Ask The Brewmasters

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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Hi Kolton, 
I work at Steel Bender Brewyard, and used to be the lab manager at Santa Fe Brewing Co for 8 years.  I ran into this same issue trying to create a yeast management program at Steel Bender, but once it was implemented we got a ton of great data that is not only helping with fermentation, but also cost effectiveness.  Feel free to email me at monica@steelbenderbrewyard.com if you would like to discuss more. 
~Monica

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Monica Mondragon
Brewery Operations Manager
Steel Bender Brewyard
Albuquerque NM
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Thank you so much! I got your email. Very interested in learning from you.

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Kolton Huerta
Brewer
Ex Novo
Albuquerque NM
------------------------------

Original Message:
Sent: 11-29-2022 14:25
From: Monica Mondragon
Subject: Yeast cell counting in a slurry.

Hi Kolton,
I work at Steel Bender Brewyard, and used to be the lab manager at Santa Fe Brewing Co for 8 years.  I ran into this same issue trying to create a yeast management program at Steel Bender, but once it was implemented we got a ton of great data that is not only helping with fermentation, but also cost effectiveness.  Feel free to email me at monica@steelbenderbrewyard.com if you would like to discuss more.
~Monica

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Monica Mondragon
Brewery Operations Manager
Steel Bender Brewyard
Albuquerque NM
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Am I missing something here? 1 g of 0.1% MB diluted to 100 g by addition of 99 g water yields 100 g of 0.001% MB, not 0.01%.

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Roger Barth
Author (with M Farber) of Mastering Brewing Science ISBN 9781119456056
Retired
West Chester PA
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Ah! My dilution was off. How embarrassing. So it would be a 1:10 solution of .1% MB, not 1:100. Correct? It still seems weird to me that a waker solution of Methylene blue would stain more cells making my viability seem lower.

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Kolton Huerta
Brewer
Ex Novo
Albuquerque NM
------------------------------

Original Message:
Sent: 11-30-2022 11:32
From: Roger Barth
Subject: Yeast cell counting in a slurry.

Am I missing something here? 1 g of 0.1% MB diluted to 100 g by addition of 99 g water yields 100 g of 0.001% MB, not 0.01%.

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Roger Barth
Author (with M Farber) of Mastering Brewing Science ISBN 9781119456056
Retired
West Chester PA
------------------------------

Hi Kolton,

Don't listen to brewers telling you it is unimportant to count cells, check viability and vitality of pitching yeast.  They are wrong and probably just don't know how it is done, or don't care to make a consistant product.   

First off, I know you used distilled water to make up the methylene blue, but did you also use the distilled water for yeast slurry dilution?  If so, that is your problem.  Distilled water could cause osmotic pressure to the cells. This would explain the low viability yeast problem observed.  Try using 0.9% Saline Buffer as a diluent instead of the distilled water to dilute the yeast slurry and see what happens.

My guess is that your viability will be significantly higher.

As a side note: I have noticed when making methylene blue from crytals a difference in batches and how much dilution water must be added to make a usable solution for viability testing.  Maybe the same is true with methylene blue solution, due to evaporation of the stock solution.  I don't know.  But I always checked viability of the solution with a yeast slide culture made with wort agar for a while after making the working solution. This is a quick, sloppy test that works well and is more accurate than the methylene blue method.  But it takes 8 or 9 hours.  Email me for how yeast slide cultures are done if interested.

Anyway, hope this helps and let me know how it goes with the saline solution used as  diluent.

Cheers,
Ken

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Kenneth Jennings
Brewing Science Services, llc
Portsmouth NH
(603) 812-7332
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This is all very helpful. I am interested in learning about the yeast slide cultures. What is your email?

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Kolton Huerta
Brewer
Ex Novo
Albuquerque NM
------------------------------

Original Message:
Sent: 11-30-2022 12:47
From: Kenneth Jennings
Subject: Yeast cell counting in a slurry.

Hi Kolton,

Don't listen to brewers telling you it is unimportant to count cells, check viability and vitality of pitching yeast.  They are wrong and probably just don't know how it is done, or don't care to make a consistant product.

