Articles, Blog

Counting Cells with a Hemocytometer

August 20, 2019


A hemocytometer is a device that is used for
counting cells. It’s a modified microscope slide, containing
two identical wells, or chambers, into which a small volume of a cell suspension is pipetted. We have already removed 100 µL of our cell
suspension and placed it in a micro-centrifuge tube. Dilute the suspension by adding 100 µL of
Trypan blue. Trypan blue is a dye that helps us distinguish between living and dead cells.
The dye passes through the membranes of dead cells so they will appear blue under a microscope. Living cells exclude and will appear mostly
clear. Load both chambers by pipetting the suspension
under the cover slip. Now place the hemocytometer under the microscope. Each chamber is divided into a grid pattern,
consisting of 9 large squares. Each square has the same dimensions and contains
10 to the negative-fourth power mL of suspension. The rules for counting cells sometimes differ
from lab-to-lab. In our lab, we count cells in the 4 large
corner squares and the center square. Let’s count the cells in the first square. One, Two, Three, Four, Five, Six, Seven, Eight… So what about the cells that are touching
the outside boundaries of the square? In our lab we count the cells that touch the
top and left boundaries, and we ignore the cells that touch the right and bottom boundaries. Nine. Ten. We need to count the number of both living
and dead cells. Remember, the dead cells will appear blue. Occasionally you will see artifacts – objects
or debris that appear blurry and don’t have a well-defined shape. This is an example of an artifact. We won’t
include it in our count. Proper storage, cleaning, and handling of
the hemocytometer will minimize the number of artifacts. There are 10 viable cells and 1 non-viable
cell in the first square. Now, the top-right square. There are 9 viable cells and no non-viable
cells. Next let’s count the bottom-right square. There are 11 viable cells and no non-viable
cells. And now the bottom-left square. There are 10 viable cells and 2 non-viable
cells. And finally, let’s count the cells in the
center square. Sometimes cells will appear as clumps or small
groups. It may be difficult to determine exactly how
many cells are in a group. The method of counting clumps of cells differs
from lab to lab, so be sure to follow the procedure in your lab. We will count this clump as 2 cells. There are 14 viable cells and no non-viable
cells in the center square. The total number of viable, or living cells
from all 5 squares is 54. The total number of non-viable cells is 3. Now that we have counted our cells, there
are several calculations we need to perform. First, let’s calculate the percentage of
viable cells. Here’s the formula. 54 viable cells, divided
by 57Éthe total number of cellsâ gives us 0.947. Multiply by 100 and the percentage of viable
cells is 94.7%. Next, let’s determine the average number
of cells-per-square. We counted 54 viable cells. We divide 54 by
5, because we counted in 5 squares. The average number of cells-per-square is
10.8 cells. Now let’s calculate the dilution factor. The dilution equals the final volume divided
by the volume of cells. Our final volume is 200 µL, because we started
with 100 µL of cells and added another 100 µL of trypan blue. 200 divided by 100 is 2. Therefore the dilution
factor is 2. Next we need to calculate the concentration
of viable cells – the number of living cells/mL Our average count-per-square is 10.8. The dilution factor is 2. 10.8 times 2 times 10-to-the-fourth-power
equals 216,000 cells/mL. We can write the concentration using scientific
notation as 2.16 times 10-to-the-fifth power cells/mL. From our calculations, we now know the concentration
of cells in our culture is 216,000 cells-per-milliliter and approximately 94.7 percent of those cells
are viable, living cells.

84 Comments

  • Reply SonOfZionReBorn August 27, 2012 at 11:52 pm

    where did the 10^4 come from???

  • Reply Microbial Zoo September 28, 2012 at 12:54 pm

    I have similar question. Should it be 10^3 (the factor that converts ul to ml)?

  • Reply Microbial Zoo September 28, 2012 at 12:59 pm

    Thanks

  • Reply Eric Sondu October 11, 2012 at 1:45 pm

    how do i repay you?

  • Reply sadly November 6, 2012 at 12:37 am

    Nice

  • Reply Dikshya Pokhrel November 29, 2012 at 1:16 pm

    it was very helpful

  • Reply vigirl340 December 14, 2012 at 2:20 am

    How do you prepare the cell suspension?

  • Reply DrMegaster January 31, 2013 at 2:22 pm

    thanks for this– very helpful!

  • Reply Brandon Rosenstrauch February 4, 2013 at 6:52 am

    Wow, this was well put together. Great job!

  • Reply Ateeb Hussain February 20, 2013 at 2:53 pm

    Amazing

  • Reply Mike Stout February 22, 2013 at 9:19 am

    Thank you. This is just what I needed.

  • Reply SurvivalSquirrel April 20, 2013 at 12:33 pm

    Very good !

  • Reply Liliana Baldé May 14, 2013 at 7:09 pm

    This is the best video about the topi… so easy to understand 🙂 Thank you so much!

