Cellcountingwithanhemacytometer.

Accuracy of manual counts with an hemacytometer depend on:

• accurate mixing of the sample (no bubbles!)

• number of chambers counted

• number of cells counted (practical: 200-500 per 0.1 mm3)

The chamber of various composition, all have a common unit of 1x1mm squares, which may divided in nine smaller squares. The height of the chamber, delimited by the coverslip is 0.1 mm.

Suppliers:

HYCOR: Kova system for standardized urinalisys N^o 87144 (expect 10% variability or more) 
Hycor Biomediacal Inc. 
Garden grove CA 92841 
Hemacytometer Fisher scientific N^o 02671 5

Dilute your cell sample in Trypan Blue dye exclusion medium. Dead cells will be blue, live cells will be birefringent. Suggested dilution 1:20 (5μl cell suspension in 95 μl Trypan Blue). 
Carefully and continuously fill evenly at once the hemacytometer chamber. 
Leave undisturbed for 1-2 min. (If you need to leave for longer, use a humid chamber). This will allow deposition of the cells on the counting plane. 
Count under the microscope two 1x1x0.1 mm areas (delimited by a double line; nine small squares), from two chambers. Ideally you should count more than 200 cells and less than 500 per chamber.

The number of cells per 1x1x0.1 mm areas = cells x 1*10^-4 cm³ (1*10^-4 ml). 
With a 1:20 dilution the easy count is: 
number of cells in two 1mm squares divided by 10 = cells x 10⁶ / ml. 
 (2 squares / 2 x 20 x10,000 / 1,000 = 10⁶ cells / ml) 
 (2 squares x 10 x 10,000 / 1,000 = 10⁶ cells / ml) 
 (2 squares x 100 = 10⁶ cells / ml) 
 (2 squares / 10 = millions / ml)

If your cell suspension is way above 500 / square, count as many smallest squares you can, calculate the mean: this number multiplied by 0.9 will approximate the number of millions / ml.

Useful refence:

Use of the Hemacytometer for the Determination of Cell Numbers 
http://www.imgen.bcm.tmc.edu/molgen/labs/bradley/hemacyto.txt