The hemacytometer is an instrument used to determine the concentration of microscopic particles, such as cells, in a liquid sample. This glass slide provides a standardized method for counting by containing the sample within a precisely defined volume. By counting the number of cells visible under a microscope within this known space, researchers can calculate the cell density of the original suspension. The device was originally developed in the 19th century to count blood cells but is now used across many biological disciplines. It remains a widely used, inexpensive, and reliable method for cell quantification, providing a quick check on cell health and concentration before experiments.
The Counting Chamber and Grid System
A hemacytometer consists of a thick glass slide featuring a central rectangular indentation that creates a chamber. This chamber is engraved with a laser-etched grid of perpendicular lines, which is the core of the counting mechanism. When a flat coverslip is placed over the chamber, the raised edges of the slide hold it a fixed distance above the engraved grid, typically \(0.1\) millimeters.
This fixed depth, combined with the known area of the grid squares, creates a chamber with a precise and known volume. The most common pattern is the Improved Neubauer ruling, which features a large \(3\) millimeter by \(3\) millimeter grid subdivided into nine \(1\) square millimeter sections. The central square is further divided into \(25\) smaller squares, which are themselves subdivided, allowing for counting cells of varying sizes and densities. The volume over each large \(1\) square millimeter section is \(0.1\) cubic millimeters, which translates the raw count into a volume-based measurement.
Step-by-Step Procedure for Use
Proper preparation of the cell sample is the first step, often involving dilution with a solution like saline or a viability stain such as Trypan Blue. The sample must be well-mixed to ensure the cells are evenly distributed before counting. For viable cell counts, the Trypan Blue dye selectively enters and stains only the dead cells blue, allowing the live, unstained cells to be counted separately.
The hemacytometer and its coverslip must be clean and positioned correctly before loading the sample. The cell suspension is carefully introduced into the V-shaped well at the edge of the coverslip using a micropipette. Capillary action then draws the liquid into the chamber, filling the space between the slide and the coverslip without creating air bubbles or overfilling the area.
After placing the slide under a microscope, a few minutes are allowed for the cells to settle onto the grid surface. To ensure an accurate count and avoid double-counting, a consistent rule is applied for cells that touch the lines defining the square. The common practice is to count any cell touching the top or left boundary lines, while ignoring cells touching the bottom or right boundary lines. Counting is typically performed in the four large corner squares and sometimes the central square for large cells, or a selection of the smaller central squares for dense suspensions.
Converting Count to Final Concentration
The goal of using the hemacytometer is to convert the raw number of cells counted into a meaningful concentration, usually expressed as cells per milliliter (cells/mL). This calculation relies on three variables: the raw number of cells counted, the volume of the area counted, and the dilution factor of the sample. For a standard Improved Neubauer chamber, the volume of each of the nine large squares is \(0.1\) cubic millimeters, which is equivalent to \(1.0 \times 10^{-4}\) milliliters.
The general formula to determine concentration is: Concentration (cells/mL) = (Total cells counted / Number of squares counted) \(\times\) Dilution Factor \(\times\) \(10,000\). The \(10,000\) is a conversion factor derived from the inverse of the volume of the counted area in milliliters (\(1 / 0.0001\) mL). For example, if a researcher counted \(100\) cells across four large corner squares, and the sample was diluted \(1:1\) with Trypan Blue (a dilution factor of \(2\)), the calculation would be \((100 / 4) \times 2 \times 10,000\), resulting in a final concentration of \(500,000\) cells per milliliter. This step translates the microscopic observation into a practical number used to plan subsequent experiments.
Primary Applications in Science and Medicine
The hemacytometer remains a standard piece of equipment across various scientific and medical settings due to its versatility. Historically, it was developed for performing manual blood counts, which involved quantifying red blood cells, white blood cells, and platelets. Today, the device is widely used in the modern cell culture laboratory, where researchers frequently determine the concentration and viability of mammalian cell lines before splitting or plating for experiments.
The chamber is used in several other applications:
- To assess the concentration of motile cells, such as in sperm counts for fertility testing.
- In microbiology to count yeast cells for applications like brewing.
- To quantify bacteria or other small microorganisms in a suspension.
- For preparing samples for downstream molecular analyses, such as flow cytometry or polymerase chain reaction, which require a specific number of cells for a valid result.