Question

1. in this exercise there must be enough bacteria inoculated to produce a lawn of growth. why is this important?

2. consider the adsorption step. how might the results be altered if you had skipped the absorption phase?
how might the results be altered if you extended it?

3. consider the water broth. why was it set at 50 degrees Celsius?
what might be some consequences of raising it's temp? of lowering it?

4. considar the soft agar. why was soft agar used for the overlay?
what would you expect to see if standard nutrient agar or trypticase soy agar had been used instead?

Answer 1

1. It is important to have lawns of the bacterial growth in order to do antibiotic susceptibility testing or for bacteriophage typing. The uniform growth of bacteria in the form of a lawn has several advantages including:

• Detection of distinct 'zones of inhibition' for checking the susceptibility of an antibiotic or a combination of it. Zones of inhibition are the regions where the specific bacteria are not able to grow because of their antibiotic susceptibility. It's diameter is measured to check the extent of inhibition.

2. In the plaque assay, the ability of the infectious virus to form a plaque onto the monolayer of cells is measured.

Pre-adsorption and adsorption phase is the one where the virus particles are allowed to adsorb on the surface of the soft agar, without allowing their growth to occur.

If this absorption phase is skipped, then the phages will absorb at different time-points and form plaques of varied sizes and at different depths of the agar.

If the absorption phase is extended, then the plaque formation is distorted as the excessive growth of the phages occurs.

3. The water broth at 50 degree celsius is kept in order to maintain the agar in its liquid state at a particular density.

• Lowering the temperature below it will make the agar solidified and hence, will not let the phage diffusion to happen.
• Increasing the temperature of the water bath beyond 50 degrees, will either denature the bacteria or phages, resulting in no plaque formation.

4. Soft agar overlay is used in the phage diffusion assay as soft agar is dense enough to immobilize the bacterial cells onto it, while allowing the bacteriophages to diffuse into it.

If any other agar inclluding the standard nutrient agar or trypticase soy agar is used in this assy instead of the soft agar, then the phage diffusion will not take place and no plaque formation. Hence, Phages would have been put in the one spot and remain undifferential.