protocol for Hoescht Staining - (Aug/13/2006 )
For detection of mycoplasma contamination, we are doing PCR technique. Now we want to do Hoescht staining for mycoplasma detection. so if any body know what is the procedure for Hoescht staining then please send us. thanks
have you tried google??
loads of methods for it on there
Hoescht Staining will only show mycoplasma positive cells when they are massively contaminated. It is recommended to get Direct culture done in combination with Hoescht Staining (Direct and Indirect). DO NOT USE PCR AS A METHOD FOR DETECTION. This is the message I give all the staff in the Institute I work in. Having coordinated Mycoplasma detection for over 20 years we have seen many false negatives using PCR detection. The main reason why people are gravitating towards PCR is that they get results within a couple of hours. The PCR method is INSENSITIVE i.e the cells again have to be massively contaminated and is still not FDA approved for use as a method of testing for vaccines and drugs etc.
It is good that you want to test, BUT DO THE RIGHT ONE. It is also useful to do the direct culture method as the cells have to be without antibiotics for at least 4 days. This also shows up any CRYPTIC contaminations that is there, hidden by the antibiotics many culturist's use.
If you are based in Great Britain, use the company MYCOPLASMA EXPERIENCE. They have been doing testing for over 30 years, mainly for big drug companies. The money they charge/cell line is nothing in comparison with the money you will waste on growing and experimenting on contaminated cells.
Although I heard of, I have never done a direct culture test myself. How do you do that? Does that take long time?
As stated earlier, we send our cells away to a commercial company that are EXPERTS in the field of mycoplasma testing and the DIRECT GROWTH method. We have had ocassions when scientist's have used PCR and got negatives,only to find months later that the cells ARE contaminated and have been identified as positiev by the growth test. The reason why people do not like the test is:
i) It takes 4 weeks to get a definitive answer. In our experience if the cells are positive it shows up in the first week of the test.
ii) It is technically more difficult than the easier PCR test which gives a result in a few hours. However in our experience PCR gives alot more false negatives.
Since initiating mycoplasma testing in my department of the university, the % of positives has dropped from 30% to 4%/year. Having come from the pharmaceutical industry into academia, I have shocked at how little is known about mycoplasma's and the different types of testing. I remind all the academics here that data will not be accepted for publication if cells have been shown to be positive. The usual response is that our cells are growing so they cannot be contaminated......how stupid is that.
CRUK have a testing centre for all groups funded by them and they perform the direct growth test.