Hemocytometer
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The hemocytometer (British spelling haemocytometer) is a device which was originally used to count blood cells. It is now used to count other cells and many types of microscopic particles as well. It 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. Therefore it is 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.
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[edit] Principles
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 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 0.1mm 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 to be counted are those which 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 haemocytometer (common medium) the total number of cells per ml can be discovered by simply multiplying the total number of cells found in the haemocytometer grid by 4000.
[edit] Usage
When a liquid sample containing immobilised cells is placed on the chamber, it is covered with a cover glass, and capillary action completely fills the chamber with the sample. Looking at the chamber through a microscope, 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 or density of the cells in the mixture from which the sample was taken: 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:
concentration of cells in original mixture =
Hemocytometers are often used to count blood corpuscles, organelles within cells, blood cells in cerebrospinal fluid after performing a lumbar puncture, or other cell types in suspension. Using a haemocytometer to count bacteria results in a 'total count' as it is difficult to distinguish between living and dead organisms unless methylene blue is used to stain the non-respiring cells.
[edit] Usage Tips
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- Mix the original mixture thoroughly before taking a sample. This ensures that the sample is representative, and not just an artifact of the particular region of the original mixture from which it was drawn.
- Use an appropriate dilution of the mixture with regard to the number of cells to be counted. 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. Naturally, a rough idea of the concentration must be known before beginning in order to guess an appropriate dilution. If the mixture is colored, it may be helpful to memorize a particular intensity of that color at which the mixture tends to be easy to analyze.
- Analyze multiple chambers. 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). Take the average of the results for a more accurate calculation.
- Make sure to put enough liquid on the instrument that some leaks out of the cover glass when it is placed over the chamber. Otherwise, it is uncertain whether the space under the cover glass is completely filled with liquid. This volume should be the same every time the instrument is used.
- Do not use a paper wipe to dry the excess liquid. The same capillary action that filled the chamber will then dry it out. If using trypan blue, rinse the hemocytometer with distilled water to remove the dye and allow it to dry. Methylated spirits or alcohol (pref ethanol) may also be used to cleanse the haemocytometer, and it is safe to use lens tissue to wipe the excess away, as long as great care is taken not to warp the haemocytometer grid. For this resason, it is not safe to autoclave (sterilize) a haemocytometer however sterilization should not be essential.
- Watch out for the objective lens. Remember that the hemocytometer is thicker than a normal microscope slide. If focussed too closely, the objective lens may contact the instrument. This may affect the choice of objective lens used.
- Count across the rows or down the columns. Use the gridlines to help remember which areas' cells have already been counted.
- There is no need to count the whole chamber. If there are lot of cells, perform a count in a section of the chamber and use the grid to determine what proportion of the chamber that is. Then extrapolate to estimate how many cells are in the chamber, and use that figure in the final calculation. This gives speed at the expense of potential accuracy; if possible, using a more appropriate dilution is better.
- Are the lines in or out? Some cells inevitably fall on top of the outside gridlines that mark the edges of the chamber. The usual practice is to include cells overlapping the top and left lines, but not those overlapping the bottom or right lines - this has the advantage of eliminating redundant counting if adjacent regions are counted.
