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.

What should I count?

Blood irrigates our whole body and therefore contains many types of cells that effect different tasks: red blood cells that carry oxygen, lymphocytes that fight infection by adaptive mechanisms (i.e. they find a tailor-made “cure”), leukocytes that constitute the innate response to infection (i.e. they display a universal “cure” and contribute to the healing process of wounds etc…). When we do a blood cell count, all of those will appear. However, because of their size, red blood cells will be distinguishable from white blood cells (all the others). That’s the way we are going to tell the difference between them when we count.

Diluting the blood

You can count blood cells with as little as a drop of blood. Because the cell density is very high, you have to dilute so much that you could do over 200 cell counts!

The dilution that is usually performed is 1:200 blood:isotonic solution. So for example, we could take 1μL of undiluted blood and add 199μL of isotonic solution (or do serial dilutions).

Loading the sample in the hemocytometer

To prepare the hemocytometer, make sure that you clean it properly with a tissue and ethanol, and place a clean glass slide on top. Once you have diluted the sample, you can additionally add a viability dye such as erythrosine B or trypan blue, on a 1:1 proportion. So you can take 10μL of the diluted blood you already have and add another 10μL of erythrosine B. This is going to be your counting solution. Introduce it with the pipette in the gap between the hemocytometer and the slide, taking care not to overfill the chamber while covering all the elevated surface of the chamber.

Counting the cells

White blood cells: because they’re bigger, you are going to count those in the four corner squares. As a reminder, you should establish a rule for the cells that are touching the peripheral lines: you can count the ones touching the top and left and skip the ones on the bottom and right, or any other combinations of two consecutive lines that you want. Note down your counts (discriminating between live and dead if you added a dye). Red blood cells: zoom into the central square, where smaller squares have been drawn. Count the cells in the four small corner squares and the small central square, and do as with the counts of the WBC.

Calculating cell density

You can proceed with the counts in the same way as in here, but this time remember to multiply by 200 due to the initial dilution you made, and additionally by 2 because of the viability dye. For faster counts, check out HemocyTap, the hemocytometer app.

Comments

  3. i am asking that why we only count wbcs in four side corners and platelets or rbcs in central 5 squares???????????? explain please

    Reply

    1. Hi Manish,
      Platelets and RBCs are smaller therefore: (1) you will probably use higher magnification which means you will not be able to see 100% of a corner square, but you will be able to see individual inner squares in the central square, which have additional lines that help you count; (2) Because you’ll be counting a smaller area, the cell density required will be larger making it impossible to count all cells in a large (corner) square (i.e. if you count 5 smaller squares in the central square you should get to 100 pretty easily). WBCs are larger so it’s easier to see them and count them with lower magnifications therefore it can be done on the corner squares (and also in the central square if you want but as a large square, not counting individual small squares).

      Hope that helps!

      Maria

      Reply

    1. Hi Will,

      You can find all the information here. Basically, when counting red blood cells (RBCs) you need to keep them from undergoing hemolysis (cell dissociation) so an isotonic solution is used. When counting white blood cells (WBCs), they become hard to see with all the RBCs present so the RBCs need to be lysed and the acetic acid in the solution helps with that.

      Hope that was useful!

      Maria

      Reply

  6. Thank you. Thanks for your usefull website and post.
    I am not good writing with English. So i am Sorry for my wrong typing and grammar.
    Anyway I have question.
    When we count wbc. We use acetic acid for lysing rbc.
    My question is “Why acetic acid lyse rbc but don’t lyse wbc?”
    Wbc have something ability that Rbc don’t have?
    I am waiting your comment.
    Thanks.

    Reply

    1. Hi Eunho,
      Glad it was helpful and no worries about your English!
      Acetic acid (and some other acids) in the lysing solution modifies the pH inside RBCs, causing ionic imbalance and internal structure changes.
      See references here and here.
      Cheers,
      Maria

      Reply

    1. Hi Humaira,

      Because the dilution to perform for RBCs is larger than the one for WBC, so a larger volume of dilution fluid is needed. RBCs are way more abundant in blood so it’s easier to count them if you dilute them more. See slides 20-21 here for more info on pipette volumes.

      Hope that helps!

      Maria

      Reply

  9. hello

    if we are using isotonic solution for Rbcs counting, so than in counting of Rbcs we will be confusing in counting of Rbcs with Wbcs…..
    my question is that what is the effect of RBCs counting solution on WBCs?
    i am waiting for your answer

    Reply

  11. it is a nice website to know a person have normal or ubnormal rbc and wbc.but ihave quetion how many % we need acetic acid use to lyse the rbc?

    Reply

  12. If I have a heterogeneous solution of cells including blood cells and other mammalian cells (digestion of a tissue), would the RBC’s still only be able to be seen in the center due to their size or should I be concerned that some of the cells in the outer corners are RBC’s?

