Vezeaufoods
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Anyone using a microscope to do cell counts before pitching yeast?
Microscopes for cell count?
- Thread starter Vezeaufoods
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Maybe if you're a microbiologist, like Dr. Denard. @loveofrose
Nope but have done yeast cell counts after pitch. (No reason other than it was a fun exercise.)
Curious - Why the desire to do cell counts prior to yeast pitch?
Curious - Why the desire to do cell counts prior to yeast pitch?
To know the pitch rate.
What I do is let the yeast flocculate and self compact in the refrigerator and then measure the volume of the resulting yeast cake. Then, make some assumption about how many billions per ml you have. Seems like 1-5 Billion per ml is the range most often discussed, which is a pretty wide range I'll admit. Anyone know of tighter bounds than that? Maybe it depends on yeast "vitality", and I'd be curious as to exactly how that gets measured.
You only need a 500x microscope to see yeast. 1000x to see bacteria. You can get even a 2000x microscope for pretty cheap these days. I've thought about it, but I think the yeast compaction may just be easier. I suppose you could centrifuge it to speed up the process, and centrifuges are pretty cheap too.
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Vezeaufoods
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Yes...to know the pitch rate. Thanks for the suggestions.
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Yes. I did my first yeast cell counts from a starter today actually. I was hoping to get some questions answered about it and was going to start a thread, but it looks like this is a good spot to start if anyone is particularly experienced with the subject.
loveofrose
Well-Known Member
You need one of these: https://www.amazon.com/dp/B00SCOGY56/?tag=skimlinks_replacement-20
A Haemocytometer gives you a grid with a known volume so that you can acutely calculate cells/ml. Take an average of 3-5 grids.
A Haemocytometer gives you a grid with a known volume so that you can acutely calculate cells/ml. Take an average of 3-5 grids.
jack13
Well-Known Member
What magnification is needed?
400x for counting / stain viability etc. That means 40x objective lens coupled with the normal 10x eyepieces.
1000x is where you want to be to look at cell structure, but you would probably be looking at oil immersion for that, a lot more light, etc.
Do you count a budding yeast cell as one or two?
More importantly: from start to finish, how long does it take to do a proper count? I suppose it's a function of how many there are to be counted, but is there some way to give a ballpark on how long it generally takes?
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With a haemocytometer it doesn't take long at all. You just need to count the cells in one square of haemocytometer, then do some "simple" math. It's best to count several and average.
Getting the right dilution so that you don't have too many, or two few, cells is the trick. But after a few times it's simple.
I made got a nifty spreadsheet that makes the math pretty easy. I'll see if I can attach it here. It might seem daunting at first, but you'll understand quickly once you do it.
Getting the right dilution so that you don't have too many, or two few, cells is the trick. But after a few times it's simple.
I made got a nifty spreadsheet that makes the math pretty easy. I'll see if I can attach it here. It might seem daunting at first, but you'll understand quickly once you do it.
Looks like this. Spreadsheet attached. Click the zip filename at the bottom of this post and it will DL to your computer.
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@RoyalGallon
Well-Known Member
Also if you add a drop of Methylene blue dye https://en.m.wikipedia.org/wiki/Methylene_blue
To your diluted yeast sample the dead cells will take up the dye and the live ones won’t so you’ll be able to work out how viable your yeast is.
To your diluted yeast sample the dead cells will take up the dye and the live ones won’t so you’ll be able to work out how viable your yeast is.
I've done three counts so far. I think the first one took me over half an hour. The second one probably about a half hour. Half the time is in preparing the sample. I'm using the method described in the yeast book to dilute and then using methylene blue as the final dilution at 1:2.
For both counts so far I have counted all 25 squares in the hemocytometer just to help me learn and mitigate errors due to my inexperience. So far the 4 corners and center method is within 2% of counting the whole chamber. When I get more comfortable with the process and just do the 5 squares I'm sure the count will take under 10 minutes + prep time.
