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Viewing Unstained Living Preparations

10/17/16


Viewing Unstained Living Preparations

There are several problems associated with microscopy of living preparations such as cell suspensions protozoa, or living bacteria. First, the cells aren't stained, so you can't see them well in bright field mode. Second, they are often quite small. The combination of factors makes it difficult to find them in the focal plane. Third, they may be moving. Fourth, the suspension is likely to dry up from the heat of the lamp before you have a chance to do much observing.

Preparing a Vaseline chamber easily solves the fourth problem. Simply take a single cover slip and hold it between thumb and forefinger by the edges. Pick up some Vaseline on the other forefinger and rub it over your thumb to make a film Scrape your thumb carefully on each edge of the coverslip to make a continuous Vaseline ledge. Place a drop or two of suspension on a clean slide, and turn the coverslip over on top of the drop. Press down the edges to seal the chamber against evaporation.

When preparing a Vaseline mount, keep in mind that the image becomes degraded with thicker mounts, especially at high powers in dark field or phase contrast. Unless the specimen is large and fragile enough to be damaged by pressing down too hard on the coverslip, keep the chamber depth very shallow.

The hardest part is finding the correct focal plane. Since the cells are unstained and usually unpigmented, the most convenient mode is dark field. Every speck of dust, and of course every cell, will show up if it is in focus. Movement can be readily detected, so that you can actually take advantage of having moving organisms. With the lowest power, in dark field mode, focus on the edge of an air bubble or the Vaseline layer, to reach the focal plane between top of slide and bottom of coverslip. You should see some Brownian motion or other evidence that you are looking at a liquid layer.

Staying in dark field mode, move to 1OOx final magnification. Even the smallest protozoa should be evident at this magnification, and bacteria can be distinguished by their movement. Focus on something that resembles a specimen of interest. It takes practice to recognize a small target at low power, so be patient.

Center the target, switch to the high dry objective, and use either dark field or phase contrast modes to observe. You may even try bright field, adjusting the aperture diaphragm to provide adequate contrast.


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