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

    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

    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

  1. 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.

    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

    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

  2. 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

  3. 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?

  4. 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?

    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

    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

  5. 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

    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

    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

    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

  6. 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

    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

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

    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

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

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