Question

What are some reasons a bacteriophage plaque assay might not be successful?

Answer 1

Over 99% of phages detected using microscopy have not been cultured. This article explores factors that influence plaque formation and if addressed may help in phage isolation.

Difficulties in getting phages to form plaques -

Some phages form very tiny or micro plaques. These can sometimes be so small that it is almost impossible to see them. Frequently 'new' phages can be observed using e.g. electron microscopy under conditions where there is strong evidence of a potential host yet it can be very time consuming or in some instances not possible to get the phage to form plaques. Less than 1% of the phages observed using microscopy have ever been grown in culture, this is sometimes called "the great plaque count anomaly" e.g. Serwer et al. (2007).

Sample preparation prior to analysis

The need to prepare samples prior to phage analysis has been discussed. In particular consideration should be given to the presence of host growth inhibitors in samples and their neutralisation if required. Additionally, consideration should also be given to the selection of membrane filters including their apparent size exclusion limits to minimise phage titre reduction effects; some phages may absorb to the filter matrix.

Some phages, particularly very large viruses, may aggregate significantly reducing their titre after either low speed centrifugation or membrane filtration (Serwer et. al., 2007). This is relatively easy to check if researchers have direct access to electron microscopy facilities but is difficult to do otherwise.

Replacing the gelling agent, agar, with agarose in the soft agar overlay

Agar is a mixture of polysaccharides some of which may contain both host-growth and virus inhibitors. It is widely used as the gelling agent in plaque assays. However, there is increasing evidence of problems using agar in virus assays. Agarose is purified agar in which most of the agaropectin, the component containing most of the sulphate and carboxyl groups which are considered responsible for inhibition of viruses, has been removed.

Many phages, particularly lactococcal phages, plaque well regardless of whether agar or agarose is used as the gelling agent in the overlay layer. However, plaque size may be improved if the agar is replaced with agarose for some phages. It would seem prudent to use agarose as the gelling agent in studies with ‘new’ phages.