The double agar method as described by Adams (1959) is widely used to enumerate phages. In this method a small volume of a dilution of phage suspension and a small quantity of host cells grown to high cell density, sufficient to give 107-108 CFU/ml, are mixed in about 2.5 ml of molten, 'soft' or 'top' agar at 46°-50°C. It is important to avoid over mixing the soft agar since that could result in air bubbles forming in the soft agar and potential misidentification of the bubbles as plaques. The resulting suspension is then poured on to an appropriate 'nutrient' basal agar medium e.g. M17 (Terazaghi and Sandine, 1975) for lactococci to form a thin 'top layer' which hardens and immobilises the bacteria. Refer to figure 1 below.
The basal agar should be free of surface moisture to ensure that the soft agar 'sticks' or adheres to the bottom agar. If low agar or agrose concentrations are used in the soft agar it may be necessary to dry the bottom agar plates prior to use otherwise it can be difficult to get the top agar to set properly. Solid, thermostatically-controlled heating blocks are useful for maintaining the soft agar, see figure 2, at the correct temperature and are more useful than water baths. The assay can also be undertaken by adding phage, host and calcium ions to a sterile test tube at ambient temperature. After several minutes molten soft agar at 46°-50°C is added and the mixture poured on to the basal agar as discussed previously. During incubation usually at 30°C, but other temperatures may be required, uninfected bacteria multiply to form a confluent film of growth over the surface of the plate.
Each infected bacterium bursts after a short time and liberates progeny phages that infect adjacent bacteria, which in turn are lysed. This 'chain' reaction spreads in a circular motion until brought to a halt by a decline in bacterial metabolism. The result is a visible, circular area of clearing in the confluent bacterial growth known as a plaque. By counting the number of plaques, and multiplying by the appropriate PSM dilution the number of plaque forming units (PFU) in the original phage suspension can be calculated. Plates containing 30-300 plaques are generally counted. Numbers used will depend to some extent on the size of the plaques. Below are examples of typical lactococcal phage plaques. Øc2(w) exhibits halo formation around plaques and Ø712 produces very small plaques.
The morphology of some lactococcal phages on M17 agar is shown in figure 3; the composition of M17 agar is described here. Plaques are generally visible after 6 hours incubation and haloes, zones
of secondary lysis around plaques, are present after 12-14 h. They are usually well developed after 48 h. incubation. Of the phages in figure 3, Øc2(w) produces haloes around plaques, Ø712 gives small 'pin hole' sized plaques and Øsk3 gives large plaques that are not surrounded by zones of secondary lysis.
Efficiency of plating
Not every phage particle will produce a plaque. Nor will every plaque be produced by one phage. For these reasons, the plaque count does not give the absolute number of phage particles present in a PSM. Careful choice of assay media and conditions can give accurate and reproducible results. However, incorrect choice of assay media or failure to supplement media with the correct level of calcium will markedly reduce the plaque count of many phages or not result in plaque formation. Such observations have led to the concept of efficiency of plating (EOP); EOP may be defined as the plaque count obtained under a certain set of conditions relative to the plaque count obtained under standard conditions. For comparative purposes, it is customary to use as standard those parameters that have been found to give the highest plaque count. The author has used a most probable number technique (MPN) that is useful as a reference method for the determination of EOP.
Note that there is also a possibility, particularly if testing environmental samples, that there could be two or more different phages capable of attacking the host strain used. This possibility should be considered when purifying environmental phages.
In the double agar method, in which the phage, susceptible cells, and agar are combined prior to pouring the overlay, more thorough mixing is achieved and greater uniformity of plates are obtained than in plating methods which do not utilise overlays. Plaque size is also increased because of greater rates of phage diffusion in the overlay. Diffusion is increased because the overlay contains a lower concentration of agar than the basal medium.
Plaque formation is also extremely useful in purifying phages or isolating phage mutants. This technique is also useful in studies of phage transduction and plasmids, for the development of new strains.