Phage quantification on a plate

The Phage Cookbook by Dominik Refardt

The double agar overlay method and its variants

The classic method

Advantage: Accurate counts, plaques of similar size. Disadvantge: Only one sample per plate.

This is the method to begin with. Once you mastered it, you can proceed.

Adsorb phage to indicator bacteria, suspend in liquid soft-agar, pour on a plate, let grow overnight.

Spotting phages on soft agar

Mix your bacteria with soft agar and pour the mixture on a plate. Now place spots of a dilution series in a grid-shaped pattern on the plate. If you divide the center of the plate into a grid of 4 x 4 squares, you can spot about 5-10 μL per square and you can fit to 8-step dilution series on a single plate. If you set up a grid of 6 x 8 squares (54 mm x 72 mm), it's exactly one half of a 96-well plate and you can even use an 8-channel pipette to spot 8 6-step dilution series. Spot 2 μL per square.

Note that you can use the same method to spot bacteria. Because those are spotted directly on the hard agar, you can use slightly larger volumes (20 μL for 4 x 4 squares, 5 μL for 6 x 8 squares).

Be careful when counting plaques or colonies: You cannot reliably count more than 15 plaques in 9 x 9 mm square. If you count more, chances are that you underestimate the real number because the plaques are on top of each other. If you count very few, chances are that you overestimate the number if some of the replicates have no plaques at this dilution step. Factor those in as well!

When quantifying bacteria in this way, grow them at 30 °C overnight. Like that the colonies are still fairly small and you can reliably count many more per spot.

Spotting phages on hard agar

Simply spread bacteria on normal plate with LB and 1.5% agar and let soak in. Then proceed as above. Plaques will be much smaller and it is easier to count high numbers. The only difficulty is to spread the bacteria evenly.