Contamination? or Debris from dead cells - Urgent! (Mar/08/2005 )

hello ppl,

I would need some advice urgently! I have these small black particles in my cell cultures. At first i thought they were just cell debris.. but after reading the forum and searching through the web sites I became unsure of what i have thought the black spots might have been.

In fact I have noticed these black spots in 1 of my primary cell culutre (human periosteal )for almost 3 months... but have never been troubled by it. the reason being my cells are growing ok, the media is clear. Recently, when i got a new primary cell (Pig bone marrow) i observed the same thing. after trypsinizing my pig bone marrow culture i left them overnight in the flask to reattached. the next morning when i observed them, the media looks cloudy and turns slightly pink and there are lots of dead cells. Which is why i am not sure whether the cloudiness is caused by the dead cells or contamination. after i change the media and left it overnight... this time round the media looks clear.???

As you might have read from 2 other questions posted in this forum, both the posters described the tiny black particles as highly mobile. I was looking or shld i say STARING at my cells at 100X mag, i can't see the black spots moving... ocassionally they seems to be wriggling or is it all my imagination? One of the websites also suggested that bacteria contamination appears as tiny black spots.

Can anyone describe the characteristic of these Blacky irritating spots, which i might suspect that its bacteria? Are the really mobile? do they cause media to become cloudy over 24 hours? and any other information regarding these spots will be welcome.

If these are really contamination by bacteria what steps do i use to eradicate them? I heard that we can use high doses of antibiotics... does anyone have the protocol to this method? i uses pen/strep & ampho in my media. Whats is high doses? at which concentration should i start with?

Really appreciate if anyone out there could give me some suggestions and informations. Thanks a million! PLEASE HELP!!!

The best and easiest way (that's how we like our ways isn't it!?!?) to find out if those are bacteria is to take a drop of potentialy contaminated media and put it in a petri dish with fresh media, without transfering any cells. If the black spots accumulate, it is automatically contamination, since debris don't proliferate!!
As how to eradicate it, the only way I know is to thouroughly rince your cells with ethanol or javel, throw them in the garbage and thaw new ones... sorry!!

Simon

Hi simon,

Well thank you for the suggestion. i ll try it out.

hi
i don't want to be negative, but a media that becomes cloudy is one of the major signs of a contamination....
Fred

hi. the observed black particles in ur plates might be bacteria (80% confidency)...reasons are three: ur medium turned cloudy and different color, ur cells didnt get attached and died, and u observed that the balck particles were mobile.

those mobile blacky particles might be swimming fast across ur culture cells cause them to lose the viability via some biochemical reactions. ur cultured cells definite do not survive, or in extremely low growth. further more, there might be some morphology differences.

if u wanna save them, u might have to use buffered saline with higher antibiotic concentration. but, lastly i would say so, ur chance to save them will be very low (5%)...that only if u really wanna save them.

but, i would suggest that u redo ur experiment, clean ur culture room, make them all under renewed sterility.

hope those help!!