Microscopic Characteristics of Colonies of Shigella Flexneri 2a and 2b and Their Relation to Antigenic Composition, Mouse Virulence and Immunogenicity
Authors: Cooper Merlin L. , Keller Helen M. , Walters Edward W. 
Published: January 2023

Summary
Oblique illumination was used for the microscopic study of colonies in surface cultures of mouse virulent parent strains of Shigella flexneri 2a and 2b. This method affords a means of detecting differences in colonial characteristics not seen by the conventional method of illumination. The appearance of the colonies was characterized by certain colors and color combinations, degrees of translucency or opacity, degrees of granularity, brilliance of reflective granules and smoothness or irregularity of the surfaces and edges. Shigellae highly virulent for mice were obtained from translucent colonies in which soft red-gold or gold-green colors predominated and whose surfaces appeared cross-hatched and irregular. Shigellae of low virulence for mice were obtained from opaque colonies whose colors were dull shades of pink-blue or gray and whose surfaces were smooth and edges slightly lobulated or entire. The virulent parent strain was usually composed of three or four different colony forms and the virulent translucent colony forms predominated. The translucent red-gold and gold-green colony forms did not vary from their parent forms in agglutination reactions while the opaque blue-pink colony forms had lost their specific or type antigen. Vaccines of the virulent translucent colony forms were highly immunogenic. Parent strains of Shigella flexneri 2a and 2b and the translucent colony forms isolated from each stimulated a high degree of homologous immunity in mice and a lower degree of heterologous immunity. [Vaccines of the non-virulent opaque colony variant were not immunogenic.] Parent strains of Shigella flexneri 2a and 2b, and translucent colony forms isolated from each, stimulated the production in rabbits of antisera of high agglutinin titers and high passive protection for mice while opaque colony variants stimulated production of antisera of low agglutinin titers and no passive protection for mice. A high degree of cross agglutination and cross passive protection between Shigella flexneri 2a and 2b was demonstrated. Reciprocal agglutinin absorption studies with the virulent translucent red-gold and gold-green colony forms of Shigella flexneri 2a and their antisera showed that these two colony forms were identical. Similar colony forms of Shigella flexneri 2b were also found to be identical. Reciprocal agglutinin absorption studies with virulent translucent gold-green and nonvirulent opaque blue-pink colony variants of Shigella flexneri 2a indicated that these colony forms were not identical. Similar results were obtained with the same forms of colonies from Shigella flexneri 2b and their absorbed antisera. Reciprocal agglutinin absorption studies with virulent translucent gold-green colony forms of both Shigella flexneri 2a and 2b indicated that these two were related but not identical, consistent with the antigenic formulae of these two sub-types. Similar studies with the nonvirulent opaque colony variants of Shigella flexneri 2a and 2b showed that they were identical. Correlations have been made between colony texture and color, serological findings, acriflavine reactions, mouse virulence and immunogenicity of colony forms isolated from parent strains of Shigella flexneri 2a and 2b. The microscopic method of studying colony forms comprising parent strains of Shigella flexneri 2a and 2b makes it possible to select parent strains in which virulent immunogenic translucent colony forms predominate or to isolate these colony forms for the preparation of highly immunogenic vaccines.