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How do you isolate a bacteriophage (phage) and obtain a pure phage preparation? This is achieved by plating a phage suspension using the double agar method, and a susceptible host strain, to obtain plaques and further purifying the phage contained within the plaque.

 The double agar method has been described previously but will be summarised again.  In this method a small volume of a dilution of phage suspension and host cells are mixed in molten, 'soft' agar.  The resulting suspension is then poured on to an appropriate 'nutrient' basal agar medium to form a thin 'top layer' which hardens and immobilises the bacteria.

During incubation the uninfected bacteria multiply to form a confluent lawn of bacterial growth over the surface of the plate.  Each infected bacterium bursts after a short time and liberates progeny phages that infect adjacent bacteria, which in turn are lysed.  This 'chain' reaction spreads in a circular motion until brought to a halt by a decline in bacterial metabolism.  Plaques are zones of bacterial lysis caused by phage action and appear as circular zones of lysis on lawns of bacterial cells.

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 all propagate on the host used in the plaque assay!  Hence there is a need to obtain pure strains since a plaque might contain more than one type of phage.

In milk plants phage can be isolated from raw milk, pasteurised milk, cheese whey, air, starter cultures, CIP solutions and a range of environmental samples including soil, silage and sewage.

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

Phages are purified by removing a well isolated plaque using either a Pasteur pipette or a wire loop

 

If a loop is used, 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 0.45 mµ syringe-mounted, filtration unit- figure 3-to remove any bacteria including phage-resistant host bacteria. Note if the phage aggregates some phage may be retained by the filter. With coli-phages chloroform treatment is generally used to kill any bacteria present and the filter-sterilisation step is sometimes omitted. Chloroform may inactivate some phages and should be used with caution and proper controls. Sodium azide can also be used for some phages.

 

The phage suspension is then filter-sterilised through a 0.45 mµ syringe-mounted, filtration  unit- figure 3-to remove any bacteria including phage-resistant host bacteria

A second cycle of purification is required to ensure a single phage-strain population. Since the suspension should contain around 102 to 105 PFU/ml, dilutions 10-1 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 may need to be optimised for particular phages e.g. large isometric phages can be fragile and the possibility that the phage aggregates resulting in apparently low titre preparations should be considered.

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


How to cite this article

W.M.A. Mullan (2001). [On-line]. Available from: https://www.dairyscience.info/index.php/isolation-and-purification-of-bacteriophages.html . Accessed: 29 September, 2016. Updated June 2010, December 2012, January 2015.

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