Hemocytometer
Encyclopedia
Cell counting
Cell counting is a general name for various methods for the quantification of cells in molecular biology and in medicine.- The need for cell counting :...
of blood cell
Blood cell
A blood cell, also called a hematocyte, is a cell normally found in blood. In mammals, these fall into three general categories:* red blood cells — Erythrocytes* white blood cells — Leukocytes* platelets — Thrombocytes...
s. It is now also used to count other types of cells
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
as well as other microscopic particles.
The hemocytometer was invented by Louis-Charles Malassez
Louis-Charles Malassez
Louis-Charles Malassez was a French anatomist and histologist born in Nevers, department of Nièvre.He studied medicine in Paris, where he worked as an interne from 1867...
and consists of a thick glass
Glass
Glass is an amorphous solid material. Glasses are typically brittle and optically transparent.The most familiar type of glass, used for centuries in windows and drinking vessels, is soda-lime glass, composed of about 75% silica plus Na2O, CaO, and several minor additives...
microscope slide
Microscope slide
A microscope slide is a thin flat piece of glass, typically 75 by 25 mm and about 1 mm thick, used to hold objects for examination under a microscope. Typically the object is placed or secured on the slide, and then both are inserted together in the microscope for viewing...
with a rectangular indentation that creates a chamber. This chamber is engraved with a laser-etched grid
Grid (spatial index)
In the context of a spatial index, a grid is a regular tessellation of a manifold or 2-D surface that divides it into a series of contiguous cells, which can then be assigned unique identifiers and used for spatial indexing purposes...
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 ruled area of the hemocytometer consists of several, large, 1 x 1 mm (1 mm2) squares. These are subdivided in 3 ways; 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 central, 0.20 x 0.20 mm marked, 1 x 1 mm square 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. This gives each square a defined volume.| 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 |
The cell-sized structures counted lie between the middle of the three lines on the top and right of the square and the inner of the three lines on the bottom and left of the square.
In an improved Neubauer hemocytometer (common medium), the total number of cells per ml can be discovered by simply multiplying the total number of cells found in the hemocytometer grid (area equal to the red square in picture on right) by 104 (10000).
Usage
Ensure that the special coverslip provided with the counting chamber (thicker than standard coverslips and with a certified flattness) is properly positioned on the surface of the counting chamber. When the two glass surfaces are in proper contact Newton's ringsNewton's rings
The phenomenon of Newton's rings, named after Isaac Newton who first studied them in 1717, is an interference pattern caused by the reflection of light between two surfaces - a spherical surface and an adjacent flat surface...
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
Capillary action
Capillary action, or capilarity, is the ability of a liquid to flow against gravity where liquid spontanously rise in a narrow space such as between the hair of a paint-brush, in a thin tube, or in porous material such as paper or in some non-porous material such as liquified carbon fiber, or in a...
which completely fills the chamber with the sample. Looking at the chamber through a microscope
Microscope
A microscope is an instrument used to see objects that are too small for the naked eye. The science of investigating small objects using such an instrument is called microscopy...
, the number of cells in the chamber can be determined by counting. Different kinds of cells can be counted separately as long as they are visually distinguishable. The number of cells in the chamber is used to calculate the concentration
Concentration
In chemistry, concentration is defined as the abundance of a constituent divided by the total volume of a mixture. Four types can be distinguished: mass concentration, molar concentration, number concentration, and volume concentration...
or density of the cells in the mixture
Mixture
In chemistry, a mixture is a material system made up by two or more different substances which are mixed together but are not combined chemically...
the sample comes from. It is the number of cells in the chamber divided by the chamber's volume (the chamber's volume is known from the start), taking account of any dilutions and counting shortcuts:
In the most common design, the volume of each large square (made up from 4x4 green squares in the picture) is 0.1 mm3. The cells in four large 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 squares/4 × 10,000.
Hemocytometers are often used to count blood
Blood
Blood is a specialized bodily fluid in animals that delivers necessary substances such as nutrients and oxygen to the cells and transports metabolic waste products away from those same cells....
corpuscles, organelles within cells, blood cells in cerebrospinal fluid
Cerebrospinal fluid
Cerebrospinal fluid , Liquor cerebrospinalis, is a clear, colorless, bodily fluid, that occupies the subarachnoid space and the ventricular system around and inside the brain and spinal cord...
after performing a lumbar puncture
Lumbar puncture
A lumbar puncture is a diagnostic and at times therapeutic procedure that is performed in order to collect a sample of cerebrospinal fluid for biochemical, microbiological, and cytological analysis, or very rarely as a treatment to relieve increased intracranial pressure.-Indications:The...
, or other cell types in suspension. Anchorage-dependent cells can also be counted if subjected to trypsinization
Trypsinization
Trypsinization is the process of using trypsin, a proteolytic enzyme which breaks down proteins, to dissociate adherent cells from the vessel in which they are being cultured. When added to a cell culture, trypsin breaks down the proteins which enable the cells to adhere to the vessel...
prior to counting. Using a special hemocytometer with a depth of 0.02mm smaller particles such as sperm, yeast or bacteria can be counted. Using the ruling described above the volumes are only 1/5 compared to the standard 0.1mm deep chamber. As it is difficult to distinguish between living and dead organisms unless particular stains are used to distinguish viable from non-viable cells this results in a 'total count' of the bacteria. In case of bigger cells the number of dead (permeabilised) cells in the sample can be obtained by adding Trypan blue
Trypan blue
Trypan blue is a vital stain used to selectively colour dead tissues or cells blue. It is a diazo dye.Live cells or tissues with intact cell membranes are not coloured. Since cells are very selective in the compounds that pass through the membrane, in a viable cell trypan blue is not absorbed;...
. Fluorescent dyes give better discrimination particularly when looking at bacteria.
A common mistake is to add the sample to the counting chamber before adding the cover glass. This risks that the cells could sediment / stick to the glass or some volume to evaporate before the coverslip is placed on top resulting in an overestimation of the cell concentration. Sedimentation is less of a problem with bacteria but evaporation has to be kept to a minimum.
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 dilutionConcentrationIn chemistry, concentration is defined as the abundance of a constituent divided by the total volume of a mixture. Four types can be distinguished: mass concentration, molar concentration, number concentration, and volume concentration...
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 inferenceInferenceInference is the act or process of deriving logical conclusions from premises known or assumed to be true. The conclusion drawn is also called an idiomatic. The laws of valid inference are studied in the field of logic.Human inference Inference is the act or process of deriving logical conclusions...
s 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).