Mycoplasma contamination?

[Apoptosise]

Hi everyone,
My PC12 cells frequently got contaminated recently. I usually use 320x invert microscope to look at the cells, and if they are contaminated by conventional bacteria (with cell wall) I can clearly see those "rods". But these days the medium remains clear for 5-6 days and then suddenly turns turbid overnight. I observe the flask under 320x magnification again, but cannot see the usual bacteria. Instead I vaguely see very tiny "-"shaped vibrating things. They are densely present in the culture, while the PC12 cells detach and I suppose they're dead. I suspect if it's mycoplasma contamination? Many websites say they can be hardly detected by conventional microscopy, so I doubt whether what I saw are really mycoplasma... Has anybody actually seen the mycoplasma before?
Thanks in advance for any comments!

[Thessi75]

Hi!

We have had mycoplasma infections in our cell cultures quite often. It keeps coming back, unfortunately... But we have never ever seen it. We detect it using PCR.

Thessi

[Apoptosise]

Hi Thessi, do you observe turbid medium if your cell cultures are contaminated with mycoplasma? And would it be so serious that the cells all die, or they just coexist? Thanks for your response!

[Thessi75]

Hi!

The cells tend to look normal and so does the medium. Cells that have tested positive for mycoplasma seem to coexist well with the infection (we have leukemic cell lines in our lab). We test and treat all our cell cultures for mycoplasma regularly.

From time to time our cells have looked rather unhappy, but we are not sure whether it has been due to the presence of mycoplasma or not. Recently I experienced that several cell cultures just more or less disintegrated (lots of dead cells and very turbid medium), without any apparent infection.

Hope you'll get rid of whatever is growing in your cultures. Soon!

Thessi

[jappy]

There are some kits and even easy DAPI staining for detecting mycoplasma in your cell cultures.
You can search on main commercial life science providers what product is the best suitable for your lab.

[jappy]

There are some kits and even easy DAPI staining for detecting mycoplasma in your cell cultures.
You can search on main commercial life science providers what product is the best suitable for your lab.

[garlic]

I've had a similar issue with the cultures once. I doubt it is micoplasma, it is usually barely noticable and often coexists with cultures. Often the only way you know something is wron, is when your experiment control down't work as supposed to. I would say scrap the cultures, clean/autoclave the incubator, get new media, new sera, etc, and get new cells from ATCC. Do you use antibodies in your media?
Good Luck!

[garlic]

I've had a similar issue with the cultures once. I doubt it is micoplasma, it is usually barely noticable and often coexists with cultures. Often the only way you know something is wron, is when your experiment control down't work as supposed to. I would say scrap the cultures, clean/autoclave the incubator, get new media, new sera, etc, and get new cells from ATCC. Do you use antibodies in your media?
Good Luck!

[Apoptosise]

Thanks everybody, now I guess it's normal bacteria since the medium gets really turbid and cells all die... I did use 0.5% PSN but maybe that's not enough? Anyway I've cleaned the incubator and made new medium, now waiting for new cells from the postgrads in my lab.

[researcher4life]

I've used the mdbioproducts PCR mycoplasma detection kit with success.

This company has also had good technical support when I needed help or had questions.

Just google their name to find them online.