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.
- Fresh culture of cyanobacteria (Lyngbya, Plectonema, Phormidium, Nostoc or Anabaena)
- Nutrient agar
- Pond water
- Sterile Petri dishes
- Sterile culture tubes
- Sterile membrane filtering apparatus
- Membrane filter
- Grow cyanobacterial culture for 1-2 days on suitable agar slants.
- Scrap the culture with a loop in a tube in broth, mix and vortex it.
- Add 1 ml of the broth culture and 1 ml of the pond water in a separate tube and incubate overnight at 35ºC.
- Prepare a plate by pouring the agar (8 ml) into Petri dish and wait to solidify
- 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.
- Dilute the filtrate in nutrient broth to obtain the dilution : 10-1, 10−2, 10−3, 10−4 and 10−5.
- Add 4 ml molten nutrient agar (45ºC), 0.5 ml freshly prepared pure culture of cyanobacterial suspension, and 1 ml filtrate (diluted/undiluted).
- Mix thoroughly and pour as the second layer into a Petri dish containing the solidified agar.
- Allow the top layer to solidify, invert the plate and incubate at 35ºC for 24 hour.
- Examine the bacterial green lawn for clear areas i.e. plaques that represent for the presence of cyanophages released after bursting the cyanobacterial cells.
- Record the number, shape and size of plaques.
Clear plaque formation on the medium shows the presence of cyanophages in the cells of cyanobacteria.
Dr. R. C. Dubey – Practical Microbiology