Aortic smooth muscle cell contamination - (Jul/02/2012 )

I currently culture both aortic and mesenteric primary smooth muscle cells from rat. The Aortic smooth muscle is cultured in DMEM + 10% FCS, 10ug/ml Pen/Strep and 10ug/ml amphoteracin B. However the cells from the aorta have recently been getting infected periodically two days into culture. Interestingly, this does not happen to the mesenteric cells.

I have tried all the usual solutions to remove cell culture contamination, such as cleaning incubators and hoods with 10% microsol and performing the dissection in a sterile manner, with antibiotics in the dissection solutions, however, none of this is helping. The contaminant has been identified as a bacillus type bacteria by a bacteriologist in our Immunology dept. Does anyone have any suggestions to help with this problem?

Many thanks

The bug could easily be in your stocks of cells at a very low concentration if you are using thawed stocks.

Silly question, but have you thrown out all your currently open culture medium/PBS/trypsin/collagenase/antibiotics/etc? If not, that would be the first place to start.

Make sure you sterilize your dissection equipment by autoclaving and only open in the hood.