My Back is Sore or I've Been Doing Cell Counts for Too Long - Home Brew Forums

 Home Brew Forums > My Back is Sore or I've Been Doing Cell Counts for Too Long

08-09-2009, 10:18 PM   #1
NotALamer
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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.

08-09-2009, 10:22 PM   #2
Professor Frink

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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
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08-09-2009, 11:21 PM   #3
NotALamer
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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.

08-09-2009, 11:32 PM   #4
Professor Frink

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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.
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08-10-2009, 12:05 AM   #5
NotALamer
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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