INTRODUCTION Concentration procedure separate parasites from fecal debris and increase the chances of detecting parasitic organisms when these are in small numbers. If number of organisms in stool specimen is low, examination of a direct wet mount may not detect parasites. Thus, whenever possible, the stool should be concentrated. Advantages Maximizes the numbers of organisms detected which may be too scanty to be seen by direct microscopy alone. Worm eggs, larvae, and protozoan cysts may be recovered. Disadvantages Destroys trophozoite stages. Most concentration methods destroy trophozoites stages. Concentration techniques can be classified as the floatation or sedimentation methods. Floatation technique Here solutions with higher specific gravity than the organisms to be floated so that the organisms rise to the top and debris sink to the bottom. Principle This technique involves suspending the specimen in a medium of greater density than that of the helminthic eggs and protozoan cysts. Eggs and cysts float to the top and are collected by placing a glass slides on the surface of the meniscus at the top of the tube. Floatation Methods includes: Saturated salt solution technique Zinc sulfate centrifugal floatation Sugar floatation technique Saturated salt solution technique Procedure: About half tea spoon (about 4 gm) of fresh stool or preserved stool in a flat bottomed container with 20 ml capacity. Now, few drops of saturated salt solution (specific gravity 1.20) is added and stirred to make a fine emulsion. More salt solution is added with stirring throughout to fill the container up to the brim, until a convex meniscus is formed. A glass slide (3”*2”) is carefully laid on the top of the container so that the center is in contact with the fluid. This preparation is allowed to stand for 20 minutes after which the glass slide is quickly lifted and examined under microscope after putting coverslip. Zinc sulfate centrifugal floatation Procedure Make a fine suspension of about 1 g of feces in 10 m L of water and strain through gauze to remove coarse particles. Collect the liquid in a small test tube and centrifuge for 1 minute at 2,500 revolutions per minute. Pour off the supernatant, add water, resuspend, and centrifuge in the same manner, repeating the process, till the supernatant is clear. Pour off the clear supernatant, add a small quantity of zinc sulfate solution (specific gravity 1.18- 1.2) and resuspend the sediment well. Add zinc sulfate solution to a little below the brim and centrifuge at 2,500 revolution per minute for 1 minute. Take samples care fully from the surface, using a wire loop, transfer to slide and examine under the microscope. A drop of dilute iodine helps to bring out the protozoan cysts in a better way. This technique is useful for protozoan cysts and eggs of nematodes and small tapeworms, but it does not detect unfertilized roundworm eggs, nematode larvae, and eggs of most trematodes and large tapeworms.

1. CONCENTRATIONS
TECHNIQUES IN
PARASITOLOGY
Presented by : Shreya Yadav
Roll No. : 29
2nd sem, B.Sc.Mlt - Nobel College

2. INTRODUCTION
 Concentration procedure separate parasites from fecal debris
and increase the chances of detecting parasitic organisms
when these are in small numbers.
 If number of organisms in stool specimen is low, examination
of a direct wet mount may not detect parasites.
 Thus, whenever possible, the stool should be concentrated.

3. Advantages
 Maximizes the numbers of organisms detected which may be
too scanty to be seen by direct microscopy alone. Worm
eggs, larvae, and protozoan cysts may be recovered.
Disadvantages
 Destroys trophozoite stages. Most concentration methods
destroy trophozoites stages.

4.  Concentration techniques can be classified as the floatation
or sedimentation methods.

5. A: Floatation technique
Here solutions with higher specific gravity than the organisms to
be floated so that the organisms rise to the top and debris sink
to the bottom.
Principle
This technique involves suspending the specimen in a medium
of greater density than that of the helminthic eggs and protozoan
cysts.

6. Eggs and cysts float to the top and are collected by placing a
glass slides on the surface of the meniscus at the top of the
tube.
Floatation Methods includes:
1. Saturated salt solution technique
2. Zinc sulfate centrifugal floatation
3. Sugar floatation technique

7. 1. Saturated salt solution
technique
Procedure:
1. About half tea spoon (about 4 gm) of fresh stool or preserved
stool in a flat bottomed container with 20 ml capacity.
2. Now, few drops of saturated salt solution (specific gravity
1.20) is added and stirred to make a fine emulsion.
3. More salt solution is added with stirring throughout to fill the
container up to the brim, until a convex meniscus is formed.

8. 4. A glass slide (3”*2”) is carefully laid on the top of the
container so that the center is in contact with the fluid.
5. This preparation is allowed to stand for 20 minutes after
which the glass slide is quickly lifted and examined under
microscope after putting coverslip.

