# Microscopic Views & Staining



## Rocker1986 (15/6/17)

One of my friends was recently selling her microscope at a reasonable price, so I thought I'd pick it up to have a look at some yeast cells, mostly for my own curiosity. I also picked up some methylene blue stain to test viability, although I've read that it's not entirely accurate so I'm waiting on some trypan blue to compare with.

Anyway, I grabbed this photo of some Wyeast 1469 cells on a slide earlier today, this came straight out of a starter so there are only one or two stained ones (was testing a method of making a slide with staining), but I was interested to see some pentagonal shaped cells in there. What make the yeast experts of these cells?


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## Danscraftbeer (15/6/17)

You took that photo? How?


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## Rocker1986 (15/6/17)

Danscraftbeer said:


> You took that photo? How?


The microscope has an LCD screen for viewing which has a photo/video function on it, rather than a traditional eyepiece


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## MHB (15/6/17)

Pretty sweet bit of kit, just need a cell counting chamber....
When you get the new stain, it might be interesting if you put the photo into a decent editor and have a play around with the RGB, can give way better separation between stained and unstained cells.
Mark

PS
About the pentagonal cells - I'm far from an expert - but I suspect its just from crowding, the cells clumping and pressing up against each other,
M


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## GalBrew (15/6/17)

MHB said:


> Pretty sweet bit of kit, just need a cell counting chamber....
> When you get the new stain, it might be interesting if you put the photo into a decent editor and have a play around with the RGB, can give way better separation between stained and unstained cells.
> Mark



Download ImageJ or FIJI as it seems to be called now to manipulate the images and perform cell counts. The user interface is awful but it is quite a powerful tool.


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## Rocker1986 (15/6/17)

At the moment for counting the cells I just use a program called ImageJ which has a click counter function, so I can open the photos in that and click each cell and it counts it. 

I'll have to give the editing a go as well, assuming that program can do that too.. might have a look now actually.


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## GalBrew (15/6/17)

Rocker1986 said:


> At the moment for counting the cells I just use a program called ImageJ which has a click counter function, so I can open the photos in that and click each cell and it counts it.
> 
> I'll have to give the editing a go as well, assuming that program can do that too.. might have a look now actually.



You can do many wonderful things with ImageJ, the key to it I working out how. I went to a training course when I was a working at Melbourne Uni and it is very powerful, but awful at the same time.


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## Rocker1986 (15/6/17)

Certainly looks like it has a hell of a lot of functions.. .I might save that for a time other than now though. No doubt it will be useful if I can edit the pictures to better differentiate between stained and unstained cells, just gotta figure out how to do it.

Also that makes sense on the pentagonal cells too Mark. I didn't notice it in the 2000 Budvar yeast I previously looked at, but certainly is in a few of the pictures I took of the 1469 slide today.


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## GalBrew (15/6/17)

1469 is a high floccultor, I would take a yeast sample and spin it on a stir plate in some fresh wort or try and agitate it somehow to break the clumps up a bit. Then have a look under the microscope again.


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## Rocker1986 (16/6/17)

This one was made from tipping a small amount of mixed up starter into a medicine/shot glass, then diluting this sample with distilled water before adding a few drops of the methylene blue stain. It was swirled around then left to sit for a minute or two, then swirled up again. I then tipped the whole thing down the sink and used one of the last drips on the slide. 

I should note though that the whole slide wasn't clumps like that, some areas had more clumping and some areas had a lot less clumping. In any case, they're easy enough to count although I do try not to use areas where the whole picture is just a big clump of yeast cells because it takes bloody ages to count them all.


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## Rocker1986 (22/6/17)

Well, after some trouble getting the trypan blue through customs, it arrived here this morning, and just for shits and giggles I decided to rehydrate some W34/70 that had been sitting in the fridge unopened for probably about 4 years. I made up two slides, one stained with TpB and the other with methylene blue, and found in both that there were hardly any stained cells at all. I looked in various places around the slides and they were all pretty much white except one or two here and there. I must admit I was quite surprised by this.

