Role of Microscopy

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Role of Microscopy

The basic flow of procedures involved in the laboratory diagnosis of infectious diseases is as follows:

For certain infectious diseases, this process may also include measuring the patient’s immune response to the infectious agent.

Microscopy is the most common method used both for the detection of microorganisms directly in clinical specimens and for the characterization of organisms grown in culture (Box 6-1). Microscopy is defined as the use of a microscope to magnify (i.e., visually enlarge) objects too small to be visualized with the naked eye so that their characteristics are readily observable. Because most infectious agents cannot be detected with the unaided eye, microscopy plays a pivotal role in the laboratory. Microscopes and microscopic methods vary, but only those of primary use in diagnostic microbiology are discussed.

The method used to process patient specimens is dictated by the type and body source of specimen (see Part VII). Regardless of the method used, some portion of the specimen usually is reserved for microscopic examination. Specific stains or dyes applied to the specimens, combined with particular methods of microscopy, can detect etiologic agents in a rapid, relatively inexpensive, and productive way. Microscopy also plays a key role in the characterization of organisms that have been cultivated in the laboratory (for more information regarding cultivation of bacteria, see Chapter 7).

The types of microorganisms to be detected, identified, and characterized determine the most appropriate types of microscopy to use. Table 6-1 outlines the four types of microscopy used in diagnostic microbiology and their relative utility for each of the four major types of infectious agents. Bright-field microscopy (also known as light microscopy) and fluorescence microscopy have the widest use and application within the clinical microbiology laboratory. Dark field and electron microscopes are not typically found within a clinical laboratory and are predominantly used in reference or research settings. Which microorganisms can be detected or identified by each microscopic method also depends on the methods used to highlight the microorganisms and their key characteristics. This enhancement is usually achieved using various dyes or stains.

TABLE 6-1

Microscopy for Diagnostic Microbiology

Organism Group Bright-Field Microscopy Fluorescence Microscopy Dark-Field Microscopy Electron Microscopy
Bacteria + + ±
Fungi + +
Parasites + + ±
Viruses + ±

image

+, Commonly used; ±, limited use; –, rarely used.

Bright-Field (Light) Microscopy

Principles of Light Microscopy

For light microscopy, visible light is passed through the specimen and then through a series of lenses that bend the light in a manner that results in magnification of the organisms present in the specimen (Figure 6-1). The total magnification achieved is the product of the lenses used.

Resolution

To optimize visualization, other factors besides magnification must be considered. Resolution, defined as the extent to which detail in the magnified object is maintained, is also essential. Without it everything would be magnified as an indistinguishable blur. Therefore, resolving power, which is the closest distance between two objects that when magnified still allows the two objects to be distinguished from each other, is extremely important. The resolving power of most light microscopes allows bacterial cells to be distinguished from one another but usually does not allow bacterial structures, internal or external, to be detected.

To achieve the level of resolution desired with 1000× magnification, oil immersion must be used in conjunction with light microscopy. Immersion oil has specific optical and viscosity characteristics designed for use in microscopy. Immersion oil is used to fill the space between the objective lens and the glass slide onto which the specimen has been affixed. When light passes from a material of one refractive index to a material with a different refractive index, as from glass to air, the light bends. Light of different wavelengths bend at different angles creating a less distinct distorted image. Placing immersion oil with the same refractive index as glass between the objective lens and the coverslip or slide decreases the number of refractive surfaces the light must pass through during microscopy. The oil enhances resolution by preventing light rays from dispersing and changing wavelength after passing through the specimen. A specific objective lens, the oil immersion lens, is designed for use with oil; this lens provides 100× magnification on most light microscopes.

Lower magnifications (i.e., 100× or 400×) may be used to locate specimen samples in certain areas on a microscope slide or to observe microorganisms such as some fungi and parasites. The 1000× magnification provided by the combination of ocular and oil immersion lenses usually is required for optimal detection and characterization of bacteria.

