Selected Methods for Antiboby and Nucleic Acid Probes COUNTING CELLS USING A HEMOCYTOMETER ‘A hemocytometer (see Figure 1.1, top) can be used to count cell in a suspension. First, the supplied cover glass is placed on the cover glass rest, A small volume (~10-12 ul) of cell suspension is then applied to each side of the hemacytometer by touching the tip of a pipette to the filling ports. Capillary action will draw the Quid into the counting ‘chambers. Excess fluid will drain into the overflow chamber. To avoid inaccurate cell counts, do not overfill the hemocytometer: Each counting chamber of the hemocytometer has a 5 x 5 central rid and four 4 x 4 comer grids (see figure 1-1, bottom). The volume.in the central grid and in each of the comner grids is 10% ml. To obtainthe . =, greatest accuracy, the cells in at least five of the 25 central squares should be counted on each side of the hemocytometer and the counts averaged. (The deviation between these duplicates should be less than 20%). This average count is then multiplied by 25 to determine the mum- ‘ber of éells in 10 ml To determine the number of cells present in the original sample, calculation should then be made to account for the dilution of the sample during the counting procedure (e.g., the 1:1 dilu- tion with trypan blue; see p.16) and for the original sample volume. Cells can also be counted in any of the 45-4 comer grids. The total volume of each 16-square corner grid is 10 ml. The average of the counts in 5 of the 16 squares on each side is the multiplied by 16, in- stead of 25, to determine the number of cells in 10°*ml of the sample. Each of the 25 squares in the 5x5 central grid is further divided into 16 smaller squares. If very small cells are being counted or if the cell density of the suspension is very high, the cell in the 4 x 4 grids within one of the squares of the central 5 x 5 grid should be counted. The aver- age of the counts from 5 of these small squares on each side is then maul- tiplied by 16 and by 25 to determine the number of cells in 10 ml. FIGURE 1:1 The Hemocytometer (Top) Side and top views of the hemocytometer (Bottom) enlarged view of a counting square viewed with phase contrast optics. Each of the 4 x 4 comer grids (e.g., the cross- hatched square in the upper left-hand cornet) and the central square (5 x 5 grid) contains 10 ml. Therefore, the cells in any of the four comer squares or the cells in the central square can be counted to determine how many cells are present in 10% ml of cell suspen- sion.Selected Methods for Antiboby end Nucleic Acid Probes 2) The following is an example of the results obtained from counting the cells in a suspension. The calculations required to determine the A nal cell number are shown. An aliquot from a $0-ml cell suspension was stained with trypan blue. (1:1) and applied to a hemocytometer. Five of the squares in each central grid on each side of the hemocytometer were counted and were found to contain 7, 8, 5, 7, and 8 cells on one-side and 5, 7, 6, 9," and 8 cells on the other side. These-counts.were then averaged. Since the average number of cells per square in the.central grid is 7, 25 squares (Le., one central grid) contain 175 cells. Sience 25 squares contain a voluine of 10°* ml there are 175 cells/10"* mi, or 1.75 x 10 cells, or 3.5 x 10* cells/ml. Since the trypan blue diluted the sample by half, the original suspen- sion contained 2 x 1.75 x 10° cells/ml, or 3.5 x 10° cells/ml. Since the initial volume of the suspension wes $0 ml, the total number of cells was 3.5 x 10° cells/ml x $0 ml, or 1.75 x.10* cells.1 Celt Culture Side View “counting chamber cover glasS™ tounting square filling port . Figure 1:1 See facing page legendSome species of Tetrahymena