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 diagnostic techniques used to detect gastrointestinal parasites, none is more accurate and reliable than centrifugal fecal flotation when it is performed properly. I think it is safe to say that if you or the commercial laboratory you submit samples to is not using centrifugal flotation procedures, you are probably underdiagnosing parasites.

Byron L. Blagburn, MS, PhD

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.

WHAT YOU'LL NEED

For effective centrifugal flotation, you need at least 1 g of formed feces, which is a cube that is 1/2 in on each side, or 2 g of soft feces. 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 (Sheather's sugar flotation solution).

As for the centrifuge, use one with either a swinging bucket or fixed-angle rotor.

Swinging bucket

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. In my experience, 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.

Fixed-angle rotor

If your centrifuge has a fixed-angle rotor, add the mixture of feces and flotation solution to a tube, filling it to within about 1 in of the top. Spin the preparation at about 1,200 rpm for five minutes. Remove the tube, add flotation solution to form a reverse meniscus, and carefully place the coverslip, as described above. Allow the preparation to stand for an additional 10 minutes, remove the coverslip, and examine the sample.

AN IN-CLASS EXPERIMENT

So what proof do we have that centrifugal flotation is better than passive flotation? I perform an interesting exercise every year in my parasitology class 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 my 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.

Byron L. Blagburn, MS, PhD, Department­ of Pathobiology, College of Veterinary Medicine, Auburn University­, Auburn, AL 36849. Dr. Blagburn is immediate­ past-president of the Companion­ Animal Parasite Council.

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.