Neubauer improved cell counting chamber

Neubauer improved cell counting chamber
Usage of Neubauer improved cell counting chamber

The frame of the counting chamber consists of 9 large squares each with a 1 mm2 area.

The large central square (which can be seen in its entirety with the 10X objective), is divided into 25 medium squares (with the 40X objective the medium squares can see completely), each with 16 small squares inside. The four large squares in the corners of the frame (not shown in the figure) are formed by 16 medium squares.

The counting can be done either in the central large square or in the corner squares, depending on the size of the cells under study. In this exercise we will count the yeast present in the central large square.

When we put the sample under the coverslip, the cell suspension reaches a height of 0.1 mm. Taking these data into account, and considering one of the large squares, the volume will be:

1 x 1 x 0,1 = 0,1 mm3 = 10-4 ml

With the 10X objective of the microscope the counting area must be located (one of the large squares). To count the cells the microscope must be switched to 40X objective. All the cells in the medium squares (25 medium squares if the central large square is chosen or 16 if we chose a large square in corner) must be counted according to the following criteria:

All the cells within each medium square and those that are over the top and right sides of the square (even when they are partially out) are counted. Following this approach, in the figure the cells in green will be counted, but not the cells in red.

If we have counted N cells in one of the large squares (that is, in 25 medium squares), the concentration of our sample will be:

N x 104 cel/ml

When prior to counting we concentrated or diluted the initial sample, we must take into account the concentration-dilution factor (f):

N x 104 x f cel/ml

initial cell suspension = diluted cell suspension x concentration-dilution factor