Über-Concentrated Freezer Stocks of M. marinum

Gregory W. Broussard

Revised: 7/10/08

(Please use, change and disseminate freely!)

1. Inoculate 5 mL M7H9/ADC/CHX/CB/Tween80 with your strain of M. marinum. Incubate at 30°C shaking until turbid.
2. Inoculate from 50-100 mls broth (in 250-500 ml non-baffled flask) with the 1 ml culture. Place culture shaking at 30°C until O.D.₆₀₀ = ~0.6-0.9.
3. Transfer to 50 ml conical tubes and centrifuge 5,000 rpm for 10 min.
4. Resuspend the pelleted cells in 1/10^th original volume of M7H9/ADC.  (5mls for 50 mls original vol.)
5. Add the same volume of 40% glycerol and mix.
6. Aliquot 200µl per 1.5ml tube.
7. Freeze at -80°C.
8. The following day streak one tube onto a M7H10 plate to check for purity.