Cell Counting 101 – Tip 1: What Is a Hemocytometer & How Do You Use It?

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Tip 1 of 6

– 5 min read

A hemocytometer, or cell counting chamber, is your primary tool used for manual cell counting. As implied in its name, the hemocytometer was originally invented with the aim of quantifying blood cells in patient blood samples1. Now, the hemocytometer is used to count many different types of cells. It can be used to determine the total cell count and viability of a sample. In this blog post, we will explore:

  • The detailed structure of a hemocytometer’s counting grid
  • How to count cells correctly using a hemocytometer

Structure & Purpose of Hemocytometers

A hemocytometer is a specialized slide used for cell counting. The most-used type of counting chamber is the improved Neubauer chamber. At the center of the slide, there is an H-shaped indent separating the space into two counting chambers. Each chamber has a 3×3 mm counting grid engraved into the glass surface, with a total of nine 1×1 mm squares. These grids are used to make the cell counting process easier and more precise.

The corner squares are further divided into 16 squares, the center square is divided into 400 squares and the remaining four large squares are divided into 100 squares each. The various grids present on the improved Neubauer chamber are used to count cells of different sizes. Lastly, a glass cover is located on the top of the chambers to cover the samples1, so they do not evaporate.

hemocytomer - etched counting grid

Details That Are Hidden From the Naked Eye

To find out the number of cells in a whole population, taking epithelial cells as an example, you should start by counting cells in a sample of epithelial cells. It’s crucial to load a representative sample from a population into the hemocytometer to obtain a reliable count. To ensure you get a representative sample, resuspend the solution before taking a sample from it and ensure frequent mixing when preparing dilutions.

Before counting the cells, stain them with a dye that makes it easier to differentiate between viable and non-viable cells. Some commonly used stains are trypan blue, propidium iodide (PI), erythrosine B, acridine orange (AO) and DAPI. Learn more about these stains and some of their properties.

How to Count Cells With a Hemocytometer

In brief, the cell counting process is divided into the following steps2:

  1. Clean the hemocytometer and the cover glass with ethanol. Make sure it evaporates completely, so it does not affect your cells
  2. Place the cover glass on top of the hemocytometer’s chambers
  3. Load 10 µl of the 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 in the sample
  6. Count the cells using a tally counter (Post 3 will explain how to count living and dead cells, and determine the sample’s viability)
  7. Clean the hemocytometer and the cover glass with ethanol

Stay Tuned…

Counting Strategies

Cell counting strategies differ from lab to lab. Some labs count the cells in the corner squares and middle square and include the cells that touch the top and left grid borders, while excluding cells that touch the bottom and the right grid border from the count.

Much More to Learn

After you obtain the cell count, you can determine values such as the total population’s cell count, cell viability and variance. We will cover these topics in future blog posts.

cell counting strategies 1000x1000 rgb
Talking about cell counting strategies on the team, we realized there are MANY ways to approach this. Want to know which is better? Read Post 3 on how to count both live and dead cells.

Read the next blog post in this series on how to properly dilute a sample and how to calculate the dilution factor.

Further Reading

References

  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.

By Christina Psaradaki, Student Assistant at ChemoMetec
Christina Psaradaki studies Human Life Science Engineering at the Technical University of Denmark. At ChemoMetec, she writes for the Cell Counting Blog.

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