Learn About Counting Chambers

What is a counting chamber?

A counting chamber, or haemacytometer, is a medical device used in microscopy to count any type of cell. Each counting chamber has a grid that is etched on with extremely precise diamond etchers, capable of drawing lines less than a micrometer apart. The result is a grid the same or similar to the image. 

Counting Chambers allow for samples of cells to be examined when under a microscope. This is done by placing a piece of glass (cover slip) which is a standardised thickness over the sample, and pipetting the sample between the counting chamber and the coverslip; which ensures that the liquid is suspended in a constant depth. By doing this cell quantities can be analysed, %'s of cells to other types of cells can be identified, and (in the case of spermatozoa) qualitative analysis of motility and morphology can be assessed. 

How to use a counting chamber

Moisten the external supports of the counting chamber with distilled water and then place the cover glass upon the chamber by pushing with gentle pressure from the front of the counting chamber.

Caution: The cover glass is fragile! The formation of interference lines (Newton rings) between the outer webs and cover glass shows that the cover glass is placed correctly.

Take a well-mixed pipette from the shaker and discard the first drop. Wipe the outside of the pipette dry and tilt at an angle until a small droplet is formed at the pipette tip. This drop is then placed between the cover glass and the counting chamber.

Capillary action fills the gap between cover glass and the chamber base. Before the liquid reaches the edges of the chamber base, the tip should be pulled away again.

Cell counting assumes that a user possesses an exact knowledge of the limit lines of the counting chamber grids. In order to ensure that the cells lying on or near the limit lines are not double counted or ignored in the count, one must count using certain rules.

The figure illustrates all the counted cells defined within the measuring range of the one large square. Cells are counted as being in the large square if they are touching or resting over the left and upper dimension lines on completely within the enclosed smaller squares.

The cells that are valid to be counted are marked in black. One should count the cells within the large squares in the manner shown to the right.

The count starts at the upper left corner of the arrow and continues through the large squares in aback and forth fashion

Example of Use

Traditionally, Haemacytometers were made for assessing blood cells hence the name: Hemo (Blood) Cyto (Cell) Meter (Count). These days, most Haematology Labs use automated equipment and the primary utility of this medical device has changed. Nowadays, it is more common to see a counting chamber in active use at a fertility laboratory (testing sperm quantities), a biology field lab (analysing worm eggs), a microbiology lab (observing enumeration of bacteria and fungal spores), an undergraduate university module, or in a brewery (measuring yeast amounts).

Haematology

As mentioned, originally the counting chamber was used in medical laboratories in blood analysis. Users would differentiate between leucocytes, erythrocytes, & thrombocytes. 

By assessing quantities of different cells, haematologists can easily establish the likelihood of various illness (due to higher concentrations of white-blood-cells) or conditions arising from high or low red-blood-cells or platelets (blood clots or haemophilia). To a trained Haematologist, different combinations and quantities of blood cells, demonstrated in the image below, will paint a picture of their patients health.

Andrology

The World Health Organisation regulations on Andrology, stipulate to this day that a Neubauer Improved Counting Chamber should be used for many types of basic semen analysis. Due to significant challenges in digitally tracking cells as mobile as spermatozoa, manual cell counting (or a hybrid) is still preferable to automation. As well as assessing sheer volumes of sperm in a semen sample, morphology and motility can be observed.

Brewing

With the boom of the craft beer industry, many brewers like to monitor as many key elements of their brews as possible, which gives them increased control over the final flavour of their product. Counting chambers can assist in this goal, by allowing brewers to observe yeast quantities and %'s of live to dead yeast cells.

Understanding Different Types of Counting Chambers

Counting chamber have 4 changeable options; Chamber Depth, Chamber Grid, Number of Cell-Rulings, and Type of Etching.

Number of Cell-Rulings

Double chamber-central bar divided (two counting nets)

Single chamber–central bar un-divided (one counting net)

Type of Etching

Counting Chamber manufacturers can offer two types of etched grids, standard (or darkline) and metalised (bright-line) versions.

