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Thread: Different Staining Methods for Determining Yeast Viability

  1. #1
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    Different Staining Methods for Determining Yeast Viability

    I have recently heard that despite methylene blue being so widely used, it may not actually be the best at determining yeast viability.

    I am curious if anyone has used other dyes with success, or if anyone has a certain dye preference to methylene blue.

    Some dyes I am curious about are:

    Trypan blue

    Phloxine B

    Rhodamine B

    Alkaline methylene blue (or violet)

    Crystal violet

    Erythrosin B

    Any feedback would be appreciated!

  2. #2
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    Methylene Blue is not very accurate if the viability is less than 85%, so it is best used as a pass/fail indicator for your yeast slurry and not for accurate counts. If >85% viability the yeast passes and the counts will be accurate. If <85% viability the viability will not be accurate, but it doesn't matter what the specific count is since it is too low to use anyway (who cares if it is 65% or 75%, all that matters is that it is too low).

    Trypan Blue is another great option with good contrast between live and dead cells (dead are very blue, live are crystal clear).
    Lallemand is a global leader in the development, production and marketing of yeast, bacteria and specialty ingredients.

  3. #3
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    I don't mean to hijack this thread!

    Eric - if using Trypan Blue are there any other considerations that need to be taken (acidification, etc)? Or is it a straight replacement to Methylene Blue? We currently use MB and do struggle occasionally with shades-of-blue and the subjectivity of who is counting resulting in different results.

    Thanks for any insight you can offer!

    -Tom

  4. #4
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    Quote Originally Posted by Lallemand Eric View Post
    Methylene Blue is not very accurate if the viability is less than 85%, so it is best used as a pass/fail indicator for your yeast slurry and not for accurate counts. If >85% viability the yeast passes and the counts will be accurate. If <85% viability the viability will not be accurate, but it doesn't matter what the specific count is since it is too low to use anyway (who cares if it is 65% or 75%, all that matters is that it is too low).

    Trypan Blue is another great option with good contrast between live and dead cells (dead are very blue, live are crystal clear).
    Thank you for your response, Eric!

    I have a few followup questions:

    As I do some research on Trypan Blue - it seems the cost of this process is pretty substantial (Which I expected, but I suppose I wasn't sure to what extent).

    Everything I am seeing is saying the yeast cells need to be suspended in PBS - whereas now we used Methylene Blue in cells suspended in diluted water.

    Do you mind if I ask your exact procedure with Trypan Blue? Do you buy the 0.4% solution already made, or powder? Do you use PBS? If so - do you dilute multiple times with it? or can the final dilution be a mixture of PBS and diluted water?

    Thanks so much.

    Kate

  5. #5
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    I too have re-invented the wheel a few times. Oddly it never gets any rounder.

    The trypan is good for easier identification (and viability outside acceptable levels), but as noted cost is more, and a tiny bit more process involved. I find it much easier to just use a slightly more dilute Methelyne blue that helps contrast living and dead cells better. Too much blue makes everything look blue. Less than 85% viability is trash to me anyhow.

    If you are really that concerned about accurate counts, viability and staining, then you should consider a cellometer. I believe both Moxiflow and Nexcelom offer viability and counts that use a larger sample size and would be more consistent. I’m sure there are others too.

    I think the forefront of dyes is more on Acridine Orange and Propidium Iodide now, but I digress. It doesn’t change your sample results in any way. It’s just an easier to see accurate measuring device. Accuracy is great, but it’s only as good as what you do with it.

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