# HEMOCYTOMETER QUESTION - HEMOCYTOMETER (Oct/19/2006 )

Its been a while since i used a hemocytometer - whats the best calculation for determining cell density? thanks

Average # of cells X dilution factor X 10,000 = (# cells/mL) / 1000 = # cells/ uL

this is how I count.

count the 16 small squares, there r 8 of these - count them all

average them

multiply x 10,000 = # of cells in a ml.

Method A

Count the number of cells in the 4 outer squares (see the left panel of Figure 2).

The cell concentration is calculated as follows:

Cell concentration per milliliter = Total cell count in 4 squares x 2500 x dilution factor

Example: If one counted 450 cells after diluting an aliquot of the cell suspension 1:10, the original cell concentration = 450 x 2500 x 10 = 11,250,000/ml

Method B

Estimate cell concentration by counting 5 squares in the large middle square

The cell concentration is calculated as follows:

Cell concentration per milliliter = Total cell count in 5 squares x 50,000 x dilution factor

Example: If one counted 45 cells after diluting an aliquot of the cell suspension 1:10, the original cell concentration = 45 x 50,000 x 10 = 22,500,000/ml

I came across this question about a hemocytometer when my lab mate was having problems seeding the correct amount of cells on her plates. We both came from different labs so our our method of counting is a little different and we get different # of cells. She got 21.5x10^6 total # of cells and i got 20.9x10^6. so that made me wonder exactly how accurate is the # you get from the hemocytometer and whether the difference in cell # is that big of a deal?

21.5x10^6 is so close to 20.9x10^6

anyway, haemocytometer is just used to __estimate__ the number

taht's what i was thinking also.. that the 2 # were so close, but she was making a big deal of it so it just made me kinda wonder.. thanks!! =)

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 â€“

I â€˜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 â€“ 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

also, you need to make sure you know the correct way of using a hemacytometer.

there's an undergrad in our lab make a mistake by putting tooo much cell suspension into hemacytometer and thus, his calculation will not represent the correct numbers of cell.

A short guide to use hemocytometer