Hemocytometer

A counting chamber, also called a hemocytometer, the light microscopic count of all kinds of small particles that are in a particle suspension serves. Counting can be used particularly for the quantification of cells (for example, erythrocytes, leucocytes ) and micro-organisms in medicine and biology.

• 3.1 Leukocyte Count
• 3.2 erythrocyte count

• 4.1 Neubauer counting chamber
• 4.2 Other Zählkammermodelle

Principle

A counting chamber is in principle a section of a virtual space which is bounded by two plane, at a small distance parallel arranged glass surfaces. The counting chamber is formed on a glass plate similar to the slides used for light microscopic studies, but thicker ( about 5 mm) and has a special design of the surface. A thin, just flat-ground and polished glass, the so-called cover glass is placed at a certain height parallel to the thicker, larger base plate. By tick marks on the surface of the base plate located between a base and the bottom of the cover slip chamber is selected, the volume of which is determined by the selected surface area and the distance between base and cover glass bottom ( chamber height). This space of known volume is the actual counting chamber. If you count microscopically after introduction of a suspension of particles in this chamber present in the marked volume particles results from their number and volume concentration of particles.

Technical details

The base plate of the counting chamber is made of special optical glass and has the size of a normal slide for transmitted light microscopy, namely: 76 mm x 26 mm (according to DIN ISO 8037-1 ), but is about 5 mm thick. Through parallel to the narrow edge milled grooves, the surface of the base plate in two broad fields (outside) and three narrow bars (inside) is shared. In contrast to the two external fields that serve the label that supports are ground and polished. The central web ( bottom chamber ) is the amount of the height of the counting chamber is lower than the two side webs thereof lying. These two ridges form the rest of the cover glass ( girder webs ). Is placed on the cover glass, these two webs, with its bottom surface is located in the intended height ( chamber depth ) on the surface of the central web, the base of the chamber ( the chamber floor ). In this base the markings are engraved on the delimitation of the intended volume. The intermediate space between the base surface ( bottom chamber ) and the lower surface of the cover glass and the graduation markings on the bottom surface delimiting the volume of the counting chamber.

The chamber floor of the central web is 0.1 mm lower than the two outer webs ( chamber depth) in the rule. For very small particles, such as bacteria, the chamber depth is only 0.02 mm. The lateral limitation of the auszuzählenden volume is formed by the imaginary planes in a perpendicular projection onto the boundary lines of the counting grids. Coverslips for counting chambers differ from coverslips for normal light microscopy by the mirror-finish polished surfaces. Depending on the counting chamber type whose size (L x W) 24 mm x 24 mm, 20 mm x 26 mm or 22 mm x 30 mm with a thickness of 0.4 mm. Since the counting chambers are often used for determining the concentration of blood cells, they are also referred to as hemocytometer and the cover glasses as hemocytometer cover glass.

In the center of the base of the counting chamber is a different depending on chamber type line network ( counting area, counting grid ). In general, two counting grids are engraved, which are separated by a groove.

Preparation of the chamber

The chamber should be as free of dust, lint, and cells before use. For correct placement of the cover glass you push it with some pressure (Caution, risk of breakage! ) In landscape mode on the two support struts. If the cover glass is seated correctly on the girder webs, so-called Newtonian interference colors are seen. That is, the height of the gap between the support webs and the cover glass is in the range of wavelengths of light, and is therefore negligible. In this condition, the cover glass does not move when tilting the counting chamber.

Loading of the chamber

The particle suspension is pipetted auszuzählende side at under a cover glass and drawn by capillary action into the gap. The particle suspension is spread in a layer of accurately known thickness. After counting the particles lying on the counting fields under a light microscope with transmitted light their number can be calculated per unit volume. Wherein the cells can be used to increase visibility in addition to their staining and phase contrast microscopy.

Cleaning

After use, you should carefully remove chamber and cover glass with a lint-free disposable cloth of the particle suspension and cleaned with 70 % 2-propanol or a similar disinfectant and dry. Here, above all, the cover glass is treated with caution, since this ground flat and therefore much more expensive than a normal cover glass.

Counting

Depending on the type of cells auszuzählenden a certain number is counted bulk or group squares, and an average is. Multiplying this value by an appropriate factor ( inverse of the product of the square surface and chamber height ), we obtain the number of cells per unit volume. Important when counting is that you can not double count lying on boundary lines cells. For this purpose, it is usual to be counted in the counting of a square, only the cells on two boundary lines (for example, up and left) and are not lying on the other two lines. Before counting, it is advisable to look at the entire line grid with a low magnification and to examine whether the cells are distributed fairly evenly over the squares. Otherwise, the particle suspension should be shaken again and re- applied. A ungleichmäße distribution can also be seen in mind that in the counting of several squares strongly fluctuating numbers of particles per square result.

With such a small area cell density as in the example on the right, very many squares must be counted in order to get a usable result. At high cell concentrations (eg yeast suspensions ) a countability must be made via serial dilution. The aim should be about 1 cell per small square.

