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Issue on mycoplasma detection and treatment in cell culture - (Jun/04/2010 )

here's my situation, I'm workin on a project with no direct grant support, so I can't spend a lot of money to accomplish my goals...
(i.e. using "extra" reagents purchased in bulk meant for other purposes... using "extra" elisa kits that most likely were going to expire)

I need to test for mycoplasma to get good data... we've had some contamination recently, cryptic and overt...

My institution has flourescent microscopes and I have the proper training, so i think I can do the stain but i don't have a protocol, can anyone help me get some details on the exact methods to use?

I'm also planning on doing the culture, but what media should i get? ATCC sells its mycoplasma media as well right? i know its not a good idea to grow mycoplasma in lab, but we have extra incubators and some are in different rooms

-telamon-

Ciao,
well I also wanted to do an analysis and thought about DAPI, but only have ethidium bromide and some Propidium iodide, Im thinking about giving it a try though the problem may be the excitation source and possibly also filters as the spectral properties of DNA stains are quite diverse

DAPI 345nm excitation 458nm emission
Ethidium Bromide 510nm excitation 595nm emission
Propidium iodide 488nm excitation 570nm emission

so it depends on the technology/exciation source and stains available, if you can do a quick test using a fluorescence microscope to check for possible mycoplasma contamination

-Mycoplasma-


here's my situation, I'm workin on a project with no direct grant support, so I can't spend a lot of money to accomplish my goals...
(i.e. using "extra" reagents purchased in bulk meant for other purposes... using "extra" elisa kits that most likely were going to expire)

I need to test for mycoplasma to get good data... we've had some contamination recently, cryptic and overt...

My institution has flourescent microscopes and I have the proper training, so i think I can do the stain but i don't have a protocol, can anyone help me get some details on the exact methods to use?

I'm also planning on doing the culture, but what media should i get? ATCC sells its mycoplasma media as well right? i know its not a good idea to grow mycoplasma in lab, but we have extra incubators and some are in different rooms




Hi Telamon,

DAPI method is a good one to test for mycoplasma contamination.

Here is the protocol.

1. Use cells that are around 60-70% confluent.
2. Wash cells twice with buffer, and fix by using a fixative (methanol) for 30sec-1 min.
3. Decant fixative and add 1.5-2 ml of DAPI stain at 5 ug/ml in methanol. Keep at room temp for 15min.
4. Decant the stain and wash the cells 3 times with methanol, 5min/each wash. (You can also reuse the stain if you want).
5. Decant last wash, add a drop of mountant, add coverslip and view under fluorescent inverted microscope.

-EMSA1983-

thanks for your replies, once i get the stain and new media i will start growing more cells again and will be trying this. will let you know how it goes

-telamon-