Written by 6:54 am BACTERIOPHAGES, PROTOCOL • One Comment

Sewage sample processing for bacteriophage isolation

As we have discussed earlier bacteriophages are present in the environment (Read about sewage sample collection here), Sewage is one of the easiest places to find higher concentrations and diversity of bacteriophages for example Coliphages (viruses against coliforms) are present in sewage (105 – 107 per liter) wherever the coliforms are present. Coliphages in sewage can be assayed by mixing sewage and log phase Host bacteria (such as Escherichia coli) culture in top agar that is overlaid onto nutrient agar and incubated. Except in clear areas called plaques where bacteriophages have killed the bacterial population, bacteria produce a confluent lawn. As a result, the presence of coliphages in the environment can be a potential environmental indicator of sewage contamination, determining the efficacy of water and waste treatment processes and indicating the survival of enteric viruses and bacteria.

Sewage sample processing in laboratory

Requirements for collecting sewage sampleSewage sample

  • Host bacteria such as Escherichia coli broth culture (3-5 hrs)
  • Soft agar (top agar- nutrient agar with 0.7% agar)
  • Nutrient agar plates (also TSA and LBA can be used)
  • Sterile 1ml pipettes
  • 9.0ml buffered saline blanks
  • Water bath (500C) and incubator at 37°C.

The procedure of isolation bacteriophages

  1. Dilute the sewage sample at 1:10 and 1:100 in the buffered saline blank.
  2. Cool under running water four tubes containing sterile soft agar (3ml/tube) to 50°C. Label them as 1,2,3,4.
  3. Aseptically add 1ml of undiluted sewage in each tube and Host bacteria young culture to tube 1 and mix the tube contents thoroughly. Pour it immediately onto the surface of dried nutrient agar and let it spread uniformly over the entire surface by rotating the Petri plate.
  4. Repeat the experiment by adding 1ml diluted sewage (1:10 and 1:100) to tubes 2 & 3 and mixing it with 1ml bacterial culture. Treat control tube 4 similarly but add buffered saline in lieu of sewage sample.
  5. Let the agar solidify. Invert the plates and incubate at 37°C (incubation may change depending on the host used) for 4hrs.
  6. At end of incubation, count the number of plaques in each dilution and calculate the concentration of phages in the sewage sample. Record the size and shape of plaques.
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Last modified: February 27, 2021