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Using a Hemocytometer to Calculate Cell Size

I already illustrated how to calculate cell size (Determining Size in Microscopic Images). The method required you to take a picture of a ruler and then use this as a reference for cell size calculation. This system had several disadvantages: first, it only works for low magnifications (you have to be able to see 1mm of the ruler on the image), and was generally rather imprecise.

I would now like to show you a much better method of determining the size of microscopic structures. You do need a hemocytometer (counting chamber), however. These specialized slides are designed to determine the concentration of cells but they can also be used to determine size. The disadvantage is, that hemocytometers do cost quite a bit more than regular slides. There are different types of hemocytometers around, it does not matter which one you use, as long as you know the real-life size of the engraved squares.

In this case, we use the side length of one of the squares of the hemocytometer as a reference. These lines are very fine and therefore permit you to make very precise measurements and size calculations (in comparison to the picture of a ruler, see above link). The math is easy, but be careful that you use the same units.

  • Step 1: Take a picture of a square of the hemocytomer with associated cells. There are squares of different sizes, so make sure that you know the dimensions of the square that you are looking at. Read this post for more information on the different square sizes of the Neubauer improved haemocytomerter: The hemocytometer (counting chamber). Make sure that one complete side length of a square is visible.
  • Step 2: Print the micrograph. The square of the hemocytometer is out internal reference. You do not have to worry about the size of the print out. The larger the print out, the more precise the result, however.
  • Step 3: Measure the length of the side of one square and the diameter of a cell. Use the same units (generally mm is appropriate).
  • Step 4: Calculate the real-life size of a cell: You know the real-life side length of a square and the length of the square on the print out. You also know the diameter of the magnified cell. This data is enough for you to calculate the real-life size of the cell.
  • Step 5: Divide the length of a square of the print-out with the real-life side length. This gives you the magnification on paper. This magnification has nothing to do with the magnification of the objective and eye piece. We’re talking about magnification on the paper.
  • Step 6: Divide the size of the cell on paper with the magnification to obtain the real-life cell size. If you mix units, (cm, mm), then you won’t get the right result. You need to convert to the same units first. E.g. do not divide the square size in cm with the real life size in mm.

Things to watch out for:

  • Do be careful when observing cells that have a vastly different refractive index to the surrounding. In this case the cells will appear to have a thick “wall” around them, which is actually nothing more than a diffraction pattern. This may make obscure the true size of the cell. Open the condenser aperture diaphragm to minimize this artifact.
  • Counting chambers have squares of different sizes. Read the manual first so that you know the true size of the square that you are looking at.
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1 Comment

  • endoplasmicreticulum says:

    Just a tip: Photoshop (available as a 30 day trial) can record precise measurements in images. If you google “how to take measurements with photoshop” there are many good guides out there. If you use this method you don’t have to waste paper and ink to do the measurements. Also, if you buy a stage micrometer and have a camera attached to your microscope you can do these calculations and they will be the same as long as you don’t move the camera or change the lenses.

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