Spilling the secrets: How to count cells with a hemocytometer

What are hemocytometers & how do they work?

A hemocytometer is a specialized slide used for cell counting with a microscope. There are several types of hemocytometers, all with different counting grids. The most commonly used is the ‘Improved Neubauer’ chamber.

What is the improved Neubauer chamber

The Improved Neubauer has an H-shaped indent at the center of the slide that separates the space into two counting chambers. Grids are engraved onto the surface to make cell counting easier and more precise.

The 3×3 mm counting grid on the Improved Neubauer is subdivided into nine 1×1mm squares. As shown in the diagram, each square is further divided into 16, 100, or 400 smaller squares. The various grids allow you to count cells of different sizes.

How to count cells with a hemocytometer

Before you begin cell counting with your hemocytometer, you need to do some preparation. Start by taking a representative sample of your cell population. You can ensure your sample is representative by resuspending the solution by pipetting up and down a few times before taking your sample.

If you want to determine the viability of your cells as well as the total cell count, you can use a dye exclusion test. This involves staining your cells with a dye that can differentiate between viable and non-viable cells. Some commonly used stains are trypan blue, propidium iodide, erythrosine B, acridine orange, and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI). In our previous blog post, you can learn more about these stains, including their properties, advantages, and disadvantages.

Do you need to dilute your cells?

If there are too many or too few cells in your counting chamber, this will impact your cell count. If there are too many cells, counting can be more difficult, resulting in errors. However, you can expect more random errors if there are too few cells. The recommended cell concentration for counting with a hemocytometer is around 106 cells/ml. Using an Improved Neubauer chamber, the optimum cell concentration range is 2.5 × 105 to 2.5 × 106 cells/ml.² As a result, you may need to dilute or concentrate your sample before counting.

If you dilute your cells, note down your dilution factor so you can calculate the concentration of cells in the original sample later on. Determining the dilution factor is simple using the following formula:

How does the hemocytometer method work?

In brief, the hemocytometer cell counting method involves the following steps:²

1. Clean the hemocytometer and the cover glass with ethanol. Ensure the ethanol evaporates completely, so it does not affect your cells.
2. Place the cover glass on top of the hemocytometer’s chambers to stop your sample from evaporating.
3. Load 10 µl of your stained sample into one or both counting chambers with a micropipette. Capillary action ensures even distribution of the suspension within the chamber.
4. Place the hemocytometer under the microscope.
5. Adjust the microscope’s focus until you can clearly see the cells.
6. Count the cells using a tally counter (see below for details about the rules of cell counting using a hemocytometer). Keep track of the total number of cells and the number of dead cells.
7. When you are finished, clean the hemocytometer and the cover glass with ethanol.

Are there any rules for counting cells with a hemocytometer?

The rules and strategies used to count cells with a hemocytometer can vary from person to person and lab to lab. Before counting the cells in your hemocytometer grid, you should decide which squares you will count, and which rules you will use to avoid counting the same cell twice. Staying consistent with your chosen strategy is essential for producing precise, reliable results, so choose carefully!

Which squares should i count?

Before you start counting, you need to decide which squares of the hemocytometer counting grid you will count. Choosing squares that give a good overall representation of the cells on the slide is crucial. For example, avoid only counting cells in the top three squares of the grid, as this may not be representative if the liquid did not spread evenly throughout the slide surface when dispensed from the pipette. Some common strategies reported by our in-house scientists include:

Based on the perspectives of our in-house Field Application Scientist team, we found these three methods to be the most used:

The logical count

A common and representative approach is to count the cells in the four corner squares and the middle square of the hemocytometer’s grid. This is called a ‘logical count.’

The quick count

Lastly, if you’re in a rush, you might be tempted to do the ‘quick count.’ With this method, you only count the cells in two squares that are diagonally opposite each other. If you use this approach, your results won’t be as representative, but it can be a good way to spot-check your cell culture if you are in a hurry.

Which cells are inside the counting square?

The results are in – calculating cell concentration & viability

Each of the nine squares in the Improved Neubauer grid has a volume of 0.1 mm³. The multiplication factor of 10⁴ in the formula above converts the count from cells per 0.1 mm³ to cells per ml. Most hemocytometer squares have a volume of 0.1 mm³, so the multiplication factor will be 10⁴ in most cases. The table to the left shows the multiplication factors for other counting chambers.

When you know the total number of cells and the number of dead cells in your sample, you can calculate cell viability using the following formula:

What are the common errors in cell counting with a hemocytometer?

Cell counting with a hemocytometer is typically error-prone, with errors often as high as 20-30%.2 Common sources of error range from human error with procedures and calculations, to errors caused by non-uniform cell staining and cell debris.

You can minimize errors by ensuring you work as consistently and precisely as possible. Take care to prepare dilutions correctly, pipette carefully, set clear counting guidelines, and be diligent about what you count as a cell. Remember, consistency is king! Performing additional counts can also ensure that your results are reliable and may help you catch errors before it’s too late. Typically, we repeat all cell counts three times, but “when in doubt, do another count!”

If you want to learn more about reducing error in manual cell counting, stay tuned for our next blog post, ‘Revealing the precision of your manual cell counts’
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Further reading

• Journal post: Is trypan blue toxic? The dark side of the popular stain
• Journal Post : All your burning cell counting questions answered
• Podcast: Standardizing cell counting
• Journal post: Why working with trypan blue is not a good idea
• The NucleoCounter® range: Automated cell counters & analyzers

Reference

1. Vembadi A, Menachery A, Qasaimeh MA.: Cell Cytometry: Review and Perspective on Biotechnological Advances. Front Bioeng Biotechnol. 2019;7:147.
2. Electron Microscopy Sciences: Neubauer Haemocytometry.
3. Stoddart MJ.: Cell viability assays: Introduction. Methods Mol Biol. 2011;740:1-6