5 Identification

Question 1

What is the significance of identification of microorganisms?

Answer 1

1) Enables identification and elimination of sources of contamination in the manufacturing process

2) Enables selection of appropriate antimicrobial agents for the treatment of infections

Question 2

What are the different methods of identification of bacteria?

Answer 2

SBSC
Staining and Microscopic examination
Biochemical reactions
Serological methods
Cultural Techniques

SUP
Spectrophotometry
Use of Bacteriophage
Protein Analysis

BANG
Base Composition of Nucleic acids
Animal inoculation
Nucleic acid hybridisation
Genetic recombination

Question 3

What is the principle behind Staining and microscopic examination?

Answer 3

Each type of microorganism possesses a particular set of characteristics which is subjected to specific staining methods and examined under the microscope to determine its characteristics

Question 4

What are the difference culture medias?

Answer 4

Solid
1) Plate
2) Slope
3) Stab

Differential
1) Blood agar
2) Eosin Methylene Blue agar (E. coli, C. albicans)
3) Cetrimide agar (P. aeruginosa)
4) Baird Parker agar (S. aureus)

Liquid
1) Colour and odour
2) Evolution of gas
3) Surface pellicle or deposit
4) Nature and intensity of turbidity

Question 5

What is the principle of biochemical reactions for identification?

Answer 5

• Each type of microogranism has a particular set of abilities to carry out biochemical reactions
• Determine the biochemical reactions that the microorganism is capable of carrying out

Question 6

What are the types of biochemical reactions?

Answer 6

1) Carbohydrate fermentation, Sugars
- Produce acid and/or gas
- Indicator used to detect acid
- Invert the Durham tube to see if there’s a bubble indicating gas produced

2) Protein breakdown
- Cooked meat medium — Blackening of medium and foul odour Clostridium botulinum
- Gelatin, egg and serum medium — Medium becomes liquid after breaking down gelatin (solidifying agent)
- Peptone water — Indole test, Add Kovac’s reagent, Yellow turns Pink E. Coli

3) Nitrate reduction

4) Presence of enzymes
- Urea —> Ammonia + Carbon dioxide + Water (Urease) — Colour change from yellow to pink using pH indicator due to Ammonia
- Hydrogen peroxide —> Oxygen + Water (Catalase) — Dip wire loop of bacteria into media containing hydrogen peroxide, effervescence is observed from oxygen
- Fibrinogen —> Fibrin (Coagulase) — Blood plasma clotting, observe precipitation

Question 7

What is the principle behind serological methods for identification?

Answer 7

Antibodies act on specific microorganisms

Using agglutinin-containing serum, complement fixation, fluorescent or radioactive antibodies to detect for a particular microorganism

Fluorescent — fluorescein isocyanate
Radioactive — radioactive isotope
Fix bacteria smear, add antibody, wash, check for antibody

Question 8

What is agglutinin-containing serum?

Answer 8

Agglutinin reacts with the microorganism giving flocculation.

Control must not have flocculation.

Question 9

What is the complement-fixation method?

Answer 9

Interaction betw antibodies and microorganisms is not manifested as flocculation and thus cannot be seen

A limited amount of complement is then added which is needed for interaction betw the antibody and microorganism

If used up, indeed there is interaction.

Question 10

What is the principle behind animal inoculation for identification?

Answer 10

Bacterial infection of animals is associated with characteristic features or symptoms

Question 11

What is the principle behind the use of bacteriophages for identification?

Answer 11

Many phages are host-specific

Using plate agar seeded with test bacterium along with a few drops of phage suspension, results in clear spots due to the lysis of bacterial cells