Hemocytometer troubles

[NotALamer]

This is a repost from a few months ago on another forum, but I didn't really get much help there so I figured I'd try again.

I've been messing around with my new hemocytometer today attempting to estimate the cell density of the slurry in the bottom of my starter. I haven't been getting the results I expected so I figured someone with more experience might be able to point out where I'm going wrong.

This starter started as a Wyeast propagator into 3L of 1.04 SG wort. While aerating it with some airstones it bubbled over like crazy and I lost probably 2/3 of it down the drain. The next day I cooked up 2L of wort and added that, so it was back up to ~3L. I let that ferment for about three days, refrigerated, decanted off the wort, and added 3L more of wort. I let that ferment for about three days and then refrigerated.

Today I poured off the wort and diluted 0.1ml of slurry into a liter of water. Going with Jamil's estimate of 3 billion cells per ml, I figured this should give me about 30 cells per 0.0001 ml hemocytometer square.

3*10^9 * 1*10^-4 * 1*10^-4 = 30

I tried to do a count with this solution but there were barely any yeast cells around. I dumped it out and diluted 1ml of slurry into a liter of water.

I counted three chambers (4 * 1mm^2 squares) of this solution and got these results:

11 + 21 + 21 + 19 / 4 = 18 =
1.8*10^8 / ml

8 + 16 + 21 + 11 / 4 = 14 =
1.4*10^8 / ml

32 + 19 + 13 + 15 / 4 = 19.75
2.0*10^8/ml

My results for the three different chambers were relatively consistent, but all less than 1/10th of Jamil's estimates. I'm not going to have any stain until Tuesday, so these counts were all unstained. Any ideas why I ended up so far off from Mr. Malty?

I'm going to go do another dilution and some more counts, hopefully someone has some ideas by the time I get back.

INTERMISSION

I did two more chambers with a new dilution and got:

10 + 28 + 24 + 13 = 75 * 2500 = 187500 * 1000 = 1.9*10^8

15 + 28 + 18 + 22 = 83 * 2500 = 207500 * 1000 = 2.1*10^8

So except for that 1.4*10^8 I'm getting somewhere around 1.8-2.0*10^8 cells/ml fairly consistently. That's about 1/10th of the estimate from the pitching rate calculator. I must be doing something wrong because at this rate I'd need like seven cups of thick slurry for my DFH 90 min clone.

[BayouBrew]

If your counts are that consistent I would say that they are correct. Your problem is probably the fact that you lost 1/3 of your initial starter which should have had the highest cell density. After that loss, it would take a while to build your counts back up.
I'm too tired to really look at your math, but I use hemocytometers daily. We dilute the cells to count 1 to 10 (10ul in 90ul=100ul or 1ml in 9ml=10ml) then count the 4 quadrants. Add them up and divide by 4 to get the average like you did, multiply by 10 to account for the dilution, and then multiply by 10000 which is the constant for the volume of the hemocytometer. If that is what you are doing your math should be right. Try again without the bubble over and see if you get closer to expected numbers.
Also, I don't really think that you need to crash and decant before stepping up. If you don't crash long/cold enough you will select for the more flocculant yeast and lose the less flocculant ones. I just add the whole starter to the next step up and haven't had any problems.

[NotALamer]

I refrigerated before I did the counts so I think the stuff that settled to the bottom should have been a pretty dense yeast slurry. I was pulling from the sludge at the bottom with a pipette. I was doing 3L and then 3L and the vessel I use for starters is only a gallon so I really had to decant off most of it before the second stage. How long should I refrigerate before decanting? I usually just do it overnight, I think I read that that was long enough in HTB. I did notice after washing some yeast that it seemed to continue settling out for a couple of days, so maybe that wasn't long enough.

[BayouBrew]

I don't really know how long you should chill to drop the yeast. I'm sure it depends on the temp. I usually crash overnight too before decanting to pitch, so I guess that that would be fine. It has been for me at least.

[boobookittyfuk]

are you using 400X on your scope?

counting yeast cells is pretty hard on a hemacytometer (which is meant to count blood cells that are 10-50 times larger than yeast).

[NotALamer]

100x objective * 10x ocular

[jamilz]

Going with Jamil's estimate of 3 billion cells per ml, I figured this should give me about 30 cells per 0.0001 ml hemocytometer square.

Where do you get that number from? This is the problem with your count. You're not going to get that density unless there is no non-yeast material and you pack it extremely tight. The White Labs vials reach this kind of density only once the yeast is packed hard in the bottom of the vial.

Also, like someone else suggested, make sure you're using the right dilution and be very careful in your measurements. You don't mention what size pipette you used, but accuracy is everything in counting. The calculator numbers are going to be pretty close. They were verified independently, so they're not off by that much.

[NotALamer]

I guess the pitching rate calculator defaults to 2.4 billion/ml, but I was off by a factor of 10 anyway. I was mostly interested in doing the counts because the calculator has a pretty wide range and I wanted to get a better idea of the density for a jar sitting in my fridge. I think I used a 5ml pipette when I was diluting 1000:1 and a 1ml pipette for 10000:1.