Microscopic Views & Staining

[Rocker1986]

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.

 

[Glomp]

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

 

[Rocker1986]

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.

 

[Danscraftbeer]

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Eg: one of Rocker's test examples was W-34/70 well out of date. That's the same strain as yeast slurry I started up after months out of date I kept bottled and refrigerated.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.

 

[Rocker1986]

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.

 

[Danscraftbeer]

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

 

[Rocker1986]

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.