For both counts so far I have counted all 25 squares in the hemocytometer just to help me learn and mitigate errors due to my inexperience. So far the 4 corners and center method is within 2% of counting the whole chamber. When I get more comfortable with the process and just do the 5 squares I'm sure the count will take under 10 minutes + prep time.
So, vitality is simply a measure of dead or alive? i.e. If a yeast cell is "tired" but not dead, then it is labelled with the same vitality as a fresh cell that's healthy and ready to work? [I know, this is silly anthropomorphism, but I lack the proper vocabulary. Feel free to correct me with the proper nomenclature].
10 minutes doesn't sound bad at all. It's motivating me to try this. How much prep time?
This all definitely sounds like thee gold standard we should all aspire to. Thank you all for the informative posts!
10 minutes doesn't sound bad at all. It's motivating me to try this. How much prep time?
This all definitely sounds like thee gold standard we should all aspire to. Thank you all for the informative posts!
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I'm close to fooling with this stuff; I have a 400x microscope, and bought this kit:
https://www.amazon.com/gp/product/B076ZT949V/?tag=skimlinks_replacement-20
It contains the hemocytometer, some stains, a few other items that are needed. I have not baptized it yet, though I do have a starter in the refrigerator that I did not pitch into a fermenter; it's there awaiting my first attempt with this.
https://www.amazon.com/gp/product/B076ZT949V/?tag=skimlinks_replacement-20
It contains the hemocytometer, some stains, a few other items that are needed. I have not baptized it yet, though I do have a starter in the refrigerator that I did not pitch into a fermenter; it's there awaiting my first attempt with this.
Blacksmith1
Captain Cheap
does this count as yeast porn?
I didn't time myself prepping the sample. I think if I got good at it I could do it in 15 minutes. Of course, there are often other time factors. I've been sterilizing my pipette in the pressure cooker ahead of time. And it takes 5-10 minutes to wash and dry the graduated cylinders and pipettes I use for the dilution and loading the hemocytometer. Again, I'm pretty inexperienced at counting yeast...and laboratory practices. So I'm sure one could get a lot faster than I do it.
I don't do it, but you can download (free) Image J and use it to automatically count cells if you can get them onto a digital image. See the following vid. My son uses the same to count cells in his grad program at school (getting published
). I don't do it, but you can download (free) Image J and use it to automatically count cells if you can get them onto a digital image. See the following vid. My son uses the same to count cells in his grad program at school (getting published
Cool! That sounds like the bee's knees to me. Why are you using it?
John Palmer, in his "How to Brew" book, wrote that he's never seen a bad outcome from overpitching, but he has from underpitching. So, lately that's how I've been approaching it: building a large enough yeast starter that I'm sure to have enough viable yeast, and very probably much more than enough. It seems to work. I'm still very intrigued though by this more scientific approach that you all are doing.
I'm not using it. I thought about it at one time, but it was just as easy to count manually. My son uses it to measure the width of mutant cells in rat's eyes in order to assess something that I don't understand.
My apologies. I meant to ask, "Why aren't you using it?" but you answered the question anyway.
BTW, I call en passant on your passed pawn.
BTW, I call en passant on your passed pawn.
You think I never heard that before
You chess nerd.
This is the one I got in December. I've been very happy with it. It has a lot of quality components for the price. It is honestly probably overkill for counting yeast.
https://www.amazon.com/dp/B005TJ5CEG/?tag=skimlinks_replacement-20
https://www.amazon.com/dp/B005TJ5CEG/?tag=skimlinks_replacement-20
search the yeast and fermentation forum I recalling seeing a post for recommended scopes.
this might help
http://braukaiser.com/wiki/index.php/Microscope_use_in_brewing
I would really like one, but by the time you add all of the other pieces required I have a hard time justifying the purchase.
Do you folks do much oil immersion, or pretty much not? I'm guessing not, in which case I'd rather have a 60x objective lens, which doesn't require oil immersion rather than a 100x that does(e.g. https://www.amazon.com/gp/product/B00AEJ9FJ4/?tag=skimlinks_replacement-20). I went looking for a microscope that came with a 60x objective as standard. However, they all seem to include the 100x lens, as far as I can tell.
