Formal-Ether Concentration Technique: Introduction, Principle, Test Requirements, Test Procedure and Uses

Formal-Ether Concentration Technique: Introduction, Principle, Test Requirements, Test Procedure and Uses

Formal-Ether Concentration Technique

The formal-Ether Concentration Technique is the recommended concentration technique. Most types of worm eggs (roundworms, tapeworms, schistosomes, and other fluke eggs), larvae, and protozoan cysts may be recovered by this method.

Principle of Formal-Ether Concentration Technique

This type of concentration technique clears its name using formalin and ether. Sedimentation techniques use solutions of lower specific gravity than parasitic organisms, thus concentrating the latter in the sediment. The formal-ether concentration technique takes advantage of the high specific gravity of protozoan cysts and helminth eggs compared to water. Their natural tendency to settle out in aqueous solutions can be accelerated by light centrifugation. 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.

 Test Requirements for  Formal-Ether Concentration Technique

  • Beaker
  • Wire sieve
  • Centrifuge tube ( 15 ml capacity)
  • Centrifuge
  • Physiological saline (0.85% w/v NaCl)
  • 10% buffered formalin
  •  Diethyl ether or ethyl acetate
  • Test tubes with stopper
  • Vortex
  • Glass rod
  • Iodine
  • Microscope
  • Positive specimen  (optional for quality control)

Procedure for Formal Ether Sedimentation Technique

  1. First, wear gloves when handling stool specimens.
  2. In a suitable container, thoroughly mix a portion of stool specimen approximately 1 ml or the size of a walnut into 10 ml of normal saline. Mix thoroughly with the help of a vortex.
  3. Filter the emulsion through fine mesh gauze or alternatively wire sieve into a conical centrifuge tube as shown above picture.
  4. Centrifuge the suspension at 2000 rpm for 10 minutes. Note: The suspension should yield about 0.75 ml of sediment for fresh specimens and 0.5 ml for formalized feces.
  5. Decant the supernatant and wash the sediment with 10 ml of saline solution. Centrifuge again and repeat washing until the supernatant is clear.
  6. After the last wash, decant the supernatant and add 10 ml of 10% formalin to the sediment. Mix and let stand for 5 minutes to effect fixation.
  7. Add 1 to 2 ml of ethyl acetate, Stopper the tube, and shake vigorously.
  8. Centrifuge at 1500 rpm for 10 minutes. Four layers should result as a top layer of ethyl acetate, plug of debris, layer of formalin, and sediment respectively.
  9. Free the plug of debris from the side of the tube by ringing with an applicator stick. Carefully decant the top three layers.
  10. Mix the remaining sediment with a pipette
  11. Transfer one drop each to a drop of saline and iodine on a glass slide and mix.
  12. Cover with a coverslip and observe first for the presence of parasitic forms under low power (10X) objective, and then high power  (40X) objective under the microscope.

Quality Control for Formal Ether Sedimentation Technique

Check solutions whether are free from contamination or not.
Run known positive specimens as positive control through the procedure to verify organism recovery. This should be done at least two times per year.

Uses  of Concentration techniques

  1. The purpose of concentrating feces is to increase the possibility of finding ova, cysts, or larvae in samples that are not be able to seen by direct microscopy.
  2.  The concentration method may be used to see whether the treatment of the parasites has been successful or not.
  3. To find ova of S. mansoné or Taenia is a few of other ova and cysts if they have not been seen in routine examination  ( due to being very few) and are suspected to be present.
  4. To examine stool specimens from patients who do not come from an area where a particular parasite is found.
  5. If the number of organisms in stool specimens is low, the examination of a direct wet mount may not detect parasites. Thus, whenever possible, the stool should be concentrated.
  6. The concentration procedure is indicated when the initial wet mount examination is negative despite the clinical symptoms indicating a parasitic infection of a patient.

Further Reading on Formal Ether Sedimentation Technique

  1. Merkell and Voge’s medical parasitology
    9th edition.
  2. Parasitology: 12th edition
    By K. D. Chatterjee
  3. District laboratory practice in Tropical countries –Part-I.
    By Monica Chesbrough.
  4. Isenberg clinical microbiology procedures Handbook
    2nd edition. Vol. 2
  5. Atlas of Medical Helminthology and protozoology -4th edn  -P.L.  Chiodini, A.H. Moody, D.W. Manser
  6. Medical Parasitology by Abhay R. Satoskar, Gary L. Simon, Peter J. Hotez and Moriya Tsuji
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