    Reply

    1. Hi Cristina,

      The RBC can be seen anywhere, the only reason to count them in the central square is that the grid is smaller, so when counting them it is a lot easier to get a smaller subset into view on the microscope. When you add the counting solution to the hemocytometer, the RBC should be equally distributed everywhere, so you should find there are approximately as many in every square (corner and central).

      In summary, keep counting them on the central square, as it is easier due to their size – the averaging calculations in the hemocytometer take care of accounting for counts being made on the central square (i.e., multiply by 250,000).

      Hope that was helpful!

      Maria

      Reply
  13. Pingback: Hemocytometer applications • Hemocytometer

    1. Hi Masood,

      So there are more RBC than WBC in blood. For an average adult the cell density is:

      • 4-6 x 109 cells/mL RBC
      • 4-10 x 106 cells/mL WBC

      That means that you need to dilute RBCs more than WBCs to get to a cell number that is neither too high nor too low to count them on the hemocytometer.
      If you dilute RBCs 200 times, you will get to a cell density of 2-3 x 107 cells/mL RBC.
      If you dilute WBCs 20 times, you will get to a cell density of 2-5 x 106 cells/mL WBC.
      Now, because you count RBCs in the small squares in the center (there are 25 of those in the central square) and WBC in the large corner squares (or the whole central square), the number of cells counted will end up being similar (2-3 x 107 / 2-5 x 106 = 4-15 times more RBCs, but these are counted in an area that is 25 times smaller).

      Does that clarify?

      Maria

      Reply

  15. Hello , ds website is really helpyfull ! Tnx a lot . My question is how does over/ under diluting the blood will affect your experiment result ? Anticipating to hear from you

    Reply

    1. Hi Raheedah,

      Thanks for the feedback!

      Well it depends on the experiment you want to run. Usually blood cells need to be seeded at a specific density, so you need to dilute to reach that density.

      If what you meant is how dilution will have an impact on the counts:

      • if you overdilute, you will count less cells so your counts might be more inaccurate as not enough cells are counted
      • if you underdilute, you will count a lot of cells so it might be different to visually tell if you are counting one or two cells as they might overlap, and generally you might get lost in terms of which cells you had counted already or not.

      Hope that helps!
      Maria

      Reply

    1. Hi Khadija,

      Glad you find it useful!

      RBCs are produced from blood stem cells in the bone marrow, the process is called erythropoiesis. Blood stem cells can give rise to any type of blood cells that circulate in the body, like lymphocytes, RBCs or platelets. For blood stem cells to differentiate into RBCs specifically, erythropoietin (EPO) needs to be supplied. This is the natural signalling hormone that is produced in the body, however it is also supplied artificially in the lab to induce those stem cells to turn into RBCs only. There are other factors that affect RBC production, like the 3D environment and the supply of nutrients, oxygen etc.

      Let me know if you have other questions.

      Cheers,
      Maria

      Reply

    1. Hi Benjamin,

      Because of their size, WBC can be counted when focusing on a whole corner square with the microscope. In reality, all 9 squares that make up the hemocytometer have equal dimensions, so you could count these cells in any of the 9 squares, as long as you then take the average. Always try to count in symmetrical squares, e.g., if you count the top left one, you should also count the bottom right one. The corner squares are more common to count because they have helper lines (but not as dense as any of the other squares). Many people also use the central square, in addition to the corner squares, and thus count 5 squares per chamber.

      Hope that helped!
      Maria

      Reply

  19. Thank you for this excellent website. We are interested in finding an alternative to the expensive hematology analyzers. We want to obtain a CBC from whole blood and a platelet concentration from PRP (platelet-rich plasma) that we obtain from the patients in our clinic where we perform knee injections. Is it possible to do this quickly with a hemocytometer?

    Thank you,
    Peter

    Reply

    1. Hi Peter,

      Glad you find it helpful!

      Hemocytometers were originally designed to count blood cells, so they are definitely suitable for that purpose. You can count WBCs in large squares and RBCs and platelets in small squares (the ones inside the central square). Hemocytometers are a manual counting method, so by default each count will take about 5-10 min including preparation, counting and cleaning. The price ranges $50-$200, and you will also require special coverslips ($50) and a microscope ($100-$1000). If you want something faster (30s/sample), you can get an automated counter, that will still not be as expensive as a hematology analyser (I believe), but there will be a consumables cost ($50-$100/month) and the up-front cost of the machine ($3000-$10000).

      Let me know if you have further questions.

      Maria

      Reply

  20. Please why shouldn’t we overfill the counting chamber. Also why shouldn’t the chamber be filled with air bubbles

    Reply

    1. Hi there,

      When you overfill the chamber, 2 things may happen: (1) the fluid does not enter anymore by surface tension so the volume of fluid between the chamber and the coverslip is not the standard one used to do the calculations; (2) when more fluid is pushed in when there is fluid already, cells might be displaced in a heterogeneous way so the cell distribution might not be even. In both cases the counts will not be accurate.

      The chamber shouldn’t have air bubbles because air replaces fluid, so anywhere where a bubble is found is a “void”, where cells can’t be counted (resulting in a lower measured concentration than the actual one).

