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Old 01-29-2013, 05:38 PM   #1
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Default Proper Haemocytometer Usage

I am getting ready to start doing some cell counts. I have the Chinese improved nebular Haemocytometer. The only references I can find online for the proper way to do a count use the fancier triple line slide.

I am curious about which cells that lay on the perimeter to count.

Would this be correct?

cell.png



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Old 01-29-2013, 07:58 PM   #2
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I am getting ready to start doing some cell counts. I have the Chinese improved nebular Haemocytometer. The only references I can find online for the proper way to do a count use the fancier triple line slide.

I am curious about which cells that lay on the perimeter to count.
You need to decide what your cutoffs are, then count it the same way every time. I use a 50% cutoff, if half the cell is within the line of the grid area that I am counting, I count it.

Your picture is a tad confusing, but you should count the ones marked X, that are within the 4x4 square.

Here's a useful link:
http://www.animal.ufl.edu/hansen/protocols/hemacytometer.htm


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Old 01-29-2013, 08:38 PM   #3
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Your picture is a tad confusing, but you should count the ones marked X, that are within the 4x4 square.
I can see how that would be.

I ran into this picture a lot when looking up the 'standard' protocol for counting.

counting_of_cells_01.png

Basically, it says count anything that is touching the middle line on the top and left, but do not count anything touching the middle line on the bottom or right. I modified that a bit, cause to me not counting the top felt better. From: http://amrita.vlab.co.in/?sub=3&brch=188&sim=336&cnt=2

BUT - I was not sure what to do about the double line vs triple line.


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You need to decide what your cutoffs are, then count it the same way every time.
Surely there is a SOP, otherwise me and you sharing a cell count would be pretty worthless. If I decided anything touching the line and you said anything 50% in, that could make a pretty huge difference . Or is it all within the margin of error anyway?
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Old 01-29-2013, 08:39 PM   #4
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This is a good image:

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Old 01-29-2013, 08:47 PM   #5
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I was not sure what to do about the double line vs triple line.

Surely there is a SOP, otherwise me and you sharing a cell count would be pretty worthless. If I decided anything touching the line and you said anything 50% in, that could make a pretty huge difference . Or is it all within the margin of error anyway?
Ha funny, posting the same picture at the same time...
And yes sorry, I see the line question now.

There is a ton of variability of cell counts based on the counter, this is why the same person is used for all cell counts in a particular experiment. There is also a pretty large margin of error, which can be reduced when more cells are counted.
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Old 01-29-2013, 08:49 PM   #6
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Seems to me that you would count cells touching the outer lines on the upper/left sides, but not count those touching the outer lines in the lower/right sides. However, I'm not a biologist and would never play one on TV.

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Old 01-29-2013, 09:01 PM   #7
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I don't think it matters too much so long as you are consistent and the area you are counting is correct. That is, upper left/lower right whatever doesn't matter. It's the same as rounding numbers...you can use whatever system you like so long as you apply it consistently. There are varying conventions that people use (even/odd) but the most important thing is that it is unbiased and consistent.

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Old 01-29-2013, 09:14 PM   #8
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Seems to me that you would count cells touching the outer lines on the upper/left sides, but not count those touching the outer lines in the lower/right sides. However, I'm not a biologist and would never play one on TV.
Hmmm, so if the cell is 100% outside the grid, but touching the outside line, you would count it?

Refer to the 2nd image I posted. Left side, bottom cell with a green check mark. That would be counted.

Basically, you are saying follow that 2nd image I posted and ignore the fact that there are only 2 lines instead of three.
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Old 01-29-2013, 09:17 PM   #9
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I don't think it matters too much so long as you are consistent and the area you are counting is correct. That is, upper left/lower right whatever doesn't matter. It's the same as rounding numbers...you can use whatever system you like so long as you apply it consistently. There are varying conventions that people use (even/odd) but the most important thing is that it is unbiased and consistent.
I guess I was trying to learn what is consistent with the 'standard' method of counting a 2 line. I have found most everyone uses the same method for the 3 line, so I would feel confident that 2 people would look at the same picture and get the same count.
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Old 01-29-2013, 09:33 PM   #10
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If you understand how the hemacytometer is ruled it is pretty clear what you should do but it is not clear from the diagram how one is ruled. In the attached photograph I drew a rectangle over one of the center squares and then dragged it to its current position. From this it is clear that it is the center line of the triplet that defines the square in which the cells are to be counted. If your rule is that any cell that touches, even if tangentially, a left or upper square boundary is considered within the square and that any cell that touches, tangentially or otherwise, a lower or right square boundary is not within the square then you will, assuming a uniform distribution of cells, count each cell on an interior boundary only once (in the square that is above or to the left of the line on which it lies) and include all cells that are on the larger (4 x 4 small cell) square boundary at top or left assigning them to the 4 x 4 array you are counting and exclude all cells that are on the right or lower boundary effectively assigning them to the 4x4 square to the right of or below the one you are counting. In this way, you will, on average, exclude as many as you include and, on average, will get the 'correct' count. The most important thing about this is to understand that it is the middle line that defines the boundaries. The rectangles and squares defined by the inner lines are smaller than the standard squares.





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