Stool Specimens - Centrifugal Flotation for Intestinal Parasites
Notes on method: Centrifugal flotation effectively concentrates fertilized eggs of Ascaris species, Trichuris species, hookworm, Hymenolepis species and Enterobius vermicularis. Cysts of protozoa and some helminth eggs may become distorted in the hyperosmolar environment after 20 minutes. Larvae of Strongyloides species, eggs of Taenia species, schistosomes and many other cestode and trematode eggs may not be detected by this method. Each flotation solution have variable efficacy for individual parasite species and references should be consulted before choosing the ideal solution for detecting specific parasites.
- For fresh, unpreserved stool, dilute the specimen 1:1 in physiological saline.
- Mix the specimen well.
- Strain 5 mL of the fecal suspension (more or less depending on its consistency) through wetted cheesecloth-type gauze placed over a disposable paper funnel into a 15 mL conical centrifuge tube. (Conical paper cups with the tips cut off are sufficient). Remaining suspension can be stirred and pressed gently with an applicator stick through the gauze.
- Add 0.85% saline or 10% formalin through the debris on the gauze to bring the volume in the centrifuge tube to 15 mL. Distilled or tap water may be used; however this may deform or destroy Blastocystis species.
- Centrifuge at 500 × g for 10 minutes.
- Decant supernatant. Add 10 mL of flotation solution to the sediment and mix thoroughly with wooden applicator sticks.
- Centrifuge at 500 × g for 5 minutes, allow the centrifuge to stop without using the brake.
- Carefully remove the tube from the centrifuge without disturbing the pellet, place in a rack.
- Avoiding disturbing the surface, add sufficient flotation solution to completely fill the 15 mL tube, ensuring a slightly convex meniscus is formed.
- Place a 15 mm x 15 mm coverslip over the meniscus.
- Set a timer for 10 minutes.
- After 10 minutes flotation, carefully remove the cover slip, ensuring that a drop of solution remains hanging from the coverlsip, and place this on a microscope slide.
- Perform microscopy for parasites within 15 minutes of preparing the microscope slide.
Preparation of Flotation Solutions
- Mix salt in distilled water using a heat block and magnetic stirrer until no more salt will dissolve. Allow to cool and any remaining undissolved salt to sediment in the bottle prior to use. For Sheather’s sucrose, formaldehyde or phenol can be added to a concentration of 1.5% for preservation.
| Solution |
Zinc Sulfate (ZNSO4) |
Sodium Nitrate (NaNO3) |
Saturated Salt (NaCl) |
Sheather’s Sucrose (C12H22O11) |
Magnesium Sulfate (MgSO4) |
|
Volume in 1L H2O |
330 g | 315 g | 350 g | 1,278 g | 350 g |
|
Specific Gravity |
1.2 | 1.2 | 1.2 | 1.27 | 1.28 |
Table: Volume of flotation salt solution required for the preparation of specific flotation solutions. The flotation solution a laboratory chooses to use should be informed by the relative sensitivity of that solution in detecting the parasitic pathogens sought.
Quality Control
- Use a hydrometer to test the specific gravity of solutions.
- Perform this QC once weekly or whenever a new batch of flotation solution is prepared.
DPDx is an educational resource designed for health professionals and laboratory scientists. For an overview including prevention, control, and treatment visit www.cdc.gov/parasites/.