We are usually able to pour NGM plates (using a Wheaton pump) on the lab bench, but we have a contamination problem that has reared its ugly head again. I was wondering if anyone else had problems with a similar contamination.
The contamination is small white spots that appears throught the NGM in the plates. It is relatively slow growing as it does not usually appear on the plates for at least a week (and sometimes longer.) It does not grow very well at all at 4C (where we store our plates long term) - often we will not be able to detect the contamination until the plates have been at room temperature for a few days (after cold storage) at which time the contamination takes over the plates with multiple spots. (They almost look like small yeast colonies to me.) We autoclave our batches of media and the tubing for the pump. (Phosphate buffer is added after autoclaving the media.)
Any suggestions for combatting this problem would be most welcome.
Assuming these are non-innoculated NGM plates and that it is a yeast contamination, then it could be ending up on your NGM plates via one of several possible routes.
Have you tried making up a small batch of NGM and pouring plates by hand at the bench, this might indicate whether your pump/tubing is still contaminated after autoclaving?
In a similar vein, you could pour the plates in a sterile hood instead of at the bench. This would indicate whether or not your lab/bench area is contaminated.
You could change your Ca, Mg and phosphate buffer solutions which are added after autoclaving and see if the contamination goes away.
You could change the pipettes that you use to add these solutions.
Do you dry your plates thoroughly before storage at 4 degrees?
That said, you say that the contamination is all over the plate, suggesting that it is something added rather than airbourne.
Thank you for the great tips. Some of the suggestions are things that we have tried in the past and others, we will be sure to incorporate into our protocol for the future. Our colleague who works with pathogenic yeast strains took a sample from our contaminated plates and determined that we have a staph, not yeast, problem. I suspect that the problem is with one of the solutions that is added after autoclaving so we are remaking the solutions (in much smaller aliquots) and trying again.