Phage Solution Recipies

These media solutions will work for propagating M. smegmatis and mycobacteriophages.  There are only 3 solutions that you need! 

MP Buffer (Mycobacteriophage Buffer)

Solution:                           Concentration:                For 500ml

Tris (pH 8)                          50 mM                           

NaCl                                 150 mM                            4.35g

MgCl2                                 10 mM                            .47g

CaCl2                                   2 mM                            .11g

Middlebrook 7H9 Liquid Medium (For growing liquid cultures of M. smegmatis)

Distilled water                                                                1000 mL

Middlebrook 7H9 powder (Difco catalog no. 0713-01-7)       4.7 g

Glycerol                                                                          5 mL

Autoclave at 15 lb/in2 for 20 min.  Ready to use once cooled. 

Middlebrook 7H9 Solid Medium (For growing M. smegmatis)

Distilled water                                                                1000 mL

Middlebrook 7H9 powder (Difco catalog no. 0713-01-7)       4.7 g

Bacto-Agar

Glycerol                                                                          5 mL

Autoclave at 15 lb/in2 for 20 min.  Let cool until the solution can be touched-to-the cheek without squinting (55C), then pour your plates (25 mL/petri dish is ideal).    Let cool on bench, stirring for 30 min.

Pour plates.  Some use a 25 mL pipette, I prefer to be more expedient and pour directly from the flask. 

Middlebrook Noble Top Agar (When attempting to make plates that contain mycobacteriophage plaques.)

Use 0.6% Noble Agar (Difco catalog no. 0142-17-0) with 0.5% 7h9 powder (Difco Catalog no. 0713-01-7).  I.E., use 3.0 g Noble Agar and 2.5 g 7h9 powder per 500 mL water.  Autoclave the solution, and leave in a 55C bath until ready to pour (the bottle can just be touched to your cheek without wincing).  Remember, if too hot, the solution will kill your microbes!  (If too cold, the solution solidifies and can't be used unless it has been put in the microwave for a minute.)