Spilling the Secrets: How to Count Cells with a Hemocytometer
A hemocytometer (also known as a haemocytometer or a cell counting chamber) is a tool used for manual cell counting. As the name implies, the hemocytometer was originally invented for quantifying blood cells.1 Now, hemocytometers are used to determine the total cell count and viability of many different cell types in various applications.
In this blog post, we answer common questions about hemocytometers, including:
- What are hemocytometers and how do they work?
- How do I count cells using a hemocytometer?
- What rules and strategies should I use for manual cell counting?
- How do I calculate dilution factor, cell count, and viability?
- What common cell counting errors should I be aware of?
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 hemocytometer, all with different counting grids. The most commonly used is the ‘Improved Neubauer’ chamber.
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). You can learn more about these stains, including their properties, advantages, and disadvantages, in our previous blog post.
You May 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, if there are too few cells, you can expect more random errors. The recommended cell concentration for counting with a hemocytometer is around 106 cells/ml. If you are 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 do dilute your cells, note down your dilution factor so you can calculate the concentration of cells in the original sample later on. Determining dilution factor is simple using the following formula:
Using Your Hemocytometer
In brief, the hemocytometer cell counting method involves the following steps:2
- Clean the hemocytometer and the cover glass with ethanol. Ensure the ethanol evaporates completely so it does not affect your cells.
- Place the cover glass on top of the hemocytometer’s chambers to stop your sample from evaporating.
- 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.
- Place the hemocytometer under the microscope.
- Adjust the microscope’s focus until you can clearly see the cells.
- Count the cells using a tally counter (see below for details about the rules of cell counting using a hemocytometer). Keep track of both the total number of cells and the number of dead cells.
- When you are finished, clean the hemocytometer and the cover glass with ethanol.
Rules for Counting Cells with a Hemocytometer
The rules and strategies used when counting cells with a hemocytometer can vary from person to person and from 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. It’s important to choose squares that give a good overall representation of the cells on the slide. 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 Absolute Count
Alternatively, you can count the cells in all nine squares of the hemocytometer. In this method, known as an ‘absolute count,’ you count the cells in all the squares while following a zig-zag pattern. This counting method is advantageous when there’s a high cell concentration in the sample because it is a pattern that’s easy to follow so you are less likely to get lost and have to restart.2
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?
Rules about which cells are counted as inside a square help ensure that you don’t count the same cell twice. Some labs include the cells that touch the top and right grid borders while excluding cells that touch the bottom and the left grid border (see diagram). Other labs use different rules, so check with your colleagues if you aren’t sure.
Regardless of your chosen strategy, the most important thing to remember is to be consistent throughout your counting procedure,2 so your results are as precise as possible, and you can compare your data over time.
The Results Are In – Calculating Cell Concentration & Viability
Once you have counted your cells, you can use your cell count and your dilution factor to determine the total cell count or concentration in your original sample using the following formula:
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 count cells using a cell stain, you can also determine cell viability. Tracking cell viability can aid everyday cell culture maintenance and help you understand how your cells respond to different environments.3
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:
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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- Blog post: Is Trypan Blue Toxic? The Dark Side of the Popular Stain
- Blog Post : All Your Burning Cell Counting Questions Answered
- Podcast: Standardizing Cell Counting
- Mini Review: Why Working with Trypan Blue is Not a Good Idea
- The NucleoCounter® range: Automated Cell Counters & Analyzers
- Vembadi A, Menachery A, Qasaimeh MA.: Cell Cytometry: Review and Perspective on Biotechnological Advances. Front Bioeng Biotechnol. 2019;7:147.
- Electron Microscopy Sciences: Neubauer Haemocytometry.
- Stoddart MJ.: Cell viability assays: Introduction. Methods Mol Biol. 2011;740:1-6
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