This picture is from the TpB slide, and it has been edited to try to show the contrast better between the one stained cell toward the top left, and the other unstained cells. Now I just have to work out a way to take non blurred pictures


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## Alchomist (23/6/17)

Amazing stuff Rocker.
Is the little stained guy dead/different strain/or the beginnings of an off flavour do you think?


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## Rocker1986 (24/6/17)

Alchomist said:


> Amazing stuff Rocker.
> Is the little stained guy dead/different strain/or the beginnings of an off flavour do you think?



Thanks mate  yes the blue stained one in that latest post is a dead one. From my reading, the trypan blue only stains dead cells, whereas methylene blue can stain live cells, but dead cells can appear unstained if they still have enough of the enzyme left to reduce the dye.


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## Danscraftbeer (24/6/17)

This again raises my suspicions about yeast viability loss by age. Say a yeast calculator like MrMalty will consider older saved yeast to be zero viability but I've activated old yeast on the stir plate no worries at all.
Why do yeast calculators consider yeast as such a short life?


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## MHB (24/6/17)

You're not the only one with concerns about MrMalty. Unfortunately (trying to find a substitute for "but" which keeps showing up in brewing conversations) there is more to yeast than the number of "live cells" the other term we hear a lot is "Vitality" which is roughly just how healthy the yeast is.
You can have a lot of unhealthy yeast which is no better or perhaps even worse than fewer healthy cells.
There can be a lot of old cells that are working much less efficiently than younger cells (after about 20 buddings the cell is too scared to respire properly)
Lots of dead cells can produce off flavours,
Cells with low glycogen reserves may fail to adapt to a new environment, produce fewer offspring and less well flavoured beer.
Increased mutations with age...

Fair to say that there is a lot more to the subject than just Live Cells.
If you have a old culture it would be best practice to run the yeast through a full life cycle, population growth, fermentation and sedimentation - a couple of times, dropping/racking off any yeast that is playing sluggard and lying on the bottom, to revitalise the yeast, build up the population and reduce the overall age of the population.

Mark


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## Jack of all biers (24/6/17)

+1 to MHB's advice above. Once again, sage words.


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## Rocker1986 (26/6/17)

I should note that I'm not planning to use that W34/70 to ferment anything, I was just curious about how many dead cells would be in it after spending a few years in the fridge, unopened. It would appear hardly any. 

I also realise viability obviously isn't the be all and end all of yeast health, however it is interesting to me to compare my own results to the viability predictions that the calculators give. It could be of some use in calculating starter sizes down the track.


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## Rocker1986 (28/6/17)

Last night I decided to kill the yeast in the jug and do another slide just to see what the stained cells looked like, mainly just to give me a better idea of what I'm looking for when I do a proper test next. It sounds obvious but sometimes it's hard to tell if one is stained or not. The editing of the pictures does make it easier though, looks like 100% death rate in this sample


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## Rocker1986 (30/6/17)

So, it turns out I wasn't using quite enough trypan blue stain in the samples to get the cells to actually stain properly. I don't need to use a shitload of it but a little more than I had used in the initial slides. Last night I made up a slide from some 1272 yeast I'd had sitting in its harvested jar in the fridge since April 23, and today did a count on the pictures I took. The result was 88.6% viability from 2019 total cells counted (231 of them stained), compared to the calculator prediction of 56%. While I understand viability isn't the be all and end all, 32.6% is a huge difference between the calculator and the actual real world results.

I like this stain better too, it gives a better contrast between stained and unstained cells, as this portion of one of the pictures shows. The photo had been edited to better show the contrast but even without editing it was pretty clear.


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## Glomp (30/6/17)

Hi Rocker.

I recently purchased a trinocular microscope with a usb camera and will start counting cells pretty soon.

Where did you get the trypan blue from?


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## Rocker1986 (30/6/17)

Nice! I got the trypan blue from a top secret location in America smuggled into Brisbane under the cover of darkness... or at least that's what it felt like with all that customs bullshit thinking it had biological matter in it. I bought it on Amazon because the only places I could find that sold it locally only sold it to companies, the shipping price was bloody ridiculous but I figure the stuff will last a long time so it's not a huge expense in the scheme of things.


