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 sub sequent 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 1). 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.

Fig1. Smear preparations by swab roll (A) and pipette deposition (B) of patient specimen on a glass slide. 

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 f lamed, 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 airdry 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 airdried 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, airdried, 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. Airdry the slides before staining.

Smear preparation varies depending on the type of specimen being processed 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 1, light microscopy has applications for bacteria, fungi, and parasites. However, the stains used for these microbial groups differ extensively.

Table1. Microscopy for Diagnostic Microbiology

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 urea plasma), 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 airdried 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 1).

Fig2. Gram stain procedures and principles. A, Gram-positive bacteria observed under oil immersion appear purple. B, Gram-negative  bacteria observed under oil immersion appear pink. (Modified from Atlas RM: Principles of microbiology, St Louis, 2006, Mosby.)

Principle. The difference in composition between gram-positive cell walls, which contain thick peptidoglycan with numerous teichoic acid cross-linkages, and gram-negative cell walls, which consist of a thinner layer of peptidoglycan, and the presence of an outer lipid bilayer that is dehydrated during decolorization, accounts for the Gram staining differences between these two major groups of bacteria. Presumably, the extensive teichoic acid crosslinks contribute to the ability of gram-positive organisms to resist alcohol decolorization. Although the gram-positive organisms may take up the counterstain, their purple appearance will not be altered.

Gram-positive organisms that have lost cell wall integrity because of antibiotic treatment, dead or dying cells, 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 10× objective (100× magnification). The microbiologist should scan the slide looking for white blood cells, epithelial cells, debris, and larger organ isms such as fungi or parasites. Next the smear should be examined using the oil immersion (1000× magnification) lens. When clinical 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., cocci or bacilli), and arrangements (e.g., chains, pairs, clusters) of the cells seen (Figure3). This information often provides a preliminary diagnosis regarding the infectious agents and frequently is used to direct initial therapies for the patient.

Fig3. Examples of common bacterial cellular morphologies,  Gram staining reactions, and cellular arrangements. 

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). 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 a true negative and not likely the result of loss of specimen during staining (Figure4). Other ways that Gram stain evaluations of how direct smears are used are discussed throughout the chapters of Part VII that deal with infections of specific body sites.

Fig4. Gram stains of direct smears showing squamous cells  and bacteria (A), proteinaceous debris (B), and proteinaceous  debris with polymorphonuclear leukocytes and bacteria (C). 

Several examples of Gram stains of direct smears are provided in Figure 5. Basically, whatever is observed is also recorded and is used to produce a laboratory report for the physician. The report typically includes the following:

• The presence of host cells and debris.

• The Gram reactions, morphologies (e.g., cocci, bacilli, coccobacilli), 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 number of bacteria for any normally sterile body site and to describe the quantity as rare or few based on microscopic observation may be understating their significance in a clinical specimen. On the other hand, noting the relative amounts seen on direct smear may be useful laboratory information to correlate smear results with the amount of growth observed subsequently from cultures.

Fig5. Gram stain of direct smears showing polymorphonuclear leukocytes, proteinaceous debris, and bacterial morphologies (arrows),  including gram-positive cocci in chains (A), gram-positive cocci in pairs (B), gram-positive cocci in clusters (C), gram-negative coccobacilli  (D), gram-negative bacilli (E), gram-negative diplococci (F), and mixed gram-positive and gram-negative morphologies (G). 

Although Gram stain evaluation of direct smears is routinely used as an aid in the diagnosis of bacterial infections, unexpected but significant findings of other infectious etiologies may be detected and cannot be ignored. For example, fungal cells and elements generally stain gram-positive, but they may take up the crystal violet poorly and appear gram variable (e.g., both pink and purple) or gram-negative. Because infectious agents besides bacteria may be detected by Gram stain, any unusual cells or structures observed on the smear should be evaluated further before being dismissed as unimportant (Figure 6).

Fig6. Gram stains of direct smears can reveal infectious etiologies other than bacteria, such as the yeast Candida tropicalis. 

