25] Chromogenic method to screen very large populations of bacteriophage plaques for the presence of specific antigen

Abstract

Detection of specific macromolecular species associated with individual bacterial colonies or phage plaques is of crucial importance in molecular cloning. Existing methods for detecting either specific nucleic acid sequences or antigens are adequate for many situations, but leave much to be desired when one is searching for the rare clone. The basic detection method explained in this chapter may be adapted to screen very large numbers of plaques per unit area of agar surface if one reduces the diameters of both plaques and immunoprecipitate. This may be accomplished by increasing the initial density of indicator cells (D 0 ) in the soft agar layer. The bacterial density at which cells can no longer sustain phage replication (D f ) is reached much sooner, the period of plaque formation is diminished, and the plaques are therefore smaller. At values of D 0 greater than about 3 × 10 9 cells/ml, plaques become virtually invisible to the unaided eye. However, the local concentration of HRP-immunoprecipirate remains high and can easily be detected by chromogenic reactions. A model of plaque formation is also described that provides a theoretical basis for these phenomena. The method in its present form should be widely applicable and should permit isolation of rare λ clones that might go undetected by other methods.