In the standard version, the counting grid is etched directly into the glass.

In the bright-line version, the chamber base is coated with a very thin metallic mirror. The counting grid is etched into the metalized surface. Using a phase-contrast microscope a colour conversion is possible, so that the counting grid can be seen as either dark or light.The bright-line design is generally preferred as it is more comfortable to work with.


There are other advantages with a metallised chamber. Filling is easier, as the blood cells are evenly distributed and clearly defined against the darker background. Microscope adjustments are less critical and the greater contrast between the lines, as well as the background, results in rapid identification of boundary line cells. The metallised area needs no special care beyond that given to an ordinary counting chamber.

Thickness of the metal film is kept within rigid limits assuring light transmission of the correct value. After coating the chamber is heated and annealed to harden the metal and bond it to the glass surface.

Chamber Grid

There are hundreds of grid options that have been designed for various purposes over the last century. Many have been trialled, but few have stood the test of time. When you look at our Counting Chamber range, you will notice that they are named after the creators of the etch design; Neubauers, Fuchs-Rosenthal, Burker, etc. Below are example of two grids that are commonly made at Hawksley.

The reason for such a range of choices is that different scientific disciplines will recommend a certain grid type, allowing for a standardisation of results domestically and globally. For example, the most recent World Health Organisation guidelines for Andrologists stipulated that a Neubauer Improved Counting Chamber is preferable over a Makler or Horwell counting chamber. Therefore, the Neubauer Improved is used by all UK andrologists.

Chamber Depth

The most obvious variable, chamber depth will vary the quantity of fluid in each sample. The depth of a counting chamber will vary and can be drilled to a depth of 10um, 20um, 50um and 3milimetres (in the case of McMaster Slides).

How a Haemacytometer is Made

All counting chambers follow the same construction principle. The actual manufacturing process includes several steps, between which are strict controls. The inner support (chamber base) and the two external supports are machined by grinding and polishing the surfaces glass.

We take a thick base plate made of a special optical glass which is the size of a microscope slide. To this glass, we cut four longitudinal grooves into the centre of the glass. The two major external surfaces are unfinished and used for labelling. The central bar and its two outer supports are ground and polished for over 48 hours. The purpose of this process is to bring the two outer support surfaces into the same level plane and to lower the inner central bar (the chamber base) relative to those outer supports by exactly the specified dimension.

After these operations the corresponding counting grid (system) is engraved using a diamond dividing machine. This bar is deeper in relation to the outer supports, typically by 0.1 or 0.2 mm.

When a flat-ground cover glass is placed onto the outer supports there is a gap of 0.1 or 0.2 mm thickness, respectively, between it and the chamber base. The imaginary vertically projected boundary lines of the counting grids form the lateral volume limit.

Lastly comes the printing and baking. All counting chambers must pass a rigorous final inspection, which is in accordance with the requirements of DIN or BS 748 standards and certification regulations. Only then is the distinction made between certified and certifiable counting chambers, with a portion being sent to the office of weights and measures and the others passing directly into sales.

NOTE: Counting chambers are precision measuring instruments, and therefore are made to stringent regulations. In Europe, counting chambers are made to the DIN standard; in the United Kingdom, the British Standard 748 is used.

Quality Control Requirements for all Chambers

The tolerances are:

- Chamber depth to ± 2% of the nominal value as per the grid requirement.

- For distances of less than 0.4 mm between any net lines ± 0.002 mm of the nominal value.

- For distances of 0.4 mm or more between any net lines ± 0.5% of the nominal value.

- Net division angle ± 1 °. The width of the lines shall not be greater than 0.005 mm. 

- The flatness tolerances for the chamber base in the counting net are 0.0002mm.

- For the cover slip contact surfaces in the region of a counting net: 0.002mm.

- For the cover slip surfaces 0,003 mm.The cover slips shall meet the requirements of the BS 748.