Yeast Cell Counting And Slurry Volumes...

[Sammus]

Alright, so I whip up an IIPA the other day, and after all my efforts of building up a starter for this monster (which actually happened to come out much smaller than expected...stupid inefficiency) I had about 1cm of compacted white yeast on the bottom of a 500ml jar (after washing).

OK, so I'm thinking all these people talk about needing 1-2L of slurry for a good start. At the time I was thinking "damnit I couldnt get anywhere near 1-2L, this is only like 50ml! hopefully this will get it started before an infection sets in". Well needless to say, it took off like a rocket.

My question is why the hell do people talk in "yeast slurry"? that has to be the most ambiguous useless descriptor ive come across in brewing. I'm guessing a 1L of slurry is pitching a yeast into 1L of wort and shaking it up once its fermented out. Thats bad enough in itself, because depending on how much yeast you start with, and if/how you step up to 1L, the cell count would have to vary lot (not even mentioning type of yeast and temperature and other environmental factors).

That off my chest, I get to my question. Can someone tell me the approximate number of cells in the compact white sludge you get after putting a washed yeast/water mixture in the fridge for a few days? I can't seem to find it anywhere (I thought this would be the most common way of approximating yeast cell count?)

I did find this topic, but that relies on the percentage of solids after settling. I can't see how that makes it any better? when I wash my yeast, I'm sure I add a different amount of water each time I fill the jar a shake it, and then once the yeast has packed down in the fridge, I keep changing the water on top till it remains clear. once its clear I only bother keeping it under about 1cm of water. So the percentage of solids doesnt make any sense here.

I am aware that the tight packed white layer isnt pure viable yeast, but surely its going to have a roughly similar percentage of viable yeast from culture to culture. Even if it isnt all that similar, itd sure as hell have to closer than unwashed trub shaken up in the starter method.

 

[mfdes]

Hi Sammus,

Can't answer about the yeast cell concentration in slurry, but what I normally see written here (which makes sense) is stuff like 'pitch the slurry from a 2L starter...' which means make a 2L starter, let it settle and toss out the supranatant (you don't want all that oxidised beer in it, especially if you used a magnetic stirrer), then pitch the slurry.

From my last 1L starter on a stirrer I got approximately 100mL of slurry, but this was hefeweizen yeast. Others may yield less.

MFS.

 

[Keifer]

Sammus, have you seen the mr malty calculator? it may help a little...

http://www.mrmalty.com/calc/calc.html

Theres also the wyeastlabs one, which sorta looks like the mr malty one (or vice versa)

http://www.wyeastlab.com/hb_pitchrate.cfm

 

[Sammus]

Sammus, have you seen the mr malty calculator? it may help a little...

http://www.mrmalty.com/calc/calc.html

Theres also the wyeastlabs one, which sorta looks like the mr malty one (or vice versa)

http://www.wyeastlab.com/hb_pitchrate.cfm

Yeah I've seen them, not really useful, as it again relies on "non-yeast percentage". Something which In my circumstances (using clean compacted yeast) varies significantly. I could have my 50ml of compacted yeast stored under 50ml of water or 500ml, its the same amount of yeast, but the non yeast percentage varies dramatically. Thats why these calculators are no use - I think they mean after a starter has fermented out put it straight in the fridge to settle, then measure the yeast/non-yeast percentage...

edit: a closer inspection showed that I also need to know the yeast concentration, which is what im trying to work out...

 

[Steve]

Sammus you need 1-2 litre starter from the original wyeast pack to pitch into a new brew.....you only need a cup full of slurry from the bottom of primary for your next brew.
Cheers
Steve

 

[Sammus]

Sammus you need 1-2 litre starter from the original wyeast pack to pitch into a new brew.....you only need a cup full of slurry from the bottom of primary for your next brew.
Cheers
Steve

The problem is, that I like to wash my harvested primary sludge before pitching it - this process undoubtedly will cause you to lose a few million yeast cells along the way, and I'd like to know how much yeast I have left afterwards.

Also when I propagate yeast before pitching it into a brew, I like to wash it and split it up, and would like to know how much yeast I have at the end of it...

 

[Steve]

Also when I propagate yeast before pitching it into a brew, I like to wash it and split it up, and would like to know how much yeast I have at the end of it...

maybe have a look on the Wyeast or Whitelabs websites. Or send them an email......Why do you wash brand new yeast before splitting it?

