Optimization of Plaque Forming Conditions for an Aeromonas hydrophila Lytic Bacteriophage

Abstract

Recent emergence of antibiotic resistant bacteria has led to renewed interest in the phage research for control of aquatic bacterial pathogens. Plaque assay is an important part of phage research. For some phages, the plaques could be very much turbid and small in size resulting in difficulties during phage isolation and enumeration. Present study investigated the effect of soft agar as well as Ca++ and Mg++ ion concentrations on plaque forming ability of a Aeromonas hydrophila lytic phage. At 0.2% soft agar, with 10 mM of CaCl2 and MgSO4, mean ± SD plaque size of 5.55 ± 0.52 mm and counts of (58 ± 4) were significantly higher the other concentrations. However, increase of agar concentration by value of just 0.1% reduced the plaque diameters to less than one millimeter. No plaques could be observed at soft agar concentration of ≥4%. High CaCl2 and MgSO4 concentrations also affected plaque morphology, as plaques were found to be very much uniform and clear up to 25 mM, but turned to turbid and irregular size at 100 mM of CaCl2 and MgSO4. This study provides important insights to optimize plaque assay for phages forming turbid and micro sizes plaques leading to their efficient utilization in phage therapy in aquaculture.