Isolation of Arthrobacter Bacteriophages from a Soil Sample

Abstract

Source: Cross, T., et al. An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates. J. Vis. Exp. (2015).

This video demonstrates the isolation of Arthrobacter bacteriophages from a soil sample using enrichment and plaque assay techniques. It highlights the infection, amplification, and visualization of phages as clear lysis zones formed on a bacterial lawn.

Protocol

1. Phage Isolation

1. Add 1 to 2.5 ml of late exponential/early stationary phase bacteria culture to each of the flasks. For an OD₆₀₀ from 0.5-0.7, use 1 ml of cells. For an OD₆₀₀ higher than an OD of 0.7, use 2.5 ml of cells.
   NOTE: Cells in exponential phase growth typically yield more phages and higher titers than cells in stationary phase growth.
2. Shake flasks at 250 rpm at 30 °C for approximately 24 hr in a shaking incubator.
3. After a 24 hr incubation period, remove enrichment flasks from the shaking incubator and dilute the enrichment samples tenfold in phage buffer.
4. Set up culture tubes with 0.5 ml of late exponential/early stationary phase bacteria culture and add various amounts of the enrichment culture (5 µl to 500 µl of a 10-1 dilution in PB) to the culture tubes.
   NOTE: It is advisable to test a wide range of concentrations since each soil sample generates different phage titers after enrichment.
5. Add CaCl₂ to culture tubes to make a final concentration equal to the concentration present in the original enrichment flask. Use a range of different CaCl₂ concentrations to select for many phages with varying CaCl₂ dependencies.
   NOTE: Most phages will be isolated within the CaCl₂ range of 1-2.5 mM, but there are phages isolated most optimally anywhere from 0-50 mM CaCl₂ concentrations. Because of this, it is best to check a range of CaCl₂ concentrations to maximize the number of unique isolated Arthrobacter phages.
6. Mix 4.5 ml of LB top agar in the culture tube and pour the mixture onto an LB agar plate. Swirl gently to distribute across the plate evenly and allow for the top agar to solidify for approximately 15 min.
   NOTE: Top agar can be prepared in larger batches (250 ml) and extra top agar can be stored at RT and reused for subsequent experiments for at least two weeks. Although there are different formulas used to make top agar, we make top agar using 7g/L of agar mixed with LB. Place the top agar in a 60 °C water bath to retain the liquid state during the course of an experiment.
7. Invert the plate and incubate at the desired temperature (temperature to 30 °C) O/N to 48 hr. Vary these conditions to optimize for the phages present. Most isolated phages come from plates incubated at 30 °C at 1-2.5 mM CaCl₂ concentration after a 24 hr incubation period.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
LB Broth powder	Fisher	BP9722-2	It's best to order these in bulk.
Granulated Agar	Fisher	BP1423-2	It's best to order these in bulk.
0.22 um Syringe filters	Fisher	09-719A
0.22 um Buchner filters	Fisher	430320	More than 50 ml of liquid can be obtained by carefully swapping the receiving tube.
Eppendorf Tubes	Fisher	05-408-129
5 ml Pipets individual	Fisher	13-678-11D
50 ml Conical tubes	Fisher	76002844
15 ml Conical tubes	Fisher	76002845
10 ml Pipets individual	Fisher	13-676-10J
25 ml Pipets individual	Fisher	13-676-10K
Whatman qualitative filter paper	Fisher	1001-824