      Hope that helps! Feel free to ask any other questions.

      Maria

      Reply

  21. please give me the implications of overfilling the chamber, and also the trapping of air bubbles in the chamber

    Reply

  22. Hi,
    Can you please suggest other ways to lyse RBC and keep platelets intact. Actually i want to work with whole blood, i mean no dilution and want to count platelets. please suggest ways.

    Thanks,
    Ruchi

    Reply

  24. Hi Maria,

    Is there a recommendation of how soon after the blood draw WBCs and platelets should be counted? We will lysed the RBCs with acetic acid. If we are collecting a large number of samples, can we wait up 4 hrs to do the counting?

    Thanks,
    Mary

    Reply

    1. Hi Mary,

      Ideally, you should count them as soon as possible so that cell viability remains high. 4 hours shouldn’t be an issue, especially if you’re just interested in cell count and are not doing any other tests on the cells. I used to count blood samples during my PhD, and had to travel to collect the sample, prepare the lab setup and do the count, so I easily took 4h; I never saw a viability lower than 99%, so you should be on the safe side there. However, I wouldn’t lyse the RBCs until a few minutes before counting.

      Let me know if you have other questions.

      Best,
      Maria

      Reply

  26. Dr Fuentes, what can you do if have been given a dilution of 1:400, and after doing the calculations you are told to work back to the appropriate dilution of 1:200?

    Reply

    1. Hi Kangwa,

      So let’s say you had calculated that in order to dilute to 1:400 (sample:total volume format), you had to add 399mL to 1mL of original solution. 1:200 means that it’s going to be twice as concentrated than what you had originally calculated, so you are going to end up with half the amount of final solution of what you had originally planned, or 200mL, for the same amount of original sample. So for 1mL of original solution, 199mL of added liquid.

      Hope that helps!

      Cheers,
      Maria

      Reply

  27. I teach a high school veterinary class. I have been using hemacytometers (with great difficulty, we hardly get good data) for the hematology unit. Is it worthwhile to continue teaching how to use it with everything automated today? Please give me feedback. I want to teach relevant topics.

    Reply

  29. Thank you, Your explanations were very clear and easy to understand. I now feel like I know what I am doing. The answers to the questions also helped a lot.
    🙂

    Reply

  34. Hi Maria,
    We use Haem’s dilution fluid which is isotonic and doesn’t have any stain so neither does it lyse RBCs or WBCs nor does it stain RBCs or WBCs . Then why is that we don’t see WBCs while counting RBCs? Please help ☺️

    Reply

  35. Suggest TWO reasons why cells that touch top or left lines are counted but cells that touch right or Botton lines are not counted?

    Reply

    1. Hi Masood,

      This sounds a lot like a homework question, so I’ll just give you a hint: You’re trying to calculate cells per area. The square has a particular area. If you counted every cell that was outside the area but touching a line, would that area be the same? How would that affect your calculation?

      -Nick

      Reply

    1. The calculation requires you to count the number of cells in an area. If cells were all tiny points and the box border extremely thin, you could just count all the points inside the border.

      But we have to make an adjustment when the border is wide and the cells are big. If you only counted cells that were all inside the border, you would be undercount; if you counted all cells that touched the border, you would overcount! And deciding whether a cell is mostly inside or outside the border is tedious and subjective.

      So instead, you can essentially ensure you’re counting consistent areas by deciding to include all cells touching the top and left borders and exclude all cells touching the bottom and right borders. And this choice of top and left allows you to count adjacent squares, because each square exclusively “owns” all the cells along a particular border.

      Reply

  37. Hi,

    I have performed an experiment in my Cell and Molecular Biology course on cell growth using a hemocytometer. During the week that I have performed the experiment there was an increase, then decrease and then increase of the cell count. Any possible explanation?

    Reply

  38. Good day!

    May I ask, why do we need to exclude counting cells touching any of the right hand and bottom borders of the small squares?

    Reply

    1. You could choose any sides, if you wanted. It’s just a convenient and easy-to-apply rule to account for the size of cells. Not excluding sides would essentially make the square bigger than you intended, and could also result in double-counting if you count adjacent squares.

      Reply

  39. Hey my question is why platelet diluting ammonium oxalate reagent should store in the refrigerator ??? And why platelets take more time to settle in the hemocytometer than RBC and WBC???

    Reply

    1. Nope! The cells and grid are on the same surface. There are some grids that fit on the objective or eyepiece, but those are not common. Feel free to use the highest magnification that allows you to see the cells and keep track of where you are. Just be careful that you know which size squares are which as you increase magnification, so you don’t get confused.

      Reply

  41. Hi! can I ask, when the WBC count is very low or very high, what are the resolutions that we can do to increase the accuracy of results of the WBC count?

    Reply

    1. Hi Louiz,

      If it’s high, you can do a dilution of the sample. If it’s low, you count more of the large squares instead of just the corners. In both cases, you can also use multiple chambers to get an average measurement.

      -Nick

      Reply

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