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 1).

Fig1. 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.)

C. Dark-Field Microscope

The dark-field microscope is a light microscope in which the lighting system has been modified to reach the specimen from the sides only. This is accomplished through the use of a special condenser that both blocks direct light rays and deflects light off a mirror on the side of the condenser at an oblique angle. This creates a “dark field” that contrasts against the highlighted edge of the specimens and results when the oblique rays are reflected from the edge of the specimen upward into the objective of the microscope. Resolution by dark-field microscopy is quite high. Thus, this technique has been particularly useful for observing organisms such as Treponema pallidum, a spirochete that is smaller than 0.2 µm in diameter and therefore cannot be observed with a bright field or phase-contrast microscope (Figure 2A).

Fig2. A: Positive dark-field examination. Treponemes are recognizable by their characteristic corkscrew shape and deliberate forward and backward movement with rotation about the longitudinal axis. (Reproduced with permission. © Charles Stratton/ Visuals Unlimited.) B: Fluorescence photomicrograph. A rod-shaped bacterium tagged with a fluorescent marker. (© Evans Roberts.) C: Scanning electron microscope of bacteria—Staphylococcus aureus (32,000×). (Reproduced with permission from David M. Phillips/Photo Researchers, Inc.)

D. Fluorescence Microscope

 The fluorescence microscope is used to visualize specimens that fluoresce, which is the ability to absorb short wave lengths of light (ultraviolet) and give off light at a longer wave length (visible). Some organisms fluoresce naturally because of the presence within the cells of naturally fluorescent sub stances such as chlorophyll. Those that do not naturally fluoresce may be stained with a group of fluorescent dyes called fluorochromes. Fluorescence microscopy is widely used in clinical diagnostic microbiology. For example, the fluorochrome auramine O, which glows yellow when exposed to ultraviolet light, is strongly absorbed by the cell envelope of Mycobacterium tuberculosis, the bacterium that causes tuberculosis. When the dye is applied to a specimen suspected of containing M. tuberculosis and exposed to ultraviolet light, the bacterium can be detected by the appearance of bright yellow organisms against a dark background.

The principal use of fluorescence microscopy is a diagnostic technique called the fluorescent-antibody (FA) technique or immunofluorescence. In this technique, specific antibodies (eg, antibodies to Legionella pneumophila) are chemically labeled with a fluorochrome such as fluorescein isothiocyanate (FITC). These fluorescent antibodies are then added to a microscope slide containing a clinical specimen. If the specimen contains L. pneumophila, the fluorescent antibodies will bind to antigens on the surface of the bacterium, causing it to fluoresce when exposed to ultraviolet light (Figure2B).

E. Differential Interference Contrast Microscope

Differential interference contrast (DIC) microscopes employ a polarizer to produce polarized light. The polarized light beam passes through a prism that generates two distinct beams; these beams pass through the specimen and enter the objective lens, where they are recombined into a single beam. Because of slight differences in refractive index of the substances each beam passed through, the combined beams are not totally in phase but instead create an interference effect, which intensifies subtle differences in cell structure. Structures, such as spores, vacuoles, and granules, appear three dimensional. DIC microscopy is particularly useful for observing unstained cells because of its ability to generate images that reveal internal cell structures that are less apparent by bright-field techniques.

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مواضيع ذات صلة

Identification of Microorganisms Grown in Culture

Direct Detection of Microorganisms

Laboratory Manifestations of Iron Overload

Differential Diagnosis of Iron Deficiency

Laboratory Manifestations of Iron Deficiency

Staining Techniques for Light Microscopy

Selection of Culture Media

Sterilization and Disinfection

Biochemical Assays for Metabolic Diseases

Laboratory Diagnosis of Urinary Tract Infections

Identification of Mycoplasma Infections

Laboratory Diagnosis of Infections of the Eyes