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Hoechst for mycoplasm testing


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#1 culturelady

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Posted 29 June 2009 - 06:24 AM

Hey everyone,

Yes I know everyone is sick of hearing about mycoplasm contamination, but after reading through the forums it seems that Hoechst staining to test for mycoplasm is the way to go. I was just wondering though if anyone had a general protocol for doing this?

And a few questions:

We mostly use primary cell culture and so incubate our cells in 5% pen/strep - can the hoechst staining by done on cells treated in this way?

Should I be testing every primary culture we prepare?

How long after prepping should I stain for mycoplasm? (we usually let cells grow for 2 weeks before performing expt)

Any help would be much appreciated!

#2 rhombus

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Posted 30 June 2009 - 02:10 AM

[quote name='culturelady' date='Jun 29 2009, 03:24 PM' post='27832']
Hey everyone,

Yes I know everyone is sick of hearing about mycoplasm contamination, but after reading through the forums it seems that Hoechst staining to test for mycoplasm is the way to go. I was just wondering though if anyone had a general protocol for doing this?

And a few questions:

We mostly use primary cell culture and so incubate our cells in 5% pen/strep - can the hoechst staining by done on cells treated in this way?

Should I be testing every primary culture we prepare?

How long after prepping should I stain for mycoplasm? (we usually let cells grow for 2 weeks before performing expt)

Any help would be much appreciated


A couple of points:

Hoechst staining should be done IN COMBINATION with agar growth assay i.e. One direct and one indirect assay.

The main reason for this is SENSITIVITY....hoechst staining will only show a massive mycoplasma contamination. The agar test is far more sensitive and is the GOLD standard for mycoplasma detection.

To do ANY test, the cells have to be antibiotic free for at least 4 days BEFORE testing commences.

It is ALWAYS best to do independent testing i.e. use a commercial company who test REGULARLY and are EXPERTS in testing. It maybe a little more expensive....but you save money in the long term. Approximate cost in UK is 75 -90/ cell line.


Hope this is useful

Kindest regards

Rhombus

#3 culturelady

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Posted 06 July 2009 - 05:43 AM

Thanks for your help! I dont think our lab would be able to do agar growth plates so independent testing might be the way to go!




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