Working Of Counting chamber

1.
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 thrombocytes) and to count cells of

liquor. Counting chambers are also used, however, to count bacteria and fungus
spores.

2.
Design principle

All counting chambers have the same basic design
principle.

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 coverglass 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 ground smooth and 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.

3.
Design and identification of a counting chamber

Design

Single net ruling: middle support without division
(one counting net)

Double net ruling: middle support with one division (two counting nets)

Standard: the counting net is directly engraved in the glass

Bright-lined: the chamber base is initially coated
with rhoidum and the counter net is then etched into the coating of rhodium. By

shifting the contrast, colour inversion under the microscope is possible so that the counter net
can be viewed either in light or dark colouring.

Identification

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

• counting net system

• name and trademark of the manufacturer

• chamber depth in mm

• area of the smallest square in mm^{2}

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 started again.

4.
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 (eg. see illustration to the right).

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 (eg. see illustration to the right).

The count should be started at the top left-hand
corner and follow the direction shown by the arrow.

Notes
on counting

a) The trim of the capacitor on the microscope must
be almost closed for all chamber counts.

b) The difference of the counter cells in the large squares and the group
squares must not exceed 10 cells.

c) 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.

d) 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.

5.
How to clean the counting chamber

Immediately after completing the count the cover
glass is to be removed and the counting chamber has to be cleaned with water or
(if

necessary) with a mild cleaning solution. Afterwards, the chamber is to be
dried with a soft cloth or rinsed with acetone.

6.
Short description of the mostly used counting chamber.

The various systems used for counting chambers differ in the design of the
counting net and the chamber depth. The counting net is

made up of a square net
division which is not visible until it is placed under a microscope (approx.
100 times magnification).

Neubauer-improved

Largest square size : 1 mm˛

Group square : 0,04 mm˛

Smallest square size : 0,025 mm˛

Depth of chamber is 0.100 mm. The net division of
these chambers has 3 times 3 large squares, each with an area of 1 mm˛

The four corner squares are used for leucocyte
counts.

The large square in the middle is also divided into five times five group
squares with an edge length of 0.2 mm each and an area of

0.04 mm^{2}
each. The group squares in turn are divided into sixteen very small squares
each with an area of 0.0025 mm^{2}.Five of these

group squares are used
for erythrocyte counts.

Special attention should be given to the fact that
the chamber has triple lines on all sides, of which the central line is to be
regarded as

the actual dimension line. This is important for deciding whether cells
in the border area are to be counted or not.

7.
Why are counting chambers still used in laboratories although there are
electrical counters?

• For smaller laboratories this equipment is too
expensive

• For special requirements e.g. research and non-routine examinations (counting
of liquor or effusion,

worm eggs, bacteria and fungus spores) counting chambers are required

• Counts are less accurate in case of small number of cells (e.g. liquor or few
thromocytes).

Possible
sources of error:

• counting chamber is not clean

• cover glass is not placed correctly onto the chamber

• chamber is not filled without bubbles

• chamber is overfilled

• not enough time for sedimentation of the cells

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