Group No. 6 Leader: Mejia, Kirsten Hazel M.

Date: 06/22/12

Experiment No. 3 Collection and Preservation of Stool Specimen

1. Describe proper stool specimen collection for the laboratory diagnosis of parasitic infections with emphasis on quality assurance. The ability to detect and identify intestinal parasites, particularly protozoa, is directly related to the quality of the specimen submitted to the laboratory. Certain guidelines are recommended to ensure proper collection and accurate examination of specimens. These are: Collection of fecal specimens for intestinal parasites should always be performed before radiological studies involving barium sulfate. Because of the excess crystalline material in stool specimen, the intestinal protozoa may be impossible to detect for at least 1 week after use of barium. Certain medications may also prevent the detection of intestinal protozoa; these include mineral oil, bismuth, non-absorbable and some antibiotics. The organisms may be difficult to detect for several weeks after medication is discontinued. Fecal specimens should be collected in clean, wide-mouthed containers; most laboratories use a waxed, cardboard half-pint container with a tight-fitting lid. The specimen should not be contaminated with water that may contain free-living organisms. Contamination with urine should also be avoided to prevent destruction of motile organisms in the specimen. All specimens should be identified with the patients name, physicians name, hospital number if applicable, and the time and date collected. Every fecal specimen represents a potential source of infectious material (e.g., bacteria, viruses, parasites) and should be handled accordingly. The number of specimens required to demonstrate intestinal parasites will vary depending on the quality of the specimen submitted, the accuracy of the examination performed and the severity of the infection. For a routine examination for parasites before treatment, a minimum of three fecal specimens is recommended: two specimens collected from normal movements and one specimen collected after cathartic, such as magnesium sulfate or Fleet PhosphoSoda. Many organisms do not appear in fecal specimens in consistent numbers on a daily basis; thus collection of specimens on alternate days tends to yield a higher

percentage of positive findings. The series of three specimens should be collected within no more than 10 days and a series of six within no more than 14 days. The number of specimens to be examined after therapy will vary depending on the diagnosis; however, a series of three specimens collected previously outlined is usually recommended. A patient who received treatment for a protozoan infection should be checked 3 to 4 weeks after therapy. Patients treated for helminth infections may be checked 1 to 2 weeks after therapy and those treated for Taenia infections 5 to 6 weeks therapy. Since the age of the specimen directly influences the recovery of protozoan organisms, the time the specimen was collected should be recorded on the laboratory request form. Freshly passed specimens are mandatory for the detection of trophic amebae or flagellates. Liquid specimens should be examined within 30 minutes of passage (not 30 minutes from the time they reach the laboratory), or the specimen should be placed in polyvinyl alcohol (PVA)fixative or another suitable preservative. Semiformed or soft specimens should be examined within 1 hour of passage; if this is not possible, the stool material should be preserved. If these limits cannot be met, portions of the sample should be preserved. Stool specimens should not be held at room temperature but should be refrigerated at 3 to 5 C and stored in closed containers to prevent dessication. At this temperature, eggs, larvae and protozoan cysts remain viable for several days. Fecal specimens should never be incubated or frozen before examination. When the proper criteria for collection of fecal specimens are not met, the laboratory should request additional samples.

References: Baron E., Peterson, L. Finegold,S(1994). Diagnostic Microbiology 9th edition. Missouri:Von Hoffman Press, Inc. 2. Describe various stool preservation methods and cite the advantages and disadvantages of each. 1. PVA Fixative (Polyvinyl Alcohol) It is consists of polyvinyl alcohol, glycerin, glacial acetic acid, and Schaudinns solution. The PVA method of preserving and fixing fecal smear on slides was introduced by Brooke and Goldman in 1949.

