I just bought a hemacytomoer and am trying to figure out the formula for calculating # of cells per mm of semen, but my grade 9 math skills are not doing the job! I get an average of 87 cells on one collection based on counting 5 squares on both ends of the hemacytomer. From there you x by 101 giving 8787 but after that I am lost! Please help!
You need to dilute the raw semen with a formalin solution to kill the sperm and make the counting easier.
Take 1 ml of raw semen and add it to 10 ml of formalin. Mix. Take 1 ml of that mixture and add it to a further 10 ml of formalin (note - if you want, you can use extender for the first mixture, thereby also diluting the sperm to allow removal of a sample for better motility evaluation). Mix again and use a sample from the second mixture to load the hemacytometer. Diluting in this manner gives a 1:99 dilution ratio, or 100 dilution.
After loading the hemacytometer, allow a few minutes for the dead sperm to settle to the bottom (it makes counting easier), and count the number of sperm in 25 squares. This should be achieved by counting 13 squares on the grid on one side and 12 on the other side (hemacytometers have 2 grids, one each side of the polished surface). If you want maximum accuracy, count all 50 squares (25 on each side) and then divide the resulting number by 2. Note that these squares are the middle-sized squares that are 5 across and 5 down on each side, not the smallest quares which are 4x4 inside the squares you are counting. You should see something like this under the 'scope at 100x power:
The squares to count as "one" are the 5x5 squares depicted (so you're seeing 25 squares in the image above}. Once you have the 'scope focused, it is often easier to take it up to 400x magnification and count each of the squares individually.
The joy of diluting the semen as described above is that the number of sperm that you count in the 25 squares is the raw concentration of sperm in millions/ml. In other words, if you count 150 sperm, the raw concentration is 150 million/ml. If you counted all 50 squares, divide the number of sperm counted by 2 and you have your concentration.
Note that this dilution and counting method will only work in hemacytometers that have a grid that is 1 mm2 and 0.1 mm deep. That is however most hemacytometers that will be used for counting sperm...
I got a dilution kit ( Unopette Dilution System : 1:100 dilution) , for semen counting with my hemacytometer so that all I do is dip the end of a tiny pipette into the raw semen then put it in the small amount of premeasured formalin. Gently mix then add a drop to both ends of the h meter. The instruction say to count only 5 of the 25 squares? on both sides then divide by 2 to get an average which was 87. So not sure if your math formula will apply with my dilution system, or is it the same? This is my brand of H meter (Hemacytometer : Calibrated glass slide for counting sperm cells. 2 counting areas, each with double Neubauer ruling grids. Includes 2 coverglass. that would be so much easier than all the funky formula they give, but why then say to do the formula? I can type out the instructions given with it if you are curious to see?
The more squares you count, the more accurate your count will be. We always count a minimum of 25 squares, and usually the whole 50.
If the Unopette is 100 dilution (99:1) - which it probably is - you can apply the same math. Note that Unopettes will tend to evaporate over a period of time, and the accuracy will therefore be decreased.
I can't comment on the model hemacytometer that you have specifically as I am not familiar with it, but it should tell you somewhere in the accompanying literature that came with it what the dimensions are.
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