Hemocytometer Cell Counting Procedure

Hemocytometers have been around since the beginning of the nineteenth century, when they were designed to accurately count blood cells for medical purposes. The technique revolutionized the field of hematology by providing a standardized and reliable method for quantifying cellular components in biological samples. Hemocytometers employ innovative concepts from various scientific disciplines, combining principles of physics, chemistry, and biology. By utilizing surface tension, cells are evenly distributed across the counting area of the hemocytometer, ensuring representative sampling. The device incorporates a grid pattern, usually etched onto a glass surface, enabling precise measurement of cell concentrations and densities. This allows researchers and clinicians to assess the health of an individual by analyzing various parameters, such as red and white blood cell counts, platelet levels, and cell morphology. Additionally, the statistical significance of the results obtained through hemocytometry plays a crucial role in drawing accurate conclusions. The use of hemocytometers has expanded beyond blood analysis, finding applications in diverse fields such as microbiology, veterinary medicine, and industrial research. This underscores the versatility and enduring relevance of hemocytometry as a fundamental tool in scientific investigation and medical diagnostics

The hemocytometer, an essential tool in cell counting and measuring cell density, offers more than just carefully etched lines delimiting nine 1 x 1 mm (1mm²) sectors. To provide even greater accuracy, each sector is further divided into 16 or 25 smaller squares of equal size, depending on the specific version of the hemocytometer being used. These smaller squares facilitate precise counting by creating a grid pattern that ensures consistent distribution of cells for analysis.

During the counting process, a specially designed slide is positioned on top of the chamber, leaving a narrow 0.1mm gap between the surface of the chamber and the bottom of the slide. This deliberate spacing serves a crucial purpose: it establishes a known volume for analysis. By considering the dimensions of the chamber and the gap, a precise volume of 0.1 mm3 (equivalent to 1E-4 mL) is obtained.

When cells are introduced into the chamber, they naturally occupy the available space within the gap. Consequently, any visible cells within each of the 1mm² sectors are representative of the cell population present in the 1E-4 mL volume. This ingenious design enables researchers and scientists to extrapolate cell density at a small scale, providing a quantifiable measure of cells per milliliter (cells/mL).

You count cells in 4x1mm2 sectors and you get the following counts 31,25,40,33 – averages to 32.25 cells per 1mm2. You know that area corresponds to a 1E-4 mL volume, so 32.25 cells exist in 1E-4 mL, resulting in a density of 322,500 cells/mL. No formulas involved – we just deduced what the cell density is based on hemocytometer dimensions. Extending this to the general case, the formula would be cell density = (total cells counted / number of squares counted) / volume of a single square – that doesn’t account for any dilutions carried out.

For 1mm2 squares, the volume of a single square was 1E-4, but depending on the square size chosen to count cells, this may be different. For example, small squares inside the central square are used for red blood cell counting. In that case, the square volume is 1E-4 mL / 25 (there are 25 smaller squares in the 1mm2 central square), leading to a 4E-6 mL small square volume.

There’s another particularly useful metric that can be obtained from a hemocytometer count: viability. Viability gives a measure of the amount of live cells among all cells in culture. It can be measured by adding a dye to the counting solution that stains dead cells but not live cells – those are then recorded separately during the count and viability is calculated as live cells / total cells (as a percent).

Cell density is critical to maintain a healthy culture, subculturing, creating cell banks, preparing samples for flow-cytometry and ELISA, and simply to keep track of your numbers for publications. On the other hand, viability is inherently a marker for culture health; it is also useful in toxicity tests to understand how many cells are killed in a specific time frame given a cytotoxic agent.

Hemocytometer usage has also expanded into other fields. It is now routinely used in brewing to assess the density and quality of yeast during fermentation and in honey production when determining the density of pollen in the final product. Technology has also evolved the hemocytometer to allow automated cell counting with disposable chambers, providing almost immediate results. This new approach works fairly well for a wide range of common cell lines (and saves tons of time!) but may need fine-tuning for more custom applications such as patient cells with rare diseases.

Cell counting with a hemocytometer can be very repetitive and time consuming. Especially when you need to go through 10-20 samples a day, count viable cells and perform calculations for all of them on the fly.

HemocyTap, is an app used to help tally-count cells and carry out those daily calculations automatically. Count live & dead cells, calculate the cell density, viability & new culture volume, save and send your data via email and plot the evolution over time. The main features of this app are:

1. Make use of two tally counters: one for living cells and one for dead cells.
   2. Each emits a distinct sound, letting you know which button you should have pressed.
   3. Records every cell you've counted in every square.
   4. Alter the square you're counting in the same way that you alter the square visible in the microscope.
   5. Select the default squares you want to count; they will be highlighted.
   6. You can decide whether to count the large or little squares inside the centre square.
   7. Computes for you the average of your counts.
   8. Tells you how much volume to add to get the required cell density when subculturing
   9. Shows cell viability and live cell number.                              
   For more cell counting information or to download the hemocyTap App visit www.hemocytometer.org