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Methylene Blue Solution and Glycine Buffer Questions

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  • Methylene Blue Solution and Glycine Buffer Questions

    So I am trying to make sure I am properly testing for viability when harvesting and repitching. Some sources I read say that a .1% methylene blue solution is all that is needed. White Labs has a solution that takes the .1% methylene blue solution and dilutes it further with a glycine buffer of 10.6 pH .1M at 10ml methylene blue solution per 100ml. What is the glycine buffer for? These methods contain drastically different concentrations of methylene blue? Just looking for some clarification. Cheers

  • #2
    Yeast counting anxiety

    I too know the worries of making sure my sample prep for yeast samples doesn't kill the yeast leading to unreliable counts. As to your specific questions regarding the use of glycine buffer, the purpose of any buffer is to allow a solution to resist changes in pH, different buffering agents are used depending on the pH range. The ASBC methods stipulates a phosphate buffered methylene blue, also that methylene blue conc. is .01% not .1%. The idea being that pH swings can kill yeast before you count them. In general the buffering of your methylene blue is a good idea but I'm a stickler for using ASBC protocols since others have gone through the trouble of verifying them so I'd look up ASBC Yeast-3 and follow those instructions. The other things to keep in mind are: 1) keep incubation with methylene blue consistently between 1 and 5 min. not more not less 2) count enough cells in total, 500 is ok but 1000 is recommended by ASBC 3) only dark blue cells are dead, light blue are considered viable and don't count buds that are smaller than 50% the size of the mother.

    Good luck!

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    • #3
      Thanks!

      Originally posted by R.Cockerell View Post
      I too know the worries of making sure my sample prep for yeast samples doesn't kill the yeast leading to unreliable counts. As to your specific questions regarding the use of glycine buffer, the purpose of any buffer is to allow a solution to resist changes in pH, different buffering agents are used depending on the pH range. The ASBC methods stipulates a phosphate buffered methylene blue, also that methylene blue conc. is .01% not .1%. The idea being that pH swings can kill yeast before you count them. In general the buffering of your methylene blue is a good idea but I'm a stickler for using ASBC protocols since others have gone through the trouble of verifying them so I'd look up ASBC Yeast-3 and follow those instructions. The other things to keep in mind are: 1) keep incubation with methylene blue consistently between 1 and 5 min. not more not less 2) count enough cells in total, 500 is ok but 1000 is recommended by ASBC 3) only dark blue cells are dead, light blue are considered viable and don't count buds that are smaller than 50% the size of the mother.

      Good luck!
      That was a huge help! Thanks for your reply. Working on getting my yeast management and micro program dialed in. I have plenty of equipment but zero lab experience. It's a daunting task especially when you are anal retentive and short on time. haha Cheers!

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      • #4
        Online lectures for yeast viability and vitality

        If you are just setting up a yeast management and micro program you might benefit from some of the online courses and lectures offered from the Siebel Institute. This one in particular is relevant to your questions about yeast viability:

        https://www.siebelinstitute.com/cour...ty-measurement
        Lallemand is a global leader in the development, production and marketing of yeast, bacteria and specialty ingredients.

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