Phage Discovery Guide: Enriched Isolation Protocol

For POLS Labs

There are two options described in the phage discovery guide for extracting bacteriophage from your environmental sample: enriched isolation (shown in this video) and direct isolation.  The enriched isolation is different from the direct isolation because the enriched isolation protocol is designed to add a step for bacteriophage replication before testing for infectious bacteriophage in your sample.

In this video, you will see that both bacteriophage extraction protocols begin the same way.  An environmental sample is mixed with some media with the goal of releasing the bacteriophage from the particulate and into the liquid.  A filtration step separates the liquid containing bacteriophage from soil particles, bacteria, and other “large” objects in the original sample.

The liquid that flows through the filter is called a filtrate.

The filtrate may contain bacteriophage that infect Mycobacterium smegmatis (or whatever host bacteria that you are using in your phage hunting experiments).  As mentioned above, the enriched isolation is different from the direct isolation because it is designed to allow the bacteriophage to replicate.  To do this, the filtrate is mixed with additional bacterial medium and the host strain of bacteria.

If the bacteriophage causes a lytic infection, there will be more bacteriophage over time in your sample.

If the bacteriophage is lysogenic, the genome integrated into the infected bacterium will replicate along with the chromosome of the bacterium as it divides to become two daughter cells.  It will continue to replicate in this manner in each generation of the bacterial cultures growth.

Non-HHMI approved protocol:

Improvised Enrichment Protocol