Isolation and Enumeration of Bacteriophages (PFU) from Water Sewage Sample Using Double Agar Layer technique

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BACKGROUND

One of the critical components of biological system are bacteriophages (PFU) and one of the abundant biological entity on earth. They are vital because they help in controlling the bacterial populations, the processes of food web, biogeochemical cycles and also increases prokaryotic diversity.

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They are very effective surrogate indicators that are mostly disease-causing enteric viruses and are transmitted fecally but are very difficult to assay. The bacteriophages are very easily enumerated for which they are used as an effective tool for assessment of fecal contamination in most of the environmental samples. A soft agar overlay is used because for restricting the physical diffusions of the viruses.

The basic objective of this test is to study about the isolation and enumeration of Bacteriophages (PFU) from water sewage samples by using a double agar layer technique.

REQUIREMENTS

Sample:

Sewage water

Broth culture of E.coli

Chemical:

Tris buffer

Soft agar

Miscellaneous:

Petri dish

Tubes

Paper towel

Water bath

Autoclave

PROCEDURE

Bring a sewage water sample containing coliphage and dilute it with Tris buffer in a ratio of 1:10 and 1: 100. It means you have to transfer 1 ml of culture to 9 ml of tris buffer and then make a second 10-fold dilution. Now melt three tubes containing soft agar with the help of an autoclave or a steam bath.¹

After that place this agar in the water bath at 45-48^oC for about 15 min so that the temperature of the agar is adjusted to 45^oC. Now to the first tube, add log phase broth culture of E. coli 1 ml and undiluted sample 1 ml. Now take out the tune from the water bath and rock it between hands very gently for proper mixing of the suspension for about 2-3 seconds.

Wipe the tube with the help of a paper towel to remove all water and now pour the agar in a previously prepared petri dish that contains bottom agar.² Now rotate the plate for spreading the top agar. Ensure that agar is covering the entire surface.

Isolation and Enumeration of Bacteriophages (PFU) from Water Sewage Sample Using Double Agar Layertechnique

Repeat the steps for other two tubes, by using the same 1 ml of the bacterial culture and 1 ml of the sample dilution. Once the agar gets solidified, invert the petri dish and keep it for incubation at 37^oC for about 48 hours.³ Clean any moisture if present from the lid, as it can cause spreading of the virus across the agar surface.

After incubation its time to calculate the number of plagues on each dilution and calculate the concentration of the phage in original sample. Meanwhile you can also mark any major differences in the size or the appearance of the plaques.⁴

CONCLUSION

Bacteriophages (PFU) as environmental factor have many important uses like indicator of contamination in sewage, water efficiency and also in wastewater treatment. Moreover, for measuring the survival of enteric viruses and bacteria in the environment.

REFERENCES

Agabian-keshishian, n. & Shapiro, l. 1971 bacterial differentiation and phage infection. virology 44,4653.
Archibald, a.r. & Coapes, h.e. 1976 bacteriophage sp50 as a marker for cell wall growth in bacillus subtilis. journal of bacteriology 125, 1195-1206.
Beher, m.g. & Pugsley, a.p. 1981 coliphage which requires either the lamb protein or the ompc protein for adsorption to Escherichia coli k-12. journal of virology 38,372-375.
Belfort, g., rotem, y. & Katznelson, e. 1974 virus concentration using hollow fiber membranes. water research 9,79-85.

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