9 replies to this topic

[justwonder]

Can you read this link and tell me what you think of methods A and B? It does not make sense for me why method A formula is;Cell concentration per milliliter = Total cell count in 4 squares x 2500 x dilution factor
and method B formula is; Cell concentration per milliliter = Total cell count in 5 squares x 50,000 x dilution factor. Where did he get 2500 in A and 50,000 in B?

http://www.animal.uf...macytometer.htm



Thanks for any inputs.

[methylnick]

hi justwonder,

the haemocytometer takes up a fixed volume.

There are squares of different sizes reflecting a fixed fraction of the volume.

Depending on which squares you use to count the cells a multiplication factor is required. Put simply, method B uses the smaller squares for cell counting hence the larger multiplication factor, while in method A the large squares are used.

Nick

[justwonder]

hi justwonder,

the haemocytometer takes up a fixed volume.

There are squares of different sizes reflecting a fixed fraction of the volume.

Depending on which squares you use to count the cells a multiplication factor is required. Put simply, method B uses the smaller squares for cell counting hence the larger multiplication factor, while in method A the large squares are used.

Nick

<{POST_SNAPBACK}>

What do you think about the methods? Usually we take the average of the counted squares, but these methods don't do it. Any ideas?

Thanks.

[methylnick]

What do you think about the methods? Usually we take the average of the counted squares, but these methods don't do it. Any ideas?

Thanks.

<{POST_SNAPBACK}>

yeah sure it's more accurate to count multiple cells and average. I usually use the squares in method A, you can count four of them and then take the average, not so easy for method B though, you need to wash haemocytometer and then reload a fresh sample for counting!

good luck

Nick

[nmdr]

Hi,

I have a very basic question regarding cell counting.
Once i centrifuge and get the pillet, I add 1ml media to the pillet and mix it.
Using hematocytometer if i count five small squares and get an average of 100 cells.
What will be the total number of cells I have in the 1ml media.

Can u please help me...
Thanks

[bob1]

Here's the guide I made up for teaching students...

Attached Files

•  Haemocytometer_counting.pdf   456.41KB   354 downloads

[scifistudent]

hi all

i also use haemocytometer. i do 10 fold dilution for the cells in Trypan blu and then count the 4 periphery corner boxes and then take the average.

it is very simple

all the best for your experiment

[memari]

http://en.wikipedia....i/Hemocytometer


-----
Babak

[ReemooN]

well i guess the issue is not in how to use and calculate the concentration of cells, but the issue is after that when we want to figure out the number of cells we needed in each well and the volume needed in each well.

this calculation is really confusing, at least for me.

[bob1]

The calculations are very simple in the end - there are a lot of confusing methods of doing the calculations but if you have a couple of basic formulae, and use those consistently you shouldn't have any problems.

The two formulae you want to use are: c=n/v (concentration = number of cells/volume) and C1V1=C2V2 (C= concentration, V=volume, 1 and 2 are before and after (or "now" and what you want), use this formula for diluting). These are the same formulae that you would use for making up solutions.