Calculations for Sperm Concentration Determination using a Hemocytometer

Important Information:

• Dilution Factor = Volume Water (µl) + Volume Semen (µl) / Volume Semen (µl)
• Count = # of sperm in 5 large squares on hemocytometer
• Average count on both sides of the hemocytometer
• 5 squares have 1/50,000 of a ml volume above them
• 50,000 = 0.05 X 10^6
• 1 ml = 1000 µl

Formulas for concentration:

• sperm /ml = Count * Dilution factor * 0.05 X 10^6
• to use this equation must have calculator with exponents or lots of decimal places
• sperm X 10^6/ml = Count * Dilution Factor * 0.05
• this equation is useful with calculators that do not have ability to use exponents
  (we use this one the most)

Problems often encountered that causes errors in the count:

• Semen is not mixed before removing sample
• Pipeting errors
• operator did not put tip on pipet tight enough
• pipeting too fast and getting air bubbles in solutions
• Not mixing semen diluted in water before placing on hemocytometer
• Over filling the hemocytometer with the semen solution
• When changing objectives on microscope, moving the coverslip on the hemocytometer
• Damaged or inoperative counting devices

General Difficulties:

• Can not focus microscope on hemocytometer
• Inability to use both eye pieces on a binocular microscope