Formol–ether concentration technique

Principle

• A sedimentation method: formalin fixes parasites, and ether (or safer ethyl acetate) extracts fat and debris.
• Centrifugation concentrates eggs, cysts and larvae in the sediment while fatty debris rises into a plug.
• Recovers most parasite stages, including operculated and schistosome eggs that may not float in flotation methods.

Specimen

• Fresh stool or stool preserved in 10% formalin (or SAF).

Reagents and equipment

• 10% formalin, diethyl ether or ethyl acetate (the CDC formalin–ethyl acetate method), saline, iodine.
• Conical 15 mL centrifuge tubes, gauze or sieve, funnel, centrifuge, applicator sticks.

Procedure

• Emulsify about 1–2 g of stool in 10% formalin and strain through wet gauze into a centrifuge tube.
• Centrifuge about 500 × g for 10 minutes (conditions vary by procedure) and decant the supernatant.
• Resuspend sediment in 10% formalin (about 9 mL), add about 4 mL ether or ethyl acetate.
• Stopper and shake vigorously for 30 seconds, releasing pressure carefully; centrifuge again.
• Four layers form: solvent on top, debris plug, formalin, sediment at the bottom.
• Free the debris plug with an applicator stick, decant the top three layers and wipe the tube walls.
• Mix the sediment with the remaining fluid and prepare saline and iodine mounts.

Results

• Report helminth eggs, larvae and protozoan cysts; trophozoites are usually not recognized after formalin concentration.

Quality control and pitfalls

• Include known positive material periodically and check centrifuge speed.
• Ether is highly flammable and forms explosive peroxides; ethyl acetate is preferred; work away from flames in a ventilated area.
• Too much stool gives a thick, poorly cleaned sediment; too little lowers sensitivity.
• Excess centrifugation can distort some organisms.

Clinical use

• Standard concentration step of the ova and parasite (O&P) examination, increasing sensitivity for light infections.