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Old 01-22-2013, 05:23 PM   #1
WoodlandBrew
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Default Yeast - 6

I'm looking to explore some other methods of testing viability of yeast. Is any one familiar with the slide culture method? Or even better, have experience with the Yeast - 6 method as described by the American Society of Brewing Chemists? (Or if anyone has a copy of it that would be wonderful)

When preparing slides the agar seems to tare pretty easy, and also has bubbles in it. To make counting more consistent I have tried to prepare a hemocytometer with a culture, but the 0.1mm depth doesn't allow much space for the sample and the agar.

What type of slide should I use? Any tips for working with small amounts of agar or gelatin?

This link is the best I could dig up so far:
http://kim.bio.upenn.edu/~simola/kimlab/lab/yeast_slide_culture.html

Any better ones out there?



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Old 01-23-2013, 05:31 PM   #2
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Sooooo far over my head at the moment, but I sincerely wish you the best of luck in gaining a more effective way at testing viability. Your endeavors greatly interest me and I can see getting into testing like you have; just not much extra time/money at the moment. If nothing else, I just bumped this post up (shamelessly ).



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Old 01-24-2013, 02:07 PM   #3
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May i ask what your objectives are?.......i am very interested in yeast strains and properties, especially vitality and viability which are two different but very important qualities of our yeasts..

Though the correct testing is usually beyond the scope, time and patience of most home brewers, staining yeast cells after growing them under the correct conditions just to see whats dead and whats alive, storing yeasts under perfect conditions, stressing the yeasts to see how they perform under certain conditions of temp, alcohol tolerance and other environmental factors is very hard to do correctly without a laboratory....though essential to know when wanting to get the best out of them i just wouldn't know where to start...

I admire your desire to determine yeast viability ....keep us posted how you get on..here's a few links to articles that may interest you..but i doubt they would be of much help

I love the idea of this test!..all you need is some diacetate-ethidium bromide!

''The fluorescein diacetate-ethidium bromide (FDA-EB) fluorescence method, primarily used to determine viability of mammalian cells, was applied to several fungi species. Living fungi cells produced fluorochromasia, i.e., an intracellular accumulation of fluorescein which could be easily visualized as a green color under the U.V. microscope. Dead cells showed a red bright color due to ethidium bromide penetration. The FDA-EB test can be successfuly employed to assay yeast and yeast like cells viability since a good correlation was observed between this assay and the colony count technique. The main advantages of FDA-EB test are its speed, high sensitivity and simplicity.''


http://link.springer.com/article/10.1007%2FBF00683967?LI=true



And:
There is some interesting info on procedures and comparisons of techniques used here

http://jcm.asm.org/content/16/1/209.full.pdf+html

Good luck!

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Old 01-24-2013, 03:07 PM   #4
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stpug,

Thanks for the bump!


daveb123,

Thanks for the links!

Most people will agree that Methylene Blue staining is not a accurate way to asses viability, but It's a very processes dependant. Different strains also take dye very differently. My intent is to compare MB staining to other methods to see how accurate the number I get with MB really are. Armed with that information I can better assess when it can be applied, and when more accurate methods are required.

I've seen some comparisons between MB and Florescent staining, but because I have seen how processes dependant staining is I can't be sure if my counts are as accurate as these comparisons.

What I really want to know is the accuracy of the MB cell counts that I do.

Yes, I agree viability and vitality are two very different things. I'm working on a blog post about that, but it's probably not going to be until the middle of February when it gets published.

I have looked into florescent staining, and may give it a try at some point. One concern I have is the UV light as my microscope is a simple bright field. It would be easy to get UV light to shine from under the stage, but focusing it concerns me. Cost is another concern. It's not exactly trivial. A 10ml of a 1% solution from Fisher Scientific cost about $60. If I were using ImageJ to automate the cell counts I could see using a more easily distinguished dye such as the florescent type, but for the couple of dozen manual counts that I might do in a week for home brewing florescent dye might be over kill.

But florescent dye does sound exciting!

Here is the most common one I have seen: (with some nice pictures)
http://www.invitrogen.com/site/us/en/home/References/Newsletters-and-Journals/BioProbes-Journal-of-Cell-Biology-Applications/BioProbes-Issues-2012/bioprobes-67-june-2012/tali-image-based-cytometer-yeast-viability.html

Here is a comparison of a different florescent technique. It looks like a technical paper, but reads more like an advertisement for the "Easy Count" method that they developed.

Figures 4 and 5 show that MB staining tracks very well with the florescent technique.
http://www.genprime.com/downloads/EasyCountTechPaper.pdf

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Old 01-25-2013, 01:35 PM   #5
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Under no circumstances should you shine UV light into a microscope!!!!! You will go blind!!!!!

Flourescent microscopes are actually illuminated from the side, and therefore the only light that escapes vertically is the flourescing of the sample. This is why flourescent photomicrographs are always black background. These microscopes are very expensive peices of equipment.

Another thing, repeatedly pouring agar into a hemocytometer is going to ruin the gridlines on the hemocytometer. The gridlines are not necessary as you are only looking for a % viability, not an absolute value.

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Old 01-25-2013, 09:12 PM   #6
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Quote:
Originally Posted by theredben View Post
Under no circumstances should you shine UV light into a microscope!!!!! You will go blind!!!!!

Flourescent microscopes are actually illuminated from the side, and therefore the only light that escapes vertically is the flourescing of the sample. This is why flourescent photomicrographs are always black background. These microscopes are very expensive peices of equipment.

Another thing, repeatedly pouring agar into a hemocytometer is going to ruin the gridlines on the hemocytometer. The gridlines are not necessary as you are only looking for a % viability, not an absolute value.
Thanks! These are just the kind of tips I was hoping for!
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Old 01-25-2013, 09:57 PM   #7
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Quote:
Originally Posted by WoodlandBrew View Post
Thanks! These are just the kind of tips I was hoping for!
Be careful man. Don't rely on forum posts to keep you from hurting yourself.

There is a lot of cool analytic stuff out there but keep in mind that some of the chemicals or procedures can hurt you if not done correctly.

Kai
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Old 01-27-2013, 03:26 PM   #8
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Since we're on the topic of ways you could hurt yourself trying this, you also want to be pretty careful with ethidium bromide. It gets used all the time in thousands of labs everyday, but it can be a very strong mutagen. Definitely make sure that anything that comes into contact with it doesn't get used for anything else that's going into your beer.

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Old 01-27-2013, 03:45 PM   #9
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Methylene/evans blue stain is a very easy way to determine viability. For a staining and scope method it is great, the only way you will get great accuracy is with flow cytometry, which is a bit out of reach for the homebrewer.

Also, there shouldnt be any reason to have agar on your hemocytometer. You can take a colony sample and resuspend it in a small amount of water, then look at a sample of that. You just have to keep track of your dilutions to get an accurate count of the original sample.

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Old 01-27-2013, 09:16 PM   #10
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The idea behind Yeast-6 is that the ultimate test of viability is the ability to reproduce. You spread a thin layer of agar over a normal, i.e. doesn't have to be a cytometer, slide and drop dilute slurry on that then incubate. Every cell you see on the slide after incubation that didn't form a colony is not viable. Every colony you see came from a viable cell.



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