3 replies to this topic

[hari256]

Hello all,

I have transfected HuEmbryonic cell lines going. They appear to do fine in DMEM + 10% FBS media but as soon as I change the media in 70 - 95% confluent cell lines to SFM4 (commercially available), The cells die due to bacterial contamination.

I see this trend in almost all my transfected cell lines. I do not use any antibiotics. But the DMEM plates are healthy and show no trace of contamination even when passaged. And this is not an isolated incident, it happened 4 times already viz. as soon as media is changed bacteria crops up.

I have done bacterial assays against the SFM4 media control, cells in DMEM + 10% FBS and the contaminated cells in SFM4. Only the contaminated show high readings everyhting else is negative for bacterial contamination.

Any help or suggestions would be greatly appreciated.

Thanking you all in advance.

Hari

[bob1]

It sounds like an intracellular bacterium of some sort.  What sort of assays have you done?

[hari256]

Hello Bob1,

I have solely used this assay to detect bacterial contamination. -- -- http://www.gen-probe.../104574RevC.pdf --- --

It does pick up the contaminated cell lines (in SFM4) as positive.

Thank you.

Hari

[bob1]

Ah, that's a mycoplasma test. Mycoplasma don't usually kill your cells and the test that you linked has quite a low sensitivity.  It is quite likely (actually certain) that the cells are contaminated before they show up as positive.  Your best bet is to throw out your cells and probably any recent stocks and get some more in from a reliable source (e.g. ATCC).

You should also go through the incubators and give them a good clean, change your lab coat, throw out any opened stocks of medium, PBS, trypsin, and other reagents you are currently using.  Appraisal of your sterile technique would be a good thing to do as well, especially if you are new to cell culture.