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## Glomp (30/6/17)

Thanks rocker.

I have sent an email to a small craft knitting dye supplier as trypan blue is also known as niagara blue and diamine blue.. i was googling those words and came up with knitting and inks. It is probably the same stuff.

I also found a hemocytometer slide with bright lines here.

http://www.perthscientific.com.au/p...-neubauer-improved-brightline/?v=fdd13832cd81

I have the cheap ebay versions from china but these are made in germany. The chinese ones are pretty ordinary


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## Rocker1986 (30/6/17)

I'll have to look into that next time I need the stain. I have a bottle of Quink blue ink that I use in my pen, I might do a muck around slide with some of that next time and see if it does the same thing as the trypan blue. Of course, it'd be impossible to really know if it's working the same way, but it'll be interesting to look at anyway.

I thought about getting some of the haemocytometer slides as well, but at this stage I'm mostly focusing on viability counts, which doesn't really need a specialised slide, rather than numbers of cells per mL. That's not to say I won't do some proper cell counting later on though, so those German made ones I will keep in mind for sure.

It's all been pretty interesting so far, and soon I'll be saving some excess yeast so I can do daily viability tests on it to see just how much really does die off over time, at least for my own storing methods and situation anyway. I've been sceptical of the rate of decline on the calculators for a while now, and the couple of tests I've done so far has appeared to back that up.


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## Danscraftbeer (30/6/17)

Science the shit out of it dudes. 

I'm now thinking yeast calculators like Mr Malty take an average of yeast viability. Different strains like hops have differing ageing profiles. Storing variables etc.
Eg: one of Rocker's test examples was W-34/70 well out of date. That's the same strain as yeast slurry I started up after months out of date I kept bottled and refrigerated. Maybe its a good shelf life etc.
I actually made a lager beer with that starter yeast cake and it was good beer. Talking about yeast slurry in a bottle for 6+ months.
It worked and had the character of w-34/70 that I know.


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## Rocker1986 (1/7/17)

I've done another slide with that W34/70 tonight, just about to edit the pictures but I might leave the counting until another day. From eyeballing them though there are a lot more dead cells than the first lot I did, I'd estimate viability around 70-75%, but the packet has obviously been sitting unsealed so it's not a fair comparison. It's still in the fridge but rolled up in a rubber band. In any case, the culture is nowhere near completely dead.

I do have a packet each of Nottingham and Windsor yeast that are unopened in the fridge as well and are possibly even older, so I'll do a slide of those next week too. 

I think the yeast calculators are based on methylene blue staining, which apparently isn't all that accurate once the viability drops below about 80-85%. I read a Braukaiser post on it where he'd stained a culture with methylene blue that showed 100% stained cells and it still grew when wort was fed to it. Obviously not 100% dead. 

From my reading so far, viable or live cells flat out reject trypan blue from passing through the cell membrane, and so remain unstained, whereas the dead ones obviously can't do this and become stained. Methylene blue works differently; it enters all cells, but the blue dye is reduced by an enzyme in the cell to appear clear. The problem is that dead cells can sometimes still have enough of this enzyme left to reduce the dye, and compromised cells may not be able to reduce it, and that's where the inaccuracies occur in regard to viability percentage. When I get a culture that is shown to be largely dead with trypan blue staining, I'll test it with methylene blue as well to compare the results. I'll be interested to find out if there is a difference there.


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## Danscraftbeer (1/7/17)

Try a very fresh and vital sample as well just to get that comparison maybe.


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## Rocker1986 (1/7/17)

That's also on the cards yes, next starter I make up. I'm guessing there won't be much difference between the two stains with a highly viable sample, but it's always good to test it properly since I have the means to now. I tested a 4 month old harvested yeast recently that came out at 81.4% or something, but it was with methylene blue so how accurate it was I'm not sure of. This is why I want to harvest some extra to do daily tests with trypan blue over a period of months, maybe even a year, to see how far it drops. I'll record each result and create a graph at the end of it all. It's a big undertaking but the data could be useful going forward.


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