8.1 Fecal Flotation and Fecal Analysis Basics

Fecal Flotation and Fecal Analysis Basics

Intestinal parasitism is a common and clinically significant issue in small and large animal medicine. The veterinary assistant plays a vital role in collecting, storing, and processing fecal samples. Accurate fecal analysis is critical because many internal parasites are zoonotic—meaning they can be transmitted from animals to humans (e.g., Ancylostoma hookworms causing cutaneous larva migrans, and Toxocara roundworms causing visceral larva migrans). Understanding the mechanics of fecal flotation and sample handling ensures high-quality diagnostic results.

Fecal Sample Collection and Storage

To obtain accurate diagnostic results, veterinary assistants must follow strict protocols for sample collection, handling, and storage.

• Sample Volume: A diagnostic fecal sample should consist of 2 to 4 grams of feces. This is roughly equivalent to the size of a human thumb, a large marble, or 1 to 2 teaspoons. Samples that are too small may not contain enough parasite material to yield a representative result, leading to false negatives.
• Sample Freshness: Feces should be as fresh as possible, ideally collected immediately after defecation. Old samples that have been sitting outdoors are often contaminated with soil nematodes, insect larvae, or free-living mites, which can confuse diagnosis. Furthermore, fragile protozoal trophozoites will die and disintegrate rapidly, and helminth eggs may embryonate or hatch into larvae, making identification of the original ova impossible.
• Labeling: Every sample must be clearly labeled immediately upon collection. The label must include the client's last name, patient's name, animal species, date and time of collection, and the initials of the veterinary assistant who collected or received the sample.
• Storage and Preservation: If a fecal sample cannot be processed within 2 to 4 hours of collection, it must be stored to prevent dehydration (desiccation) and parasite development.
  • Refrigeration: Store the sample in a sealed, airtight container in a dedicated laboratory refrigerator at 35°F to 40°F (1.6°C to 4.4°C). Refrigeration preserves cell morphology and halts the development of eggs for up to 24 hours.
  • What to Avoid: Never freeze a fecal sample. Freezing causes water inside protozoal cysts and helminth ova to expand and form ice crystals, which ruptures and distorts their structures, rendering them unidentifiable. Additionally, do not store samples in warm areas or leave them in direct sunlight.

Flotation Solutions and Specific Gravity

Fecal flotation is based on the principle of relative density, measured as specific gravity (SG). Specific gravity is the ratio of the density of a substance to the density of water (which has an SG of 1.000).

Most common parasite ova have a specific gravity between 1.05 and 1.15. Fecal debris, such as undigested plant fiber and hair, has a much higher specific gravity (usually greater than 1.30). To separate the eggs from the debris, we use a flotation solution with a specific gravity of 1.18 to 1.25. In these solutions, the lighter parasite eggs float to the top, while the heavier fecal debris sinks to the bottom.

Several flotation solutions are commonly used in veterinary practice:

1. Zinc Sulfate (SG 1.18 - 1.20): This is the solution of choice for detecting protozoal cysts, particularly Giardia. Zinc sulfate causes minimal osmotic distortion of delicate cysts and trophozoites, preserving their morphology for microscopic identification.
2. Sodium Nitrate (SG 1.18 - 1.20): This is the most common solution used in commercial diagnostic kits (such as standard passive flotation devices). While highly effective at floating common nematode eggs (roundworms, hookworms, whipworms), it has a major drawback: it crystallizes rapidly on the slide. If the slide is not read within 15-20 minutes, sodium nitrate crystals will cover the field and distort the ova.
3. Sheather's Sugar Solution (SG 1.20 - 1.25): A dense solution made of sucrose and water. Because of its high specific gravity, it is excellent for floating heavier eggs (such as Trichuris vulpis whipworms and tapeworms) and Cryptosporidium oocysts. It does not crystallize, allowing slides to be kept and read hours later. However, it is highly sticky and messy, attracting insects if not cleaned up promptly.

Passive vs. Centrifugal Flotation Techniques

Veterinary practices utilize two primary methods for floating parasite eggs: passive (simple) flotation and centrifugal flotation.

Passive (Simple) Flotation

Passive flotation relies entirely on gravity.

• Procedure: 2 to 4 grams of feces are thoroughly mixed with a small amount of flotation solution in a diagnostic vial. The mixture is strained through gauze or a strainer to remove large debris, and the remaining liquid is poured into a clean vial. The vial is filled to the very top until a positive (convex) meniscus forms. A glass coverslip is carefully placed on top of the meniscus. The setup is left undisturbed for 10 to 15 minutes.
• Limitations: Because it relies only on gravity, heavier eggs take a long time to rise. If the coverslip is removed too early, these eggs are missed. Passive flotation has a higher rate of false-negative results, particularly in samples with low parasite loads.

Centrifugal Flotation

Centrifugal flotation uses centrifugal force to accelerate the separation of eggs from debris.

• Procedure: Feces are mixed with flotation solution and strained into a centrifuge tube. The tube is placed in a centrifuge. The centrifuge must be balanced by placing a tube of equal weight directly opposite it. The sample is spun at 1,200 to 1,500 RPM for 3 to 5 minutes.
  • Swing-head centrifuge: The tube can be filled to a meniscus and a coverslip placed on top prior to spinning.
  • Fixed-angle centrifuge: The tube is spun without a coverslip. After spinning, the tube is placed in a rack, topped off with solution to form a meniscus, a coverslip is placed on top, and it sits for 5 to 10 minutes before reading.
• Advantages: Centripetal forces drive heavier fecal debris to the bottom of the tube while rapidly pushing lighter eggs and cysts to the meniscus. Centrifugal flotation is the gold standard of veterinary parasitology because it is significantly more sensitive than passive flotation, recovering more eggs and cysts in less time.

Slide Preparation and Microscope Examination

Once the flotation time is complete, the veterinary assistant prepares the slide for microscopic examination by the technician or veterinarian.

• Flotation Slide Prep: Lift the coverslip straight up from the meniscus in a swift, vertical motion to capture the film of liquid containing the floated eggs. Place the coverslip gently onto a clean glass slide, lowering it at an angle to minimize the entrapment of air bubbles.
• Direct Smear Prep: Used specifically to identify motile protozoal trophozoites (such as Giardia or Tritrichomonas foetus) which would be destroyed by the high osmotic pressure of flotation solutions.
  • Procedure: Place a tiny drop of saline or water on a glass slide. Using an applicator stick, mix in a tiny amount of fresh feces (about the size of a match head). The smear must be thin enough to read newspaper text through it. Place a coverslip on top and examine immediately under the microscope.
• Microscopic Scanning: The slide is examined under the microscope starting on low power (10x objective) to scan for larger ova, and then switching to high-dry power (40x objective) to identify smaller ova and cysts. The examiner must scan the entire coverslip systematically using a 'zig-zag' pattern to avoid missing any areas.