Counting cells by use of a hemacytometer is a convenient and practical method of determining cell numbers in suspension culture or from dispersed monolayer cultures.
The hemacytometer consists of two chambers, each of which is divided into nine 1.0 mm squares. A cover glass is supported 0.1 mm over these squares so that the total volume over each square is 1.0 mm × 0.1 mm or 0.1 mm3, or 104 cm3. Since 1 cm3 is approximately equivalent to 1 mL, the cell concentration per mL will be the average count per square × 104.
Hemacytometer Counts Are Subject to the Following Sources of Error:
- Unequal cell distribution in the sample
- Improper filling of chambers
- Failure to adopt a convention for counting cells in contact with boundary lines or with each other
- Statistical error.
With careful attention to detail, the overall error can be reduced to about 15%. It is assumed that the total volume in the chamber represents a random sample. This will not be a valid assumption unless the suspension consists of individual separated cells. Cell distribution in the hemacytometer chamber depends on the particle number, not particle mass. Thus, cell clumps will distribute the same as single cells and can distort the final result. Unless 90% or more of the cells are free from contact with other cells, the count should be repeated with a new sample. Cells that are difficult to obtain in uniform suspensions, or in which extensive clumping cannot be avoided, may be counted by separating nuclei. This method is more time-consuming than direct counting and is subject to additional error if the population contains multinucleate cells. A sample will not be representative if the cells are permitted to settle before a sample is taken. Always mix the cell suspension thoroughly before sampling.
With a 10X objective and a 10X ocular, one square (1 mm2) will approximately fill the microscope field (the circle on the representation of a hemacytometer grid). The cell suspension should be diluted so that each such square has between 20 and 50 cells (2–5 × 105 cells/mL). A total of 300 to 400 cells should be counted since the counting error is approximated by the square root of the total count. A common convention is to count cells that touch the middle line (of the triple lines) to the left and top of the square, but not to count cells similarly located to the right and bottom (see diagram).