A counting chamber can determine the cell concentration in a liquid suspension. This device is also known as a hemocytometer, as it was originally designed to count the number of blood cells in a clinical sample. Samples are loaded into a V-shaped well, placed under a microscopic lens and cells are counted manually against a background marked with a grid. In short, the cells in several squares of the grid are counted and the total sample size can be calculated for the entire volume.
Typically, cultures can contain millions of cells per milliliter. In a serial dilution, a measured volume of cell suspension is removed from a tube containing the initial concentration and transferred into another test tube loaded with a premeasured volume of liquid. For example, remove one mL from the original tube and place into a second tube containing nine mL of the liquid solvent. This represents a ten-fold dilution. Repeat this process until a limited number of cells are available to be counted using a hemocytometer.
Light diminishes as it passes through any medium, such as a liquid suspension containing cells. Based on this characteristic, it is possible to measure cells by optical density (OD), or the change in light emitted and detected. The amount of light scattered by the cell suspension, measured in an apparatus called a spectrophotometer, can be interpreted to represent cell concentration. Readings are determined at certain wavelengths of light, measured in nanometers (nm), and are reported, for example, as OD at 600 nm. The OD reading is inversely proportional to the concentration. In other words, the lower the OD reading, the greater the cell concentration.
Using a microscope to determine cell concentrations is rather laborious and often inaccurate. Cell concentrations will first have to be diluted by the serial dilution method explained previously. A known volume of the cellular solution is then mounted onto a slide and the cells are counted manually against a grid to acquire an estimate of the sample. When using this method, it is important to keep the sample well mixed so that cells do not settle on the bottom of the tube. If this occurs, there will be a large discrepancy in cell numbers from the upper layer of the sample as compared to lower layers.