Cell counting/hemacytometer

Abstract
The exact number of cells in a culture or preparation may be very important for many reasons, consistency being a major one. One of the easiest way to count cells is under a light microscope with the hemacytometer (aka erythrocytometer, hemocytometer). Moreover the viability of the cells can be simultaneously determined with simple stains.

The hamacytometer is basically a thick glass slide with a counting chamber. The central portion of this chamber is the counting platform with one or two extremely precise grids. The counting platform is covered by an even surfaced coverslip. This gives a very precise volume in the space delimited by the grid and the coverslip.

Reagents

 * 70% Ethanol
 * Cell suspension
 * Viability stain
 * RBC lysis solution

Equipment

 * Light microscope
 * Counter
 * Hemacytometer with coverslip
 * Micropipette

Procedure

 * 1) Clean the surface of the hemacytometer and coverslip with 70% ethanol.
 * 2) *[[Image:Difficult step.png]] Be careful not to scratch the surface of the coverslip, use microscope lens cleaning paper and a "light touch" so not to break it into pieces.
 * 3) Place the coverslip over the counting platform, pressing on the elevated reidges of the hematocytometer, not the center.
 * 4) *[[Image:Optional step.png]] Some place a small amount of water on the ground glass area to improve the seal.
 * 5) Mix the cells thoroughly to disperse clumps and produce a uniform suspension.
 * 6) *[[Image:Critical step.png]]
 * 7) Either use directly the cell suspension or mix with a staining solution and/or lysis buffer. Exact dilution should be determined for the type of cells and initial concentration. A maximum cell count of 20 to 50 cells per 1mm2 is recommended.
 * 8) Transfer the sample to the edge of the hemacytometer and let it be drawn under the coverslip by capillarity.
 * 9) *[[Image:Critical step.png]] Do not overfill nor underfill the chamber or it will alter the distance between the hemacytometer surface and the coverslip and thus an altered volume and calculated concentration.
 * 10) Allow a few minutes for the cells to settle and the viability stain to workbefore counting.
 * 11) View the slide at 100X magnification (10X ocular with 10X objective). The central area of the grid should occupy the center of the microscope field and cells should be evenly distributed and without any clumps.
 * 12) *[[Image:Critical step.png]] If cells are not evenly distributed or clumped, wash the hemacytometer and coverslip and start over.
 * 13) Use the counter to record the number of cells in the grid.
 * 14) * Most often the center field and four corners are counted but certain types of cells/applications use more or less of the 1mm2 squares as needed for the desired precision. The more squares and cells, the better the precision but the more time consuming.
 * 15) * Count the cells touching the middle line of the triple line on the top and left of the grid but do not count the cells touching the line on the bottom or right.
 * 16) Analysis. Derive the concentration using the general formula: cs = df x c = df x (n / v) / sq were
 * 17) * cs is the concentration of the sample
 * 18) * c is the concentration of the solution on the hemacytometer
 * 19) * df is the dilution factor of the sample
 * 20) * n is the number of cells per 1mm2 squares
 * 21) * v is the volume under the coverslip as delimited by a 1mm2 squares, 1x10-4mL for a 0,1mm depth under the coverslip
 * 22) * sq is the number of 1mm2 squares

Critical steps
Most of the errors in this procedure occur by incorrect sampling and transfer of cells to the chamber, i.e.:
 * Clumps
 * Not uniformed suspension
 * Incorrect volume in mixing with staining solution
 * Overfilling/Underfilling
 * Presence of lint between coverslip and hemacytometer

Troubleshooting

 * Inconsistent counts
 * Hemacytometer counting is cheap, do not hesitate to repeat counts of a single sample to eliminate abnormal counts. Using the average of both grids when available is good if both counts are relatively similar.

Specific Protocols

 * Stevej:Cell_counting/hemocytometer
 * Marceau:DBT cells
 * Marceau:Mouse spleen cells-with ACK lysis
 * Marceau:Mouse spleen cells-without ACK lysis

Discussion
You can discuss this protocol.