Standard inoculation protocol

I use 20% glycerol stocks. We do not have dry ice so I use regular ice boxes to transfer the tubes from the -80 to the bench and back, which means they become quite a bit warmer/softer compared to using dry ice. With disposable inoculation loop, I transfer a small scoop of material to 2 mL of NYC glucose medium in 12 mL Greiner tubes (ref 164162). I close the tubes of with the caps but do not screw them tight to leave air flow to allow for the liquid to equilibrate with the anaerobic atmosphere.

These tubes are transferred to an Oxoid transparent jar. After closing the jar I apply three cycles of: pulling a vacuum and filling with N2+5%CO2 gas. I incubate at 37 degrees.

I regularly inoculate all strains on Thursday and use them on the next Monday. This is twice as long as the recommended growth times of the slowest growing bacteria in my practice (L. iners DSM strain). Optimally, I would use 48 hours however, this is impractical due to the experiments I am running and the time I have. Furthermore, I have not experienced any problems using this preculturing time.


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