Contrast

The third key component to light microscopy is contrast, which is needed to make objects stand out from the background. Because microorganisms are essentially transparent, owing to their microscopic dimensions and high water content, they cannot be easily detected among the background materials and debris in patient specimens. Lack of contrast is also a problem for the microscopic examination of microorganisms grown in culture. Contrast is most commonly achieved by staining techniques that highlight organisms and allow them to be differentiated from one another and from background material and debris. In the absence of staining, the simplest way to improve contrast is to reduce the diameter of the microscope aperture diaphragm increasing contrast at the expense of the resolution. Setting the controls for bright field microscopy requires a procedure referred to as setting the Kohler illumination (see Procedure 6-1 on the Evolve site).

Procedure 6-1   Kohler Illumination

Method

1. Turn on the microscope, and adjust the light source so that it is approximately at a maximum of 50% strength.

2. Place a microscope slide containing a specimen on the stage, and secure in place with the slide clips.

3. Adjust the eyepiece for comfort and proper alignment for interpupillary distance.

4. Using the 10× objective for a total magnification of 100×, focus the specimen.

5. Adjust each individual eyepiece. To focus the left eyepiece, close the right eye and use the fine focus to adjust the image. Close the left eye and use the Diopter ring on the right eyepiece to adjust the focus for the right eye.

6. Close the field diaphragm and the condenser aperture. A small circle of light should be visible.

7. If no light is visible, open the field diaphragm until a circle of light is present.

8. Adjust the condenser screws as needed to center the light in the field of view.

9. Adjust the condenser focus knob until the light appears as a sharp circle.

10. Remove the eyepiece and look down the cylinder. A circle of light should be visible in the center of a dark field.

11. Open the diaphragm until the circle of light fills three fourths of the field of view.

12. Place the eyepiece back into the cylinder and record the condenser diaphragm setting for the 10× objective.

Staining Techniques for Light Microscopy

Smear Preparation

Staining methods are either used directly with patient specimens or are applied to preparations made from microorganisms grown in culture. A direct smear is a preparation of the primary clinical sample received in the laboratory for processing. A direct smear provides a mechanism to identify the number and type of cells present in a specimen, including white blood cells, epithelial cells, and predominant organism type. Occasionally an organism may grow in culture that was not seen in the direct smear. There are a variety of potential reasons for this, including the possibility that a slow-growing organism was present, the patient was receiving antibiotic treatment to prevent growth of the organism, the specimen was not processed appropriately and the organisms are no longer viable, or the organism requires special media for growth. Preparation of an indirect smear indicates that the primary sample has been processed in culture and the smear contains organisms following purification or growth on artificial media. Indirect smears may include preparation from solid or semisolid media or broth. Care should be taken to ensure the smear is not too thick when preparing the slide from solid media. In addition, smear from a liquid broth should not be diluted. Liquid broth cultures result in smears that more clearly and accurately represent the native cellular morphology and arrangement in comparison to smears from solid media. Details of specimen processing are presented throughout Part VII, and in most instances the preparation of every specimen includes the application of some portion of the specimen to a clean glass slide (i.e., “smear” preparation) for subsequent microscopic evaluation.

Generally, specimen samples are placed on the slide using a swab that contains patient material or by using a pipette into which liquid specimen has been aspirated (Figure 6-2). Material to be stained is dropped (if liquid) or rolled (if on a swab) onto the surface of a clean, dry, glass slide. To avoid contamination of culture media, once a swab has touched the surface of a nonsterile slide, it should not be used for subsequently inoculating media.

A slide may also be presterilized to avoid contaminating the swab when only a single specimen is received for processing of slides and cultures. Sterilization can be performed by thoroughly flaming the slide using a Bunsen burner and allowing it to cool before use. The slide may be alternately dipped in absolute ethanol and flamed, allowing the alcohol to burn off and thereby killing contaminating organisms. These techniques, although useful, may be limited by increasing safety regulations and the removal of open flame equipment such as Bunsen burners within the clinical laboratory.

