Yeast contamination - (May/22/2008 )

Hello, is it possible to have mycoplasma contamination of yeast cell (S. cerevisiae) culture? Because, when I do immunofluorescence and I stained DNA with DAPI I saw the labelling not only in the nucleus but also in the cytoplasm (maybe mitochondria DNA staining). But how can I be sure of that it is not mycoplasma staining? My cells are grown in YPD medium + adenine.

Interesting! I don't think mycoplasma can infect yeast cells. For that matter, do viruses infect yeast?

As far as the cytoplasmic staining with DAPI is concerned, mitochondrial DNA does not bind to a perceptible level wioth DAPI so as to be detectable with fluorescent microscopy. So, what you are seeing is not mitochondria.

Mycoplasma have 10 times more DNA content that of mitochondria, and so they appear as tiny blocks. Viral DNA may also stain with DAPI or if you have any extra-chromosomal DNA (other than mito).

Maybe its some RNA which got stained by DAPI? I saw a similar staining in my yeast cells, but didn't think a lot about it (put the intensity/exposure time down until only nucleus was visible). Maybe its some completely unspecific staining.
If your staining is that high, that you can't detect any specific staining of the nucleus, check in DIC/Brightfield if the cells are intact. If not the nucleus starts fragmenting and the DNA gets cleaved in small pieces and spread in the cell. You can detect dead or nearly dead cells if they have very big vacuoles or a dark grainy structure.

Don't you guys ever look at the literature? Mycoplasma have not been reported to "infect" yeast cells. Fungi are subject to "killer" viruses.

The 1st question we should ask is how the controls looked - the most likely explanation is that the experiment was screwed up.