Thursday, October 16, 2008

Counting yeast

So, as promised, here's some detail on my yeast counting adventure the other night...

I ordered a smack pack of the Wyeast Danish Lager yeast about two months ago. I knew that I'd need to build up a starter in order to have enough healthy yeast to pitch into my 5.5 gallons of 1.054 wort. So, about four days before brew day I made a starter:

3.5 liters of 10˚ Plato wort boiled with 1/2 tsp. of yeast nutrient. Cooled it to room temperature, pitched the smack pack, and set it on the stir plate to ferment out and build up the yeast count. Once fermentation was finished, I crashed it to about 32˚ until brew day. Decanted the "beer" off the top of the yeast slurry (by siphoning, not pouring) and was left with about 300ml of yeast slurry to pitch into my wort.

Here's everything needed to do the job:
  1. 4000ml erlenmeyer flask - I have 1000ml and 2000ml flasks, but they just don't do the job. No room for enough wort to build big yeast counts, and more importantly, not enough room for krausen. Had too many overflow problems with the smaller flasks.
  2. 10˚ Plato Wort - I take my last runnings from brew day and boil them down to about 10˚ brix on the refractometer (brix/Plato, close enough). I then fill up mason jars with the near-boiling wort, cap, bring them down to room temp, then refrigerate until needed.
  3. Yeast nutrient - the yeast nutrient gets boiled for about 10 minutes in the flask with the wort to sanitze before making the starter.
  4. Foam stopper - these dudes are great. I get them from Northern Brewer, and they're perfect for yeast starters. easier than doing an airlock because I can keep it in the flask while I boil and sterilize the whole bit.
  5. Stir bar - I've used a few different sizes and seem to have the best luck with the smaller ones. They don't get as much of a vortex in the flask, but they don't get thrown nearly as often as the larger ones. Throw it in the boiling starter wort shortly before it's finished to sanitize it.
  6. DIY stir plate - the standard CPU fan and magnet project.
So on brew day I wanted to know about how much yeast was in that 300ml or so of slurry. How? Gotta count it on a hemocytometer under a microscope. Not exactly the easiest thing for most folks to do in their basement or garage or spare bedroom, but when you take a hobby too far....

Equipment:
  1. 10ml pipette
  2. Pipette pump (10ml)
  3. (2) 100ml graduated cylinders
  4. Hemocytometer and slide cover
  5. Methylene blue dye
  6. Microscope
I filled each of the 100ml cylinders to 90ml with distilled water. Used the pipette and pump to take a 10ml sample from the yeast slurry and add it to one of the cylinders. Now I had a 10:1 dillution of slurry. Then I took 10ml from that cylinder and added it to the other one. Now I had a 100:1 dillution - this makes it easier to count.

The theory behind the hemocytometer is that it has a 0.0001 cubic mm chamber that you can view under a microscope and count the amount of stuff that's in there. Usually blood or sperms, but brewers use them to count yeast cells. So try not to get yourself a used hemocytometer.

I added a drop of methylene blue stain to the cylinder (more on that later), used a dropper to take a sample from it, put it on the hemocytometer, and put the slide cover on. Then I viewed it under the microscope to count the cells at 400X magnification.

The 0.0001 cubic mm hemocytometer chamber is divided up into a five by five grid. You could count what you've got in all 25 boxes, but that's a lot of work. Standard practice is to count five of the boxes - the four corners and the one in the middle.

So I counted the yeast cells in five of the boxes. (My hemocytometer has two 0.0001 cubic mm chambers, so I counted both.) I counted 239 in one and 286 in the other. So now how do we figure out how much yeast is in the slurry? Multiply the cell count (239 or 286) by the proportion of boxes counted (5:1) by the dillution factor (100:1) and divide by the hemocytometer chamber volume (0.0001 cubic mm = 0.0001 ml).

(239cells*5*100)/0.0001ml = 1,195,000,000 cells/ml

or

1.195x10^9 cells/ml

or

1.430x10^9 cells/ml for the second chamber counted with 286 cells

Now, the rule of thumb is that you're supposed to pitch 1,000,000 (1.0x10^6) cells per ml of 1˚ Plato of wort. I had 5.5 gallons of 13.0˚ brix wort. 5.5 gallons = 2.08x10^4ml, and we can assume that 13.0 brix = 13.0 Plato. So I needed:

13.0˚ x (1.0x10^6 cells per ml/1˚) = 1.3x10^7 cells/ml

2.08x10^4ml * 1.3x10^7 cells/ml = 2.7x10^11 cells

So how much of my yeast slurry would give me 2.7x10^11 cells?

2.7x10^11 cells / 1.195x10^9 cells/ml = 226 ml

or

2.7x10^11 cells / 1.430x10^9 cells/ml = 189 ml

Hope you love math. Oh, and what about that methylene blue stain? The idea is that healthy yeast will reject it, and dead yeast will be stained blue under the microscope. I don't know if I didn't add enough dye or what, but I couldn't see any stained cells. So I skipped that part of the exercise.

And how exactly is this supposed to help me when my erlenmeyer flask only has 500ml marks? Not much. I guesstimated about 300ml of slurry in the flask, but it easily could have been 200. Or 250. Or 350. And then what about my 5.5 gallons of wort? That could have easily been 5.25, which would throw my calculations off by about 5%.

So what's a poor homebrewer to do? Just pitch the whole damn slurry! RDWHAHB!

3 comments:

Angela said...

What type of Microscope are you useing

Tom E said...

I'm not sure what brand it is, I got it off eBay. Here's an auction from the same seller (bh542) for the same microscope. I've been very happy with it:

http://cgi.ebay.com/40x-1600x-Binocular-Compound-Microscope-Free-Shipping_W0QQitemZ170273252098QQcmdZViewItemQQptZLH_DefaultDomain_0?hash=item170273252098&_trksid=p3911.c0.m14&_trkparms=72%3A1205%7C66%3A2%7C65%3A12%7C39%3A1%7C240%3A1318

thatguy314 said...

I'm not sure you need to make the math that complicated.

There are 9 big squares on the hemocytometer, each of them is representative. You should count at least 4 to be statistically accurate, but you can count as many of them as you want. Decide what you want to count before you take a look otherwise that affects it.

Either way, divide the # of cells by the # of big squares you counted. Each cell you count is 100,000 cells per mill. So if I count 4 squares and get 200 --> 200/4 = 50 x 10^5 = 5 million cells per ml. If you diluted the sample, then multiple by that factor.

Anyways, thinking like that makes it easier todo in your head and seems less intimidating.