The light microscope is one of the most useful and often used type of microscope available to the microbiologist. Although the limit of resolution and magnification capabilities are much less when compared to an electron microscope, its affordibility, ease of use, and multifunctionality make it a popular tool in most research laboratories. Several types of microscopy can be practiced on light microscope. Three such microscopy principles are brightfield, phase contrast, and fluorescence.
Brightfield Microscopy
Brightfield microscopy requires that the sample be stained with special chemicals to be viewed. In brightfield microscopy, the light passes from the illumination source through the sample into the eyepiece. All wavelengths of visible light travel through the specimen but because bacteria naturally have low contrast, the image is often of very poor quality. Therefore, chemical stains are used to treat the bacteria before viewing them in the microscope.
Simple Staining
Simple stains act by indiscriminately binding to the negatively-charged surface of the bacterial cell. Some simple stains, such as crystal violet and methylene blue, allow all types of bacteria to be viewed under the light microscope.
Differential Staining
Differential stains act just as their name implies-- they differentiate different kinds of bacteria from other kinds. Like simple stains, differential stains improve the contrast so that cells may be seen from the background, but not all cells look the same.
One of the most important differential stains is that of the Gram stain. For historical reason, the result of the Gram stain (negative or positive) is often one of the first characteristics determined for new bacterial isolates.
Functionally, this stain differentiates cells with a thick cellular envelop (Gram +) from those with thinner cell walls (Gram -).
The first step of this popular staining procedure involves staining with crystal violet. This positively charged compound binds to the negatively charged components on the outside of the cell envelop. At this point, all cells look deep purple.
Next, the sample is flooded with a mordant (iodine) which forms an insoluble precipitate with the crystal violet. This is followed by a decolorizing rinse (usually acid- alcohol), which dehydrates the cell envelop. In bacteria with a thick cell wall, the crystal violet-iodine complex gets trapped inside the cell, and the cell remains purple (Gram +). In bacteria with a thin cell wall, the crystal violet-iodine complex leaks out, and the cells become colorless once again.
The final step is a counter stain (in this case, safrinin, a pink stain) which serves to stain the Gram(-) cells to give them contrast above the background. The end result is purple-colored Gram (+) cells and pink-colored Gram (-) cells.
Structural Staining
Structural stains are also available for use with brightfield microscopy. These stains emphasize certain structural components of bacterial cells such as flagella, capsules, and spores. A major problem with using these stains is the difficulty in seeing the faintly-stained structures which are often below the resolution limit of the microscope.
Phase Contrast Microscopy
Phase contrast microscopy is extremely useful since it takes advantage of fact that structures of different refractive indices bend the light in different directions. With special phase optics, this difference in light bending translates into a difference in contrast. For example, light would be able to travel more easily though the glass slide alone than it would be able to travel through a bacterial cell. The cell retards the light as it passes through, causiong the contrast difference between a cell and its background. For the microbiologist, this is extremely important since no physical or chemical alteration (staining) of the bacterial cell is required. This means that bacteria (and other microorganisms) can be viewed alive. Simple wet mounts can be made and such properties as motility and growth can be observed.
Fluorescence Microscopy
Fluorescence microscopy requires a special type of powerful illumination source, usually a mercury lamp. The light from the lamp passes through special colored filters which only allow light with distinct wavelengths to pass. This narrow band of light hits the bacterial specimen. Certain compounds in the specimen (either natural compounds or fluorescent stains) capture the light and reflect it back up as light with a lower energy. This reflected light is detected either with the viewer's eye, or with sensitive detectors. Because this type of microscopy uses reflected light on a dark background, very small amounts of light (and of your sample) can be seen. Flourescent compounds include natural compounds such as chlorophyll, as well s certain DNA-binding dyes such s ethidium bromide and DAPI. Sometimes fluorescent stains are attached to the constant region of antibodies, to generate very specific, very sensitive bacterial tags.