A hemocytometer is a specialized device used to accurately count cells or other microscopic particles within a liquid sample. This tool has become a fundamental instrument across various scientific fields, including biology, medicine, and industry. Accurate cell counts are essential for experiments, diagnostics, and quality control, ensuring reliable results.
Essential Components
A hemocytometer system consists of a thick glass slide, also called a counting chamber, which features a precisely etched grid. The most commonly used grid is the Improved Neubauer, characterized by nine large squares, each measuring 1 square millimeter (mm²). This chamber is designed with a specific depth, typically 0.1 millimeters, creating a known volume above the grid when covered.
A specialized coverslip is placed over the counting area, forming the chamber where the cell suspension is held by capillary action. A light microscope is necessary for visualization and counting. A micropipette is used for sample loading, and a manual tally counter assists in keeping track of counted cells.
Sample Preparation
Preparing the cell sample accurately is crucial for obtaining reliable cell counts. Cells must be uniformly suspended and diluted to an appropriate concentration for counting. An ideal cell density for counting with an Improved Neubauer chamber ranges from 2.5 × 10^5 to 2.5 × 10^6 cells per milliliter. If the sample is too concentrated, cells will overlap, making counting difficult; if too dilute, there may be insufficient cells for a statistically significant count.
Dilution is achieved using a suitable diluting fluid, which can also serve specific purposes. For instance, trypan blue dye is commonly added to differentiate between live and dead cells, as it only enters cells with compromised membranes, staining them blue. Other diluents like isotonic saline or dilute acetic acid may be used, with acetic acid specifically lysing red blood cells to facilitate counting of other cell types. Before loading, the diluted cell suspension must be thoroughly mixed to ensure an even distribution of cells, preventing settling and ensuring the sample accurately represents the population.
Loading and Counting Cells
Loading the prepared sample onto the hemocytometer requires careful technique to ensure proper distribution and avoid air bubbles. With the special coverslip firmly in place, a small volume of the well-mixed cell suspension, typically around 10 microliters, is introduced into the V-shaped notch at the edge of the counting chamber. The liquid fills the chamber by capillary action, spreading evenly under the coverslip without introducing air bubbles. Allowing the sample to settle for a few minutes helps ensure the cells are stationary on the counting grid.
Once settled, the hemocytometer is placed on the microscope stage and focused to clearly visualize the grid lines and cells. For most cell counting applications, cells within the four large corner squares and the central square are counted. A consistent counting rule must be applied to avoid double-counting cells on boundary lines; generally, cells touching the top and left lines of a square are counted, while those on the bottom and right lines are excluded. This systematic approach ensures an unbiased and accurate count.
Calculating Cell Concentration
After counting the cells in the designated squares, the next step involves calculating the cell concentration of the original sample. This calculation accounts for the number of cells observed, the volume of the counted area, and any dilution performed during sample preparation. The standard formula used is: Cells per milliliter = (Total cells counted / Number of squares counted) × Dilution Factor × 10,000.
The “Total cells counted” refers to the sum of cells observed across all the chosen squares. The “Number of squares counted” is typically five, representing the four corner squares and the central square of the Improved Neubauer grid. The “Dilution Factor” accounts for any dilution made to the original sample. The constant “10,000” is a conversion factor that translates the cell count from the hemocytometer’s volume to cells per milliliter. For instance, if 325 cells were counted in 5 squares after a 1:1 dilution, the concentration would be (325 / 5) × 2 × 10,000, resulting in 1,300,000 cells per milliliter.
Cleaning and Care
Proper cleaning and maintenance of the hemocytometer and coverslip are essential to ensure their longevity and maintain accuracy. After each use, thoroughly clean them to remove any residual sample or staining agents. Rinsing with distilled water is an initial step, followed by cleaning with 70% ethanol.
Avoid using harsh detergents, as their residues can interfere with cell counts. After cleaning, gently dry the hemocytometer and coverslip using a lint-free cloth or lens paper to prevent scratches and water spots. Store the hemocytometer in a protective case or a clean, dry environment to prevent dust accumulation and physical damage.