The Fish Disease Diagnostic Laboratory (FDDL) at Kentucky State University is used as an Extension tool to give advice to fish/pond owners experiencing fish mortalities. Bacteria isolated from sick fish samples submitted to the FDDL are tested for antibiotic susceptibility to the antibiotics legal for use on fish. Variability in antibiotic susceptibility testing results, not previously described in fish, was discovered in a study at the KSU College of Agriculture, Communities, and the Environment. Antibiotic susceptibility testing (AST) results were not always consistent between bacterial colonies from the same bacterial isolate. The study showed that 20 percent of the time, antibiotic susceptibilities among colonies from the same bacterial isolate were not consistent with each other. For example, among four colonies of Aeromonas hydrophila bacteria isolated from internal organs of a channel catfish, one or two of the colonies were sometimes resistant to the antibiotic being tested while the other colonies (from the same bacterial isolate) were susceptible. This inconsistency is a cause for concern when making treatment recommendations to fish farmers. If only one or two bacterial colonies are selected from a single isolate, a colony having bacteria resistant to an antibiotic may be overlooked/missed. If this happens, then AST results may indicate that a particular antibiotic will be effective in treating the bacterial infection in that fish population, when, in fact, it will not. We conclude that if multiple bacterial colonies are used for antibiotic susceptibility testing, the chances of including a colony resistant to the antibiotic are increased. Results, then, should indicate that resistance exists to that antibiotic, and it would not be recommended to the fish farmer for adding to medicated feed for the sick fish. This procedural modification has been implemented in the KSU FDDL to help assure more reliable antimicrobial resistance results, increasing the chance of selecting an effective antibiotic for the medicated feed used to treat sick fish.