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INTRODUCTION

This chapter discusses the basic structure and function of the components that make up eukaryotic and prokaryotic cells. It begins with a discussion of the microscope. Historically, the microscope first revealed the presence of bacteria and later the secrets of cell structure. Today, it remains a powerful tool in cell biology.

OPTICAL METHODS

The Light Microscope

The resolving power of the light microscope under ideal conditions is about half the wavelength of the light being used. (Resolving power is the distance that must separate two point sources of light if they are to be seen as two distinct images.) With yellow light of a wavelength of 0.4 µm, the smallest separable diameters are thus about 0.2 µm (ie, one-third the width of a typical prokaryotic cell). The useful magnification of a microscope is the magnification that makes visible the smallest resolvable particles. Several types of light microscopes, which are commonly used in microbiology, are discussed as follows.

A. Bright-Field Microscope

The bright-field microscope is the most commonly used in microbiology courses and consists of two series of lenses (objective and ocular lens), which function together to resolve the image. These microscopes generally employ a 100-power objective lens with a 10-power ocular lens, thus magnifying the specimen 1000 times. Particles 0.2 µm in diameter are therefore magnified to about 0.2 mm and so become clearly visible. Further magnification would give no greater resolution of detail and would reduce the visible area (field).

With this microscope, specimens are rendered visible because of the differences in contrast between them and the surrounding medium. Many bacteria are difficult to see well because of their lack of contrast with the surrounding medium. Dyes (stains) can be used to stain cells or their organelles and increase their contrast so that they can be more easily seen in the bright-field microscope.

B. Phase-Contrast Microscope

The phase-contrast microscope was developed to improve contrast differences between cells and the surrounding medium, making it possible to see living cells without staining them; with bright-field microscopes, killed and stained preparations must be used. The phase-contrast microscope takes advantage of the fact that light waves passing through transparent objects, such as cells, emerge in different phases depending on the properties of the materials through which they pass. This effect is amplified by a special ring in the objective lens of a phase-contrast microscope, leading to the formation of a dark image on a light background (Figure 2-1).

FIGURE 2-1

Using the phase contrast illumination technique, this photomicrograph of a wet mount of a vaginal discharge specimen revealed the presence of the flagellated protozoan, Trichomonas vaginalis. (Courtesy of Centers for Disease Control and Prevention, Public Health Image Library, ID# 5238.)

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