Laboratory Determination of Bacteriophage Activity
Bacteriophage presence in the investigated sample can be revealed by drop of the filtrated material on Petri dish with appropriate medium, pour plated with the susceptible bacterial culture. After incubation for 24-48 h at 37°С the infection of bacteria by phages results in a plaque formation, a clear area in the bacterial lawn. The plaque is formed due to phage lytic activity.
Quantitative phage determination is possible by titration methods.
Two main variations of these methods are based on phage titration in liquid or solid nutrient medium.
In first case tenfold dilutions of the phage-containing material are prepared and then inoculated into liquid nutrient medium. The susceptible microbial culture is added to the broth. After incubation for 24-48 h at 37°С the growth inhibition is evaluated. The phage titer is established as the last dilution of the phage culture, which is able to cause complete inhibition of visible microbial growth.
Similar agar titration method is performed as follows: mixture of susceptible bacterial culture with tenfold phage dilutions is poured on different Petri dishes with solid nutrient medium. After incubation for 24-48 h at 37°С the growth inhibition with plaque formation is estimated. The last phage dilution resulting in isolated plaque formation is considered to be the endpoint (titer) of the reaction. Total phage quantity is calculated by multiplication of last plaque count by the dilution of the sample.