Black dots in cell culture - (Feb/05/2011 )

Hello everyone!

I feel what I am going to ask is not really new or difficult. I have seen this topic discuss several times but I never got to see a clear decision or advice on the issue.

Problem: I am growing ME180 cells in McCoys medium without antibiotics for the last 7 months ad been working on experiments. Everything was fine until since last month, I have been getting to see some black spots, that look like bacteria (and similar to other observations discussed in these forums from time-to-time)

I know many claim these to be debris from the cell and not bacteria or other live contaminant as expected or tested by others. So I will not go pulling on what it might be...or what i did next to get rid of it (bcos i really havent got rid of it and its still out there in the culture)

My simple question is: Can I use these cells to perform any experiment at all? I am planning to infect these cells with bacteria and then see at gene expression for various genes of interest.

Everyones advice will help me decide if I should order new cells or try to solve the problem (We are currently tight on funds and really cannot afford on getting a new cell line)

Regards
Roshan

If they are bacteria, you should be able to culture them in the medium you are currently using. Take a little of the medium from a flask with the spots and add it to a fresh flask, incubate as you would for the cells (i.e. same CO₂, temperature etc.) and see if they proliferate.

Hi Bob,

I have tried growing them on plates and media...nothing grows on them! I am sure they are not the bacteria! but wondering what they cud be.

bob1 on Sun Feb 6 22:51:46 2011 said:

They could be debris from the cells dying (as they will in any cell population) or debris from the FCS. There is a chance that they are still bacteria of some sort though, they could be bacteria that require the presence of the cells (or something that the cells secrete) to grow. Do they appear to proliferate at all with extended culture of the cells? How about if you remove the medium from some cells and put that medium in the incubator - do they still proliferate?

Thanks agains for the reply Bob.

Yes I have tried removing the supernatant and letting them in incubator to see if they grow...but they did not! I also see a lot of round cells with large nucleus...then someone told me they coiuld be undergoing apoptosis. Actually I have no idea what is the actual passage number fo this vial i removed from the -80. We are a total mcirobiology lab and very less culture work and hence did not invest in a liquid nitrogen tank!

I do not know if something is happening to cells bcos of high passaging! But when I spread the pellets after spining there was no growth bacterial media even after 2 days.

Just wonderign shud I simply do my expts or wait till they clear out...big question is when will it happen?

thanks again
roshan

bob1 on Tue Feb 8 01:58:11 2011 said:

Can you post a picture of the cells? If the vials have been stored in the -80 for more than 6 months (or less for some cell lines) then they could well be very unhappy and many of the cells will die or enter senescence (large flattened cells with visible nuclei, won't move and will never proliferate again). The dying cells will also rupture and release particles which you may be seeing as your black dots.

Hi Bob,

I can sure try to take some snaps of how they are looking! Might take me some time since we dont have a inverted microscope too! The cells were surely stuck in the -80 freezer for more than a year, hence I too am doubting if the cells are happy at all!

Some forums had dsicussed that when cells are extensively washed with PBS the dots might reduce a bit. I tried that and it looks like a good amount of dots are gone..but I am waiting to see if they can come back after few days of culture.

Hoping to get back to you with images at the earliest.

Thanks
Roshan

roshandinesh on Sat Feb 5 23:09:28 2011 said:

You indicate that you will be using these (contaminated) cells to examine changes in gene expression, but you do not indicate if you will be examining bacterial or ME180 gene expression. If you plan on examining changes in ME180 gene expression, my question would be how do you know that the changes you observe are not the result of the contaminant? Changes observed simply between the uninfected and infected cells would not be sufficient because there is no way to know if there was an additive or synergistic effect between your organism of interest and that of the unidentified contaminating organism. It very well could be that a gene you identify as being up- or down-regulated in the contaminated ME180 cells would not be similarly regulated in the absence of the contaminant. The contaminant could be subverting any effect triggered by your pathogen. It might be that a constituent of your organism reacts with a constituent of the contaminant to induce the changes observed. Having said that, we have received several batches of contaminated cells (of various types) from several different biotech sources (including the ATCC). This problem is obviously very wide-spread, and you might have trouble finding cells that are not contaminated. Of course, I cannot say that the contaminant you have in your cells is the same as that reported by others, although it seems likely given the limited options available to researchers in terms of commercially available sources for cells (the ATCC supplies cells to most biotech companies).