Enumeration of lactococcal 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 the concentration of airborne phage.
• To determine if cultures are contaminated.
• To obtain information on phage/culture relationships.
• Determine the efficiency of virucidical filters.
Materials tested for phage are generally called phage suspect materials (PSM). These include starters; raw milk; cheese milk; whey; the clean-in-place solutions and rinse water; pipelines, vat surfaces, valves, cheese-making implements; plant atmosphere; curd and cheese.
Although the bulk of cheese factories in the UK no longer propagate their own starters, only 17 % in 2000 ( Boyle and Mullan, unpublished), examination of starters for phage contamination is important for those producing their own bulk starter. Examination of the milk in cheese vats immediately prior to starter addition is important since the concentration of any phage present provides a good indicator of the effectiveness of rotations, the insensitivity of cultures to phage and the effectiveness of the CIP system and the level of plant hygiene.
Cheese whey is perhaps the most commonly used PSM in cheese plants. In those plants that have effective phage monitoring systems it is usual to test whey at running and at milling for phage initially with new starters and to move to testing at milling with stable starter systems.
Lactococcal phages can be relatively resistant to detergents and sterilisers particularly in the presence of milk proteins. It is therefore prudent to consider periodic testing of CIP solutions and rinse waters for phage.
Internal vat surfaces, valves, and cheese-making implements can be swabbed and examined for phage. Particular attention should be given to hollow agitators and 'double -skinned' vessels in case of 'pin holing'. The author has investigated phage problems that were caused by whey leaking into hollow vat agitators and the phage in the whey contaminating the starter subsequently used in the vat.
Agitation of whey produces aerosol particles. Some operations such as whey separation using centrifuges generate large volumes of very small particles. Since whey can contain high concentrations of phage and the smaller particles can take, many hours to sediment the atmosphere in plants can become contaminated and the use of fumigation/ fogging systems may be required to as part of the phage control system. Under these conditions, it is sometimes useful to obtain information on phage levels in the atmosphere.
Curd and cheese are generally only tested if acid production has failed during cheese manufacture. High phage levels (109 - 1011 PFU/ml) in the curd combined with the absence of starter lactococci (microscopic examination of curd smears) indicate mass lysis of starter by phage.
Treatment of phage suspect materials and transport to laboratory
If there is likely to be a delay between sampling and phage enumeration consideration should be given to the possibility of phage proliferation during transport/ storage and also the possibility of a reduction in phage concentration which might occur during transit.
If CIP or equipment rinse waters are to be analysed, potential antiphage compounds such as chlorine, hypochlorite, iodophors, quaternary ammonium compounds should be inactivated by adding effective neutralisation agents after sampling e.g. sodium thiosulphatefor hypochlorite residues.
In general all samples should be stored at <5°C, or frozen until analysis.
Specialist equipment is required to quantify phage concentrations in the atmosphere. Essentially phage levels can be quantified by several methods including:
(a) Passing a known volume of air through a 'trapping solution'. Essentially this can be a broth or milk solution containing a stabiliser e.g. gelatine to increase the viscosity and reduce foaming. The solution is titred for phage.
(b) Passing a known volume of air through a column containing a material to which the phage absorbs or that 'traps' the phage. Phage can be released by washing or solublising the filter material and the 'wash' or solublised material titred for phage;
(c) impinging a known volume of air into a revolving petri-dish containing M17 agar layered with soft agar seeded with test culture.
With the above methods the results are expressed as PFU/ per cubic metre (m3) of air or other desired volume e.g. 100 m3.
Pre-treatment of PSM prior to analysis
Frequently treatment of PSM is required prior to analysis. In some e.g. cheese, curd, starter, whey, high levels of phage-resistant starter cells may be present. These should be removed to avoid erroneous results. Centrifugation followed by membrane filtration can be used to provide cell free filtrates for starters.
Methods of phage assay
Conventional methods of phage enumeration measure phage directly, by its ability to lyse infected cells in agar and so giving rise to plaques, zones of clearing in confluent lawns of host cells or, indirectly by assessing culture activity that is inhibited in the presence of phage. There is growing interest in the development of ELISA and other methods including the use of emerging novel procedures based on molecular biology approaches.
Search for literature cited in this website
How to cite this article
Mullan, W.M.A. (2002).
[On-line]. Available from: https://www.dairyscience.info/index.php/enumeration-of-lactococcal-bacteriophages.html . Accessed: 23 September, 2017.