Exercise 6

Concentration Technique:
Sedimentation
I. Introduction
A concentration procedure is performed mainly to separate the parasites from fecal debris. The
concentration procedure not only increases the numbers of parasites in the sediment but it also
unmasks them, making them more visible by removing organic and inorganic debris.
Sedimentation methods (using centrifugation) lead to the recovery of all protozoa, oocysts,
spores, eggs, and larvae present; however, the preparation contains more debris. If one technique is
selected for routine use, the sedimentation procedure is recommended as being the easiest to perform
and least subject to technical error.
II. Procedure
Simple Sedimentation
Add about 10 grams of stool in
a 150 mL container

Add 0.5% glycerinated water

enough to make it 3/4 full

Filter the solution in a funnel

with a wet gauze

Again Add 0.5% glycerinated

water enough to make it 3/4 full

Pour about 3/4 of the

supernatant

Allow to settle for 1 hour

Allow to settle for 1 hour

Carefully pour the supernatant

without spilling the sediment

Suck sediments from the top,

middle and bottom portion

Examine under LPO.

Make 3 different smears from the

different layers of the sediment

Formalin-Ethyl Acetate Method

Make a solution: 10 parts water
and 1 part stool

Filter the solution in a funnel

with a wet gauze

Add NSS till it is 3/4 full

Add 7 mL 10% formalin, stand

for 5-10 minutes

Repeat process until

supernatant is clear

Centrifuge for 2 minutes @

1,500 rpm

Add 3 mL ethyl acetate and

mix thoroughly

Centrifuge for 2 minutes @

1,500 rpm

Pour the 3 layered supernatan

until sediments remain

Examine under the microscope

Add a drop of NSS and a drop

of sediment on a slide

III. Results
Patient: Karen Franco
Age: 8 yrs.old
Sex: Female
RBC
WBC
Parasites:
Ascaris - many (actual eggs counted - 34)
Trichuris - many (actual eggs counted - 41)
IV. Discussion
Simple Sedimentation Method
This method uses 0.5% glycerinated water to wash the fecal sample and clear any fecal debris, but
before that, this technique also follows the principle of centrifugation which would allow the ovas to
remain in the sediments and would not be lost during decantation.
Formalin-Ethyl Acetate Sedimentation Concentration
By centrifugation, this concentration procedure leads to the recovery of all protozoa, eggs, and larvae
present; however, the preparation contains more debris than is found with the flotation procedure.
Ethyl acetate is used as an extractor of debris and fat from the feces and leaves the parasites at the
bottom of the suspension. The formalin-ethyl acetate sedimentation concentration is recommended as
being the easiest to perform, allows recovery of the broadest range of organisms, and is least subject
to technical error.
The specimen must be fresh or formalinized stool (5 or 10% buffered or nonbuffered formalin or
sodium acetate-acetic acid-formalin [SAF]). Polyvinyl alcohol (PVA)-preserved specimens can also
be used.
V-A. Questions for Research
1. Describe the principle of sedimentation technique
The principle of this technique is based in specific gravity. Ethyl acetate is added to a salinewashed formalin-fixed sample and the tube is then centrifuges. Parasites are heavier and settle in the
sediment whereas fecal debris is usually higher and rises to the upper layers of the tube
2. Give the advantages and disadvantages of sedimentation concentration techniques.
Advantages
Disadvantages
Easy to Perform
Preparation Contains more fecal debris than a
floatation technique
Provides good recovery of most parasites
More challenging to the microscopist
Worms, eggs and larvae may be recovered
Destroys trophozoite stages

3. Give other methods of sedimentation concentration technique other than the ones performed,
describe their difference and/or similarities with the ones performed.
A. Modified formalin ether sedimentation
- The morphology of most parasites is retained for easy identification.
- requires several apparatus which makes it difficult
B. Acid-ether method
- uses 15% Hydrochloric acid instead of Formalin
V-B. Guide Questions
1. When is concentration techniques applied or done in the lab?
Concentration techniques are done when DFS shows a negative result for parasite, this then
would be confirmed by performing concentration techniques because this would allow the recovery of
smaller eggs and larvae.
2. Where do we recover the parasites or ova when present using sedimentation technique?
We recover the paarasites in the bottom where ther would be sediments because parasites tend to
be heavier than the solution used.
VI. Update
Title: Factors Affecting Recovery of Strongyloides stercoralis Larvae: an Approach to a Newly
Modified Formalin-Ether Concentration Technique for Diagnosis of Strongyloidiasis
Authors: Witthaya Anamnart,Attarat Pattanawongsa,Pewpan Maleewong Intapan and Wanchai
Maleewong
Abstract: To improve the diagnosis efficiency of human strongyloidiasis by using formalin-ether
concentration technique (FECT), the effects of various factors on the recovery rates of Strongyloides
stercoralis larvae were comparatively evaluated. Fresh stool and a short time exposure of larvae to
formalin yielded significantly higher numbers of larvae than preserved stool and 10-min exposure.
Likewise, straining through wire mesh yielded a significantly higher number of larvae recovered than
straining through gauze did. In addition, centrifugation for 5 min for separation of larvae from debris
yielded a significantly greater number of larvae recovered than centrifugation for 2 min did. The
efficacies of the five versions of FECT with different factors-FECT 1, FECT 2, FECT 3, FECT 4, and
FECT 5-were then compared. It was found that FECT 5 was 1.8, 2.0, 1.9, and 1.4 times more
effective than FECT 1, FECT 2, FECT 3, and FECT 4, respectively. FECT 5 is a modified FECT
method, whose modifications included using fresh stool without a preservative substance; a short-time
rather than 10-min formalin exposure; and the use of wire mesh instead of gauze.
VII.Reference
http://jcm.asm.org/content/48/1/97.short
Zeibig, Clinical Parasitology: A practical approach, 2nd ed., 2013
Garcia, Diagnostic Medical Parasitology, 5th Ed.,