6 replies to this topic

[distalless]

Hi,

I have been trying to start cultures from my frozen stocks of cells but they turn out to be contaminated when I revive them. I have checked and the media is not contaminated.

I am suspecting that the DMSO that the cells were frozen in could be the culprit because all the cells were frozen in the same DMSO. And the lab technician confessed today that she made the mistake of opening the DMSO outside the hood so that it is no longer sterile.

So my question is "Can DMSO be the source of the contamination or is it too toxic to be contaminated?" Thanks!

[powertoeki]

Of course, DMSO can get contaminated.  ANY TC (Tissue culture) work have to be done using aseptic technique and sterilized supplies.
DMSO being toxic and its sterility is a separate issue. For instance, many of cancer treatments are toxic yet that does not mean it won't get cells  contaminated.  Any non-sterile cancer chemical treatments could get cancer cells contaminated.

In order to have DMSO in sterile condition, I open the new  glass cell culture grade DMSO inside the hood and transfer its content into an amber glass vial.  In the future, write STERILE to all the cell culture supplies and avoid its usage if you suspect its sterility.

Happy TC!

[bob1]

Think about it this way - you can freeze eukaryotic cells in DMSO, and bacteria are a lot more resilient than eukaryotic cells... so they can easily survive the DMSO.

[ThibautEBV]

Also, your cells frozen stock can be initally contamined. Ask if anyone who used the same DMSO had the same problem.

Thibaut

[scolix]

keep your own supplies for tissue culture. others using them can contaminate them including DMSO.

[leelee]

Also, the likelihood of the DMSO being the source is pretty slim if the technician opened the bottle outside of the hood only briefly and was using proper aseptic technique otherwise.

Keep in mind tc used to be performed on the bench before the routine use of hoods, also bacteria work is done outside of a hood and bacteria broth won't usually become contaminated just because its opened once....

I think Thibaut has a good point, ask if anyone else is having the same problem with that batch of DMSO. I also agree with scolix, always better to have your own stocks of reagents in future.

[klinmed]

I do not sterilize DMSO and have never experienced contamination from this source. In fact, I believe 100% DMSO is probably self-sterilizing.
However, air oxidation of DMSO is relatively rapid and produces products that can significantly reduce viability on thawing. For this reason, I aliquot a new bottle of DMSO into sterile glass tubes and store these at -20oC. These aliquots are used only once.