A double layer plaque assay using spread plate technique for enumeration of bacteriophage MS2

Bacteriophage MS2 is used widely as a model organism to estimate pathogenic virus survival in various environments, and is usually quantified by plaque assay. Although current plaque assays work well in enumeration of MS2 in environmental samples, quantification of MS2 calls for better visibility and higher consistency. In an attempt to improve the visibility and consistency of the current plaque assay, spread plate technique was introduced, instead of the pour plate technique used commonly in existing methods. Other parameters that influence the outcome of the plaque assay were also compared. Using spread plate technique resulted in an increase of plaque size by approximately 50% and contributed to a better visibility. Addition of supplements (glucose, CaCl2 and thiamine); reduction of agar thickness and hardness, also contributed to enhanced plaque visibility and increased plaque count. Among all the conditions tested, a supplemented thin bottom agar (10 ml 1% agar) and a supplemented thin top agar (10 ml 0.45% agar) with spread plate technique gave the maximum countable plaques with a minimum standard deviation. When compared to other methods, it produced significantly higher plaque count and lower variation. The optimized plaque assay significantly improved visibility and consistency of the existing plaque assay methods and could be used in quantification of MS2.