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 squares. These are subdivided in 3 directions; 0.25 x 0.25 mm, 0.25 x 0.20 mm
and 0.20 x 0.20 mm. The centralsquare is further subdivided into 0.05 x 0.05 mm squares.
The raised edges of the hemocytometer hold the coverslip 0.1 mm off the marked grid,
giving each square a defined volume. 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:
where the volume of the diluted sample is the volume of the sample after dilution and
the volume of the original mixture in the sample is the volume of the actual cell suspension
undiluted. For example, if the volume of the original mixture was 20μL and it was diluted
once, then the second term in parentheses is 40μL/20μL. The volume of the squares
counted is the one shown in the table at the top, depending on the size. The number of
cells counted is the sum of all cells counted across squares in one chamber. The proportion
of the cells counted applies if not all inner squares within a set square are counted.
For most applications, the four large corner squares are only used. The cells that are
on or touching the top and left lines are counted, but the ones on or touching the right
or bottom lines are ignored.
Requirements
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. 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, 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.
References
External links Online exercise - Yeast counts in Neubauer
improved or Thoma counting chambers Hemocytometer calculator online