Fecal Examination Using the Ethyl Acetate Sedimentation Method
Below is description of how to test a fecal sample using the Ethyl Acetate Sedimentation Method:
- Pass a piece of feces (about 5x5 mm) through a sieve into about 9 ml of water, then pour solution into a 15 ml centrifuge tube.
- Add about 3 ml of ethyl acetate, and plug the tube with a rubber stopper. Shake the tube vigorously, and then centrifuge it.
CAUTION: Test materials before placing Ethyl Acetate into them. This solvent will dissolve many types of plastic!! The white plastic centrifuge tubes (Polypropylene) used in the lab are OK, but clear plastic tubes (polystyrene) and the disposable polystyrene cups will dissolve.
- Using a stick, "ring" the plug of fat at the water-ethyl acetate interface (the plug adheres to the side of the tube and must be detached before the liquid contents of the tube can be poured off).
- Pour off the supernatant, being careful to leave the pellet at the bottom of the tube intact.
- Transfer some of the sediment from the bottom of the tube to a slide and examine. The sediment can be transferred in several ways: 1)If some liquid remains, the pellet can be resuspended and a drop transferred with a pipette. 2) Add a drop of iodine to the pellet to resuspend it and then transfer with a pipette. 3) Use a stick to remove some of the pellet and smear it on a slide as you would when making a direct smear.
NOTE: When removed from the centrifuge, your tube will have clearly defined layers:
A. An ethyl acetate layer on top.
B. A plug of dissolved fat in the middle.
C. A layer of water.
D. A pellet of sediment at the bottom.
Because formalin fixed eggs and cysts may not float (they may now have a specific gravity of greater than 1.2) this technique is preferred for formalin fixed samples.
NOTE: If you did this technique just to remove fat, you can resuspend the pellet in flotation solution, centrifuge, and remove the material from the top of the float to examine for eggs.
