HHMI002 – Phage Enrichment for Isolation (Direct Isolation, too)

Submitted by Joel Graff

This is a modified version of protocol 5.5 from the Phage Discovery Guide.  In this experiment, phages were released from soil particles by incubating 15ml of soil with 20 mL 7H9 medium supplemented with calcium chloride.  Inversions of tubes were followed with shaking at 250 rpm for a couple of hours.  Following centrifugation to pellet soil particulates, the supernatants were filtered using a 0.22 uM 50 mL conical tube system.  Pre-filtration through paper towels was problematic because the paper towels soaked up much of the sample.  If I overdid this, I would need to squeeze out a few milliliters from the saturated paper towels.

Anyway, about 10 mL of filtrate was combined with 1 mL of M. smeg culture for the “enrichment isolation” method.  (Used vented 50 mL tubes here to allow aeration of the culture as it shook overnight at 37C.)  A couple mL of filtrate was stored overnight to be used in “direct isolation” follow-up experiments.