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I am trying to create a long-term stock for Bacillus subtilis (Bacterial glycerol stocks). I have looked online for guides and step-by-step tutorials and I found that they don't say where the sample originally comes from. In my case, I am using Bacillus subtilis subsp. subtilis (ATCC® 23857™) pure from the supplier bottle.

What is the proper procedure to create a long-term stock for Bacillus subtilis using this product?

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  • $\begingroup$ I worked with B. subtilis WB800 and just used 50% glycerol / 50% LB. I imagine the same would work fine with wild-type. $\endgroup$
    – canadianer
    Jul 14, 2021 at 1:48
  • $\begingroup$ Do you add the bacteria in the LB before mixing with the glycerol? $\endgroup$ Jul 14, 2021 at 1:58
  • $\begingroup$ No I would prepare the glycerol/LB mixture and autoclave it then create the stocks from log-phase (approximately) plate cultures. $\endgroup$
    – canadianer
    Jul 14, 2021 at 2:04
  • $\begingroup$ @canadianer Wouldn't you want to use liquid cultures as the source so you can count the number of cells? Or does B. subtilis not grow in suspension? I come from the world of eukaryotic (mostly mammalian) cell culture, so I honestly don't know... $\endgroup$
    – MattDMo
    Jul 14, 2021 at 3:37
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    $\begingroup$ @canadianer Can you turn your comments into an answer so that I can vote it up? $\endgroup$
    – jakebeal
    Jul 14, 2021 at 14:36

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I worked with Bacillus subtilis WB800 and just used 50% glycerol / 50% LB. I imagine the same would work fine with wild-type. I would prepare the glycerol/LB mixture and autoclave it then create the stocks from log-phase (approximately) plate cultures. This method was sufficient for my application (protein expression).

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