Ethyl Acetate Sedimentation Method
1. Pass a grape-sized piece of feces through a sieve into about 9 ml of water and pour into a 15 ml centrifuge tube.
2. Add about 3 ml of ethyl acetate, plug the tube with a rubber stopper and shake the tube vigorously.
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 hard plastic tubes and the disposable polystyrene cups will dissolve.
3. Remove the rubber stopper and centrifuge the tube (1500-2500 rpm) for 1 to 2 minutes.
4. 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).
5. Pour off the supernatant, being careful to leave the pellet at the bottom of the tube intact. (Flush the ethyl acetate down the sink with plenty of water.)
6. 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: For this technique to be as sensitive as a flotation method, you must examine the entire pellet!
NOTE: When removed from centrifuge, your tube will have clearly defined layers:
1. ethyl acetate layer on top
2. plug of dissolved fat in the middle
3. layer of water below fat
4. pellet of sediment on bottom
USES: Used when the feces contain a large amount of fat (which make use of a flotation method difficult) or when you are specifically looking for trematode or cestode eggs.
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.
Click here for a video demonstration of a Ethyl Acetate Sedimentation
TIP: 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.