Protocol for stab and glycerol handling
1. Agar preparation (scale if necessary)
LB medium (1 liter: Bacto yeast extract 5 g; Bacto trypton 10 g; NaCl 10 g; H2O up to 1 Liter) + 1-1.5% of agar. 21.
Boil the mixture for 1 minute (or in a microwave). Remove from heat.
Monitor the temperature until it falls to approximately 45 - 50° C (or: cover and let stand a few minutes).
Pour enough melted agar into each sterile plastic petri dish or to Eppendorf tubes (for stabs)
Stabs: Using a sterile straight wire, pick a single colony from a freshly streaked plate or from a fresh culture,
and stab it deep down into the soft agar several times (Figure 1).
Incubate the vial at 37°C for 812 h leaving the cap slightly loose.
Seal the vial tightly and store in the dark, preferably at 4°C.
In steril conditions add 400 ml autoclaved 100% glycerol and 1.4 ml of bacteria (LB medium after ON growth) in a 2 ml eppendorf tube (-80oC resistent). Mix (invert) and store it at -80oC.