NEUBAUER CHAMBER: HISTORY, CHARACTERISTICS, USES - BIOLOGY - 2025

The Neubauer chamber, hematometer or hemocytometer, is a laboratory instrument consisting of a special thick glass plate. This camera is used to count some cell types such as red blood cells, white blood cells and platelets, although it can be used to count spores, sperm, parasites, etc.

It presents some very peculiar characteristics, as it consists of 3 zones, a central one for counting and two support zones. Each chamber has two counting zones or crosshairs, one at the top and one at the bottom.

Neubauer chamber. Source: SantiBadia. Edited image.

These have multiple divisions in a grid form. The counting areas are the medium squares found at the 4 corners of both graticules, plus the central square.

The assembly of the camera must be done with great care, as any detail influences the cell count. There are many mistakes that can be made, but if any of them occur, the camera must be disassembled, cleaned and reassembled. The main errors include the following:

Overflowing the chamber or underfilling, letting the chamber dry, attempting to remove excess liquid with gauze, tipping the chamber when transporting it, filling a dirty or wet chamber, not mixing the dilution or sample well, among others. All these errors will result in an unreal value.

History

The Neubauer chamber is a precision instrument, and the manufacturing process undergoes strict quality control. It was created for the precise counting of particles or formed elements per mm ³, such as cells in various liquids. Its delicate graphic is carved with a diamond pencil.

Neubauer chamber characteristics

The entire chamber is the size of a normal slide so that it can be placed on the microscope stage.

The chamber consists of three central rectangular surfaces (a, b, c). In zone “b” is located the R zone or counting zone, also called a reticulum. One on each side of the camera, separated by zone "d".

Graphical diagram of the Neubauer Chamber. Source: Practical Guide to Hematology. School of Bioanalysis of the University of Carabobo, Venezuela.

Each graticule is a polished area that contains the counting area engraved. It consists of a square with a surface area of ​​9 mm ² and is internally divided into 9 squares with a surface area of 1 mm ² each. The four corner squares are divided into 16 smaller squares (0.0625 mm ² in area).

These grids are formed by a series of millimeter lines that intersect with each other, constituting perfectly graphed grids delimited to the measurements that have been specified. These lines have been engraved with a diamond tip.

Improved Neubauer reticulum. Source: Practical guide of Hematology of the School of Bioanalysis of the University of Carabobo, Venezuela.

The four sides correspond to the counting area. It is on these sides or corners that most of the cells (red blood cells and leukocytes) are counted, while platelets are counted in the central area.

The central zone has more divisions, it consists of a 1 mm ² square divided into 25 squares that have a surface area of ​​0.04 mm ² each. These in turn are divided into 16 grids with an area of ​​0.0025 mm ².

Description of the improved Neubauer reticulum. a) Square of the corners, b) central square, c) median square of the central square. Source: Practical guide of Hematology of the School of Bioanalysis of the University of Carabobo, Venezuela.

Zone “a” and “c” serve as support to place a special cover object called a hematometric slide or hematimeter cover.

The height between the foil and the counting surface is 0.1 mm. Measurements of the surface area of ​​the tally boxes, as well as the height of the chamber and the dilution of the sample, are data necessary to make the final calculations.

Applications

It is used for cell counting. It is especially helpful in the hematology area, since it allows the counting of the 3 blood cell series; ie red blood cells, white blood cells, and platelets.

However, it can be used in other areas, for example to count sperm, spores, bacteria or other items of importance depending on the type of sample.

How to use?

Sample preparation

To perform the cell count, it is generally started from a previous dilution. Example: to count white blood cells, prepare a 1:20 dilution with Turk's liquid. The dilution is mixed well before loading the pipette and mounting the Neubauer chamber.

There are times when a 1:20 dilution is not enough to count. For example, in patients suffering from certain types of chronic leukemias. In these cases, higher dilutions such as 1: 100 should be made.

If, on the other hand, the count is very low, as in severe leukopenias, smaller dilutions can be made to concentrate the sample. Example: you can make a 1:10 dilution.

The changes that are made influence the calculations.

Neubauer chamber mounting

The Neubauer chamber is assembled by placing the hematometric slide in the central area. Both must be very clean and dry. To place the lamella, it is taken by the edges and gently dropped onto the camera.

This is filled by placing the tip of a Thoma automatic pipet or pipet at a 35 ° angle at the edge of the loading zone. The liquid is discharged smoothly and the loading area is filled by capillarity. This is done from both sides to load the two crosshairs.

The reticles should not be overloaded and neither should they be denied liquid. The load must be exact. It is important that the filling is done homogeneously, that is, there should be no bubbles.

Once the chamber is assembled, it is left to rest for 2 minutes so that the cells precipitate to the bottom and their visualization and counting are easier.

After the time of rest, it is mounted on the stage of the light microscope for observation. First it is focused with a 10X objective and if necessary then it goes to 40X.

To improve its visualization, the passage of light from the microscope is reduced. To do this, the condenser is lowered and the diaphragm is slightly closed.

