1 Posted: Thursday, June 01, 2017 9:27 PM

As I posted in my other yeast thread, I recently acquired a microscope from a friend who was selling it cheaply, in order to do a bit of yeast staining to get an approximate idea of the viability of the yeast I'm pitching into my starter worts, and how closely it correlates with the estimations on the yeast calculators. I'd been interested in doing this for some time, but didn't really want to drop a heap of money on it, so it was just pure luck that my friend was selling hers at a cheap price and I decided to give it a go. This microscope has an LCD screen for viewing specimens rather than an eyepiece, which also takes photos which I can transfer to the computer to view properly.

I did a practice run tonight to get a bit of an idea of how big of a drop to put on the microscope slide so the cover glass doesn't squeeze it all out the edges, literally one drop is enough. I transferred some of the methylene blue solution into a dropper bottle to make that easier to deal with too.

I'd had an FG sample from the latest pilsner sitting on the kitchen bench since Monday, so I decided to tip most of the beer out, swirl up the little bit of yeast in the bottom and use that as my sample. As close to one drop as I could get trying to tip it out of the hydrometer sample tube was placed on the slide, and one drop from the bottle of methylene blue was dropped onto it. The cover glass was then placed over the top to “squash” the sample and get it all on relatively the same plane for analysis. The sample was then looked at, and I got a few pics too. I could also see cells moving around in the sample at times as well.

These are some photos I took from the practice run tonight. The first one is a view from further out, to give an idea of how many viable cells to blue stained cells there are, while the other two pics are the close ups of two different “sites” on the slide. I didn't bother counting them on this occasion but as you can see, the viability is very high, which is to be expected really.







I'll be doing this again with some 1469 yeast that's been in the fridge since February when I make up its yeast starter soon. It will be interesting to see how that one is looking.

Cheers

Kelsey



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2 Posted: Thursday, June 01, 2017 10:11 PM

Interesting stuff !

3 Posted: Thursday, June 01, 2017 10:37 PM

Bloody good stuff mate!
Are the dark blue ones dead?
I'd really like to try it with my 4 month old stored yeast to see what the viability is. It is still cranking strong in my last 2 brews especially the 1469 that wants to climb out of the top of the FV.

4 Posted: Thursday, June 01, 2017 10:57 PM

So good

5 Posted: Friday, June 02, 2017 6:27 AM

Thanks guys! Morrie, yes the blue ones are dead, perhaps damaged but mostly dead. There are a few cells in the bottom left corner of the second photo that have a bluish tinge to them, but these are fine, it's the ones that are strikingly blue all over that are the dead ones.

I have read some articles online about the reliability of methylene blue for determining yeast viability. Apparently dead cells can still have enough of the enzyme or whatever it is that breaks down the blue dye to appear to be live cells, and that the stuff can stain live cells as well. An alternative is Trypan Blue or Safranin.

Trypan Blue is pretty hard to get, but Safranin I can find on eBay. The latter stains dead cells red. At this point though, I'm not that worried. I also found an article where the guy had tested the three stains to determine yeast viability across a few different yeast strains, and with the Saccharomyces yeast all three stains pretty much yielded the same results. There was a large difference when looking at Brettanomyces, with the methylene blue not performing very well, but I don't use Brett so it's a non issue really.

Cheers

Kelsey

6 Posted: Friday, June 02, 2017 7:06 AM

Kelsey - I've read somewhere that you can determine your pitching rate if you have a microscope to determine the yeast cell count per ml. I guess that is what the microscope is ultimately for, and if so can you please give us a run down on this process if you are already familiar with it?

The microscopic pictures are impressive to say the least.

7 Posted: Friday, June 02, 2017 8:12 AM

Yes, very cool Kelsey, great score.

Cheers,

Christina.

8 Posted: Friday, June 02, 2017 9:15 AM

Good post!

What is the magnification on the microscope?
Are you able to differentiate between yeast species at this level?

John

9 Posted: Friday, June 02, 2017 12:11 PM

I want one. What brand is it ?

10 Posted: Friday, June 02, 2017 12:27 PM

Who says you need a hemocytometer to count yeast? Just draw some grid lines on the pictures with MS Paint and away you go! I took more photos than the ones posted last night, 10 all up, so I decided to do a count. In total there were 1830 cells counted, with 264 of them stained blue, giving a viability of 85.6%. This isn't a great surprise given the yeast came out of the fermenter after it had been at FG for at least two days and then sat on the kitchen bench for another 3 days before I did these samples. If it had been in the fridge the whole time the viability would probably be higher.

Morrie:

Kelsey - I've read somewhere that you can determine your pitching rate if you have a microscope to determine the yeast cell count per ml. I guess that is what the microscope is ultimately for, and if so can you please give us a run down on this process if you are already familiar with it?

The microscopic pictures are impressive to say the least.

Cheers mate. Yes, you can but I guess you'd need to get exactly 1mL of slurry on the slide and then work out how much of that mL each view is actually looking at. Probably do a count of 5 or 10 different views around the slide and get an average to extrapolate up to 1mL. I haven't looked into it though so I'm just guessing that's how it done, at this point I'm happy enough with the estimated cell counts from the calculators, but more interested in the viability measures.

