Procedure for DrawingBoard III tablet
After liquefaction, an aliquot of 11.5 μL of thoroughly mixed, either undiluted or diluted fresh semen is placed on a microscope slide and covered with a standard coverslip of 24 x 24 mm, to give a uniform depth of 20 μm (11.5 μL divided by 576 mm2 is equal to 0.02 mm or 20 μm). Alternatively, an aliquot of 8 or 10 μL semen can be covered with a coverslip of 20 x 20 or 22 x 22 mm respectively.
Analysis is performed at room temperature (between 20 and 24°C) using the 40x objective.
Bring the sperm into focus. Move the tablet to locate the grid in the center of the visual field. Analysis starts from the small square (A1, Figure 1) at the upper left corner of the large square grid. Each sperm present in the small square must be classified. Motile spermatozoa are tracked by moving the cursor on the digitizing tablet in such a way that the superimposition of the red light follows sperm movement as closely as possible. While doing so button  of the cursor is pressed. After a predefined period of time you hear a computer generated beep and you release the button. Since it is impossible to track the lateral head movement, the sperm has to be tracked at its midpiece. The superimposition of the light point of the cursor on the microscopic field must follow the movement of the sperm-midpiece as accurately as can be done by hand.
Spermatozoa presenting non-progressive motility are identified by pressing button  of the cursor and leaving the cursor on the spot.
Immotile spermatozoa are recorded by pressing button  of the cursor.
It is of the utmost importance to record each sperm which is present in a particular square at a given moment of time. Do not wait for spermatozoa to enter the square, nor skip any present spermatozoa, as this will cause unreliable results.
Figure 1: A diagrammatic representation of the sequence of analysis.
Analysis is continued until all spermatozoa in the first small square (A1) are characterized. Then you proceed to the next small square (A2), following a well defined sequence. When all spermatozoa within the large square are analyzed, press button  of the cursor. If, at this point, less than 60 spermatozoa are characterized, the preparation is shifted to another visual field to continue the analysis of another large square. The analysis is completed when a sufficient number of large squares has been evaluated to characterize at least 60 spermatozoa. This is indicated by a computer generated triple beep.
It is possible to interrupt the procedure before 60 spermatozoa have been characterized. First complete counting the cells in least 1 large square, press button  to indicate end-of-square, and next click the Stop button in the dialog box on the screen.
You can cancel the analysis by pressing the [Esc] key or clicking the Cancel button.
The procedure described above yields optimal results in semen samples with sperm concentration between 5 and 50 x106/mL. If the number of spermatozoa per visual field is less than 5, corresponding to severe oligozoospermia, you can classify each sperm present in the large square without scanning through the small squares. If sperm concentration is high, it is suggested either to reduce chamber depth, or to dilute the sample. In the former case only 8.6 μL of semen is placed under a 24 x 24 mm coverslip, resulting in a chamber depth of 15 μm (8.6 μL divided by 576 mm2 is equal to 0.015 mm). It is, however, not recommended to reduce the chamber depth bellow 15 μm, since this will hinder the free movement of sperm and artificially reduce linearity. Alternatively, the sample can be diluted before being evaluated. Dilution is preferentially done by means of cell free seminal plasma of the particular semen sample, obtained by centrifugation. The reduction of chamber depth or the dilution factor must be recorded, and the system automatically calculates sperm concentration taking into account these factors.