Isolation Of High TItre Of Bacteriophage

The titre is defined as high titre of phage which is required for plaque bioassay. In this procedure initially a plaque is cut from a plate and suspended in tryptone broth. After vortexing for 2-3 minutes it allowed to transfer in a overnight grown culture of specific bacteria (E. coli, etc.) and result in the appearance of numerous plaques containing high titre of plaques.

Requirements

• Tryptone broth
• bacterial culture
• Nutrient agar plates with plaques
• Sterile needle or forceps
• Sterile pipette
• Incubator

Procedure

1. Procure a plate containing plaques
2. Scoop out a plaque from the plate
3. Meanwhile, prepare an overnight grown bacterial culture
4. Mix 0.1 ml plaque broth in 10 ml culture of E. coli .
5. Incubate the mixture at 37ºC for 6-8 hours.
6. Centrifuge the mixture at 10,000 rpm for 20 minutes to remove the bacterial cells.
7. Add a few drops of chloroform for lysing the bacterial cells, if any, present in the suspension.
8. Pour the suspension (1-2 ml) on each plate containing nutrient agar.
9. Incubate the plates at 37ºC for 24 hours in inverted position.
10. Observe the plate for plaque formation.

Results

Numerous plaques (clearings) of uniform shape and size will be visible against the dark back ground of agar.

Reference:

Dr. R. C. Dubey – Practical Microbiology