Understanding Bacterial Differentiation Flashcards
Describe how bacterial biochemistry can differ and how this can be used for selective culture.
How do you distinguish between bacteria?
Morphology.
Motility.
Cell wall composition – i.e. Gram type.
Respiration.
(Aerobic/ Anaerobic/ facultative anaerobe/ microaerophilic)
Colony morphology on specific plates.
Growth on specific media.
Haemolysis.
Substrate utilisation.
(i.e. what can the bacteria metabolise a big list)
Presence and absence of specific enzymes (i.e. catalase, oxidase).
Tolerance to compounds (Enterobacteriaceae tolerate bile salts).
What is biochemical separation?
Once a pure culture has been established, various test can be carried out to asses biochemical differences which could be checked on media alone
The use of multiple tests can categorise bacteria into genera and in some cases species
What are API test strips?
Combine a number of biochemical test & extensive data base for interpretations
Established reference technique
List common tests and how they can support diagnosis.
- Haemolysis
- Catalase Test
- Oxidase Test
- Coagulase Test
- Urease Test
-Fermentation Assays
Describe the Haemolysis test
Tests the ability to lyse erythrocytes
(when on blood agar some lyse or damage blood cells to access iron
Blood Agar used
Three common descriptions for haemolysis:
Alpha - incomplete produces green zone of lysis.
Beta - complete destruction clear zone of lysis.
Gamma - absence of any haemolysis.
Describe the Catalase and Oxidative tests
Illustrate how components relating to respiration can be present or absent in different bacteria and used to help discrimination
Catalase test
-Catalase catalyses the decomposition of hydrogen peroxide to water and oxygen.
-It helps protect cells from oxidative damage by reactive oxygen species (ROS).
-Catalase trigger decomposition of hydrogen peroxide so it fizzes as it produces oxygen.
Oxidase
-Oxidase test identifies bacteria that produce cytochrome c oxidase, Cytochrome c is an enzyme of the bacterial electron transport chain used by bacteria growing aerobically.
-The colour change to blue comes from reduction of test compound.
Describe the Coagulase Test
Shows how properties can impact on the patient
Identify the presence of bound coagulase or clumping factor, which is attached to the cell walls of the bacteria.
Bound coagulase reacts with the fibrinogen in plasma, causing the fibrinogen to precipitate.
Main clinical use:
To separate coagulase positive /negative Staphylococcus aureus and also separate other groups of non-coagulase positive staphylococci S. epidermidis.
Coagulase is often associated with virulence but some coagulase negative strains can cause disease.
Describe the Urease test
pH based indicator test.
In this case urease breaks down urea to CO2 and ammonia.
The indicator is more red the more alkaline it is. A more yellow the more acidic.
Rapid diagnostics.
Describe the fermentation tests
Ability to ferment different sugars
Liquid fermentation
Vial is to collect gases produced within the tube
Assays can be changed for different sugars. For example the sugars lactose, fructose, galactose etc.
All assays are based on a pH change more acid if the sugar can be fermented
There is a pH indicator phenol red and is red colour at neutral pH.
At low pH the phenol red turns yellow
Another pH indicator used is neutral red which appears as pink to red when pH is low
The inverted Durham tube in the assays can collect CO2 which is produced by some bacteria when fermenting the sugars.
MacConkey agar does a fermentation test for lactose in a solid format but as you will see multiple tests can be combined and different bacteria have a almost a bar code of which sugars they can or can not ferment.
Describe how susceptibility to antimicrobials compounds can be used for selective media.
Selective media contain antimicrobial compounds which inhibit groups of bacteria you do not want allowing those you want to isolate to grow.
Antimicrobials are compounds which inhibits bacterial growth.
Some are selective only acting on certain groups of bacteria.
Example: Certain enteric bacteria (intestinal bacteria) tend to be resistant to bile
salts. If you add bile salts to media it will inhibit n a lot of non-enteric bacteria. This
means the media can be used to select for intestinal bacterial.
Antibiotics can also be used to selected for certain groups of bacteria as some bacteria have intrinsic resistances certain antibiotic groups lacking targets for the drugs.
A whole range of supplements is available to add to media to allow you select out bacteria you do not want.
What is Typing?
Separation of bacteria within the same species
Allows linkage between bacteria derived from the same lineage or source
What are methods of Typing?
Recognition of variable surface structures
–Serotyping
–Phage typing
DNA variation
–Ribosome sequencing
–Multi locus Sequence Typing (MLST)
–Probe based tests
–PCR
Other
–Mass spectometry profiles
Explain how phage typing can be used to differentiate between bacteria
Uses lytic bacteriophage – virus that infects bacteria.
Pattern of susceptibility of a bacterial isolate establishes it phage type (PT).
Phage attaches to surface proteins
These structures can subtly vary between different isolates of the same species or
simply be present or absent
Multiple phage are assessed for their ability to infect the bacteria
The pattern of susceptibility leads to a phage type
Explain how use of antibodies can be used to differentiate between bacteria
Serotyping
Use of antibodies that recognize variable structures. Antibody-antigen interaction is very specific!
Example antigen:
O-antigen is the lipopolysaccharide (LPS) of Gram-negative bacteria
Very subtle structural changes between the O-antigen can lead to changes in binding.
Some O-antigen core structures are more conserved so define genera/species of bacteria.
Some more variable used to define different lineages within the same species.
Antibody targets are specific to groups of bacteria.
