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General Category => Yeast and Fermentation => Topic started by: anje on March 01, 2012, 01:22:19 pm

Title: Determining yeast numbers?
Post by: anje on March 01, 2012, 01:22:19 pm
I've only made a few batches of beer so far (first was quite good, second's still conditioning but seems promising, third's still fermenting), all with dry yeast. From reading here, it seems like one of the modifications I'm going to want to make soonest is to make yeast starters and start pitching them in larger quantities.

So I keep reading that it's important to pitch the correct amount of yeast for a good flavor profile, and I see calculators.  But how do you determine how much yeast you have? I don't have a spectrophotometer in my kitchen, and by the time I'd done plate counts the numbers would change significantly, even if I had the patience and equipment.  Surely numbers vary substantially based with the gravity of the starter wort and any limiting yeast nutrients, so we can't go by volume of a stationary phase culture when the yeast have flocculated, can we?
Title: Re: Determining yeast numbers?
Post by: narvin on March 01, 2012, 01:26:28 pm
I've only made a few batches of beer so far (first was quite good, second's still conditioning but seems promising, third's still fermenting), all with dry yeast. From reading here, it seems like one of the modifications I'm going to want to make soonest is to make yeast starters and start pitching them in larger quantities.

So I keep reading that it's important to pitch the correct amount of yeast for a good flavor profile, and I see calculators.  But how do you determine how much yeast you have? I don't have a spectrophotometer in my kitchen, and by the time I'd done plate counts the numbers would change significantly, even if I had the patience and equipment.  Surely numbers vary substantially based with the gravity of the starter wort and any limiting yeast nutrients, so we can't go by volume of a stationary phase culture when the yeast have flocculated, can we?

For cell counts, you'd use a microscope and a hemacytomer -- basically a slide that holds a known volume and has a grid etched into it for counting.  It's not all that expensive, but most of the time unnecessary.  Other people have done the work and made it pretty easy to estimate your cell count by starter size or slurry volume.  Obviously, you're still just estimating, and not taking into account things like the viability, but it's a repeatable process that you can use to get results you like over and over again.  Check out mrmalty.com for Jamil's calculator.
Title: Re: Determining yeast numbers?
Post by: anje on March 01, 2012, 01:53:53 pm
True, there are direct counts, though acquiring a respectable microscope seems easily as costly as a (basically indestructible) Spectronic 20. Slightly less bulky, though.
Title: Re: Determining yeast numbers?
Post by: tschmidlin on March 01, 2012, 11:53:21 pm
True, there are direct counts, though acquiring a respectable microscope seems easily as costly as a (basically indestructible) Spectronic 20. Slightly less bulky, though.
I should get one of those, maybe they have one at UW surplus, a spec is way faster. :)

If you're going to stick with dry yeast, I think it is better to just buy an extra pack than to make a starter.  If it's not cheaper (and it might be) it is way easier and not worth it to save a little money.

If you're going to use liquid though, since that is more expensive, it makes more sense.  The online calcs are fine, but if you have access to a spec at work you can always take some samples of your finished starter in and measure them.  Once you have a feel for how your starters are performing you won't need to take samples any more.
Title: Re: Determining yeast numbers?
Post by: thcipriani on March 02, 2012, 10:55:26 pm
Any good links to resources for using a spec in brewing?
Title: Re: Determining yeast numbers?
Post by: tschmidlin on March 03, 2012, 01:07:18 am
Any good links to resources for using a spec in brewing?
I've got a sheet somewhere that correlates the spec reading with the density of the culture and thus cell counts.  It is not necessarily that accurate, but it will get you in the ballpark and if you want you can dial it in better for a specific strain by doing some plating and staining/scope work at the same time.  The sheet I have was generated for a common lab strain (S288C) and is good enough for a lot of lab work.  It's probably good enough for pitching, but I'd probably test it.

The only other use I can think of for a spec is determining beer color.  It's the ASBC standard method last time I looked.
Title: Re: Determining yeast numbers?
Post by: thcipriani on March 03, 2012, 08:18:12 am
The other application that I think would be really awesome is determining FAN content for meads.

Looks like you could also do IBU: http://beersensoryscience.wordpress.com/2011/04/05/the-ibu-assay/

Also, looks like, could be used for carbon-filtered water chlorine, diacetyl, etc http://www.probrewer.com/vbulletin/archive/index.php/t-18167.html

I have to say that the lab part is one of my favorite parts of the process – almost as addictive as the brewing   ;)
Title: Re: Determining yeast numbers?
Post by: dimik on March 03, 2012, 11:10:27 am
I would trust microscope with hemocytometer rather than specs or plates.
Title: Re: Determining yeast numbers?
Post by: tschmidlin on March 03, 2012, 04:00:54 pm
The other application that I think would be really awesome is determining FAN content for meads.

