5 replies to this topic

[El Crazy Xabi]

Hi guys,

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?

Thanks!!

[El Crazy Xabi]

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...).

Edited by El Crazy Xabi, 14 November 2012 - 09:52 PM.

[bob1]

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.

[El Crazy Xabi]

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...

[El Crazy Xabi]

Tested the FDA/PI and works pretty well... the red maybe a little bit weak but... it's sooo pretty!!!

[bob1]

Yeah, that should look good.  I like fluorescene, but it does fade quickly.