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.

Easily calculate your cell density, cell numbers and viability! Check out this page for more details on the calculation steps and this app for endless cell counting capabilities.

1. Leila says:

I’v counted micoalgae by hemocytometer in this way:
diulution :100 microliter of culture in 1900 microliter distilled water.
counting : all cells in the corner squers.
I dont know how report the number of cells per ml?

1. biolabprotocols says:

Hi Leila,
Ok, the way you would do it in the calculator is as follows:
Live cells: total number of microalgae counted in all corner squares
# squares counted: 4 (as per your comment)
Dilution factor: 100uL culture in 1900uL of water is equivalent to setting the dilution factor to 20.
Which squares have you counted: Big squares in the corner

You then get the number of cells per mL under Cell Density in the results section.

Hope that was useful!

Maria

2. m summer says:

i need numericals on haemocytomeer

1. biolabprotocols says:

See here for details on hemocytometer calculations.

3. Debra says:

In the hemacytometer calculator, is the “volume” field the total volume of the fluid measured (i.e., 5ml of synovial fluid)?

1. biolabprotocols says:

Hi Debra,
The volume field is the total volume before any dilutions specific to the process of counting cells are carried out. For example, if you start with 100mL of cell suspension, you take 0.1mL for cell counting, you dilute with 0.1mL water and you count, the volume field should read 100mL.
Hope that helps!
Maria

4. pasthana says:

I want to feed cells in 96 well plate. A total of 2000 cells per well for 46 wells. I have 10ml of cell suspension with 56000 cells per ml. How to feed cells in my plate if I only want to transfer 100ul solution to well each time for the ease of use.

1. Hi there,

So there’s essentially two things you need to calculate: 1) What’s the concentration you need in each well; 2) How much you need to dilute the original cell suspension to get to that concentration.

1) is quite easy. In each well, you’re going to have 2000 cells in 100uL (0.1mL) so the concentration will be 2000 cells/0.1mL = 20000 cells/mL.

2) needs a bit more calculations. The total number of cells you will need is 2000 cells x 46 wells = 92000 cells. But you probably want to have some extra just in case, so let’s say you need to have 100000 cells at the final concentration of 20000 cells/mL (which means you will end up with (100000 cells) / (20000 cells/mL) = 5mL of diluted solution) . You use the formula c1 x v1 = c2 x v2 (where 1 means the initial cell suspension and 2 the diluted one): 56000 cells/mL x v1 = 20000 cells/mL x 5mL and you solve for v1: v1 = (20000 cells/mL x 5mL) / (56000 cells/mL) = 1.786mL. This gives you the volume you need to take from the original 56000 cells/mL suspension, and you need to dilute it into 5mL-1.786mL = 3.214mL of PBS (or medium, or whatever you are using for preparing your solution).

I hope that was helpful, let me know if you have any other questions.

Maria

5. Savid says:

My total cell suspension volume is 6mL.. I take 10uL and dilute it in 90uL PBS (making it 10x diluted). Then I take 10uL of the diluted sample and mix with 10uL of Tryphan blue(2x diluted, so final dilution factor is 20) Am I right? I use a Fuchs Rosenthal Cell counting Chamber, the cell depth is 2mm and it has 16, one mm squares. I counted cells in five squares and take average of it. (example: 111 is average), so My calculation is ((average number of cells counted x dilution factor)/(2 x 10^-4)). which is (111 * 20 * 10000)/2 = 11,100,000 cells per mL.

Please let me know if my dilution factor and calculations are correct. Because I’m seeing a huge difference between manual hemacytometer count and automated cell count. Your reply will be appreciated. Thanks.

