Counting chamber
A counting chamber , also known as a hemocytometer , is used for the light microscopic counting of all types of small particles that are in a particle suspension. Counting chambers are used in particular for the quantification of cells ( e.g. erythrocytes , leukocytes ) and microorganisms in medicine and biology .
principle
In principle, a counting chamber is a virtual section of a space that is delimited by two flat glass surfaces that are arranged in parallel at a short distance. The counting chamber is formed on a glass plate similar to the microscope slides used for examinations . However, this is thicker (about 5 mm) and has a special surface design. A thin, precisely ground and polished pane of glass, the so-called cover glass , is arranged parallel to a certain height above the thicker, larger base plate. Line markings on the surface of the base plate mark a space located between the base area and the underside of the cover glass, the volume of which is determined by the marked base area and the distance between the base area and the underside of the cover glass ( chamber height ). This space of known volume is the actual counting chamber. If one counts the particles present in the marked volume microscopically after introducing a particle suspension into this chamber, the concentration of the particles results from their number and the volume.
Technical details
The base plate of the counting chamber is made of special optical glass and is the size of a normal slide for transmitted light microscopy, namely: 76 mm × 26 mm (according to DIN ISO 8037-1), but is about 5 mm thick. With grooves milled parallel to the narrow edges, the surface of the base plate is divided into 2 wide fields (outside) and 3 narrow bars (inside). In contrast to the two outer fields, which are used for lettering, the bars are ground flat and polished. The middle bar ( chamber bottom ) is lower than the two bars on the side by the amount of the height of the counting chamber. These two bars form the support of the cover glass ( carrier bars ). If the cover slip is placed on these two bars, its lower surface is at the intended height ( chamber depth ) above the surface of the middle bar , the base of the chamber (chamber floor). The markings to delimit the intended volume are engraved in the base area. The space between the base (chamber floor) and the lower surface of the cover glass and the line markings on the base define the volume of the counting chamber.
The chamber floor of the middle bar is usually 0.1 mm lower than the two outer bars (chamber depth). For very small particles, such as bacteria, the chamber depth is only 0.02 mm. In the middle of the base area of the counting chamber there is a line network ( counting area , counting network ) that varies depending on the type of chamber . As a rule, two counting nets are engraved, separated from each other by a groove. The lateral boundary of the volume to be counted is formed by the imaginary planes in a vertical projection onto the boundary lines of the counting networks. The cover glasses for counting chambers differ from cover glasses for normal light microscopy in that they are ground and polished. They are also a little thicker than normal cover slips so that they are not bent by capillary forces. Depending on the type of counting chamber, their size (L × W) is 24 mm × 24 mm, 20 mm × 26 mm or 22 mm × 30 mm with a thickness of 0.4 mm each.
Because the counting chambers are often used to determine the concentration of blood cells , they are also known as hemocytometers and the coverslips are called hemocytometer coverslips .
Preparing the Chamber
The chamber should be as free of dust, lint and cells as possible before use. To put the cover glass on correctly, push it with a little pressure (caution, risk of breakage!) In landscape format onto the two support bars. If the cover slip is correctly seated on the support bars, so-called Newtonian interference colors can be seen. This means that the height of the space between the carrier webs and the cover glass is in the order of magnitude of the light wavelengths , so it is negligible. In this state, the cover glass does not slip when the counting chamber is tilted.
Loading the chamber
The particle suspension to be counted is pipetted onto the side with the cover slip in place and is sucked into the gap by capillary force. The particle suspension is spread out in a layer with a precisely known thickness. After counting the particles lying on the counting fields under the light microscope with transmitted light, their number per volume unit can be calculated. In the case of cells, phase contrast microscopy can also be used in addition to their staining for better recognition .
cleaning
After use, the chamber and cover glass should be carefully freed from the particle suspension with a lint-free disposable cloth, cleaned with 70% 2-propanol or a similar disinfectant and dried. The cover slip, in particular, should be treated with care, as it is ground flat and therefore a lot more expensive than a normal cover slip.
Counting method
Depending on the type of particle to be counted, a certain number of large or group squares is counted and an average is calculated from this. If you multiply this value by a corresponding factor (reciprocal of the product of the square area and the chamber height), you get the number of particles per unit volume. When counting, it is important that particles lying on borderlines are not counted twice. When counting a square, it is customary to only count the particles on two boundary lines (e.g. top and left) and not count those on the other two lines. Before counting, it is advisable to look at the entire grid of lines at a low magnification and to check whether the particles are reasonably evenly distributed over the squares. Otherwise the particle suspension should be shaken up again and reapplied. An uneven distribution can also be recognized by the fact that counting several squares results in strongly fluctuating particle numbers per square.
With such a low surface density of the particles as in the example on the right, a large number of squares have to be counted in order to get a usable result. In the case of high particle concentrations (e.g. yeast suspensions), countability must be established by dilution. The aim is to have about 1 particle per small square.
Leukocyte count
For this purpose, 4 large corner squares are counted and this number is divided by 4 to get an average value per corner square. This value is multiplied by 10 and this gives the number of cells per µl (microliter) . The factor 10 results from the fact that each corner square has an area of 1 mm² and the chamber height is 0.1 mm, i.e. a corner square corresponds to a volume of 0.1 µl (1 mm² × 0.1 mm = 0.1 mm³) . So the number of cells per corner square, i.e. 0.1 mm³ each, is multiplied by 10 the number of cells per mm³. A corresponding dilution, which was (inevitably) made before applying the cell suspension to the chamber, must of course also be taken into account.
