Yeast Staining

[Otto Von Blotto]

That has been magnified 200 times under certain light conditions... obviously they don't look like that to view them with the naked eye

[Beerlust]

Interesting stuff Kelsey.

 

Well, if you hadn't qualified already, I think the acquisition of a microscope should definitely tip you over the edge.

 

You may now sign your name as "Otto Von Blotto (YD)"

 

YD = Yeast Daddy.

 

 

Cheers,

 

Lusty.

[Otto Von Blotto]

That's a good point you make Lusty.

 

Whenever I get commercial beers with yeast sediments it would be interesting to look at them and see what the viability is too. Might do that next time I get one.

[Otto Von Blotto]

I found this nifty little Javascript program called ImageJ last night, which allows me to count the cells much more easily than drawing grids on pictures and adding them all up. It has a counter in it whereby you simply click the mouse on the picture somewhere and it counts it, as per the image below. I did another count on the 10 pictures I did the other day and the counts were practically the same (1830 then vs. 1836 tonight).

 

It doesn't automatically count it just by reading the image or whatever but it's a hell of a lot quicker than the other way.

 

 

Cheers

 

Kelsey

 

 

[Morrie]

I can see a few instances where there are small cells attached to larger ones. I'm guessing that has been the birth of a new cell growing off a parent cell, so to speak? I'm not sure if that is the process or does a cell equally divide in half and then split off into two?

[Otto Von Blotto]

Yeah, those little ones attached to the bigger ones are budding cells. These do get counted as cells too.

[Otto Von Blotto]

Hi guys,

 

I did another staining and viability test tonight, this time with 1469 yeast that's been in the fridge since February 20th. The yeast calculator suggested a viability of 39% when I put the date into it earlier today.

 

I took 20 pictures from various locations around the slide to try to get a good cross section of the sample for counting. 3018 cells were counted, with 546 of them being stained. This gave a viability of 81.9%.

 

I'm not entirely convinced of this figure just yet though. Tomorrow I'm going to order some trypan blue stain which is a lot more accurate at determining viability at any level compared to methylene blue. It should arrive before I make my next starter so I will do two slides, one with MB and one with TB, and compare them. I'll probably do this on all yeast samples for a while, but I'll use the TB figures as my actual ones.

 

In any case, even if the MB staining isn't entirely accurate, a difference of nearly 43% in viability compared to the calculator is significant, and even if the real viability isn't as high as 82%, it's probably not as low as 39% either, supporting my theory (and that of others) that the calculators overestimate how much yeast actually die over time.

 

This is one of the pictures I used for counting, in this picture the viability measured about 80%

 

 

Cheers

 

Kelsey

[Otto Von Blotto]

Quick update, the starter had a nice krausen on it this morning when I got up for work, so whatever the actual viability of the yeast is, it's obviously still fine after 4 months in the fridge. This gives me confidence for future periods where I don't use a strain for a while.

 

I decided to go with a middle ground viability figure of around 60% just for working out the approximate cell count. According to the calculator with this new viability number, I'll get to 399bn cells from this starter, harvest 145bn, leaving me with 254bn for the batch. This figure is somewhere between the standard ale pitching rate and the hybrid pitching rate. Using the MB stain viability number the cells increase to 432bn, harvesting 157bn, leaving me with 275bn for the batch, which is right on the hybrid pitching rate.

 

Once I get enough data I might even try to make a little calculator for myself to approximate the viability over a period of time rather than relying on other calculators that are obviously on the very conservative side of things. If I do work out some way to make a calculator, I'll keep testing viability each batch and see how closely it lines up with its predictions.

 

Cheers

 

Kelsey

[Otto Von Blotto]

I harvested my yeast starter earlier, so I decided to try another method for preparing a slide for viewing the yeast under the microscope.

 

This method I tipped a small amount of mixed up starter into a shot glass, then diluted it with distilled water, and then added the methylene blue stain. I let it sit for a couple of minutes then swirled the whole thing up and tipped it down the sink, saving one drop for the microscope slide. This method proved much easier; when I do it properly I won't use as much yeast because it will just be dregs from the harvested jar/smack pack, probably a few drops worth with a few drops of distilled water and then the stain.

 

I took some more pictures to do a viability count even though it's gonna be pretty much 100% anyway, but just for interests' sake. I'll do the count later. I was interested in the shape of some of the cells though, as you can see in this picture there are a few pentagonal shaped cells in there.

 

Edit: the viability test came out at 98.25% viable yeast, which was expected but nice to confirm. I would think by the time it's pitched into the batch tomorrow it wouldn't be much different.

 

 

Cheers

 

Kelsey

[Titan8]

Well i just ordered the one i posted earlier. Really fascinating stuff. Thanks for the thread Kelsey.

[Otto Von Blotto]

Nice one. I find it fascinating too, currently waiting on the trypan blue to arrive which it probably will in the next few days. Then I can do a viability comparison between it and the methylene blue when I make up the next yeast starter.

