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 mm^{2}.
This creates a region of known volume (0.1 mm^{3}) 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 10^{4}
gives the number of cells per ml.

** **

**Procedure:**

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 1mm^{2} 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 10^{4} where C =
cells per ml

Ñ = average of cells counted

10^{4} = volume conversion factor for 1mm^{2 }

Total Yield = C x V where V = total volume of cells (ml)

**Example:-**

Count1 = 182 cells/1mm^{2} Count2 = 175 cells/1mm^{2}

Volume of Cells = 55 ml

Average cells counted = __Count1 + Count2 __ __182 +
175__ = 178.5

2 2

C
= 178.5 x 10^{4} = 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 10^{5} 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.