Counting

To count the white blood cells or leukocytes, the entire surface of the four median corner squares and the central square of each reticulum must be counted.

Counting starts in the square in the upper left corner. You start from the first square of the first row, that is, from left to right until you reach the opposite end.

There you go down and look back from right to left until you reach the other end, and so on, the cells within each grid are counted in a zigzag fashion. The 16 grids of each median square are counted.

To avoid counting a cell twice, there are rules about the cells that are located on the boundary lines of each grid. Cells on the left and top lines are counted and cells on the right and bottom lines are ignored.

A manual cell counter must be available so that the operator presses the device key as many times as there are cells observed. With the use of the counter, the operator can count without having to look up from the microscopic field. At the end of the count you will see the total number of cells counted.

Calculations

For the calculations you can proceed in several ways. A single graticule can be counted or both can be counted and both are averaged. In these two situations, the counted cells must be multiplied by a factor, which in this case would be 40. And thus the total count per mm ^{3 is obtained}.

But if the two grids are counted and the average is not taken, it must be multiplied by a different factor, in this case by 20.

-Multiplication factor

Here's how the multiplication factor is calculated.

Various data are taken into consideration for the calculations, including the dilution titer, the height of the chamber and the area counted.

Dilution

The standard dilution used is 1:20 for the leukocyte count.

Chamber height

The height between the chamber and the blood cell sheet is 0.1 mm.

Counted area

If 5 squares of 1mm ² area are counted, it means that the total counting area is 5mm ². This data must be multiplied by the height of the chamber to obtain the total volume counted. That is, 5mm ² x 0.1mm = 0.5mm ³.

Formulas and calculations

With the data we have it is said:

If in 0.5 mm ³ --there are --- number of cells counted

In 1 mm ³ --- there will be - X n ° of cells

X no. Of cells = (no. Of cells counted x 1) / 0.5 mm ³

But dilution must also be taken into account. Therefore, the formula is as follows:

(number of cells counted x 1) x 20 / 0.5 mm ³

Finally, to summarize, the number of cells counted can be multiplied by 40. Thus, the value of leukocytes per mm ^{3 is obtained}.

If the two reticles are counted, the data for the counted area is changed, which in this case would be 10 squares, that is, 10 mm ². And a total counted volume of 1 mm3. The formula would be:

(number of cells counted x 1) x 20/1 mm ³

Therefore, in this case the multiplication factor would be 20.

Mistakes

-If when loading the camera it is exceeded or exceeded with liquid, the height of the camera will vary. This results in the count being higher than the real thing. If you try to remove the excess with gauze or cotton, this is a huge mistake. This action will cause the cells to concentrate, increasing the count.

-If it is loaded poorly, the count will be lower than the real one.

-If the camera is mounted and allowed to dry, it is no longer possible to count because it will give wrong results.

-If the dilution of the sample is not mixed well before loading the chamber, there is a risk of an error in the reading, as the cells will not be homogeneously distributed. Therefore, there will be a lower or higher concentration of cells, depending on whether the sample is taken from the surface of the liquid or from the bottom of the tube respectively.

-The presence of bubbles reduces the amount of liquid that must enter the reticulum, interfering with the correct visualization and distribution of the cells. All of this significantly affects the results.

-During counting, do not look up from the microscope until each large square is completed to avoid getting lost.

-One reason for error is tilting the camera after mounting. For this reason, the stage of the microscope must be carefully raised.

Recommendation

If for any reason you detect an abnormality in the filling of the chamber, it is recommended that you disassemble that preparation, clean the chamber and reassemble from scratch.

Take great care when cleaning the camera to avoid scratching the cross hairs. On the other hand, be aware that the hematometric slide is delicate and fragile. Improper handling can break it.

Before you start counting, make sure the cells are well distributed. An uneven distribution of cells occurs from poor sample mixing or dilution. If this happens, the assembly must be repeated.

One way to know if the cells are well distributed is by comparing the count of each large square, the number of cells counted by each square should not be exaggeratedly different from one to the other.

-If the white blood cell count is above 50,000 mm ^{3, it} is advisable to repeat the count, making a greater dilution.

-If you change the dilution, you must recalculate the multiplication factor, as this influences the formula.

References

1. Cardona-Maya W, Berdugo J, Cadavid A. Comparison of sperm concentration using Makler's chamber and Neubauer's chamber. Actas Urol Esp 2008; 32 (4): 443-445. Available in: scielo.
2. Neubauer chamber. (2018, March 27). Wikipedia, The Free Encyclopedia. Consultation date: 04:10, June 23, 2019 from es.wikipedia.org
3. Meneses A, Rojas L, Sifontes S. Application of an alternative counting method in the Neubauer Chamber to determine the concentration of Trichomonas vaginalis. Rev. Cub Med Trop 2001; 53 (3): 180-8. Available at: researchgate.net
4. Gómez-Pérez Roald E. Analysis of the Spermogram. Rev. Venez. Endocrinol. Metab. 2007; 5 (2): 19-20. Available in: ve.scielo
5. Hematology practical guide of the School of Bioanalysis of the University of Carabobo. Venezuela. 1998