My Back is Sore or I've Been Doing Cell Counts for Too Long

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, washed it, 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.

There are multiple ways to count using a hemocytometer, but what I do is count the total number in the four large corner squares, then multiply by 2500. That will give you your concentration in cells/ml. Here's a link that might help:
hemacytometer

Yeah that gives me the same numbers. For the first chamber in my last post:

11 + 21 + 21 + 19 = 72 * 2500 = 180000 * 1000 = 1.8*10^8

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.

Your dilution is off. If you diluted 0.1 ml to 1 liter, it's a 10,000x dilution, not a 1000x dilution. It looks like your starter should be right on.

I started with a 10,000:1 dilution but there weren't enough cells around to count. I did two 1000:1 dilutions and I was able to count those.

75 * 2500 = 187,500 * 1,000 = 1.9 * 10^8 cells/ml