Yeast cell counting vs required amount

So I count 400 cells in my hemocytometer using the simple method 5 squares of the 1 x 1 x .10 mm volume. The yeast was diluted in a 100 : 3 dilution (multiplier of 33). And I measure 175 ml of pure yeast volume - assuming 100% viability.

Doing this math give me 115 billion cells (400 x 5 x 33 x 175 x 10000).

I pitched this in a 19.5 plato wort of 11 gallons ale. Mr Malty's calculator says I need 608 billion cells.

I've got 5 times less than what is needed but the fermentation starts within 12 hours going strong. And this is what I have been seeing the last (3) batches where the amount of yeast cell that I have are a factor of 2 to 5 times less than what we are told they should be.

The wort was o2'd with pure oxygen for about 2 minutes at a low pressure (don't have a gas flow meter on the bottle). And then again about 3 hours after initially pitching.

When you look at Mr. Malty's calculator, it says 150 ml of yeast will be sufficient. But my initial calculation is well under the # of cells needed.

Any comments or suggestions from others would be welcomed as I'm scratching my head.

I'm not sure what you are looking for. You can under pitch and the yeast will still ferment. A proper pitch will give you repeatable results - optimal flavors and a timely finish.

Yeah, I think there is some skepticism among some home brewers about the pitching rates recommended on mrmalty and yeastcalc, but there is pretty widespread agreement with them too. It sounds link you want the skeptics to weigh in. I would just point out that you're mixing cases, insofar as you are counting actual yeast cells and then switching to talk about when fermentation starts and how vigorous you think it is (as if this is objectively measurable) as if those last things are dispositive. Even if we grant that your observations are valid, pitching rates are about things other than fermentation start time and vigor.

My post was intent on asking the question of what I could be doing wrong. As we see so much in home brewing there isn't necessarily a right or wrong but preferred method.

I did make the leap on my pitching rate vs 'subjective' evaluation of air lock activity. My intent once again wasn't to look for a skeptic to reply but to ask the question to the folks who might be doing / asking these questions. If I am 5 times under the 'preferred' quantity where else could I be missing it?

When I dilute my 100 drops of water to 3 drops of yeast, I'm using a glass pipet with a very thin tip. The drop size seems to be similar between the (2). Just wondering is my multiplying factor might be off with respect to this?

BB

The factor 5 puzzles me. Shouldn't there also be an additional factor 4 or 5, depending on which and how many squares you count?

The 5 multiplier is from the using the center grid of 25 squares. It is 1 mm x 1 mm. I count the corners (4) and the center (1) so I have to multiply by 5 to have the full area of (25).

[URL="https://www.homebrewtalk.com/photo/hemocytometer-grid-61305.html][/URL]

OK, I got ya. I see nothing wrong with your multipliers then. There could be somewhat variation when taking and dropping the yeast slurry, but nothing over say 5-10% from calculated, if that much.

The bigger question is how to assess the density of the yeast slurry. If Mr. Malty suggests 150ml should be appropriate and, while you've got even more at 175ml, at the same time you actually count way fewer cells than Mr. Malty predicted, something is off.

It is an interesting experiment, indeed.

We don't stand alone in finding Mr. Malty quite conservative in both calculating viability and estimating needed yeast cells for a good and active fermentation. That doesn't mean it's wrong, those could well be optimum amounts, scientifically proven and all. At the same time, it doesn't negate the fact that with less than half you can still blow off your airlock, or fail to taste stressed yeast syndrome. People have done qualitative side by side comparisons with pitching rates. I'm not sure what the outcomes were, and they'd still be highly subjective by any standard.

The 2 injections of pure O2 surely helped in the growth phase, and those may have contributed more than a larger starter would have. The yeast coming out of the starter is used to 1.040 while the yeast grown in your fermentor has adapted to your 1.081 wort.

Just checked the intermediate gravity @1.054 using the hydrometer. Still actively burping away at a control fermentation chamber temperature of 68f. BTW, i used Denny's Favorite 50 1450. My mash efficiency was 80% and rested at 150 to 152 for 75 minutes.

bbbrew said:

My post was intent on asking the question of what I could be doing wrong. As we see so much in home brewing there isn't necessarily a right or wrong but preferred method. I did make the leap on my pitching rate vs 'subjective' evaluation of air lock activity. My intent once again wasn't to look for a skeptic to reply but to ask the question to the folks who might be doing / asking these questions. If I am 5 times under the 'preferred' quantity where else could I be missing it? When I dilute my 100 drops of water to 3 drops of yeast, I'm using a glass pipet with a very thin tip. The drop size seems to be similar between the (2). Just wondering is my multiplying factor might be off with respect to this? BB

Click to expand...

Why are your units in drops and not milliliters? Your dilution should be 1ml of yeast solution into 9ml water. Continue this dilution until you can accurately count cells. You counted 400 cells in 5 squares. I would recommend another dilution.

Make sure cells are evenly spread across the entire grid. Count the four corner and center squares. Then do the same for the other grid. If there is more than a 10% difference between the two grids; take another sample from your dilution and count again. It just takes practice.

Indianaroller, I used drops just because I don't have a small graduated cylinder for 10 ml. My glass pipet has a long nose (for not knowing the technical term).

I decant off all the liquid (or as much as I can) from the starter and then just pull in yeast though that long nose of the pipet. It seem to be really consistent (creamy white) and since I only need one drop on the hemocytometer I didn't want to make a large amount. I thought this was a pretty good way of doing it.

