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  • Commonly used; ±, limited use; -, rarefy used.
  • Commonly used; ±, limited use; -, rarefy used.

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 lOOOx magnification, oil immersion must be used in conjunction with light microscopy. Immersion oil is used to fill the space between the objective lens and the

Magnified image

Ocular lens -

Objective lens Specimen-

Condenser lens

Ocular lens -

Objective lens Specimen-

Condenser lens

Ocular lens

Light

Immersion oil Stage —-

Condenser lens

Light source

Ocular lens

Immersion oil Stage —-

Condenser lens

Light source

Microscope components

Magnification

Light path

Microscope components

Figure 6-1 Principles of bright-field (light) microscopy. (Modified from Atlas RM: Principles of microbiology, St Louis, 1995, Mosby.)

glass slide onto which the specimen has been affixed. The ofl enhances resolution by preventing light fays 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 lOOx magnification on most light microscopes.

Lower magnifications (i.e., lOOx or 400x) 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 lOOOx 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.

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. Details of specimen processing are presented throughout Part VII and in most instances the preparation of every specimen includes application of some portion of the specimen to a dean 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 6r2). Material to be stained is dropped (if liquid) or rolled (if on a swab) onto the surface of a dean, 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.

For staining microorganisms grown in culture, a sterile 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

Figure 6-2 Smear preparations by swab roll (A) and pipette deposition (B) of patient specimen on glass microscope slide.

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 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. To examine organisms grown in liquid medium, an aspirated sample of the broth culture is applied to the slide, dried, and fixed before staining.

Smear preparation varies depending on the type of specimen being processed (see 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 suffident material must be applied to the slide so that chances for detecting and distinguishing microorganisms are maximized. At the same time, 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 being sought

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 49 and 50, respectively. The stains for microscopic examination of bacteria, the Gram stain and the add-fast stains, are discussed in this chapter.

Gram Stain

The Gram stain is the prinapal stain used for microscopic examination of bacteria. 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„ grampositive bacteria), and those that allow the crystal violet dye to wash out easily with the decolorizer alcohol or acetone (i.e., gram-negative bacteria).

  1. 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<elIs, as well as bacteria, and is especially useful for examining bloody spedmen 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 deared of crystal violet. Addition of the counterstain safranin will stain the dear gram-negative bacteria pink or red (Figure 6-3).
  2. The difference in composition between gram-positive cell walls, which contain thick peptido-glycan with numerous teichoic add cross-linkages, and gram-negative cell walls, which consist of a thinner layer of peptidoglycan, accounts for the Gram staining differences between these two major groups of bacteria. Presumably, the extensive teichoic add cross-links contribute to the ability of gram-positive organisms to resist alcohol decolorization. Although the counterstain may be taken up by the gram-positive organisms, their purple appearance will not be altered.

Gram-positive organisms that have lost cell wall integrity because of antibiotic treatment, old age, or action of autolytic enzymes may allow the crystal violet to wash out with the decolorizing step and may appear gram-variable, with some cells staining pink and Others staining purple. However, for identification purposes, these organisms are considered to be truly gram-positive. On the other hand, gram-negative bacteria rarely, if ever, retain crystal violet (e.g., appear purple) if the staining procedure has been properly performed. Host cells, such as red and white blood cells (phagocytes), allow the crystal violet stain to wash out with decolorization and should appear pink on smears that have been correctly prepared and stained.

Gram Stain Examination. Once stained, the smear is examined using the oil immersion (lOOOx magnification) lens. When dinical material is Gram-stained (e.g., the direct smear), the slide is evaluated for the presence of bacterial cells as well as the Gram reactions, morphologies (e.g., coco or bacilli), and arrangements (e.g., chains, pairs, dusters) of the cells seen (Figure 6-4). This information often provides a preliminary diagnosis regarding the infectious agents and frequently is used to direct initial therapies for the patient.

The direct smears should also be examined for the presence of inflammatory cells (e.g., phagocytes) that are key indicators of an infectious process. Noting the presence of other host cells, such as squamous epithelial cells in respiratory specimens, is also helpful because the presence of these cells may indicate contamination with organisms and cells from the mouth (for more information regarding interpretation of respiratory smears see Chapter 53). Observing background tissue debris and proteinaceous material, which generally stain gram-negative, also provides helpful information. For example, the presence of such material indicates that specimen material was adequately affixed to the slide. Therefore, the absence of bacteria or inflammatory cells on such a smear is •real" and not likely the result of loss of specimen during staining (Figure 6-5). Other ways that Gram stain evaluations of direct smears are used are discussed throughout the chapters of Part VII that deal with infections of specific body sites.

Several examples of Gram stains of direct smears are provided in Figure 6-6. Basically, whatever is observed is also recorded and is used to produce a laboratory report for the physician. The report typically indudes:

  • The presence of host cells and debris
  • The Gram reactions, morphologies (e.g., coed, bacilli, coccobadlli), and arrangement of bacterial cells present. Note: Reporting the absence of bacteria and host cells can be equally as important.
  • Optionally, the relative amounts of bacterial cells (e.g., rare, feW, moderate, many) may be provided. However, it is important to remember that to visualize bacterial cells by light microscopy, a minimum concentration of 105 cells per 1 ml of specimen is required. This is a large

Gram+ bacteria

S in purple

S in purple

Mordant (Gram's iodine)

Remain purple

Steps for staining

Cells on slide

Primary stain {crystal violet)

Mordant (Gram's iodine)

'iram bacteria fWt

Stain purple

Stain purple

Remain purple

Remain purple f

Remain purple

Remain purple

Remain purple

Decolorizer, (alcohol and/or acetone)

Secóme colorless

Remain purple

Counterstain (safranin)

Remain purple

Stain pink

Fix material on slide with methanol or heat. If slide is heat fixed, allow it to cool to the touch before applying stain. Flood slide with crystal violet (purple) and allow it to remain on the surface without drying for 10 to 30 seconds. Rinse the slide with tap water, shaking off all excess. Flood the slide with iodine to increase affinity of crystal violet and allow it to remain on the surface without drying for twice as long as the crystal violet was in contact with the slide surface (20 seconds of iodine for 10 seconds of crystal violet, for example). Rinse with tap water, shaking off all excess. Flood the slide with decolorizer for 10 seconds and rinse off immediately with tap water. Repeat this procedure until the blue dye on longer runs off the slide with the decolorizer. Thicker smears require more prolonged decolorizing. Rinse with tap water and shake off excess. Flood the slide with counterstain and allow it to remain on the surface without drying for 30 seconds. Rinse with tap water and gently blot the slide dry with paper towels or bibulous paper or air dry. For delicate smears, such as certain body fluids, air drying is the best method. Examine microscopically under an oil immersion lens at 10OOx for phagocytes, bacteria, and another cellular material.

Stain pink

Figure 6-3 Gram stain procedures and principles. A, Gram-positive bacteria observed under oil immersion appear purple. B, Gramnegative bacteria observed under oil immersion appear pink. (Modified from Atlas RM: Principles of microbiology, St Louis, 1995, Mosby.)

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Bacterial Vaginosis Facts

Bacterial Vaginosis Facts

This fact sheet is designed to provide you with information on Bacterial Vaginosis. Bacterial vaginosis is an abnormal vaginal condition that is characterized by vaginal discharge and results from an overgrowth of atypical bacteria in the vagina.

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