I did my PhD in the Department of Chemical Engineering at Imperial College London. My research focused on mathematical modeling of the cell cycle in leukemia and involved experiments with cell lines. During that time, I had to count cells with a hemocytometer so often to track growth that I got tired and decided to build an app, HemocyTap, and share my knowledge on the topic here to help as many people as possible.

Once you have counted cells in each of the squares, you perform the hemocytometer calculations based on your total counts, dilution factor, initial volume and desired final density.

Now, here’s what you have to do to calculate your cell density manually or with Hemocytap, the hemocytometer app.

Manually:

Take the average of cells per square (sum of all cells in each small square you have counted, divided by the total number of squares you have counted), multiply it by the dilution factor (if you haven’t diluted your sample, multiply by 1) and divide by the volume (in mL) of a small square, following the equation:

hemocytometer equation cell density

The volume of a small square is specific to the hemocytometer. It is calculated by multiplying the width by the height (which are the same – usually 1mm each) by the depth (usually 0.1mm) of a small square. In the most common case, this would be (check here to find out the volume of other squares):

Hemocytometer volume equation

With the measured cell density obtained, you are going to calculate how much more medium you need in order to reach the manufacturer’s recommended cell density.

Volume to add equation

If you have already suspended the cells in some new medium, you will need to substract this from the final volume to add:

hemocytometer equation resuspend

As Monsieur Malassez would say, “Voilà!”. For faster calculations, use our free hemocytometer calculator online:

Or better still, our app:

If clicking on”subculture”, introduce the dilution, target density (recommended cell density) and initial volume. Get all the calculations above done for you and read the volume you need to add. Check here for a detailed video on how to do it.

If clicking on “cell density”, introduce the dilution and the initial volume (only if you want to know the total cells). You will get the cell density (and the cell number if you gave the initial volume) as per the calculations below.

Save for your records. Ta-da!

Comments

  1. Ho do you find what dilution is the most accurate to calculate cell concentrations for your original sample, from the density??

    1. Hi Samuel,
      The more you dilute, the less cells from the original sample remain in the diluted volume. To account for this, you multiply by the number of times you have diluted. For example, you have 100,000 cells/mL in the original sample. You dilute once (let’s say 50uL in 50uL of trypan blue; this is a 1:1 dilution or dilution factor equal to 2), the concentration should be half right? So you dilute once, the concentration in your diluted solution is 50,000 cells/mL. Now let’s say you dilute once your diluted solution: you had 50,000 cells/mL, you take 50uL from there and 50uL trypan blue or water (this is a 1:1 dilution or dilution factor equal to 2 from the diluted solution, or 1:3 dilution / dilution factor 4 from the original sample), you get 25,000 cells/mL. As you can see, in the first dilution you had a dilution factor of 2 and concentration of 50,000 cells/mL while in the second you had a dilution factor of 4 (from the original) and a concentration of 25,000 cells/mL. To calculate the original concentration backwards, you would multiply the dilution factor by the concentration. For the first dilution, this is 2 x 50,000 cells/mL = 100,000 cells/mL. For the second dilution, this is 4 x 25,000 cells/mL = 100,000 cells/mL.

      Therefore, the original cell concentration is always the same for the same sample. Depending on how many times you dilute, the dilution factor will change.

      Was that clear? Cheers

      Maria

  2. Hi maria, I have a question Why some equation should to multiply by 10,000 cell/ml and multiply dilution factor?

    for example this equation
    [ Total cell/ml= Total cells counted x (dilution factor/# of squares) x 10,000 cell/ml ]

    why they multiply by 10,000 cell/ml?

    1. Hi there,

      You multiply by the dilution factor if you want to find out the original cell concentration, i.e., previously to any dilutions you have performed specifically for counting cells. The 10,000 factor is not in cell/mL but in mL^-1 (or 1/mL). It represents the inverse of the volume of one of the corner squares, which is calculated as the area: 1 mm x 1 mm = 1 mm^2 times the height of the space between the hemocytometer and the coverslip (0.1mm), or 1 mm^2 x 0.1 mm = 0.1 mm^3 = 0.0001 mL. When you do the inverse, 1/0.0001 mL^-1 = 10,000 mL^-1 which is the factor you are using. You can find more details about these calculations in my other post on hemocytometer sizes.

      Hope that helped!

      Maria

  3. How will you calculate the dilution for salivary Nutrophil
    50ml of saliva collected,centrifuged, supernant discarded. To pellet 5ml of HBS was added. 1ml taken from and to that 20microliter of acridine orange was added. What is the dilution factor for this

  4. Hi! so i’m trying to calculate the total amount of cells under to coverslip.
    We put 20ul of blood into 5ml of saline. Therefore I calculated the dilution factor to be 251.
    We counted the amount of RBC in a square at 40X on the microscope and got an average of 76 RBC.
    Using the volume of 0.0001… the measured cell density is 190760000…? and also where does the recommended cell density come from?

    My question is, how do I calculate the number of RBC under the coverslip AND does it matter how much of the solution I put on the slide. If so how does that work into the equation. (we put 5ul of the solution on the slide.

    Sorry if that is really jumbled thoughts, im very confused.

    1. Hi Danielle,

      Glad you asked! Here’s the step-by-step of your calcs:

      • Dilution factor: 20uL->5mL (=5000uL) therefore dilution factor = (5000 + 20) / 20 = 251 ✓
      • 76 cells per square (I assume this is in the corner square or in the whole of the central square, not in the small squares inside the central)
      • Cell density: (76 cells x 251) / 0.0001 mL = 190,760,000 cells/mL ✓
      • Recommended cell density: this is only used if you are putting cells back into culture. When dealing with RBCs, you most likely just wanted to do a cell count so by this point you are done
      • Number of cells under the coverslip: this is something that confuses a lot of people. You don’t really need to know the number of cells under the coverslip, as the calculation is giving you the cell density which applies to blood (and that’s all you care about really). However, if you really want to know then the way to calculate it is to not multiply by the dilution factor (as now you are seeking the density of the diluted solution): 76 cells / 0.0001 mL = 760,000 cells/mL (cell density), and because you have added 5uL, the total number of cells under the slide is: 760,000 cells/mL x 0.005 = 3,800 cells
      • Does it matter how much of the solution I put on the slide: not really. As long as the chamber is completely filled (and you don’t force the solution into it, try to let it in by capillarity), your counts will be accurate. Never overfill the chamber.
      • Cell number in blood: the last thing that you may want to know is how many RBCs were in the overall volume of blood you took the sample from. Let’s say you had 20mL of blood; then the total number of RBCs would be: 190,760,000 cells/mL x 20 mL = 3.815 x 109

      Did that clear up your confusion? 🙂

      Maria

Leave a Reply

Your email address will not be published. Required fields are marked *