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Old 01-28-2013, 04:47 PM   #11
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Methylene/evans blue stain is a very easy way to determine viability.
unfortunately it is not very accurate.

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Old 01-29-2013, 04:03 PM   #12
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unfortunately it is not very accurate.

Kai
Relative to what? MB staining (and other stain conversion assays that depend on cellular enzymes) is an incredibly common laboratory procedure to test cell viability before downstream procedures that require particular cell counts. If it works in the lab, it works for the homebrewer. Many of the issues related to MB staining are user error, such as sample collection and replicates.

Is there a better assay for use on the homebrewing scale that is as easy and requires little technical skill and equipment?
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Old 01-29-2013, 04:19 PM   #13
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I agree with Kai that there is error, but I also agree with ColoHox, the inaccuracies I have seen seem to fall into the "user error" catagory you mention.

I've seen inaccuracies due to insufficient or excessive staining. Every strain seems to be a little different. WLP004 stains fine with 0.01%MB (the concentration recommended by White Labs, but WLP566 dosen't stain well with 0.01%. It does stains fine with 0.03%. EC-1118 doesn't seem to really stain well until the level is about 0.1% MB. WLP004 stained with 0.1% MB seems to lead to cell death. Especially when incubated for more than a minute.

Then there are dilution errors, and counting errors, inaccuracies due to clumping. (These all fall into the user error category)

I would really like to see where my "user error" is by comparing the way I count viability with MB to other methods. Or to find out if it is just inherent in MB staining that there can be 25% error on viability below 50%, and there is nothing I can do about it.

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Old 01-29-2013, 07:10 PM   #14
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One aspect that's often over looked is the simple error inherent when anything is counted. If you count 100 cells for example and the true viability is 50 percent 68% of the time you do the experiment you will get a number between 45 and 55 viable cells but 5 % of the time you will get a number less than 40 or greater than 60. To cut those bands in half (i.e. down to 48 to 52 and 45 to 55) you have to quadruple the number of cells counted (i.e. to 400).

I've seen detailed explanations as to why the cells may or may not take up the dye in situations not reflective of whether they are viable or not. OTOH it's hard to argue that a cell which spawns a colony is not viable.

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Old 01-29-2013, 07:33 PM   #15
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Old 02-01-2013, 10:49 PM   #16
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Quote:
Originally Posted by ColoHox View Post
Relative to what? MB staining (and other stain conversion assays that depend on cellular enzymes) is an incredibly common laboratory procedure to test cell viability before downstream procedures that require particular cell counts. If it works in the lab, it works for the homebrewer. Many of the issues related to MB staining are user error, such as sample collection and replicates.

Is there a better assay for use on the homebrewing scale that is as easy and requires little technical skill and equipment?
relative to plate counts where you count the cells that have the ability to reproduce and grow colonies on a plate. But that takes more effort and time than staining which is why staining is so popular.

I once had a very old sample of yeast and it showed a viability of at least 10 % according to MB. When I plated this sample nothing grew. All the cells were dead but not all the cells stained.

check out the pics I have at the bottom of this section: http://braukaiser.com/wiki/index.php..._blue_staining

when I kill cells with heat they readily stain. But when they die of starvation they don't necessarily stain when they are dead.

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Old 02-02-2013, 12:12 AM   #17
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Quote:
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relative to plate counts where you count the cells that have the ability to reproduce and grow colonies on a plate. But that takes more effort and time than staining which is why staining is so popular.

I once had a very old sample of yeast and it showed a viability of at least 10 % according to MB. When I plated this sample nothing grew. All the cells were dead but not all the cells stained.

check out the pics I have at the bottom of this section: http://braukaiser.com/wiki/index.php..._blue_staining

when I kill cells with heat they readily stain. But when they die of starvation they don't necessarily stain when they are dead.

Kai
Plating would be an ideal method, but indeed requires more effort. We routinely discard samples that show <50% viability via MB staining. This is mostly due to the specificity of the test, as most small samples report false positives at that low level. The condition of the cells is another issue. Heat killing cells deactivates the enzymes involved in neutralizing the stain, so heat killed cells will readily stain. Starvation does not always deactivate cellular processes. The cells, although not able to reproduce, are able to prevent the stain from entering the membrane either through survival mechanisms or active enzymes.
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