Isolation of Bacteriophages from a Soil Sample

To isolate phages of soil-dwelling bacteria, take a soil sample collected from a desired location.

Add a phage buffer to preserve the integrity of the phage particles and gently stir to suspend the soil in the buffer.

Allow the heavier soil sediment to settle, while the phages and other microorganisms remain suspended in the soil extract.

Take a filtration unit with a filter paper and pass the soil extract through it to remove debris.

To the filtered extract, add nutrients to support the growth of host bacteria later during specific phage isolation.

Transfer the nutrient-rich solution to a vacuum filtration unit.

Pass the solution through a fine filter to remove all bacterial and fungal contaminants, leaving only phage particles in the filtrate.

Divide the sterile filtrate into culture flasks.

To each flask, add calcium chloride, which promotes phage-host interactions later during specific phage isolation.

The solution with purified phages is now ready for further experiments.

Since bacteria in the Arthrobacter genus are common soil bacteria, they and the phages that infect them can be found in most types of soil.

Gather 200 to 400 grams of soil from the desired location. Add at least 400 milliliters of phage buffer to the soil in a large enough beaker so that at least 200 milliliters of phage buffer is in the supernatant and is able to be extracted, mixed by gently stirring or swirling until the soil is suspended, and allow the soil sediment to settle, typically for 30 minutes. Next, remove soil sediment debris by passing the soil extract through a filter paper by vacuum filtration.

Add 4 grams of LB powder to 200 milliliters of the filtered soil extract and mix to make a 2% solution. Pass the solution through a 0.22 micron filter by vacuum filtration to obtain a sterile filtrate. If possible, continue on to the next step immediately.

Nevertheless, if need be, the filtered samples can be stored at 4 degrees Celsius for up to a week before proceeding. However, the number of viable phage particles will be reduced. Using aseptic techniques, aliquot multiple 50 milliliter portions of the filter sterilized LB soil extract mixture into individual 250 milliliters sterilized shaking, baffled culture flasks. Add sterile 2.0 molar calcium chloride solution to the final desired concentrations, since different Arthrobacter phages grow optimally under different calcium chloride conditions.