Isolation of Coliphages from Sewage | Microbial Biotechnology

In this article we will discuss about the principle, requirements and procedure for isolation of coliphages from sewage.

Principle:

Samples like raw sewage when inoculated with a susceptible host on an agar medium will form plaques of the phage.

There are various steps involved in isolation of phages:

(i) Collection of phage containing samples from its source and addition of a sus­ceptible culture of host cell to increase the number of particles.

(ii) Incubation and centrifugation.

(iii) Filtration of supernatant through a bac­teria retaining membrane filter.

(iv) Grow susceptible host cells on a soft agar medium and inoculate bacteria free fil­trate.

(v) Inoculate and observe phage particles seen as plaques.

Requirements:

Part– I:

1. Gloves.

2. 5 ml-24 hr broth culture of E. coli and 5 ml phage nutrient broth.

3. Medium.

4. Bacteriophage nutrient broth (pH 7.6):

5. 45 ml raw sewage sample in 250 ml Erlenmeyer flask.

6. Incubator.

Part II:

1. 100 ml centrifuge bottle.

2. Centrifuge and tubes.

3. 125 ml flask.

4. Membrane filter assembly.

5. Tryptone soft agar:

6. Tryptone agar broth:

7. Bunsen flame.

8. 1 ml sterile disposable pipettes.

9. Sterile Pasteur pipettes.

10. Mechanical pipetting device.

11. Test tube rack.

12. Glass marking pencil.

Procedure:

Part I: Enrichment of sewage:

Aseptically add 5 ml of bacteriophage nutrient broth, 5 ml of E. coli broth culture and 45 ml of raw sewage sample to a labelled 250 ml Erlenmeyer flask. (Handling of raw sewage should be done with extreme precau­tion because it may transmit human patho­gens). Don’t pipette by mouth. Use auto­matic (mechanical) pipetting device.

Incubate the culture for 24 hrs. at 37°C.

Part II: Filtration and seeding:

a. After incubation pour the phage infected culture into a 100 ml centrifuge bottle or several centrifuge tubes and centri­fuge at 2500 rpm for 20 minutes.

b. Decant supernatant into 125 ml flask.

c. Pour this through a sterile membrane filter apparatus.

d. The vacuum flask below will have me­dium which has bacteria-free phage.

e. Melt five soft tryptone agar slants, cool to 45°C and label the tryptone agar slants and plates as 1, 2, 3, 4 and 5 re­spectively.

f. Aseptically transfer 0.1 ml of E. coli cul­ture to all molten soft agar tubes.

g. With a sterile Pasteur pipette add 1, 2, 3, 4 and 5 drops of the filtrate to the molten soft agar in tubes. Mix, pour each tube of inoculated soft agar into labelled (1 to 5) plates.

h. Allow agar to solidify and incubate all plates at 37°C for 24 hours.

i. Depending on the concentration of in­oculum (1-5 drops) plaques appear in the plate.