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UCL Home  /  Geography  /  Resources  /  Laboratory  /  Light Microscopy  /  Using a high powered microscope

Using a high powered microscope

Highmag DFWith a compound microscope, dark field is obtained by placing an occulting disk in the light path between source and condenser. A cheap set of occulting disks can be prepared by cutting circular pieces of black electrical tape ranging from about the size of a five pence piece up to a diameter that equals the width of the slide, and sticking them to the slide in a row. The circles should be spaced well apart. A specimen is placed on the microscope stage as usual, and the illumination should be made as uniform as possible. If there is an aperture diaphragm in the condenser (contrast lever), it should be opened up wide. After focusing at low power, the slide with occulting disks is placed in the light path between source and condenser, bringing it as close to the bottom of the condenser as it will go.

Start with the largest disk, sliding it around until it is directly in the center of the light path. Increasing the illumination should then produce a good dark field effect. To optimize, first try stopping down the field diaphragm to get the best contrast between background and specimen. Try to match the size of the occulting disk to the field diameter, so that the edge of the disk is just outside the field of veiw - smaller disks are appropriate for higher power objectives. Vertically, the disk should be a close to the condenser as possible, to make the contrast the greatest. On microscopes with built-in dark field equipment, the view is so impressive because the occulting disk is built into the condenser - very close and focused. After testing the set-up this way, a stand might be rigged to fit under the microscope, so the slide can be placed in position without holding it. Something that 'grabs' the condenser and supports the occulting disks would be ideal. The less you have to mess with, the better.

Suspensions of cells and samples of pond water look spectacular in dark field. While specimens may look washed out and lack detail in bright field, protists, metazoans, cell suspensions, algae, and other microscopic organisms are clearly distinguished and their details show up well. At 100x you can readily see bacteria, even distinguish some structure (rods, curved rods, spirals, or cocci) and movement. Non-motile bacteria look like vibrating bright dots against a dark background. Motile bacteria can be seen moving in a definite direction, sometimes remarkably fast. In pond water samples you may find Spirillum volutans, a very large (up to 0.5 mm) motile spiral bacterium.