Formal Ether Sedimentation techniques

If the number of parasites in the stool specimens is low, examination of a direct wet mount may not detect them, hence the stool should be concentrated to increase the sensitivity of the microscopic detection method.  Concentration technique increase the sensitivity Formal ether sedimentation technique is a commonly used concentration technique.

Fig 1: Formal Ether Sedimentation Technique

Principle of Formal ether (Formalin-Ethyl Aceatate) sedimentation technique

Formalin fixes the eggs, larvae, oocysts, and spores, so that they are no longer infectious, as well as preserves their morphology. Fecal debris is extracted into the ethyl acetate phase of the solution. Parasitic elements are sedimented at the bottom. 

Materials required for Formal Ether Sedimentation techniques are

1.                   Glass container

2.                   Guaze

3.                   Funnel

4.                   Centrifuge tube (15ml capacity)

5.                   Centrifuge

6.                   Physiological saline (0.85% w/v Nacl)

7.                   10% formal dehyde

8.                   Ether (ethyl acetate)

9.                   Test tubes with stopper

10.               Glass rod

11.               Iodine

12.               Microscope

Procedure for Formal Ether Sedimenation Technique

1.                   Transfer half teaspoonful of faeces in 10 ml of water in a glass container and mix thoroughly.

2.                   Place 2 layers of gauze in a funnel and strain the contents into a 15 ml centrifuge tube.

3.                   Centrifuge for 2 minutes at about 500 g.

4.                   Discard the supernatant and resuspend the sediment in 10 ml of physiological saline. Centrifuge at 500 g and discard the supernatant.

5.                   Resuspend the sediment in 7 ml of 10% formaldehyde (1 part of 40% formalin in 3 parts of saline).

6.                   Add 3 ml of ether (or ethyl acetate).

7.                   Close the tube with a stopper and shake vigorously to mix. Remove the stopper and centrifuge at 500g for 2 minutes.

8.                   Rest the tube in a stand. Four layers now become visible the top layer consists of ether, second is a plug of debris, and third is a clear layer of formalin and the fourth is the sediment (Fig 1).

9.                   Detach the plug of debris from the side of the tube with the aid of a glass rod and pour off the liquid leaving a small amount of formalin for suspension of the sediment.