calculations for sperm concentration determination using a hemocytometer important information: * dilution factor = volume

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 106
*
1 ml = 1000 µl
Formulas for concentration:
*
sperm /ml = Count * Dilution factor * 0.05 X 106
*
to use this equation must have calculator with exponents or
lots of decimal places
*
sperm X 106/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

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