viability/integrity stain of fungal cells - (Nov/12/2012 )
Do you know about stainings that could be used to evaluate the cell integrity or viability of fungal cells in bright field or fluorescence microscope?
In case that somebody may be interested, now or in the future, I found some stains that can be used as vital stains for fungi:
<*>Fluorescein diacetate (FDA)/Propidium iodide (PI): live cells show green fluorescence, dead ones are bright blue. Calcein-AM is a better alternative than FDA as it stays longer inside the live cells. BCECF is also better than FDA.
<*>Trypan blue, methylene blue, methylene violet: Trypan blue gives better results. Methylene blue usually overestimates viable counts when viability is <95%.
<*>Neutral red, another clasical dye can be used as vital fluorescent stain but I didn't find much about.
Now looking for a protocol for FDA/PI staining (luckily both are in the lab so...).
I was going to suggest trypan blue, but wasn't sure that it would get past the cell wall.
Note - fluorescene is usually green and PI is usually red, so I don't see how you get the blue stain above, unless you are using DAPI instead of PI.
Neutral red is a viable uptake dye, it is red, so should be visible with light microscopy, though individual cells may not be particularly brightly stained.
Yeah, I know. Most of this techniques are common for other type of cell but the cell wall... it took me a while to find cites about using this on fungi.
That's true, PI is bright red not blue, I mix up the notes while copying
I don't know if I can get trypan blue, but we have PI and FDA in the lab so I'm trying to find a protocol for dual staining but I couldn't find any yet.
PD: PI/FDA and neutral red can be used with plant cells too, even given the cell wall and such...
Tested the FDA/PI and works pretty well... the red maybe a little bit weak but... it's sooo pretty!!!
Yeah, that should look good. I like fluorescene, but it does fade quickly.