LM - Counting Chambers

What is a counting chamber and what is it used for?

A counting chamber is a precision measuring instrument made of special optical glass. It is used to count cells or other particles in suspensions under a microscope.

Counting chambers are mainly used for blood analysis (counting leucocytes, erythrocytes and thrombozytes) and to count cells of liquor. Counting chambers are also used to count bacteria and fungus spores.

Design principle

All counting chambers have the same basic design principle. (Pic. 1)

There are four longitudinal grooves in the central third of a rectangular and thick base plate made of special optical glass. The grooves are parallel to the short sides of the base plate and the central third has the same size as the cover glass used with the counting chamber. The two larger external surfaces are unfinished and are used for marking purposes.

The central support and the two external supports are smooth and highly polished. The surface of the central support is deeper than that of the two external supports. The counting nets are engraved in the central support (chamber base).

If a cover glass is placed on the external supports, a capillary gap is produced between the underside of this cover glass and the central support of the counting chamber.

Designs and identification of counting chambers

Two types of Counting Chambers:
Single grid ruling: middle support without division (one counting grid) - Pic. 2
Double grid ruling: middle support with one division (two counting grids) - Pic. 3

Furthermore there are two different designs of grids:

• standard: the counting grid is directly engraved in the glass.
• bright-lined: the chamber base is initially coated with rhodium and the counter net is then etched into the rhodium coating. By shifting the contrast, colour inversion under the microscope is possible so that the counter net can be viewed either in light or dark field illumination.

The bright lined chamber is preferred, since viewing is less strained.

Identification:

The following details are printed on both unworked surfaces of the counting chamber:

• counting net system
• chamber depth in mm
• area of the smallest square in mm²

Production and quality descriptions

Counting chambers are precision instruments. They are predominantly used in medical laboratories. All counting chambers are manufactured in compliance with the relevant Calibration Ordinance and DIN standard.

Production:

The production of counting chambers includes several individual processes, each of these is followed by stringent checks.

The internal support (chamber base) as well as the two external supports are ground and polished. The flatness and the accuracy are the most important requirements. They are described in the standard DIN 12874. The central support (chamber base) is ground to requirements, for example the depth of the Neubauer must be 0.1 mm. Besides the standard depth there are special depths available (f.e. 0.2 mm / 0.5 mm).

After these processes the counting net is engraved in the chamber base which is followed by the lettering on the un - worked surfaces and by a heat treatment. The production of the counting chamber is now completed with the final check which ensures that the counting chamber is in accordance with the DIN standards for weights and measures.

Requirements on quality controls:

The maximum deviations allowed according DIN 12847 are as follows:

• for the chamber depth in the area of a counting net ± 2% of the nominal value
• for distances of less than 0.4 mm between any net lines ± 2µm
• for distances of 0.4 mm or more between any net lines ± 0.5% of the desired value
• for the angle of the net division ± 1°
• the width of the division marks must not be greater than 5 µm

The flatness tolerance acc. to DIN 7184 Part 1 is as follows:

• for the chamber base near the counting net 2 µm
• for the support areas 2 µm
• for the cover glasses 3 µm (according to DIN 58 884)

Filling the counting chamber

Sliding on the cover glass

The external supports are to be moistened with distilled water and the cover glass then is gently pushed onto the counting chamber from the front.

Important: The cover glass is fragile!

The formation of interference lines (Newton rings) between the external support and the cover glass shows that the cover glass is correctly positioned.

Feeding:

Take a well mixed pipette from the shaker and dispose off the first few drops. Wipe the pipette dry on the outside and then hold it at an angle until a small drop has arisen at the tip of the pipette.

This drop is then to be placed between the cover glass and the counting chamber.

As a result of the capillary effect the gap between the cover glass and the chamber base fills up. Before the thinned blood solution can overflow at the edges of the chamber section, the tip of the pipette must be removed. If any air bubbles are visible or if the liquid has overflowed over the edges and into the grooves, the chamber must be cleaned and feeding must be repeated. (Pic.5).

Blood Mixing pipettes to be used:

a.) Erythrocyte pipette (red bulb)

b.) Leukocyte pipette (white bulb)

Counting the particles

Counting technique:

Counting assumes precise knowledge of the limit lines of the counting chambers used. These are shown in the illustration.

To ensure that cells which are on or along the limit lines are not counted twice or are not missed during the count, certain rules have to be observed (see illustration Pic. 6.).

The count should be started at the top left-hand corner and follow the direction shown by the arrow (Pic 6.1). Counting may be enhanced with the microscopes illumination reduced.

Notes on counting:

• Use reduced microscope illumination for all chambers.
• The difference of the counter cells in the large squares and the group squares must not exceed 10 cells.
• Double checks must be performed for all cell counts. After counting the two counting nets the bottom counting net is to be counted in the same way as a check. When doing this it is to be ensured that the chamber has not dried out. This can be prevented by filling the bottom chamber only shortly before the count and the counting after the sedimentation time.
• The difference between the totals of the counts for the two counting nets must not exceed 10 cells. The average value of the counts is then used in the calculation formula or multiplied by the corresponding factor.

Calculation

Formula:

 counted cells x dilution factor ____________________________________ = cells per ml of blood mm2 of counted area x chamber depths

Chamber: Neubauer improved

1. Leucocytes
   1. Counted cells 156 leucocytes
   2. Counted area: four squares (= 4 x 1mm²)= 4mm²
   3. Chamber depth 0.1mm
   4. Dilution factor 1:20
      
      156 x 20 ___________ = 7800 leucocytes /ml of blood 4 x 0.1
2. Erythrocytes
   1. Counted cells 528 erythrocytes
   2. Counted area: five squares (= 5 x 0.04 mm²)= 0.2mm²
   3. Chamber depth 0.1mm
   4. Dilution 1:200
      
      528 x 200 ___________ = 5.28 million erythrocytes /ml blood 0.2 x 0.1

Cleaning the counting chamber

Immediately after completing the count, remove the cover glass. The counting chamber and cover glass has to be cleaned with water or, if necessary, with a mild cleaning solution. Then dry the chamber with a soft cloth or Kimwipe lint free wiper. Chambers may be dried with acetone.