Detection of polysaccharide, teichoic acid, and protein antigens in bacterial colonies on an agar surface.

An improved method of using fluorescein-labeled antibody for the detection of polysaccharide, protein, and teichoic acid antigens synthesized by streptococcal colonies on an agar surface is described. The bacteria were grown on the surface of an agar medium contained in the shallow well of an immunodiffusion slide. An agar overlay containing the fluorescein antiserum was dispensed over the colonies, excess antiserum was washed out of the overlay agar, and the fluorescent colonies were observed under an ultraviolet microscope. The shallow well in the immunodiffusion slide prevented the agar from floating loose during washing, and the agar overlay prevented the fragmentation and loss of colonies. The thin layer of agar facilitated microscopic examination and the counting of fluorescent and nonfluorescent colonies. Colonies producing an antigen against which the antiserum was directed could readily be distinguished from colonies not producing the antigen. The specificity of the method was shown by using mixtures of streptococci representing six serological groups and five types. Those not known to possess cross-reacting antigens were specific in their reaction to the fluorescein antibody. Cross-reactions between the group antigens of A, C, and G, as reported previously by fluorescent staining of streptococcal suspensions, were also seen. Group A colonies reacted weakly with fluorescent E antibody and vice versa. The extraction of this antigen with cold trichloroacetic acid indicates it was related to the teichoic acids. Colonies possessing polysaccharide, protein, and teichoic acid antigens gave equally strong fluorescent reactions. This procedure permits detection of the synthesis of antigen which could not be observed by the use of a selective medium; it also eliminates the necessity for subculture of each colony and testing by appropriate serological means. Such a technique has value for studies in classification and biochemical genetics, and should be applicable to other genera of bacteria.