 

Cheers

 

Kelsey

[Otto Von Blotto]

After a bit of a delay with customs, my Trypan Blue stain arrived earlier this morning. Because it had American Bioinnovations logos on it, they thought it might have contained biological matter so I had to get a declaration from the company that it didn't before they'd release it, but all done now so all good.

 

Today I'm going to re-hydrate some old dry yeast that's still in the fridge, and make up a slide with the trypan blue to see just how dead it actually is. This won't form any part of my testing (below), I'm just curious to see how much of it is still alive after a few years sitting in the fridge minding its own business.

 

Now that I have this more accurate stain, I plan to test the decrease in viability over time based on my normal yeast storage conditions of a mixed up starter being allowed to settle out in the fridge with the yeast sitting under the beer.

 

I'll make up a small starter, or even overbuild my next one by a larger amount than usual and save some of it in a jar in the fridge as normal, and then test its viability each day over a period of probably 6 months, to see just how much it really does drop over time in those storage conditions, and record this data as well.

 

I'll probably do this on a few different strains to see how or if they behave differently, and eventually when I have enough data I'd like to construct a calculator to approximately estimate the viability of a stored yeast, depending on what the data tells me. It might line up with the ones currently online, but it might not either, and if it doesn't then I'll try to get something happening. This will obviously take some time to gather all the data, so I'm not expecting to do anything in regards to a calculator for at least 2-3 years.

 

I also plan to do a comparison test between the TB and the methylene blue stain, but I'll have to use a yeast that is half dead, as MB is quite accurate with high viability yeast as well. With lower viability yeast it can give numbers that aren't correct; I saw a Braukaiser experiment where the MB staining suggested 100% cell death, but the yeast still grew, so obviously it wasn't all dead.

 

The reason for the inaccuracy is that MB can stain live cells and it can also make dead cells look live if those dead cells still have enough of the enzyme left which reduces the dye. TB works differently and will only stain cells that are dead. I'm looking forward to embarking on this little experiment, even though it'll take forever, even if just to prove to myself that the yeast doesnt die as quickly as the calculators suggest it does.

 

Cheers

 

Kelsey

 

 

[joolbag]

Fascinating stuff Kelsey. I'll be following this one and watching this thread over the coming months. Very interested to see the results and if they prove/disprove your theories

[Otto Von Blotto]

Yes it will be interesting to see what the data says over the coming months. The first strain I'll test will be Wyeast 1272, as that one is next in line to be built up in a starter to ferment an SNPA clone batch due in the FV after this current batch is kegged.

 

Methylene blue works by entering all the cells, but live cells contain the enzyme that reduces the dye and turns it colorless, and they appear unstained, while the cells minus the enzyme (dead cells) remain blue. As mentioned, accuracy problems occur when dead cells still contain enough enzyme to reduce the dye and/or live cells are damaged in a way that prevents them from reducing the dye.

 

Trypan blue is a different type of stain, which the live cells block from entering; it will only enter dead cells with ruptured cell walls and for this reason is a lot more accurate at determining the percentage of live and dead cells.

 

Obviously neither stain will be able to tell you what condition the cells are actually in other than live or dead but this is a measure of viability, not vitality. I'll do the dry yeast testing when I get home from work later as it's a short break today and I have no time now, but a nice early (for me) finish at 5pm.

 

Cheers

 

Kelsey

[Otto Von Blotto]

Some interesting data tonight, not quite sure what to make of it. I rehydrated some of a packet of W34/70 which had been sitting unopened in the fridge since probably late 2013/early 2014, the date on it was 10/2013 which I think is the manufacture date?? Either way it's past its 2 year best before, that is until I made up a couple of slides and stained it.

 

I made two slides, staining the yeast with methylene blue on one and trypan blue on the other. Then I looked at them each under the microscope, going across the slide to all parts. To my surprise, there were barely any stained cells at all in either slide. I did take a couple of pics that I'll post soon; I haven't done a proper count but the viability from eyeballing it would be above 95% easily.

 

I also did a slide with some yeast that has been sitting in the hydrometer sample but that was pretty pointless really as the viability would be high anyway, there were a few stained cells but not very many.

 

I've kept the re-hydrated yeast in its jug and tomorrow I'm gonna microwave it and kill it all, then do another stained slide just so I know what I'm looking for in terms of stained cells. It sounds obvious but sometimes it's hard to tell whether a cell is truly stained or not. The stained cell or two can be pretty clearly seen in the couple of pics I got though.

 

Cheers

 

Kelsey

[Otto Von Blotto]

These are the two pictures I took of the W34/70 slides, note that I have edited them to give a better contrast between the white and stained cells. They're a little blurred too but still good enough to see the difference.

 

It's a tentative theory from my angle at this point, but this testing tonight does appear to show that dried yeast can survive extremely well in the fridge for long periods of time, and also that re-hydration ensures that almost full viability is retained, rather than the 75-80% I have seen reported previously. Note that this is obviously only a single data point and may not be repeated so it's not definitive evidence, although I will be testing the other dried yeasts in the fridge which are all of a similar age to see if they are like this one. Even then, it's not conclusive, but a starting point.

