Cell Counting 101 – Tip 3: Cell Counting Strategies When Using a Hemocytometer

Tip 3 of 6

– 5 min read

As mentioned in our first blog post in this series, you obtain manual cell counts with a hemocytometer to determine the total cell count and the number of viable and dead cells in a population. This can be challenging because you need to consider dilution factor (see this blog post), and you might doubt which parts of the hemocytometer grid to count. Today, we’ll cover these points to make the process more manageable:

  • Different cell counting strategies – don’t go for the quick count
  • How to calculate the total cell concentration in a sample based on your manual cell count
  • When to do another dilution

It All Comes Down to Strategy!

There are many different cell counting strategies centered around the hemocytometer grid. When you count the cells, it’s important to choose the squares that give the best overall representation of the cells in the sample. For example, you shouldn’t only count cells in the three top squares of the grid as this leads to results expressing a trend that isn’t recognized in the rest of the grid if the liquid did not spread evenly throughout the slide surface when dispensed from the pipette. Therefore, this would not be representative of the whole sample.

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 strategy

The Logical Count

A common and very representative approach is to count the cells in the four corner squares and the middle square of the hemocytometer’s grid (illustrated by an improved Neubauer chamber). By doing this, you can predict the cell count of the rest of the squares.

To make sure that the count is consistent and that cells aren’t counted twice, you should only count cells on two of the square sides, e.g., the left and top borders of the square. Others choose the opposite where cells on the right and bottom are included in the count whereas those on the left and top aren’t. Regardless of the strategy, the most important thing to remember is to be consistent throughout your counting procedure and to apply the same strategy every time1, so you can compare your data.

The Absolute Count

Alternatively, you can count the cells in all nine squares of the hemocytometer. This strategy ensures that you count everything and avoid error from the generalization-assumption mentioned above. For this type of cell count, you count the cells in the squares while following a zig-zag pattern.

Here, the same rule applies as above to avoid counting cells twice. This counting method is particularly useful when there’s a high cell concentration in the sample because of a pattern that’s easy to follow1.

The Quick Count

Lastly, if you’re in a rush to do a cell count, you might get tempted to do the quick count. With this strategy you only count the cells in the top left square as well as the cells in the bottom right square. With this approach, you won’t get a result that is as representative as the two methods mentioned above, but it can be a good way to spot-check your cell culture when in a hurry.

These are some of the approaches you can take. The most important thing for a successful cell count is that the same strategy is applied every time to get as few inconsistencies as possible.

Calculating the Cell Concentration

You use the number of counted cells, the dilution factor as well as the number of counted squares to determine the total cell concentration in the sample in cells/ml. Within each of the nine squares of the improved Neubauer grid, the volume is 0.1 mm3 and you therefore multiply with 104(*) to convert this volume to ml.

To determine the total cell count in a sample from an improved Neubauer chamber, use the following formula:

* In most cases, the multiplication factor will be 104 since most hemocytometer squares have an area of 1 mm2 and a depth of 0.1 mm, i.e. a volume of 0.1 mm3. Please refer to the table above for examples of different hemocytometers and their multiplication factors.

You May Need to Dilute

Continuous and precise cell counting is important for many applications such as monitoring trends in a cell population or when performing research studying cell culture responses to different stimuli. However, for the cell counts to be reliable you need to meet certain requirements.

A rule of thumb is that a sample’s concentration should be below 2.5×106 cells/ml, ideally around 1×106 cells/ml. If you exceed this 2.5×106 cells/ml, you need to further dilute the sample1. For more on this topic, read our blog post on how to calculate the dilution factor.

When In Doubt, Do Another Count

When you need precise results for further decision making, do another cell count to validate your data. This way you ensure that you have reliable results because you catch possible errors before it’s too late.

Keep your eyes open for the next blog post which covers what cell viability is and how to calculate it.

Further Reading

Reference

  1. 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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2 comments

  1. Suzanna Long | N/A |

    Hello,

    In your blog you mention the mazimum amount of cells /ml, after which one needs to dilute.
    I am often working with the opposite scenario, I have a very low number of cells (4-10 cells/square) which leads to inconsistencies in my cell counts. Do you have any recommendations that could help me get more consisten counts?
    Thank you

    1. Janny Marie Peterslund | Scientific Affairs Manager, ChemoMetec |

      Dear Suzanna,

      Thansk a lot for this question. In a case of having too few cells in your sample, we would recommend spinning down the cells, and diluting them in a smaller volume of culture medium. Watch this webinar about why we recommend culture medium and not PBS here: https://chemometec.com/webinar/webinar-is-pbs-affecting-your-cell-counts/

      You risk losing some of your cells during the spin procedure, and if so, this will add to the variance you see in your final cell counts. So it depends very much on what you need the cell count data for: Is it to give you a rough idea of how much to dilute your cell culture at a split and precision is not crucial? Or do you need the data to inform accurately of cell number as you want to know the average protein output per cell in a production-like setup? You will have to take this into account and you can validate your cell counting procedure, including the sample concentration step, to know how it affects your data. Perhaps read this blog post on data validation in cell counting: https://chemometec.com/how-the-meticulous-researcher-validates-their-cell-counting-method/

      I hope this answers your questions?
      All the best,
      Janny Marie.

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