Formal-Ether (Formalin-Ethyl Acetate) Sedimentation Technique: Principle, Procedure, and Results

A district laboratory in rural Ethiopia receives a stool specimen from a child with chronic intermittent diarrhoea. A direct saline wet mount is prepared — negative. The clinician is not convinced. The technician then runs a formal-ether sedimentation: after centrifugation, a single Giardia cyst is visible in the sediment. Treatment is started.

This scenario is the reason concentration techniques exist. Direct wet mount examines roughly 2 mg of stool from a 10–20 g sample. Concentration techniques like formal-ether sedimentation process a much larger volume, physically accumulate parasites into a small sediment, and increase detection sensitivity by 3 to 5 times. For light infections — exactly the patients most commonly misdiagnosed and undertreated — they are essential.

Figure: Fig-1: Formal Ether Sedimentation Technique

The number of parasitic forms of both protozoan and/or helminthic parasites) in fecal specimens are often too low to be observed microscopically in direct wet mounts or in stained smear preparation. In such cases, the use of the concentration technique increases the chances of detecting parasitic organisms, thus increasing the sensitivity of copromicroscopic technique.

The two most commonly used stool concentration techniques are sedimentation and flotation. Sedimentation techniques are performed commonly in general diagnostic laboratories because they are easier to perform and less prone to technical errors.

When to Use Formal-Ether Sedimentation

Key advantage over flotation: Formal-ether detects all organisms regardless of density — heavy eggs (Fasciola hepatica, Schistosoma, unfertilised Ascaris) that do not float in zinc sulfate still sediment and are recovered. This is why sedimentation is the workhorse method in general diagnostic laboratories.

Principle of Formal Ether 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.

Materials required

• Glass container
• Guaze
• Funnel
• Centrifuge tube (15ml capacity)
• Centrifuge
• Physiological saline (0.85% w/v NaCl)
• 10% buffered formalin
• Ether (ethyl acetate)
• Test tubes with stopper
• Glass rod
• Iodine
• Microscope

Procedure for Formal Ether Sedimentation Technique

1. Wear gloves when handling stool specimens.
2. In a suitable container, thoroughly mix a portion of stool specimen about the size of a walnut into 10mL of saline solution. Mix thoroughly.
3. Filter the emulsion through fine mesh gauze into a conical centrifuge tube.
4. Centrifuge the suspension at a 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.
5. Decant the supernatant and wash the sediment with 10 mL of saline solution. Centrifuge again and repeat washing until 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 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 sediment
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. 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.

What each layer contains and why it forms:

Clinical point: The sediment is the only layer examined. The debris plug must be freed from the tube wall and discarded cleanly — if it falls back into the sediment before decanting, it obscures the parasites below.

Quality Control

1. Check solutions with each use to be sure they are clear and free of any bacterial contamination.
2. 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 the more detailed morphology of the object in question.

Figure: Eggs (ova) of various intestinal parasites

Procedure Notes-sedimentation technique

1. 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.
2. 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.
3. 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.
4. 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

1. Certain parasites, such as Giardia lamblia, hookworm eggs, and Trichuris eggs 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.
2. 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.
3. Cryptosporidium oocysts: Cryptosporidium parvum oocysts are very small (4–6 μm) and may not sediment reliably if centrifugation time or speed is insufficient. Additionally, even when present in the sediment, oocysts are not reliably identified by standard wet mount — they require a modified acid-fast stain applied to the sediment smear. If Cryptosporidium is suspected (HIV/AIDS patient, traveller's diarrhoea, young child), specifically request modified acid-fast staining of the formal-ether sediment.

Where Students Actually Get Confused

1. "Formal-ether detects trophozoites." No — formalin fixation kills trophozoites and destroys motility. Formal-ether concentration is for cysts, eggs, and larvae only. To detect trophozoites, a direct saline wet mount from fresh stool processed within 30 minutes is required.

2. "The debris plug is part of the sediment to examine." The debris plug must be cleanly discarded with the other upper layers. Allowing it to fall back into the sediment obscures the parasites. The procedure note about "ringing with an applicator stick" is specifically to free the plug before decanting.