Looks like you could also do IBU: http://beersensoryscience.wordpress.com/2011/04/05/the-ibu-assay/

Also, looks like, could be used for carbon-filtered water chlorine, diacetyl, etc http://www.probrewer.com/vbulletin/archive/index.php/t-18167.html

I have to say that the lab part is one of my favorite parts of the process – almost as addictive as the brewing   ;)
Good points, I was thinking about things you could do that didn't require hard to find or expensive reagents.  The need for iso-octane and octanol makes IBU determination a pain.  I don't know what might be required for the other assays.
Title: Re: Determining yeast numbers?
Post by: tschmidlin on March 03, 2012, 04:02:00 pm
I would trust microscope with hemocytometer rather than specs or plates.
Sure, it will be more accurate.  But it takes a long time, and the spec should be accurate enough while only taking a few seconds.
Title: Re: Determining yeast numbers?
Post by: anje on March 03, 2012, 08:39:09 pm
I would trust microscope with hemocytometer rather than specs or plates.
Plate counts would be the most accurate, since they'd give a viable count. However, you have to have decent technique (change pipettes/tips at each point or your numbers end up completely screwed up, and good ways to accurately measure volumes) and plenty of plates. Easy enough for me to manage in my lab with lots of micropipettes and access to an autoclave, but I'd hate to attempt it in the tiny kitchen in my apartment.  In an ideal world you do growth curves correlating the optical density (probably at 600nm) with the plate counts in a growing sample -- again, this requires lots and lots of plates and seems like a hassle for someone like me who lacks dedicated space and equipment to do work with food organisms.
Title: Re: Determining yeast numbers?
Post by: tschmidlin on March 03, 2012, 09:18:21 pm
I would trust microscope with hemocytometer rather than specs or plates.
Plate counts would be the most accurate, since they'd give a viable count. However, you have to have decent technique (change pipettes/tips at each point or your numbers end up completely screwed up, and good ways to accurately measure volumes) and plenty of plates. Easy enough for me to manage in my lab with lots of micropipettes and access to an autoclave, but I'd hate to attempt it in the tiny kitchen in my apartment.  In an ideal world you do growth curves correlating the optical density (probably at 600nm) with the plate counts in a growing sample -- again, this requires lots and lots of plates and seems like a hassle for someone like me who lacks dedicated space and equipment to do work with food organisms.
Not to mention that for a plate viability assay you've got to let the plates go overnight at a minimum, in order for the colonies to get big enough to see.  It's easier to count if you wait another night though.
Title: Re: Determining yeast numbers?
Post by: dimik on March 03, 2012, 10:16:15 pm
Yeah but if you screw up your dilutions, there goes your count. And the time it takes... I'd just go trypan blue + hemocytometer route or just count and reduce to 80% as a general rule. If you're really into yeast counting and ranching you'll have to invest in a microscope any way you look at it. I don't think that a few seconds you might save with OD, if any at all since you have to calibrate with the same pre-fermentation starter, is worth sacrificing the accuracy.
Title: Re: Determining yeast numbers?
Post by: tschmidlin on March 04, 2012, 12:01:12 am
Yeah but if you screw up your dilutions, there goes your count. And the time it takes... I'd just go trypan blue + hemocytometer route or just count and reduce to 80% as a general rule. If you're really into yeast counting and ranching you'll have to invest in a microscope any way you look at it. I don't think that a few seconds you might save with OD, if any at all since you have to calibrate with the same pre-fermentation starter, is worth sacrificing the accuracy.
Few seconds?  How fast can you count with a hemocytometer?  A lot faster than me apparently. :)

Running two samples on a spec will take less than a minute.  Besides that, if you can't dilute it without messing it up then your hemocytometer counts will be off anyway.

I think you can totally get by without a microscope.  Once you eliminate counting what do you really need it for?  That's not to say it's not awesome to have one, but you really don't have to have one for yeast ranching.
Title: Re: Determining yeast numbers?
Post by: dimik on March 04, 2012, 12:22:58 am
Meh, maybe so. I've been counting cells for a long time and it's really easy after a while. You just count 40 and then directly convert by 0.1 into E6.
But on the other hand, would you really get a spec just to count cells? I mean I can see a use for a scope to look at stuff at home, but a spec? I guess you could use it for gravity measurements after playing around with it...
Title: Re: Determining yeast numbers?
Post by: tschmidlin on March 04, 2012, 03:04:37 am
I probably wouldn't get one, but scopes hurt my eyes.  i don't do much scope work anyway, but I've been spoiled by the ones with video cameras hooked up to computers so I can just look on the screen and see everything.
Title: Re: Determining yeast numbers?
Post by: bluesman on March 04, 2012, 09:13:00 am
As much as I'd like to get a scope, I find that I get headaches from eye strain. Now a scope with video is another story but that's out of my price range for sure. The question of acquiring a scope and hemocytometer is a decision that's still up in the air for me.

Tom...do you know of any good resources for using a spec in brewing?
Title: Re: Determining yeast numbers?
Post by: tschmidlin on March 05, 2012, 12:09:30 am
The assays we talked about earlier are the main ones, but there might be others.  Don't expect to be able to test IBUs unless you spend a few hundred dollars on reagents - iso-octane, octanol, and HCl come to mind.  The nice thing is once you have those reagents you'll be able to run a LOT of samples, it doesn't take much per sample.  But it's a pretty big initial outlay.  Plus you need a quartz cuvette, a plastic one isn't going to hack it with those solvents in there.
Title: Re: Determining yeast numbers?
Post by: thcipriani on March 06, 2012, 07:14:41 am
The question of acquiring a scope and hemocytometer is a decision that's still up in the air for me.

