Yeast Cell Counts and Hemocytometer Dilution

[Bainbridge]

So I've been using distilled water to dilute our yeast 100:1 for cell counts and viability tests using a hemocytometer. It occurs to me that being distilled, the lack of ions in the water might be hard on the yeast, what with osmosis and all. If it kills some, then the methylene blue will tag them as dead and my viability will be off. Using tap water would solve that, but who knows what critters are in there, plus chlorine. Anyone out there bother using a buffered solution, or something else? I've got one biologist friend saying absolutely yes, and another saying naw, yeast are tough little buggers.

[ColdFusion]

Use buffered saline. You should be able to find it at any pharmacy and you don't have to worry about your yeast swelling up and popping if you leave them in there for a bit. Alternatively you could just add 9 grams of salt to a litre of water to make it isotonic.

[DBrewer]

Distilled water works well and matches our readings on a salometer for secondary verification...

[LongLiveLagers]

Russ,

If they're going to die in distilled water, they're going to die in wort, too.

Kevin

[a10t2]

Using tap water would solve that, but who knows what critters are in there, plus chlorine.

Even if there is something in your water, why does the hemocytometer sample have to be sanitary?

[kyle.carbaugh]

Even if there is something in your water, why does the hemocytometer sample have to be sanitary?

I think the OP is less concerned about the sample medium being sanitary, and more concerned with it tainting the results of cell count viability.

Unless the tap water has a considerably high/low mineral concentrations (in which case you're probably seriously treating or manipulating for brewing), it seems that tap water would suffice for cell counts. As far as critters are concerned, bacterial contamination of the sample wouldn't be visible at the same level of magnification as you would be counting, and I'm certain that you don't intend to introduce the sample back into your yeast pitch/colony.

Brewing Science Institute has a great 'Lab Handbook' which includes a protocol for performing cell counts (http://www.brewingscience.com). Dilutions are referenced in the protocol, but no mention is made as to the specific dilution media -- seems like if it made a difference they would have specified.

I agree with LongLiveLagers' sentiments -- if you're concerned about them dying in distilled water or the base water you're for brewing, I think you'd want to exclude them from the viability count anyways as they aren't really the cells you'd want to rely on doing the job anayway. Just my 2 cents.

[ColdFusion]

Yeast, in fact any mammalian cell, will die in distilled water. It is only a matter of time before the amount of water that diffuses into the cells by osmosis will rupture the cell membrane. This will happen to all cells regardless of how good they might happen to be at fermentation.

If you happen to be counting your cells immediately after dilution, you are probably fine using whatever you like. If you dilute your cells at 1:1000 and then forget them for 30 min, you might be shocked at your counts.

Tap water might be ok depending on your source but where we are, water from the tap isn't that different from distilled.

Finally, I would have hoped that it would be obvious that wort and water are different. They have vastly different osmolalities, in fact, some wort can be so concentrated (hypertonic) that it can also kill yeast.

[LongLiveLagers]

well, it looks like you got a test to do. Dilute your yeast to 1:1000 and check viability. Wait for a few hours and check it again and you can see how distilled water fries those mammilians

[CaptainEBC]

Methylene blue will also start to kill some of the good cells after a little while, so you want to count them as soon as you prepare the solutions.

[Bainbridge]

Hah, now I'm going to think of them as little mammalians...

Our protocol right now is dilute 10ml of homogenized (i.e. stirred and de-gassed) yeast slurry into 90 ml of distilled water. Then take a ml of that and dilute it with 9 more of water. (100:1) Then one drop of methylene blue and let it work while I get the microscope set up, then take a count.

Early on I put too much blue in and apart from being hard to read, it also fried the yeast. But a drop into 10ml seems to be working well now.

Main reason I asked the original question was we seem to be getting high cell counts but low viability (sometimes 50-30% after a week or so in the brink), so I was wondering if they were actually that sad, or if it was something I was doing to the sample, or if we need to adjust our harvesting practices.

[kyle.carbaugh]

Hah, now I'm going to think of them as little mammalians...

Our protocol right now is dilute 10ml of homogenized (i.e. stirred and de-gassed) yeast slurry into 90 ml of distilled water. Then take a ml of that and dilute it with 9 more of water. (100:1) Then one drop of methylene blue and let it work while I get the microscope set up, then take a count.

Early on I put too much blue in and apart from being hard to read, it also fried the yeast. But a drop into 10ml seems to be working well now.

Main reason I asked the original question was we seem to be getting high cell counts but low viability (sometimes 50-30% after a week or so in the brink), so I was wondering if they were actually that sad, or if it was something I was doing to the sample, or if we need to adjust our harvesting practices.

Just checking/curious: are you using the aqueous methylene blue solution?

[LongLiveLagers]

Russ,

Don't go to heavy on the mammalian comment; yeast are still firmly in the fungi kingdom last time I checked. However, maybe I have no idea what mammalian means-I never done gone to college!

Anywho, check your sampling technique. Maybe you're killing some of the sample? or maybe your pressure gauge on the brink is bad, keep it under 5psi. Make sure your brink is cold enough, etc. etc.

If your viability is bad and you need a boost and you have a few days: autoclave some wort or dme in some clean containers and feed the brink. It can get a little complicated, but I know it works.

[ColdFusion]

sorry... should have said eukaryotic not mammalian, but that probably doesn't help.

Back to the OP, as long as you aren't osmotically stressing the cells and/or subjecting them to shear stress (high speed centrifuge, vigorous mixing, hard pipetting), you should be getting accurate values.

I've personally always used trypan blue for staining. Both methylene blue and TB should give you the same results though.

[a10t2]

Main reason I asked the original question was we seem to be getting high cell counts but low viability (sometimes 50-30% after a week or so in the brink), so I was wondering if they were actually that sad, or if it was something I was doing to the sample, or if we need to adjust our harvesting practices.

I suspect this is due to the long contact time with the methylene blue giving a lot of false positives. My preferred procedure is to drop the methylene blue onto the edge of the cytometer/cover slip, let diffusion carry it through the sample (you can watch it happen!), then start counting.

I really doubt your viability is actually anywhere near 50%. I think the lowest I've ever seen for recently-harvested slurry is ~85%.

[Bainbridge]

Hah yeah, I suppose yeast are eukaryotic, not being plants and all. I'm just thinking of one of the Patrick O'brian 'Master and Commander' novels where Stephen is keeping a beehive in the captain's cabin and Captain Aubrey keeps referring to the bees as "reptiles". </geek>

We're using a standard aqueous methelyne blue.

We've got two brinks, converted kegs with pressure gauges, co2 barbs for pushing/venting, etc.. We try to keep it below 5psi. Sometimes though we'll have to put a third strain in a keg and I rig up a sanke tap with some shutoff valves and then vent it whenever I walk by it in the cold room, or we have a 5 gallon corny that gets used sometimes. Fun fact, if you forget to vent that one it becomes a yeast cannon.

I'm wondering whether our crashing regimen is hard on them in the cone. Double jacketed 10bbls. We crash to 45 using only the top jacket, then 35 with the bottom open full and the top cracked a little. This 'flips' the beer and mixes the temps well, otherwise we find we often get stuck at 38 or so. (The thermoprobe is in-between the jackets and if it doesn't mix it just keeps running the glycol without actually cooling the area where the probe is.) We'll usually let it do this overnight and typically will harvest the next day before fining. Maybe the cold down in cone is too much for em.