One problem in cell counting is that the cell culture needs to be evenly suspended in its volume and any cell clumps need to be broken up. That is not an issue with poorly flocculating yeasts like German ale yeast. But heavy flocculators like English Ale yeast (WLP 002) provide a challenge. I knew that any yeast can be un-flocculated in the presence of maltose since maltose inhibits flocculation. This makes sense since it it more advantageous for the yeast cells to float freely when food is available. However, adding fresh wort to a yeast slurry simply for counting its cells seems a bit wasteful.
So I asked White Labs about this and their response was to use sulfuric acid or EDTA (Ethylenediaminetetraacetic acid). A search on EDTA revealed that it is a chelating agent. This makes sense in the context of preventing yeast flocculation since it is able to chelate the calcium necessary for flocculation. This gave me ideas for other chemicals that might work.
Tonight I spent some time in my lab/fermentation room to test a variety of chemicals for their ability to un-flocculate WLP002, the heaviest flocculator I have encountered so far. I had some WLP 002 sediment from the fast ferment test for a pale ale I brewed last weekend.
The experimentation set-up was simple. I added a little bit of WLP002 sediment, water and the de-flocculation agent I wanted to test to test tubes. Then I closed the test tubes and sloshed the contents around to see if the clumps are breaking up. When the sample was un-flocculated I inspected a sample under the microsope to check if there are truly no clumps of cells left. Here is what I found:
- water: this was the control. While the yeast clump did break up it only broke up into small floccs of yeast. This was not good enough for counting.
- fresh wort: it didn’t take long for the yeast clumps to break up into individual cells after brewing wort was added. This is the best option if the yeast sediment needs to be suspended anyway for pitching or as another stage of yeast propagation. This method does not kill the yeast and the viability can be assessed with methylene blue.
- glucose: clumps broke up slowly. This method does not kill the yeast either, but it is not very practical since there are better options.
- sulfuric acid: works very well. Clumps broke up very quickly and it doesn’t kill the yeast which allows for methylene blue staining. But sulfuric acid is a hazardous chemical and needs to be handled with care
- PBW: Yes, Five Star’s Powdered Brewery Wash. I got the idea to use this since it also contains chelating agents. It works very well and the cells were quickly suspended as individual cells. It does not dissolve the yeast immediately but kills them. When stained with methylene blue a significant number of cells stained blue while the culture had a known viability of 95+%. PBW is much safer to handle than sulfuric acid and many brewers have it at hand.
- GH test solution: since calcium chelation is the key for many of these agents and general hardness (GH) tests do just that I also gave this a try. It worked very well but is not practical due to its cost. The amount that can be found in a simple GH/KH test kit for $6 would only be enough to un-flocculate a few yeast sediment samples.
- disodium EDTA: I haven’t tried this yet but plan to test it when I order from a place that sells it. I expect that it works and that it does not kill the yeast either.
- Phosphoric acid: (mentioned by Northwestbeer in the comments for Yeast pitching by weight) Works very well, is generally available in a home brewer’s lab and does not kill the yeast. When I tried it I used about 2% phosporic acid (1 ml 10% phosphoric acid, 3 ml water and 1 ml yeast sample)
For now I’ll use fresh wort when I need to re-suspend the yeast in wort anyway and I’ll use PBW when I only need to count the cells in a given slurry of yeast without plans to use the yeast later.
And what was the cell count of WLP002 sediment, about 2.5 B/g like another ale yeasts?
I did not count cells during this experiment. But when I used this yeast for my last beer I weighed the slurry before it was re-suspended with fresh wort. The cell density turned out to be 3.5 B/g
Outstanding research Kai. Do you have any information that would serve as a primer for those wishing to try their hand at microscopy? This subject has been in my radar, maybe it is the science nerd in me. Having another test set to compare results with is never a bad thing either 😉
Looks like I just wrote that primer: http://braukaiser.com/wiki/index.php?title=Microscope_use_in_brewing
But this is no coincident. The experiments for un-flocculating yeast were done to support that article. I plan to use PBW to count the cells in a sample of a WLP002 slurry that I harvested last night.
That coincidence had very good timing. Yeast counting is something I have wanted to learn for a little while now. Just the thing I needed from someone with experience like yourself. Thanks for all the great info Kai. Keep it coming!
Great. Let me know if you have questions that left unanswered.
Any thoughts on other common brewing acids like lactic or phosphoric? In school they told us that acid washing yeast will change the flocculation characteristics of the yeast.
Thanks for the suggestion. I have not tried other acids since I was assuming that sulfuring acid does have some special chelation properties. But I’ll try hydrochloric acid, lactic acid and vinegar next time I have a sample of flocculated yeast. To what extent these agents change yeast flocculation behavior permanently I don’t know. It seems that once they are removed and calcium is supplied again they should flocculate again. Too bad that I don’t have a cetrifuge to effectively wash and rinse yeast. That would be an interesting experiment.
So far the use of PBW works very well for me.
What are your dose rates for PBW? I have some kolsch yeast that I can never count because it clumps together so badly, even in the starters it almost forms little balls.
A quick search found this from White labs. Near the bottom it talks briefly about acid washing and flocculation, not that it matters much for cell counting anyway, but you may find it interesting.
http://www.wyeastlab.com/hb_clarification.cfm
Great site by the way, one of the most useful brewing sites I have seen, for both pros and home brewers.
Can you tell us the final concentration of sulfuric acid that reversed the flocculation and the concentration of the other agents you used whether they worked or not.?
For sulfuric acid I an simply refer to the Chris and Jamil’s yeast book. There they mention that a 0.5 % H2S04 solution should be used for dilution instead of water to break up clumps. To use EDTA they recommend centrifuging the yeast and replacing the liquid with a 10% w/w EDTA solution.
I don’t think you have to be all that precise. When I did the experiments I was mostly interested in qualitative results. When I use PBW to break up yeast I add less than 0.1 g per liter. For a 10 ml sample that amounts to maybe a dozen grains.
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If you use solid EDTA, you will need to add base to water to get it to dissolve around pH 8.
That sounds like one extra step that I’d like to avoid. Although I should be able to make a stock solution.
If you want I can send you some EDTA solution. As Roy stated, you’ll need a slightly basic solution. The 10% w/w concentration would be ~270 mM based on the disodium salt (not free EDTA). We usually prepare 0.5M stock. Let me know!
Sure, I’ll take some. Thanks.
Where do you want it shipped to? I can send you a PM on HBT if you want….
Does dead yeast un-flocculate as well as healthy yeast?