3.1: Observing microorganisms with light microscopy

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Light microscopes have a maximum resolution of ~0.2 μm, which is sufficient to resolve individual yeast cells and provide rough infomation about their intracellular organization. (More detailed information about subcellular structure requires an electron microscope.) Compound light microscopes use a system of lenses to gather and focus light passing through a specimen and to project the image on the viewer’s retina. The specimens used for light microscopy are usually stained to increase their contrast prior to observations. Today, a large number of specialized reagents and protocols for staining cells have been described, and investigators select stains to suit the purposes of their individual experiments.

In this lab, you will learn how to adjust the light microscope using a slide containing a stained human blood smear. The blood cells were stained with hematoxylin and eosin (H&E). With H&E staining, it is possible to observe large organelles, such as the cell nucleus, which stains a dark purple. You will also use a simple iodine solution to stain living yeast and bacterial cells. The iodine stains glycogen particles, thereby increasing the contrast of the cultures.

Our labs are equipped with Leica DM500 light microscopes (see the following page). Light from an LED source at the base of the microscope enters a condenser that focuses the
light that will reach the specimen on the microscope stage. Users are able to control the amount of light reaching the specimen by opening or closing an iris diaphragm on the condenser. The microscope has four, interchangeable objective lenses, with magnifications of 4X, 10X, 40X and 100X. Ocular lenses in the eyepieces magnify specimens an additional 10-fold, producing final magnifications of 40X, 100X, 400X and 1000X. The lenses on the DM500 are parfocal, meaning that specimens remain reasonably well-focused when the lenses are changed. When working with the microscope, always begin with the lowest power objective, which is easiest to focus, and work your way up to the higher power objectives.