Colony Counter / Test Chambers

Q.How do I Determine the Sperm Concentration using a Haemocytometer Counting Chamber

A: A Haemocytometer can be used to determine the abundance of most types of cell, whether it be red cells, white cells, yeasts or spermatozoa. It’s important to know which squares you need to count and the area under the cover slip to determine the correct counting method.

Neubauer counting chamber

The rails of the haemocytometer are designed to hold a cover slip 0.1mm above the mirrored surface of the counting chamber. This area has a grid etched into the mirrored surface.

When measuring spermatozoa its important to immobilise them by killing them with water and diluting them sufficiently to be able to measure them. A general guide is set out below:

Bovine Species: 101 dilution of 1.0ml of water to 10µl of bovine semen.

Equine Species: 21 dilution of 1.0ml of water to 50µl of equine semen.

Human Species: 20 dilution of 1.oml of water to 19µl of human semen.

Both sides of the haemocytometer should be used as the test needs to be performed in duplicate.

When using a haemocytometer make sure that the sample is kept in an incubation chamber and slightly moist using a dampened filter paper beneath the counting chamber. A petri dish is ideal housing the wetted paper under the chamber.

Method:

  • Place 10-15µl of diluted sample under the cover slip on both sides of the haemocytometer
  • Place the counting chamber on the microscope stage ensuring the bottom is dry
  • Visualise using the x40 objective with phase rings out and close down the aperture on the condenser so you can visualise the spermatozoa. (raising or lowering the condenser will also help).
  • Be careful when focusing not to smash the cover slip.
  • Count the spermatozoa in 5 of the large squares on each side of the counting chamber.
  • Average the two results before the final calculation
  • Count the sperm where more than half is in the counting area

Calculation:

The hemocytometer is 0.1 mm deep and the 25 large squares represent an area of 1 square mm. The volume above the 25 squares shown is 0.1 µl. As you are only counting 5 squares, you counted the sperm that settled out of 0.02 µl (0.1/5=0.02). Therefore you must multiply your count in 5 squares by 50,000 in order to determine how many sperm would have been in 1.0 ml (1000/0.02=50,000; we usually express sperm concentration in terms of numbers/ml). To get the concentration of the original sperm sample we must however also multiply by the dilution factor. The equation that follows would be used to convert the counts in 5 squares to concentration/ml:

Concentration/ml = (Dilution Factor)(Count in 5 squares)(0.05 X 106)

Conventionally, spermatozoa concentration is usually expressed in terms of sperm X 106/ml.

Normal ranges can be found online for each species.

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