[narvin]

Anyone do cell counts on their starters?  I've started out of curiosity, but the results are different than what I'd expect.

I've been growing up yeast for a 10 gallon batch of Pilsner this week.  The cell counts I'm getting are lower than what I expect based on both the starter size and the volume of yeast I ended up with.  I know these are both guesstimates, and I was wondering if anyone else had similar experience or if my counts just aren't accurate.

Starter specifics:

- Began with 1 vial of WLP830
- Starters contain 1 g/10ml DME.
- 2 stages: 1.6L for 48 hours, then chilled, decanted, and pitched into 4.4L
- Aerated with pure O2 for 60 seconds, fermented on stir plates at ~ 75dF, breathable foam stopper

I chilled the final starter and the compressed cake at the bottom looks to be 200-250 ml based on rough eyeballing.  According to Jamil's calculator using the "repitching from slurry" tab, this should be about 700 - 900 billion yeast cells even if it has a high amount of non-yeast material (break).  This is the amount that his and the wyeast calculator predicted for me as well.

Problem is, every time I do a count I'm consistently estimating that there are 400 - 500 billion cells in the starter. The starter was still on the stir plate and I didn't see a lot of flocculation.  I took a decent sample from near the middle of the flask using a thief, diluted 10x, and counted.  I can post the specifics of my calculation for a Neubauer-improved hemacytomer when I get home if that would help.

[tom]

that sounds like a lot of oxygen

[narvin]

You're right, and I misspoke... it was more like 20 or maybe 30.  60 is for a carboy of beer, and I specifically dialed it back for the starter.

One more thing to note is that I stained with methylene blue when counting and the viability was around 90-95%.  Is this low for a fresh starter?  5-10% non-viable in the final starter could have been more like 40-50% if they all came from the White Labs vial.

Still, the biggest thing that's throwing me off is the discrepancy between the large volume of yeast cake I have and the cell count.  But perhaps yeast volume is just not an accurate measurement.

[tom]

How long did you give it to flocculate?  It takes a while to really get to "thick" yeast slurry.

[a10t2]

A little more detail on your counting procedures might help. At a 10:1 dilution I'd expect you to be counting a bare minimum of 1000 cells. Maybe you just have more patience than I do.

Another factor could be that you pulled from the top of the flask and got the thinnest portion of the slurry.

How old was the yeast? If it really was at ~50% viability then that would explain things. 90-95% viability coming out of a starter doesn't sound too bad though.

I've done four or five counts on starters and they all came up within about 15% of the MrMalty predictions.

[narvin]

I'm only counting 5 of the 25 "group" squares -- the four corners and the center one.  These are the squares that have 16 smaller squares inside.  I get about 200 cells total.  

200 cells / (0.2 mm^2 * 0.1 mm * (1/10) )     = 100,000 cells / uL = 100 billion cells per L

This is from 4.5 L of liquid, so that would be 450 billion cells.


This is the yeast cake in the fridge after 2 days:



You may be right... even though it was on the stir plate and I tried to pull from the middle of the flask, there may have been more yeast at the bottom and the sample wasn't representative.

[jjflash]

While the mash is going I decant the cold erlenmeyer flask and transfer the yeast with the stir bar into a one liter beaker.
I put the beaker on the stir plate letting it warm up and re-suspend the yeast.
Once I establish the wort final gravity I then pull 10ml out of the spinning slurry to a 1:100 dilution for a yeast count.
Calculate the pitch rate at this point and toss it in the fermenter.
Works quite well for an accurate yeast count for me!

[narvin]

While the mash is going I decant the cold erlenmeyer flask and transfer the yeast with the stir bar into a one liter beaker.
I put the beaker on the stir plate letting it warm up and re-suspend the yeast.
Once I establish the wort final gravity I then pull 10ml out of the spinning slurry to a 1:100 dilution for a yeast count.
Calculate the pitch rate at this point and toss it in the fermenter.
Works quite well for an accurate yeast count for me!

I was thinking about trying to count again after decanting and before pitching... this sounds like an easy way to do it.  I'll try it with my next batch.