Really great advices!! thanks missele
Great tips for beginner.
i pm fred asking him to pin it.
QUOTE (spanishflower @ Nov 27 2006, 07:31 PM)
i pm fred asking him to pin it.
This thread is pinned.
I would like to use Cipro (Sigma), could you please tell me which concentration can I use?
QUOTE (kcerione @ Aug 30 2006, 01:51 PM)
This contamination has been an aweful problem for our lab since the beginning of the year and has virtually eliminated our ability to perform informative tissue culture experiments. We work with several immortal cell lines (i.e. HeLa, 3T3, Cos, SkBr3, P19, etc.) and have found this contamination present in every circumstance. As described by others, its very small, slow-growing, doesn't change medium pH, no turbididty, doesn't kill cells but makes them sick. These bugs seem to live on or in the cells. We have also noticed that serum starvation causes the cells to detach. We are a cell signaling lab and find that many signaling proteins are constitutively active in the presence of this contamination. We sent three samples to our diagnostic laboratory and two times, they said there wasn't any contamination and one time we got three species back, so we were never able to make a positive I.D. on what the organism is. We find it to be resistent to Pen/strep, neomycin, G418, etc. The only antibiotic we have had any success at all with is Cipro. Scarey. Given that we couldn't come up with a common reagent that was contaminated (given the many different medias and serum we were using),and that sterile filtering was of no use, we suspected that the contamination was environmental. However, I began testing the serum, FBS and newborn CS. Suprisingly, everything I tested from Invitrogen, Cellgro, Atlanta Biological and Gemini was coming up contaminated. This seemed highly suspicious given the different sources of the sera and that they are coming (apparently) from different processing plants. Recently, I tried some gamma-irradiated FBS from Invitrogen and the results were amazing. I brought up 3 new cell lines from the ATCC and there was absolutely no signs of contamination. Furthermore, our functional assays showed proper regulation of signaling molecules. We have since purchased large lots of irradiated FBS and CS and hope this will be the end to the problems. I really worry, however, that this is a widespread problem in the scientific community and that it is escaping the notice of many laboratories. Invitrogen (where we usually purchase serum from) claims that there have been no complaints about this contamination. I would encourage anyone with this contamination to test the serum (beware, it takes at least 2 weeks- the bugs don't grow well without the cells) and then complain to the company supplying the serum if you find a problem.
I have observed these particles in my cell cultures. I plated some media from the cultures onto agars to see if there is any bacterial growth, but it came out negative. they can't be mycoplasma because they are not detectable under the microscope. I don' know whether the movements seen are due to them being motile or its just vibration. they did not kill my cells or hinder thier growth.
I don't know, could they be debris?
I always look carefully at my cells and have noticed these small swimmers in all my cultures recently.
Like everyone else, they are small, slow growing and do not see to affect the cells growth or morphoplogy. However, I was optimizing transfections with Genejuice which worked brilliantly until these things appeared - and now trasnfections don't work at all.
The worst thing about this contamination is that cells can look clean for up to a week then the bacteria appear!!
We have these buggers too - looks like they've been around a long time from this paper which recommends how to get rid of them....
I am working with CHO cells, serum free and serum supplemented cultures.
I have always wondered what are the reasons for turbidity in the culture flasks?
Is it only due to contamination or can it be also due to very high cell density( that will coincide with media going primarily yellow dur to low pH) ??