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Old 12-19-2012, 05:06 PM   #21
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I'm not a microbiologist, but this is what I have gathered:

Plate counts:
- take time
- do not show you if cells are clumping together. Each clump will be one colony. You'll have to inspect the diluted sample to check for clumps
- only counts cells that are actuallt viable. You are able to establish a correct viability count by matching cell density from hemocytometer with growth colonies.

MB staining
- quick
- you are able to see clumps of cells since you have to look through a microscope
- cell viability can be grossly overestimated compared to pate counts. This is b/c methylene blue may not be able to enter a dead cell. I had old cultures that hardly stained with MB but nothing grew on a plate.

MB staining is useful to check the health of a culture. If you find that less than 10% stain, you can assume that the culture is fairly viable, If more than 10% stain you have an old culture that you should not pitch anyway.

Kai

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Old 12-19-2012, 06:59 PM   #22
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Kai,

This is the first time I noticed your location. You're only about 40 minutes from where I work. Small world.

I recently plated some WLP004 that measured 10% viability with MB and got approximately the number of colonies I was looking for. This was a pretty rough experiment so I want to re-run it, but I wanted to mention it. I also fermented a sucrose wort with 10% viability EC-1118 recently without a problem.

What I have noticed about Methylene Blue is that different strains take different amounts of dye to stain well. EC-1118 works well with equal parts 0.1% solution and diluted cells, while WLP566 stains fine with only one tenth of that concentration at 0.01%. If there is significant protein material it may take a little more stain as well. Normally I start with a 2:1 dilution of cells by 0.03% MB and if the dead cells are just a little bit blue I'll measure what is left in the test tube and add that much of 0.1% MB. This gives me a 4:1 dilution of about 0.06% MB. If you have too much MB the slide will look dark under the scope.

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Old 12-20-2012, 02:06 PM   #23
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[quote=WoodlandBrew;4698693]Kai,
This is the first time I noticed your location. You're only about 40 minutes from where I work. Small world.
[quote]

Cool. I'm in the Woburn area on a regular basis.

Quote:
What I have noticed about Methylene Blue is that different strains take different amounts of dye to stain well. EC-1118 works well with equal parts 0.1% solution and diluted cells, while WLP566 stains fine with only one tenth of that concentration at 0.01%. If there is significant protein material it may take a little more stain as well. Normally I start with a 2:1 dilution of cells by 0.03% MB and if the dead cells are just a little bit blue I'll measure what is left in the test tube and add that much of 0.1% MB. This gives me a 4:1 dilution of about 0.06% MB. If you have too much MB the slide will look dark under the scope.
I have not played around with determining the reliability of MB staining or improving its accuracy. I'm citing from the literature sources and this is one of the charts I have come across in a German presentation from the Weihenstephan brewing school:
yeastvitalitymethods.jpg  
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Old 12-20-2012, 03:21 PM   #24
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That is very interesting. Your results and this citation have definitely brought this to a new light for me. This will certainly be in my mind when I measure low viability.

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Old 12-20-2012, 04:01 PM   #25
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This has been a well researched topic: Google search for "methylene blue yeast accuracy"

and I wish it was more reliable.

Kai

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Old 12-26-2012, 05:15 PM   #26
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Quote:
Originally Posted by jkendal View Post
Why? If you're using fresh yeast and pitching the amount that Mr. Malty recommends, you really can't go wrong. I haven't heard anyone yet complain that it didn't produce good results. You could always pitch a little more than what Mr. Malty recommends if it's a concern. Do you want a microscope just to see how many are actually there?

I'm just curious as to why you're looking for something other than a pitching rate calculator.


Edit: After reading this, the tone didn't come through - I really am just curious. No disrespect intended....
That's just it, I don't buy very much fresh yeast. I wash a lot of yeast and top crop sometimes. I am also a very scientific person, and my studies are going in this direction anyways so I figured I might as well dive in head first. Great discussion so far, thanks for the input, I had forgotten about this thread.
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Old 12-26-2012, 08:23 PM   #27
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I use a violet stain which supposedly is better. I got it from White Labs, but sadly I've be so busy lately I haven't been able to do a good comparison between it and the methblue for low viability.

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