Leukocyte count

For this purpose, 4 large corner squares are counted and dividing this number by 4 to get an average per corner square. This value is multiplied by 10, and thus we obtain the number of cells per ul ( microliters). The factor 10 arises from the fact that each corner square has an area of ​​1 mm ² and the chamber height is 0.1 mm, so a corner square a volume of 0.1 ul (1 mm ² x 0.1 mm = 0.1 mm ³) corresponds. The number of cells per square you have to share or simply multiply by the reciprocal of 10 now by the volume of 0.1 ul. An appropriate dilution, the one before applying the cells to the chamber (inevitably ) has made, of course, must also be taken into account.

Erythrocyte count

To determine the number of small cells such as erythrocytes or CHO cells in cell cultures, four times 5 group squares are counted and calculated as the mean value for 5 group squares. This number by 50 gives the number of cells per microliter (1 / (5 × 0.04 mm ² x 0.1 mm) = 50). Again, it should be a possibly previously made ​​thinner.

Zählkammertypen

The Zählkammertypen essentially differ by the type of Zählgitters

Neubauer counting chamber

The counting grid is at the Neubauer counting chamber from 3 x 3 large squares of 1 mm edge length and thus an area of 1 mm ². The central large square is subdivided 5 smaller group squares, each with 0.2 mm edge length in the Improved Neubauer counting chamber in 5 x. The surface of such a group is so square 0.04 mm ². A special feature of the Improved Neubauer 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 ( Thoma counting chamber corresponds, see below), the average large square into 4 x 4 squares group of also 0.2 mm edge length is divided. These are separated by triple boundary lines, of which only the first left and right is to be regarded as the actual limit. In addition, the triple lines are only found on 2 pages.

Furthermore, each group square is divided into 4 x 4 smaller squares each 0.05 mm edge length with each 0.0025 mm ².

Other Zählkammermodelle

• Improved Neubauer: double ruling. The ruling shows 9 large squares of 1 mm ². The four large squares in the corners are divided into 16 squares with side length of 0.25mm. They are used for counting leucocytes. The large square in the center is divided into 25 squares each with a side length of 0.2mm. Each group square into 16 smaller squares, each with side length of 0.05 mm and an area of ​​0.0025 mm ². The ( from top left) diagonal 5 group squares are used for platelet and red cell count. Deserves special attention that all group squares have sides triple boundary lines. The middle line is the boundary line and decides whether cells in the boundary region are counted or not.
• Improved Neubauer, bright-lined: double ruling. The same counting grid as Neubauer improved but chamber floor mirror with rhodium. Rulings are engraved on the rhodium and appear bright under normal microscope settings By contrast it is under the microscope, a color reversal possible, so that the rulings appear brighter or darker depending on the requirement *
• Thoma: double ruling. The power division corresponds to the large square located in the center of the Neubauer chamber. The area of ​​the small square is 0.0025 mm ². Since the outer large squares are not, the Thoma - chamber system is only used to count of thrombocytes and erythrocytes.
• Thoma ( new): (equals Neubauer improved without running corner squares and with other boundary representation).
• Door ( corresponding 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 version
• Bürker: double ruling, the ruling shows 9 large squares of 1 mm ². They are used for counting leucocytes. Each square is divided by double lines ( in 0.05 mm pitch) in 16 squares each with a side length of 0.2mm. The group squares correspond in size to those of the Neubauer counting chambers, but without further subdivision. They are used for the platelet and red cell count. The double lines, least squares result with an area of ​​0.0025 mm ².
• Bürker door: double ruling ( combination of systems Bürker and Thoma ). The ruling shows 9 large squares of 1 mm ². Each square is divided into 16 squares each with a side length of 0.2mm. In the central square of each major group square is divided into 16 smaller squares each with side length 0.05 mm ( = 0.0025 mm ²).
• Fuchs -Rosenthal: double ruling. The ruling differs from the chamber systems as they are customary for the blood cell count, with its large surface area of 16 mm ². The ruling shows 16 large squares of 1 mm ². Each square is divided into 16 smaller squares, each with side length of 0.25mm and an area of ​​0.0625 mm ². These counting chambers are commonly used, including for counting cells in cerebrospinal fluid ( spinal fluid ).
• Jessen in Standard and Bright Lined version.
• Lemaur in Standard and Bright Lined version ..
• Malassez: double ruling. The ruling is rectangular and covered 5 mm ². The large rectangles have an area of ​​0.25 mm x 0.20 mm = 0.05 mm ². They are each divided into 20 small squares with an area of ​​0.0025 per mm ². These counting chambers, inter alia, used for the cell count in the cerebrospinal fluid ( spinal fluid ) or for counting nematodes.
• McMaster with 3 fields, size approx 127 mm x 26 mm, depth 1.5 mm, for counting worm eggs.
• Nageotte: double ruling. The chamber depth is 0.5 mm. The square area of ​​100 mm ² is divided into 40 rectangles with an area of 0.25 mm x 10 mm = 2.5 mm ². These counting chambers, inter alia, used for counting cells in cerebrospinal fluid ( spinal fluid ) or for counting nematodes.
• Petroff - Hausser with special depth ( Petroff ) for counting of bacteria, platelets, sperm, etc., dark line, for use in dark field microscopes. The depth of the counting compartment is 0.2 mm with a thickness of 1.5 mm.
• Schilling: Cross network of 3 x 3 large squares, divided into 4 x 4 mini squares with an area of ​​each 0.0025 mm ² and 4 rectangles with an area of ​​0.01 mm ² and unit mesh of 9 large squares, which are the central square of the crossover network is equal.