Mycoplasma - (Mar/18/2011 )

Hi, I need some information about Mycoplasma.
Is Mycoplasma only located in the cells or are Mycoplasmas also "swimming" in the supernatant?
Mycoplasmas are small (0.1-1um), is it posible to see them with a normal microscoop (40x)?
By using DAPI or Hoechst it is posible to see them, what kind of magnification do I need?
I have a cleaning protocol but before starting cleaning I want to be sure that there is Mycoplasma, what kind of infection can you see by eye (except infetions like funghi, yeast, bacteria and other infections that easy are to identify)? The RPMI is clear after 4 days culturing, but there is something moving between the cells...
Thanks for help,
Richard

Mycoplasma are not normally visible under light microscopy unless the culture is very heavily contaminated. They are visible as small bright dots under fluorescence with DAPI or other DNA binding dye, and may be seen in both the medium and inside the cell, but more commonly inside the cell. You should use fluorescence microscopy to detect them and a soft agar assay to confirm the presence.

Dots in the medium are usually not mycoplasma, they are much more likely to be other sorts of bacteria or cell debris from dying cells.