This protocol is usually used in conjunction with serial dilution of a stock solution of phage for enumeration of phage titer. But it can also be used with any phage-containing input. With thanks to the Wichman lab for setting me up with a good protocol for this early on.
The usual media for plating is lysogeny broth (LB). Remember that this is not a defined media (so you should attempt to minimise batch effects for reproducibility) and it comes in Lennox and Miller varieties. The appropriate type is Miller which contains 10g/L of sodium chloride (versus 5g/L in Lennox). Both plate (base layer) and top agars should be prepared. For one litre:
10 g Tryptone
5 g yeast extract
10 g NaCl
15g agar (if plate agar) | 7g (if top agar)
Make up to 1 litre in distilled water and autoclave. After autoclaving:
CaCl2 to a final concentration of 2 mM,
MgCl2 to a final concentration of 10 mM.
(for these use hydrated crystals as anhydrous powders do not dissolve effectively. Matching concentrations should prevent diffusion of salts away from one layer).
Top agar can be retained at 50-55 centigrade for medium-term storage and deployment. It is recommended to prepare top agar in small batches (4ml required per plate) to prevent cross-contamination. Microwaving is possible but not extensively tested in our lab.
Please note, you will need one extra bacteria-only control plate on the day.
1. Pour plates in the usual way using the plate agar solutions only (no top agar yet). No antibiotic is usually required so you can do this with quite hot agar taking precautions to avoid scalding.
2. Allow the plates to cool for 5 minutes and then move to an incubator. Slightly displace the lids to allow excess water to evaporate. (This prevents streaks from forming on the plates and for titering applications perfect sterility is not required.)
3. Move plates to +4 centigrade storage.
We usually work with a dense culture of overnight-grown bacteria. They should re-enter exponential phase upon dilution into the top agar.
100ul are required per plate, so a 4ml overnight is usually sufficient for most applications.
On the Day
First Steps and Top Agar Preparation
1. Set up a water bath to 48 centigrade
2. Take out plates, equilibrate to room temperature (too cold and the top layer will solidify too quickly) and label them. 3. Add as many tubes as required (+ some extras), each capable of holding 4ml of top agar (you can use 15ml centrifuge tubes). These do not need to be labelled.
4. When the tubes are warm, use a serological pipette to add 4ml of molten top agar to each tube. (You can use a 10ml pipette and overload to 12ml = 3 * 4ml to dispense)
5. Allow the top agar to equilibrate to the water bath's temperature. (Hotter and you may kill the bacteria. Cooler and the agar may prematurely solidify.)
1. Set up your working space for ergonomics. All within easy reach you need the dry plates (base layer only), the water bath with aliquoted top agar, the overnight bacterial culture and the phage solutions to be plated. You will also need a P200 pipette set to 100ul. We recommend using filter tips and two pipettes (one for phage, another for bacteria).
2. Working quickly but calmly, add 100ul of phage followed by 100ul overnight bacteria to a single tube of top agar.
3. Immediately add invert the tube to mix, tap the lid and dispense onto a plate.
4. Rock and rotate the plate gently to distribute the top layer.
5. Set aside and allow to solidify
6. Move on to the next plate. We recommend doing one at a time to minimise the time bacteria and phage spend at high temperatures in the hot top agar.
7. Remember to prepare one plate with bacteria only. This control for contamination is recommended and should be your last prepared plate.
8. Incubate the plates at 37 centigrade. For phiX174, 4 hours is the recommended time, with plaques appearing from ~3.5 hours on.
I recommend checking:
Kropinski, A.M., Mazzocco, A., Waddell, T.E., Lingohr, E. and Johnson, R.P., 2009. Enumeration of bacteriophages by double agar overlay plaque assay. Bacteriophages: Methods and Protocols, Volume 1: Isolation, Characterization, and Interactions, pp.69-76.
for more details of the plaque assay and variations thereon.