Detection of Bacteriophages: Statistical Aspects of Plaque Assay

Abstract

Approaches to enumerating bacteriophages, as with microorganisms generally, tend to be limited by the small size of the subjects, which prevents unaided visualization of individuals. For phages, visualization is further hampered by our inability in most cases to view individual viruses even employing standard brightfield microscopy, which in any case would supply total rather than viable counts. A standard solution in microbiology towards obtaining viable counts involves plating, typically using Petri dishes to hold the plating medium and plated organisms. For cellular microorganisms we thus enumerate not individual viable cells but instead macroscopic clumps of cells as derived from the growth of individuals, that is, what we refer to as colonies. For phages, as well as viruses generally, such clumps not only cannot form on their own accord when employing solely abiotic growth media but also would tend to be invisible to the naked eye even given such concentrating. Instead, towards phage enumeration as well as viability determination, along with characterization of other properties, often what one counts are phage plaques. These too are areas of high organism concentration, though here as generated by localized phage replication rather than bacterial population growth. Plaques are not directly visible due to local phage presence, but instead as a consequence of a full or partial local absence of cells, that is, reduced cell numbers due to this localized phage propagation. Here we consider especially statistically derived best practices of plaque-based phage enumeration.