I only count the center 1 mm x 1 mm square (25 small squares in all). And from that, I count the 4 corners and center. That's where my (5) multiplier comes in.
see here:
https://www.homebrewtalk.com/photo/hemocytometer-grid-61305.html

I wish I knew how to insert a picture directly.


BB

I checked your math and it seems correct, but I have one question and one comment.

Are you adding your yeast suspension to a dye before loading it onto your haemocytometer? When counting cells I typically add trypan-blue at a 1:1 dilution to my cells so I have the additional 2X in my equation.

An just a comment, from a perspective of a person who works in a laboratory and counts cells a lot. You dilution technique is most likely giving you your error. A single small dilution into a large volume can really exaggerate any small pipetting error that is made. And because you are not using a calibrated precision pipette I bet you are not getting a 1:33 dilution like you are thinking probably closer to 1:50-1:200 at the end of it all. You might be better off doing serial dilutions with larger volumes you can more accurately measure.

According to Chris White from white labs, and wyeast , If you have a yeast pack that is 33-40% by volume that will be approximately 1x10^9 cells/ml. So next time you are going to count yeast go to Target and get some breast milk containers that have graduated markings, and add your slurry to it and let the yeast settle then calculate how much yeast/ml you should see, then do your counting protocol and see if you are close or off.

Also, there are precision pipettes on ebay for fairly cheap. Id recommend getting a p200 or a p1000 and some eppendorf tubes so you can perform more accurate dilutions.

Edit...This popped in my head last night... If you have 175ml of pure yeast slurry that you are pulling your sample from its probable close to 3 billion cells/ml or thats what you should be expecting, and you are counting 0.6 billion/ml you are counting 5X under what is there.

That was exactly my thought. 5x less. The fermentation shows it also as its only down to 1.030 from starting at 1.081 (8) days ago.

The 5x less is what I calculated. So I have come across a pipet that has 0.20 ml increments on it. I'm going to try this out the next time I hit the smack pack.

Thanks for your suggestion.

I'm hoping that the original Denny's 50 Favorite makes it to 1.024. Even though it might have taken 12 days to get there the beer should still be good. I just want to understand the 'best' healthy yeast required to have a strong fermentation.

BB

You know, as long as you are going through the effort of counting yeast, you might want to add a fast forced fermentation test to your protocol. This test will give you the real concentration of fermentables available in the wort of the beer you are fermenting. With this test you can be certain if fermentation has ended, or stalled.

B^2,
Thanks for the suggestion. I did pick up a graduated pipet with 0.1 ml increments and used that this time to measure a smack pack directly.

I got 1.7 x 10E9 cells after doing the math so it seems that using this equipment is in the ball park of what Wyeast predicts.

Coming back to the prior issue, my American Stout that fermented out with Denny's 50 is down to 1.022 now. So it did obtain attenuation even though it took 12 days. We'll have to see how it tastes but the initial tasting from the gravity reading seems to be spot on, if you can get beyond some of the yeast flavor.

I still believe that my initial calculations for # number of yeast cells was well below what was expected and hence the slower fermentation process.

BB

Hey Bsquared, I have a pipette question you may be able to answer.

Do you suck up to a bigger graduation than you need and then drain down to a lower graduation that gives you the quantity you want, or do you suck up to the graduation you need and squirt out to empty? I hope that made sense!

Also, do you need to wet the inside of the pipettes a few times with your intended injection? Needless to say I am not an expert at this, but have graduated pippettes and two of those plastic handles with the thumb wheels.

Do you fancy doing an instructive post on this, or a nice easy to follow YouTube video for all us numpties who don't do this for a living.

Or, where can peeps get good reading or viewing info on how to be a biochemist without actually owning a brain.

Silver Brewer,
I found a 2 ml in 1/10 on ebay for 5 bucks. It's made by fisherbrand#13-665. The graduations start at the bottom and decrease as you go up. So I'm assuming you should fill it and dispense as you count down (back to the tip).

I did find an old bulb for the end. It looked clean inside but hopefully it wasn't used somewhere that it shouldn't have been. Even so, this instrument is only going to be used for measuring cells and the liquids will never go back into the fermentation population.

I figured that I should error on the low side of cells if anything, so I filled it 10 times getting an exact 20 ml of water and then added to it 0.2 ml of yeast. This way I got a factor of 100 x (20 ml / 0.2 ml).

BB

silverbrewer said:

Hey Bsquared, I have a pipette question you may be able to answer.

Do you suck up to a bigger graduation than you need and then drain down to a lower graduation that gives you the quantity you want, or do you suck up to the graduation you need and squirt out to empty? I hope that made sense!

Also, do you need to wet the inside of the pipettes a few times with your intended injection? Needless to say I am not an expert at this, but have graduated pippettes and two of those plastic handles with the thumb wheels.

Do you fancy doing an instructive post on this, or a nice easy to follow YouTube video for all us numpties who don't do this for a living.

Or, where can peeps get good reading or viewing info on how to be a biochemist without actually owning a brain.

Click to expand...

When using graduated pipets with cells, I will pipet up and down a few times to mix the cells and then just draw it up to the amount I need. There is no need to wet the inside of the pipet when dealing with cells and supernatant.

No, I don't have any instructionals or videos or anything. I think most everyone develops their own technique. I think the Yeast book from BA has a good section on sanitary pipetting techniques and yeast handling if you are looking for a good reference.

The bigger graduated pipets (1-100ml) are good for dispensing, but are not as accurate. If you are using these for pipetting yeast dilutions for counting be sure to realize the pipetting error. Ideally you should be using something like a pipetman, or other micropipet in the .001-0.10ml , 0.20-0.200ml or, 0.2-1.0ml range to get a good accurate yeast count.