commonly used bacterial characterisation tests.

Answer 1

Colonial morphology

Gram stain

KoH

Catalase

Oxidase

Question 2

Why are characterisation tests carried out?

Answer 2

To construct a preliminary identification of unknown organisms

Question 3

Why is the KoH test carried out?

Answer 3

It is used to separate gram-positive and gram negative bacteria

Question 4

What reaction results from using a gram-negative bacteria in the KoH test?

Answer 4

They will test positive for the KoH test

Question 5

What reaction results from using a gram positive bacteria in the KoH test?

Answer 5

They will test negative for the KoH test

Question 6

Describe a positive KoH result.

Answer 6

A gelatinous sticky solution results

Question 7

Describe a negative KoH result.

Answer 7

No gelatinous sticky solution results

Question 8

Describe why a gelatinous sticky solution results for a positive KoH test.

Answer 8

This is because gram-negative cells lyse in the presence of KoH and release their internal constituents including their DNA (this is sticky)

Question 9

Why do gram positive bacteria not lyse in the presence of KoH?

Answer 9

Due to the difference in their cell wall compares to gram negative cells

Question 10

How do you carry out a KoH test?

5

Answer 10

Flame a loop

Add 5-6 drops of KoH to a labelled slide

Flame loop

Collect 4-5 well isolated colonies and emulsify in th KoH solution

Observe for the production of a gelatinous sticky solution

Question 11

What does the catalase test test for?

Answer 11

It tests for the presence of catalase

Question 12

What is catalase?

Answer 12

An enzyme that breaks down hydrogen peroxide into water and oxygen gas

Question 13

Why is the catalase test used often?

Answer 13

It is used to determine whether a gram-positive cocci is a staphylococcus or a streptococcus spps

Question 14

How do you carry out a catalase test?

3

Answer 14

Add 4-5 drops of hydrogen peroxide to a pre-labelled test tube

Collect a single well isolated colony and place into the test tube containing the hydrogen peroxide

Observe for immediate bubble formation

Question 15

What is the chemical formula for hydrogen peroxide?

Answer 15

H2O2

Question 16

What is a positive result for the catalase test?

Answer 16

Bubble formation

Question 17

What is a negative result for the catalase test?

Answer 17

No bubble formation

Question 18

Why is the oxidase test carried out?

Answer 18

It is used to identify microorganisms containing the enzyme oxidase cytochrome C

Question 19

Why is the oxidase test carry out often?

Answer 19

It is used in screening for bacteria species which belong to the Enterobacteriaceae (oxidase negative) ir the Pseudomonas (oxidase positive) genus

Question 20

What does a positive oxidase test mean?

Answer 20

The bacteria are part of the Pseudomonas genus

Question 21

What does a negative oxidase test mean?

Answer 21

The bacteria are part of the Enterobacteriaceae genus

Question 22

How does the oxidase test work?

Answer 22

Cytochrome oxidase transfers electrons from the electron transport chain to oxygen (final electron acceptor) and reduces it to water

During the test when the electron donor is oxidised by cytochrome oxidase a colour change occurs

Question 23

How do you carry out the oxidase test?

3

Answer 23

Place 2-3 drops of oxidase reagent onto a sterile swab

Touch the swab against a single well isolated colony

Observe for colour change within 20 seconds

Question 24

What is a positive oxidase reaction?

Answer 24

The appearance of a dark red colour within 20 seconds

Question 25

What is a negative oxidase reaction?

Answer 25

The absence of a colour change within 20 second