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 ÂµL, because we started

with 100 ÂµL 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

where did the 10^4 come from???

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

Thanks

how do i repay you?

Nice

it was very helpful

How do you prepare the cell suspension?

thanks for this– very helpful!

Wow, this was well put together. Great job!

Amazing

Thank you. This is just what I needed.

Very good !

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

good

great job guys! love your video!

THANK YOU SO MUCH!!! viable for learning

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.

Well spotted, sir.

thanks a lot..

really help me a lot

thank you so much… its really awesome…

wow! excellent job

thnx a lot! i learned so much!

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

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

amaaaaaaaaaaaaaaaaaaaazing ,,,,, really amazing

excellent photographing … direction…. sound effect… commentary … subtitle .. perfect timing.

science is different actually

Thank you

good！

impressive

Great but I think you have to close your labcoat

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

Thank you, Finally I understand it

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

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

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

thnku finally I understand it

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

how would you work out number of cells per ul ?

Great explanation !!!

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

very clear and easy to understand.

thanks a lot!

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

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.

nice

Thank u !!!

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

thank you so much!

Thank you, this is the best presentation in this regards

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 ?

very good presentation

A great explanation!

neat

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

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.

Great video!

Easy to understand, Thanks

aa

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!!!

0:33 oh the bubble! oh nooooessss

I always mess this up

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

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!

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

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

How do you know how many volume to seed?

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.

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

This was the best video ever

I always has a shaking hand when doing this

The virtual hemocytometer isn't accessible :((

Thank you so so much!!

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

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

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.

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

Hi

Is this suitable for sample has oil??

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

Thanks for such a great explanation …really worthy enough

the surround sounds makes me wanna kms

awesome video, thank you very much!

is this the same as couting red cells??

I love it…Good refresher.

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.

Amazing