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Old 01-29-2013, 10:12 PM   #11
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Originally Posted by Huff360 View Post
Basically, you are saying follow that 2nd image I posted and ignore the fact that there are only 2 lines instead of three.
Yes, taking into account ajdelange's advice about the "ruling" of the slide, and adjusting appropriately to use the inner or outer line as your limit.
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Old 01-29-2013, 10:21 PM   #12
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- Drags out scope and cheap Chinese Haemocytometer-

It looks as though the double line ones omit the inner set of lines, making all the blocks the same area, and not creating rectangles.

So one should treat the inner line on a double line as if it were the middle line on a triple line unit. Which actually makes the first graphic I posted correct also. Right?

2013-01-29_17-12-04_333.jpg

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Old 01-30-2013, 04:16 PM   #13
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There is also a pretty large margin of error, which can be reduced when more cells are counted.
It turns out this is very simple to quantify. The coefficient of variation (multiply by 100 to get percentage error) is simply 1/sqrt(number of cells you count). This if you count 1200 cells total your Cv is 2.88%. If you only count 300 it's twice this.

More details at http://www.wetnewf.org/pdfs/hemocytometer.html
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Old 01-30-2013, 05:35 PM   #14
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It turns out this is very simple to quantify. The coefficient of variation (multiply by 100 to get percentage error) is simply 1/sqrt(number of cells you count). This if you count 1200 cells total your Cv is 2.88%. If you only count 300 it's twice this.

More details at http://www.wetnewf.org/pdfs/hemocytometer.html
This is by far the absolute best explanation of how and why to decide when and where to count a cell that I have seen.

Thank you very much for taking the time to write that up.
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Old 01-31-2013, 09:48 AM   #15
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Good points. Consistency is key in counting. The rule I use is that if it is touching the top or left box boarder it gets counted. If it is touching the bottom or right it does not. I also have an inexpensive hemocytometer and these lines are the ones that made the boxes the most square.

AJ's statistical analysis is a good way to decide how many boxes to count. I also look at box to box standard deviation and make sure it is reasonable. keep in mind that dilutions and homogenization are also critical. One thing that I have found recently is that a pipette can retain 5% of the previous sample clinging to it's walls. So if you wash your pipette and then pull a sample it could be 5% diluted.

A little acidic acid helps with un clumping the cells, but seems to make staining difficult. Glycine work well for declumping and seems to work better with stains.

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Old 01-31-2013, 12:57 PM   #16
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A little acidic acid helps with un clumping the cells, but seems to make staining difficult. Glycine work well for declumping and seems to work better with stains.
I'm fighting that right now. I got a sample of 002 from a 60bbl conical. It is extremely thick and clumped worse than cottage cheese! Based on Kai's site I did the first dilution with wort. That seemed to help some, but after 20 minutes the cells were still clumping. I dropped a few grains of PBW into the tube and agitated. That solved the clumping issue immediately! But Kai says PBW interferes with staining.
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Old 01-31-2013, 01:04 PM   #17
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I'm fighting that right now. I got a sample of 002 from a 60bbl conical. It is extremely thick and clumped worse than cottage cheese! Based on Kai's site I did the first dilution with wort. That seemed to help some, but after 20 minutes the cells were still clumping. I dropped a few grains of PBW into the tube and agitated. That solved the clumping issue immediately! But Kai says PBW interferes with staining.
That's the same place I started when looking for ways to un-flocculate the cells! Kai has some very good information on his site. Top notch.

The other day I did a count on some WLP002. It clumps more than all the yeasts I have seen.

See here for pictures:
http://woodlandbrew.blogspot.com/201...b-results.html

I did the viability count with MB diluted with water, and the cell density count with a 5% acetic acid solution. (actually just distilled white vinegar)


Glycine seems to do the trick and doesn't seem to interfere with the staining, although I haven't tried WLP002 with it. I just got some off Amazon here: Glycine
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Old 01-31-2013, 01:38 PM   #18
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I did the viability count with MB diluted with water, and the cell density count with a 5% acetic acid solution. (actually just distilled white vinegar)
Thanks for that. I didn't think of using vinegar.

Lab standard seems to be sulfuric acid but I'd like to avoid that.

Kai
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Old 01-31-2013, 02:18 PM   #19
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I am tempted to do the viability with them clumped and just kinda wing it. For a count though, the PBW worked like a charm. I added about 10 grains into a test tube with a 1:500 diluted sample. Gave it a couple seconds on the vortex mixer and it immediately went from chunky to just a nice haze.

I am redoing it now with the PBW in the first 1:10 dilution so hopefully the serial dilution gets more accurate.

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Old 01-31-2013, 02:19 PM   #20
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I did the viability count with MB diluted with water, and the cell density count with a 5% acetic acid solution. (actually just distilled white vinegar)

Rough volume? Did you just do all your dilutions into vinegar instead of water?
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