Enhancing Cell Counting Accuracy with a Haemocytometer: A Comprehensive Guide

Enhancing Cell Counting Accuracy with a Haemocytometer: A Comprehensive Guide

Cell counting is an important part of many different scientific experiments, enabling researchers to gather crucial data about cell populations and their characteristics. In this step-by-step guide, we will delve into the utilization of a hemocytometer, coupled with trypan blue staining, to achieve accurate cell counting and determine cell viability.

A Neubauer cell chamber, also referred to as a hemocytometer, stands as a versatile and indispensable tool in the realm of cell counting and viability assessment. Comprising two integral components, namely the counting chamber and the cover slip, the hemocytometer offers a comprehensive solution for precise cell quantification. The counting chamber exhibits a distinctive square shape, which is further subdivided into a grid of smaller squares or rectangles. These grid lines help in maintaining a systematic and organized arrangement of cells during the counting process, enabling efficient and accurate quantification.

The cover slip, a transparent piece of glass or plastic, is positioned atop the counting chamber, serving as a protective barrier and holding the cells in place. It prevents cell movement and displacement, ensuring that the cells remain within the designated counting area throughout the analysis. This controlled environment is crucial for obtaining reliable and reproducible cell counts.

By utilizing the hemocytometer and trypan blue staining, researchers can gain valuable insights into cell viability. Trypan blue, a vital dye, is commonly employed to distinguish between live and dead cells. The dye selectively penetrates the membranes of non-viable or damaged cells, staining them blue, while live cells with intact membranes exclude the dye and remain unstained. This staining technique provides a means to assess the proportion of viable cells within a given population.

Processing cells for loading into the hemocytometer

The initial step involves preparing the cell suspension for accurate counting. In the case of liquid samples like blood or culture media, centrifugation is necessary to consolidate the cells. Following centrifugation, the resulting cell pellet can be resuspended in a small quantity of saline or water. Conversely, for solid samples such as tissue slices or cultured cell monolayers, directly placing the sample on the counting chamber suffices.

  • Transfer the cells to a tube, ensuring a sufficient quantity for clear visibility and counting (at least 25-50 cells).
  • Centrifuge the cell suspension at 1000 RPM for five minutes, facilitating the settling of cells and their adherence to the tube's bottom.
  • Carefully remove the supernatant (the liquid above the cell pellet) through decantation, taking care not to disturb the cell pellet at the tube's bottom.
  • Add 500 μL of PBS (phosphate-buffered saline) to resuspend the cells, gently swirling to ensure thorough mixing.

Hemocytometer preparation

A hemocytometer serves as a cell counting device featuring a microscope slide with a precisely etched grid on its surface. This grid provides a framework for counting cells within a defined area.

  • Begin by filling the Neubauer cell counting chamber with a trypan blue solution. The trypan blue solution should be filled up to the line labeled "0.04mm" on the Neubauer cell counting chamber.
  • Subsequently, introduce your cell sample into the Neubauer cell counting chamber. Ensure an adequate number of cells to enable accurate counting (at least 25-50 cells).
  • Gently swirl the Neubauer cell counting chamber, facilitating the mixing of the cell sample and the trypan blue solution.

Cell counting procedure

  • Begin by delicately positioning the Neubauer cell counting chamber onto a microscope slide, ensuring that the microscope lens is oriented toward the chamber's grid lines.
  • Adjust the microscope focus to clearly visualize the grid lines within the Neubauer cell counting chamber. Individual cells should become discernible within the grids, allowing for precise counting.
  • Systematically count the number of cells present within a designated area of interest, such as a quadrant or sector, ensuring accuracy and consistency in the counting process.
  • Proceed to repeat this meticulous counting procedure for the remaining areas within the Neubauer cell counting chamber, systematically covering all relevant regions.

For estimating the number of White blood cells (WBCs) in a given volume:

  • Calculate the WBC dilution factor (DF). This involves dividing the total number of cells in the Neubauer cell counting chamber by the number of WBCs counted within that chamber.
  • Proceed to count the number of WBCs present in each Neubauer cell counting chamber. Multiply the count of WBCs observed per chamber by the WBC dilution factor. This calculation provides you with the number of cells/mL in your original sample.

For estimating the number of Red blood cells (RBCs) in a given volume:

  • Count the overall number of cells present in the Neubauer cell counting chamber, encompassing both white blood cells and red blood cells.
  • Calculate the percentage of RBCs in your sample by dividing the number of RBCs counted by the total number of cells observed.
  • Multiply the percentage of RBCs in your sample by the volume of your original sample. This calculation will yield an estimation of the total number of RBCs present in your original sample.

Trypan blue cell viability assay

Trypan blue is a dye that is used to stain live cells. When the dye binds to a cell, it causes the cell to take on a blue color. This makes it easy to distinguish between live and dead cells. Live cells will take up trypan blue, while dead cells will not.

- To calculate cell viability, you will need to know the total number of cells counted and the number of live cells (stained with trypan blue).

- The formula for cell viability is: % Cell Viability = (Number of Live Cells/Total Number of Cells) x 100.

- For example, if you counted 100 cells and there were 30 live cells, the cell viability would be: (30/100) x 100 = 30%.

- To calculate the percentage of dead cells, you would use the same formula, but substitute "Number of Dead Cells" for "Number of Live Cells".

Trypan blue assay

Tips for improving efficiency of cell counting in a hemocytometer

- Count cells in multiple areas of the Neubauer cell counting chamber to get a more accurate estimate of the number of cells in your sample.

- Try to use the same area of the Neubauer cell counting chamber for all of your counts. This will help you to be more accurate and consistent in your measurements.

- When using the neubauer cell counting chamber to count cells in a sample it is important to use a microscope with at least 40x magnification. This will allow you to see individual cells within the grid.

- To stain the cells, mix a small volume of trypan blue with your sample and let it sit for at least five minutes. Be careful not to disturb the cells on the cover slip.

- Use a microscope with a digital camera to take pictures of the neubauer cell counting chamber. This will allow you to save time by not having to count cells manually.

- Use a cell counting software program to automate the cell counting process. This can be a real time saver, especially if you are counting a large number of cells.


The Neubauer cell counting chamber is a handy tool that can be used to count cells and determine cell viability. We hope that this article was helpful and now you feel confident using a hemocytometer! Have any tips of your own? Share them with us.

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Written by Pragna Krishnapur

Pragna Krishnapur completed her bachelor degree in Biotechnology Engineering in Visvesvaraya Technological University before completing her masters in Biotechnology at University College Dublin.

11th Jun 2023 Pragna Krishnapur, Msc

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