Demonstration For The Presence Of Cyanophages

Cyanophages are the viruses that attack the members of cyanobacteria. There are several cyanophages (LPP-1, LPP-2, LPP-3, AS-1, etc) which have been isolated from polluted (eutrophicated) ponds, rivers, lakes, any other water bodies. Such water has the possibility of having cyanophages.

Requirements

• Fresh culture of cyanobacteria (Lyngbya, Plectonema, Phormidium, Nostoc or Anabaena)
• Nutrient agar
• Pond water
• Chloroform
• Sterile Petri dishes
• Incubator
• Sterile culture tubes
• Centrifuge
• Sterile membrane filtering apparatus
• Membrane filter

Procedure

1. Grow cyanobacterial culture for 1-2 days on suitable agar slants.
2. Scrap the culture with a loop in a tube in broth, mix and vortex it.
3. Add 1 ml of the broth culture and 1 ml of the pond water in a separate tube and incubate overnight at 35ºC.
4. Prepare a plate by pouring the agar (8 ml) into Petri dish and wait to solidify
5. Mean while centrifuge the sample (obtained in three steps) and pass the supernatant through the membrane filter or treat the sample with chloroform for 5-10 minutes.
6. Dilute the filtrate in nutrient broth to obtain the dilution : 10-1, 10−2, 10−3, 10−4 and 10−5.
7. Add 4 ml molten nutrient agar (45ºC), 0.5 ml freshly prepared pure culture of cyanobacterial suspension, and 1 ml filtrate (diluted/undiluted).
8. Mix thoroughly and pour as the second layer into a Petri dish containing the solidified agar.
9. Allow the top layer to solidify, invert the plate and incubate at 35ºC for 24 hour.
10. Examine the bacterial green lawn for clear areas i.e. plaques that represent for the presence of cyanophages released after bursting the cyanobacterial cells.
11. Record the number, shape and size of plaques.

Results

Clear plaque formation on the medium shows the presence of cyanophages in the cells of cyanobacteria.

Reference

Dr. R. C. Dubey – Practical Microbiology