Erythrocyte count
To increase the number of smaller cells such as B. to determine erythrocytes or CHO cells in cell cultures , four times 5 group squares are counted and the mean value for 5 group squares is determined. This number multiplied by 50 gives the number of cells per µl (1 / (5 × 0.04 mm² × 0.1 mm) = 50). Any previously made dilution must also be taken into account here.
Counting chamber types
The counting chamber types differ essentially in the type of counting grid
Counting chamber according to Neubauer
The counting grid in the Neubauer counting chamber consists of 3 × 3 large squares with an edge length of 1 mm each and thus an area of 1 mm² each . In the improved Neubauer counting chamber, the central large square is divided into 5 × 5 smaller group squares, each with an edge length of 0.2 mm. The area of such a group square is 0.04 mm². A special feature of the Neubauer improved are the triple boundary lines of the group squares, in which the middle line represents the actual boundary between two fields.
In the old Neubauer counting chamber (corresponds to the Thoma counting chamber, see below), the central large square is divided into 4 × 4 group squares with an edge length also 0.2 mm. These are also separated by threefold border lines, of which only the left and right are to be regarded as the actual delimitation. In addition, the triple lines can only be found on 2 sides.
| Difference Middle Square Neubauer old and improved | |
|---|---|
| (Two group squares are marked with yellow or gray) | |
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| Neubauer improved | Neubauer and Thoma counting chambers |
Furthermore, each group square is divided into 4 × 4 small squares with an edge length of 0.05 mm each and an area of 0.0025 mm² each.
Other counting chamber models
- Improved Neubauer : double network division. The counting grid shows 9 large squares of 1 mm² each. The 4 large squares in the corners are divided into 16 squares each with a side length of 0.25 mm. They are used for leukocyte counts. The large square in the middle is divided into 25 group squares with each 0.2 mm side length. Each group square consists of 16 small squares with a side length of 0.05 mm each and an area of 0.0025 mm². The 5 diagonal group squares (from top left) are used for counting platelets and erythrocytes. It should be noted that all group squares have triple border lines on all sides. The middle line is the boundary line and decides whether cells in the border area are to be counted or not.
- Improved Neubauer, light-lined : double grid division . Same counting network as improved Neubauer, but the chamber bottom is mirrored with rhodium. The counting grid is engraved in the rhodium layer and appears light with normal microscope settings. By shifting the contrast, a color reversal is possible under the microscope, so that the counting grid appears light or dark as required *
- Thoma : double network division. The grid division corresponds to the large square in the middle of the Neubauer Chamber. The area of the smallest squares is 0.0025 mm² each. Since the outer large squares are not executed, the Thoma chamber system is only used to count platelets and erythrocytes.
- Thoma (new) : (corresponds to Neubauer improved without implemented corner squares and with a different representation of the boundaries).
- Türk (corresponds to the old Neubauer or Thoma chamber with the difference that the corner squares are divided with double lines)
- Agasse-Lafont in standard and bright-lined versions
- Bürker : double grid division, the counting grid shows 9 large squares of 1 mm² each. They are used for leukocyte counts. Each large square is divided by double lines (0.05 mm apart) into 16 group squares, each 0.2 mm on a side. The size of the group squares correspond to those of the Neubauer counting chambers, but without any further subdivision. They are used for counting platelets and red blood cells. The double lines result in tiny squares with an area of 0.0025 mm².
- Bürker-Türk : double network division (combination of the Bürker and Thoma systems). The counting grid shows 9 large squares of 1 mm² each. Each large square is divided into 16 group squares with a side length of 0.2 mm each. In the middle large square, each group square is divided into 16 small squares, each with a side length of 0.05 mm (= 0.0025 mm²).
- Fuchs-Rosenthal : double mesh division. The counting network differs from the chamber systems used for counting blood cells in that it has a large surface area of 16 mm². The counting grid shows 16 large squares of 1 mm² each. Each large square is divided into 16 small squares, each with a side length of 0.25 mm and an area of 0.0625 mm². This counting chamber is used very often, u. a. for the cell count in the CSF (lumbar fluid).
- Jessen in standard and bright-lined versions.
- Lemaur in standard and bright-lined versions.
- Malassez : double network division. The counting grid is rectangular and covers 5 mm². The large rectangles have an area of 0.25 mm × 0.20 mm = 0.05 mm². They are each divided into 20 tiny squares with an area of 0.0025 mm² each. This counting chamber is u. a. Used for counting cells in CSF (lumbar fluid) or for counting nematodes.
- McMaster with 3 fields, size approx. 127 mm × 26 mm, depth approx. 1.5 mm, for counting worm eggs.
- Nageotte : double mesh division. The chamber depth is 0.5 mm. The square base area of 100 mm² is divided into 40 rectangles with an area of 0.25 mm × 10 mm = 2.5 mm² each. This counting chamber is u. a. Used for counting cells in CSF (lumbar fluid) or for counting nematodes.
- Petroff-Hausser with special depth (Petroff) for counting bacteria , blood platelets, sperm, etc., dark-lined, for use in dark-field microscopes . The depth of the counting chamber is 0.02 mm with a thickness of 1.5 mm.
- Schilling : Cross network of 3 × 3 large squares, divided into 4 × 4 small squares with an area of 0.0025 mm² each and in 4 rectangles with an area of 0.01 mm² and a unit network of 9 large squares, which are equal to the middle square of the cross network .
Individual evidence
- ↑ Cell Counting with a Hemocytometer . Retrieved July 15, 2012.