This is a cached page for the URL (http://icg.cpmc.columbia.edu/cattoretti/Protocol/FlowCytometry/Hemacytometer-counting.html). To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
Hemacytometer counting

Cell counting with an hemacytometer.

Accuracy of manual counts with an hemacytometer depend on:

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 No 87144 (expect 10% variability or more)
Hycor Biomediacal Inc.
Garden grove CA 92841
Hemacytometer Fisher scientific No 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