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Some of the following information has been adapted from Clinical Microbiology Procedures Handbook, published by the American Society for Microbiology (1). Additional information on the following topics can also be found in that publication. A number of excellent documents are also available for information and guidance (2–4).
Good, clean microscopes and light sources are mandatory for the examination of specimens for parasites (Fig. 11.1). Organism identification depends on morphologic differences, most of which must be seen under stereoscopic microscopes (magnification, ×50) or regular microscopes at low (×100), high dry (×400), and oil immersion (×1,000) magnifications. The use of a 50× or 60× oil immersion objective for scanning can be very helpful, particularly if the 50× oil and 100× oil immersion objectives are placed side by side. This arrangement on the microscope can help avoid accidentally getting oil on the 40× high dry objective. Calibration of the microscope is discussed later in this chapter. Although 5× oculars are acceptable, most laboratories select 10× oculars, preferably with a binocular, tilting head.
Types
Stereoscopic Microscope. A stereoscopic microscope is recommended for larger specimens (arthropods, tapeworm proglottids, and various artifacts). The total magnification usually varies from approximately ×10 to ×45, either with a zoom capacity or with fixed objectives (0.66×, 1.3×, and 3×) that can be used with 5× or 10× oculars. Depending on the density of the specimen or object being examined, you must be able to direct the light source either from under the stage or onto the top of the stage (may require a separate simple laboratory light directed at the top of the stage). Current uses include the examination of larger specimens (arthropods, artifact removed from clinical specimens, whole mounts, etc.).
Figure 11.1 Microscopes routinely used in clinical laboratories. (Top row) Regular compound microscopes. (Second row, left) Microscope with tube for photography; (right) stereomicroscope. (Bottom) Fluorescence microscope. doi:10.1128/9781555819002.ch11.f1
Regular Light Microscope. The light microscope should be equipped with the following:
1. Head. A binocular head is recommended and should be equipped with a diopter adjustment to compensate for focus variation in the eyes. A tilt head is highly recommended to accommodate various users.
2. Oculars. 10× oculars are required; 5× oculars can be helpful but are optional.
3. Objectives. 10× (low power), 40× (high power), 100× (oil immersion). Some laboratories are currently using 50× or 60× oil immersion lenses for screening permanent stained smears. Examination with a combination of the 50× oil or 60× oil and the 100× oil immersion lenses allows screening to proceed more quickly and eliminates the problem of accidentally getting oil on the high dry objective lens when switching back and forth between the 40× (high dry) and 100× (oil immersion) objectives.
4. Stage. A mechanical stage for both vertical and horizontal movement is required. Graduated stages are mandatory for recording the exact location of an organism on a permanent stained smear and recommended for any facility performing diagnostic parasitology procedures. This capability is essential for consultation and teaching responsibilities. However, remember that calibration numbers for the exact location of an organism may not be the same for different microscopes and different stages.
5. Condenser. A bright-field condenser equipped with an iris diaphragm is required; however, an adjustable condenser is not required with the newer microscopes. The condenser numerical aperture should be equal to or greater than the highest objective numerical aperture.
6. Filters. Both clear blue glass and white ground-glass filters are recommended.
7. Light source. The light source, along with an adjustable voltage regulator, is usually contained in the microscope base. This light source should be aligned as specified by the manufacturer. If the light source is external, the microscope must be equipped with an adjustable mirror and an adjustable condenser containing an iris diaphragm. The light source should be a 75- to 100-W bulb.
8. Current Uses. Routine microscopy in the microbiology laboratory, and parasitology in particular, includes the examination of direct wet mounts, concentration sediment and surface flotation layers, stained permanent smears, blood specimens, aspirates and small samples or smears/scrapings from any body site, and cytologic and histologic preparations (stained or unstained).
Fluorescence Microscope. The fluorescence microscope should be equipped with the following:
1. Head. A binocular head is recommended and should be equipped with a diopter adjustment to compensate for focus variation in the eyes. A tilt head is highly recommended to accommodate various users.
2. Oculars. 10× oculars are required; 5× oculars can be helpful but are optional.
3. Objectives. 10× (low power), 40× (high power), 100× (oil immersion).
4. Stage. A mechanical stage for both vertical and horizontal movement is required. Graduated stages are mandatory for recording the exact location of an organism on a permanent stained smear and recommended for any facility performing diagnostic parasitology procedures. This capability is essential for consultation and teaching responsibilities. However, remember that calibration numbers for the exact location of an organism may not be the same for different microscopes and different stages.
5. Light source. Mercury-free microscopy (MFM) provides a new approach that encourages the selection of modern mercury-free light sources to replace more traditional mercury-based arc lamps. Microscope performance is enhanced with new solid-state technologies; they offer a more stable light intensity output and have a more uniform light output across the visible spectrum. Solid-state sources eliminate mercury, eliminate the cost of consumable bulbs (lifetime ~200 hours), use less energy, reduce the instrument downtime when bulbs fail,
