Blood Cell Counting Using Haemocytometer

A haemocytometer is a thick, specially ruled glass slide used to count blood cells directly under a microscope. At its centre sits a counting chamber containing two identical grids, each ruled to the 'improved Neubauer' pattern: a 3 mm by 3 mm primary square, itself divided into nine 1 mm by 1 mm secondary squares. The four corner secondary squares are further subdivided into 16 tertiary squares each and are used for counting white blood cells, while the central secondary square is divided into 25 still-smaller tertiary squares (each 0.2 mm by 0.2 mm, further subdivided into 16 smaller squares) - the five black squares of this central square together with the shaded squares in the middle are used for platelet counting, and the five black squares alone are used for counting red blood cells. Before counting, blood must be diluted in a fluid chosen to keep the cells intact and undamaged: for a total RBC count, the diluting fluid is Hayem's fluid, which is isotonic with blood so it does not cause the red cells to burst (haemolyse) - blood is diluted 1:200 with this fluid and the cells are counted under the microscope's 45x objective. For a total WBC count, the diluting fluid is Turk's solution, a mixture of glacial acetic acid and gentian violet dye - the acetic acid lyses (bursts) the red blood cells so they do not interfere with the count, while the gentian violet stains the nuclei of the remaining white blood cells (leucocytes) so they stand out; blood for a WBC count is diluted 1:20 and counted under the 10x objective, with the resulting total expressed per cubic millimetre (mm3). The general procedure is to draw blood into the pipette up to its 0.5 graduation mark, then draw the appropriate diluting fluid up to graduation 11 (for the WBC pipette) or 101 (for the RBC pipette); the pipette is then rotated horizontally several times to mix the blood and dil …