Exercise 6 Concentration Technique: Sedimentation I. Introduction A concentration procedure is performed mainly to separate the parasites from fecal debris. The concentration procedure not only increases the numbers of parasites in the sediment but it also unmasks them, making them more visible by removing organic and inorganic debris. Sedimentation methods (using centrifugation) lead to the recovery of all protozoa, oocysts, spores, eggs, and larvae present; however, the preparation contains more debris. If one technique is selected for routine use, the sedimentation procedure is recommended as being the easiest to perform and least subject to technical error. II. Procedure Simple Sedimentation Add about 10 grams of stool in a 150 mL container
Add 0.5% glycerinated water enough to make it 3/4 full
Filter the solution in a funnel with a wet gauze
Again Add 0.5% glycerinated water enough to make it 3/4 full
Pour about 3/4 of the supernatant
Allow to settle for 1 hour
Allow to settle for 1 hour
Carefully pour the supernatant without spilling the sediment
Suck sediments from the top, middle and bottom portion
Examine under LPO.
Make 3 different smears from the different layers of the sediment
Formalin-Ethyl Acetate Method Make a solution: 10 parts water and 1 part stool
Filter the solution in a funnel with a wet gauze
Add NSS till it is 3/4 full
Add 7 mL 10% formalin, stand for 5-10 minutes
Repeat process until supernatant is clear
Centrifuge for 2 minutes @ 1,500 rpm
Add 3 mL ethyl acetate and mix thoroughly
Centrifuge for 2 minutes @ 1,500 rpm
Pour the 3 layered supernatan until sediments remain
Examine under the microscope
Add a drop of NSS and a drop of sediment on a slide
III. Results Patient: Karen Franco Age: 8 yrs.old Sex: Female RBC WBC Parasites: Ascaris - many (actual eggs counted - 34) Trichuris - many (actual eggs counted - 41) IV. Discussion Simple Sedimentation Method This method uses 0.5% glycerinated water to wash the fecal sample and clear any fecal debris, but before that, this technique also follows the principle of centrifugation which would allow the ovas to remain in the sediments and would not be lost during decantation. Formalin-Ethyl Acetate Sedimentation Concentration By centrifugation, this concentration procedure leads to the recovery of all protozoa, eggs, and larvae present; however, the preparation contains more debris than is found with the flotation procedure. Ethyl acetate is used as an extractor of debris and fat from the feces and leaves the parasites at the bottom of the suspension. The formalin-ethyl acetate sedimentation concentration is recommended as being the easiest to perform, allows recovery of the broadest range of organisms, and is least subject to technical error. The specimen must be fresh or formalinized stool (5 or 10% buffered or nonbuffered formalin or sodium acetate-acetic acid-formalin [SAF]). Polyvinyl alcohol (PVA)-preserved specimens can also be used. V-A. Questions for Research 1. Describe the principle of sedimentation technique The principle of this technique is based in specific gravity. Ethyl acetate is added to a salinewashed formalin-fixed sample and the tube is then centrifuges. Parasites are heavier and settle in the sediment whereas fecal debris is usually higher and rises to the upper layers of the tube 2. Give the advantages and disadvantages of sedimentation concentration techniques. Advantages Disadvantages Easy to Perform Preparation Contains more fecal debris than a floatation technique Provides good recovery of most parasites More challenging to the microscopist Worms, eggs and larvae may be recovered Destroys trophozoite stages
3. Give other methods of sedimentation concentration technique other than the ones performed, describe their difference and/or similarities with the ones performed. A. Modified formalin ether sedimentation - The morphology of most parasites is retained for easy identification. - requires several apparatus which makes it difficult B. Acid-ether method - uses 15% Hydrochloric acid instead of Formalin V-B. Guide Questions 1. When is concentration techniques applied or done in the lab? Concentration techniques are done when DFS shows a negative result for parasite, this then would be confirmed by performing concentration techniques because this would allow the recovery of smaller eggs and larvae. 2. Where do we recover the parasites or ova when present using sedimentation technique? We recover the paarasites in the bottom where ther would be sediments because parasites tend to be heavier than the solution used. VI. Update Title: Factors Affecting Recovery of Strongyloides stercoralis Larvae: an Approach to a Newly Modified Formalin-Ether Concentration Technique for Diagnosis of Strongyloidiasis Authors: Witthaya Anamnart,Attarat Pattanawongsa,Pewpan Maleewong Intapan and Wanchai Maleewong Abstract: To improve the diagnosis efficiency of human strongyloidiasis by using formalin-ether concentration technique (FECT), the effects of various factors on the recovery rates of Strongyloides stercoralis larvae were comparatively evaluated. Fresh stool and a short time exposure of larvae to formalin yielded significantly higher numbers of larvae than preserved stool and 10-min exposure. Likewise, straining through wire mesh yielded a significantly higher number of larvae recovered than straining through gauze did. In addition, centrifugation for 5 min for separation of larvae from debris yielded a significantly greater number of larvae recovered than centrifugation for 2 min did. The efficacies of the five versions of FECT with different factors-FECT 1, FECT 2, FECT 3, FECT 4, and FECT 5-were then compared. It was found that FECT 5 was 1.8, 2.0, 1.9, and 1.4 times more effective than FECT 1, FECT 2, FECT 3, and FECT 4, respectively. FECT 5 is a modified FECT method, whose modifications included using fresh stool without a preservative substance; a short-time rather than 10-min formalin exposure; and the use of wire mesh instead of gauze. VII.Reference http://jcm.asm.org/content/48/1/97.short Zeibig, Clinical Parasitology: A practical approach, 2nd ed., 2013 Garcia, Diagnostic Medical Parasitology, 5th Ed.,
