In general, microscopy is used in microbiology for two basic purposes: the initial detection of microbes and the preliminary or definitive identification of microbes. The microscopic examination of clinical specimens is used to detect bacterial cells, fungal elements, parasites (eggs, larvae, or adult forms), clumps of viruses (viral inclusions) present in infected cells, and histological changes in infected cells or tissues. Characteristic morphologic properties can be used for the preliminary identification of most bacteria and are used for the definitive identification of many fungi and parasites. The microscopic detection of organisms stained with antibodies labeled with fluorescent dyes or other markers has proved to be very useful for the specific identification of many organisms. Five general microscopic methods are used (Box 1).
Box1. Microscopic Methods
MICROSCOPIC METHODS
Brightfield (Light) Microscopy
The basic components of light microscopes consist of a light source used to illuminate the specimen positioned on a stage, a condenser used to focus the light on the specimen, and two lens systems (objective and ocular) used to mag nify the image of the specimen. In brightfield microscopy the specimen is visualized by transillumination, with light passing up through the condenser to the specimen. The image is then magnified, first by the objective lens and then by the ocular lens. The total magnification of the image is the product of the magnifications of the objective and ocular lenses. Three different objective lenses are commonly used: low power (10-fold magnification), which can be used to scan a specimen; high dry (40-fold), which is used to look for large microbes such as parasites and filamentous fungi; and oil immersion (100-fold), which is used to observe bacteria, yeasts (single-cell stage of fungi), and the morphologic details of larger organisms and cells. Ocular lenses further magnify the image (generally 10- to 15-fold). Thus using an oil immersion lens (100-fold) with a 10-fold ocular lens provides a total magnification of 1000-fold that is generally necessary to see bacteria, yeasts, and single-cell parasites in a specimen.
The limitation of brightfield microscopy is the resolution of the image (i.e., the ability to distinguish that two objects are separate and not one). The resolving power of a microscope is determined by the wavelength of light used to illuminate the subject and the angle of light entering the objective lens (referred to as the numerical aperture). The resolving power is greatest when oil is placed between the objective lens (typically the 100× lens) and the specimen because oil reduces the dispersion of light. The best brightfield micro scopes have a resolving power of approximately 0.2 μm, which allows most bacteria, but not viruses, to be visualized. Although most bacteria and larger microorganisms can be seen with brightfield microscopy, the refractive indices of the organisms and background are similar. Thus organisms must be stained with a dye so they can be observed or an alternative microscopic method must be used.
Darkfield Microscopy
The same objective and ocular lenses used in brightfield microscopes are used in darkfield microscopes; however, a special condenser is used that prevents transmitted light from directly illuminating the specimen. Only oblique scattered light reaches the specimen and passes into the lens systems, which causes organisms to be brightly illuminated against a black background. The advantage of this method is that the resolving power of darkfield microscopy is significantly improved compared with that of brightfield micros copy (i.e., 0.02 vs. 0.2 μm), which makes it possible to detect extremely thin bacteria such as Treponema pallidum (etiologic agent of syphilis) and Leptospira spp. (leptospirosis). The disadvantage of this method is that light passes around rather than through organisms, making it difficult to study their internal structure.
Phase-Contrast Microscopy
Phase-contrast microscopy enables the internal details of microbes to be examined. In this form of microscopy, as parallel beams of light are passed through objects of different densities, the wavelength of one beam moves out of “phase” relative to the other beam of light (i.e., the beam moving through the more dense material is retarded more than the other beam). Through the use of annular rings in the con denser and the objective lens, the differences in phase are amplified so that in-phase light appears brighter than out of-phase light. This creates a three-dimensional image of the organism or specimen and permits more detailed analysis of the internal structures.
Fluorescent Microscopy
Some compounds called fluorochromes can absorb short wavelength ultraviolet or ultrablue light and emit energy at a higher visible wavelength. Although some microorgan isms show natural fluorescence (autofluorescence), fluorescent microscopy typically involves staining organisms with fluorescent dyes and then examining them with a specially designed fluorescent microscope. The microscope uses a high-pressure mercury, halogen, or xenon vapor lamp or laser that emits a shorter wavelength of light than that emit ted by traditional brightfield microscopes. A series of filters are used to block the heat generated from the lamp, eliminate infrared light, and select the appropriate wavelength for exciting the fluorochrome. The light emitted from the fluorochrome is then magnified through traditional objective and ocular lenses. Organisms and specimens stained with fluorochromes appear brightly illuminated against a dark background, although the colors vary depending on the fluorochrome selected. The contrast between the organ ism and background is great enough that the specimen can be screened rapidly under low magnification, and then the material is examined under higher magnification once fluorescence is detected.
