This protocol uses M. smegmatis to amplify the phage found in soil.
1.) Place a test-tube cap-full of soil into a test tube. If the soil sample will adhere strongly to the test tube cap, fill the test tube approximately 1/4 full of soil. Add 5 mL MP phage buffer. Flick the capped test tube with your finger, mixing well. It is difficult to suspend the soil in the MP buffer, and therefore I recommend sealing the surface of the tube with plastic wrap/wax paper and inverting the tube to mix. In our lab we sealed the tubes with parafilm for inversion. Other groups might want to use 15 mL falcon screw-tap tubes or a plastic snap-cap tube.
2.) Add 50 uL M. smegmatis culture, and mix the culture again.
3.) Incubate the culture for 0.5 hour at 37 C in a heating block (or incubator). Mix the tubes every 5 minutes.
4.) Transfer 2 mL liquid (the soil should have sedimented to the bottom, do not resuspend) into centrifuge tubes. Centrifuge for 5 minutes at low rpm in order to pellet the debri. (we used 2 microcentrifuge tubes per sample, using a p1000 pipettman, transfering 1 mL solution into each tube). Centrifuge the samples at 2000 g for 5 minutes in a centrifuge. [The point is to remove the soil, and if a centrifuge is not available, filtering with a 0.45 u syringe filter before using a 0.22 u filter may suffice.] If you do not have a centrifuge, try carefully pipetting phage buffer from the soil and filtering with only a minimum amount of soil added to the filter since the soil will rapidly clog the filter. We have had success with either technique.
5.) Transfer the clear phage solution into a syringe with a 0.2 u filter attached, and filter the sample. Only about 1 mL sample will be obtained. Split the filtrate between two tubes, and reserve the other tube for another day.
6.) Using sterile technique, add 100 uL of the phage solution to 100 uL M. smegmatis culture. Incubate at 37°C for 30 minutes.
7.) Sterilely pipet 4 mL of 55°C 0.35% top agar into the tube containing the phage-smegmatis mixture. Pull up the entire amount back into the pipet (to mix them together). Immediately expel the mixture onto a 7H9 agar plate. Very gently rotate the plate with the lid on so that the agar spreads evenly. Let the plate cool on the countertop.
8.) Once cooled, invert the plate and incubate at 37°C for 24-48 hours.
9.) Observe for plaques. Plaques are clearings in the lawn of bacteria in the top agar. Most are visible at 48 hours.
Data to Collect: (complete for all samples tested).
Sample # Sample Source: Date plated: Date Observed: Plaque Description: