Can I repeatedly autoclave a buffer solution?
July 6, 2012 1:56 PM   Subscribe

Can I repeatedly (2-3 times) autoclave a buffer solution without altering it?

Essentially, it is similar to a phosphate buffered saline (it has NaCl, KCl, CaCl, and NaHCO3 in it). I am curious if repeated autoclaving changes my buffer, in any way. I am using a large enough container in the autoclave so that none of the liquid is lost in the autoclave and am autoclaving on the liquid setting for ~25 minutes (and letting it exhaust for 15 minutes).
posted by pwb503 to Science & Nature (10 answers total)
I have never heard anyone say that you can't do this, nor can I think of any particular reason why a simple buffer solution such as the one you describe above should degrade upon repeated autoclaving. Autoclaves don't get that hot, only 121C normally – hot enough to sterilize things, but you're not burning anything except for the stripes on the autoclave tape.

Note that I have never tried actually doing this. Hopefully someone who has will come in and be able to set your mind at ease. I would totally do this though if I had some reason to, and I wouldn't give it a second thought. What is your reason for re-autoclaving the buffer, by the way? I assume that some contamination (or potential contamination) occurs between autoclavings? If there's anything that might conceivably cause a problem I expect it's most likely to be introduced there.
posted by Scientist at 2:10 PM on July 6, 2012

Response by poster: I guess I could avoid re-autoclaving if I simply transfer aliquots of my sterile buffer into 10-15 sterile conical tubes - BUT - because I run sequential assays for a series of about 2 weeks, sometimes it is nice to simply re-autoclave my working solution (the solution has already been autoclaved once for stock and a second time to make the working dilution) if I'm going to pull from it again.
posted by pwb503 at 2:21 PM on July 6, 2012

It most likely is fine, but it would probably be quicker and easier to sterile filter if you need to re-sterilize something.

Also, why do you have to sterilize the second time? Autoclaved water and sterile stock combined in a hood should still be sterile.
posted by juliapangolin at 2:27 PM on July 6, 2012

You might worry about evaporation though -- everytime you heat the buffer, you're going to lose some amount to steam because you can't tightly cap the bottle while it's in the autoclave or immediately after. Whether it's a trivial amount or not, I don't know.

Also, from wikipedia's sodium bicarbonate page:

Above 70 °C, sodium bicarbonate gradually decomposes into sodium carbonate, water and carbon dioxide. The conversion is fast at 200 °C:[9]
2 NaHCO3 → Na2CO3 + H2O + CO2
Most bicarbonates undergo this dehydration reaction. Further heating converts the carbonate into the oxide (at ca. 1000 °C):
Na2CO3 → Na2O + CO2

So, likely you're having some chemical changes to the sodium bicarb as well, but of course, that's already happened the first and second times the solution was autoclaved, so I'm also unsure what impact this would have compared to those first autoclavings.
posted by Tandem Affinity at 2:34 PM on July 6, 2012

I doubt it would be harmed and wouldn't hesitate to do it if I had to, but assuming you have access to a TC hood or similar, wouldn't it be easier to just remove some working solution in the hood from the bottle each time you want it while leaving your stock sterile?

Or, if you don't have access to a hood, flaming the entrance and pouring might be good enough "sterility", depending on your needs. Or filter-sterilizing. Re-autoclaving seems excessive, if fairly harmless.
posted by randomnity at 2:38 PM on July 6, 2012

I have seen similar solutions autoclaved multiple times with no bad effects. But I think that a terminal .22 micron filter would make your life easier.
posted by kamikazegopher at 2:51 PM on July 6, 2012 [1 favorite]

NaHCO3 in aqueous solution "begins to break up into carbon dioxide and sodium carbonate at about 20C and completely on boiling." --Merck Index
posted by jamjam at 3:17 PM on July 6, 2012

I would assume that anything that is stable enough to be autoclaved in the first place can be autoclaved again. If you're worried about evaporation, maybe do some checks to see how much you're losing each time.
posted by Lt. Bunny Wigglesworth at 5:24 PM on July 6, 2012

I'm not seeing how your bicarbonate ion could have survived an initial autoclaving (and isn't it the source of the buffering action?).

Are you sure it wasn't added after the autoclaving?
posted by jamjam at 7:24 PM on July 6, 2012

How sensitive are your assays? Because you're absolutely going to lose some to evaporation and degrade at least some of what's in there and add contaminants every time you go in and out of the bottle - even if just at a very low level. Even re-filtering it can take out some of the active ingredients and cause a change, but then microbial contamination will also change your buffer over time so you're right to work to avoid it.

How much any of this matters depends on how uniform you need that series of assays to be. Re-autoclaving (or whatever) is adding a new technical parameter to your data series. It might be within the noise of the assay or it might be significant, you'll have to carefully look at the data and think about what's going on within your actual experiment to decide.

Personally when I do a series of matching experiments that take several weeks I make sure to aliquot out whatever I need at the start into amounts small enough that they don't change before they're used up (but not too small to make me crazy) with enough aliquots for the entire time series plus some extra (but not too much extra because, again, crazy). Then I use a proper randomised block design to assign treatment/analysis order to my samples and make every attempt to look at and control for technical biases in the statistical analysis. Then it's all good. But changes in reagents will definitely show up in a lot of what I do (e.g. cell assays totally change when the cells get too old so I freeze back aliquots) whereas it might not be a problem for you at all.

Assuming you're using correct sample allocation with some kind of randomisation - because why wouldn't you? - it might be totally fine to autoclave the buffers every day. We can't really tell you, you need to look at your own data and decide.
posted by shelleycat at 2:54 AM on July 7, 2012 [1 favorite]

« Older In Search of Occupy Blueprint   |   Geeky intellectual hero(ine) stories Newer »
This thread is closed to new comments.