Lecture 5 Taxonomy

Question 1

What does taxonomy try to achieve?

Answer 1

Taxonomy tries to build an overall picture of bacterial evolution from a global viewpoint

Question 2

What does identification seek to achieve?

Answer 2

Identification seeks to place a bacterium within the overall context

Question 3

How can taxonomy help with loosely classifying bacteria?

Answer 3

Based on the bacteria’s closest relatives we can determine it’s properties and behaviour

Question 4

What is medical bacteriology involved in?

Answer 4

Medical bacteriology is involved in the identification of the particular strain of bacteria. It’s easier than enviromental bacteriology as scientists don’t need to worry about how the strain fits in within the bigger picture, only statistics such as how to best treat the strain

Question 5

What are the three methods of characterisation for bacteria?

Answer 5

1) Visual e.g under the light/electron microscope, how it reacts to staining, the appearance of colonies
2) Biochemical: The ability to grow on particular substrates or media. The presence of certain characteristic chemicals e.g hydrogen sulfide with E.coli
3) Genetic characteristics: Similarities in DNA and specific genes

Question 6

What are the three procedures of classical taxonomy?

Answer 6

1) Visualisation of bacteria including microscopic staining.
2) Determination of substantial number of growth phenotypes, including the ability to grow on a range of carbon compounds/sources e.g lactose. The ability to ferment a range of sugars and amino acids. The presence of key enzymes which is dependent on pH and temperature.
3) Numerical comparison of strains by determination of similarity co-efficient (mathematically how similar they are).

Question 7

Why are phenotypes not a good way of relating bacteria?

Answer 7

Some phenotypes are exhibited by different genotypes because

1) They might have the same biochemical mechanism which is the result of convergent rather than divergent evolution.
2) Results of entirely different biochemical mechanisms i.e different pathways for the same growth substrate.
3) Advances of bacterial taxonomy resulted from methods involving structure of macromolecules, proteins and nuclei acids

Question 8

What is the evolution of bacteria dependent on?

Answer 8

It’s dependent on the gradual acquisitions of changes in DNA sequences which in then turn affect protein and amino acid sequences therefore leading to evolution.

Question 9

What is the procedure of DNA hybridisation?

Answer 9

It involves making a chromosomal DNA preparation of strain and measuring quantitively the extent to which it can hybridise with a similar preparation of other test strains. The more similarities the more cross hybridisation. If more than 70% can hybridise then it’s considered the same species

Question 10

What are the three criteria that justify using specific molecules as comparison instead of strains?

Answer 10

1) Is the molecule representative of the entire genome of the organism and therefore its evolution.
2) Is the variation introduced by evolution appropriate for the span we are trying to measure
3) Is the molecule that we are using technically easy to compare/use

Question 11

If the molecule is representative of the genome of the organism, what are the other criteria it must meet?

Answer 11

It must be a protein or gene that is a control gene that has evolved early and not transferred via genetic exchange and must be vital for cellular viability. I.e Antibiotic resistance cannot be a viable option

Question 12

What is genetically needed for two organisms to be linked ancestrally?

Answer 12

The mutation chosen must show sufficient similarity across the range of organisms to be recognised as having common ancestry.

Question 13

Why are protein sequences not used for comparison?

Answer 13

1) Technically difficult because it requires protein purification
2) Needs an amino acid sequence analysis

Question 14

Why are nuclei acid sequences used?

Answer 14

It’s technically more simple

Question 15

What is the gold standard gene?

Answer 15

16S Ribosomal RNA

Question 16

Why is the gold standard gene used?

Answer 16

1) It’s essential for ribosomal structure and function.
2) There are multiple copies of this gene in bacterial chromosomes.
3) There are parts that are highly conserved and other parts are more variable

Question 17

What is the advantage of some parts of the gene being highly conserved and other parts more variable?

Answer 17

PCR primers can be designed from conserved regions spanning variable regions. The gene can be amplified by these universal primers. Variable sequences enable species and genera comparisons

Question 18

What are the 7 identification techniques?

Answer 18

1) Plating onto selective or indicator agars
2) Staining and microscopy
3) Testing a range of growth phenotypes
4) Use of bacteria specific antibody test
5) Chromatographic determination of fatty acid composition of cells aka FAME
6) Phage typing
7) DNA methods based on hybridisation with synthetic probes

Question 19

Describe what Plating onto selective or indicator agars is

Answer 19

Lactose fermenters showing red or pink on agars

Question 20

Describe the procedure of staining and microscopy

Answer 20

1) Stain heat treated smear with crystal violet
2) Adding Potassium iodide (KI) and iodine (mordant) which then forms complexes with CV and cellular material
3) Add ethanol
4) Counterstain with safranin (red colour)

Be aware that on old culture there can be a false negative

Question 21

Describe Testing a range of growth phenotypes

Answer 21

This is linked to a colour reaction, i.e E.coli turns carbohydrate solution from red to yellow due to fermentation and a low pH, E.coli has a foul smell. Gas produced and it is shown in a small tube called the Durham tube. This can be used in commercial tests for identification

Question 22

Describe Use of bacteria specific antibody test

Answer 22

Agglutination test and ELISA test.

The antibody adheres to latex beads and complementary antibodies/antigens which causes clumping

Question 23

Describe Chromatographic determination of fatty acid composition (FAME)

Answer 23

Fatty acids are found in membrane lipids, composition characteristics of genera and species. Cells hydrated with NaOH, which makes it methylated with methanol/HCl which then goes into gas chromatography. This is used to identify.

Question 24

Describe Phage typing

Answer 24

Phages specific to species and sometimes individual strains can be used in the procedure.

1) Bacteria spread to form lawn on an agar plate
2) Drops of individual phages preparations added
3) Plaques as indicators of lysis

Question 25

Describe the procedure of DNA methods based on hybridisation with synthetic probes

Answer 25

1) Preparation of crude lysate of DNA from culture
2) Denaturation to SS DNA and binding to a nitrocellulose filter
3) Add synthetic SS Oligonucleotide probe specific to particular pathogen which is often a gene for a toxin or other virulent factor
4) Only binds to filter if bacterial DNA contains gene
5) Binding detected by label on Oligoprobe which is radioactive or enzyme linked