How to use a Hemocytometer Cell-counting Chamber

Exploring the essential process of cell counting using a hemocytometer.

The hemocytometer chamber has carefully etched lines delimiting nine 1mm2 sectors. Each sector is in turn divided into 16 or 25 smaller squares of equal size (for the most common hemocytometer version). When counting, a special slide is placed on top of the chamber, leaving a 0.1mm gap between the surface and the bottom of the slide. This results in a volume of 1mm2 x 0.1mm = 0.1 mm3 (1E-4 mL).

When cells are placed in the chamber, they fill the space available in the gap so any cells that can be seen in each of the 1mm2 sectors will be known to exist in a 1E-4 mL volume. And that’s how you measure cell density (in cells/mL) at small scale.

You count cells in 4x1mm2 sectors and you get the following counts 31,25,40,33 – averages to 32.25 cells per 1mm2. You know that area corresponds to a 1E-4 mL volume, so 32.25 cells exist in 1E-4 mL, resulting in a density of 322,500 cells/mL. No formulas involved – we just deduced what the cell density is based on hemocytometer dimensions. Extending this to the general case, the formula would be cell density = (total cells counted / number of squares counted) / volume of a single square – that doesn’t account for any dilutions carried out.

For 1mm2 squares, the volume of a single square was 1E-4, but depending on the square size chosen to count cells, this may be different. For example, small squares inside the central square are used for red blood cell counting. In that case, the square volume is 1E-4 mL / 25 (there are 25 smaller squares in the 1mm2 central square), leading to a 4E-6 mL small square volume.

Cell density is critical to maintain a healthy culture, subculturing, creating cell banks, preparing samples for flow-cytometry and ELISA, and simply to keep track of your numbers for publications. In my case, I had to use it to match the cell growth predictions my model made to the actual cell growth observed in the cultures. On the other hand, viability is inherently a marker for culture health; it is also useful in toxicity tests to understand how many cells are killed in a specific time frame given a cytotoxic agent.

Another useful metric that can be obtained from a hemocytometer count is cell viability. Calculating viable cells gives a measure of the amount of live cells among all cells in culture. It can be measured by adding a dye to the counting solution that stains dead cells but not live cells – those are then recorded separately during the count and viability is calculated as live cells / total cells (as a percent).

Hemocytometer usage has also expanded into other fields. It is now routinely used in brewing to assess the density and quality of yeast during fermentation and in honey production when determining the density of pollen in the final product. Technology has also evolved the hemocytometer to allow automated cell counting with disposable chambers, providing almost immediate results. This new approach works fairly well for a wide range of common cell lines (and saves tons of time!) but may need fine-tuning for more custom applications such as patient cells with rare diseases.

Cell counting with a hemocytometer can be very repetitive and time consuming. Especially when you need to go through 10-20 samples a day, count viable cells and perform calculations for all of them on the fly. That’s what I faced when I started my PhD as a newbie in a biological lab.

In the beginning, I was trained like everyone else and did my 10-20 manual counts a day diligently. But it got to a point where I spent more time doing calculations in the lab than taking care of the cells. I thought there had to be a better way, so putting my engineering skills into practice I developed an app, HemocyTap, to help tally-count cells and carry out those daily calculations automatically. I started using it in the lab, but then other people started using it too, and I realised it may be something others outside my lab could benefit from. I uploaded it to the App store and started getting reviews.

By that time, I also realised there was a lot of confusion around the whole counting cells topic and people were struggling to understand the protocol and the logic behind the calculations, in the same way that I had struggled. I couldn’t find any resources online that addressed the topic in depth, so I built hemocytometer.org. All of that was back in 2013, and the website has grown a lot since then. I’ve also received hundreds of questions related to counting cells and techniques around it – so if you have any after reading this post, head there and leave a comment!

For more cell counting information or to download the hemocyTap App visit www.hemocytometer.org