Method: Titering Lambda Phages

June 27 1990

C. Helms

Principle:

This procedure is used to find the number of viable phage particles in a lysate. The phage lysate is serially diluted. A bacterial host strain is inoculated with an appropriate amount of diluted phage and plated out onto growth medium. The plates are counted the following day and the concentration of the original lysate calculated. For most lambda phage clones we prepare the serial dilutions assuming a fresh lysate has approximately 2 X 10e10 pfu (plaque-forming units) per ml of lysate.

Total time is less than one hour but is spread over a three day period.

Day 1

1. Inoculate 5 ml of LBM medium with a single colony (or use 0.1 ml of -20 degrees C glycerol stock) of an appropriate bacterial host strain e. g. LE392 and grow overnight at 37 degrees C.

Day 2

1. For 100-fold dilutions dilute 10 ul phage lysate into 990 ul of sterile lambda diluent. Vortex. Carry out as many serial dilutions as required to plate out approximately 200 pfu in a 100 ul aliquot. You should plate out at least two different dilutions. If 10-fold dilutions are desired dilute 50 ul into 450 ul lambda diluent. All dilutions can be prepared in eppendorf tubes.
2. Using 5 ml sterile disposable glass culture tubes mix 0.1 ml diluted phage with 0.1 ml fresh overnight culture of bacterial host cells. Incubate 15 minutes at 37 degrees C.
3. Add 3 ml warm (50 to 55 degrees C) LBM top agar mix briefly and pour the entire contents onto an LBM plate spreading evenly over the surface by tilting the plate. Work quickly before the top agar hardens (it helps to warm up the plates to 37 degrees C before plating).
4. Allow the agar to set for at least 10 minutes on the bench top. Invert plates and incubate overnight at 37 degrees C.

   Example of dilutions required:

   We assume the lysate has a concentration of 2 x 10e10 pfu/ml. To obtain 200 pfu on the plate we will need a 10e-8 dilution on the plate. To cover the possibility that the titer is nearly 10-fold higher or lower we will also plate out 10e-7 and 10e-9 dilutions (a total of three plates of LBM required).

Day 3

1. Count the plaques on the plates. Multiply the number of pfu times the dilution factor to obtain the number of pfu /ml in the original lysate e. g. if the plates above had >1000 pfu 178 pfu and 20 pfu respectively use the plate closest to 200 (i. e. the plate having 178 pfu ) and multiply by the dilution factor in this case: 178 x 10e8 or 1.78 x 10e10 pfu / ml = lysate titer.

References: none