 

 

 

Cheers

 

Kelsey

[Otto Von Blotto]

I microwaved the yeast in the jug last night to kill it off, and then made up a slide of it stained with trypan blue to compare to the other ones while it was still alive. I must say, the smell of microwaved/dead yeast is bloody terrible... no wonder autolysis results in off flavours!

 

Anyway, as expected pretty much all the cells were stained blue this time, as can be seen in these pictures. I kept the slide made up and will have another look at it today, just to see if the extra time sitting there has resulted in better staining of the cells. These pictures have also been edited in the same way as the ones in the previous post.

 

 

 

Cheers

 

Kelsey

 

[Otto Von Blotto]

I've made up a fresh starter wort to be pitched tonight with some harvested 1272 yeast, which has been sitting in the fridge in its jar since April 23, a little over 2 months. According to the calculator, it should be at 56% viability, so I'll be interested to test it later on with some jar dregs after pitching into the starter wort.

 

I'm putting my daily testing of viability on a temporary hiatus as it's getting closer to moving in with SWMBO, and with all that going on I won't be able to do the daily tests I had planned, so I will be waiting until we're in our place before beginning that project.

 

Cheers

 

Kelsey

 

 

[Beervis]

SWMBO just came into the room and I showed her the pics of the yeast cells and explained that one of the brewers on the Coopers forum bought a microscope to count yeast and her immediate and firm reply was ----- " WELL YOU'RE NOT GETTIN ONE".

 

That would exactly the reaction I'd get. I'm having enough trouble with just a couple of fermenters and some assorted gear in the garage :P

[Otto Von Blotto]

I'd probably get the same reaction if it was 10 or 20 years into the future , but as it is at the moment I already pretty much had all my brewing and kegging gear before I met SWMBO. I only picked up the microscope recently because it was going for a good price, otherwise I wouldn't have bothered. She doesn't give me the "you're not getting that" response, it's either "you bloody nerd" or "I can't see the point but if you want it..."

[Otto Von Blotto]

The results are in from last night's yeast slide made up from some 1272 dregs out of the harvest jar, which had been in the fridge since 23/4. I worked out when making this a couple of times that I have to use more of the trypan blue stain to actually get it to stain the cells properly, not a huge amount but more than a couple of drops.

 

Anyway, the calculator suggested a viability of 56% for the date of "manufacture" given, and I've just finished counting the cells from the 25 photos I took of the slide last night. In all, 2019 total cells were counted with 231 of them stained, giving a viability result of 88.6%. That's significantly higher (32%) than the calculator prediction, and I trust it more than the methylene blue as trypan blue is known to only stain dead cells.

 

Putting this new figure into the calculator with yesterday's starter gives me more cells and more harvested as a result, up to 162bn harvested instead of 144bn. The amount pitched also goes up but not by a huge amount. Once I get my daily testing done I can hopefully more accurately build starter sizes and not end up with a shitload more cells than I need because the viability calculation was wrong to begin with.

 

I'm also liking the TB stain better too, it seems to give a greater contrast between the stained and unstained cells, as this portion of one of the pictures shows. It has been edited to enhance it further, but even without editing the difference was obvious.

 

 

Cheers

 

Kelsey

[robwalk]

Hey Otto

 

Not a frequent forum user, but drop in every couple of weeks. Been following your yeast staining topic - very interesting. Just shows me there is so much more I can explore and in so many different ways.

 

I think I can say I am no longer a beginner with 26 brews completed now. Even though I have not got past kit and extract brews, I have started crystal and other malt additions and adjustments to bitterness and flavour in my boils. Mostly excellent results with some mediocre or disappointing ones. None done the drain yet.

 

Just wanted to let you know, I enjoy your posts, support and enthusiasm.

[Otto Von Blotto]

Thanks for the kind words mate . It's mainly being done for my own purposes of course but I figured I'd document it anyway in case anyone else was interested in reading about it and seeing the results of my testing. I'm looking forward to getting started on the daily testing and data recording from that too, I think this could be useful information for everyone. When I do I'll probably do weekly updates on the data I record.

 

I've got some old Windsor and Nottingham in the fridge too.. I might do up a slide with one of them tonight and have a look at the viability too. I did obviously do the W34/70 but mucked up the staining of it, so I'm going to have another go with some unopened yeast.

 

Cheers

 

Kelsey

[Otto Von Blotto]

A culture of Nottingham dry yeast, which had been re-hydrated in warm water, then stained with trypan blue. This packet was 3 and a half years past its best before date (Jan 2014), and sat in the fridge unopened since I bought it. Still a high percentage of alive (unstained) cells at 83.7% in this particular photo.

 

Whether the viable cells are particularly healthy or not is another matter of course. I'll edit and do a count on the rest of the pictures tomorrow.

 

 

Cheers

 

Kelsey

[Titan8]

Well it arrived. Looking at the samples provided. Really great machine. Disappointed they dont privide any blank slides.