9. 2. Zinc sulfate centrifugal
floatation
Procedure
1. Make a fine suspension of about 1 g of feces in 10 m L of
water and strain through gauze to remove coarse particles.
2. Collect the liquid in a small test tube and centrifuge for 1
minute at 2,500 revolutions per minute. Pour off the
supernatant, add water, resuspend, and centrifuge in the
same manner, repeating the process, till the supernatant is
clear.

10. 3. Pour off the clear supernatant, add a small quantity of zinc
sulfate solution (specific gravity 1.18- 1.2) and resuspend the
sediment well.
4. Add zinc sulfate solution to a little below the brim and
centrifuge at 2,500 revolution per minute for 1 minute.
5. Take samples care fully from the surface, using a wire loop,
transfer to slide and examine under the microscope. A drop of
dilute iodine helps to bring out the protozoan cysts in a better
way.

11. 6. This technique is useful for protozoan cysts and eggs of
nematodes and small tapeworms, but it does not detect
unfertilized roundworm eggs, nematode larvae, and eggs of
most trematodes and large tapeworms.

12. 3. Sugar floatation technique:
Sheather's sugar floatation technique is recommended for the
detection of cryptosporidia infection.
Procedure
1. Faecal specimen + Sheather’s sugar floatation technique
2. Stir the solution
3. Vigorously centrifuge and examine the smear from the
surface.
4. Does not float on salt solution: eggs of Ascaris, Taenia spp,
operculated eggs of trematodes, Larvae of strongyloides, etc.

13. Advantages
 produce a cleaner material than the sedimentation technique.
Disadvantages
 delay in examination can result distortion. frequent checking
of specific gravity.
 walls of eggs and cysts will often collapse, hindering
identification.
 some of the parasitic eggs do not floats.

14. B: Sedimentation technique
 In this technique eggs and cysts settle down at the bottom following
centrifugation.
 Solutions of a specific gravity lower than the parasitic organisms
are used ,thus concentrating the eggs, cysts and other forms in the
sediment.
Principle
 It involves concentration of stool specimen by centrifugation . The
protozoan cysts and helminthes eggs are concentrated at the
bottom of the test tube because they have greater density than the
suspending medium.

15. 1. Formal –ether sedimentation
technique
 Formol-ether concentration method has been the most widely
used sedimentation method.
Procedure:
1. Emulsify 1-2 g feces in 10 mL of water and let large
2. particles sediment. Take the supernatant and spin at
3. 2,500 revolutions per minute for 2-3 minutes.
4. Discard the upernatant. Add 10% formol-saline, mix
5. well and let it stand for 10 minutes.

16. 6. Add 3 mL ether and shake well. Spin at 2,500 per minute for
2-3 minutes. Four layers will form
 (a) a top layer of ether
 (b) a plug of debris at the interface
 (c) the formalin-saline layer and
 (d) the sediment at the bottom

18. 7. Carefully detach the debris from the sides of the tube and
discard the top three layers.
8. Suspend the sediment in a few drops of fluid and examine
wet mount and iodine preparation.
 As ether is inflammable and explosive, its use can be
hazardous. Ethyl acetate can be conveniently used in its
place, with equally good results.
 The method is useful for all helminth eggs and protozoan
cysts.

19. Advantages
1. The sensitivity of detecting the ova or cysts increases by 8-
10 folds. Size and shape of the parasitic structures are
maintained.
2. Inexpensive, easy to perform. Fecal odor is removed.
3. As formalin kills the fecal parasites,, no risk of acquiring
laboratory acquired infection.
Disadvantages
1. Trophozoite forms are killed and hence not detected in this
method.

20. 2. Bearmann's sedimentation
method
Procedure:
1. Another method of examination of stool specimen suspected
of having small numbers of Strongyloides larvae is the use
of a modified Baermann apparatus.
2. The Baermann technique, which involves using a funnel
apparatus, relies on the principle that active larvae migrate
from a fresh fecal specimen that has been placed on a wire
mesh with several layers of gauze, which are in contact with
tap water.

21. 3. Larvae migrate through the gauze into the water and settle to
the bottom of the funnel, where they can be collected and
examined.
4. Besides being used for patient's stool specimens, this
technique can be used to examine soil specimens for the
presence of larvae.

22. Refrences:
 Paniker’s textbook of medical parasitology- 8th edition
 https://www.slideshare.net
 Wikipedia

23. THANK YOU!!