The regularly-structured protein microcapsule (A-layer) of Aeromonas salmonicida has been shown to be responsible for cell-to-cell aggregation, cell-to-tissue adhesion, and to be requisite for virulence. It has been known for some time that these traits can be lost during culture and that this loss appears to be irreversible.

However, there has never been a convenient method for distinguishing between cells or colonies which possess the A-layer (Alr+) from those missing it (Alr-).  Bacteriophages have been used by our group, and by others, to select for one or the other phenotype but the presence of phage resistant colonies makes distinguishing these phenotypes by this approach inadequate for many studies.  Therefore, another more direct approach was taken in this study. We approached the problem by trying to find a protein-specific dye which would bind to the A-layer but not to other cellular proteins.  Several dyes were found which fulfilled this function, and one was chosen for further study. The dye was readily bound to Alr+ colonies having the micro capsular protein exposed.  Colonies lacking the protein have LPS as the external component of the cell wall and specifically blocks penetration of the dye to the cell wall proteins of the other membrane.

The optimum dye concentration in several media has been determined; it has also been found that the dye is autoclaveable and does not change color with pH or redox potential.  The, medium can be used to detect one Alr- colony in the presence of 10⁴ Alr+ colonies and vice versa. Single colonies or sectored colonies can easily be picked under a dissecting microscope for further analysis.

Some preliminary studies using this medium will be discussed.