An Improved Double Layer Plaque Assay for Male Specific Bacteriophage MS2

Introduction: Plaque assay is an extremely important enumeration technique used in the study of bacteriophages. Traditional double layer plaque assay usually produces small and hazy plaques difficult to detect. A plaque assay with good visibility is critical to the accurate enumeration of bacteriophages.

Purpose: Develop an accurate enumeration technique for bacteriophage MS2.

Methods: An optimization of the double layer plaque assay for bacteriophage MS2 was developed. A double layer tryptone agar plate containing a thin layer (10 ml per 100 x 15 mm plate) of freshly prepared bottom agar (1%) with a thin layer (10 ml) of freshly prepared top agar (0.45%) was used for plaque enumeration. Both layers were supplemented with 0.1% glucose, 5 mM CaCl₂, 10 µg/ml thiamine, and the top layer also contains the 6-h culture of E.coli, the host of bacteriophage MS2. Thirty µl of bacteriophage MS2 stock was spread on top evenly and the plate was incubated at 37 °C for 12-15 h.

Results: The influences of bottom layer composition (w/, w/o supplements, 1%, 1.2% agar), bottom layer thickness (10, 20, 30 ml), top layer composition (w/, w/o supplements, 0.45%, 0.6% agar), top layer thickness (10 ml, 15 ml, 20 ml), position of bacteriophage (contained in top layer, spread on top), quantity of bacteriophage (30, 50, 100 µl) on the visibility of the plaques were investigated. Results indicated that adding supplements to both layers and decreasing top layer agar content extensively increase the plaque size. A combination of a thin bottom layer and a thin top layer produces the largest plaques. The plaque size is increased when the bacteriophage is spread on top instead of contained in the top layer.

Significance: The development of a novel double layer plaque assay ensures accurate detection and enumeration of bacteriophage MS2.