PXR-5:

What is the magnification on the microscope?
Are you able to differentiate between yeast species at this level?

The first photo is magnified 200x, and the other two more close up ones are 400x. You probably wouldn't want to go any higher really, otherwise the things become too big. The microscope can go up to 1600x with the digital zoom feature.

You can differentiate between different yeast species, not necessarily the strains themselves though. For instance, the Saccharomyces strains all look pretty much the same as one another, but Brettanomyces does look different. Interestingly I found nothing that looked like bacteria in this sample, despite it sitting on the bench with fruit flies floating in it for days.

Titan8:

I want one. What brand is it ?

It's a Celestron model 44345. Glad I didn't buy it new though, they're about $500 or more.

This has been quite fun if not a little tedious but I'm looking forward to doing it again with the longer stored 1469 yeast when it is pitched into its starter. I'm interested to see if my viable count agrees with the calculator. The calculator suggests around 40% viability. I'll update when I do the counts, probably some time next week.

Cheers

Kelsey


11 Posted: Friday, June 02, 2017 4:53 PM

found this one

This stuff fascinates me.

12 Posted: Friday, June 02, 2017 7:40 PM

That one is pretty much the same as the one I have so it will definitely be good for it. You'll obviously also need glass slides and cover glass to prepare and view the samples properly, but they're pretty cheap.

When I do the testing on the next lot I'll grab a few pics of the process I use. It's pretty simple but pictures do help sometimes.

13 Posted: Friday, June 02, 2017 8:53 PM

Otto Von Blotto:

Morrie:

Kelsey - I've read somewhere that you can determine your pitching rate if you have a microscope to determine the yeast cell count per ml. I guess that is what the microscope is ultimately for, and if so can you please give us a run down on this process if you are already familiar with it?

The microscopic pictures are impressive to say the least.

Cheers mate. Yes, you can but I guess you'd need to get exactly 1mL of slurry on the slide and then work out how much of that mL each view is actually looking at. Probably do a count of 5 or 10 different views around the slide and get an average to extrapolate up to 1mL. I haven't looked into it though so I'm just guessing that's how it done, at this point I'm happy enough with the estimated cell counts from the calculators, but more interested in the viability measures.

Cheers
Kelsey


Could you just use a drop on your slide and count the approximate cells in that and work out how many drops to a ml and multiply that out to get your pitching rate?

14 Posted: Friday, June 02, 2017 8:54 PM

What is the long term plan with the microscope Kelsey? Are you planning to start keeping more yeast varieties, storing them in the freezer etc?

Cheers,

Christina.

15 Posted: Friday, June 02, 2017 9:01 PM

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”.

16 Posted: Friday, June 02, 2017 9:03 PM

Hahahahaha Morrie, I suspect if I said the same to my wife she would use the EXACT same line!@

Kelsey, I'm really enjoying this thread, it is fascinating. Look forward to the results of the 1469 staining and counting

17 Posted: Friday, June 02, 2017 9:08 PM

“ WELL YOU'RE NOT GETTIN ONE”.
Is not a very good thing to be saying to me. It could have consequences.

P.S. - Christina, I've replied to one of your posts on - https://club.coopers.com.au/coopers-forum/topic/16675/?page=2

18 Posted: Friday, June 02, 2017 9:23 PM

I don't know if using “just a drop” on the slide would work all that well because you don't know how much volume that particular drop is, so it's a bit difficult to calculate any real cell density from it. It seems easier to me if you're aiming to calculate cell density per mL of slurry that you'd use 1mL or half a mL or at least a known volume, and then somehow work out what proportion of that volume is being viewed in the microscope.

No plans to store yeast in the freezer or any differently to how I am now Christina, just mainly curious as to my actual viabilities vs. yeast calculator viabilities. I managed to find some Trypan Blue stain on Amazon that reckons it ships to Australia so I might grab some next week, and at some point do a comparison between it and the methylene blue staining and see if the results are practically the same as each other as they were in that article I read.

As you can see from this graphic from that article, the three stains tested are all pretty much the same with Saccharomyces and bakery yeast, but vary with Brett. As I mentioned earlier I don't use Brett, so it doesn't affect me there, but I would be interested to compare between TB and MB with my own strains to see if the results are similarly close.



Cheers

Kelsey

19 Posted: Friday, June 02, 2017 9:34 PM

Morrie:

“ WELL YOU'RE NOT GETTIN ONE”.
Is not a very good thing to be saying to me. It could have consequences.


I got “You're not buying another bike” this evening. Well I want one and I found a good carbon road bike second hand on eBay (barely ridden, my kind of buy). I'm due for an upgrade from my faithful aluminium bike with carbon forks. I did just get my tax return back… and it happens to cover the cost of this bike!

20 Posted: Friday, June 02, 2017 9:51 PM

Jools - yep, tax return is a dangerous time for me too. Last year some of my tax return went on a Brau20. I'm due to get back $7.5K any day now. Hell, I haven't had a new bike since 2010, a DR650 kitted up which I rode up to Cape York and back and prior to that a road burner a R1200RT in 2008 and feel its about time I got another bike too. I've still got the RT but am eyeing off a Triumph Street Triple.