# HEMOCYTOMETER - (Oct/26/2006 )

This was really very Helpful

Thanks:)

Pooja

Then:

10^4 / 2.3 x 10^6 x Z ml = 5ml

Where Z is the amount of medium you have to add to obtain the desired 10^4 cells/ml.

Z = 5/(10^4/2.3 x 10^6) = 5 x 2.3 x 10^6 / 10^4 = 5 x 2.3 x 10^2 = 1150 ml.

So it is a 1:230 ratio (1150/5). Accordingly you can add 1µl of your initial suspension (2.3 x 10^6) to 229 µl of medium and obtain 230 µl which contain 10^4 cells.

Thanks for the information:)

I had a question, Ideally how many cells should be plated for assays such as ELISA using a 96-well plate?

Pooja

First of all 230 cells/ square (16 smaller squares) is to many cells to do an accurate cell count. You should really have between 25 and 100 cells/square. And you should count 4 squares and then average the count.

Are you doing a VIABLE count i.e. using Trypan blue as an EXCLUSION DYE. If you are you need to have the dilution factor within your calculation ......for example 100ul of cell suspension + 100ul of Trypan is a 1:2 dilution, thus the factor of 2 is put into the following calculation :

Mean Cells/square x 2 x 10,000 (10^4) = No of cells/ ml.

Your example:

230 x 10,000 = 2.3 x 10^6

You need to dilute your cell suspension by a factor of 230 to get a cell suspension of 10^4

i.e 1ml of cell suspension to 229mls of media = 10^4 cells/ml

or 0.1ml of cell suspension to 22.9mls of media = 10^4 cells/ml

or 10ul of cell suspension to 2.29mls of media = 10^4 cells/ml

or as Jou has already stated eloquently 1ul of cell suspension to 229ul of media = 10^4 cells

Hope this is clear

Rhombus.

Hi , I have a question regarding cell counting and the dye exclusion test :

I have always used the dilution factor when doing the counting , but the lab I am in right now the calculation that they do am not able to follow –

I ‘ve been told to count the number of cells in any of the 2 opposite squares take their average .Take the average cell count and multiply it with 10 ^ 4

i.e. if I get 32 n 33 in 2 squares I divide it by 2 to get = 32.5 * 10^4 cells / ml

Is this correct ?? Am not sure

Also when I mix the cell suspension n tryphan blue – I take 100 micro lt of cell susp + 100 micro lt of dye , mix it well n load 10 micro lt onto the hemocytometer .

If I do this type of procedure what sort of dilution factor I have to take into account if any?? Please let me know what to do or is this correct. !! Thanks

I have always used the dilution factor when doing the counting , but the lab I am in right now the calculation that they do am not able to follow –

I ‘ve been told to count the number of cells in any of the 2 opposite squares take their average .Take the average cell count and multiply it with 10 ^ 4

i.e. if I get 32 n 33 in 2 squares I divide it by 2 to get = 32.5 * 10^4 cells / ml

Is this correct ?? Am not sure

Also when I mix the cell suspension n tryphan blue – I take 100 micro lt of cell susp + 100 micro lt of dye , mix it well n load 10 micro lt onto the hemocytometer .

If I do this type of procedure what sort of dilution factor I have to take into account if any?? Please let me know what to do or is this correct. !! Thanks

that is correct. We count all 4 squares so we divide by 4 and then multiply by 10,000.

If you dilute your sample 1:2, then i will multiply the final cell count by 2.

eg. you have counted 50 and 60 in 2 squares.

110/2 = 55 / square

55 x 10,000 x 2 = final cell density.

Hello

I just came across this site which does all the calculations for you if you put in your numbers. Here is the address

http://www.changbioscience.com/cell/hemo.html

I hope it was helpful

Thanks

dear all..

facing some more curious abt hemacytometer...

if for budding yeast cell, we count the old cell and the new budding cell (they are stick together) as 1 group or jus as 2 cell..??

if counted as 1 group, is it ok if some group got 3 to 4 budding...??

if counted as 2 cell (according to number of cell we get to see), then it regardless of its size??

anyway, thanx a lot for ur help..

This protocol describes a method for estimation of mammalian cell number in a defined volume of medium using a hemocytometer. Automated methods using cell-counting devices such as those produced by Coulter are desirable when large numbers of individual samples are to be counted.

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reetha.

http://www.protocol-online.org/forums

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reetha.

http://www.protocol-online.org/forums

sori... may i know which protocol that u mean?? thanx a lot..