Detection of Bacteriophages in Environmental Samples

1. Obtain a sample of sewage or water containing coliphage.
2. Dilute the sample 1:10 and 1:100 using Tris buffer. Do this by transferring 1.0 mL of culture to 9 mL of Tris buffer, and then making a second 10-fold dilution.
3. Melt three tubes of soft agar (0.7% nutrient agar or trypticase soy agar per 3 mL tube) by placing them in a steam bath or autoclave.
4. Place the agar in a water bath at 45-48 °C for 15 min to allow the temperature of the agar to adjust to 45 °C.
5. To the first tube, add 1 mL of a log phase broth culture of E. coli¹ and 1 mL of undiluted sample.
6. Remove the tube from the water bath and gently rock between hands to mix the suspension for 2-3 s.
7. Wipe the water from the tube with a paper towel and pour the agar over a previously prepared Petri dish containing bottom agar (regular nutrient agar or trypticase soy agar).
8. Quickly rotate the plate to spread the top agar. Be sure the agar covers the entire surface.
9. Repeat Steps 5-8 with the other two tubes of soft agar, using 1 mL of bacteria and 1 mL of each sample dilution (Figure 2).
10. After the agar has solidified, invert the Petri dishes and incubate at 37 °C for 48 h. Knock any moisture off the lid of the Petri dish. If a drop of moisture falls on a plaque it will cause the virus to spread across the agar surface.
11. After the incubation, count the number of plaques on each dilution (Figure 3) and calculate the concentration of phage in the original sample. Record any major differences in the size or appearance of the plaques.

Figure 2. Procedure for the preparation of a bacterial lawn using top agar for coliphage enumeration.

Figure 3. Phage plaques on a bacterial lawn.

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.