Use of the Improved Neubauer Haemocytometer
The haemocytometer is a specialised microscope slide on which 2 grids have been engraved, in a central region that is 0.1 mm lower than the rest of the slide. Each grid comprises 25 large squares, each containing 16 smaller squares of area 1/400 mm2. This creates a region of known volume (0.1 mm3) when a special coverslip is correctly placed over the central region (get someone to show you how to do this - and take care as the coverslips are easily broken!). Ten µl of culture are then pipetted under the coverslip and cells counted in a proportion of the grid squares (count as many as is convenient). Multiplying the total number of cells in the entire grid by 104 gives the number of cells per ml.
1. Clean the coverslip and the haemocytometer with 70% ethanol.
2. Moisten the sides of the coverslip by breathing on the surface and align the coverslip over the counting chamber using gently downward pressure. “Newton’s rings” should be produced between the coverslip and the slide.
3. Take a well mixed 20-50µl aliquot of the dissociated cell suspension using either a Pasteur pipet or a micropipettor only drawing the cells into the tip. Immediately transfer the cell suspension to the chamber by placing the tip of the pipet at the edge of the chamber and allowing the chamber to fill completely via capillary action.
4. Repeat this procedure using another aliquot sample for the second chamber on the opposite side of the haemocytometer.
5. Place the haemocytometer on the microscope stage and, using the 10X objective, focus on the counting chamber grid lines. Adjust the contrast as needed to clearly see both grid and cells.
6. Adjust the field area by slowly moving the slide to obtain a central grid bounded by three lines on all sides (see fig. above). Count the total number of cells present in this 1mm2 area including those cells which are on the top and left borders and excluding those on the right and bottom borders.
7. For accuracy count at least 100-500 cells. Depending upon yield and density more or fewer areas may be counted.
8. Repeat the count for the second chamber. If no second chamber exists, the slide should be cleaned and the process repeated.
C = Ñ x 104 where C = cells per ml
Ñ = average of cells counted
104 = volume conversion factor for 1mm2
Total Yield = C x V where V = total volume of cells (ml)
Count1 = 182 cells/1mm2 Count2 = 175 cells/1mm2
Volume of Cells = 55 ml
Average cells counted = Count1 + Count2 182 + 175 = 178.5
C = 178.5 x 104 = 1,785,000 cells/ml
Total yield = C x V = 1,785,000 x 55 = 98,175,000 cells
Note: For best results the cell density should be at least 105 cells per milliliter. Common errors occur by improper mixing of the cell suspension prior to sampling and/or by allowing the cells to settle in the pipet prior to loading the haemocytometer counting chamber. Avoid the counting of multiple cell aggregates; the presence of aggregates indicates incomplete dissociation which may require further optimization of the isolation parameters. A single cell suspension provides the best results.