A hemocytometer is a specialized device for manually counting cells and other microscopic particles in liquid samples. Originally developed to quantify blood cells, its application has expanded across various scientific disciplines. It is now employed in fields like cell biology, microbiology, and medicine, where accurate cell concentration is important for experimental consistency and diagnostic purposes.
Understanding the Hemocytometer
A hemocytometer is typically constructed from a thick glass slide with a rectangular indentation that forms a precision volume chamber. This chamber features an etched grid of perpendicular lines on its surface. The most commonly used design, known as the Improved Neubauer chamber, includes two separate counting areas, each with a detailed grid pattern.
The grid on an Improved Neubauer hemocytometer consists of nine large squares, each measuring 1 millimeter by 1 millimeter. These large squares are further subdivided into smaller squares, defining precise areas for counting. The coverslip, which is specifically designed to be thicker than standard coverslips, rests on raised edges of the hemocytometer, creating a fixed depth of 0.1 millimeters above the counting grid. This precise depth, combined with the known area of the grid squares, allows for the calculation of the exact volume of liquid within each square.
Step-by-Step Cell Counting Procedure
Before beginning the counting process, ensure both the hemocytometer and its specialized coverslip are clean. Use lens paper or a soft, lint-free cloth to remove any dust or debris, then clean them with 70% ethanol and allow them to air dry completely. Carefully place the coverslip over the counting chambers, ensuring it is properly seated and adheres to the raised edges, which can sometimes be indicated by the appearance of Newton’s rings.
Prepare your cell suspension, ensuring the cells are evenly distributed by gently mixing the sample. If assessing cell viability, a dye such as Trypan Blue can be added to the cell suspension, typically at a 1:1 ratio. This dye selectively stains dead cells blue, allowing for their differentiation from live cells. Using a micropipette, carefully load a small volume of the cell suspension, typically around 10 microliters, into the V-shaped well at the edge of the counting chamber. Allow capillary action to draw the sample evenly under the coverslip, filling the chamber without overfilling.
Once the chamber is loaded, place the hemocytometer on the microscope stage and bring the grid lines into focus, usually starting with a 10x objective lens. Systematically count the cells within specific squares of the grid. A common practice is to count cells in the four large corner squares and the central large square of the nine-square grid. When counting, consistently apply a boundary rule: count cells that touch the top and left boundary lines of a square, but exclude those that touch the bottom and right boundary lines. This method prevents double-counting cells located on the grid lines, contributing to more consistent results.
Calculating Cell Concentration
After counting the cells in the designated squares, the next step involves calculating the cell concentration of the original sample. The volume of each large square (1 mm x 1 mm x 0.1 mm deep) is 0.1 cubic millimeters, which is equivalent to 10-4 milliliters.
The general formula for cell concentration is:
Cells/mL = (Total cells counted / Number of squares counted) x Dilution factor x 10,000
The “10,000” factor converts the count from cells per 0.1 mm³ to cells per milliliter. For instance, if you counted a total of 325 cells across five large squares (four corner squares and the center square) and your sample was diluted 1:2 with Trypan Blue (dilution factor of 2), the calculation would be: (325 cells / 5 squares) x 2 x 10,000 = 1,300,000 cells/mL.
Ensuring Accurate Cell Counts
Achieving accurate cell counts with a hemocytometer relies on careful technique and attention to potential sources of error. Improper loading of the chamber is a common issue; this can include overfilling, which alters the chamber volume, or introducing air bubbles, which obstruct the counting area. Uneven distribution of cells within the chamber, often due to insufficient mixing of the sample or allowing cells to settle, can lead to skewed counts.
Cell clumping can also complicate counting, as it becomes difficult to distinguish individual cells, potentially leading to underestimation. Incorrect focusing of the microscope, or the presence of debris mistaken for cells, can also introduce inaccuracies. To improve the reliability of counts, ensure the cell suspension is thoroughly mixed before loading and load the chamber promptly to prevent settling.
It is also beneficial to perform multiple counts from the same sample, perhaps by loading both chambers of the hemocytometer or preparing duplicate samples, and then averaging the results. Consistent application of counting rules, such as the boundary rule for cells touching grid lines, helps reduce human variability between counts.