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  • Yeast cell counting, viability, staining..

    Hi!

    Took up yeast counting again been a long time since I did it.
    I'm helping out a small brewery and have sourced the needed material.

    I got methylene blue 2% solution which I have diluted with distilled
    water to 0.5%.

    I took 1ml of diluted yeast solution and 1ml water and finally 1ml 0.5% methylene blue solution with a total dilution
    factor of 300. Added a drop to the hemocytometer and into the microscope with 400x magnification.

    Now to my problem, all cells are blue and blue-ish..

    Which concentration of methylene blue are you using?
    How much do you usually dilute your samples?

    I will redo counting and take a bigger sample from the yeast-brink
    and see if that helps..

    Cheers, R

  • #2
    What is the yeast strain that you are counting? We have had various English-type yeasts not respond well to viability counts - everything is stained blue. Also, are there lots of budding cells? These will typically retain dye in a different fashion than single cells.

    Comment


    • #3
      Yep, English Ale, London Ale III, second generation.
      Any tips on how to do counts with this kind of yeast?

      Comment


      • #4
        Haha - that is the one English yeast that we have had great luck counting!

        When we look at London Ale III we usually see a good amount of budding cells, all of which are usually some shade of blue. If a cell is budding we count it as viable regardless of its color, as long as it is at least half the size of the mother cell. Where it gets difficult with english yeasts is when you see a massive cluster of flocc'd cells. When we run into this we will usually do 3 or more separate counts, from the same dilution, and average the results hoping that this offsets any extremes in the numbers.

        Regarding harvesting of this yeast, we have found it easier to work with (count) if we only drop the fermenter to 60°F for a single day, dump the trub, and harvest at that temp. This gets us a thinner slurry but is much easier to work with under the microscope and still ends up being 1.2+ billion cells per ml. If we crash the beer to the low 30's the slurry ends up being super thick and that much more of a pain to count.

        Hope this helps!

        Cheers,
        Tom

        Comment


        • #5
          I've been thinking some more on your methylene blue dilution as well - my math brain isn't working today but I'm curious what color your final dyed yeast solution is? We do larger dilution samples and do them by mass rather than volume, using a digital lab scale:

          90g water:10g slurry
          90g water:10g previous dilution
          15g water + 3 drops 1% MB dye:15g previous dilution

          This results in a 200:1 dilution that is deep blue in color but still nice and transparent - not dark royal blue or purple-ish.

          We dilute by mass rather than volume to mitigate errors made with foamy/thick slurries when trying to measure precise volumes in a pipette. And we chose larger sample sizes to further dilute measurement errors. (i.e. it's more forgiving to be off by .1g in a 100g sample than .1ml in a 1ml sample)

          Just some further thoughts on counting!

          -Tom

          Comment


          • #6
            BemidjiBrewing, thanks for taking the time to help out! There is a lot of good info there for me to test!

            Regarding the color the first sample was dark blue and not very transparent. The other tests I did was
            trying to match the color of a image I found here on the forum by adding drop by drop just to test and
            see what kind of

            Doing it by weight seems a lot easier than by volume, I use pipettes to measure everything.

            My dilution strategy:
            • 10ml slurry to 90ml water (x10)
            • 1ml of above to 9ml water (x10)
            • 1m of above +1ml metylene blue solution (0.5%) +1ml water (x3)


            I took a photo with my phone from the first test, when I used 1ml of 0.5% Methylene blue solution for staining.

            Click image for larger version

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ID:	191450

            Cheers!

            Comment


            • #7
              Honestly, that photo looks pretty standard for what we see with London Ale III. We would count all of those cells pictured as alive and healthy. They all appear to be budding and have metabolized some or all of the dye (assuming your dyed solution is deep blue in color). I've always operated under the notion that budding cells are occupied with the metabolic task of reproducing and as such don't metabolize the dye as efficiently as healthy, not-budding cells. But they are still viable and capable of healthy fermentations and should be counted towards that in determining pitching rates.

              If there were single, lone cells not budding that were stained deep blue we would count them as dead.

              Here's our brewery's cell counting procedure. I made these documents based upon the BSG presentation by Peter Hoey (he gets all the credit for this info!). I have these all printed out and posted above our lab sink/microscope to ensure that our brewers and cellarfolk are using a standardized counting procedure. They were made in Google Presentation, so the graphics aren't stellar...

              Awesome BSG article:


              Helpful set of slides to post near microscope:
              Viability Counting-4.pdf

              Helpful cell counting worksheet for staff just getting used to counting large numbers of cells:
              Cell Count Worksheet - Sheet1-3.pdf

              Cheers,
              Tom

              Comment


              • #8
                That is very helpful! Thank you! I will do a new count during the weekend.

                Cheers!

                Comment


                • #9
                  M.Blue

                  I am running a lab at a local brewery and I recently moved away from Methylene Blue and started using Trypan Blue instead. Trypan Blue takes into account cell wall integrity as it pertains to a healthy yeast cell, so the first thing you'll notice is that your viability percentage will jump substantially. I use a 0.8% solution to stain with and an overall 100 dilution factor when counting. If you use any kind of acidic de-flocculant, you'll see your M.Blue stains go super blue, as the acid lets more stain in. You'll not see the same change with Trypan blue as long as the cell's walls are still intact (yeast cell walls are pretty resilient).

