Microscopic Fecal Exam Procedures
Fecal examination procedures likely to be accepted and implemented in most veterinary practices include flotation (centrifugal or passive), sedimentation, and direct examination (direct smear). Only flotation and sedimentation are concentration procedures. Direct smears have poor sensitivity because of the small amount of feces examined, but may be useful for demonstrating motile organisms. CAPC recommends that feces be routinely screened by a centrifugal flotation method, which is consistently more sensitive than simple flotation. Accuracy of centrifugal flotation techniques depends on procedural details and specimen attributes.
- Gross examination. Specimens should be examined grossly for the presence of blood, mucus, intact worms, or tapeworm segments.
- Sample size and preparation. Specimen size should be at least 1 gram of formed feces (1 cubic centimeter or a cube about one-half inch on a side). If feces are soft, sample size should be 2 grams. If it is slurry-like, the sample should be 4 grams. For liquid feces, a sample of 6 grams or greater might be appropriate. Inadequate sample size (e.g., fecal loop sample) may result in false-negative results. To remove large fecal debris, sieving is recommended prior to centrifugation. The sample is sieved through cheesecloth or a tea strainer after mixing with water or flotation solution. Passive flotation kits typically include a device that prevents larger particles from floating to the surface.
- Flotation solution. Both the type and concentration of sugar or salt solutions used can affect recovery of diagnostic stages of parasites from feces. Common flotation solutes include sodium nitrate, zinc sulfate, sucrose (usually granulated sugar), magnesium sulfate, and sodium chloride. These solutes can be mixed at varying concentrations with water to achieve flotation solutions with different densities. Flotation solutions with higher densities are capable of floating heavier (denser) parasite stages. However, higher density flotation solutions also float many other fecal particles that can render preparations more difficult to examine and can collapse thin-shelled parasite stages, making them difficult to identify or causing them to float poorly. More viscous solutions, such as Sheather's sugar (sucrose) solution, are more efficient for centrifugation. Most salt solutions dry very quickly, crystallizing on slides and obscuring observation.
- Centrifugation. Centrifugation of sieved feces may be performed in flotation solution either with a coverslip placed on top of a filled tube or with the coverslip added after the centrifuge has stopped. In the latter case, the tube is spun near-full, and then the tube is filled to form a reverse meniscus, the coverslip is added, and the tube is allowed to sit a few minutes longer. Centrifugation with the coverslip on the tube works best when a sugar flotation medium is used. Alternate methods for sampling the reverse meniscus include loops or glass rods that can be flamed between samples; however, this approach is less efficient than centrifuging with the coverslip in place.
- Slide examination. The entire area under the coverslip should be examined. It is helpful to focus on a small air bubble to obtain the correct focal plane. The edge of the coverslip can be sealed with nail polish to prevent drying and to allow examination of the specimen under oil immersion. Sucrose preparations can be stored in high humidity in a refrigerator for hours to days without significantly altering the morphology of most common helminth eggs.
Although routine fecal examination should always include centrifugation, at times, other examination methods are needed to reach a diagnosis. For example, motile trophozoites and nematode larvae can be observed using the direct smear method. Certain nematode, trematode, and tapeworm eggs will not float in less dense flotation solutions and are better demonstrated using sedimentation. The Baermann funnel method may aid in diagnosis of a feline lungworm (Aelurostrongylus abstrusus) infection. Stained direct smears are useful for diagnosis of a protozoal infection such as giardiasis or trichomoniasis. Specimens to be examined for protozoa can first be fixed using a commercial fixative such as Proto-fix™ or a fixating stain such as MIF (merthiolate-iodine-formalin). Fecal antigen detection tests and PCR are useful for diagnosis of many species of parasites.