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?

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 ).

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/BF00683967?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!

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/e...li-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

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.

theredben said:

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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Thanks! These are just the kind of tips I was hoping for!

WoodlandBrew said:

Thanks! These are just the kind of tips I was hoping for!

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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

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.

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.

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.

ColoHox said:

Methylene/evans blue stain is a very easy way to determine viability.

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unfortunately it is not very accurate.

Kai

Kaiser said:

unfortunately it is not very accurate.

Kai

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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?

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.

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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ColoHox said:

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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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?title=Microscope_use_in_brewing#Methylene_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

Kaiser said:

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?title=Microscope_use_in_brewing#Methylene_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

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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.