How to count cells with a haemocytometer

Counting cells manually might seem like an endless task, but it’s surprisingly easy and a routine procedure.  

All you need is a handy tool called a haemocytometer—a slide covered in squares that makes the process easy.

Why cell counting matters

Cell counting is essential in various fields, from medical diagnostics to biological research. Understanding the number, concentration and viability of cells is key to tracking disease progression, checking microbial growth rates, assessing pharmaceutical efficacy, and much more.

Although counting cells manually may seem daunting, using a haemocytometer offers a simple, accurate and cost-effective solution. With its reliable and reproducible results, it’s an excellent choice for quick cell counting in small volumes.

What's a haemocytometer, anyway?

A haemocytometer is a specially designed slide featuring 2 separate counting chambers.

Each chamber is marked with a grid pattern that creates 9 large squares that are further divided into smaller squares. Each large square represents a specific volume of sample solution (0.0001ml), making it easy to calculate the number of cells in any given volume.

What you’ll need

• Microscope
• Haemocytometer and coverslip
• 70% ethanol (for cleaning)
• Dilution solution (such as saline buffer)
• Pipette
• Stain (if needed—more on this later!)
• Containers or well plates for your dilutions

Step-by-step guide to cell counting

1. Prepare your sample

Start by preparing a solution that contains your cells. Dilute the solution according to your specific requirements, aiming for approximately 50 to 100 cells per large grid square. If you find you have too many or too few cells, adjust the dilution and try again.

If you want to assess cell viability, consider adding a stain like Trypan Blue. This will colour any dead cells blue, making it easier to count the living ones. Other staining options include acetic acid with methylene blue, propidium iodide or erythrosine B.

Be sure to note your dilution rations, as you’ll need them for calculations later.

2. Load your haemocytometer

Now it’s time to prepare your haemocytometer for counting:

a)    Clean the counting chambers and coverslip using 70% ethanol.

b)    Once dry, carefully place the coverslip on the haemocytometer.

c)    Using a pipette, gently add 10 μL of your cell solution into one (or both) of the V-shaped wells.

d)    Observe how the solution spreads across the chamber(s) through capillary action.

3. Set up your microscope

Position your loaded haemocytometer on the microscope stage and focus on the counting grids at 10x magnification. If you’re working with smaller cells, you may prefer to use a higher magnification for better visibility.

4. Time to count

You’re ready to start counting. Fortunately, you don’t need to count every cell in every square—just focus on the 4 large corner squares and the centre square. Count the cells in each smaller square within these larger squares and sum them up to find the total number of cells per large square.

When counting cells that touch the gridlines, only include those touching the top or right sides of the square to avoid double counting.   

5. Crunch those numbers

Now for the fun part—calculating your cell concentration! Use this simple formula:

Total cells/ml = Average count from large squares × 10,000 × Dilution Factor

a)  Calculating average count

Add together your 5 cell counts and divide by 5 to get your average cell count.

b)  Multiplying by 10,000

Each large square holds 0.0001ml of solution, so multiply your average count by 10,000 to find your cell count per ml.   

c)  Determining dilution factor

Remember that you diluted your sample with a buffer solution and possibly a stain. For instance, if you diluted your original cell solution to a 1:10 ratio (1 part sample solution to 9 parts diluent), your dilution factor would be 10.

You diluted your sample with a buffer solution and perhaps a stain, and we need to compensate for that.

That wasn’t so difficult, was it? With a little practice, you’ll be counting cells like a pro in no time!