Cell counting with a Thoma cell counting chamber

Apply coversheet to cell counting plate with a small drop of water on the sidelining

glass bridges. The glass has to really adhere to the surface, not a lot of water is

required (like half a drop).

Cut off a small square of parafilm, place it under the hood and pipette 10 µl Trypan

blue on it.

Take 10 µl cell suspension sample and pipette it into the drop of trypan blue. Pipette

up and down to mix.

Bring the counting chamber to the edge of the laminar flow under the hood. Gently

insert the mixed fluid at the edge between coverglass and counting chamber.

Fill the chamber and hopefully there are no bubbles left underneath, otherwise the

chamber has to be cleaned and the whole step repeated.

Take the counting chamber to the microscope and take a tick-counter.

Count the cells in the four checkered fields. Cells on the tripled sideline are counted

on two of the four borders.

Take the median of the four counts, double it (1:1 mix with trypan blue). Multiply by

10000. (Each of the squares has the surface area of 1 mm2, the gap between

coverslip and chamber base is 0,1mm. Multiplying the cell count with 10 gives the

number of cells in 1 µl. Multiplying by 1000 then gives the number of cells in 1 ml.)

(81 + 96 + 83 + 80)

4= 85

85 × 2 = 170

170 × 10000 = 1700000

1,7 × 106𝑐𝑒𝑙𝑙𝑠

𝑚𝑙