How to Use a Hemocytometer to Count Cells
For example, count the cells on the top and left lines of a square (marked by the green line), but do not count the cells on the bottom and rights lines of a square (marked by the red line). Move the hemocytometer to the next set of 16 corner squares and continue to . Apr 06, · Very large numbers of Red Blood Cells are present in the Blood Specimen. Practically, counting this amount of Red cells directly under the microscope is highly impossible. So, the Red Blood cells are counted by using a special type of chamber, designed for the counting of blood cells in the specimen, known as Hemocytometer or Neubauer’s chamber.
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Be a Microscope Master!
calculation Count the number of cells in the small 5 squares in the center of hemocytometer These 5 squares equals to 1/5 of the whole center square. OR: number of cell counted x dilution factor x 5 volume () = cell/µLFile Size: KB. Adjust the microscope to 10X magnification and focus on the cells. Using a hand tally counter, count the cells (stained nuclei) in each of the four outside squares of the hemocytometer (Figure 1A), including cells that lie on the bottom and left-hand perimeters, but not those that lie on the top and right-hand perimeters (Figure 1B). If the number of cells per 1 mm 2 exceeds 50, dilute the sample and count again. If the number of cells per 1 mm 2 is less than 15, use a less diluted sample. If less dilute samples are not available, count cells on both sides of the hemocytometer (8 x 1 mm 2 areas). Keep .
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. 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.
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.
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 cell counts! The dilution that is usually performed is blood:isotonic solution. 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 proportion.
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. 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.
You can proceed with the counts in the same way as in here , but this time remember to multiply by 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. Thanks, so many sites are not just talking about microorganisms or stem cell rather than blood. This is very helpful. 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.
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. I am waiting your comment.
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. Because the dilution to perform for RBCs is larger than the one for WBC, so a larger volume of dilution fluid is needed. See slides here for more info on pipette volumes. There is a more detailed explanation on how to prepare samples from blood tests here.
Hi Faisal, Isotonic solutions do not lyse either type of cell. More info here. Does that help? 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. 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. Hello , ds website is really helpyfull! Tnx a lot.
Anticipating to hear from you. 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. Thanks alot we are really gaining from dis website,my question is dat what are d factors that affect RBC synthesis.
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. 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. 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. 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. 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? Hemocytometers were originally designed to count blood cells, so they are definitely suitable for that purpose. Hemocytometers are a manual counting method, so by default each count will take about min including preparation, counting and cleaning. 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. Have a look at the answer I gave to the same question here. 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. There are different RBC lyse reagents available, see for example here. 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? Ideally, you should count them as soon as possible so that cell viability remains high. Dr Fuentes, what can you do if have been given a dilution of , and after doing the calculations you are told to work back to the appropriate dilution of ? So for 1mL of original solution, mL of added liquid. 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. 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. Suggest TWO reasons why cells that touch top or left lines are counted but cells that touch right or Botton lines are not counted? 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? 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.
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?
May I ask, why do we need to exclude counting cells touching any of the right hand and bottom borders of the small squares?