Isolation of Arthrobacter Bacteriophages from a Soil Sample

Take a soil extract containing bacteriophages, including Arthrobacter phages, pre-mixed with a growth medium.

Add Arthrobacter bacteria and incubate under agitation.

Arthrobacter phages attach to the bacterial surface and inject their DNA to produce new phages.

The host cells lyse to release progeny phages, enriching the medium with Arthrobacter phages.

Dilute this medium to reduce the phage concentration.

Add fresh Arthrobacter cells and calcium ions, which aid in phage infection.

Mix with a soft agar and pour onto a nutrient agar plate. The solidifying soft agar immobilizes the bacteria and restricts the phage spread.

Upon incubation, each phage infects a nearby host cell. It then produces progeny phages that infect neighboring bacteria, allowing localized infection.

The uninfected bacterial cells grow, while the infected bacterial cells lyse, detected as clear zones called plaques over a bacterial lawn.

These plaques contain progeny phages, ready for downstream use.

Begin this procedure by adding 1 to 2.5 milliliters of late exponential or early stationary phase bacteria culture to each of the flasks containing filter sterilized LB soil extract mixture. If the OD 600 of the culture is 0.5 to 0.7, use 1 milliliter. If the OD₆₀₀ is higher than 0.7, use 2.5 milliliters.

Shake the flasks at 250 RPM at 30 degrees Celsius for approximately 24 hours in a shaking incubator. After the 24 hour incubation period, remove the enrichment flasks from the shaking incubator. Dilute the enrichment samples tenfold in phage buffer.

Set up culture tubes with 0.5 milliliters of late exponential or early stationary phase bacteria culture. Add various amounts from 5 microliters to 500 microliters of the diluted enrichment culture to the culture tubes.

Add calcium chloride to the culture tubes to make a final concentration in 5 milliliters equal to the concentration present in the original enrichment flask. Use a range of different calcium chloride concentrations to select for many phages with varying calcium chloride dependencies. Add 4.5 milliliters of LB top agar to each culture tube, and pour the mixture onto an LB agar plate.

Swirl gently to distribute evenly across the plate and allow the top agar to solidify for approximately 15 minutes. Invert the plates and incubate at 30 degrees Celsius overnight to 48 hours. Vary the incubation times to optimize for the phages present.