First off, I know you used distilled water to make up the methylene blue, but did you also use the distilled water for yeast slurry dilution?  If so, that is your problem.  Distilled water could cause osmotic pressure to the cells. This would explain the low viability yeast problem observed.  Try using 0.9% Saline Buffer as a diluent instead of the distilled water to dilute the yeast slurry and see what happens.

My guess is that your viability will be significantly higher.

As a side note: I have noticed when making methylene blue from crytals a difference in batches and how much dilution water must be added to make a usable solution for viability testing.  Maybe the same is true with methylene blue solution, due to evaporation of the stock solution.  I don't know.  But I always checked viability of the solution with a yeast slide culture made with wort agar for a while after making the working solution. This is a quick, sloppy test that works well and is more accurate than the methylene blue method.  But it takes 8 or 9 hours.  Email me for how yeast slide cultures are done if interested.

Anyway, hope this helps and let me know how it goes with the saline solution used as  diluent.

Cheers,
Ken

------------------------------
Kenneth Jennings
Brewing Science Services, llc
Portsmouth NH
(603) 812-7332
------------------------------

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.com
https://www.doublestrandconsulting.com/
ASBC Alternative Beverage Subcommittee Chair
ASBC Technical Committee
MBAA Webinar Committee Vice-Chair
------------------------------

Thank you so much for the info. I am also interested in your cell-counting workshop. Would you be able to send me some info on that? My email is brewer.mrkolton@gmail.com

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Kolton Huerta
Brewer
Ex Novo
Albuquerque NM
------------------------------

Original Message:
Sent: 11-30-2022 13:04
From: Kevin McCabe
Subject: Yeast cell counting in a slurry.

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

------------------------------
Kevin McCabe, PhD
Founder
Double Strand Consulting
kevin@doublestrandconsulting.com
https://www.doublestrandconsulting.com/
ASBC Alternative Beverage Subcommittee Chair
ASBC Technical Committee
MBAA Webinar Committee Vice-Chair
------------------------------

Hello Kolton,
  While methylene blue is the industry standard it does have its pitfalls when it comes to accuracy, especially when there is oxidation involved.  We've found that methylene violet is a more accurate and stable dye for measuring viability than methylene blue.  It is even somewhat comparable to more expensive molecular counting techniques that utilize genetic dyes. If you'd like we can send you a few milliliters of stabilized methylene violet for testing and can also provide you with an SOP for utilizing it.  Feel free to reach out to our technical support team anytime at, 719-482-4895 ext.3.  Cheers!

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Kory Davis
QC Propagation Specialist
Brewing Science Institute
Woodland Park CO
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I've heard that the methylene violet solution is more accurate. I will give you a call to try it out sometime. Thank you so much for the advice.

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Kolton Huerta
Brewer
Ex Novo
Albuquerque NM
------------------------------

Original Message:
Sent: 11-30-2022 12:54
From: Kory Davis
Subject: Yeast cell counting in a slurry.

Hello Kolton,
  While methylene blue is the industry standard it does have its pitfalls when it comes to accuracy, especially when there is oxidation involved.  We've found that methylene violet is a more accurate and stable dye for measuring viability than methylene blue.  It is even somewhat comparable to more expensive molecular counting techniques that utilize genetic dyes. If you'd like we can send you a few milliliters of stabilized methylene violet for testing and can also provide you with an SOP for utilizing it.  Feel free to reach out to our technical support team anytime at, 719-482-4895 ext.3.  Cheers!

------------------------------
Kory Davis
QC Propagation Specialist
Brewing Science Institute
Woodland Park CO
------------------------------

Hey Kolton, I ran into a very similar issue last year when we bought some new methylene blue dye. Suddenly our viabilities took a nose dive. The new dye that we had bought was a different manufacturer than we had been using before. I went back to the dye from the old manufacturer and our numbers went back to normal. I confirmed that our counts were accurate by sending samples to our local yeast supplier and having them count them with their Cellometer as well. I have never been able to explain why the different manufacturer's dye messed with our readings. I visited some other breweries around town and had them try that dye and they did not see a difference in their counts.

Otherwise, your SOP looks very similar to the one that we use, which I developed based on the ASBC method.

So, maybe you switched brands of methylene blue and your yeast did not appreciate it?

DJ Mares
Brewing Manager
Denver Beer Company

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DJ Mares
Brewing Manager
Denver Beer Company
Denver CO
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