3. "Ethyl acetate and diethyl ether are the same." The original procedure used diethyl ether, which is highly flammable and explosive. Ethyl acetate (ethyl acetate concentration technique) replaced diethyl ether in modern laboratories for safety reasons. The principle is identical — both extract fats. The current standard name is "formalin-ethyl acetate sedimentation" (FEAS); "formal-ether" is the legacy name retained because of its historical prevalence in curricula.

4. "Centrifugation time doesn't matter." Too short a centrifugation fails to pellet small oocysts (Cryptosporidium). The recommended minimum is 10 minutes at 450–600g. Always wait for the centrifuge to reach full speed before starting the timer.

5. "PVA-preserved specimens cannot be processed by formal-ether." PVA-preserved specimens can be processed by formal-ether sedimentation using the modification described in the procedure notes. However, Giardia cysts, hookworm eggs, and Trichuris eggs may not concentrate well from PVA — fresh or formalin-preserved specimens give better results.

Key Exam Facts in One Table

Self-Check Questions

1. After formal-ether centrifugation you observe four distinct layers. From which layer do you make your examination slide, and what do the other layers contain?
2. A technician suspects Cryptosporidium in an HIV patient's stool. She runs formal-ether sedimentation and examines the sediment on a saline wet mount — negative. Has she excluded Cryptosporidium?
3. Why has ethyl acetate replaced diethyl ether in modern sedimentation protocols?
4. A preserved (PVA) stool specimen is sent for formal-ether concentration. The result shows no Giardia cysts despite strong clinical suspicion. What procedural factor may explain this?
5. What is the single most important advantage of formal-ether sedimentation over zinc sulfate flotation?

Answers

1. The sediment (bottom layer) is examined. The top layer contains ethyl acetate with dissolved fats and lipids; the debris plug contains non-lipid organic debris; the formalin layer is the aqueous phase. All three upper layers are discarded after rimming the debris plug free with an applicator stick.
2. No. Cryptosporidium oocysts (4–6 μm) require a modified acid-fast stain for reliable identification — they appear pink/red against a blue background. Standard saline or iodine wet mount of the sediment is insufficient for Cryptosporidium. She must request a modified acid-fast stain on a smear from the sediment.
3. Diethyl ether is highly flammable and explosive, posing a serious laboratory safety hazard. Ethyl acetate performs the same function (dissolving fecal fats) without the explosion risk and is now the standard in all modern laboratories.
4. Giardia cysts do not concentrate reliably from PVA-preserved specimens by formal-ether sedimentation. The PVA preservation affects the surface properties of cysts. Fresh stool or formalin-preserved specimens give significantly better Giardia recovery.
5. Formal-ether sedimentation recovers heavy-density eggs — including Fasciola hepatica, Schistosoma species, and unfertilised Ascaris eggs — that have specific gravity greater than the flotation solution and do not float in zinc sulfate. These organisms would be missed entirely by flotation methods.

References and further readings

1. Garcia, L. S. (2016). Diagnostic Medical Parasitology (6th ed.). ASM Press. (Updates the current article's incomplete "Gracia L."
2. Winn, W. C., Jr., Allen, S. D., Janda, W. M., Koneman, E. W., Procop, G. W., Schreckenberger, P. C., & Woods, G. L. (2006). Koneman's Color Atlas and Textbook of Diagnostic Microbiology (6th ed.). Lippincott Williams & Wilkins.
3. Cheesbrough, M. (2006). District Laboratory Practice in Tropical Countries (2nd ed., Part 1). Cambridge University Press.
4. World Health Organization. (2012). Bench aids for the diagnosis of intestinal parasites (2nd ed.). WHO. https://apps.who.int/iris/bitstream/handle/10665/37323/9789241544764_eng.pdf
5. Ritchie, L. S. (1948). An ether sedimentation technique for routine stool examinations. Bulletin of the US Army Medical Department, 8(4), 326.
6. Allen, A. V. H., & Ridley, D. S. (1970). Further observations on the formol-ether concentration technique for fecal parasites. Journal of Clinical Pathology, 23(6), 545–546. https://doi.org/10.1136/jcp.23.6.545
7. CDC – DPDx: Laboratory Identification of Parasites of Public Health Concern. https://www.cdc.gov/dpdx/index.html