SAMPLE COLLECTION AND PROCESSING

The method of fecal sample collection can affect which portion of the rectum is represented (luminal vs. mucosal) and the cellular (eucaryotic and procaryotic) content of the sample. The ideal sampling method, desired amount to collect, and subsequent sample handling depends upon the intended fecal testing (Broussard, 2003). Important goals of sampling for fecal cytology include examination of a fresh sample (less than 5 minutes old) that is representative of the mucosal surface and preparation of a fecal thin film preparation that is not excessively dense. Excessively dense areas of fecal thin film preparations are prone to detach during staining or, if they do not fall off during staining, obscure cytologic details. Because the cellular content of fecal material is dynamic and will likely continue to change after sample collection, immediate examination following collection is recommended.

For fecal cytology, there are a few possible sampling methods, including collection of unadulterated fecal material, rectal saline lavage, and rectal scraping. A small amount of fresh fecal material may be obtained during digital rectal examination or using a moistened, cotton-tipped applicator or fecal loop if digital rectal exam is not possible. Immediately collected, voided feces may also be used; however, voided samples may be more representative of the luminal portion of the rectum, which is not ideal for fecal cytology. Voided fecal material is a preferred sample for other diagnostic tests, such as fecal flotation, fecal sedimentation, or the Baermann technique, since it is more copious and may be rich in parasitic eggs and cysts (Broussard, 2003). Usually, if a small amount of fecal material is obtained using a moistened, cotton-tipped applicator, the cotton tip of the applicator can be gently rolled on the slide to prepare a direct thin film. Otherwise, slight dilution of the feces may be used to prepare a thin film preparation by placing a drop of sterile, normal saline on a clean, glass microscope slide, adding a very small amount of fecal material (no larger than a match-head), mixing with a sterile, wooden applicator, and spreading as for other fluid thin film preparations. Prior to and after spreading, newsprint should be legible though the preparation.

Rectal saline lavage enriches for mucosal material, including mucus, motile protozoa, and bacteria. Lavage fluid contains relatively less luminal fecal material and is ideal for fecal cytology (Broussard, 2003). Properly collected lavage fluid should have a mudlike consistency, and a single drop can be used to prepare a thin film preparation.

Material obtained by rectal scraping is often submitted as a sample for dry-mount fecal cytology, though feces and rectal scrape material are not synonymous. Rectal scraping with a swab or blunt spatula is a somewhat more invasive method of sampling the rectal mucosa by direct scraping of its surface. Rectal scraping is typically required to identify some infections that localize to the deeper portion of the mucosa (e.g., histoplasmosis, protothecosis) or to characterize deep mucosal cellular infiltrates.

Dry-mount fecal cytology can be useful to examine the microorganism flora and any host cells that may be present (e.g., epithelial, inflammatory) and to detect other pathogens that may be present (e.g., bacterial, fungal, algal, oomycetal, or protozoal). Occasionally, evaluation of dry-mount fecal cytology is diagnostic. More often it aids in ruling selected causes of diarrhea to be less likely.

NORMAL OR INCIDENTAL MICROSCOPIC FINDINGS

The background microbial flora should be predominantly comprised of an extremely polymorphic population of several different bacterial bacilli (Fig. 8-1). There should be fewer than 5 spore-forming bacilli per 100× objective field (Broussard, 2003). Cocci should be absent or only rarely observed. A low number of extracellular, circular or ovoid, 5 to 10 μm diameter, stippled, variably basophilic yeast with a thin, colorless capsule may occasionally be seen (Figs. 8-2, 8-3, 8-4). While these yeast structures are frequently found in diarrheal stools, it is not certain whether there is a direct causal relationship.

FIGURE 8-1 Normal bacterial flora. A highly polymorphic, mixed bacterial flora that is comprised predominantly of several different bacilli is expected to be observed in feces and is typically found on an amorphous, pale basophilic background that contains either none or few host cells. (Wright-Giemsa; HP oil.)

FIGURE 8-2 Incidental yeast. A few circular or ovoid, 5 to 10 μm diameter, stippled, basophilic yeast with a thin, colorless capsule may occasionally be incidentally observed extracellularly within fecal smears. (Wright-Giemsa; HP oil.)

FIGURE 8-3 Incidental yeast. Closer magnification extracellular fecal yeast that can be observed incidentally in fecal smears. (Wright-Giemsa; HP oil.)

FIGURE 8-4 Incidental yeast. Cat. These yeast were present in an adult cat with diarrhea. They appear both as single and budding forms with both clear and focally dense staining areas within the structure. The significance of the yeast is unknown in causing the diarrhea; however, they occur more commonly in watery stools possibly by producing a wash effect that releases the organisms more readily. (Wright; HP oil.)

(Courtesy of Rose Raskin, Purdue University.)

Cyniclomyces guttulatus (formerly Saccharomycopsis guttulata) is part of the normal flora of some rodents and lagomorphs (rabbits, guinea pigs, and chinchillas) (Zierdt, 1988). Occasionally, a low number of individual or doublet Cyniclomyces may be observed as an incidental finding in canine feces and are thought to represent a nonpathogenic result of coprophagia (Fig. 8-5). However, there is uncertainty about whether this yeast is entirely nonpathogenic in dogs. A few preliminary research or anecdotal reports have associated this yeast with clinical cases of chronic diarrhea (Houwers and Blankenstein, 2001; Mandigers, 2007; Saito et al., 2000). During evaluation of some patients with chronic diarrhea, the only abnormality detected in dry-mount fecal cytology may be the presence of an extremely large number of Cyniclomyces both individually and in large mats of numerous budding organisms (Figs. 8-6, 8-7). The observation of many apparently replicating organisms in a fresh fecal sample may represent a factor contributing to the ongoing diarrhea or may simply represent abnormal flora due to underlying disease, physiologic processes, or prior antimicrobial treatments. At this time, the pathogenicity of this yeast is unproven, though it is occasionally observed in large number in the feces of dogs with chronic diarrhea; in such cases, this observation should be reported as an abnormal finding.

FIGURE 8-5 Yeast. An abnormal bacterial flora, containing numerous diplococci, is present on a pale basophilic background that contains a small amount of irregularly shaped, refractile, amber material and a single incidental, extracellular fecal yeast (left) and two Cyniclomyces guttulatus organisms. (Wright-Giemsa; HP oil.)

FIGURE 8-6 Yeast. Dog. Several budding Cyniclomyces guttulatus organisms observed in the feces of a dog with chronic diarrhea. (Wright-Giemsa; IP.)

FIGURE 8-7 Yeast. Large mat of budding Cyniclomyces guttulatus associated with amorphous grey-brown ingesta and squamous epithelial cells. (Wright-Giemsa; IP.)

It is common to see a variable amount of irregularly shaped, colorless or amber material and green-blue material with parallel cell walls, representing digesta and ingested plant matter, respectively (Fig. 8-8). The background also commonly contains a variable amount of amorphous basophilic material consistent with mucus. The amount of mucus observed depends on whether the underlying disease is associated with rectal oversecretion of mucus and whether the sampling method is more representative of the rectal mucosa or the rectal lumen. Being more representative of the rectal mucosa, samples obtained by rectal saline lavage or rectal scraping may contain more mucus than samples collected by other means.

FIGURE 8-8 Plant material. Green-blue ingested plant matter with parallel cell walls is commonly seen in canine and feline fecal smears. (Wright-Giemsa; HP oil.)