Fecal examination procedures likely to be accepted and implemented in most veterinary practices include centrifugal flotation, 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.

Why Fecal Centrifugation is Better

Gastrointestinal parasites are not only primary disease agents in companion animals, some are also transmissible to people. Of all the microscopic diagnostic techniques used to detect gastrointestinal parasites, none is more accurate and reliable than centrifugal fecal flotation when it is performed properly If you or the commercial laboratory you submit samples to is not using centrifugal flotation procedures, you are probably underdiagnosing parasites.

Fecal Flotation Basics

Fecal flotation separates parasites and objects in feces based on their differential densities. Flotation solutions are soluble preparations of either sugar or salt in water. When sugar or salt is dissolved at increasing concentrations, the density (measured as specific gravity) increases. When passive or tabletop flotation is used, parasite ova or cysts whose densities are less than that of the flotation solution will overcome gravity and rise to the surface (buoyant force). Objects that are of greater density than the solution will sink to the bottom. However, when flotation preparations are spun in a centrifuge, a much greater force is placed on the heavier objects, allowing for a more rapid and efficient separation of parasites and debris.

1. Gross examination. Specimens should be examined grossly for the presence of blood, mucus, intact worms, or tapeworm segments.

2. 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.

3. 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.

Use a flotation solution with a density (specific gravity) between 1.18 and 1.27. Veterinary practices often choose sodium nitrate (specific gravity 1.18 to 1.20) because it is easily obtained commercially. Many parasitology laboratories prefer to use a sucrose solution prepared at a specific gravity of 1.27. You can obtain sucrose flotation solution in 500-ml and 1-gal containers from Jorgensen Laboratories - Jorvet.com (Sheather’s sugar flotation solution).

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.

4. Centrifugation. As for the centrifuge, use one with either a swinging bucket or fixed-angle rotor. Centrifugation of sieved feces may be performed in flotation solution either with a coverslip placed on top of a filled tube (swinging bucket) or with the coverslip added after the centrifuge has stopped (fixed angle). 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.

To use a swinging bucket centrifuge, mix the feces and flotation solution in centrifuge tubes, and place the tubes in opposing buckets in the rotor. Carefully add flotation solution to the tubes to create a reverse meniscus. Then gently apply a coverslip to each tube by first contacting one side of the tube and then slowly lowering the coverslip, reducing the angle over the meniscus. Next, gradually increase the rotor’s speed to a maximum of 800 rpm. To do this, the centrifuge must have a dial, knob, or digital entry button that allows incremental increases in speed The sucrose solution retains the coverslip on the tube better than less viscous solutions such as sodium nitrate will. Spin the sample for 10 minutes and allow the machine to stop without touching the rotor. Remove the coverslip, place it on a slide, and scan it for parasites.

5. 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. Salt preparations will dry out faster and should be examined quickly.

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 also useful for diagnosis of many species of parasites.

An In-Class Experiment

So what proof do we have that centrifugal flotation is better than passive flotation? For several years, an interesting exercise was performed in parasitology classes by using a fecal sample from a dog with a hookworm burden typical of what practitioners would see in pet dogs. The students are divided into three groups. One group performs a direct smear, another group mixes 2 g of feces with flotation solution and performs a passive flotation procedure, and the third group uses 2 g of feces and performs the centrifugal flotation procedure.

Each year the results are graphic. Usually only 25% of the students performing the direct smear recover hookworm eggs. About 70% of the students performing the passive flotation procedure report seeing hookworm eggs. And every year, without exception, 100% of the students performing the centrifugal flotation procedure report recovering hookworm eggs. This simple exercise convinces students of the improved sensitivity of centrifugation. Improved recovery rates using centrifugal flotation procedures are also substantiated by published studies.^1-4

Conclusion

Now that prepared flotation solutions and high-quality, inexpensive swinging bucket centrifuges can be purchased from several commercial sources, it is much easier to adopt centrifugal flotation techniques. It doesn’t require much cost, effort, or time to improve your parasite detection technique with this important diagnostic procedure

References

1 Blagburn BL, Butler JM. Optimize intestinal parasite detection with centrifugal fecal flotation. Vet Med 2006;101(7):455-464.

2 Dryden MW, Payne PA, Ridley R, et al, Comparison of common fecal flotation techniques for the recovery of parasite eggs and oocysts. Vet Ther 2005;6:15-28.

3 Dryden MW, Payne PA, Smith V. Accurate diagnosis of Giardia spp and proper fecal examination procedures. Vet Ther 2006;7:4-14.

4 Zajac A, Johnson J, King S. Evaluation of the importance of centrifugation as a component of zinc sulfate fecal flotation examinations. J Am Anim Hosp Assoc 2002;38:221-224.