Fine. You have your counts. Now, here’s what you have to do with and without Hemocytap, the hemocytometer app.

**With the app:**

If clicking on”subculture”, introduce the dilution, target density (recommended cell density) and initial volume. Get all the calculation below 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!

**Without the app:**

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:

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):

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.

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

As Monsieur Malassez would say, “Voilà!”.

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## Samuel

February 6, 2015 at 12:44 pm

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

## biolabprotocols

February 6, 2015 at 1:30 pm

Thanks for your question. Please see my answer here

## Samuel

February 9, 2015 at 12:47 pm

Hi Maria, I have a question why does the Original cell concentration (ml^-1) increase as the dilution increases ??

## biolabprotocols

February 9, 2015 at 1:40 pm

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

## Samuel

February 9, 2015 at 1:52 pm

Yes its very clear thank you 😀