The first stage in the isolation of phage lysin is the production of lysates containing high concentrations of phage. Because lysin concentration is correlated with phage concentration, this objective can be achieved by obtaining lysates containing >1 x 10 ¹⁰ pfu/ml (Mullan and Crawford, 1985a). Information on the production of high tire phage lysates has been discussed previously.

To obtain significant purification several purification strategies are generally employed. Ion-exchange chromatography is usually employed at an early stage.

WHY ENUMERATE BACTERIOPHAGES?

There are many reasons why information on the concentration of bacteriophage in a sample may be required. These include:

 • To determine the level of phage contamination of dairy processing plant.
 • To determine the effectiveness of cleaning and sterilising programmes.
 • To determine levels of airborne phage.
 • To determine if cultures are contaminated.
 • To obtain information on phage/culture relationships.
 • Determine the efficiency of virucidical filters.

The double agar method as described by Adams(1959) is widely used to enumerate lactococcal and other phages.  In this method a small volume of a dilution of phage suspension and a small quantity of host cells grown to high cell density, sufficient to give 10⁷-10⁸ CFU/ml, are mixed in about 2.5 ml of molten, 'soft' agar at 46°C.  The resulting suspension is then poured on to an appropriate 'nutrient' basal agar medium e.g. M17 (Terazaghi and Sandine, 1975) to form a thin 'top layer' which hardens and immobilises the bacteria. Refer to figure 1 below.

 The major factors that influence plaque formation by lytic lactococcal phages were reviewed by the author in 1979, and in 1986.  These papers and the references to the literature can be downloaded by the author's students, this facility is currently not available to other site users. Work reported by Dag Lillehaug (1996) who has studied plaque formation by six lysogenic lactococcal phages is also briefly summarised. Dag Lillehaug's paper is available as a free download from the publisher's website.

Phage activity can also be assessed indirectly by measuring culture activity, the premise being that the presence of disturbing phage will inhibit starter growth.

Several methods are available, and include-

• Modified starter activity tests

• Failure of infected cultures to coagulate milk and/ or to reduce redox dyes.

The original activity test was reported by Anderson and Meanwell (1942).  This test required sterile milk to be inoculated with a phage free starter and a 0.01% inoculum of PSM added.  The acidity produced after incubation at 30°C or 37°C for 6 hrs was compared with a control infected with heat-treated PSM.  A reduction of titratable acidity of 10% or more was taken to indicate the presence of phage.  Interestingly this very basic test was still being used, as one of several methods of phage detection, in the UK in the 1980s (Walker et al., 1981).  Activity tests in this form are limited in value since

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.

It is frequently necessary to produce and use high titre phage preparations.

This section provides summary information on the production and storage of high-tire lactococcal phage preparations.

Preparation of phage lysates

Purified suspensions of phages (genetically homogeneous phage populations) may be isolated by research workers using the technique described previously, obtained from research centres or culture/phage collections. They are generally in freeze-dried form or suspended in broth, whey or other liquid. It is more convenient to work with liquid preparations than freeze-dried preparations. Note freeze-drying almost invariably results in a significant drop in titre.