Determination Of One-Step Growth Curve Of Bacteriophage
There are several steps occurring during multiplication of bacteriophage. The phage comes in contact with suitable bacterial host cell wall (adsorption), its genome (DNA) is injected inside the bacterial cell (penetration), where the vegetative phages synthesise its own machinery with the support of host cell to organise viral particles (synthesis and assembly) leading to lysis of bacterial cell resulting into cell release of several hundred phages (burst size). This process is called lytic cycle of bacteriophage. The duration from adsorption until release of viral particles is called latent period. The time between the end point of latent period and plaque formed is called the rise period.
- T4 phage suspension
- Pure culture of E. coli
- Nutrient agar
- Soft agar
- Test tubes
- Transfer 2 ml overnight grown culture of E. coli into a test tube and add 0.1 ml of T4 phage suspension.
- After 5-10 minutes, take out 0.1 ml suspension and transfer to 9.9 ml of nutrient broth in first tube.
- Take out 0.1 ml from first tube and transfer in the second test tube containing 9.9 ml of the nutrient broth (to obtain 10 2 and 10 4 dilution respectively).
- Mix the contents well and incubate the second tube at 37ºC.
- Meanwhile, prepare double agar plates by making first the nutrient agar plate and pouring over it the molten soft agar containing well mixed 3-4 drops of E. coli suspension.
- Take out 0.1 ml sample from the second tube (step iv) at different duration (0, 15, 30, 45, and 60 minutes) and mix with 0.1 ml soft nutrient agar containing E. coli and pour the contents on Petri dishes and incubate them overnight at 37 C. Observe the plates for plaque formation.
Many clear plaques of different size and shape are formed. Count the plaque number and calculate the PFUs by multiplying with dilution factor.
Dr. R. C. Dubey – Practical Microbiology