Bacterial Characterisation Flashcards
List the five most commonly used bacterial characterisation tests.
Colonial morphology
Gram stain
KoH
Catalase
Oxidase
Why are characterisation tests carried out?
To construct a preliminary identification of unknown organisms
Why is the KoH test carried out?
It is used to separate gram-positive and gram negative bacteria
What reaction results from using a gram-negative bacteria in the KoH test?
They will test positive for the KoH test
What reaction results from using a gram positive bacteria in the KoH test?
They will test negative for the KoH test
Describe a positive KoH result.
A gelatinous sticky solution results
Describe a negative KoH result.
No gelatinous sticky solution results
Describe why a gelatinous sticky solution results for a positive KoH test.
This is because gram-negative cells lyse in the presence of KoH and release their internal constituents including their DNA (this is sticky)
Why do gram positive bacteria not lyse in the presence of KoH?
Due to the difference in their cell wall compares to gram negative cells
How do you carry out a KoH test?
5
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
What does the catalase test test for?
It tests for the presence of catalase
What is catalase?
An enzyme that breaks down hydrogen peroxide into water and oxygen gas
Why is the catalase test used often?
It is used to determine whether a gram-positive cocci is a staphylococcus or a streptococcus spps
How do you carry out a catalase test?
3
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
What is the chemical formula for hydrogen peroxide?
H2O2
What is a positive result for the catalase test?
Bubble formation
What is a negative result for the catalase test?
No bubble formation
Why is the oxidase test carried out?
It is used to identify microorganisms containing the enzyme oxidase cytochrome C
Why is the oxidase test carry out often?
It is used in screening for bacteria species which belong to the Enterobacteriaceae (oxidase negative) ir the Pseudomonas (oxidase positive) genus
What does a positive oxidase test mean?
The bacteria are part of the Pseudomonas genus
What does a negative oxidase test mean?
The bacteria are part of the Enterobacteriaceae genus
How does the oxidase test work?
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
How do you carry out the oxidase test?
3
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
What is a positive oxidase reaction?
The appearance of a dark red colour within 20 seconds
What is a negative oxidase reaction?
The absence of a colour change within 20 second