  • Reply MrPrabhubct June 13, 2013 at 5:12 pm

    good

  • Reply Damir Maričić June 18, 2013 at 1:04 am

    great job guys! love your video!

  • Reply Rebecca Mae Patricio June 19, 2013 at 12:40 am

    THANK YOU SO MUCH!!! viable for learning

  • Reply hellofriends40 July 2, 2013 at 4:31 am

    Very good video, but one mistake. At the end, she says that 94.7% of the 216,000 cells/mL are viable. However, she uses the number of viable cells (54) to calculate average number of cells/square. If she had used the number of total cells (nonviable + viable, or 57) to calculate cells/square, then in the end it would make sense to take the final number and say that 94.7% of those cells are viable.

  • Reply JayJ October 12, 2013 at 12:49 pm

    Well spotted, sir.

  • Reply sasiti nugrahaningsih November 10, 2013 at 12:02 pm

    thanks a lot..

  • Reply 王海东 November 28, 2013 at 3:47 pm

    really help me a lot

  • Reply Pearl Rodrigues November 30, 2013 at 4:13 pm

    thank you so much… its really awesome…

  • Reply Sarah Hussain December 1, 2013 at 7:27 am

    wow! excellent job

  • Reply Thathsara Abeywardhana February 9, 2014 at 1:14 pm

    thnx a lot! i learned so much!

  • Reply saleh obaidi March 7, 2014 at 5:00 pm

    Can we use this method to count human sperms and differentiate between viable and non viable sperm

  • Reply Matt Cartwright June 4, 2014 at 8:10 am

    Thank you for the help. This video has now made my understanding of Hemocytometers even greater and therefore has also made my day better.

  • Reply ahmed salah July 7, 2014 at 10:27 am

    amaaaaaaaaaaaaaaaaaaaazing ,,,,, really amazing
    excellent photographing … direction…. sound effect… commentary … subtitle .. perfect timing.
    science is different actually

  • Reply MeDo August 3, 2014 at 4:29 am

    Thank you

  • Reply 何糖糖 September 8, 2014 at 5:40 am

    good!

  • Reply 魏语呈 September 10, 2014 at 9:52 pm

    impressive

  • Reply Jules Guittard November 7, 2014 at 5:44 am

    Great but I think you have to close your labcoat

  • Reply EDUARDO12348 November 20, 2014 at 6:20 am

    Thank you very much, now am ready for lab class tomorrow here at MD anderson

  • Reply Ranim Mira February 5, 2015 at 5:55 pm

    Thank you, Finally I understand it

  • Reply sadafbenaf March 17, 2015 at 4:53 pm

    This is THE best explanation, but what would the approximate RBC# be for a sheep?

  • Reply TheTextralian May 13, 2015 at 3:17 pm

    Why fill both squares?  You've only counted one.

  • Reply Ismael Ramos June 29, 2015 at 5:14 am

    @BioNetwork  Thank you very much!  i was able to understand this perfectly!!!!

  • Reply vaibhav nagar August 23, 2015 at 9:24 pm

    thnku finally I understand it

  • Reply Lajos Winkler October 3, 2015 at 2:12 pm

    The fact she's wearing unnecessary goggles is making me cringe.

  • Reply Sam O'Brien October 28, 2015 at 11:35 pm

    how would you work out number of cells per ul ?

  • Reply sazan Ismael January 2, 2016 at 2:17 am

    Great explanation !!!

  • Reply Trevor Booth February 21, 2016 at 4:26 am

    I have also seen Hemocytometer spelt Haemocytomer and Hemacytometer. Can anyone please confirm the correct spelling? Thanks

  • Reply albert ng July 5, 2016 at 3:54 pm

    very clear and easy to understand.

  • Reply Natalia Maruri Saucedo July 30, 2016 at 5:55 pm

    thanks a lot!

  • Reply Shajedul Islam August 4, 2016 at 8:09 pm

    But when we do subculture how much amount of media or cell suspension will be required ..please explain that one also…and thanks for your good explanation

  • Reply Lemya finder August 10, 2016 at 9:51 am

    please where are you doing this manip? is it possible to realise a work there…i'm looking for a cell culture lab for an internship.

  • Reply Mudassar Ali September 16, 2016 at 12:54 pm

    nice

  • Reply Y.X. Han September 26, 2016 at 1:48 am

    Thank u !!!

  • Reply Rafal October 11, 2016 at 8:58 pm

    it was 10^-4 suspension (negative) meaning 1ul right? so 10.8*2*1000ul =21600 why do we multiply with 10^4?

  • Reply luna October 24, 2016 at 9:32 pm

    thank you so much!

  • Reply Ahmad Alsaadi November 20, 2016 at 11:53 am

    Thank you, this is the best presentation in this regards

  • Reply NahLa Ebaied November 25, 2016 at 2:55 pm

    how does doing this tells me if there is a certain disease? like what is the average number of cells that i should be comparing my results to ?

