Tut 4 : Confirmation and identification Flashcards
After enumerating the number of colonies of the target MO (e.g. E.Coli) on agar plate, we still need to confirm that the MO is indeed E.Coli and identify which strain of E.Coli is it. Why is it important to confirm he identity of E.Coli?
No method is totally accurate, and it may not be E.Coli that we detected, the agar plate could have detected another microorganism but with similar growth conditions as E.Coli / produces similar observations to E.Coli (colour of colonies etc)
DEFINITION
What is confirmation?
Using a limited number of (biochemical/immunological/molecular) tests to test the presumed identity of target organisms
DEFINITION
What is identification?
To establish umambiguously the identity (genus, species) of the microorganism
DEFINITION
What is isolation?
separating (isolating) the target microorganism from a mixed population / matrix
DEFINITION
What is phenotyping?
Subtyping based upon phenotypic (observable) characteristics such as :
- growth & survival charactersitics,
- antibiotic resistance,
- ferementation of carbohydrates etc
DEFINITION
What is genotyping?
To determine an isolate to subspecies level based upon genome fingerprint of a microorganism
Gram staining is a type of ____ method
phenotyping
The gram staining agent is crystal violet (purple) at first. It undergoes iodine treatment, decolourisation and a counter stain with Safranin, which is pink in colour.
What is the final colour of gram stain solution for
1. gram positive
2. gram negative bacteria?
Explain
- Purple. The crystal violet staining agent does not get decolourised as the stain droplets are entrapped in between the thick peptidoglycan layer
- Pink. The crystal violet staining agent gets decolourised, making the molecule white, before getting stained pink with Safranin
What are the 3 kinds of commonly used biochemical tests?
- Oxidase test
- Catalase test
- Commercial kits
What is the purpose of oxidase test ?
To determine if a bacteria produces certain cytochrome c oxidases
What do commercial kits do?
Identify microorganisms based on biochemical reactions
What kind of bacteria is serotyping used for?
- Gram-negative bacteria, because their outer membrane of cells walls contain lipopolysaccharides (LPS) which serve as antigens
Different variations in LPS structures result in different serotypes, allowing for the differentiation of strains.
(esp have a lot of O [somatic, cell surface] antigen!!)
- Gram negative bacteria is also virulent and it is important to identify and track the specific serovar that caused the outbreak in the context of epidomological diseases
What is the standard method for serotyping?
Aggutination , using DEFINED SETS of polyclonal / monoclonal antibodies
- Polyclonal : ability of molecule to identify different epitopes (part to which an antigen attaches to itself), probably contains serum of several animals
Is serotyping phenotyping / genotyping?
Does it distinguish at the species or subspecies level?
Phenotyping but at subspecies level
(usually phenotyping is at species level)
How are monoclonal/polyclonal antibodies obtained for serotyping?
Using immunological methods.
Inject an animal with the specific bacteria w the specific antigen, and animal produces the specific antiboy. Antibody is then extracted as serum from the animal
Serum : in blood of animal, blood is aseptically extracted from animal and then allowed to clot –> serum that does not clot is extracted
What is the limitation of serotyping?
Context : Salmonella has 2k types of serovars and you want to determine the specific serovar of 1 sample.
It is costly to do so, since you will have to collect 2k types of antibodies for each serovar from animals
Thus, it may impossible to identify specific serovar for bacteria with too many serovars
Why is genotyping mainly used for diffentiating between subspecies / different strains within a species, but not to differentiate different species of bacteria?
Genotyping aims to compare the different variations of nucleotide bases at specific genetic loci among different strains / subspecies
However, different species show broader variations across the WHOLE gene and not just at that loci, and different species may have same combination of bases at the same particular loci. Thus genotyping methods is not broad enough to discriminate at the species level.
(Conserved Genes: Bacteria share many genes that are essential for basic cellular functions, such as DNA replication, transcription, translation, and metabolism. These genes are often highly conserved across different bacterial species, meaning their sequences remain relatively unchanged over evolutionary time. Therefore, it is not uncommon for different species to have identical or highly similar sequences at these loci.)
- analogy : like how different humans share 99.9% genetic similarity, but different subspecies (races) of humans may have different variation of alleles at a specific gene loci (for eye colour)
Genotyping : Pulsed-field gel electrophoresis (PFGE)
What is the process of PFGE? (3 steps)
- DNA is extracted
- DNA is digested with specific enzymes to get certain segments of gene loci
- Electrophoresis is used to separate gene fragments
Genotyping : Pulsed-field gel electrophoresis (PFGE)
What is the principle behind PFGE?
Large DNA fragments require more time to reverse direction in an electric field than small DNA fragments
Genotyping : Multi Locus sequencing typing (MLST)
WHat does MLST measure?
It measures DNA sequence variations (of nucleotide base) in a set of housekeeping genes
(e.g. 7 specific housekeeping genes)
Genotyping : Multi Locus sequencing typing (MLST)
What are the 2 steps involved in MLST?
- PCR amplification
- DNA sequencing
Genotyping : Multi Locus sequencing typing (MLST)
What is meant by housekeeping genes?
Genes involved in basic functions of bacteria cells and needed for sustenance of cell
(under relative low level of selective pressure, i.e. do not change much through evolution)
