Detection of Bacteriophages in Environmental Samples

Bacteriophages are studied in environmental science because they are a critical component of biological systems. They are the most abundant biological entity on earth and are important because they help control bacterial populations, food web processes, biogeochemical cycles, as well as enhance prokaryotic diversity via horizontal gene transfer. There is also evidence that phages are reliable surrogate indicators for disease-causing enteric viruses that are also fecally-transmitted but difficult to assay. The availability of relatively quick and inexpensive methods to enumerate bacteriophages makes them an attractive tool for the assessment of fecal contamination in environmental samples.

Coliphages in water are assayed by addition of a sample to soft or overlay agar along with a culture of E. coli in the log phase of growth. The phage attach to the bacterial cell and lyse the bacteria. The bacteria produce a confluent lawn of growth except for areas where the phage has grown and lysed the bacteria. These resulting clear areas are known as plaques. A soft agar overlay is used to restrict the physical diffusion of the viruses so that, upon lysing out of a bacterium, they can only spread to neighboring bacterial cells.

To obtain optimal plaque formation it is important that the host bacteria is in the log stage of growth. This ensures that all the phage attach to live bacteria and produce progeny. This requires that a culture of host bacteria be prepared each day that an assay is performed. Usually, a culture is incubated the day before the assay so that it will be in the stationary phase. On the day of the assay, the culture is used to inoculate a broth, which is incubated to obtain enough host bacteria in the log phase for the assay (this usually requires 2-3 h of incubation in a shaking water bath at 35-37 °C).

Dilution of sewage sample = 10^-1

Number of plaques obtained = 9

Therefore, phage concentration in sewage sample
= 10 x 9 ÷ 1 mL
= 90 plaque-forming units / mL

Raw sewage typically contains 10³ – 10⁴ coliphage per mL, with a range of 10² – 10⁸ per mL.

There are many potential applications of coliphages as environmental indicators. These include their use as indicators of sewage contamination, efficiency of water and wastewater treatment, and survival of enteric viruses and bacteria in the environment. The use of bacteriophages as indicators of the presence and behavior of enteric bacteria and animal viruses has always been attractive because of the ease of detection and low cost associated with phage assays. In addition, they can be quantified in environmental samples within 24 h as compared to days or weeks for enteric viruses.

¹E. coli strain ATCC 15597 usually will produce the greatest number of plaques from sewage samples. It should be grown overnight in a 250-mL Erlenmeyer flask containing 100 mL of nutrient or trypticase soy broth and incubated under shaking conditions at 35 °C. 3 h before the phage assay inoculate one mL of this culture into a fresh flask containing 100 mL of nutrient or trypticase soy broth and place in a shaking water bath at 35-37 °C. This will ensure that the bacteria are in the log phase of growth.