Hemocytometer

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A hemocytometer. The two semi-reflective rectangles are the counting chambers.
Load a chamber
The parts of the hemocytometer (as viewed from the side) are identified.
Hemocytometer grid:
red square = 1 mm2, 100 nl
green square = 0.0625 mm2, 6.25 nl
yellow square = 0.04 mm2, 4 nl
blue square = 0.0025 mm2, 0.25 nl
at a depth of 0.1 mm.

The hemocytometer is a device used to count cells. It was originally designed for the counting of blood cells.

The hemocytometer was invented by Louis-Charles Malassez and consists of a thick glass microscope slide with a rectangular indentation that creates a chamber. This chamber is engraved with a laser-etched grid of perpendicular lines. The device is carefully crafted so that the area bounded by the lines is known, and the depth of the chamber is also known. It is therefore possible to count the number of cells or particles in a specific volume of fluid, and thereby calculate the concentration of cells in the fluid overall.

Principles

The gridded area of the hemocytometer consists of several 1 x 1 mm (1 mm2) squares. These are subdivided in 3 directions; 0.25 x 0.25 mm (0.0625 mm2), 0.25 x 0.20 mm (0.05 mm2) and 0.20 x 0.20 mm (0.04 mm2). The centralsquare is further subdivided into 0.05 x 0.05 mm (0.0025 mm2) squares. The raised edges of the hemocytometer hold the coverslip 0.1 mm off the marked grid, giving each square a defined volume.[1]

Dimensions Area Volume at 0.1 mm depth
1 x 1 mm 1 mm2 100 nl
0.25 x 0.25 mm 0.0625 mm2 6.25 nl
0.25 x 0.20 mm 0.05 mm2 5 nl
0.20 x 0.20 mm 0.04 mm2 4 nl
0.05 x 0.05 mm 0.0025 mm2 0.25 nl

Usage

To use the hemocytometer, first make sure that the special coverslip provided with the counting chamber is properly positioned on the surface of the counting chamber. When the two glass surfaces are in proper contact Newton's rings can be observed. If so, the cell suspension is applied to the edge of the coverslip to be sucked into the void by capillary action which completely fills the chamber with the sample. The number of cells in the chamber can be determined by direct counting using a microscope, and visually distinguishable cells can be differentially counted. The number of cells in the chamber is used to calculate the concentration or density of the cells in the mixture the sample comes from. It is the number of cells in the chamber divided by the chamber's volume, which is known from the start, taking account of any dilutions and counting shortcuts: {\mbox{concentration of cells in original mixture}}=\left({\frac {{\mbox{number of cells counted}}}{({\mbox{proportion of chamber counted}})({\mbox{volume of chamber}})}}\right)\left({\frac {{\mbox{volume of sample dilution}}}{{\mbox{volume of original mixture in sample}}}}\right) [2]

In the most common design, the volume of each large square is 0.1 mm3. The cells in four large (red in figure above) squares are counted and cells over or touching the lines on top and on the left are counted, but cells over or touching the right or bottom lines are ignored. The concentration in cells per ml = cells in four red, large squares/4 × 10,000.[3]

Requirements

Empty hemocytometer grid at 100x power.
  • The original suspension must be mixed thoroughly before taking a sample. This ensures the sample is representative, and not just an artifact of the particular region of the original mixture it was drawn from.
  • An appropriate dilution of the mixture with regard to the number of cells to be counted should be used. If the sample is not diluted enough, the cells will be too crowded and difficult to count. If it is too dilute, the sample size will not be enough to make strong inferences about the concentration in the original mixture.
  • By performing a redundant test on a second chamber, the results can be compared. If they differ greatly, the method of taking the sample may be unreliable (e.g., the original mixture is not mixed thoroughly).

Applications

  • Blood counts: for patients with abnormal blood cells, where automated counters don't perform well.
  • Sperm counts
  • Cell culture: when subculturing or recording cell growth over time.
  • Beer brewing: for the preparation of the yeast.
  • Cell processing for downstream analysis: accurate cell numbers are needed in many tests (PCR, flow cytometry), while some others require high cell viability.
  • Measurement of cell size: in a micrograph, the real cell size can be inferred by scaling it to the width of a hemocytometer square, which is known.[4]

References

  1. ↑ "Hemocytometer: square sizes". 
  2. ↑ "Basic Hemocytometer usage". 
  3. ↑ Strober W (2001). "Monitoring cell growth". In Coligan JE, Bierer BE, Margulies DH, Sherach EM, Strober W. Current Protocols in Immunology 5. USA: John Wiley & Sons. p. A.2A.1. doi:10.1002/0471142735.ima03as21. 
  4. ↑ "Measuring cell size with a hemocytometer". 

External links

  • Online exercise - Yeast counts in Neubauer improved or Thoma counting chambers
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