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There is a book that says to store Bacillus spores in 50% glycerol at -70 degrees Celsius (doesn't mention if the 50% is final concentration or not). But from what I know, the cells themselves can be stored in any % glycerol as long as the final glycerol concentration is 15% (v/v) at -80 degrees Celsius.

Is there a difference between preserving spores and cells? Because the book said to preserve the cells at a certain glycerol concentration and the spores at a different glycerol concentration.

Also, say I scrape some B. subtilis out from the cryovial for reuse, and they are frozen. Do I wait for the scraped cells to thaw and then plate them, or plate them while they are still frozen?

And if I want to sporulate these cells, should I recover these frozen cells by plating and incubating overnight, or can I just drop them directly into the sporulation medium? I want to get the details down properly.

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  • $\begingroup$ Welcome to Biology! You wish to store B stubtilis and be able to grow them again later, or to what purpose are you asking this question? Also, why do you think a standard glycerol stocking procedure for E. coli will not work for you? Background info helps folks to answer your question. $\endgroup$ – AliceD May 28 '15 at 1:36
  • $\begingroup$ I advise you to edit your original question and add these details $\endgroup$ – AliceD May 28 '15 at 2:00
  • $\begingroup$ Thanks for the edits. I just added a few spaces here and there $\endgroup$ – AliceD May 28 '15 at 2:35
  • $\begingroup$ This is just anecdotal and I don't know the best method, but I used to store B. subtilis cells in 50% glycerol at -80C. For protein production, I'd thaw the stock completely, plate them to obtain log phase cells and then inoculate the growth medium. Worked every time. $\endgroup$ – canadianer May 28 '15 at 3:41
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While I have never done bacterial stock cultures from spores, I don't think it is necessary, as the standard procedure for making bacterial stocks should work without problems.

For that you grow your bacteria in liquid culture until you reach the late log phase (so you have a lot of bacteria in your media), take 1 ml of the culture, mix it with 100% glycerol to reach a final glycerol concentration of 50% and then freeze it a -80°C. At this you can store it for long times (probably not indefinitely but for years is possible).

When you want bring your cultures back to life, take the tube and either use an inocculation loop or a pipette tip to scrape a bit of the frozen culture and plate it on an appropriate agar plate. If you want to scale up, pick one colony from the plate into liquid media.

Since glycerol is a pain to pipette (and can also have sterility problems), I have switched over to DMSO as a cryo-protective agent for bacterial cultures as well. Simply add 15% (end concentration) DMSO into the cryo vials (so 150ul DMSO to 850 of culture), mix and then freeze the cells. In my experience this works as good as glycerol.

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  • $\begingroup$ +1 - just out of philosophical curiosity, you say When you want bring your cultures back to life; do you regard your cells as dead at -80°C? $\endgroup$ – AliceD May 28 '15 at 6:04
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    $\begingroup$ At least they are not growing and doing metabolism. I would say dead, resting fits probably better. $\endgroup$ – Chris May 28 '15 at 6:09

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