1. Faculty of Medicine and Health Sciences Parasitology Lab Second semester 2013/2014 prepared by: Mohammad Al-Qadi E-mail: m.qadi@najah.edu

2. Concentration methods ( Sedimentation & Floatation techniques)

3. Examination of Stool Specimens Stool specimens can be examined either 1- Fresh or 2- Preserved. A- Examination of fresh specimens: Permits the observation of motile trophozoites, but this must be carried out without delay.  Liquid (diarrheic) specimens (which are more likely to contain trophozoites) should be examined within 30 minutes of stool sample collection (not within 30 minutes of arrival in the laboratory!)  Soft specimens (which may contain both trophozoites and cysts) should be examined within one hour of stool sample collection (not within 1 hour of arrival in the laboratory). Important note: If delays cannot be avoided, the specimen should be preserved to avoid disintegration of the trophozoites. However, preserved specimens can be kept for up to one day, with overnight refrigeration if needed, prior to examination.

4. B- Examination of specimens preserved in formalin: Such stool specimens can be tested directly (wet mount, immunoassay, chromotrope stain, UV fluorescence) or can be concentrated prior to further testing.

5. Concentration procedure: Used to separate parasites from fecal debris and increase the chances of detecting parasitic organisms when these are in small numbers. Concentration procedure are divided into: 1- Flotation techniques and 2- Sedimentation techniques

6. 1- Flotation techniques: Principle: Use solutions which have higher specific gravity than the organisms to be floated so that the organisms rise to the top and the debris sinks to the bottom.  The main advantage of this technique is to produce a cleaner material than the sedimentation technique.  The disadvantages of most flotation techniques are that the walls of eggs and cysts will often collapse, thus hindering identification. Also, some parasite eggs do not float.

7. Flotation method can be used using: 1.Saturated Salt Solution technique 2.Sheather’s Sugar Centrifugal Flotation technique 3.Zinc Sulphate Centrifugal Flotation technique Raed Z. Ahmed, Medical Parasitology Lab.,2012

8. 1- Flotation procedure using Saturated Salt Flotation Method: A small amount of feces (a pea to grape size, about 1 gram) is mixed with about 10 ml of flotation medium and poured into a tube so that the liquid comes just over the top of the tube. The mixture is allowed to sit for about 15 min while the eggs float to the top and the rest of the fecal matter sinks to the bottom. A cover slip can be placed on the top of the tube before the incubation period starts or can be applied at the end. The cover slip is then transferred to a microscope slide.

9.  Boil granular sodium chloride in excess in water to produce a saturated solution which when cooled has a specific gravity of 1.18 - 1.2.  Half fill a wide- mounted flat bottomed container with the saturated salt solution.  Emulsify 1gm of feces in the solution and strain it to remove the debris from the surface.  Pour the filtrate into meniscus and fill it to the top with saturated salt solution.  Lay a glass slide or large coverslip over the top, avoiding any bubbles being trapped.  Leave for 20 min before quickly inverting the slide.  Microscopic Examination. Raed Z. Ahmed, Medical Parasitology Lab.,2012 Procedure Saturated Salt Flotation:

10. 2- Floating procedure using Sheather’s sugar solution: – Table sugar -------------------------------------- 500gm – Distilled water ---------------------------------- 320ml – Phenol crystal ( melt in hot water bath) ----- 6.5gm Raed Z. Ahmed, Medical Parasitology Lab.,2012

11. Procedures 1.Soften 1gm of feces with water to a soft. 2.Strain the aqueous suspension through a wire sieve. 3.Mix 1 part aqueous suspension with 2 part of Sheather's sugar solution. 4.Pour into a centrifuge tube, centrifugation 1500 rpm for 10 minutes. 5.Pour the supernatant into a meniscus and add a sufficient solution to bring the meniscus to the top. 6.Place a coverslip and wait for 10 minutes. 7.Examine under microscope. Raed Z. Ahmed, Medical Parasitology Lab.,2012

12.  Advantages: – Reveals most nematode eggs and protozoan cyst.  Disadvantages: – Flukes eggs and tape worm eggs are not demonstrate well. – Also most nematode larvae are not demonstrate well. Raed Z. Ahmed, Medical Parasitology Lab.,2012 Sheather’s Sugar solution Floating technique

17. 2- Sedimentation techniques: Use solutions of lower specific gravity than the parasitic organisms, thus concentrating the latter in the sediment. Example: formalin-ethyl acetate technique

18. Formalin-Ethyl Acetate Sedimentation Concentration Procedure: 1- Mix the specimen well with normal saline. 2- Pour 5ml of the fecal suspension through wetted gauze placed over a disposable paper funnel into a 15 ml conical centrifuge tube. 3- Add 0.85% saline (or 10% formalin) through the debris on the gauze to bring the volume in the centrifuge tube to 15 ml. 4- Centrifuge at 500 × g for 10 minutes. 5- Decant supernatant. Add 10 ml of 10% formalin to the sediment and mix thoroughly with wooden applicator sticks. 6- Add 4 ml of ethyl acetate, stopper the tube, and shake vigorously for 30 seconds.

19. 7- Centrifuge at 500 × g for 10 minutes. 8- Decant the top layers of supernatant. 9- Add several drops of 10% formalin to resuspend the concentrated specimen. 10- Add a drop on a glass slide. Place a cover slip on the drop 11- Microscopic examination

21. Thank you