In addition to counting yeast cells, I would like to know if a particular ferment has has a serious bacteria problem though. Occasionally I get a bad ferment that smells funny (maybe like bluecheese or something odd like that), and I'd like to be able to confirm whether or not it's a bacteria problem rather than an unhappy yeast problem if perhaps far more bacteria appear than usual. So, would I need the 100x objective after all in order to check that, or would I be able to tell just using a 60x, which doesn't require oil immersion?
In addition to counting yeast cells, I would like to know if a particular ferment has has a serious bacteria problem though. Occasionally I get a bad ferment that smells funny (maybe like bluecheese or something odd like that), and I'd like to be able to confirm whether or not it's a bacteria problem rather than an unhappy yeast problem if perhaps far more bacteria appear than usual. So, would I need the 100x objective after all in order to check that, or would I be able to tell just using a 60x, which doesn't require oil immersion?
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Reporting back: I ordered this microscope: http://rover.ebay.com/rover/1/711-5...0001&campid=5338413729&icep_item=381347257727 It uses DIN lenses.
It's not phase contrast, but I can add a 60x objective lens for fairly cheap (around $35, including shipping). This should be good enough for checking living yeast vitality (plump vs football shaped), and if it looks vital but I find funny smells, then I guess I'll just infer bacteria. 1.25 numerical aperature on the 100x lens means I might be able to see bacteria (I hope) if I do oil immersion.
As far as yeast counts goes, from what I've read, apparently you should only expect to get order of magnitude anyway, so, aside from checking vitality, I'm not sure it offers any more accuracy than measuring the volume of self-consolidated yeast cake.
It's not phase contrast, but I can add a 60x objective lens for fairly cheap (around $35, including shipping). This should be good enough for checking living yeast vitality (plump vs football shaped), and if it looks vital but I find funny smells, then I guess I'll just infer bacteria. 1.25 numerical aperature on the 100x lens means I might be able to see bacteria (I hope) if I do oil immersion.
As far as yeast counts goes, from what I've read, apparently you should only expect to get order of magnitude anyway, so, aside from checking vitality, I'm not sure it offers any more accuracy than measuring the volume of self-consolidated yeast cake.
Blacksmith1
Captain Cheap
That's pretty cool. And a lot less than I expected at first glance.
What agent are you guys using to unflocullate the yeast to get a more accurate cell count? Sulfuric acid, or something else?
This guy found a number of different unflocculators: http://braukaiser.com/blog/blog/2012/10/03/yeast-un-flocculation-for-cell-counting/
The trick is finding one that doesn't also kill the yeast cells.
Ideally, I'd like to get a bottle of pre-made solution (whether it be sulfuric acid or something else) that I can use on Day 1 to unflocculate the yeast without killing them. Anyone know of such a bottle with the right stuff already in it that I can simply buy on, say, amazon.com, or similar?
This guy found a number of different unflocculators: http://braukaiser.com/blog/blog/2012/10/03/yeast-un-flocculation-for-cell-counting/
The trick is finding one that doesn't also kill the yeast cells.
Ideally, I'd like to get a bottle of pre-made solution (whether it be sulfuric acid or something else) that I can use on Day 1 to unflocculate the yeast without killing them. Anyone know of such a bottle with the right stuff already in it that I can simply buy on, say, amazon.com, or similar?
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Yup, I placed an order for the same kit.
I ordered phosphoric acid as a deflocculator.
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Reporting back: I received the microscope today, and I prepared a yeast slide from a mead that I'm brewing. I can see the yeast. It's fairly boring to look at, though, because there is no action or even any visible movement.
Um, I'm pretty sure the yeast's job is not to entertain you. You might want to think about puppies as a hobby.
Um, I'm pretty sure the yeast's job is not to entertain you. You might want to think about puppies as a hobby.