 

[Trent]

Sammus
When wyeast put together their packages, they work on about 1.2 billion cells per mL. I believe that the whitelabs ones are around 4.5 billion cells per mL. I emailed Jamil about this very subject, regarding his calculator, and he said that "yeast solids", calculated on his thing to be 4.5bil/mL, are basically gotten by pure yeast that has been vibrated down to what you see in a whitelabs vial.
According to him, washing your yeast and getting some pure white stuff in the bottom of your jar, could be around 2.4 billion cells per mL, though he also said it would be VERY hard for him to estimate without seeing it.
As far as the "non yeast" component on the calculators, they are not talking about the water, but say you have 50mL of yeast in the bottom, there may be 25% trub, dead yeast cells, whatever, and hence, you do not have 100% yeast. The calculator is very good, it is just up to you to estimate what percentage of viable yeast is in your sample there, and take really good notes on the fermentation, flavours, esters, etc... and see how you fare. Heaps of esters means you probably underpitched a little, no esters can mean that you overpitched, etc...
I would work on 2 billion cells per mL, and if you wanna pitch into a 1084 IIPA (20Plato) you will want about 15 million yeast cells per mL of wort (0.75 million cells, per degree plato, per mL of wort)
15,000,000 x 23,000 = 345 billion cells you will need. If you have 2 billion cells per mL, then you want about 170mL of that yeast slurry (sorry, but that is what I call it) sitting on the bottom of your 500mL container. If you think you dont have enough, then pitch that yeast into a 2L starter, let it ferment out and THEN pitch the slurry (what has settled onto the bottom) from that.
Either that, or just pitch what you have, aerate really well, and hope for the best.
Hope this made sense, if ya have any other questions, just PM me.
Hate to try and say that you are wrong about the calculators, but once you have them dialled in for your system, they should work fine.
All the best
Trent
EDIT Spelling

 

[Sammus]

maybe have a look on the Wyeast or Whitelabs websites. Or send them an email......Why do you wash brand new yeast before splitting it?

I don't wash the yeast straight out of the packet, I propgate it first buy pitching into wort. At the end of there is always brown crap all through the yeast that settles that I like to get rid of.

Sammus
When wyeast put together their packages, they work on about 1.2 billion cells per mL. I believe that the whitelabs ones are around 4.5 billion cells per mL. I emailed Jamil about this very subject, regarding his calculator, and he said that "yeast solids", calculated on his thing to be 4.5bil/mL, are basically gotten by pure yeast that has been vibrated down to what you see in a whitelabs vial.
According to him, washing your yeast and getting some pure white stuff in the bottom of your jar, could be around 2.4 billion cells per mL, though he also said it would be VERY hard for him to estimate without seeing it.
As far as the "non yeast" component on the calculators, they are not talking about the water, but say you have 50mL of yeast in the bottom, there may be 25% trub, dead yeast cells, whatever, and hence, you do not have 100% yeast. The calculator is very good, it is just up to you to estimate what percentage of viable yeast is in your sample there, and take really good notes on the fermentation, flavours, esters, etc... and see how you fare. Heaps of esters means you probably underpitched a little, no esters can mean that you overpitched, etc...
I would work on 2 billion cells per mL, and if you wanna pitch into a 1084 IIPA (20Plato) you will want about 15 million yeast cells per mL of wort (0.75 million cells, per degree plato, per mL of wort)
15,000,000 x 23,000 = 345 billion cells you will need. If you have 2 billion cells per mL, then you want about 170mL of that yeast slurry (sorry, but that is what I call it) sitting on the bottom of your 500mL container. If you think you dont have enough, then pitch that yeast into a 2L starter, let it ferment out and THEN pitch the slurry (what has settled onto the bottom) from that.
Either that, or just pitch what you have, aerate really well, and hope for the best.
Hope this made sense, if ya have any other questions, just PM me.
Hate to try and say that you are wrong about the calculators, but once you have them dialled in for your system, they should work fine.
All the best
Trent
EDIT Spelling

Excellent reply Trent, cheers. I never thought the calculators were wrong or bad, I just didnt think they could be used for what I want, but I guess they can.

And I guess I must lose a lot of yeast when washing, because my 50 odd ml compacted at the bottom of a jar came from washing the entire yeast cake from a 1.050 APA which had fermented out, and I imagine that it wouldve had to to contain enough yeast to do something slightly bigger Which brings me to my next question...

The pitching rate from those calculators? Do you (or anyone reading this) know where that rate comes from? surely the pitching rate depends on the type of yeast you are using, and the type of beer your a brewing (more specific than ale, lager or hybrid).
I did head somewhere that zero lag time is bad - it's good to underpitch all the time, as during the yeast growth phase their metabolism will break down/change certain compounds and it is important to the flavour profile of any beer that this happens.

S

 

[jamilz]

The pitching rate used in the Mr Malty calculator is 0.75 for ales and 1.5 for lagers. I think I originally got that rate from George Fix, many years ago. I'm not sure if George worked that out himself or got those numbers from somewhere else. However, I did a number of experiments with pitching rates at that time, trying lower numbers (down to the amount in the 50 ml Wyeast and the original amount in the White Labs vials 30-50 Billion) and higher numbers (up to the theoretical number needed to ferment the wort with no cell growth). What I found was that (for my brewing) the .75/1.5 numbers were very close to ideal in the majority of cases. George was right.

Certainly there are different worts (or beer styles) that require tweaking that number. For example, a number of folks, including Dave from Wyeast, feel that a lower pitching rate on Bavarian Hefe is important, as it will create more of the esters and phenolic compounds key to that style. (I don't like too bold a hefe, so I stick with the higher rate.) On really big beers, you may need a higher rate. It really depends on the flavor/aroma profile you're striving for in a beer and the yeast being used, fermentation temp, oxygen available, etc. Many, many factors.

