Protocol for stab and glycerol handling

Stab preparation

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 8–12 h leaving the cap slightly loose.
Seal the vial tightly and store in the dark, preferably at 4°C.

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 Glycerol preparation

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 (-80^oC resistent). Mix (invert) and store it at -80^oC.