Phage Discovery Guide: Direct Isolation Protocol

For POLS Labs

In the direct isolation protocol, the goal is to determine whether any bacteriophage can be detected in an environmental sample that is directly added to the host bacterial strain, such as Mycobacterium smegmatis.  This method is different than the enriched isolation protocol because no replication of the virus is allowed to occur before mixing your sample with host bacteria for a plaque assay.

The environmental sample (usually soil, pond water, etc.) is mixed with bacterial growth medium to allow for bacteriophage to release from particulates in the sample.  The following step involves filtering out soil, bacteria and other “larger” particulates in your sample.  Bacteriophages are too small to get caught in the filter, so they will flow through with the medium.  This filtered liquid is called the filtrate.

The filtrate is mixed with bacteria to allow for infection to occur.  This assumes that there is bacteriophage that will infect the host bacteria in your filtrate.  Proceeding to a plaque assay will allow you to determine whether any infections did occur.

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