Author Topic: Counting Yeast Slurry with Hemacytometer  (Read 981 times)

Offline Tristan

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Counting Yeast Slurry with Hemacytometer
« on: March 19, 2011, 06:57:25 AM »
When you count yeast from a slurry do you sample from the thickest portion, or mix it all up and sample that?

Here is why I ask.  I racked a Munich Helles yesterday in the middle of my Baltic Porter brew day.  Originally, the helles was pitched at the proper rate of 1.5 million cells X 11.25 Plato X 23000ML of WLP830.  The Helles is at FG, 1.010 from 1.045; 14 days into fermentation.  My fermentation was picture perfect.  5 days at 49, krausen started to drop and I ramped to 60 for 5 days, then slowly reduced temperature to 50.  The beer had cleared fairly well for this stage and there was a nice yeast cake on the bottom.

Here is the rub, I had about 2L of beer left after racking to a keg.  I mixed together the slurry and the beer and poured into a sanitized gallon jug.  I had 3.5L by volume.  I mixed thoroughly and took 1ML of that mix and diluted it 10:1 to do a count.  I hoped to determine how much slurry to pitch into my 6 gallon batch of 1.089 Baltic Porter.  My count was 139 cells in 5 squars * 25 * 10,000 times my dilution of 10.  This gives me 69 Million cells per ML of the original mixed slurry/beer.  This seems pretty low.  I had originally taken 1L of this and pitched it into the Baltic Porter after oxygenating.  I realized that this was grossly underpitching, so I pitched the remainder of the yeast cake into the porter.

Each time I mixed the slurry, after a few minutes I saw a nice compact layer at the bottom.  I'm not sure that my mixture is showing a true count of cell density.  Hence the question above.  Is it best to stick a pipet directly down into the bottom of the slurry and obtain the thickest part for counting? 

I feel I made an error in not letting the yeast cake cold crash over night so I could decant the beer and get a better picture.  Am I way off base?  Is it possible that I only got 241 billion cells from the yeast cake?  That seems incredibly low!  I'm guessing my count was way off and I ended up over pitching.  I'd rather do that then not pitch enough.  Either way, it will make beer, hopefully good beer.  Brew day went perfect until the yeast got involved.   ;D
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Offline narvin

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Re: Counting Yeast Slurry with Hemacytometer
« Reply #1 on: March 19, 2011, 07:48:11 AM »
You'll get the same total number of cells whether you have 69 million cells per ml X the volume of the large, diluted sample or a higher concentration X the smaller volume of the yeast cake.  If you sampled from the yeast cake, you just have to dilute by a larger factor to make it countable.

It could be that you didn't agitate enough and all of the yeast wasn't in suspension.
« Last Edit: March 19, 2011, 07:54:16 AM by narvin »
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Offline a10t2

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Re: Counting Yeast Slurry with Hemacytometer
« Reply #2 on: March 19, 2011, 08:17:48 AM »
Are you sure you're using the correct multiplier for your hemocytometer? Mine is 50,000, for example.

Then again, for a count of 139 cells in a 10:1 dilution, you would have to have a very very thin slurry. That's roughly what I'm used to seeing for a 100:1 dilution, and the thinnest slurry I've ever measured was still >100 million/mL.
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Offline Tristan

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Re: Counting Yeast Slurry with Hemacytometer
« Reply #3 on: March 19, 2011, 08:49:32 AM »
Thanks for the input you guys.  My guess is that I didn't mix well enough and I have more yeast than what the count revealed.  I would think 6 gallons of 1.045 munich helles would yield more than enough yeast for a 1.089 Baltic Porter.  Atleast, that's what I'm hoping.  It's hard to tell how much I actually pitched into the porter.  Here are my plans to fix the problem next time:

1)  Collect the yeast 1-2 days prior and let the sample cold crash, collect in 4L erlenmeyer flask, that's been heat sanitized with a bit of water on the stove, with stir bar inside
2)  Decant the liquid
3)  Run the sample on the stir plate for 10-15 minutes prior to collecting sample
4)  Dilute 100:1 - 1 ML diluted with 99ML of RO water

Does that plan sound appropriate?

On the multiplication factor, 50,000 * dilution factor = density/ml is the same as the formula in the OP.  That is a simplification from 5 squares counted (138) * 5(to equal total number of squares in the counting area) * 10,000 (volume adjustment to get you up to density/ml).  It works out the same for density in ML.  The formula in the OP is from Chris White's yeast book.  The formula with 50,000 is what the Brewer's Lab Guide had in it.  At first I thought this might cause the discrepancy, but it's just a different way of working the numbers with the same results.
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Offline Tristan

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Re: Counting Yeast Slurry with Hemacytometer
« Reply #4 on: March 20, 2011, 10:09:44 AM »
Fermentation is going perfect on the Baltic Porter.  High Krausen is beginning already.  With a small amount of headspace available I may have to attach a blowoff tube!  I have the feeling that my counts were artificially low and that I ended up pitching more than the ideal rate.  Worse case scenario is that there isn't as much yeast character.  This is a good lesson for next time!  In the meantime, I'm going to follow Charlie's advice, RDWHAHB.
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Offline a10t2

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Re: Counting Yeast Slurry with Hemacytometer
« Reply #5 on: March 20, 2011, 10:25:36 AM »
Sounds pretty similar to what I do. I don't bother with a stir plate though; I just use sample jars with lids and shake them thoroughly before diluting or pulling a sample. Sorry for the confusion on the multipliers. I couldn't tell for sure what the overall factor was originally.
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Offline Tristan

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Re: Counting Yeast Slurry with Hemacytometer
« Reply #6 on: March 20, 2011, 10:44:34 AM »
Sounds pretty similar to what I do. I don't bother with a stir plate though; I just use sample jars with lids and shake them thoroughly before diluting or pulling a sample. Sorry for the confusion on the multipliers. I couldn't tell for sure what the overall factor was originally.

No problemo and no apology needed!  Thanks for the advice man!  It's good to think about these equations and to understand the purpose of each piece. 

I'm pumped to have access to these tools to make my beer better!  I wonder what percentage of home brewers are using microscopes and hemacytometers.  I would guess it's 1% or lower?  Which is why I'm thankful for this forum.  It seems to have a higher percentage of home brewers that fall into the "10th level beer nerd" category  ;D

Also, looking at the chart from Chris White's yeast book, it looks like 2 yeast packages in 20 liters (which is close to what the helles was) would yield the equivalent of 8.5 yeast packages.  Hopefully, I wasn't too far off on my pitching rate using almost the whole yeast cake!
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