For staining microorganisms grown in culture, a sterile loop or needle may be used to transfer a small amount of growth from a solid medium to the surface of the slide. This material is emulsified in a drop of sterile water or saline on the slide. For small amounts of growth that might become lost in even a drop of saline, a sterile wooden applicator stick can be used to touch the growth; this material is then rubbed directly onto the slide, where it can be easily seen. The material placed on the slide to be stained is allowed to air-dry and is affixed to the slide by placing it on a slide warmer (60° C) for at least 10 minutes or by flooding it with 95% methanol for 1 minute. Smears should be air-dried completely prior to heat fixing to prevent the distortion of cell shapes prior to staining. To examine organisms grown in liquid medium, an aspirated sample of the broth culture is applied to the slide, air-dried, and fixed before staining.

A squash or crush prep may be used for tissue, bone marrow aspirate, or other aspirated sample. The aspirate may be placed in the anticoagulant ethylenediaminetetraacetic acid (EDTA) tube and inverted several times to mix contents. This prevents clotting of the aspirated material. To prepare the slide, place a drop of the aspirate on a slide and then gently place a second slide on top, pressing the two slides together and crushing or squashing any particulate matter. Gently slide or pull the two slides apart using a horizontal motion. Air-dry the slides before staining.

Smear preparation varies depending on the type of specimen being processed (see the chapters in Part VII that discuss specific specimen types) and on the staining methods to be used. Nonetheless, the general rule for smear preparation is that sufficient material must be applied to the slide so that chances for detecting and distinguishing microorganisms are maximized. At the same time, the application of excessive material that could interfere with the passage of light through the specimen or that could distort the details of microorganisms must be avoided. Finally, the staining method to be used is dictated by which microorganisms are suspected in the specimen.

As listed in Table 6-1, light microscopy has applications for bacteria, fungi, and parasites. However, the stains used for these microbial groups differ extensively. Those primarily designed for examination of parasites and fungi by light microscopy are discussed in Chapters 47 and 60, respectively. The stains for microscopic examination of bacteria, the Gram stain and the acid-fast stains, are discussed in this chapter.

Gram Stain

The Gram stain is the principal stain used for microscopic examination of bacteria and is one of the most important bacteriologic techniques within the microbiology laboratory. Gram staining provides a mechanism for the rapid presumptive identification of pathogens, and it gives important clues related to the quality of a specimen and whether bacterial pathogens from a specific body site are considered normal flora colonizing the site or the actual cause of infection. Nearly all clinically important bacteria can be detected using this method, the only exceptions being those organisms that exist almost exclusively within host cells (e.g., chlamydia), those that lack a cell wall (e.g., mycoplasma and ureaplasma), and those of insufficient dimension to be resolved by light microscopy (e.g., spirochetes). First devised by Hans Christian Gram during the late nineteenth century, the Gram stain can be used to divide most bacterial species into two large groups: those that take up the basic dye, crystal violet (i.e., gram-positive bacteria), and those that allow the crystal violet dye to wash out easily with the decolorizer alcohol or acetone (i.e., gram-negative bacteria).

Procedure Overview.

Although modifications of the classic Gram stain that involve changes in reagents and timing exist, the principles and results are the same for all modifications. The classic Gram stain procedure entails fixing clinical material to the surface of the microscope slide, either by heating or by using methanol. Methanol fixation preserves the morphology of host cells, as well as bacteria, and is especially useful for examining bloody specimen material. Slides are overlaid with 95% methanol for 1 minute; the methanol is allowed to run off, and the slides are air-dried before staining. After fixation, the first step in the Gram stain is the application of the primary stain crystal violet. A mordant, Gram’s iodine, is applied after the crystal violet to chemically bond the alkaline dye to the bacterial cell wall. The decolorization step distinguishes gram-positive from gram-negative cells. After decolorization, organisms that stain gram-positive retain the crystal violet and those that are gram-negative are cleared of crystal violet. Addition of the counterstain safranin will stain the clear gram-negative bacteria pink or red (Figure 6-3). See Procedure 6-2 on the Evolve site for detailed methodology, expected results, and limitations.