  • Reply Venkat Karri December 12, 2016 at 7:01 pm

    very good presentation

  • Reply Spawn February 18, 2017 at 8:43 am

    A great explanation!

  • Reply Mak Maker March 1, 2017 at 7:04 am

    neat

  • Reply Benjamin Mustonen March 6, 2017 at 2:43 am

    Is there a specific type of pipette you have to use?

  • Reply Nilesh M May 1, 2017 at 9:02 am

    can someone please explain me why they multiply with 10^4 at the end? I dont get it why to multiply with 10^4 to calculate number of cells per ml..
    Many Thanks for the video and for explanation.

  • Reply TheHypercasual May 12, 2017 at 4:13 pm

    Great video!

  • Reply Ataur Rahman Bhuiyan August 15, 2017 at 1:20 pm

    Easy to understand, Thanks

  • Reply Radhha Chaulagain September 11, 2017 at 8:02 am

    aa

  • Reply Muhammed Ismail September 18, 2017 at 1:48 am

    i wish every single labs out there had beautifully edited constructed with great explanations like this… would definitely make uni life way more interesting… amazing video, hats off!!!

  • Reply bonnolog October 11, 2017 at 6:33 pm

    0:33 oh the bubble! oh nooooessss

  • Reply ZULU ZERO December 7, 2017 at 11:16 pm

    I always mess this up

  • Reply Sukanya Roychowdhury January 23, 2018 at 12:04 am

    for cyanobacteria or blue gree algae also the dead cell appear blue?

  • Reply Nathan Stephens January 26, 2018 at 4:39 pm

    We use this video in an introductory cell biology lab course, it's very informative and helpful! However, the linked portion where you can print off a certificate at the end appears to be broken. This has been a great mechanism for giving students credit for watching the video. Do you have any plans to make that part active again? Cheers!

  • Reply Afida fatma March 27, 2018 at 8:09 am

    How to count sel density microalgae that has spiral form like Spirulina with haemocytometer?

  • Reply shingonakai April 6, 2018 at 4:30 am

    Your videos have given me great teaching aids to students who are new to cell culture! Thank you!

  • Reply Nichole Rojas May 9, 2018 at 9:59 am

    How do you know how many volume to seed?

  • Reply Rum Ham May 30, 2018 at 11:31 pm

    Is that a standard light microscope?
    In my readings it says that a fluorescent microscope is required to detect the dye that distinguishes living from dead cells.

  • Reply Jarrod C July 16, 2018 at 9:27 pm

    why multiply by 10000 at the end? You never explain that.

  • Reply Adarsh Patel August 7, 2018 at 5:57 am

    This was the best video ever

  • Reply Shin Chen October 23, 2018 at 1:02 pm

    I always has a shaking hand when doing this

  • Reply Gam November 10, 2018 at 2:32 am

    The virtual hemocytometer isn't accessible :((

  • Reply Alex Chan November 26, 2018 at 8:37 am

    Thank you so so much!!

  • Reply navjot singh December 6, 2018 at 5:05 pm

    thanks you for such a beautiful video , its an interesting task.

  • Reply Gritsana Thunyaluxdecho December 18, 2018 at 12:45 pm

    Why does it have 2 chamber but counted only 1 chamber? So what's about the other one?

  • Reply Vlad the Inhaler December 22, 2018 at 2:17 am

    My lab does body fluid cell counts on Fuchs Rosenthal hemocytometer and we use CSF diluting fluid to lyse RBC's. We use Neubauer hemocytometers for manual platelet and manual WBC counts.

  • Reply Nivedita Sagar January 22, 2019 at 7:40 am

    How much grams of trypan blue in how much ml of distilled water you have taken

  • Reply za1996 Aljoman January 29, 2019 at 1:24 pm

    Hi
    Is this suitable for sample has oil??
    And the trypan blue solution is suitable for sample has oil??

  • Reply AISHWARYA RAY January 29, 2019 at 4:06 pm

    Thanks for such a great explanation …really worthy enough

  • Reply owen wright February 5, 2019 at 3:22 am

    the surround sounds makes me wanna kms

  • Reply The thinker 03 February 10, 2019 at 2:57 pm

    awesome video, thank you very much!

  • Reply MAISSA MEFTAH February 22, 2019 at 11:53 pm

    is this the same as couting red cells??

  • Reply ddneb February 23, 2019 at 6:47 pm

    I love it…Good refresher.

  • Reply nicky sharma February 25, 2019 at 8:05 pm

    Really easy to understand and great use of simple graphics, I'm just looking into yeast counts for brewing. If I can follow this anyone can, thank you.

  • Reply MONICA SIMON August 6, 2019 at 7:24 pm

    Amazing

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