Isolation of Bacteriophages from a Soil Sample

Resumo

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 bacteriophages from a soil sample. A soil sample is collected and suspended in a phage buffer and allowed to settle the sediment. The extract is filtered, and nutrients are added. A vacuum filtration is done to remove microbial contaminants. The filtrate with phages is aliquoted into flasks, and calcium chloride is added to be used for further phage isolation.

Protocolo

1. Collection of Phage from Soil Samples
   1. Gather 200-400 g of soil from the desired location. 
      Note: Since bacteria in the Arthrobacter genus are common soil bacteria, they and the phages that infect them can be found in and are pervasive in most types of soil.
   2. Add at least 400 ml of Phage Buffer (PB) [68 mM sodium chloride (NaCl), 10 mM magnesium sulfate (MgSO₄),10 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-Cl) (pH 7.5)] to the soil and mix in a large enough flask so that at least 200 ml of PB is in the supernatant and is able to be extracted.
   3. Mix by gently stirring or swirling until the soil is suspended, and allow the soil sediment to settle, typically 30 min.
   4. Pass the “soil extract” through filter paper by gravity filtration to remove soil sediment debris.
   5. Add LB powder and mix to make a 2% solution (4 g LB powder in 200 ml soil extract).
   6. Pass the solution through a 0.22 µm filter by vacuum filtration to obtain a sterile filtrate. If possible, continue on to step 1.7 immediately. If need be, store filtered samples at 4 °C for up to a week before proceeding; however, the number of viable phage particles will be reduced.
   7. Aliquot multiple 50 ml portions of the filter-sterilized LB/soil extract mixture into individual 250 ml sterilized shaking baffled culture flasks using aseptic techniques. Add sterile 2.0 M calcium chloride (CaCl₂) solution to the final desired concentrations (0-50 mM) since different Arthrobacter phages grow optimally under different CaCl₂ conditions.
      NOTE: We find that the majority of the Arthrobacter phages identified by us are within the range of 1 mM-2.5 mM CaCl₂. However, several of the isolated phages grew by us exclusively at 0 mM CaCl₂ or at very high CaCl₂ concentrations. Proceed immediately to phage isolation.

Materiais

Lista de materiais utilizados neste artigo

Nome	Empresa	Número de catálogo	Comentários
LB Broth powder	Fisher	BP9722-2	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