Hi Kolton,
Here's a review article summarizing different methods for assessing vitality and viability.
https://academic.oup.com/femsyr/article/14/7/1068/531490 Methylene blue is the industry go-to unless you get into fluorescence with a cellometer. Methylene blue is taken up into both "living" and "dead" cells, but active metabolism in "living" cells reduces the dye to a colorless form. If there is too much of the dye, some "living" cells may be unable to reduce all of the methylene blue and may appear "dead." What this means is that the effective concentration of the dye is incredibly important for accurate results. Higher concentrations of dye will make stain more cells blue, making your "viability" appear low. Too low of an effective concentration of dye will artificially inflate your "viability." I'd double check your dilutions and the concentration of the dye as Roger noted, both the new stuff versus how the old stuff was prepared if you know for certain. Also, your note that the old stuff was expired may be a contributing issue if it was well past its expiration date. I put live, dead, and viability in quotes because it isn't a direct measurement because you're measuring the ability of the cells to metabolize the dye, not their ability to reproduce when pitched to fresh wort, but that doesn't mean it isn't a useful measurement. As Monica notes, the data you get is incredibly useful, helping with consistent fermentations and costs.
Therefore, whomever these many brewers are that are saying yeast cell counting is a waste of time, ignore them, or better yet, educate them. I volunteer my time to teach a cell counting workshop for District NW's Spring meeting every year to introduce brewers to yeast counting and methylene blue staining, and give attendees a cheap hemocytometer, methylene blue, some slides, and counters because I think it is the most important first step toward quality and consistency in a brewery, big or small. Knowing your way around a microscope is also helpful for so many other reasons.
Cheers,
Kevin
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Kevin McCabe, PhD
Founder
Double Strand Consulting
kevin@doublestrandconsulting.comhttps://www.doublestrandconsulting.com/ASBC Alternative Beverage Subcommittee Chair
ASBC Technical Committee
MBAA Webinar Committee Vice-Chair
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Original Message:
Sent: 11-29-2022 12:55
From: Kolton Huerta
Subject: Yeast cell counting in a slurry.
I have been trying to create a better yeast management system at work. I've attempted to determine viability and count yeast cells in a slurry. The issue I ran into with this is the methylene blue solution. I mixed 1g of .1% methylene blue solution with 99g of distilled water making my solution .01%. This seemed to work fairly well for a while then I noticed that the solution was expired. When we ordered more and used the same solution, the new methylene blue threw off everything for some reason. All viabilities now are seemingly very low.
I used the BSI lab book online as a reference for counting cells. I created my own SOP which is attached if anyone cares to take a look. I was wondering if anyone has a better method for using methylene blue. Perhaps an SOP or some literature could help me out. I've been told by many brewers that I shouldn't even bother trying to count cells. If that's the case, what is the best thing to determine yeast health and viability in your opinion?
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Kolton Huerta
Brewer
Ex Novo
Albuquerque NM
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