BACTERIOPHAGE ISOLATION AND PURIFICATION

When isolating phage in environmental samples it is important to realise that the phage population may consist of several phage strains with one common characteristic; they propagate on a particular host. Hence there is a need to obtain pure strains.

While the material below is particularly relevant to lactococcal phage isolation and purification it should be applicable to bacteriophages generally.

Phages are purified by removing a well isolated plaque using either a Pasteur pipette or more crudely, but just as effectively, a wire loop-figure 1. Using a wire loop, the area surrounding the plaque is cut carefully with the loop (excised) and the piece of 'soft agar' containing the phage is added to 9 ml of 25% Ringers solution or other diluent-figure 2.

The agar should be gently broken into smaller pieces with the wire-loop, mixed briefly with a vortex-mixer and left for 5-10 minutes at ambient temperature.

The phage suspension is then filter-sterilised through a 0.45mµ syringe-mounted, filtration unit-plate-figure 3-to remove any bacteria including phage-resistant host bacteria. With coli-phages chloroform treatment is generally used to kill any bacteria present and the filter-sterilisation step is sometimes omitted.

A second cycle of purification is required to ensure a single phage-strain population. Since the suspension should contain around 10⁴ to 10⁵ PFU/ml, dilution's 10^-3 to 10^-4 should give plates with adequate plaque numbers for further work.

The above procedure should work with most phages and give reliable results;it is a simple method for isolating phages. It may need to be optimised for particular phages; large isometric phages can be fragile and difficult to isolate.

For information on how to produce and store high tire phage lysates click here.

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Discovery | Phages for lactic acid bacteria | Morphology | Bacteriophage lysins |Enumeration | Isolation and purification | Storage lactococcal lysates | Industrial significance | Control

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