Stool Concentration Technique: Sedimentation

Prepared by: Chason Neri 2IMT

Microscopic Examination

The microscopic examination of feces is required

for the recognition and identification of
intestinal parasites:

Advantages
Useful for the observation of motile
protozoan trophozoites.
Disadvantages
May not detect ova, cysts and
larvae which are present in scant
numbers.
Stool Concentration Methods
A concentration technique is performed mainly to
separate the parasites from fecal debris.
The concentration procedure not only increases
the number of parasites in the sediment but
it also unmasks them, making them more
visible by removing organic and inorganic
debris.

Advantages
Maximizes
the
numbers
of
organisms detected which may be
too scanty to be seen by direct
microscopy alone.
Separate the parasites from fecal
debris.
Worm eggs, larvae, and protozoan
cysts may be recovered.
Disadvantages
Destroys trophozoite stages. Most
concentration
methods
destroy
trophozoites stages.
The purpose of concentrating feces is to
increase possibility to finding ova, cyst, or
larvae in samples that not be able to seen by
direct microscopy.
Where heavy infestation
method is not needed.

is

present,

this

Sedimentation Concentration Methods

In sedimentation methods, the parasites are not
floated but deposited, usually by centrifuging.
Sedimentation techniques use solutions of
lower specific gravity than the parasitic

organisms, thus concentrating the latter in the

sediment.
Sedimentation techniques are recommended for
general diagnostic laboratories because they
are easier to perform and less prone to
technical errors.
Fecal sedimentation is used to detect large or
heavy ova such as many spirurid ova, many
fluke eggs, and many tapeworm eggs that will
not float in fecal flotation techniques.
The sediment contains a lot of debris
examining these slides is quite tedious.

so

You can use formalin as a preservative and

ether or ethyl acetate as an extractor of fat
and debris from faeces after filtration to leave
the parasites in a sediment at the bottom of
the tube after centrifugation.
Simple Sedimentation Method
A small piece of stool is mixed with saline in a
tube or bottle and sieved through a strainer. The
sieved contents are centrifuged and the
supernatant fluid poured off. The deposit is
resuspended in more saline, mixed, and
centrifuged.
This
is
repeated
until
the
supernatant fluid is clear. The deposit is
examined directly on a slide. By this simple
method, parasitic cysts, eggs, and free living
parasites can be concentrated.
Formol-saline Ether Sedimentation Method
This method gives a good concentration of
parasitic contents and is recommended for
routine work. This method, however, cannot be
used to concentrate free living forms as
formalin kills the parasites.
Materials
Stool samples
Glass slides
Coverslips
Pipettes
Applicator stick
Glass centrifugal tubes
Beaker
Wire sieve
Vortex or whirlimixer
Centrifuge
Compound Microscope
Reagents

Stool Concentration Technique: Sedimentation

Prepared by: Chason Neri 2IMT

10% formol saline

Saline 450 mL
Concentrate formaldehyde solution 50 mL (40%
w/v)
Add the formaldehyde solution to the saline and
mix well.

10. Cover with a coverslip and examine under

10X and 40X objectives with a reduced
condenser aperture. For the identification of
cysts; iodine, eosin, or Sargeaunts stain can be
used.

Method
1. Mix a small piece of stool in about 10 mL of
10% formol saline, in a tube or bottle.
2. Sieve the suspension into a beaker through a
strainer with small holes.
3. Pour about 6 mL of the sieved suspension into
a centrifuge tube.
4. Add about 3 mL of ether.
5. Mix well and immediately centrifuge at 3,000
rpm/min for 1 minute.
6. Four layers are seen
a. An upper layer of ether
b. Middle layer of stool particles
c. A lower layer of formol saline
d. The deposit in which parasitic contents will be
found.

Notes:
Normal (0.85%) saline is used for routine
examination of stool specimens, as it is
isotonic with living organisms. Use fresh
uninfected saline.
Use of 1% Eosin provides a pink
background against which the cysts and
amebae stand out as clear unstained objects.
New Methylene blue or iodine solution
enhances visualization of the parasite ova
and oocytes.
Sargeaunts Stain is used to stain the
chromatid bars of cysts, and is of value
especially for E. histolytica.
A drop of stain of choice could be mixed in
before placing the cover slip.
If there is a lot of debris, water may be
added first.
Sources:
AL Hindi, A. S. (2011, February 25).
Concentration Techniques: Sedimentation.
Retrieved April 6, 2016, from
http://site.iugaza.edu.ps/mona/files/3Concentration-Techniquessedimentation.ppt

Various layers as seen after centrifugation

7. Using an applicator stick, separate the middle

layer from the sides of the tube and pour this
away together with the ether and formol saline.
8. Resuspend the deposit by tapping the bottom
of the tube with the finger.
9. Transfer the deposit to a slide using a pasteur
pipette.

Almuhana, A. (2012). Concentration methods of

fecal parasites. Retrieved April 6, 2016,
from
fac.ksu.edu.sa/sites/default/files/concentr
ation_teq._0.pptx
Mukherjee, K. L., & Ghosh, S. (2010). Medical
laboratory technology: Procedure manual
for routine diagnostic tests. New Delhi:
Tata McGraw Hill.