Despite the fact of the recent technical development of scientific laboratories, the Neubauer chamber remains the most common method used for cell counting around the world. This technical data sheet has been composed in order to help experienced, as well as non-experienced, researchers perform a proper cell counting using a Neubauer chamber or Hemocytometer. The principles described in this technical data sheet apply to any cell counting chamber, although the dimensions and volumes of each chamber may differ. The technical data sheet describes the best practices, applications, and recommendations when performing a cell count.
Figure 1. Equipment necessary for performing cell count with hemocytometer
The Neubauer chamber is a thick crystal slide with the size of a glass slide (30 x 70 mm and 4 mm thickness). In a simple counting chamber, the central area is where the cell counts are performed. The chamber has three parts: (1) the central part, where the counting grid has been set on the glass, and (2) double chambers/two counting areas that can be loaded independently.
Figure 2. Neubauer commercial chamber; Figure 3. Pile of glass covers, and box
Neubauer chamber's counting grid is 3 mm x 3 mm in size. The grid has 9 square subdivisions of width 1mm (See Fig. 4-1). In case of blood cell counting, the squares placed at the corners are used for white cell counting. Since their concentration is lower than red blood cells a larger area is required to perform the cell count. The central square is used for platelets and red cells. This square is split in 25 squares of width 0.2 mm (200 µm). See Fig. 4-2. Each one of the 25 central squares is subdivided in 16 small squares. Fig 4-3. Therefore, the central square is made of 400 small squares.
Figure 4. Neubauer-improved chamber counting grid detail
The glass cover is a squared glass of width 22 mm. The glass cover is placed on the top of the Neubauer chamber, covering the central area. The glass cover leaves room for the cell concentration between the bottom of the chamber and the cover itself. The chamber is designed so that the distance between the bottom of the chamber and the cover is 0.1 mm. It is not uncommon that the glass cover remains slightly lifted when we introduce more liquid than necessary in the chamber. To avoid this, some counting chambers have two special clamps to avoid the cover glass to avoid edge-lift. If the glass cover is lifted, the distance between the chamber and the cover will be higher than 0.1 mm, and the cell counts will not be accurate.
The pipette allows for the introduction of a precise amount of liquid into the Neubauer chamber. Historically, they have been manufactured in glass. Nowadays, glass pipettes have been replaced by micropipettes, than can be calibrated with a maximum capacity of 20,200 and 2,000 µl.
Take 10 µl of dilution prepared in step 1 with the micropipette.
The Neubauer chamber has been loaded, and is ready to perform the cell count!
Figure 5. Sample filling a Neubauer chamber; Figure 6. Count in a Neubauer chamber big square
Figure 7. High cell concentration cell count; Figure 8. Example of cell count in one of the 9 big squares of a Neubauer chamber
We apply the formula for the calculation of the concentration:
Concentration (cell / ml) = Number of cells / Volume (in ml)
The number of cells will be the sum of all the counted cells in all squares counted.
Since the volume of 1 big square is:
0.1 cm x 0.1 cm = 0.01 cm2 of area counted.
Since the depth of the chamber is 0.1mm:
0.1 mm = 0.01 cm
0.01 cm2 x 0,01 cm = 0.0001 cm2 = 0.0001ml = 0.1 µl
So, for the Neubauer chamber, the formula used when counting in the big squares is:
Concentration = Number of cells x 10,000 / Number of squares
In the case that a dilution was applied, the concentration obtained should be converted to the original concentration before the dilution.
In this case, the concentration should be divided by the dilution applied. The formula will be:
Concentration = Number of Cells x 10,000 / Number of square x dilution
For a 1:10 dilution → Dilution = 0.1
For a 1:100 dilution → Dilution = 0.01
Errors in the range of 20-30% are common in this method due to pipetting errors, statistical errors, chamber volume errors, and errors from volume of sample introduced into the chamber. Regardless, the Neubauer chamber remains the most widely used cell counting method in the world.