Staining Characteristics

With the routine Romanowsky-type stains (Wright’s, Diff-Quik, Dip-Stat) all bacteria, whether gram-positive or gram-negative, stain blue to purple with a few exceptions such as Mycobacterium. The lipid cell wall of Mycobacterium spp. prevents uptake of Romanowsky-type stains, causing the organisms to appear as nonstaining small rods.

Gram stains can be used, but it is much more difficult to find bacteria with Gram stains than with Romanowsky-type stains and Gram stains often do not give reproducible, accurate results for bacteria in exudates. Cells, exudative proteins, and bacteria (whether gram-positive or gram-negative) tend to stain red in exudates.

Primary Versus Secondary Infection or Normal Microflora

Intracellular bacteria indicate a bacterial infection (primary or secondary), whereas extracellular bacteria may represent a bacterial infection, normal microflora, or contamination. Bacterial infections often reveal bacteria intracellularly and extracellularly, whereas normal microflora and contamination yields only extracellular bacteria. Also, a monomorphic bacterial population (only one bacterial type present) suggests infection, whereas a pleomorphic population (mixture of rods and cocci or variably sized rods) may be seen with contamination, normal microflora, or a bacterial infection. Pleomorphic bacterial populations may occur with gastrointestinal infections, bite wounds, and foreign bodies.

Bacterial Cocci

Pathogenic bacterial cocci are usually gram positive and of the genera Staphylococcus, Streptococcus, Peptostreptococcus, or Peptococcus (Figure 3-1). Staphylococci usually occur in clusters of 4 to 12 bacteria, but Streptococcus, Peptostreptococcus, and Peptococcus spp. tend to occur in short or long chains of organisms. Staphylococcus and Streptococcus spp. are aerobic, and Peptostreptococcus and Peptococcus spp. are anaerobic. When cocci are identified in cytologic preparations, aerobic and anaerobic cultures and sensitivity tests should be performed to identify the organism and the optimum antibiotic therapy. Because most cocci are gram-positive, antibiotic therapy effective against gram-positive organisms should be used when it is necessary to start therapy before culture and sensitivity results are received.

Figure 3-1 A, Neutrophilic inflammation with moderate numbers of bacterial cocci present both intracellularly and extracellularly. (Wright’s stain.) B, Higher magnification showing a neutrophil containing phagocytized bacterial cocci. (Wright’s stain.) C, Scattered red blood cells, two neutrophils, and a superficial squamous epithelial cell are shown. One of the neutrophils contains phagocytized bacterial cocci. (Wright’s stain.)

Dermatophilus congolensis replicates by transverse and longitudinal division, producing long chains of coccoid bacterial doublets that resemble small, blue railroad tracks (Figure 3-2). It infects the superficial epidermis, causing crusty lesions. Cytologic preparations from the undersurface of scabs from these crusty lesions are most rewarding in demonstrating organisms. The preparations usually contain mature epithelial cells, keratin bars, debris, and organisms. A few neutrophils may also be found.

Figure 3-2 A, Skin lesions showing bacterial cocci replicating by transverse and longitudinal division, producing long chains of coccoid bacterial doublets that resemble small, blue railroad tracks. This pattern of bacterial cocci is typical of Dermatophilus congolensis. (Wright’s stain.) B, D. congolensis organisms. (Wright’s stain.) C, Group of D. congolensis organisms giving the appearance of railroad tracks. (Wright’s stain.)

Small Bacterial Rods

Most small bacterial rods are gram-negative. Some can be recognized as bipolar rods (Figure 3-3). All pathogenic, bipolar bacterial rods are gram-negative. Common small bacterial rods include Escherichia coli and Pasteurella spp. Infections with bacterial rods are usually associated with a marked neutrophilic inflammatory response. When small bacterial rods are recognized in cytologic preparations, the lesion should be cultured to identify the organism, and sensitivity tests should be performed to determine the optimum antibiotic therapy. If it is necessary to institute antibiotic therapy before the culture and sensitivity results are received, the therapy employed should be effective against gram-negative organisms because most pathogenic, small rods are gram-negative.

Figure 3-3 A, Neutrophils, red blood cells, and scattered, small bipolar bacterial rods are shown. Whereas all bacteria stain blue-black with Wright’s stain, bipolar rods indicate gram-negative bacteria. B, Many small bipolar bacterial rods are present extracellularly. (Wright’s stain.)

