Hemocytometers are cell-counting devices that determine cell density in liquid samples. Their usage is common in microbiology, cell culture, and other applications requiring the calculation of cell concentration, such as semen analysis.
A hemocytometer features a microscope slide made of thick glass with two chambers of a precise depth formed by a rectangular indentation. Perpendicular lines etched on the chamber form a grid with a defined surface area.
To calculate the cell density of a fluid sample, a user must know the number of cells or other particles to be counted in the chamber. Raised edges around the chambers serve as mounting support for the cover glass, called a coverslip, which rests on top of the sample at an exact height of 0.1 mm.
Hemocytometers feature a standard design suitable for multiple functions. However, numerous types with non-standard depth and grid spacing exist for different cell types and sizes. For instance, specialized hemocytometers are employed in totaling sperm cells due to the smaller size of the cells.
The exterior of the counting chamber and the coverslip are first cleaned using lens paper. Coverslips designed for these devices are thicker than the slides used in conventional microscopy, as they must be robust enough to withstand the surface tension of the fluids. The coverslip is positioned over the chamber before introducing the sample.
A small amount of the fluid containing the cells is applied between the edge of the slip and the chamber to cover the mirrored exterior. The loaded counting chamber is viewed under a microscope by adjusting the magnification for efficient cell distinction. This allows the calculation to be performed.
When viewing a standard hemocytometer with a Neubauer ruling pattern, an entire grid is viewable at 40x (4x objective). The grid is separated into nine squares, each having a square mm surface area, and the 0.1 mm deep chamber.
The fluid holding the cells is prepared before placement on a hemocytometer. The mixture should be a homogenous suspension to avoid clumped cells and ensure uniform distribution.
A moderate cell concentration in the sample fluid is preferred for optimal results; an excessive concentration level causes cells to overlap making it difficult to total them.
Inadequate concentration levels with a few cells per square lead to a higher statistical error ratio. This necessitates the counting of more squares, thereby lengthening the process.
One method of minimizing statistical errors and improving the count accuracy is to determine the concentration of cells per milliliter using measurements from two separate squares. A major deviation may point to an error in sampling.
Dilution using a viability dye enables the differentiation between dead cells and viable ones when viewed under a microscope. Dead cells absorb the dye, changing to a particular color while the live cells remain unstained. A test using the second chamber is done to verify the accuracy of an initial result. Varying results can indicate flaws in the process followed.
Calculating Cell Density
For the majority of applications, only the four large corner squares and the central square are considered. Special procedures apply when cells fall on a line. Cells touching the top and right lines on a square are not counted, whereas cells located on the bottom and left side are. Moving cells, such as sperm cells, must be immobilized prior to initiating the process.
When calculating cell density, the following indicators are required to obtain an accurate cell count in 1 ml of solution:
- Number of cells in a square
- Height of the sample
- Dilution factor
- Area of the square
Once these values are known, the following steps are employed:
Averaging: Unless all cells within a larger square have been counted, the results must be averaged before continuing to the next step.
Volume computation: To calculate the volume of the square, the width and height of the square are multiplied by the sample's height.
Determining how many cells are in 1 ml: The direct proportion method is used to calculate this value from the numbers arrived at in the prior steps.
Adjusting for dilution: When a sample is diluted before counting, adjustments to the final count must be made to reflect this.
Hemocytometers serve a variety of functions, such as performing blood cell counts and culturing cells in a laboratory setting where the solution containing nutrients requires periodic renewal.
Cell cultures are frequently employed in preparing yeast for fermenting beer. Hemocytometers are also useful in cell size measurement and cell processing for analysis (PCR, flow cytometry).
When selecting a hemocytometer, cost and performance characteristics should be weighed against the intended use. In case of an extremely specific nature of the application, consider acquiring a specialized device, as opposed to a generic one.
ASTM F2149 - Standard test method for automated analyses of cells - the electrical sensing zone method of enumerating and sizing single cell suspensions.