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The common method of storing bacteria long-term is to collect saturated liquid culture, such as LB, and mix it with sterile glycerol so as to obtain 10-30% final glycerol concentration. The resulting mixture is then frozen, usually at -80 or -20C. Bacteria stored in this way are said to be viable for years. I am curious as to what affects the success of such a storage strategy?

  • Obviously the temperature should be below freezing, but how exactly is viability affected? Is colder always better (assuming the tube can withstand it)? Is there rapidly diminishing returns after -80C? Is there an optimal temperature? I'm expecting something like a plot of viability after 1 year of storage vs. temperature, ideally with multiple species.
  • What is the optimal glycerol concentration?
  • How much does viability drop per freeze-thaw cycle?
  • Does it matter how quickly you freeze after adding glycerol?
  • While letting a frozen sample thawed is considered unadvisable, after it does thaw, does it matter how long? Does every second count, or is there little difference so long as it's not several multiples of the lag phase duration?

However, I found it very difficult to locate specific information about the optimal conditions for such storage. For instance, Addgene claims that optimal glycerol concentration is unknown. Moreover, a lot of the older papers on this topic are paywalled (even from my institution). But the experiment to determine it would be so trivial that I can't believe nobody has done it. I am interested primarily in data from actual experiments, and I am interested in glycerol alone as a cryopreservant.

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