Tuesday, 14 September 2010

GGS LIVE - Counting cells

Hello BioFreaks Reader,

Today in GGS LIVE section we are having some basic Tissue Culture technique:

Method: Determination of cell number or cell density in the culture.

About: Determination of cell number is essential in performing experiments where appropriate number of cells is required in order to sustain uniform conditions during the experiment.

What: Determination of cell number in suspension cell culture.

The most common technique to determine cell number in culture is to count single cells using a microscope and a hemacytometer (see picture below).

To count suspension cells we need to first mixed them well to homogenise the entire culture. After mixing 10microliteres is taken and loaded onto a hemocytometer as shown on pictures below.

To count cells using hemocytometer we need to hava a closer look at it. Under magnification smooth area of hemocytometer looks like this:

The lilquid that occupy area of a centre square (here indicated with red border) which contains 25 smaller squares has volume of 0.1microliter. After loading cell onto hemocytometer we should see something like this:

The red boarder again indicates the central square that we are going to score. Additionally red arrowheads inducate three represenatative cells. We should count cells at least twice and take an average of two as the final result. For example: count one 93 cells
                                      count two 87 cells
                                             total 180 cells
                                        average  90 cells

To get a number of cells in a 1ml of culture we need to multiply our result by 10 000. Why? because volume scored is 0.1microliter and that is 10 000 less tham 1ml, so in 1ml there is 10 000 more cells than in the volume we had investigated (if you have problem with prefixes and calculations visit this post Scientific prefixes - you will be laughing). So in our study case we have 90 x 10 000 = 900 000 cells in 1ml.

I hope you enjoyed it.


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