Sewage sample processing for bacteriophage isolation

sewage water for bacteriophage isolation
Sewage water

 As we have discussed earlier bacteriophages present in the environment (Read about sewage sample collection here), Sewage is one of the easiest places to find higher concentration 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. Bacteria produce a confluent lawn except in a clear area called plaques where the bacteriophages have killed the bacterial population. Hence, the presence of coliphages can be a potential environmental indicator of sewage contamination, determining the efficacy of water and waste treatment processes indicating the survival of enteric viruses and bacteria in the environment.

Requirements

  • Sewage 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 370C.

Procedure

  • Dilute the sewage sample 1:10 and 1:100 in the buffered saline blank.
  • Cool under running water four tubes containing sterile soft agar (3ml/tube) to 500C. Label them as 1,2,3,4.
  • 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.
  • Repeat the experiment adding 1ml diluted sewage (1:10 and 1:100) to tubes 2 & 3 and mix it with 1ml bacterial culture. Treat control tube 4 similarly but add buffered saline in lieu of sewage sample.
  • Let the agar solidify. Invert the plates and incubate at 370C (incubation may change depending on the host used) for 4hrs.
  • 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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