1. Hi Savid,

Your calculations for the dilution are correct. The cell depth in the Fuchs Rosenthal cell counting chamber is actually 0.2mm (see here) but the final value of 2×10-4 mL you’re using is the right one, and the final result as per your calculations looks good. I would recommend to check your hemocytometer counts: maybe count more than 5 squares, try with a proper glass cover (some of them are so thin that the volume between it and the hemocytometer is decreased due to surface tension of the liquid) and perhaps dilute more (you’re doing some serious counting with over 100 cells in 5 different squares). On the automated cell count side, I would ask: what kind of counting is it, image based (hemocytometer picture), flow based (Coulter or flow-cytometry) etc.. Different methods can give different results (10-20% difference) so that can also have an impact. How huge is the difference you’re seeing? The last thing I would check on the machine is the settings and the input values you are giving it. Does it ask for a dilution factor? Does it include the fluid used by the machine for counting (if any)? Is the actual hemocytometer type (and volume) configured into the machine?

I hope that helps. Do come back to me if none of the latter worked, or if you have other questions.

Cheers,
Maria

6. Savid says:

Hi Maria,
Thanks for getting back , Oh my bad it was a typo , yes you are right , the depth is 0.2mm.
I lost the coverslip which came with the counting chamber, so I’m using a normal thin glass coverslip (which may be one of the reason for the differences in cell count). The automated counter I’m using is invitrogen countess counter, I believe it uses a image based counting system. its like hemacytometer (counts four, 1mmx1mm square equal to 0.4uL volume). I don’t enter dilution factor as the instrument is programmed to use (1:1) sample:tryphan blue. And with respect to manual counting I use the dilution factor I mentioned in my original question. The difference is sometimes more than 50% between automated and manual counting. As you said counting 100 cells per square is really tough, maybe I should dilute it further. I will try to buy a replacement coverslip for my counting chamber. Also Im wondering if this type of counting chamber (fuchs rosenthal) is not the best one for cancer cell line?? Any idea. Do you think this will be a good counting chamber for any cell line? Thanks once again and appreciate your feedback.

savid

1. Hi Savid,

No prob! Yes I think if you try with a proper hemocytometer coverslip, it can make a difference.

Regarding the Countess counter, mind that the concentration given is the one prior to addition of trypan blue (see here page 2, point 1 and here, page 23), so in the case you mentioned before, you should still multiply that by 10. This is for the Countess II FL, not sure if that’s the model you’re using, but they generally have the same software.

I used human leukemic cell lines during my PhD and I did my counts with a Neubauer hemocytometer, which is probably similar to the one you are using on your Countess machine (I used to count 5x 1mm x 1mm squares and take the average). The difference lies on the layout of the squares (which doesn’t really matter too much) but more importantly on the depth of the chamber (0.1mm deep vs 0.2mm for the Fuchs Rosenthal). I am wondering if that can have an impact on how cells enter through capillarity and also maybe some cells mask others? Typically human cells are 50um at max, so two layers of cells could fit between the hemocytometer surface and the coverslip (especially since your cell suspension is quite concentrated).

Let me know how that goes. Good luck!

Maria

1. Savid says:

Hi Maria,
Yes I multiply the dilution factor with the concentration provided by the Countess. I ordered the proper coverslip, hope that can help me. But as you mentioned I really wonder if the 0.2mm depth does make a big difference, because at least with the current thin coverslips, i do see two layers of cells. I will update my reply once i use with proper coverslip. Thanks for your prompt replies and appreciate your help. Have a great day.

Savid

7. Tr says:

Hi,
My cell count is1120000cells/ml. I need to plate it in Lab Tek with 8 chambers. Need 500ul for each chambers and cell count for each chamber has to be 5000cells/ml. How do I find out how much cell suspension add to what volume of media to get enough samples for the 8 chambers with 5000cell/ml ?

Thanks

1. Hi there,

Starting from the end, you want 8 chambers filled with 0.5 mL each containing cells at a density of 5,000 cells/mL, in other words you need 8 chambers x 0.5 mL x 5,000 cells/mL = 20,000 cells. In order to dilute just the exact amount you use the formula: final cells = initial cell density x volume to be taken, which becomes: 20,000 cells = 1,120,000 cells/mL x volume to be taken, or volume to be taken = 20,000 cells / 1,120,000 cells/mL = 0.0178 mL or 17.8 uL.

