How to autoclave the supplies for a frozen yeast bank

So I have the supplies to start a frozen yeast bank (test tubes, glycerin, autoclave-able jar) and my buddy has an autoclave, but I couldn't find any posts explaining how to autoclave this stuff.

So how should I go about autoclaving the test tubes and glycerin solution?

Also, can I autoclave a regular glass jar? I will probably use this to collect the yeast after making the starter then pour from this into the test tubes (seems easier than pouring from the beaker into the test tubes, also then I can assess exaclty how much yeast I have and how much to put into each test tube).

Oh and the test tubes are specifically stated as autoclave safe and have screw on lids.

thanks in advance for any help.

Are you sure that the test tubes are not already sterile? IF they have screw on lids, there is a chance that they have been sterilized prior to shipping. They should be labeled as such.

We typically run a 30-min "liquid" cycle at 121°C in order to sterilize test tubes with 10 mL of buffer solution or tryptic soy broth medium. Make sure the caps are loose to prevent any pressure issues with the tubes.

If you are trying to sterilize your empty test tubes with the screw caps in place; loosen those as well I would also recommend placing a small amount of water (0.2 mL) into each tube in order to make sure that there is steam present in the tubes (just in case the caps don't allow for pure stems to reach the inner volume of the tubes). Most glass jars can be autoclaved, however, it is important to make sure that you keep any lids loose to allow for pressure to enter and leave the jars. This minimizes the chances of a broken/exploding jar. Reseal the lids after the cycle is complete. Foil can also make a good cover for larger mouthed vessels (beakers or flasks) without screw caps.

Cool, thanks bucketnative.

How about the glycerin solution? i assume i have to sterilize that as well.

That's the second line in my response... 30-min, 121°C cycle, liquid cycle. The liquid cycle uses less extreme pressure changes to prevent rapid evaporation or liquid bubbling over out of the tube. You could probably go even shorter in cycle time, since I imagine you are working with smaller volumes (we typically sterilize 1,000 tubes in a cycle).

Oh, I missed that, thanks a lot.

The volume of glycerin solution will be less than 500 mL, since the autoclave jar is 500 mL and I know I shouldn't fill it all the way up. How much do you think I should fill it?

Also, I will mix the solution 25% glycerin and 75% water, and I am concerned that evaporation during the autoclave might change that concentration. Any thoughts about that?

Thanks again for all your advice, really helpful.

drunkinThailand said:

Oh, I missed that, thanks a lot.

The volume of glycerin solution will be less than 500 mL, since the autoclave jar is 500 mL and I know I shouldn't fill it all the way up. How much do you think I should fill it?

Also, I will mix the solution 25% glycerin and 75% water, and I am concerned that evaporation during the autoclave might change that concentration. Any thoughts about that?

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Leave some headspace in the jar. In our autoclave, we have validated the 30-min liquid cycle for volumes up to 1-L in 12 bottles per cycle. Your mileage may vary.

As for evaporation, if you use a liquid cycle, the pressure changes are done at a slower rate to prevent boil overs and such. During an autoclave cycle, the steam is at saturation (theoretically), and you should lose minimal liquid due to evaporation. We find that with a test tube with 10-mL buffer (loosely capped), we lose less than 0.1 mL in a cycle, which is good enough for us.

awesome, thanks again for all your answers. really appreciated.