To really know out how many cells you have in a given yeast cake (when the yeast is settled in the bottom of your container) or in a slurry (when the yeast is suspended in liquid), you need to do cell counts with a microscope. Once you've done that a few times it becomes clear that cell sizes vary considerably by strain and by how the yeast were handled up to that point. They can be a lot larger or smaller than usual based on environment.

Like Trent says, you'll want to take measurements, keep notes, and then adjust from there. Start out with an assumption on the amount of cells in a yeast cake (and an assumption on the amount of trub) and see if that gives you good results. If not, adjust up or down and see if things improve. Eventually, for your brewing habits and your yeast management techniques, you'll have things dialed in to work for you.

On the calculator, if you look at the repitching tab it has a slider for adjusting the amount of non-yeast solids (this is trub, break material and hop bits, etc.) in the yeast cake. And a slider for adjusting the density of the yeast cake. The default settings are probably pretty close to what most folks have after the yeast has sat in the fridge for a couple days. If you're doing lots of washing, perhaps the non-yeast portion is a little smaller, but it is hard to know without seeing your yeast.

If you have access to White Labs vials, look at one of those. They contain 35ml of liquid and yeast. When they get packed down tight (which is usually how they are unless someone shook it up) it is the same as moving the calculator slider all the way to the thickest setting and the lowest non-yeast percentage setting. About 22 ml of yeast solids = 100 billion cells for many yeast strains.

I built both the Mr Malty calculator and the Wyeast calculators. They both use the same yeast starter growth curve, but attack the question from two different angles. The Wyeast calculators tell you how much yeast you should get when you tell it how big a starter you're making. The Mr Malty calculator tells you how big a starter you need based on the beer you want to make. (It also handles dry yeast and repitching yeast from a previous batch, which are not in the Wyeast calculators.)

I hope that helps answer some of your questions.

 

[Trent]

There ya go Sam
An answer from Jamil himself - ya cant ask for much more than that! Welcome to the site, Jamil.
All the best
Trent

 

[///]

Sammus - PM your address, I have a little yeast management to send thru.

The Brewery Yeast Management guide fro wyeast states;

Ale - 1 million cells/ml/ per degrees plato
Lager - 2 milllion cells/ml/ per degrees plato

Accurate and consistent pitch rates can be calculated after perfrming a cell count and viability test. However, without a microscope and lab equipment, yeast is usually pitched by weight or volume and involves some guesswork and trail and error. Percentage of yeast solids to volume of slurry can be found by allowing a sample to sediment under refrigeration and estimating yeast pack. Do not include trub with estimation. Average thick slurries will contain 20-40% yeast solids, corresponding to 0.75 - 1.5 x 10 to the power of 9 cells/ml.

Recommended approx amounts also differ for above and below 16 degrees plato.

 

[etbandit]

Has anyone tried yeast counting with a hemacytometer and trypan blue staining (a standard laboratory protocol for determining cell viability by distinguishing live from dead cells)?

Has anyone and experimented with pitch rates based on yeast counts with a microscope and a hemacytometer?

Interested to know what your results were.

 

[Darren]

ET,

I have done counts with hemocytometer and trypan blue. Couldn't be bothered these days. Just make a healthy starter and be done with it.

cheers

Darren

 

[Sammus]

Has anyone tried yeast counting with a hemacytometer and trypan blue staining (a standard laboratory protocol for determining cell viability by distinguishing live from dead cells)?

Has anyone and experimented with pitch rates based on yeast counts with a microscope and a hemacytometer?

Interested to know what your results were.

Theres a Slideshow on the bjcp website (here) in which they experiment with lots of the variables and evaluate the tasters reactions.

Also I the MrMalty and Wyeast Calculator were written based on extensive lab testing and tasting. Someones already done the hard work for us

 

[etbandit]

ET,

I have done counts with hemocytometer and trypan blue. Couldn't be bothered these days. Just make a healthy starter and be done with it.

Darren,

With trypan blue staining, what was the % cell viability you usually obtained?

Did you experiment with pitching different amounts after counting with a hemocytometer, and how did it change the flavour of your beer etc.?

ETB

 

[Darren]

ET,

90-95% viable. Never bothered to count/adjust pitched amounts. I am just too lazy to do that. Plus the gazzillion other variables.

cheers

Darren

 

[mobrien]

Great topic, and exactly what I'm thinking about as I prepare to reuse the first lot of yeast from my conical.

I am curious about the washing of the yeast - I've heard and read arguments that support both sides - some saw wash, some say don't.

Given the current topic and the people talking here - I'm curious what the pros and cons in your opinion are? And if you think you should wash, what is your method?

This is taking this thread off on a bit of an angle, but I think it still applies - if someone thinks this is too far off topic, I'm happy for the mods to move it, or I'll start a new thread.

Matt