detecting Mycoplasma in cultures - (Apr/13/2005 )
Hello,
I look after informations about DNA fluorochrome staining for detect Mycoplasma in cell culture.
Anyone used it?
Thanx.
unfortunatelly, i can't help you with DNA fluorochrome staining, but i', also interested in finding a reliable and simple way to detect mycoplasma in cell cultures and incubator. i was thinking about PCR based ane, but i can't find distrbuter registred in croatia.
i am aware you are still in information collecting phase, but should this DNA fluorochrome staining be reliable and simple.
I've used fluorescent staining for mycoplasma, with Hoecht's stain. It's fairly simple to do, and when it's done properly it's reliable enough to be a validated technique. It takes a few days (growing, fixing, staining, and screening), but the only really time-consuming part is the screening.
You'll need a fluorescent microscope for the screening, and you'll also need a very dark lab to do the work in. Because Hoecht's works to stain DNA in general, the cell nucleus is going to be an enormous glowing body in the middle - and if mycoplasma is present, it will show up as little glowing dots in the cytoplasm... think of a starry night. You need your eyes to be completely dark adjusted to visualize mycoplasma contamination, particularly if it's at low levels.
There are mycoplasma media available; they are extremely slow, as it can take several weeks to detect the turbidity that indicates the presence of mycoplasma contamination. They're low-tech and easy to use, but most of us want answers faster than that.
I know that a number of manufacturers have kits available for mycoplasma screening with different techniques - but I haven't used them, so I'll leave it to someone else here to discuss how well they work.
Hello,
Thanks a lot for yours answers.
The next week i'm going to try an indirect method for mycoplasma which used chemiluminescent substrate (CAMBREX). This test is very quikly because you can have your result in an hour!!.But i'm not sure it is very reliable.Of course for this test you need chemiluminescence plate and a luminometer (I've BERTHOLD luminometer).I'm going to say more the next week.
So i need an other test to confirm my result because i don't want false negatives.In my opinion DNA fluorochrome staining it's maybe the best method if you don't want direct culture ( the most effective and sensitive method to detect Mycoplasma , but it'is also the most difficult and time consuming).
Can we find positve and negative Mycoplasma control slides with DNA fluorochrome kit?
Thanx.
[Can we find positve and negative Mycoplasma control slides with DNA fluorochrome kit?
Thanx.
[
When we were doing this screening, we made all of our own materials and followed a protocol, so we didn't have any kits. We made our own positive controls - the job that NOBODY wanted!!!
I'm sure that some vendor somewhere would be happy to supply you with control slides.
hi
for detecting mycoplasma in cell culture my colleague just did a pcr on 100µL supernatant.
for detecting mycoplasma in cell culture my colleague just did a pcr on 100µL supernatant.
Bonjour,
Do you remember what sort of kit your colleague used ( the manufacturer)?
Thanx a lot.
virginie from toulouse.
Just about everyone sells a PCR based kit these days. They are great. See the following links for information on kits (I caution you to be somewhat aware that the PCR based method can give false positives and negatives so run your controls diligently):
http://www.protocol-online.org/forums/lofi....php/t1786.html
http://www.stratagene.com/products/display...ct.aspx?pid=584
http://www.sigmaaldrich.com/Area_of_Intere..._Detection.html
http://www.cambio.co.uk/l3.php?l1_category...category_id=228
http://www.roche-applied-science.com/pack-...rt/1663925a.pdf
http://www.rndsystems.com/asp/g_sitebuilder.asp?bodyID=648
This is just a fraction of what's out there. Do a Google search.