Tissue culture contamination - can't get rid of it!!! (Jul/04/2010 )

Hi!

Since a few weeks we have a serious contamination problem in our tissue culture. It started "normal" with 1-2 flasks affected by a bacterial contamination (spirilla). We proceeded as always, i.e.got rid of the cells, cleaned everything etc. But the contamination reappeared until the point that all growing cells were contaminated. I work with cell culture since 4 years, but never seen such a contamination before. The bacteria don't grow as fast as normal, i.e.the cells can look fine and clean for a few days and suddenly the bacteria start to grow over night and everythings is full of it. The contamination also seems to be dependent on cell density, because the bacteria only start to grow when the confluency of the cells is >60%.
They also seem to spread, because freshly thawed cells (old vials, so definitely clean) can get it too.
What we've done so far: cleaned incubator, waterbath, hood, aspirator, all surfaces, microscope; got rid of medium, trypsin, PBS and prepared everything fresh. We also got FCS from another group, so that's definitely clean, too.
After cleaning everything we started to grow two cell lines again (old vials, clean), they were fine for one week, but this morning I saw that one cell line is affected again. By the way, we have Penicillin/Streptamycin in our medium.

Any suggestions what to do, where is the contamination coming from? I don't know what to do any more, so please HELP!!!!!

Jeannine

Make sure your pipette-aid is clean. I've seen people stealing these from TC hoods for bacterial work and then putting them back. Get a fresh one or at least change the filter and wipe out the inside with 70% ethanol.

I haven't thought about the pipette boy yet, thanks for that!

I'm also afraid that it's in the hood. I wiped all the surfaces, but is there an option to fumigate it?

aside from filter within the pipette boy, how about the HEPA filter within the laminar flow cabinet?

Jeannine on Jul 4 2010, 06:42 PM said:

They can be fumigated with formadehyde or peroxide. There are protocols on the net for doing it but I recommend getting a proper company to do it, as it is reasonably tricky and the chemicals involved are not nice, so leaks etc are a big risk for the lab.

In this situation it is not enough to sterilize the most likely culprits. The whole "room" and all the contents needs to be sterilized and then shown to be "clean" via testing before resuming processing. And don't forget the filters in the HVAC system.

Have you determined based on the type of organisms, if the source is likely to be water, air, or human? This may guide your further investigation.

Even if your cell bank was sterility tested there is still a possibility of contamination. It might be adviseable to do random sterility testing of the vials even though you consume part of your lot.

If you haven't already, double check the COAs of all your raw materials by lot number including the culture vessel (flask, petrie disk, etc.) and other disposables to be sure they have passed the USP sterility test. This is still not a guarantee but it gives some assurance.

If you are using any reuseable equipment, has the sterilization method been validated?

Review your protocols for aseptic processing starting with thawing the cells, personal protective equipment, entering the processing area, sterilizing material and equipment placed in the processing hood with alcohol or other decontamination solution, minimize handling during processing, and decontamination at frequent intervals of your gloves (or replacement) and the work area and upon completion of processing.

Also review the air and surface sampling plans and routine cleaning/decontamination of the processing area and its contents.

In my experience, it is likely you will not identify the source of contamination but by doing all of the above you can at least eliminate the current outbreak and reduce the chance of recurrance.

You can also add Kanamycin to 50ug/ml. I know, that's cheating (and doesn't really solve your problem), but if you have precious cells... At least you'll get rid of your contamination (as long as you add Kanamycin).

Cheers,

Minna