Principle of Formal ether (Formalin-Ethyl Aceatate) sedimentation technique
Sedimentation techniques use solutions of lower specific gravity than the parasitic organisms, thus concentrating the latter in the sediment.
It 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.
- Glass container
- Centrifuge tube (15ml capacity)
- Physiological saline (0.85% w/v Nacl)
- 10% buffered formalin
- Ether (ethyl acetate)
- Test tubes with stopper
- Glass rod
- Wear gloves when handling stool specimens.
- In a suitable container, thoroughly mix a portion of stool specimen about the size of a walnut into 10mL of saline solution. Mix thoroughly.
- Filter the emulsion through fine mesh gauze into a conical centrifuge tube.
- Centrifuge the suspension at relative centrifugal force (RCF) of 600 g (about 2000 rpm) for no less than 10 minutes. The suspension should yield about 0.75mL of sediment for fresh specimens and 0.5 mL for formalinized feces.
- Decant the supernatant and wash the sediment with 10 mL of saline solution. Centrifuge again and repeat washing until supernatant is clear.
- 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.
- Add 1 to 2 mL of ethyl acetate, Stopper the tube and shake vigorously.
- Centrifuge at 450 g RCF (about 1500 rpm) for 10 minutes. Four layers should result as follows
- a top layer of ethyl acetate;
- plug of debris;
- layer of formalin; and
- Free the plug of debris from the side of the tube by ringing with an applicator stick. Carefully decant the top three layers.
- With a pipette, mix the remaining sediment with the small amount or remaining fluid and transfer one drop each to a drop of saline and iodine on a glass slide. Cover with a coverslip and examine microscopically for the presence of parasitic forms.
- Check solutions with each use to be sure they are clear and free of any bacterial contamination.
- Run known positive specimens through the procedure to verify organism recovery. This should be done at least two times per year.
Observation and Results
Systematically examine the entire surface of each coverslip with the 10x objective or, if needed for identification, higher power objectives of the microscope in a systematic manner so that the entire coverslip area is observed. When organisms or suspicious objects are seen, switch to higher magnification (40X) to see more detailed morphology of the object in question.
Procedure Notes-sedimentation technique
- The sedimentation procedure can also be used to process polyvinyl alcohol (PVA) fixed specimens. After the procedure by filing one half of a tube with the stool-PVA mixture and add 0.85% NaCl almost to the top of the tube. Then filter the mixture through wet gauze into a 15 mL centrifuge tube and follow the remaining standard procedure.
- If ethyl acetate is used, swab the insides of the tube with a cotton-tipped applicator stick after the plug of debris is rimmed and the excess fluid is decanted. Excess ethyl acetate in the sediment at the time smears are prepared will lead to bubbles that may obscure parasitic forms one is attempting to observe.
- Errors in interpretation may occur if too much or too little feces is used in the sedimentation procedure. Adhere to the recommended formula of 0.75 mL of sediment for fresh specimens and 0.5 mL for formalinized feces.
- Allow the centrifuge to reach maximum speed before the time is monitored. If the centrifugation time is too little, certain smaller parasitic forms, such as the oocysts of Cryptosporidium species, may not reach the sediment.
Limitation of Sedimentation Technique
- Certain parasites, such as Giardia lamblia, hookworm eggs, and Trichuris egg may not concentrate well from PVA-preserved specimens. The oocysts of Isospora belli do not routinely appear in concentrates. Therefore, examination of permanently stained smear is highly recommended.
- With both the sedimentation and the flotation techniques, species identification may not be possible in all cases, depending on the clarity of the forms observed. Permanently stained smears are usually required to make the final identification, particularly when attempting to confirm the identity of the Entamoeba histolytica
References and further readings
- Gracia L. Examination of Fecal Specimens. In. Diagnostic Medical Parasitology. Washignton, DC: American Society for Microbiology.
- Washington WJ et al., Koneman’s Color Atlast and Textbook of Diagnostic Microbiology. Sixth Edition:Lippincott Williams & Wilkins