Bacteriophages isolation and purification

bacteriophage plaques on a plate
plaques on a plate

 This is achieved by plating a phage suspension using the double agar method, and a susceptible host strain (any bacteria), to obtain plaques and further purify the plaque's phages.

 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. After a short time, each infected bacterium bursts 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 pipet, te the area around the plaque is stable, and soft areas 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
picking a plaque

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
filter-sterilized

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 bacteriophages to be used in phage therapy this procedure can not be jumped due to the fact that it's important to have the pure form of the bacteriophage which is fully characterized so that there will be no way of having negative effects like transfer of antimicrobial resistance genes and other unrequited effects.

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