I think everyone ought to own a scope given how cheap they are. You can get a cell count with 400x.
I use this scope and it's been great for years:
http://www.ebay.com/itm/ws/eBayISAPI.dll?ViewItem&item=380248813512

WRT eye-strain, I know you can buy adapters for your dslr or other cameras that'll replace the eyepiece.
Title: Re: Determining yeast numbers?
Post by: dimik on March 06, 2012, 06:10:31 pm
Yeah, you can get a camera easy. I have one though looking into the eye piece looks way better.
Title: Re: Determining yeast numbers?
Post by: Jimmy K on March 07, 2012, 08:09:39 am
As much as I'd like to get a scope, I find that I get headaches from eye strain. Now a scope with video is another story but that's out of my price range for sure. The question of acquiring a scope and hemocytometer is a decision that's still up in the air for me.

Tom...do you know of any good resources for using a spec in brewing?

What you need is a nice flow cytometer. They are only around $150,000 ... used.
Title: Re: Determining yeast numbers?
Post by: dimik on March 07, 2012, 08:31:24 am
Unless his yeast express GFP or something I don't think it would be a good way of cell counting. Otherwise it'll just be same as counting without determining viability. But then again he'd probably be able to get the total number of cells without projecting calculations... Hahaha, honestly I think it's the funniest post so far hehehehehe.
Title: Re: Determining yeast numbers?
Post by: a10t2 on March 07, 2012, 08:51:01 am
Few seconds?  How fast can you count with a hemocytometer?  A lot faster than me apparently. :)

After doing several a week, for several months, I'm down to <10 min total. That includes diluting the sample and methylene blue, prepping the plate, doing three counts at ~100 cells, and cleanup. Once you get a good system going, the counts are the fastest part.
Title: Re: Determining yeast numbers?
Post by: bluesman on March 07, 2012, 09:11:49 am
As much as I'd like to get a scope, I find that I get headaches from eye strain. Now a scope with video is another story but that's out of my price range for sure. The question of acquiring a scope and hemocytometer is a decision that's still up in the air for me.

Tom...do you know of any good resources for using a spec in brewing?

What you need is a nice flow cytometer. They are only around $150,000 ... used.

Maybe...if I hit the powerball.  :)
Title: Re: Determining yeast numbers?
Post by: tschmidlin on March 07, 2012, 10:16:37 am
Unless his yeast express GFP or something I don't think it would be a good way of cell counting. Otherwise it'll just be same as counting without determining viability. But then again he'd probably be able to get the total number of cells without projecting calculations... Hahaha, honestly I think it's the funniest post so far hehehehehe.
You can use propidium iodide as a viability stain for a flow cytometer.

Few seconds?  How fast can you count with a hemocytometer?  A lot faster than me apparently. :)

After doing several a week, for several months, I'm down to <10 min total. That includes diluting the sample and methylene blue, prepping the plate, doing three counts at ~100 cells, and cleanup. Once you get a good system going, the counts are the fastest part.
That's faster than me, but I don't do them regularly.  Still not as fast as a spec though. ;)
Title: Re: Determining yeast numbers?
Post by: dimik on March 07, 2012, 11:39:21 am
Heh, you're right! If he invests into flow cytometer, might as well get PI hehehe, just don't run the entire starter with it.
Title: Re: Determining yeast numbers?
Post by: anje on March 08, 2012, 02:56:48 pm
Flow cytometer is a bit out of my budget right now, unfortunately. (After all, I'm still starting out here. Haven't even gotten my own Pipettemen yet.) Though I do have a friend who was trying to develop a field-size or possibly pocket-size one for her degree....
Title: Re: Determining yeast numbers?
Post by: Jimmy K on March 09, 2012, 06:31:52 am
Pocket size flow cytometer?
Title: Re: Determining yeast numbers?
Post by: tschmidlin on March 09, 2012, 11:47:34 am
That would be awesome :)
Title: Re: Determining yeast numbers?
Post by: dimik on March 09, 2012, 01:22:25 pm
But impossible :)
Title: Re: Determining yeast numbers?
Post by: tschmidlin on March 09, 2012, 01:28:04 pm
Depends on how big your pockets are ;D
Title: Re: Determining yeast numbers?
Post by: anje on March 09, 2012, 01:37:05 pm
She wasn't extremely clear on the final dimensions she expected it to have. But it was to be field-size, using a bunch of fluidics and flow cell design that's way beyond me.
Title: Re: Determining yeast numbers?
Post by: bluesman on March 09, 2012, 06:14:01 pm
Pocket size flow cytometer?

Now that sounds like a plan!  :)
Title: Re: Determining yeast numbers?
Post by: dimik on March 09, 2012, 08:56:53 pm
Would be sweet if it was physically and financially possible.