While preparing glycerol stocks of my transformed bacterial cells (DH5alpha) the protocol I had to follow was that I would make one 50% glycerol stock (500µl culture, 500µl glycerol 50%) and another 87% glycerol stock (850µl bacterial culture, 150µl glycerol 87%). 87 seems to be a very specific number. Any particular reason why 87% glycerol concentration is used?

  • $\begingroup$ In which protocol/reference did you find the 87% glycerol? $\endgroup$ – Chris Jul 2 at 6:26
  • $\begingroup$ I think my lab protocol follows sambrook russel's molecular cloning. $\endgroup$ – Arnab Ray Jul 2 at 6:48
  • $\begingroup$ Yes I will try to provide a proper reference. $\endgroup$ – Arnab Ray Jul 2 at 11:00
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    $\begingroup$ Glycerol is typically sold at 87%, which just pushes back the question one step. Many years ago I was told that this is because of glycerol's hygroscopic nature, such that a higher concentration would tend to absorb water from the atmosphere and dilute itself anyway. Does this stop at 87%? Figure 1 from Glycerine: an overview suggests that 50% relative humidity might lead to a glycerol concentration that is in the ballpark of 87%, but 40 or 45% relative humidity looks closer. Not definitive, so I'll leave this as a comment. $\endgroup$ – iayork Jul 2 at 12:18

Typically, a glycerol stock will have a final concentration of 10-20% of glycerol. Glycerol is highly viscous. Pipetting higher concentrations of glycerol is difficult (especially for smaller volumes). Therefore a lower concentration such as a 50% solution (works for me) is convenient: add 300µl to 700µl of bacterial culture.

Regarding 87%: some vendors provide 87% glycerol along with anhydrous (>99%) glycerol. The reason is probably the same: it is easier to pipette 87%.

From a vendor's website:

Glycerol is a component of several buffers, media or reagents in biological research. It is supplied as glycerol anhydrous or 87 %, the latter containing 13 % water. Both glycerols are from the same quality, the 87 % glycerol has the advantage of a lower viscosity. Anhydrous glycerol can be pipetted with difficulty only, especially at low temperatures.

But the question is why 87% and not 90 or 80? See Michal et al. (2017) for a relationship between viscosity and concentration of glycerol-water mixtures.

enter image description here

Another graph plotted from the data given in the document mentioned by iayork in the comments. Different coloured lines denote data at different temperatures (in °C). Original data from: The properties of ordinary water substance. Compiled by N. E. Dorsey, New York (Reinhold), 1940.

enter image description here

So I am guessing that 87% must have been optimal beyond which the viscosity just shoots up rapidly with a small increase in concentration. I wish to reiterate that this is just my guess. I could not find a authoritative (in fact any) reasoning on why precisely 87% glycerol is (or was historically) supplied.

  • $\begingroup$ Thanks so much for such an well reasoned and knowledgeable reply. That ought to be the reason for using 87% glycerol stock. $\endgroup$ – Arnab Ray Jul 3 at 6:50
  • $\begingroup$ I think this is more plausible than the hygroscopicity I was told was the reason, since 87% doesn't show an obvious connection with anything on the hygroscopic chart whereas it's a better match with viscosity. The vendor's comment you quote also strongly supports this. Upvoted! $\endgroup$ – iayork Jul 3 at 14:15

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