Introduction
Bacteriophages are viruses that infect bacteria. They are specific in their activity and they infect particular species of bacteria. They can be isolated from different natural environments, but for phages infecting enteric bacteria like E. coli, raw sewage would be the best source.
OBJECTIVE
METHODOLOGY
A. Isolation
1. Obtain 15 ml of raw sewage, add 5 chloroform, mix thoroughly and allow standing at room temperature for 60mins. Chloroform will lyse almost all of the bacteria but will not affect phages. Decant chloroform.
2. Add 5 ml of the chloroform-treated sewage to 25 ml of an overnight culture of E. coli and allow standing at room temperature for 5 minutes.
3. Prepare the three tubes of soft nutrient agar (0.7%). To one tube, add one drop of the sewage culture filtrate, to the second, add 5 drops of the filtrate and to the third, add nothing.
4. Mix and then pour separately onto sterile nutrient agar plates.
5. Allow the soft agar to solidify and incubate the dishes at 370C for 24 hours. In the agar plates, round, clear areas may be formed. These clear areas are called plaques. The formation of plaques in the agar seeded with E. coli is an indication that phage particles have attacked the bacteria and caused lyses of the cells.
B. Purification
1. To obtain pure culture of phage, touch the center of a single plaque with a sterile needle and transfer to 24-hour broth culture of E. coli.
2. Incubate this inoculated culture at 370C for 24 hours and observe for clearing (lyses) in the tube.
RESULTS
1. Show to your instructor your nutrient agar plates with phage plaque.
2. Does a phage plaque grow to specific size? Why does it not clear the whole plate? Discuss how a plaque is formed.
References
