Effect of Antibiotics and Glycerol on Improving Bacteriophage Detection and Enumeration

Antibiotic-resistant bacteria are a growing issue in aquaculture, and phage therapy offers an alternative approach to the use of antibiotics. Phages for phage therapy can be isolated, identified and enumerated using a Double-Layer Agar (DLA) technique. While some phages form large and well-defined plaques that can be enumerated when plated with the DLA technique, some produce small and turbid plaques that are difficult to detect and count. A phage, Aeromonas phages-T65, was isolated from Aeromonas hydrophila, which formed small and turbid plaques making it hard to count. The use of antibiotics and chemicals have been suggested as a way to increase plaque size for easy detection and enumeration of phage plaques. To increase the plaque size of Aeromonas phages-T65, antibiotics and glycerol were added to the culture media, and tested at different concentrations. Application of penicillin-streptomycin and/or glycerol showed an increase in plaque size and the biggest improvement in plaque size were observed using either 40 U/mu l penicillin-streptomycin or a 5 % glycerol in the top agar layer of the DLA technique. These findings suggest that such modifications could be implemented into the standard DLA overlay method to investigate new Aeromonas phages for the development of phage therapy against Aeromonas spp. in aquaculture.