Counting cells using a hemocytometer

Counting cells using a hemocytometer is a fundamental technique in cell biology, enabling researchers to quantify cell populations with precision. Hemocytometers were originally developed for counting blood cells and remain essential for quantifying blood cells in blood samples as well as other cell types. This protocol outlines a reliable method for assessing cell viability using trypan blue exclusion. It covers sample preparation, chamber loading, and microscopic analysis using a Neubauer or Burker chamber, with the Improved Neubauer chamber being a widely used standard for accurate cell counting due to its detailed grid and design advantages. The method is ideal for both adherent and suspension cells and supports reproducible results across various experimental setups. Whether you’re working in oncology, microbiology, or general cell culture, this guide ensures accurate manual cell counts. With clear instructions and troubleshooting tips, it is a valuable resource for both novice and experienced scientists seeking dependable cell quantification.

Introduction

Manual cell counting remains a cornerstone of cell culture workflows, offering direct insight into cell viability and density. The hemocytometer, a specialized counting chamber, is widely used for manual cell counting and enables researchers to visually assess live and dead cells using trypan blue staining and other dyes for cell viability assessment. This protocol provides a step-by-step guide for preparing samples, staining cells, and performing counts under a microscope. The principle of trypan blue exclusion is a classic dye exclusion test for determining cell viability, distinguishing viable from non-viable cells. It is designed to enhance reproducibility and accuracy in cell-based experiments. Whether validating cell growth, preparing for downstream assays, or monitoring culture health, this method is essential for maintaining experimental integrity. The protocol is compatible with both glass and disposable hemocytometers, making it adaptable to various lab environments.

Background and principles

The hemocytometer is a precision-engineered glass slide etched with grids composed of perpendicular lines forming squares of different sizes to accommodate various cell types. Each grid square in the Improved Neubauer chamber has a specific volume, which is essential for accurate cell concentration calculations. The surface of the hemocytometer and the coverslip are designed to ensure proper sample distribution, with surface tension helping to spread the liquid evenly without leakage or bubbles during chamber loading. The principle relies on trypan blue exclusion: living cells exclude the dye, while dead cells absorb it, appearing blue. However, prolonged exposure to trypan blue can lead to cell death, affecting the accuracy of viability measurements. By counting cells in defined grid areas, researchers can systematically calculate the total number of cells in a sample. After centrifugation, the cell pellet is resuspended for viability assessment. Cell viability can be determined using various dyes, such as acridine orange and propidium iodide, in addition to trypan blue. Automated cell counters and flow cytometers are alternative methods for cell counting and viability assessment, offering increased speed and objectivity. This method is particularly useful for adherent cells post-trypsinization and suspension cultures. The protocol emphasizes aseptic technique, proper chamber loading, and consistent counting practices to minimize variability. It is a cost-effective and widely accepted approach for cell quantification, especially in labs without access to automated counters.

Stage 1 - Preparing hemocytometer

Steps

If using a glass hemocytometer and coverslip, clean with alcohol before use.

• Moisten the coverslip with water and affix to the hemocytometer.

If using a disposable hemocytometer (for example, INCYTO DHC-N01), remove it from the packet before use.

Stage 2 - Preparing cell suspension

Aseptic technique prevents contamination of cell cultures and reagents by microorganisms.

Table 1. The volume of DPBS and trypsin-EDTA required for trypsinization of adherent cells.

Steps

Gently swirl the flask to ensure the cells are evenly distributed.

Before the cells have a chance to settle, take out 0.5 mL of cell suspension using a 5 mL sterile pipette and place in an Eppendorf tube.

Take 100 µL of cells into a new Eppendorf tube.

• Take 100 µL of cells into a new Eppendorf tube and add 400 µL 0.4% Trypan Blue (final concentration 0.32%).
• Mix gently.

Stage 3 - Counting

Steps

Using a pipette, take 100 µL of Trypan Blue-treated cell suspension and apply to the hemocytometer.

• If using a glass hemocytometer, very gently fill both chambers underneath the coverslip, allowing the cell suspension to be drawn out by capillary action.
• If using a disposable hemocytometer, pipette the cell suspension into the well of the counting chamber, allowing capillary action to draw it inside.

Using a microscope, focus on the grid lines of the hemocytometer with a 10X objective.

Using a hand tally counter, count the live, unstained cells (live cells do not take up Trypan Blue) in one set of 16 squares.

• When counting, employ a system whereby cells are only counted when they are set within a square or on the right-hand or bottom boundary line.
• Following the same guidelines, dead cells stained with Trypan Blue can also be counted for a viability estimate if required.

Move the hemocytometer to the next set of 16 corner squares and carry on counting until all 4 sets of 16 corners are counted.

Stage 4 - Viability

Steps

Take the average cell count from each of the sets of 16 corner squares.

Multiply

• Multiply by 10,000 (10⁴).

Multiply by 5 to correct for the 1:5 dilution from the Trypan Blue addition.

If both live and dead cell counts have been recorded for each set of 16 corner squares, an estimate viability can be calculated.

Steps

Add together the live and dead cell count to obtain a total cell count.

Divide the live cell count by the total cell count to calculate the percentage viability.

Best practices for counting cells

To achieve precise and reproducible results when counting cells with a hemocytometer, it’s important to follow a set of best practices. Consistency is key; always use the same squares on the hemocytometer grid for each count, and establish clear rules for counting cells that touch the grid lines (for example, count cells on the right and bottom lines, but not on the left and top). This approach helps minimize human error and ensures that your cell counts are comparable across different samples and experiments.

Carefully calculate and apply the correct dilution factor when preparing your sample, as errors here can significantly affect your final cell concentration. Use a pipette to handle your cell suspension, and avoid touching the hemocytometer grid with your fingers to prevent contamination or damage. Regularly clean and inspect both the hemocytometer and the microscope to maintain clear visibility of the grid and cells.

By standardizing your counting technique and maintaining your equipment, you can reduce variability and improve the accuracy of your cell counting results. These best practices are essential for reliable cell culture work and for generating high-quality data in research settings.

Finalizing cell counting results

After completing your cell counting and calculating the cell concentration, it is important to finalize your results by recording all relevant data. This includes the total cell count, the number of viable cells (those with intact cell membranes that exclude trypan blue), and the percentage of viable cells in your sample. Make note of any observations during counting, such as the presence of cell debris, clumped cells, or unusual cell shapes, as these can provide valuable insights into the health and quality of your cell population.

These finalized results are crucial for making informed decisions in cell culture, such as determining the optimal seeding density for experiments or identifying potential issues with cell viability. Accurate documentation also supports reproducibility and troubleshooting in future research.

For larger or more complex samples or when higher throughput is needed, automated cell counters or flow cytometry can supplement manual counts. These technologies can provide rapid, objective measurements of total cell count and cell viability, further enhancing the reliability of your data. Regardless of the method, careful finalization and interpretation of your cell counting results are essential steps in any cell culture or research workflow.