Manual Counting with a Hemocytometer
The hemocytometer (also hemacytometer, haemocytometer, or haemacytometer) was originally developed for counting blood cells. It soon became popular for counting all types of cells including unicellular organisms such as bacteria, yeast and protozoa, and dispersed cells from multicellular organisms. Most hemocytometers have two Neubauer chambers on a single slide, each with an etched grid and a known volume.
To count cells manually with a hemocytometer, a cover slip is placed on the hemocytometer, and a cell suspension enters a chamber by capillary action. Using a microscope, the cells are counted individually within an area of the grid. The volume of the chosen grid area is used to calculate the cell concentration.
Hemocytometer Alternatives: Improving the Accuracy of Cell Counts
While the low cost and simplicity of a traditional hemocytometer may be appealing, manual cell counting with a hemocytometer has a number of inherent limitations. A hemocytometer does not give accurate counts for dilute cell suspensions. The lower limit for accurate counting of cells in a hemocytometer is usually considered to be 2.5 x 105/ml. At the lower limit, counting multiple aliquots will increase accuracy, but this is time-consuming and can pose a problem with small sample volumes.
Too high a concentration of cells can also lead to inaccurate determinations of cell numbers. Counting errors are more likely when there are a large number of cells within the area counted due to miscounted cells and getting lost within the grid. For cell numbers greater than 2.5 x 106/ml, it is generally recommended that the sample be diluted.
Another potential problem is that either too large or small a volume may be introduced into the chamber of the hemocytometer. Most commonly, too large a volume is introduced, resulting in the cover slip being slightly lifted. The increased volume results in overestimated cell counts.
Hemocytometer Price vs. Cell Counter Price
The low initial purchase price of a hemocytometer compared with a cell counter is attractive to many experimentalists on a limited budget; however, one must also consider the ongoing costs of the additional labor required and the ultimate price of inaccurate results over the life of the instrument.
Cell counters provide additional advantages beyond time and labor savings and increased accuracy, enabling cell biology researchers to conduct experiments using previously unavailable methods. Unlike a hemocytometer, an advanced automated cell counter such as Bio-Rad's TC20™ cell counter features user-defined gating for cell size. This feature allows for selective cell counting within a size range. This enhancement allows for the counting of subpopulations within mixed populations with multiple cell sizes. This is a valuable ability for protocols using coculture and for primary cells isolated from tissue or organs. The ability to gate for size significantly expands the utility of automated cell counters.
The TC20 cell counter uses autofocusing microscopy on multiple focal planes to identify the best plane and exclude debris and more accurately calculate the total cell count. Using autofocusing instead of subjective manual focusing is especially important when assessing cell viability because an incorrectly selected focal plane will lead to inaccurate results. The conventional method of analyzing viability using a single focal plane can lead to inaccurate conclusions because light scattering and the alignment of cells at different heights in a counting chamber can change the appearance of cells -- live cells may appear to be dead and vice versa. To determine if cells are viable, the TC20 cell counter analyzes each cell using images acquired from multiple focal planes during the focusing step.
An automated cell counter can provide accurate cell counts for a wider concentration range than a hemocytometer. Cell concentrations as low at 5 x 104/ml and as high as 1 x 107/ml can be accurately counted. Therefore, unlike cell counting with a hemocytometer, there is no requirement for many replicate counts at low cell concentrations, and dilution of samples at high cell concentrations is no longer necessary.