CONCENTRATIONS TECHNIQUES IN PARASITOLOGY PRESENTATION.pptx

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 2 nd 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: Saturated salt solution technique Zinc sulfate centrifugal floatation Sugar floatation technique

7. 1. 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.

8. 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 .

9. 2. 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.

10. 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 . T ake 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. 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 Faecal specimen + Sheather’s sugar floatation technique Stir the solution Vigorously centrifuge and examine the smear from the surface. Does not flo at 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: Emulsify 1-2 g feces in 10 mL of water and let large particles sediment. Take the supernatant and spin at 2,500 revolutions per minute for 2-3 minutes. Discard the upernatant . Add 10% formol -saline, mix well and let it stand for 10 minutes.

16. 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

17. Carefully detach the debris from the sides of the tube and discard the top three layers. 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.

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

19. 2. Bearmann's sedimentation method Procedure: Another method of examination of stool specimen suspected of having small numbers of Strongyloides larvae is the use of a modified Baermann apparatus. 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.

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

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

22. THANK YOU!!