Phage Discovery Guide: Plaque Assay Protocol

For POLS Labs

Plaque assays with bacteriophage involve overlaying “bottom agar” with “top agar” that contains bacteriophage-infected host bacteria.

This video will show you the technique for doing this with a single tube of infected bacteria.  In practice, you will likely set up infections of several tubes of bacteria using serially diluted bacteriophage sample. The purpose of plaque assays using serially-diluted bacteriophage is typically done to calculate the titer of your bacteriophage sample.  “Titer” refers to the number of infectious virions (aka plaque-forming units or pfu) per volume of viral stock.  Virus titers are most commonly described as “pfu/mL”.

Alternatively, you may use this assay in an attempt to create “webbed” plates, where the bacteriophage are diluted to 30,000 plaques per plate (give or take an order of magnitude).  This method requires that you have already determined the titer of your virus (described in the previous paragraph).  Webbed plates are those that have been infected with enough bacteriophage to end up with 90-95% of the bacteria within the top agar to be killed by the following day.  The number of plaques per plate will depend on the size of the plaques and the length of time the plates are incubated.  Rather than determining the titer of your bacteriophage, this variation of a plaque assay should provide you with the highest viral recovery possible from a single plate.

Phage Discovery Guide: Spot Tests

A good time to test for the presence of bacteriophage in your sample using this method is after you have collected your enriched isolation samples.  Rather than performing a plaque assay using serial dilutions, a spot test can be performed with just a single plate.

The spot test can be used as a yes or no assay.  Is there bacteriophage in my sample?  A variation of this assay can also be used to estimate the bacteriophage titer in the sample.  A sample that is known to have bacteriophage can be serially diluted and then spots of each dilution can be made “spotted” onto the plate.  (Remember to note the volume of sample spotted onto the plate and factor it into your titer calculation.)

Major benefit:

  • Using the spot test, you can screen several of your samples (or dilutions of a sample) for bacteriophage.

Potential problems:

  • Spots should be well-spaced so that the samples don’t flow into each other.
  • Inverting the plate for incubation can be problematic if the samples haven’t fully been absorbed into the top agar.  (Gravity will make spots move on your top agar surface.)

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.

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

Bacteriophages Videos for SEA-PHAGES Program

The SEA-PHAGES (Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science) program is designed for students to learn about bacteriophages.  The three HHMI (Howard Hughes Medical Institute) iBiology videos shown here will give students a good background knowledge for understanding the curricula and the research possibilities.

Part 1: Bacteriophages – What are they?

Part 2: Bacteriophages – Genomic Insights

Part 3: Bacteriophages – Genomic Insights

 

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.

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HHMI001 – Collecting Environmental Samples

Submitted by Joel Graff

The first step of isolating mycobacteriophage is to collect environmental samples.  Mycobacterium smegmatis is found in soil, so I attempted to find samples that were high in organic content (by extension these samples should be high in bacterial density as well).  Three samples were collected in a single “garden” but near different types of plants: coleus (annual, dicot, fresh compost), ornamental grasses (perennial, monocot), and yew tree (perennial, coniferous).  I got greedy and collected a fourth soil sample at the shoreline of a pond just north of the biology building at UBMC.  I might drop the “yew tree” sample because it was so dry…