Dear Dr Cone- In the last fortnight of yeast you mentioned the methylene blue technique. I use methylene blue, but am not sure how long it lasts or what its failure mode is - does it make live cells look dead or dead ones look alive when it goes off? It's not easy to find so I'd like to use it as long as possible.

- David Edge

RESPONSES:

My experience with Methylene blue is that it puts out with time and requires filtering. The protocol for producing the 0.01% w/v stain:

Dissolve 0.3 grams methylene blue in 30 ml. of 95% ethanol and 100 ml. citrate buffer (pH 4.6). The stain is toxic to the yeast, so the reading should be made with in 10 minutes. We made fresh supplies frequently because we used it up. We never had to test the shelf life. I would imagine that it would be many months.

Dr. Clayton Cone

It becomes more and more difficult to differentiate between dead and alive cells the older your methylene solution is. This means you will end up with more light blue cells and it depends on how the person who does the microscopic exam interprets the blue color. I don't know what kind of methylene blue you have (powder, stock solution or ready to use) but the ready to use solution should be ok for 3 month. Stock solutions can be kept for much longer; the powder can be kept almost indefinitely.

Tobias Fischborn