# Cell Count and Dilution

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#### Begin2Brew

##### Searching for the perfect beer one at a time
This year I am looking to expand my knowledge on yeast. I am looking to start counting cells and test for viability. My current project is to determine growth in a starter to be sure I have the required pitch amount.

I am planning on brewing an Marzen with a OG of 1.055. Yeastcalc states I need 423B cells. According to the calculator I should end up with 455B cells at the end of my starters for a total of 455B cells in a final 2.5 liter starter.

So for my dilution schedule:
I have 455B cells in 2.5 liters
I need a dilution to be ~150 cells per ml to count the cells
So to check my math here is what I have for my dilutions:
455B/2500ml = 1.82B per ml
10:1 dilution = 182M per ml
1000:1 dilution = 182K per ml
1000:1 dilution = 182 per ml

Does this look right?

#### Kaiser

##### Well-Known Member
When working with a lager yeast after propagation you'll have to unflocculate it before you can count it. If you still want to brew with the yeast you should use fresh wort to accomplish this.

Here is what I do:

- let the yeast settle overnight
- decant the starter wort
- weigh the flask+yeast sediment+stir bar. I know the weight of the flask (written on it) and the stir bar and are thus able to determine the weight of the sediment. That weighing step is optional but easy enough to do and will yield useful information that can allow you to skip counting in the future
- Add a known amount of wort to the flask. This would be wort from the beer you are brewing. Let's assume you add 2000 ml. That means the cell density in that wort is expected to be 450/2 = 225 M/ml
- place the yeast+wort on the stir plate and wait until it deflocculates completely. This may take 15 min or more.
- With the hemocytometer that I have a good cell density for counting is around 25 M/ml. Based on what you expect the current cell density to be with a 10:1 dilution (9 ml water + 1 ml yeast starter) you expect to be at 22.5 M/ml which is perfect.
- add a drop of 1% Methylene blue stain to the diluted sample you are counting and place it in a hemocytometer
- check if the cells are unflocculated and evenly distributed over the counting grid. You don't want to see clumps. If you have clumps you need to wait a little longer.
- I have a spreadsheet where i keep track of the wort I used to grow the yest, the amount of yeast i started out with, the final cell count and the slurry weight. That allows me to find correlations that guide my future yeast propagation. Especially knowing how many cells to expect per gram of sediment allows me to skip the counting and simply estimate yeast density by weight.

Kai

#### ldave

##### Active Member
Especially knowing how many cells to expect per gram of sediment allows me to skip the counting and simply estimate yeast density by weight.
I've begun weighing the refrigerated compacted yeast sediment for my stir plate starters as well, and I'm coming up with numbers that seem much larger than possible. As I don't have hemocytometers and microscopes and such, at this point it is simply a mystery for me.
For instance, for a 10 gal batch of O.G. 1.056 I'm aiming for pitching around 350 B cells of WLP001. Use a stir plate starter of 2.15 L of 1.040 wort created from RO water + 245 g DME. Boil 15 mins. Hot trub is decanted off with RO water top up after to arrive at the 2.15 L at 1.040. Chill and pitch 25 B cells (1/4 vial of WLP001). I use a gentle stir plate setting, where I crank it up till I hear the pinging of the vortex off the bar, then back it down a little till only occasional pinging. Ferment to completion (F.G. 1.012). Refrigerate for 2 days at 2 deg C. Decant beer completely. Add pre-weighed amount of room temp wort. Resuspend yeast cake. Pour out and weigh mixture. Then, of course, pitch this into batch.
I get 80 grams of sediment. From existing literature, I calculate that, at most, there's about 8 g of trub present. Pobably much less, given that I decant the hot trub. The yeast cake is very dense. I can upend the vessel and the sediment stays put. So, you would think this would be a high density, around 7 - 8 B/g. But this gives a total cell count around 560 - 640 B cells. Way high from target. If I have, in fact, arrived at my target, then my density is 4.4 - 4.9 B/g for WLP001, which seems too low given the level of compaction.
Could you share some of your numbers based on weight of yeast sediment from single step starter for some yeast strains so I could get an idea of the size of the ballpark?