The total volume required to fill all eight chambers is 0.5 mL x 8 chambers = 4 mL. Therefore the remaining volume of media to be added is 4 mL – 0.0178 mL = 3.982 mL.

As a recommendation, you generally try to prepare more medium than required in case you lose some while tranferring it to the chambers or you want to make another count to double check the density. For example, in this case you could prepare 5 mL using 0.0178 mL * 5 mL / 4 mL = 0.0223 mL (22.3 uL) of original cell suspension and 5 mL – 0.0223 mL = 4.9777 mL of media.

Hope that helped!

Maria

8. Sofea says:

Hi Maria,

Just need to clarify one thing, the ‘initial volume’ in the calculator refers to the volume of sample taken for counting. Right?

1. Hi Sofea,

The initial volume can be used in many different ways. It will be used in this calculation to determine the total number of cells present based on the calculated cell density ( total number of cells = calculated cell density x initial volume). So if you’re not interested in total cell numbers, only about cell density, forget about it. As you can see above, it doesn’t have a star next to it so it’s not required.

Here’s an example in case you do want to use it: you have a master cell culture of 100 mL from which you take 15 mL to subculture. From those 15 mL, you take 10 uL to perform the count and you determine that you have a cell density of 200,000 cells/mL. Now, you can use each of those three volumes to calculate the total number of cells in each of the three steps. In the master cell culture, you had 200,000 cells/mL x 100 mL = 20,000,000 cells before taking any samples, and 200,000 cells/mL x 85 mL = 17,000,000 cells after taking the sample. In the 15 mL to subculture you had 200,000 cells/mL x 15 mL = 3,000,000 cells. In the 10uL (0.01 mL) taken to perform the count you have: 200,000 cells/mL x 0.01 mL = 2,000 cells. In this case, you would enter the initial volume of the master cell culture if you want to determine the total number of cells of the master cell culture; the initial volume of the subculture sample if you want to know the number of cells there; and finally the volume of sample taken for counting if you’re interested in the number of cells you’re “wasting” for the analysis (but this is not very useful really).

I hope that was useful. Let me know if you have other questions and thanks for visiting the blog!

Maria

9. Joy says:

Hello,

I currently have 3ml of cell suspension with 546E4 cells/ml. If I would like to dilute 546E4 cells/ml into 1E6 cells/ml, how much more Media should I add into the cell suspension??

Thank you!

1. Hi Joy,

Since you are not taking away any cells, the total number of cells when diluting is conserved which means you have: 5.46E6 cells/mL x 3 mL = 16.38E6 cells at the end. Now, you want to have 1E6 cells/mL which means: 16.38E6 cells / 1E6 cells/mL = 16.38 mL which is the total volume in which the cells are found at the end of the dilution. To dilute, subtract the volume that was already present in the beginning: 16.38mL – 3mL = 13.38 mL of new media that you need to add.

Cheers!

Maria

10. hidayah says:

hello. is it ok if i not diluting the sample when using the haemacytometer? will i get the no if cells/m3 if i am using the haemacytometer and the sample was not diluted?

1. Yes, you can calculate cell density without dilution (I assume in this case you are not performing a viability count). In the calculator, just enter 1 in the dilution factor field. The output of the calculator is in cells/mL, if you want cells/m3 you will have to multiply by 1E6.

Hope that helps!

Maria

11. BHARGAV PATEL says:

Hi, Is it important to count every square of the conrners (64) or if I can just count the 1 corner (16 cell) and then multiply with other three (16×3)…does it goes same with the centre square??

1. Hi Bhargav,
For accuracy purposes, it is better to count every square of the corners because:

• when dispersing the fluid inside the chamber, it might have been distributed in a non homogeneous way (more concentrated on one side of the chamber than the other)
• when counting, you might not have enough cells in a corner square for the count to be representative (typically, the sum of cells counted in all corner squares should be no less than 100 cells)

To ease the pain of counting loads of cells, I recommend diluting the sample until you get around 25 cells per corner square. The same goes for the central square, try counting 5 of the small squares inside it and take the average.

