Eosin methylene blue agar

E. coli was isolated and identified according to Lee and Nolan [11 ]. Briefly, all the collected samples were pre-enriched in buffered peptone water (Oxoid, UK) and incubated aerobically at 37°C for 24 h. Then, a loopful of the broth culture was inoculated onto MacConkey agar (Neogen, US) and eosin methylene blue agar (LabM, UK) plates and reincubated at 37°C for 24 h. The isolated colonies were identified morphologically and biochemically (oxidase strips and triple sugar iron agar from Oxoid; urea, Simmon citrate agar, and peptone water from LabM; and Kovac reagent from HiMedia, India).

Two wastewater samples of about one L were collected from two locations of the municipal sewer system of Trabzon, Turkey. For primary isolation of gram-negative bacteria, the wastewater samples were diluted and plated on eosin methylene blue agar, tryptic soy agar, and plate count agar (Lab M, Lancashire, UK). The cultures were incubated at 37°C for 24-48 hours. The bacterial colonies with distinct morphology were purified, gram stained, and used as hosts for lytic phage isolation. The same wastewaters were used for the isolation of lytic phages using the culture-enrichment method described by Lin et al^{8 (link)} with some modifications. One hundred twenty-five milliliters of each wastewater was mixed with 125 mL of double-concentrated Luria-Bertani (LB) broth (Lab M) and incubated at 37°C for 24 hours with shaking at 150 rpm. The cultures were centrifuged at 8000 ×g for 10 minutes at 4°C, and the supernatants were filtered using 0.22 μm pore size filters. The phage lysates were stored at 4°C in the dark with one drop of chloroform until use.

Escherichia coli was isolated and identified according to Nolan et al. [1 ]. Briefly, all the collected samples were pre-enriched in buffered peptone water (Lab M, UK) and incubated aerobically at 37°C for 24 h. A loopful of the broth culture was inoculated onto MacConkey agar (Neogen, US) and eosin methylene blue agar (Lab M) plates, which were incubated at 37°C for 24 h. The isolated colonies were identified morphologically and biochemically (oxidase strips and triple sugar iron agar were from Oxoid, UK; urea, Simmons’ citrate agar, and peptone water were from Lab M; and Kovacs reagent was from HiMedia, India) [1 ]. In addition, antisera against somatic (O) antigens (Denka Seiken Co., Tokyo, Japan) were used for serotyping isolated E. coli following the manufacturer’s instructions.