Post #15 - How one counts COT sperm

It's been another long but productive day in the lab, doing a lot of water chemistry for Emily's experiments (more on that soon).  It's late and we need to get up early, but I want to get this out to you.

I showed you in Post #13 a test tube with a stock suspension of COT sperm and described how a serial dilution is made so that eggs in different culture tubes are exposed to vastly different concentrations of sperm.  As I've described previously, I count in each tube how many eggs develop normally, how many develop abnormally, and how many are unfertilized.  In order to analyze the results though, I need to know the concentration of sperm to which the eggs are exposed.  I do this by preserving three samples of each sperm stock suspension in tiny plastic microcentrifuge tubes.  Here is a picture similar to one in Post #12 that shows the tubes and the pipetter with which a tiny drop of a sperm sample is placed on the counting slide seen at the bottom.

The counting slide has a tiny grid etched into it and is designed so that a precisely determined volume of your sample is held between the cover slip and the grid.  This allows you to count the number of things (sperm in this case) in a known but tiny volume of fluid, and from this the concentration in your suspension can be calculated.  Here is what I see when counting sperm:

The picture is not real clear because I had to jury-rig a camera onto the microscope, but at least you can see the dots that are the sperm.  I count the sperm in 25 of those squares for each of 6 samples for each sperm suspension, with the data immediately going into a spreadsheet on my laptop.  Tedious but necessary, and one can't help but smile when done!