1. Medical Parasitology Lab. Concentration techniques

2. The microscopic examination of feces is required for the recognition and identification of intestinal parasites:
Direct Microscopy:
Advantages
Useful for the observation of motile protozoan trophozoites.
Disadvantages
May not detect ova, cysts and larvae which are present in scant numbers.

3. 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.
worm eggs, larvea, and protozoan cysts may be recovered by concentration but protozoan trophozoites will not be seen as they are usually destroyed during the concentration procedures. This makes direct wet mount examination obligatory as the initial phase of microscopic examination.

4. The concentration procedure is indicated when the initial wet mount examination is negative despite the clinical symptoms indicating parasitic infection of a patient.

5. Concentration techniques :
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.
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.

6. Concentration Methods
Sedimentation method
Modified Formal- Ether sedimentation technique
Acid- Ether sedimentation technique
Flotation method
Saturated Salt Solution technique
Sheather’s Sugar Centrifugal Flotation technique
Zinc Sulphate Centrifugal Flotation technique

7. Modified Formal- ether sedimentation
Sedimentation Methods
Modified Formal- ether sedimentation

8. Modified Formal- Ether Sedimentation
Formalin- Ether or Formalin- Ethyl acetate method is the recommended concentration procedures.
Most types of worm eggs (round worms, tapeworms, schistosomes, and other fluke eggs), larvae, and protozoan cysts may be recovered by this method.
Advantages:
Speed: one sample can be processed in 5 minutes.
Broad spectrum: it will recover most ova, cyst and larvae.
The morphology of most parasites is retained for easy identification.
Disadvantages:
Requires several pieces of apparatus which does not make it an easy.
The preparation contains some debris.
Ether is flammable. Formalin is an irritant.
Hymenolepis nana and Fasciola spp. do not concentrate well.

9. Materials and Method Libra Applicator stick Glass centrifugal tubes
Beaker
Wire sieve
Vortex or whirlimixer
Centrifuge.
Reagent:
Reagent I: 10% formalin solution in distilled water.
Reagent II: diethyl ether or ethyl acetate.

10. Caution
Ether is a highly flammable compound and will ignite and explode quickly if there is a flame or spark nearby.

12. Procedures
Emulsify 1 gm. of feces in 7 ml of 10% formalin in a centrifuge tube.
Strain the suspension through a brass wire sieve, and collect in beaker.
Pour the filtrate into a 15 ml boiling tube and add 3 ml of ether, then mix well 15 sec on vortex or whirlimixer or 1 min by hand.
Transfer the ether- formalin suspension back into the washed centrifuge tube, and centrifuge at 3,000 rpm for 1 min.

14. Procedures (cont.)
Loosen the fatty layer and debris at the top of the tube with an applicator stick and invert the tube quickly to discard the supernatant.
On righting the tube, a few drops only should remain with the sediment, mix the sediment well and transfer one drops onto a glass slide and cover it with coverslip.
Scan the whole coverslip using 10x objective, turning into 40x for confirmation of identification of parasites.

16. Acid- ether sedimentation technique
Sedimentation Methods
Acid- ether sedimentation technique

17. Materials and Method Libra Applicator stick Glass centrifugal tubes
Beaker
Wire sieve
Vortex or whirlimixer
Centrifuge.
Reagent:
Reagent I: 15% Hydrochloric acid.
Reagent II: diethyl ether or ethyl acetate.

18. Procedures
Mix thoroughly 1 gm. feces with 3 ml of 15% of hydrochloric acid and then mix well.
Add and additional 5-6 ml of 15% HCl and mix.
Strain the suspension through a wire sieve into beaker.
Place suspension in a glass centrifuge tube and make up to the 10 ml with distilled water.
Add 4 ml of ether, stopper the tube and shake vigorously sec using vortex.

19. Procedures (cont.)
Centrifuge 2-3 min at 1500 rpm, the suspension now will be layered.
Loosen plug of debris with applicator stick and immediately pour off liquid.
Transfer one drops onto a glass slide and cover it with coverslip.
Scan the whole coverslip using 10x objective, turning into 40x for confirmation of identification of parasites.

20. Entamoeba histolytica/ dispar
E. histolytica inhabit large intestine and cause amoebic dysentery.
There is two diagnostic stages for E. histolytica/ dispar:
Cyst is regular round measuring measure 10 – 20u in diameter with 4 nuclei, and it’s the infective stage.
Trophozoite is the motile form, measure 15-20u in diameter with large nucleus. (motility by pseudopodia).
Diagnosis:
Stool examination to see cyst stage, or trophozoite stage if the sample is fresh.

22. Entamoeba histolytica/ dispar

23. Entamoeba histolytica/ dispar Trophozoite