Lecture 2 - Bacterial Identification Flashcards
How are bacteria identified.
16S rRNA sequence comparisons - can get to strain level
Classical taxonomy - can’t get to strain level however is still used, particularly in hospital labs.
Use of differential and selective media
What are the taxonomic hierarchies?
Domain
Phylum
Class
Order
Family
Genus
Species
Strain - Each population given its own strain number
What is meant by strian differences?
For every species name there are many strains. Each strain is deposited in culture collections
* American Type Culture Collection (ATCC)
* National Collection of Type Cultures (NCTC) - Public Health England
* The National Collection of Industrial, Food and Marine Bacteria (NCIMB) - also in UK
Some strains can be in different collections and have a different strain number (but the must always cross reference)
Bacterial strains within a single species will behave differently and will have different virulence’s.
E.g. Escherichia coli
Class 1 - K12, DH5α (non-pathogenic)
Class 2 - Most others (can cause disease however it is unlikely)
Class 3 - O157:H7 O103 (defiantly cause disease)
Class 4 - There are no known strains of bacteria in class four as currently everything can be treated with antibiotics however this may not be the case in the future due to antibiotic resistance.
When handling bacteria it is important to be aware of the class as this will determine how they are handled in the lab.
Due to the fact there are different classes with a species this has allowed for misrepresentation on household products as companies can claim that their product kills various bacteria but they are testing it on class 1 strains of bacteria.
What is classical taxonomy?
Classical taxonomy and identification relies on phenotypic analyses to identify bacterial species
What morpholological features can be used to identify bacteria taxonomically?
Morphology includes cell size, shape, the presence of flagella etc. Along with Gram staining these features can be used via a taxonomic dichotomous tree to identify bacteria.
* Shapes of bacteria include: Cocci, Bacilli, Rods and others. In some cases such as Pneumococcus which forms encapsulated diplococci the genus can be identified just by its shape.
* Bacteria can also be divided into groups via their oxygen tolerance. They can be obligate aerobes, Anaerobes, facultative aerobes, Microaerophile and aerotolerant anaerobes.
* Some Gram +ve bacteria form endospores which can be seen down the microscope.
Endospores allow the survival of bacteria under unfavourable conditions. They have special proteins to protect the DNA and they are resistant to heat, drying, radiation and chemicals.
* Cell motility - The motility test involves inserting bacteria via a needle into unhardened agar which contains a dye that stains the bacteria red. If the red stain spreads it suggests that the bacteria are moving.
* Catalase activity can identify bacteria that use the enzyme catalase to break down H2O2 produced during aerobic metabolism into water and oxygen. If the bacteria are added to a drop of H2O2 and bubbles form this would be a positive result.
Oxidase activity can be tested using Kovacs reagent which detects the presence of cytochrome c and associated oxidase. When reduced the reagent is colourless and when oxidised it is purple (positive result)
How is the G+C content of DNA measured?
G-C content can be measured easily with temperature as due to the extra hydrogen bond between G and C nucleotides more energy is needed to break the bonds.
The relative absorbance of single stranded and double stranded DNA is fixed. The Tm (melting temperature) which is directly proportional to GC content is extrapolated from the absorbance where 50% of the DNA strands have been separated.
How are physiolgy and metabolism used to identify bacteria?
Ø Nutrient sources
Ø Relationship to pH, temperature, salinity etc.
Ø Products formed
Ø Presence of certain enzymatic activities
Metabolism
Example 1 - Growth on lactose (similar method for all sugars)
· Production of acid (pH changes)
· pH indicator phenol red used
If the bacteria can use lactose the pH will change and the dye will turn yellow
Example 2 - Hydrogen sulphide production (H2S)
· Formation of ferrous sulphide (which is insoluble)
In hospital labs media kits are used for the rapid identification of clinical isolates. The bacteria is simply added and left to incubate overnight, if the colour in the slot changes it indicates a positive result.
An code is generated from the results:
The tests are separated into groups of three labelled 1, 2, and 4. These numbers (for the positive results) are then added together to generate a number between 0 and 7 and form part of the code with the other groups of three tests. The code can then be input into a computer witch will give an identification and a description of how certain it is to be the bacteria generated. If the identification is poor then extra tests can be carried out.
What is defferential media.
Differential media will allow different levels of growth for different bacteria.
e.g. MacConkey’s Agar for Enterobacteriaceae (looks for lactose fermentation)
The colour where there is no bacterial growth doesn’t change. The colour changes where the bacteria has fermented and grown.
e.g. Blood Agar tests how well a bacteria can break down RBCs. If there it becomes colourless all the blood cells have been destroyed (streptococcus), partial degradation appears slightly green (E.coli)
Chocolate agar (heated blood) where blood is added to boiling hot agar: Haemophilus, Neisseria
What is selective media?
To make the medium more selective allows differentiation between genera. Add something that only one can use or add something that kills one of them. Becomes a Selective medium for a particular genus.
Selective media
E.g. Brilliance E.coli/Coliform selective agar
· Chromogenic
· Coliforms - pink
· E.coli - purple
E.g. Listeria selective agar
Non-pathogenic listeria colonies have no halo
Pathogenic listeria colonies have a halo
Knowing the metabolism of a bacterial group allow the formulation of selective media. Bu only ca. 3% of bacterial strains can be culture in the laboratory
Cultured strains are used as indicators of what the other 97% are doing in situ. However there are gaps.
