A hemocytometer is a specialized, thick glass slide designed for accurately counting cells or other particles suspended in a liquid. It features a counting chamber with an etched grid of known dimensions, which allows scientists to determine the concentration of particles within a precise volume. This guide offers a detailed, step-by-step methodology for the proper use of this tool.
Anatomy of the Hemocytometer
The hemocytometer is a robust glass slide with a central, mirrored region featuring a rectangular depression known as the counting chamber. This chamber is flanked by raised support platforms on which a specialized, thick coverslip rests. The distance between the bottom of the chamber and the coverslip is precisely manufactured, typically measuring \(0.1\) millimeters, which is the depth factor used in all calculations.
The floor of the counting chamber is engraved with a laser-etched grid pattern, most commonly the Improved Neubauer ruling. This grid consists of nine large squares, each measuring \(1 \text{ mm}^2\). The four corner squares are typically used for counting larger cells. The central \(1 \text{ mm}^2\) square is further subdivided into \(25\) smaller squares for counting smaller, more concentrated particles like red blood cells.
Preparing and Loading the Sample
Before beginning the count, the hemocytometer and its specialized coverslip must be meticulously cleaned with alcohol and dried to prevent contamination. The cell suspension must be homogeneous, requiring gentle mixing immediately before sampling to ensure an even distribution of cells. If the cell concentration is too high—ideally aiming for \(25\) to \(80\) cells per large square—the sample must be diluted with a buffer or stain, such as Trypan blue for viability assessment.
The dilution factor must be carefully recorded as it is incorporated into the final concentration calculation. To load the chamber, the coverslip is placed first, and a small volume, typically \(10\) microliters, is pipetted into the V-shaped notch at the edge of the coverslip. The liquid is drawn under the coverslip by capillary action, evenly filling the chamber without introducing air bubbles. The loaded hemocytometer should then sit undisturbed for a few minutes to allow the cells to settle onto the grid surface.
Microscopic Counting Methodology
The loaded hemocytometer is placed onto the microscope stage, and the etched grid is located and brought into focus using a low-power objective, such as \(10\text{x}\). After locating the grid, the objective can be switched to a higher magnification, typically \(20\text{x}\) or \(40\text{x}\), to clearly distinguish individual cells. A systematic counting strategy is used to ensure statistical relevance; for most cell suspensions, the four large corner squares and the central square are counted.
The most important rule for counting is the “touching rule,” a convention designed to prevent double-counting of cells that lie on the boundary lines. A standard practice is to count all cells fully within a selected square, as well as those that touch the top and left boundary lines. Cells touching the bottom and right boundary lines are excluded, as they will be counted in the adjacent squares. This consistent approach ensures that the count accurately reflects the cell density. The count proceeds sequentially through the selected squares, often using a hand tally counter.
Final Concentration Calculation
Once the total number of cells from the counted squares is determined, the final concentration of the original sample is calculated. The calculation integrates three critical factors: the total number of cells counted, the dilution factor, and the chamber volume correction factor. The total cell count is first averaged across the number of squares counted.
The volume correction factor is derived from the known volume of the counted squares, which is then extrapolated to \(1 \text{ milliliter}\). For the standard Improved Neubauer chamber, the volume of a single \(1 \text{ mm}^2\) large square is \(0.1 \text{ mm}^3\), equivalent to \(1/10,000 \text{ mL}\). Therefore, the total count is multiplied by \(10,000\) to convert the result to cells per \(1 \text{ mL}\). Finally, the concentration is multiplied by the dilution factor to account for any pre-dilution of the sample, yielding the final cell concentration in cells per milliliter.