                  Here's my basic procedure:
                  Tube A-8ml distilled water + 1ml 0.8% Trypan Blue (9ml total)
                  Tube B-9ml distilled water

                  Transfer 1ml yeast slurry (dense slurry- not fluid on top) to tube A for a total of 10ml. Mix this tube for about a minute. Then transfer 1 ml of that solution into Tube B (10ml total). This gives you a 100:1 dilution factor. Use this to place on your hemocytometer. You can do a side by side of methylene blue vs Trypan Blue and you should see a profound difference in the viability percentage (percent of stained cells to unstained cells), especially at end-fermentation (increased yeast stress from ETOH, Alpha Acids, and change in Osmotic pressure).

                  Hope this works out for you!

                  Comment


                  • #10
                    Originally posted by TheKevin View Post
                    I am running a lab at a local brewery and I recently moved away from Methylene Blue and started using Trypan Blue instead. Trypan Blue takes into account cell wall integrity as it pertains to a healthy yeast cell, so the first thing you'll notice is that your viability percentage will jump substantially. I use a 0.8% solution to stain with and an overall 100 dilution factor when counting. If you use any kind of acidic de-flocculant, you'll see your M.Blue stains go super blue, as the acid lets more stain in. You'll not see the same change with Trypan blue as long as the cell's walls are still intact (yeast cell walls are pretty resilient).

                    Here's my basic procedure:
                    Tube A-8ml distilled water + 1ml 0.8% Trypan Blue (9ml total)
                    Tube B-9ml distilled water

                    Transfer 1ml yeast slurry (dense slurry- not fluid on top) to tube A for a total of 10ml. Mix this tube for about a minute. Then transfer 1 ml of that solution into Tube B (10ml total). This gives you a 100:1 dilution factor. Use this to place on your hemocytometer. You can do a side by side of methylene blue vs Trypan Blue and you should see a profound difference in the viability percentage (percent of stained cells to unstained cells), especially at end-fermentation (increased yeast stress from ETOH, Alpha Acids, and change in Osmotic pressure).

                    Hope this works out for you!
                    Where do you get the .8% Trypan Blue, everything I have seen is 0.4%
                    Thanks!

                    Comment


                    • #11
                      Is this a picture from a cell count that you did? I think your dilution is too high. To get a more accurate measure, you should be counting around 20-50 cells per square to get an accurate count.


                      Originally posted by ridgeback View Post
                      BemidjiBrewing, thanks for taking the time to help out! There is a lot of good info there for me to test!

                      Regarding the color the first sample was dark blue and not very transparent. The other tests I did was
                      trying to match the color of a image I found here on the forum by adding drop by drop just to test and
                      see what kind of

                      Doing it by weight seems a lot easier than by volume, I use pipettes to measure everything.

                      My dilution strategy:
                      • 10ml slurry to 90ml water (x10)
                      • 1ml of above to 9ml water (x10)
                      • 1m of above +1ml metylene blue solution (0.5%) +1ml water (x3)


                      I took a photo with my phone from the first test, when I used 1ml of 0.5% Methylene blue solution for staining.

                      [ATTACH]50727[/ATTACH]

                      Cheers!

                      Comment


                      • #12
                        Originally posted by ziggy beersci View Post
                        Is this a picture from a cell count that you did? I think your dilution is too high. To get a more accurate measure, you should be counting around 20-50 cells per square to get an accurate count.
                        Yes, I have changed to 100:1 dilution and see between 20-50 cells per square, but the staining with MB
                        is hard with WY1318, much easier with other strains. I will try to get some Trypan Blue and test with that instead.
                        Any tips on where to get it in Europe?

                        Comment


                        • #13
                          Originally posted by TheKevin View Post
                          I am running a lab at a local brewery and I recently moved away from Methylene Blue and started using Trypan Blue instead. Trypan Blue takes into account cell wall integrity as it pertains to a healthy yeast cell, so the first thing you'll notice is that your viability percentage will jump substantially. I use a 0.8% solution to stain with and an overall 100 dilution factor when counting. If you use any kind of acidic de-flocculant, you'll see your M.Blue stains go super blue, as the acid lets more stain in. You'll not see the same change with Trypan blue as long as the cell's walls are still intact (yeast cell walls are pretty resilient).

                          Here's my basic procedure:
                          Tube A-8ml distilled water + 1ml 0.8% Trypan Blue (9ml total)
                          Tube B-9ml distilled water

                          Transfer 1ml yeast slurry (dense slurry- not fluid on top) to tube A for a total of 10ml. Mix this tube for about a minute. Then transfer 1 ml of that solution into Tube B (10ml total). This gives you a 100:1 dilution factor. Use this to place on your hemocytometer. You can do a side by side of methylene blue vs Trypan Blue and you should see a profound difference in the viability percentage (percent of stained cells to unstained cells), especially at end-fermentation (increased yeast stress from ETOH, Alpha Acids, and change in Osmotic pressure).

                          Hope this works out for you!
                          I finally got delivered 0.4% Trypan Blue, big difference in staining. Below is a picture of 2 weeks old beat up 1318.
                          So I guess with my dilution of Trypan Blue I would use:

                          Tube A-7ml distilled water + 2ml 0.4% Trypan Blue (9ml total)
                          Tube-B-9ml distilled water

                          And I transfer 1ml from Tube A to Tube B for a 100:1 dilution factor.

                          Click image for larger version

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                          Comment


                          • #14
                            Hi,

                            So how many dead cells would you say are in there? 12?

                            Comment


                            • #15
                              Maybe 18?

                              I count like this:
                              Click image for larger version

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                              Right or wrong?

                              Comment

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