A hemocytometer is a specialized counting chamber used to accurately count cells or other microscopic particles in a liquid sample. This instrument allows scientists and medical professionals to determine the precise concentration of cells. Its applications span various fields, including blood cell counts in diagnostics, cell culture monitoring, and yeast cell enumeration in brewing.
The Hemocytometer’s Design
The hemocytometer itself is a thick glass microscope slide distinguished by an H-shaped moat engraved on its surface. This moat creates two raised counting areas, each designed to hold a precise volume of liquid. Within these raised platforms, a grid pattern is precisely etched, forming the counting area. Several grid types exist, with the “Neubauer improved” grid being a common standard due to its specific configuration.
The coverslip, which rests on the raised edges of the hemocytometer, creates a chamber with a fixed depth, typically 0.1 millimeter (mm). This depth, combined with the known dimensions of the etched grid squares, defines a precise volume for cell counting. The Neubauer improved grid features large squares, each measuring 1 mm by 1 mm, resulting in an area of 1 square millimeter (mm²). These large squares are further subdivided into smaller squares, allowing for systematic counting.
Preparing Your Sample
Before counting, samples require careful preparation to ensure accurate and representative results. Dilution of the original sample is often necessary to achieve a countable number of cells within the hemocytometer’s grid. If the sample is too concentrated, cells will overlap, making accurate counting impossible; if too dilute, there may be too few cells to obtain a statistically significant count. The appropriate dilution factor depends on the initial concentration of cells in the sample and the desired counting range.
Various diluents are used depending on the cell type and the purpose of the count. For instance, isotonic saline solutions are common for blood cell counts, while specific media might be used for cultured cells. For viability assessments, dyes like trypan blue are added to the diluent; this dye stains only cells with compromised membranes, allowing distinction between living and dead cells. After dilution, the sample must be thoroughly mixed to ensure an even distribution of cells, preventing clumping or settling that could lead to inaccurate counts. Maintaining cleanliness throughout the preparation process is also important to prevent contamination, which could interfere with cell identification or introduce foreign particles.
The Counting Procedure
Once the sample is prepared, it is carefully loaded onto the hemocytometer by capillary action, allowing the liquid to fill the space between the coverslip and the counting surface. Care must be taken to avoid introducing air bubbles, which can obstruct the counting area and invalidate the count. After loading, the hemocytometer is placed on a microscope stage, and counting begins, typically at a magnification of 100x or 200x.
For a Neubauer improved chamber, counting typically focuses on specific large squares to ensure a representative sample. This often includes the four large corner squares and the central large square of the grid. To prevent double-counting or omitting cells that lie on the grid lines, a consistent rule is followed: cells touching the top and left boundary lines of a square are counted, while those touching the bottom and right lines are excluded. To improve accuracy, multiple squares are counted, and the cell counts from these squares are then averaged.
Calculating Your Results
After counting cells in the designated squares, the raw counts are converted into a meaningful concentration, typically expressed as cells per milliliter (cells/mL). The general formula used for this calculation combines the average cell count with factors related to dilution and the known volume of the counting area. The formula is: Cell Concentration (cells/mL) = (Average cell count per square) × (Dilution factor) × 10,000 cells/mL.
The “Average cell count per square” is derived from the total cells counted across multiple squares, divided by the number of squares observed. The “Dilution factor” accounts for any dilution performed on the original sample before loading onto the hemocytometer; for example, if the sample was diluted 1:10, the dilution factor would be 10. The constant “10,000 cells/mL” is derived from the inverse of the volume of one large counting square, which is 0.0001 mL (1 mm² area × 0.1 mm depth). This effectively scales the count to a per-milliliter basis. For example, if an average of 50 cells were counted in the designated squares of a sample diluted 1:2, the concentration would be 50 × 2 × 10,000, resulting in 1,000,000 cells/mL.