Gram Stain of Bacteria Grown in Culture. The Gram stain also plays a key role in the identification of bacteria grown in culture. Similar to direct smears, indirect smears prepared from bacterial growth are evaluated for the bacterial cells’ Gram reactions, morphologies, and arrangements. If growth from more than one specimen is to be stained on the same slide, a wax pencil may be used to create divisions. Drawing a “map” of such a slide allows different Gram stain results to be recorded in an organized fashion (Figure 7). The smear results will be used to determine subsequent testing for identifying and characterizing the organisms isolated from the patient specimen.

Fig7. Example of a slide map for staining several bacterial  colony samples on a single slide. 

Acid-Fast Stains

 The acid-fast stain is the other commonly used stain for light microscopic examination of bacteria.

Principle. The acid-fast stain is specifically designed for a subset of bacteria whose cell walls contain long chain fatty (mycolic) acids. Mycolic acids render the cells resistant to decolorization, even with acid alcohol decolorizers. Thus, these bacteria are referred to as being acid-fast. Although these organisms may stain slightly or poorly as gram-positive, the acid-fast stain takes full advantage of the waxy content of the cell walls to maximize detection. Mycobacteria are the most commonly encountered acid-fast bacteria, typified by Mycobacterium tuberculosis, the etiologic agent of tuberculosis. Bacteria lacking cell walls fortified with mycolic acids cannot resist decolorization with acid alcohol and are categorized as being non–acid-fast, a trait typical of most other clinically relevant bacteria. However, some degree of acid-fastness is a characteristic of a few nonmycobacterial bacteria, such as Nocardia spp., and coccidian parasites, such as Cryptosporidium spp.

Procedure Overview. The classic acid-fast staining method, Ziehl-Neelsen, is depicted in Figure 8. The procedure requires heat to allow the primary stain (carbolfuchsin) to enter the wax containing cell wall. A modification of this procedure, the Kinyoun acid-fast method (see Proce dure 64 on the Evolve site), does not require the use of heat or boiling water, minimizing safety concerns during the procedure. Because of a higher concentration of phenol in the primary stain solution, heat is not required for the intracellular penetration of carbolfuchsin. This modification is referred to as the “cold” method. Another modification of the acid-fast stain that is used for identifying certain non-mycobacterial species is described and discussed in Part III, Section 14. When the acid-fast stained smear is read with 1000× magnification, acid-fast positive organisms stain red. Depending on the type of counterstain used (e.g., methylene blue or malachite green), other microorganisms, host cells, and debris stain a blue to blue-green color (Figures 8 and 9).

Fig8. The Ziehl-Neelsen acid-fast stain procedures and principles. A, Acid-fast positive bacilli. B, Acid-fast negative bacilli. (Modified  from Atlas RM: Principles of microbiology, St Louis, 2006, Mosby.)

Fig9. Acid-fast stain of direct smear to show acid-fast bacilli  staining deep red (arrow A) and non–acid-fast bacilli and host cells  staining blue with the counterstain methylene blue (arrow B).

As with the Gram stain, the acid-fast stain is used to detect acid-fast bacteria (e.g., mycobacteria) directly in clinical specimens and provide preliminary identification information for suspicious bacteria grown in culture. Because mycobacterial infections are much less common than infections caused by other non–acid-fast bacteria, the acid-fast stain is only performed on specimens from patients highly suspected of having a mycobacterial infection. That is, Gram staining is a routine part of most bacteriology procedures, whereas acid-fast staining is reserved for specific situations. Similarly, the acid-fast stain is applied to bacteria grown in culture when mycobacteria are suspected based on other growth characteristics (for more information regarding identification of mycobacteria).

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مواضيع ذات صلة

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

Laboratory Diagnosis of Central Nervous System Infections

Diagnosis and Differential Diagnosis of Paroxysmal Nocturnal Hemoglobinuria

Differential Diagnosis of Pancytopenia

Diagnosis of Aplastic anemia

Diagnosis of Upper Respiratory Tract Infections

tuberculosis testing (TB testing, Interferon gamma release assay [IGRA, T-Spot.TB], QuantiFERON-TB Gold [QFT, QFT-G, TB Gold test, TB blood test], Nucleic acid amplification for TB [NAAT], TB antibody)