Looking at yeast under a microscope

[Luthierzan]

So after listening to an archived session episode, the first one when the White Bros were on, I decided to get a microscope.
This thing is lots of fun, but as I am a carpenter, and not a microbioligist I have no experience looking at things through it.

So when looking at yeast, how do you know which cells are dead and which ones are alive?

Anyone have any good websites or any other resorces for info on studying yeast through a microscope?

Thanks
David

[ajdelange]

It is part of brewing tradition to stain yeast cells with methylene blue. The enzymes in living cells turn the dye colorless this one can theoretically determine the percentage of live cells by counting the number of colorless and blue cells in a haemacytometer. The problem is that the test is not really reliable as other things than cell death can result in denatured/deactivated enzymes. Thus cells stained blue may not really be dead. Nonetheless if you have mostly unstained cells the yeast is healthy.

[ApresSkiBrewer]

My experience is that the brewing industry has become more and more fed up with methylene blue as a viability stain. It's unreliable, and there are so many cells that are *kind of* blue... or a slight tint of blue... alive or dead? It's too objective. I've heard some people have good luck with a related stain, used in the same way, methylene violet, and there's another I don't remember.

Just looking at the cells can be informative though. They should all be uniform, the cell walls should look nice and round... You'll know it when you see a cell that is most definitely dead - they look grainy, the cell walls are broken, (and with the methylene blue, these are the only cells that dye distinctly dark blue).

You can get a good microscope for a decent price, but a decent hemocytometer tends to be pretty pricy. (then again, im shitty at deal hunting)

I've been doing a ton of yeast cell counts at the new job - and you can add me to the list of fed up brewers (or cellarmen or whatever) with methylene blue.

[Luthierzan]

You can get a good microscope for a decent price, but a decent hemocytometer tends to be pretty pricy. (then again, im shitty at deal hunting)

What is the diference between a decent hemocytometer and a cheap one?

David

[a10t2]

What is the diference between a decent hemocytometer and a cheap one?

Plate hemocytometers can be very inexpensive (here's one on eBay for $20) but aren't particularly accurate - I've seen studies in which two researchers looking at the same sample came up with differences of 20%. There are also some systemic errors associated with using one, since you're diluting a sample and the errors in volume measurements, plus mixing, will come into play.

Flow cytometers, which some of the big breweries use, are much more accurate (I think rated ±1% typically) but run in the thousands of dollars.

Here's a good white paper on the differences in technique: http://fluidimaging.com/pictures/Yeast_ ... 200ppi.pdf

[brewinhard]

I've been doing a ton of yeast cell counts at the new job - and you can add me to the list of fed up brewers (or cellarmen or whatever) with methylene blue.

I think the other stain is Carmine Red

[ApresSkiBrewer]

Trypan blue - is another one I've heard kicked around.

[Charlie]

You got me interested, so I found this:

http://www.coulterflow.com/bciflow/documents/instruments/Vi-Cell/Comparison%20of%20the%20efficacy%20of%20various%20yeast%20viability%20stains%20(ta-204).doc

Hemocytometers aren't hard to use. Your bench tech skills are the most important factor in getting accurate results.

Charlie