Principles
The ruled area of the hemocytometer consists of several areas. Large one is 1 x 1 mm (1 mm2) squares. It is subdivided in 3 ways : 0.25 x 0.25 mm (0.0625 mm2); 0.20 x 0.20 mm (0.04 mm2). The central part is further subdivided into 0.05 x 0.05 mm (0.0025 mm2) squares.

The raised edges of the hemocytometer hold the coverslip 0.1 mm off the marked grid. This gives each square a defined volume.

The cell-sized structures counted lie between the middle of the three lines on the top and right of the square and the inner of the three lines on the bottom and left of the square.

In an improved Neubauer hemocytometer (common medium), the total number of cells per ml can be discovered by simply multiplying the total number of cells found in the hemocytometer grid by 10 ^4(10000).

Usage
Ensure that the special coverslip provided with the counting chamber (thicker than standard coverslips and with a certified flattness) is properly positioned on the surface of the counting chamber. When the two glass surfaces are in proper contact Newton's rings can be observed. If so, the cell suspension is applied to the edge of the coverslip to be sucked into the void by capillary action which completely fills the chamber with the sample. Looking at the chamber through a microscope, the number of cells in the chamber can be determined by counting. Different kinds of cells can be counted separately as long as they are visually distinguishable. The number of cells in the chamber is used to calculate the concentration or density of the cells in the mixture the sample comes from. It is the number of cells in the chamber divided by the chamber's volume (the chamber's volume is known from the start), taking account of any dilutions and counting shortcuts.