Indirect assay of lactococcal bacteriophages
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
•In its original form, the test does indicate the concentration of phage present.
•Inhibitory materials present in samples may influence the method.
• Raw milk phages may not be detected
• Phage that do not propagate at the test temperature will not be detected.
Errors due to inhibition caused by heat labile natural milk inhibitors can generally be controlled if the PSM is diluted a hundred fold in 25% Ringers solution prior to use. The method can be made quantitative by using a most probable number technique (MPN) and more useful by using pasteurised, phage and inhibitor free, milk and the temperature profile used in the fermentation process.
Failure of infected cultures to coagulate milk
This method is similar to the previous method except that normal acidification, and by inference, freedom from phage, is indicated by coagulation of the test culture. Sterile milk is inoculated with test culture, mixed with dilutions of PSM and incubated at 30°C or 37°C for 6 hours. The highest dilution at which coagulation does not occur is recorded and expressed as the phage titre. The method can be made more precise by using several tubes per dilution and a MPN technique.
Methods that use Redox indicators
This method is similar to the previous methods except that growth of culture is indicated by the reduction of a redox indicator. When lactococci grow in milk, they lower the redox potential and a redox indicator can be used to indicate whether growth has taken place and, to a certain extent, the extent of growth. Redox indicators used include resazurin and methylene blue.
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How to cite this article
W.M.A. Mullan (2002).
[On-line]. Available from: https://www.dairyscience.info/index.php/enumeration-of-lactococcal-bacteriophages/indirect-assay-of-lactococcal-bacteriophages.html . Accessed: 16 June, 2019.
Revised 2004, 2005, 2006, 2007, 2008. Last revision July 2008.