Electron Microscopy
Unlike other forms of microscopy, magnetic coils (rather than lenses) are used in electron microscopes to focus a beam of electrons from a tungsten filament through a specimen and onto a screen. Samples are usually stained or coated with metal ions to block the beam and show up dark on a screen, like an x-ray image of bone. Because a much shorter wavelength of light is used, magnification and resolution are improved dramatically. Individual viral particles (as opposed to viral inclusion bodies) can be seen with electron microscopy. There are two types of electron microscopes: transmission electron microscopes, in which electrons pass directly through the specimen, and scanning electron microscopes, in which electrons bounce off the surface of the specimen at an angle and a three-dimensional picture is produced. Today, electron microscopy is used more as a research tool than a diagnostic aid. However, one example where electron microscopy is still used identifies viruses in a clinical sample using virus specific antibodies to cause the viruses to clump in a technique called immunoelectron microscopy.
EXAMINATION METHODS
Clinical specimens or suspensions of microorganisms can be placed on a glass slide and examined under the micro scope (i.e., direct examination of a wet mount). Large organisms (e.g., fungal elements, parasites) and cellular material can be seen using this method, but analysis of the internal detail is often difficult. Phase-contrast microscopy can overcome some of these problems; alternatively, the specimen or organism can be stained by a variety of methods (Table 1).
Table1. Microscopic Preparations and Stains Used in the Clinical Microbiology Laboratory
Direct Examination
Direct examination methods are the simplest for prepar ing samples for microscopic examination. The sample can be suspended in water or saline (wet mount), mixed with alkali to dissolve background material (potassium hydroxide [KOH] method), or mixed with a combination of alkali and a contrasting dye (e.g., lactophenol cotton blue, iodine). The dyes nonspecifically stain the cellular material, increasing the contrast with the background, and permit examination of the detailed structures. This method is particularly useful for the rapid detection and potential identification of fungi and parasites. A variation is the India ink method, in which the ink darkens the background rather than the cell. This method is used to detect capsules surrounding organisms, such as the yeast Cryptococcus (the dye is excluded by the capsule, creating a clear halo around the yeast cell) and encapsulated Bacillus anthracis.
Differential Stains
A variety of differential stains are used to stain specific organisms or components of cellular material. The Gram stain is the best known and most widely used differential stain and forms the basis for the phenotypic classification of bacteria; yeasts can also be stained with this method (yeasts and molds are Gram-positive). The iron hematoxylin and trichrome stains are invaluable for identifying protozoan parasites, and the Wright-Giemsa stain is used to identify blood parasites and other selected organisms. Stains such as methenamine silver and toluidine blue O have largely been replaced by more sensitive or technically easier differential or fluorescent stains.
Acid-Fast Stains
At least three different acid-fast stains are used, each exploiting the fact that some organisms retain a primary stain even when exposed to strong decolorizing agents such as mixtures of acids and alcohols. The Ziehl-Neelsen is the oldest method used but requires heating the specimen during the staining procedure. Many laboratories have replaced this method with either the cold acid-fast stain (Kinyoun method) or the fluorochrome stain (auramine-rhodamine method). The fluorochrome method is the stain of choice because a large area of the specimen can be examined rapidly by simply searching for fluorescing organisms against a black background. Some organisms are “partially acid-fast,” retaining the primary stain only when they are decolorized with a weakly acidic solution. This property is characteristic of only a few organ isms (see Table 1), making it quite valuable for their preliminary identification.
Fluorescent Stains
The auramine-rhodamine acid-fast stain is a specific exam ple of a fluorescent stain. Numerous other fluorescent dyes have also been used to stain specimens. For example, the acridine orange stain can be used to stain bacteria and fungi, and calcofluor white stains the chitin in fungal cell walls. Although the acridine orange stain is rather limited in its applications, the calcofluor white stain has replaced the KOH stains. Another procedure is the examination of specimens with specific antibodies labeled with fluorescent dyes (fluorescent antibody stains). The presence of fluorescing organisms is a rapid method for both detection and identification of the organism.
