Be careful with the model of hemocytometer that you are using: Thome, Neibauer, Neubauer Improved... the calculations are different.

Thanks Juan...i am using improved Neubauer chamber.. 

Have a look, may be can be useful:

http://weis.science.oregonstate.edu/files/weis/Protocols/Symbiodinium/Cell%20Counts.pdf

thanks alot.. thats a better protocol. 

Hi Hizb

see  these links and files http://www.ansci.wisc.edu/jjp1/ansci_repro/lab/procedures/hemacytometer/hemocytometer%20use.html

Thanks Ali... The same i did by comparing all the protocols... there are total 9 squares in which central one is divided into 25 squares (each having 16 small squares). for sperms in 1/25 squres, i counted 10 out of 25 and obtained average number of sperms in 1/25 squares. now i multiplied that value with 25 to obtain sperm/25 squares (central square). this square has volume of 0.1micL so i multiplied the number with 10,000 to obtain number of sperm/1 ml. after that i multiplied the result with dilution. 

That's all. It is very simple protocol 

best of luck

Hello Friend 

regards

If care fully done RBC chamber of 

Procedure using RBC chamber of Neubauer Hemocytometer is good enough. Additionally you can get it counted by two different persons each twice and take average to avoid subjective variation. Procedure and calculation are given below. Kindly modify as per your need and experiment.  

a.       Determination of sperm cell can also be done by using RBC chamber of NH.

b.      After collection of semen it is kept in 30°C water bath.

c.       From this semen is drawn carefully up to the 05 mark of RBC mixing pipette (red bead) of the NH. To align the meniscus exactly at the mark, the tip of pipette is blotted.

d.      This is followed by drawing diluting fluid to ‘101’ level. This makes final dilution of 200 (Dilution Factor, DF).

e.       Mixing pipette is shaken vigorously for at least 2 min to ensure proper mixing and distribution of sperm cells.

f.        Discard first few drops (8-10) of the diluted sample.

g.       The diluted sample is then charged from both sides into a standard RBC counting chamber covered with a specific cover slip (25 mm thick).

h.       Allow the charged counting chamber to settle for a few minutes.

i.         Count the number of heads lying within the two sides of the subunits under 40x.

For very concentrated samples

a.       Take 0.1 mL of neat semen in a test tube and to this add 9.9 mL of diluting fluid.

b.      This makes rate of dilution 1: 100. Add this additional DF into final calculation.

Calculation:

            For counting in RBC chamber normally either all the spermatozoa in the major square E are counted (N) or only the ones in small square E1 to E5 are counted. In the latter case the value obtained is multiplied by 5 to arrive at a corresponding value for major square E (N x 5).

      Sperm concentration = No. of spermatozoa counted x VCF x DF

I mL of neat semen = N x 104 x 200 (for N)

                                = (or) N x 5 x 104 x 200 (for 5N)