Contamination Paranoia - Everything looks like bacteria to me now (Oct/20/2005 )

It was a quiet contamination problem. Apparently, the bacteria had been there all along, exisiting as little black dots in the cultures that nobody would have noticed unless looking for them. Only after a flask had been neglected for a week would we start to see alot of these dots dancing aorund. No media color change, no turbidity. Yeah, more dead cells in the media then there ought be, but, the cells were still growing to confluency. But they were bacteria all right. We scoped it at 100x to prove it to ourselves.

Ordered new cells from atcc. After first plating it and looking at it under the scope- its still there! little black dots... jiggling... localized close to the cells, and also randomly in the media....

Changed the media. Still there. black vibrating dots... there is no possible way these could be bacteria. Some great scientists looked at my cultures, assured me it wasn't bacteria (or my culture would be dead and cloudy) and said that it would be fine.

Well, the last contamination- didn't turn dead and cloudy. Are these really just exosomes? How will I know the difference between a "quiet contamination" and cell debris?

It is most likely cell debris moving with brownian motion. I have seen this before and it has freaked me out. You are likely to have lots of this when you first thaw your cell cultures since there is usually some death and destruction associated with that. If you are concerned, take an aliquot of cells and let it sit for a hwile like you did last time, then you will really know if it is contamination.

It cannot be bacteria, otherwise media colour will change. Which cell line you are with? I would check ATTCC website for morphology.

are there more and more spots when you let your culture for more long time?
and do you see them in a culture media incubated at 37C with no cell?
very strange...

Seb_

Hi Babybug,

What you have described is very similar I have seen almost a year ago.

Tell me something, did ever do a stain with Hoechst to see if these darting black dots have in any way, any DNA within them. Hoecsht by the way is a stain used for determining any mycoplasma contamination. If you did what did you see, If you haven't then please do so and let me know ewhat the outcome is. If they are bacterial (let us assume they are for the time being), then you will see te nuclie of the cells as well as their cell bodies

My second questions is, did you see any cells rounding off and becoming populated with these balck dots, almost like going through an incubation period with these black bodies within them.

I am really curious. Please let me know by e-mail if you like to bscience@hotmail.co.uk

I have not done a Hoeschst stain. We are a brand new lab- we've put in an order for a Mycoplasm testing kit- but still waiting for the paperwork for the purchase order to go through. The only means of quality control testing I currently have are tryp. broth and blood agar, which I snuck from the Pathology lab. How expensive/difficult is the Hoecsht stain to get? Where do you recommend I order it from? The labs around here have bad habbits, I doubt I'll find it in house.

To this day the black debris has not gone away. It's got jiggly brownian motion, theres lots of it, localized around my cells and in the media. Fellow lab members have it in their culture, but they choose to ignore it.

As far as my cells rounding off- well, I haven't really seen that. The majority of my cells are fine- though I do notice some cells that look apoptotic- blebbing and all that. And some cells have a whole lot more of these jiggly brown dots around them then others.... they seem attached almost.....