Happy counting!
Maria

12. Trigal Magala Velásquez says:

Dear Maria:

Thanks for the creation of this discussion space. I work with parrotfishes, quantifying zooxanthellae (coral symbionts) in their faeces. The size of this algae is between 8-12 μm. At the moment, I am using a Neubauer Improved Chamber, but I have a problem. Taking into account that zooxanthellae density is low in my samples I decided to count all the 9 squares, but for each 2 or 3 cells that fell into these squares, there are 8-10 cells out of the chamber´s counting area. I think that my method is not capturing the representativity of my variable (number of zooxanthellae).

The faeces samples are characterized by a huge concentration of organic materia, so I do not know if my problem is related with homogenization or if I need to use other chamber. I read that Fuchs-Rosenthal Cell counting chamber is more appropriated for samples with low cell concentration values; other positive aspect about Fuchs-Rosenthal Cell counting chamber is its 0.2mm deep due to I can put a higher sample volume. Maybe in a higher sample volume I have the opportunity to find more cells. I have to use just manual methods.

I use just 10-1 dilution for do not loose more cells.

I appreciate any idea for resolve this problem.

Trigal.

1. Dear Trigal,
Thanks for your question. I do not think using a Fuchs-Rosenthal chamber will have a big impact in the accuracy of your counts. As you can see here, the density ranges for Neubauer and Fuchs chambers are very similar.
There may be different reasons why your counts are not representative:

• you have other impurities in your sample (organic material)
• it seems that your cell density is extremely low (as you have mentioned).
• when inserting the sample in the hemocytometer, it doesn’t spread homogeneously

The suggestions I have for you:

• treat your sample with something that removes or minimises the organic material content. This is very important as you need to ensure what you are counting are just the cells, and not debris from the organic material.
• concentrate your sample. You can do this by centrifuging it, renmoving the supernatant and resuspending in a lower volume of fluid (do this after removing the organic material)
• ensure the hemocytometer coverslip is properly placed, and insert the sample in the chamber by capillarity slowly, without flooding the chamber.

I hope these tips help, let me know how it goes and if you have any further questions.
Cheers,
Maria

13. Sonali says:

Hi, my question is I am using neubauer hemocytometer for counting fungal cells. My procedure is serial dilution technique right from diluting 1ml of sample into 9ml of water marking it as 10-1 and again taking 1ml of sample from first tube into 2nd tube containing 9ml of water and so on upto 10-9. I am taking 10ul of sample in hemocytometer and counting 5 squares ie. 4 from corner and 1 from center. Problem arises while calculating dilution factor . Suppose I have counted 120 spores in total 5 squares using 10ul of sample from dilution tube 10-1 then what would be the dilution factorand please also clear me the formula used. Different research papers showing different formula, I dont understand which one I should use. Presentely I am doing research from CSIR. Please help.

1. Hi Sonali,
If I understand correctly, then the dilution factor is the one you had in the 10uL sample prior to doing any counting (since you are not diluting any further). I am not sure what you mean by 10-9, is it that you do a 1:9 dilution 9 times? If so, the final concentration would be 0.1×0.1×0.1…(9 times)…x0.1 = 10^-9 smaller, or the dilution factor would be 10^9.

But if you are taking the sample from the first dilution, the dilution factor would be 10, which is calculated as 1 (parts sample) + 9 (parts water) / 1 (parts sample).

Hope that helps!
Maria

1. Sonali says:

Hi
Thanks for replying but I am still not clear with calculating dilution factor, my bad!

I am using serial dilution technique from 10rais to power -1 ,10rais to power -2, uptill 10rais to power -9 . So if the dilution factor for first dilution is 10 according to your answer, then dilution factor for 10 rais to power -5 would be 100000 and 10 rais to power -8 would be 100000000. Am i right or I messed it up.

