Mycoplasma contamination - detection and de-contamination (Oct/14/2005 )

Hello,
I am suspecting mycoplasma contamination in caco2 cell culture. the culture surface appears covered with tiny little spots and they doesn´t move and they are transparent. the cells grow slow but the media doesn´t become cloudy. Another evidence is that when I perform RNA extraction of caco cells I got nice ribosomal bands but I got aditional bands that could be bacterial ribosomal bands.

I know that are commercial kits for detection of mycoplasma by PCR, but I wonder If someone of you have got a method for detection (maybe the secuence of the primers and a technique for mycoplasma DNA isolation). And I´d like to ask which antibiotics are useful against mycoplasma. Thanks.

You can find the primer sequences by looking at kit info and making some inferences


you do not need to do a DNA extraction. Simply grow your cells without abx for 4-7 days and do a PCR on 1-2 ul of the supernatant

the good thing about the kit is that you get positive and negative controls, and you get enough to check a lot of cells so the investment is good.

Good luck, that stuff is hard to get rid of

It is NOT possible to SEE mycoplasma contamination in cells even under a microscope.

I REALLY Think that what you have on your gel is tRNA (I just responded to your other post)

Of course I may be wrong, and there is nothing wrong with making sure your cells are not contaminated... However, you really can't see mycoplasma, I have been working with caco cells and like you said in the other post there is alot of stuff visible in the media of these cells, and i always saw what I would also call transparent "spots" on the surface depending on the plane of focus, but this is normal for this cell line...

Is there some other reason, besides the RNA gel and the stuff you "see" under the scope to indicate the cells are contaminated??

Good Luck, and I hope everything works out...

hi
Detection is simple. Using a pcr on 100µl of supernatant of your cells
Decontamination, if needed) by plasmocin (here is a useful webpage with a link to a graet informing pdf)

topics/links that may help :
http://www.protocol-online.org/forums/inde...?showtopic=7832
http://www.mycoplasma-exp.com/eradproc.htm
http://www.protocol-online.org/forums/inde...?showtopic=6520

from this topic :

answer from qqUss :


For: 5' GGG AGC AAA CAG GAT TAG ATA CCC T 3'
Rev: 5' TGC ACC ATC TGT CAC TCT GTT AAC CTC 3'
Product: 280 bp

They were pulled out from Vojdani et al (1999): Multiplex PCR for the Detection of Mycoplasma fermentans, M. hominism, and M. penetrans in Patients with Chronic Fatigue Syndrom, Fibromyalgia, Rheumatoid Arthritis, and Gulf War Syndrome

However, as I don't work in the area of mycoplasma detection, I won't be able to tell you if these primers are THE best. Read the paper, and maybe you'll be better informed whether you should use the sequence. Good luck!

there are three effective reagents for mycoplasma

see this article

http://www.roche-applied-science.com/PROD_...1_96/p48-51.pdf

"Currently the most often used antibiotics for mycoplasma decontamination
are the two fluoroquinolones, Mycoplasma Removal Agent (MRA) and ciprofloxacin,
and BM-Cyclin (4,11,18–23). "
They have analyzed results of antibiotic treatment (BM-Cyclin, ciprofloxacin, and
MRA) of mycoplasma contamination in mycoplasma positive cell cultures.

some more mentioned here
http://www.dsmz.de/mutz/mutzmyco.htm

We are planing to use Cambrex Mycoalert kit for Mycoplasma detection, that is luminescence based biochemical test.

Anybody has tried this? ne feedback?

We've used mycoalert in our lab on a routine basis to test the health of our maintained cultures as well as those we obtain from colleagues. In all it seems to work just fine. We detected mycoplasma in 3/5 cultures from our colleagues so it does seem to work.