Phage isolation and purification

Actinobacteriophages were isolated from soil samples with host, Streptomyces platensis JCM 4664 substrain MJ1A1. An enrichment culture was prepared from 1 g soil and 2.5 ml of S. platensis added to 15 ml of Luria-Bertani (LB) medium (for one liter: tryptone 10 g, yeast extract 5 g, NaCl 10 g, agar 15 g, pH 7.0), followed by a 2-day incubation at 30°C with shaking. Phage were isolated from a 1.2 ml volume of enrichment culture that was centrifuged at maximum speed for 3 min, 1 ml of the resulting supernatant was filtered (0.22 μM filter), and 5 μl of the filtrate was spotted and then streaked onto an LB plate containing 100 μg/ml of cycloheximide. S. platensis (0.1 ml) was mixed with 4.5 ml of LB top agar 0.7%, poured over the streak plate and incubated for two days at 30°C. Resultant plaques were re-streaked onto new LB plates containing 100 μg/ml of cycloheximide about 3–4 times for phage purification.