1. Hi again Sonali,

Sorry for my delay in responding.

The dilution factor for each step is equal to the final volume (after diluting) divided by the initial volume (before diluting). Assuming 10^-1 is the same as adding 9 parts of fluid to 1 part of sample, then yes the dilution factor for the first step is indeed 10. If you do that 8 times, you will get to 100,000,000 which will be the dilution factor to convert from the final concentration to the initial concentration (by multiplying).

Did that clarify?

1. SONALI says:

hey Maria

Thank you so much for this help. I got the point now. I hope you could solve problems in our lives too 🙂 . thanks once again for being so dedicated to help people like us.

kudos
Sonali

14. riya thomas says:

How to calculate dilution factor for the following…
Saliva 50ml collected,centrifuged, Supernant removed. The pellet resuspended in 5ml of HBSS solution. To this 20microlitr of acridine orange was added. What is the dilution factor

1. Hi Riya,

The dilution factor is the final volume divided by the initial volume (more info here, you can also use my dilution factor calculator), so in your case:

• initial volume = 50mL
• final volume = 5.02mL

Dilution factor = 5.02 / 50 = 0.1004. It is below 1 because you are increasing the concentration instead of decreasing it as in a normal dilution.

Hope that helped!

Maria

1. riya thomas says:

Hi Maria, Thank you.
There is a slight change in procedure. After addition with HBSS solution in pellet, 1ml from this was taken and 20ul of acridine orange was added. Does the addition of stain change the dilution? So has the dilution factor considered as 1 as per the calculation you gave? So the formula for calculating neutrophils would be average cells*10000*dilution factor (I.e 1)?

1. Hi Riya,
I originally replied to your first question where you were adding 20uL to the 5mL. For the case where you add 20uL to 1mL of that 5mL solution prepared:

The dilution factor stays constant as long as you don’t add anything to it or resuspend it in a different volume. Technically, the volume will not change much for the addition of 20uL to 1mL so for this kind of case you could just calculate the dilution factor for the first step and use that (5/50 =0.1). But for the sake of explaining this further, I will calculate it for you.

1. First way (intuitive): imagine instead of taking 1mL from the 5mL, you had kept the 5mL and had added the equivalent of 20uL of stain for the 1mL to the whole 5mL, or 20uL x 5 = 100uL (20uL per 1mL of solution). That would have resulted in a final volume of 5mL + 100uL = 5.1mL. So the dilution factor would be 5.1/50 = 0.102.
2. Second way (formula): the dilution factor for the first step is 0.1 as I have mentioned before. For the second step: 1.02/1 = 1.02. If you multiply the two you get the overall dilution factor: 0.1 x 1.02 = 0.102.

As you can see, the dilution factor you get when adding very small amounts to a solution (0.102 in this case) is very close the the original one (0.1 in this case).

In the cell count formula, you should use the dilution factor of the step from which you want the concentration. If you want the concentration prior to making the pellet, multiply by 0.102. If you want the concentration of the 5mL, multiply by 1.02.

I hope that was clear!
Maria

15. riya thomas says:

Thank you Maria.

16. Bob Bennett says:

Have a question regarding sperm hemocytometer calculation with a dual chamber hemocytometer. Our location has us perform a average of the count of the 2 sides to ensure that we are not overcharging/undercharging either side. But when calculating, they have us use the average as part of the calculation. However, they have us use the total number of squares counted as well. The calculation used looks like this:

(hemocytometer side 1 and side 2 average) X (dilution used)/(# of squares counted) X (depth)

Would this not essentially cut the actual cell count in half, as the average of each side is taken as well as the total # of squares? Thank you very much for input!

