Hemocytometer
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The haemocytometer is a device originally used to count blood cells (as the name suggests). It is now used to count other cells and many types of microscopic particles. It consists of a thick glass microscope slide with a rectangular indentation that creates a chamber of certain dimensions. This chamber is etched with a 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 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 haemocytometer hold the coverslip 0.1 mm off the marked grid. This gives each square a well 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.
Often known as the 'Pope of Biology', the hemocytometer is often prayed to in order to receive good results. As it is such an important instrument for many biological experiments, breaking or misuse of a haemocytometer will result in bad luck in science for 8 years, the total number of quadrants on a haemocytometer.
[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 =
Haemocytometers 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.
[edit] Usage tricks
- Mix your original mixture thoroughly before taking a sample. This ensures that your sample is representative, and not just an artifact of the particular region of your original mixture from which you drew it.
- Use an appropriate dilution of your mixture with regard to the number of cells you hope to count. If your sample is not diluted enough, the cells will be too crowded and difficult to count. If it is too dilute, your sample size will not be enough to make strong inferences about the concentration in the original mixture. Naturally, you must have a rough idea of the concentration before you begin in order to guess an appropriate dilution. If your 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, you can compare the results. If they differ greatly, your method of taking the sample may be unreliable (e.g. the original mixture is not mixed thoroughly). You can use the average of your 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 you use the instrument.
- Do not use a paper wipe to dry the excess liquid. The same capillary action that filled the chamber will then dry it out.
- Watch out for the objective lens. Remember that the hemacytometer is thicker than a normal microscope slide. If you focus too closely, your objective lens may contact the instrument. This may affect your choice of which objective lens you use - carefully figure out what will fit, before you start.
- Count across the rows or down the columns. Use the gridlines to help you remember which areas' cells have already been counted.
- You don't have to count the whole chamber. If there are lot of cells, you can just perform your count in a section of the chamber and use the grid to determine what proportion of the chamber that is. You can then extrapolate to estimate how many cells are in the chamber, and use that figure in your final calculation. This gives you 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 smack 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 you count adjacent regions.
