Comparative Evaluation of Bacteriophage Plaque Formation on Different Culture Media

Abstract

Bacteriophages are bacterial viruses with promising applications in microbial biocontrol and antimicrobial therapy. The efficiency of bacteriophage isolation and visualization can depend on the type of culture medium used for plaque formation. This study provides a comparative assessment that helps standardize plaque assays for routine phage screening. This study aimed to isolate and compare bacteriophage recovery from different agar media—Luria–Bertani Agar (LBA), Nutrient Agar (NA), Eosin Methylene Blue (EMB) Agar, and MacConkey Agar—using Escherichia coli as a host. Sewage samples from livestock facilities were processed and enriched using the double agar layer (DAL) method. Distinct plaques appeared after 18–24 hours of incubation at 37 °C, varying in size and clarity across media types. The highest phage titer and clearest plaques were obtained on LBA (4.7 × 10³ PFU/mL), followed by EMB (4.1 × 10³ PFU/mL), NA (3.6 × 10³ PFU/mL), and MacConkey Agar (3.2 × 10³ PFU/mL). LBA supported larger and more distinct plaques, while MacConkey medium produced smaller, turbid plaques. These results demonstrate that media composition significantly influences bacteriophage plaque morphology and enumeration, with LBA being the most effective for phage isolation and visualization against E. coli. Taken together, these results underscore the importance of carefully selecting culture media when designing phage isolation workflows, particularly in laboratories working on phage therapy development and environmental surveillance of E. coli phages.