1. Hi Bob,
Thanks for your question. I guess it depends on what you put in “hemocytometer side 1 and side 2 average”. If it’s the sum of the averages in each of the chambers (say, 20 and 22 cells = 42 cells total), and then you divide by the number of squares counted (like you have in the formula, so 42/2), then the calculation is correct since you are basically taking the average of the two chambers. However, if you are dividing the average on the two chambers (42/2 = 21) by two (21/2 = 10.5), you are indeed cutting the cell number in half.
Does that clarify?
Cheers,
Maria

17. Bright Junior says:

1. Hi there,
It really depends. If the dilution is only so that you can count your sample (so you don’t need the rest of your cells to be diluted), then take 10uL of cell suspension, and add 190uL of dilutant (water, PBS or whatever you use to dilute). That will give you a solution diluted 1:20 (parts sample:total parts) from the original one.
Cheers,
Maria

18. Sanjay K M says:

Guys,

I need to get final volume. I have counted 100 cells in 2 squares and I don’t use trypon blue, means no dilution factor. Initial volume is 30L and desired conc. of cells per mL is 50000. Can you please help me out to get the final voulume?

1. Hi Sanjay,
So, your inputs in the calculator would look like this:

• Live cells counted: 100
• #squares counted: 2
• Initial volume: 30000 mL (as you mention you have 30L)
• Dilution factor: 1 (no dilution)
• Which squares have you counted? I assume the ones on the corners

With these inputs, I get for the original cell suspension:

• Cell density: 500,000 cells/mL
• Total live cells: 15E9

To get the final volume, knowing that your target density is 50,000 cells/mL:
final volume = total live cells / target density = 15E9 / 50,000 = 300,000 mL = 300L

If you were counting the small squares in the center, then the live cells would be 375E9 and the final volume would be 7500L (but this looks massive so I assume you were counting cells in the corner squares).

Does that make sense? Let me know otherwise.

Cheers,
Maria

19. Kai says:

Hi Maria,

What program did you use to produce the app? I have used your app for counting cells in my 3rd year biomedical science BSc project at London Metropolitan University and wanted to make a similar program for other calculations but have not found a suitable program as many of those provided for free online as “free app tools” are too basic. Did you use a program that was open source or proprietary, or did you code it yourself and if so, which language did you use?

Thanks!

1. Hi Kai,

For the online calculator, I used Calculated Fields Form which is a free WordPress plugin you can use to create custom calculators. For the one in this page, I didn’t have to do any coding, but for the dilution factor calculator I did as the plugin also allows some flexibility with javascript (have a look at the “Select a relative notation” field, where I had to add some code to calculate the greatest common denominator).

I would love to see it in action once you’ve set up the website!

Cheers,
Maria

20. Lakshmi says:

I have calculated the cells using hemocytometer. I got 2.78X10^6 cells in 2 ml. I would like to take 1.6X10^6 cells for my assay from this. What volume should I use from the vial?

1. Hi Lakshmi,
I understand you got 2.78×10^6 cells in the solution you took the cells from (i.e., all the dilutions have been taken into account). In that case:

volume needed = cell number needed / original cell density = 1.6×10^6 cells / ( 2.78×10^6 cells / 2 mL) = 1.151 mL

Cheers,
Maria

21. Lilly says:

Hi,
I can’t access the calculator anymore, is the website updated or what?

1. Hi Lilly,

Sorry, I deactivated the calculators by mistake! All back to normal, let me know if there are any further issues.

Cheers,
Maria

1. Lilly says:

Hi Maria,

Thanks so much, all is normal now

Dr. Maria did a wonderful job. She explined the procedure of counting in the easiest way and finally made the cell counting enjoyable. But unfortunately I saw it after completion of excel based calculation of prawn blood sample for my thesis work. Thinking I had not yet completed my task. What a thrill!

23. Allison Grunkemeyer says:

Hi Maria,
I would like to know what calculation you are using to acquire the total cell count and how the dilution factor affects that number? We are counting the 5 small squares. Let me know, thanks!\

1. Hi Allison,

You can find all the details on the calculations here. Live cells are calculated as live cell density x original volume. Or if you want total cells, then multiply the total cell density by the volume. The dilution factor affects cell count proportionally: if you change your dilution factor to 4 instead of 2, your cell number will be doubled.