Filamentous Rods

Pathogenic filamentous rods that cause cutaneous or subcutaneous lesions are usually Nocardia or Actinomyces spp. Some other anaerobes, such as Fusobacterium and Mycobacterium spp., may be filamentous, but rarely are. Nocardia and Actinomyces spp., generally have a distinctive morphology in cytologic preparations stained with Romanowsky-type stains. They are characterized by long, slender (filamentous) strands that stain pale blue and have intermittent small pink or purple areas (Figure 3-4). This morphology is characteristic of both Nocardia and Actinomyces spp. and the filamentous form of Fusobacterium spp. When these features are recognized cytologically, cultures should be performed specifically for Nocardia and Actinomyces spp. and for other anaerobes.

Figure 3-4 A, A long filamentous rod staining pale blue with intermittent small pink or purple dots (arrowheads) and smaller filamentous rods (arrows) are shown. (Wright’s stain). B, Composite picture showing filamentous rods (arrows) typical of Actinamyces or Nocardia spp. (Wright’s stain.) C, Multiple filamentous rods and rupured cells are shown. (Wright’s stain.)

On the other hand, Mycobacterium spp. often does not stain with Romanowsky-type stains. As a result, negative images (Figure 3-5) may be observed in the cytoplasm of macrophages and/or inflammatory giant cells. When epithelioid macrophages and/or inflammatory giant cells are encountered in cytologic preparations that do not contain any obvious organisms, a careful search for negative images of Mycobacterium spp. should be made. Mycobacterium spp. stain with acid-fast stains; therefore when negative images are encountered, or when the character of the lesion suggests that Mycobacterium spp. be considered, an acid-fast stain can be performed to show the organism (see Figure 3-5, C), and/or cultures for Mycobacterium spp. can be performed for identification.

Figure 3-5 A, Large macrophage containing several nonstaining bacterial rods characteristic of Mycobacterium spp. (Wright’s stain.) B, Fine-needle aspirate of a consolidated pulmonary mass. An alveolar macrophage, which contains nonstaining bacterial rods identified as clear streaks through the cell (arrow), is indicative of a Mycobacterium infection. (Wright’s stain.) C, Acid-fast stain from the same dog as in A, showing reddish filamentous Mycobacterium organisms (arrows). (Acid-fast stain.)

Because these organisms are often refractory to common antibiotic therapy, and reliable culture has special requirements, cytology is very useful in indicating to the practitioner that special cultures are needed.

Large Bacterial Rods

Large bacterial rods that are pathogenic and sometimes infect the cutaneous and subcutaneous tissues include Clostridia spp., and infrequently, Bacillus spp. When large bacterial rods are thought to be pathogenic, both aerobic and anaerobic cultures should be performed. Also, the smears should be inspected for large rods containing spores (Figure 3-6). If spore formation is observed, Clostridium spp. is most likely present.

Figure 3-6 A, Scattered inflammatory cells, red blood cells (RBCs), and large spore-forming bacterial rods typical of Clostridium spp. are shown. (Wright’s stain.) B, Higher-power view from same case as in A. Scattered RBCs and two large extracellular spore-forming bacterial rods are shown. (Wright’s stain.) C, Same case as in A. Multiple phagocytized spore-forming bacterial rods are shown. (Wright’s stain.) D, A neutrophil containing a phagocytized spore-forming bacterial rod is shown. (Wright’s stain.)

Occasionally, the extremely large nonpathogenic bacterial rods of Simonsiella spp. are observed in cytologic specimens (e.g., contaminated tracheal washes). Simonsiella spp. are bacteria that divide lengthwise, therefore yielding parallel rows of bacteria that give the impression of a single, large bacterium (Figure 3-7).

Figure 3-7 A, Superficial squamous epithelial cell with adherent bacteria. Simonsiella organisms are large striated rodlike organisms. Other bacteria are present, adhered to the squamous cell and free in the background of the smear. (Wright’s stain.) B, Two superficial squamous epithelial cells with many Simonsiella organisms. (Wright’s stain.) C, Two superficial squamous epithelial cells with many Simonsiella organisms. (Wright’s stain.) D, One superficial squamous epithelial cell with many Simonsiella organisms and bacterial rods. (Wright’s stain.)

General Comments

Mycotic agents produce lesions that tend to have more macrophages present than do bacterial lesions. However, neutrophils may still be the predominant cell type, and eosinophils may be plentiful with certain hyphating fungi. Mycotic lesions often contain low numbers of lymphocytes, plasma cells, and fibroblasts. Mycotic agents tend to be phagocytized by macrophages, but may also be found in granulocytes. The infectious agent, the location of the lesion, the chronicity of the lesion, and the immune status of the animal all influence the character of the lesion. A comparison of the common fungi that form only yeasts in tissues is presented in Table 3-1.

Table 3-1 Common Fungi That Form Yeasts in Tissue