Tut 4 : Confirmation and identification

Question 1

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?

Answer 1

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)

Question 2

DEFINITION

What is confirmation?

Answer 2

Using a limited number of (biochemical/immunological/molecular) tests to test the presumed identity of target organisms

Question 3

DEFINITION

What is identification?

Answer 3

To establish umambiguously the identity (genus, species) of the microorganism

Question 4

DEFINITION

What is isolation?

Answer 4

separating (isolating) the target microorganism from a mixed population / matrix

Question 5

DEFINITION

What is phenotyping?

Answer 5

Subtyping based upon phenotypic (observable) characteristics such as :

• growth & survival charactersitics,
• antibiotic resistance,
• ferementation of carbohydrates etc

Question 6

DEFINITION

What is genotyping?

Answer 6

To determine an isolate to subspecies level based upon genome fingerprint of a microorganism

Question 7

Gram staining is a type of ____ method

Answer 7

phenotyping

Question 8

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

Answer 8

1. Purple. The crystal violet staining agent does not get decolourised as the stain droplets are entrapped in between the thick peptidoglycan layer
2. Pink. The crystal violet staining agent gets decolourised, making the molecule white, before getting stained pink with Safranin

Question 9

What are the 3 kinds of commonly used biochemical tests?

Answer 9

1. Oxidase test
2. Catalase test
3. Commercial kits

Question 10

What is the purpose of oxidase test ?

Answer 10

To determine if a bacteria produces certain cytochrome c oxidases

Question 11

What do commercial kits do?

Answer 11

Identify microorganisms based on biochemical reactions

Question 12

What kind of bacteria is serotyping used for?

Answer 12

1. 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!!)

2. 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

Question 13

What is the standard method for serotyping?

Answer 13

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

Question 14

Is serotyping phenotyping / genotyping?

Does it distinguish at the species or subspecies level?

Answer 14

Phenotyping but at subspecies level

(usually phenotyping is at species level)

Question 15

How are monoclonal/polyclonal antibodies obtained for serotyping?

Answer 15

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

Question 16

What is the limitation of serotyping?

Context : Salmonella has 2k types of serovars and you want to determine the specific serovar of 1 sample.

Answer 16

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

Question 17

Why is genotyping mainly used for diffentiating between subspecies / different strains within a species, but not to differentiate different species of bacteria?

Answer 17

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)

Question 18

Genotyping : Pulsed-field gel electrophoresis (PFGE)

What is the process of PFGE? (3 steps)

Answer 18

1. DNA is extracted
2. DNA is digested with specific enzymes to get certain segments of gene loci
3. Electrophoresis is used to separate gene fragments

Question 19

Genotyping : Pulsed-field gel electrophoresis (PFGE)

What is the principle behind PFGE?

Answer 19

Large DNA fragments require more time to reverse direction in an electric field than small DNA fragments

Question 20

Genotyping : Multi Locus sequencing typing (MLST)

WHat does MLST measure?

Answer 20

It measures DNA sequence variations (of nucleotide base) in a set of housekeeping genes

(e.g. 7 specific housekeeping genes)

Question 21

Genotyping : Multi Locus sequencing typing (MLST)

What are the 2 steps involved in MLST?

Answer 21

1. PCR amplification
2. DNA sequencing

Question 22

Genotyping : Multi Locus sequencing typing (MLST)

What is meant by housekeeping genes?

Answer 22

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)