Looking at yeast under a microscope

[Luthierzan]

Plate hemocytometers can be very inexpensive (here's one on eBay for $20) but aren't particularly accurate

That was the one I was thinking about purchasing. Is a $50 one better, if so why?

David

[BrianL]

What power magnification microscope do you need for yeast?

[thatguy314]

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.

These dies work by exclusion of dye, which is impermeable to the membranes. So if it takes up any dye, there are holes in the membrane, and it is probably dead or damaged. At least that's my experience using the (similar) trypan blue for studying (most) mamalian cells. Of course, I've noticed when I count one particular cell line the lightly blue cells tend to recover and counting them dead leads to overpopulated plates... so it's really a crapshoot.

The white labs guys have developed a protocol to acidify methylene blue which is supposed to improve accuracy.

Also my advise: IGNORE DOC. You don't need a 100x scope to look at yeast. And in fact, most 100x scopes of any quality are oil immersion and more than is worth dealing with. You only need to go 50-100x if you want to evaluate morphology carefully of look at subcellular structures clearly. 20x is my preferred counting range because you can see a whole quadrant of a hemacytometer. I use 10x for macrophages, but 20x for yeast because they're substantially smaller.

One thing to remember is that when you count you should look for things with abnormal morphologies in your count. If you have any significant number of them, either the yeast is extremely ill, or you may have a contamination. You should see only large round cells, or if it's a fresh starter where cells are still dividing, some smaller round cells. There should be no rods, no small cells growing in long chains, etc., If you see any significant number of them in your field while doing a cell count, you likely have a gross contamination and should discard it. In a pro brewery they acid wash or do something to keep their yeast clean, but it's really just not cost efficient at a homebrew scale.

[thatguy314]

What power magnification microscope do you need for yeast?

20x is a good magnification. The important thing is you want a quality scope. Quality is clarity of image, not power of magnification. Who cares if a scope can do 1000x if everything looks really grainy (like the cheap ones you had as a kid). Most laboratory scopes only go up to 100x and that's usually an oil immersion lens to provide clarity. A quality scope with 10-20x magnification will give you a good view with a wide field. i like 20x for yeast, because you can see distinct morphology but still view a whole quadrant to do a count.

That was the one I was thinking about purchasing. Is a $50 one better, if so why?

David

You want to try to find a name-brand hemacytometer. Hausser is a good brand. I got a Hausser Hy-Lyte, which silver-backed. This silvered (bright-line) hemacytometer causes increased contrast. Typically they're over $100, while the cheap ones are about 20. However, there are some people on ebay who don't know enough and they'll sell a silvered one for the same price.

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

Flow cytometers run in the 10s of thousands of dollars, even for one that's over 10 years old. http://www.labx.com/v2/adsearch/detail3.cfm?adnumb=416120. Also they require substantially more traning than a microscope to use and maintain correctly. They're also very expensive to maintain, and you need more expensive fluorescent dyes.

They also can't be used to evaluate morphology, so you need to do immunostaining to get specific identification of cells. So yes, if you have unlimited funds they are superior, but for over 100x the price. Unless you work at a facility with them (like I do) it's not a viable option. And if you do work at a facility with them (like I do) you'll still probably stick to yeast counts because of ease, time, and cost.

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

[/quote]

That's quite right. The differences of over 20% in cell counts comes from people who 1) probably aren't practiced and 2) have non-standard preparations of stain which can greatly affect it. I know within my lab we agree that reasonable people can disagree by under 10% for crystal violet (which is a much more difficult stain to count with) and for under 5% for trypan blue, which has a more obvious phenotype). I would say that getting an accurate count on a hemacytometer IS NOT EASY FOR BEGINNERS but as you do a lot of counts you can develop a careful eye that will make the count fairly easy. It's best if you learn with someone who has a practiced eye.

[ajdelange]

What power magnification microscope do you need for yeast?

Depends on what you are trying to look at. For just cell counting (hemacytometer) pretty low power (100x) should be plenty. If you want to see any details of the cell structure 400x is more like it (but note that a phase contast attachement will be required for good visualization of interior arrangement). Beyond 400x I'd say the upper limit is as high as you can go without needing immersion oil (i.e. below 1000x) because immersion oil is a PITA.

[thatguy314]

What power magnification microscope do you need for yeast?

Depends on what you are trying to look at. For just cell counting (hemacytometer) pretty low power (100x) should be plenty. If you want to see any details of the cell structure 400x is more like it (but note that a phase contast attachement will be required for good visualization of interior arrangement). Beyond 400x I'd say the upper limit is as high as you can go without needing immersion oil (i.e. below 1000x) because immersion oil is a PITA.

I find even going as high as 100x to make cell counting more difficult, b/c you can't view a whole quadrant in a field. I don't know anyone in my department (microbiology) that regularly uses that high a magnification if they're not looking at subcellular structure or distinct morphologies. That said, if you have an unpracticed eye, a lot of people feel more comfortable at high power, though it's really not necessary.

[ajdelange]

These dies work by exclusion of dye, which is impermeable to the membranes. So if it takes up any dye, there are holes in the membrane, and it is probably dead or damaged. At least that's my experience using the (similar) trypan blue for studying (most) mamalian cells.

I always thought the theory (WRT yeast anyway - no comment on mammalian cells though I own several) was that methylene blue and similar "dyes" are really redox indicators and that living healthy yeast cells, being reknowned oxygen scavengers, have interiors which are in a highly reduced state thus turning the dye colorless. A dead or dying cell does not maintain the reduced state and does not discolor the "dye".

You don't need a 100x scope to look at yeast. And in fact, most 100x scopes of any quality are oil immersion and more than is worth dealing with.

I think you have dropped a zero here or are thinking of the objective (not total magnification). I've never seen a 100x scope that requires oil immersion but I've never seen a 100x objective that doesn't.

You only need to go 50-100x if you want to evaluate morphology carefully of look at subcellular structures clearly.

and this convinces me further that you meant 1000X above

I find even going as high as 100x to make cell counting more difficult, b/c you can't view a whole quadrant in a field.

I'll admit I was reckless earlier and relied on memory so I rumaged around until I found my old hemacytometer in a drawer (it's one of the Hausser silvered - actually partially silvered - jobs mentioned here) and popped it onto my scope stage. The lowest power objective I have is 10x and the eyepieces are 10x for 100x magnification and I find a quadrant fits nicely into the field with a bit of room to spare. At 100 the yeast are plenty "big" in the field so that one can easily detect ones with buds and tell whether they cross a ruling line.

After more rumaging I did find another ancient (my father had used it with an oscilloscope camera in the 1950's) objective that appears to be 4X (though it isn't marked). Putting this on the 'scope gives me 40x and that's too small for these old eyes. I would have trouble spotting buds and the count squares are just too small to be comfortable for me. OTOH YMMV.

[thatguy314]

I think we are talking about different things. I'm talking about objective lens strength. I believe on all microscopes there is a null objective magnificaion. On the type of microscope peole typically use I think its about 10x. That would be consistent between us. I don't like explaining that to people because if I say a 100x power scope they think 100x objective which is not the same. In fact I gave a demonstration stonight and someone tried to use the 100x objective and ended up cracking my coverslip. but you're right, null objective is 10x so with a 10x lens you're looking at 100x. But I think unless you explain that people will look for a 100x lens. While you're being more accurate I worry about confusing people. but my point remains the same. if you get a scope with a 10x objective you will be fine

That said, I may be wrong about trypan blue. I just assumed it worked like propidium iodide, which works by physical membrane exclusion, since I get a 1:1 correllation between the 2 (though in my labwork I use PI almost exclusively).