 It is convenient to dispense the PVA fixative solution in screw-capped bottles, in approximately 5ml quantities. To this volume of fixative, approximately 1 g of feces may be added. To prepare slides for staining, shake the preserved specimen well or mix contents with two applicator sticks. Pour some of the PVA mixture onto blotting paper and allow to stand for a few minutes. Apply stool material to slide and dry for 2 hours at 37 C, or overnight at room temperature, then stain with trichrome. Advantage/s: It is especially effective in detecting trophozoites in diarrheic and mushy stools that would escape detection by other techniques. Even formed stools may contain trophozoites of amebae and cysts. A permanent stained smear can be easily prepared from PVA-preserved material. It is highly recommended as a means of preserving protozoan cysts and trophozoites for examination at a later time. This fixative remains stable for long periods (months to years) when kept in sealed containers at room temperature. Disadvantage/s: The fixative will keep indefinitely but must not be subjected to extremes of temperature. When placed in water or hematoxylin stain, PVA film may be lost from the slide. 2. Formalin 10% formalin is prepared by adding 900ml. of 0.85% of NaCl solution to 100 ml of Formaldehyde (USP) and 5% formalin is is prepared by adding 950ml. of 0.85% of NaCl solution to 50 ml of Formaldehyde (USP). Formalin is used in the ratio of at least three parts formalin to one part of fecal material; thorough mixing of the fresh specimen and fixative is necessary to ensure good preservation. Advantage/s: Protozoan cysts, helminth eggs, and larvae are well preserved for long periods in 5% or 10% formalin.

 Disadvantage/s: The fixative permits the examination of the specimen as wet mount only, a technique much less accurate than the stained smear for the identification of protozoa. It is impractical for most laboratories to use hot formalin but it can be used to prevent further development of helminth eggs to the infective stage.

3. MIF Solution (Merthiolate (thimerosal)-iodine-formalin) It is prepared in two separate stock solutions to be mixed immediately before use. Solution I, made up in 480 ml amounts, consists of 250 ml of distilled water, 200 ml of thinerosal (Merthiolate), 25 ml of formaldehyde U.S.P., and 5 ml of glycerol. Solution II is Lugols solution (5% iodine in 10% potassium iodide solution in distilled water. Both solutions are stored in tightly stoppered brown glass bottles. For immediate use the two solutions are combined, 94 parts of solution I with 6 parts of solution II. Into a vial containing 9 ml of MIF, 1 ml (1g) of the fecal specimen, measured by displacement, is added and mixed by stirring and shaking until completely suspended. It is introduced by Sapero and Lawless. Advantage/s: It can be used as stain preservative for most types and stages of intestinal parasites and may be helpful in field surveys. Helminth eggs, larvae, and certain protozoa can be identified without further staining in wet mounts, which can be prepared immediately after fixation or several weeks later. It can be useful for field collections. All microscopic parasite cysts, egges, and larvae are well preserved and adequately stained for diagnosis as accurately as is possible in wet smear preparations. Disadvantage/s: The fixative permits the examination of the specimen as wet mount only, a technique much less accurate than the stained smear for the identification of protozoa. MIF method has the instability of its iodine component. Although a technique of concentrating cysts and eggs of the parasites in MIFpreserved specimens has been described, experience has shown that it is unreliable for specimens that have been stored more than a few days.

4. SAF Fixative (Sodium acetate-acetic acid-formalin) It contains formalin combined with sodium acetate, which acts as a buffer. It is a liquid fixative very similar to 10% aqueous formalin. The fixative solution is made up as follows: 1.5 of sodium acetate, 2ml glacial acetic acid, 4ml 0f 40% formaldehyde and 92.5 ml of distilled water. This fixative is more liquid than PVA, and the preserved specimen must be centrifuged after straining through gauze and the sediment used to prepare smears for stainig; adherence to the glass slide may be improved if the slide is coated with albumin. After drying, the slides may be placed in 70 % alcohol. Advantage/s: A permanent stained smear can be easily prepared from PVA-preserved material. Disadvantage/s: When the sediment is used to prepare permanent stained smears, one may have some difficulty in getting material adhere to the slide.

References: Baron E., Peterson, L. Finegold,S(1994). Diagnostic Microbiology 9th edition. Missouri:Von Hoffman Press, Inc. Beaver, P. ,Jung R., Cupp,E.(1984). Clinical Parasitology 9th edition. Philadelphia: Lea and Febiger Markell,E., John,D. (1992).Medical Parasitology 7th edition.Philadelphia: W.B. Saunders Company