filtering and autoclaving solutions - anybody have any suggestions as to when and how to do it? (Nov/25/2008 )

Hello,


Recently, I've been making various solutions for future experiments, and I've been noticing that various matter--dust and other tiny particulate matter in the air, and probably all sorts of flora from myself (despite wearing a full labcoat)--is getting into the beaker in which my solution is in. This is a common enough issue and nothing can really be done about it, but somebody suggested to me to filter the solution after I'm done, using the same type of filter I use for filtering media (Nalgene 75mm 500ml filter unit). This sounds simple enough, but I'm afraid that some of the chemicals in the solution (such as tris, glycine, EDTA, or any other typical chemical that must be dissolved) would stick to the filter, and I could no longer count on the solution having the same concentration of chemical as before the filtering. Does anybody here know if this is so? is there a better method of doing this?

On a similar note, a protocol I have calls for making 0.5M EDTA and then autoclaving it--as there is nothing else to go on in the protocol, does anybody have advice regarding autoclaving solutions (parameters and conditions, how tightly I should close the bottle cap, etc...), and whether this is advisable to do for all of my buffers/solutions which will be in direct contact with living matter?.

My previous lab was situated on a third floor outside several trees, so all of our buffers constantly suffered from contamination (some sort of mold or fungus growing in the buffers after a stretch of 6 weeks or more); while my new lab has HEPA filters installed, I still want to avoid contamination and keep 10x and 50x buffers sterile rather than constantly make them anew.

Your help is much appreciated,

--Buffonidae

Hi,

I would suggest not to keep solutions in open containers of any sort... anyhow, the filtering idea sounds pretty good to me. The chemicals you mentioned can be safely filtered (0.75 is not even the smallest... you may still get contaminations with this filter), and the solution remains fine. I don't know about the autoclave for EDTA.... sorry.

Good Luck.

Virtually all of the filters sold for laboratory use are very low binding. They are already designed to allow solutes to pass through, it's only material that is larger than the pore size that gets trapped. Ions and molecules in solution are very, very small this is no problem. You can normally look up the chemical compatibility of all the various filter membranes from the manufacturer.

The only solutions I am cautious about filtering are those with biological molecules: this is because proteins are very large sticky molecules that have a tendency to stick to each other and to membranes, and because solutions such as serum sometimes form harmless precipitates after thawing that might cause your material to look cloudy, but they don't affect the performance of the solution.

Ask your autoclave technician about how to use the autoclave properly: every machine is different, and it is important that you know the machine's correct settings and operating instructions for different materials. For liquids, it is generally 30 minutes at 121°C and a high PSI, but ask someone in your department what they normally use. Lids should always be very loose during autoclaving, but they should be closed as soon as you take them out of the autoclave.

One thing I do to avoid contamination is prepare my stock solutions in lots of smaller bottles instead of one big one. For example, I might filter 250ml of solution and then aliquot it into sterile 15 ml falcon tubes. It's easier to work with a small tube than a big one, and if I have any reason to suspect contamination, I simply through out the rest of the tube and get a new one. Also for solutions with a larger volume, I might prepare 1 L but autoclave it in 2x250ml bottles.

Ginger

Do not autoclave: Detergents, Flammable solvents, heat labile chemicals.

Things like tris, EDTA, NaCl, Sucrose, bacterial media (most of them) can all be autoclaved.

Despite what Ginger said, you should not close your bottles tightly as soon as they come out of the autoclave, you should wait until they are at room temperature so as to not create a vacuum inside the bottle that will suck in air and contaminants when you open the bottle, unless of course, you are planning on using your bottles in a sterile air environment.