Intended Use

MICRO-SED™ is a concentration system for the recovery of eggs, larvae, and protozoa from preserved fecal specimens. MICRO-SED™ is designed to be used with 5% or 10% formalin, SAF, PVA, and Z-PVA preserved material. When used with Para-Fix™ collection vials, MICRO-SED™ provides a convenient and reproducible method for detecting parasites even when present in very low numbers.

Summary and Explanation

The diagnosis of parasitic infection is confirmed by the recovery of helminth larvae and eggs, protozoan trophozites and cysts, coccidian oocysts, and microsporidian spores. A concentration procedure should be performed as a routine part of a complete examination for parasites. Concentration procedures permit the detection of organisms present in small numbers, that may be missed using only the direct wet mount. Organisms that can generally be identified using a concentration procedure include: helminth eggs and larvae; cysts of Giardia lamblia, Entamoeba histolytica/Entamoeba dispar, Entamoeba coli, Endolimax nana, Blastocystis hominis and Iodamoeba butschlii; and oocysts of Isospora belli. The identification of other protozoa should be considered tentative, and confirmed with a permanently stained smear.

Kit Contents

• 50 concentrator units
• 50-50ml centrifuge vials
• 50 vial caps
• 1 bottle surfactant
• 1 instruction sheet

Materials Not Provided

• ethyl acetate
• cotton tip applicators
• centrifuge
• transfer pipettes
• microscope slides and coverslips
• microscope
• physiological saline
• applicator sticks
• Lugol’s iodine

Specimen Collection

1. A well preserved specimen is critical to the detection process. The MICRO-SED™ is designed to be used with the Para-Fix™ collection kits. For collection procedures see package insert provided with each kit.
2. For the concentration of fresh material collected in the Para-Fix™ clean vial, add 3-5g of stool sample to clean vial and then add 15ml of 5% or 10% formalin or SAF. Mix well, and allow to stand for at least thirty minutes before processing.
3. If a permanent stained slide is going to be prepared from the SAF sample, remove some of the preserved material prior to concentration or the concentrated stool can be used to prepare this smear.

Specimen Processing

1. Remove the cap from the specimen vial and add 8-10 drops of surfactant. Recap the vial, making sure lid is securely fastened.
2. Mix the contents of the vial by shaking vigorously, or vortexing for 30 seconds.
3. Place a filter funnel in one of the 15ml centrifuge vials.
4. The approximate specimen volumes will be 3ml for thick specimens to 10ml for thin specimens. Filter a sufficient volume of specimen through the funnel so that 1ml of sediment remains after initial centrifugation.
5. Do not force fecal material through the filter funnel. After filtration is complete, discard filter funnel using established laboratory procedures for fecal specimens.
6. Add physiological saline* to the 14ml mark, on the centrifuge vial. Centrifuge at 500xg (1800-2500 rpm) for 10 minute.
7. Decant the supernatant, retaining the fecal sediment at the bottom of the vial.
8. Add physiological saline* to bring the tube contents to ~9ml.
9. Add 3-4ml of ethyl acetate (or other ether substitute) to the 15ml centrifuge tube. Recap the tube with the cap provided with the kit.
10. Shake vigorously for 30 seconds. If diethyl ether is used (not recommended) pressure may build up in the vial during shaking, and the cap should be carefully loosened after shaking to release the pressure, then retightened.
11. Centrifuge at 500 xg (1800-2500 rpm) for 10 min.
12. Carefully remove the cap. The resulting solution should have four layers:
    1. Top: ethyl acetate or ethyl ether
    2. Second: debris plug
    3. Third: formalin
    4. Fourth: sediment
13. Ring the debris layer with an applicator stick to loosen the debris.
14. Invert the tube to pour off the supernatant and debris layer. While the tube is still inverted, wipe the sides of the tube with one or two cotton tip applicators to remove ethyl acetate or debris left behind. Failure to remove the excess ethyl acetate may result in the formation of solvent bubbles in the wet mount. The sediment at the bottom of the vial will contain the parasites.
15. Resuspend the pellet at the bottom of the tube with saline*. If using SAF, do not resuspend the sediment. Remove some sediment and prepare smears for permanent staining prior to resuspension of the remaining sediment for wet preparation examination.
16. To prepare a wet mount, draw a sample from the resuspended material with a capillary or transfer pipette. Place one or two drops on a microscope slide and cover with a coverslip. Examine immediately.
17. If an iodine mount is preferred, place one drop of Lugol’s iodine on a slide, and one drop of the resuspended material. Place a coverslip on the slide and examine immediately.
18. If smears will be prepared for special staining (Cryptosporidium parvum, Isospora belli, Cyclospora cayetanensis, or the microsporidia), the remaining sediment can be used for making the smears. Note: Steps 6 and 7 may be omitted if laboratory chooses to only wash specimen once.

*5% formalin, 10% formalin or SAF may be used in place of physiological saline.

Precautions

1. Ethyl acetate and diethyl ether are flammable. Use in a well ventilated area. Keep away from direct flame. Avoid contact of the solution with skin and eyes. Should contact occur flush with running water. Avoid breathing fumes.
2. Every sample should be treated as a potential source of infection. Good laboratory practice should be followed at all times. The use of gloves and hand washing is recommended.

Stability

When stored at room temperature, the product is stable for two years from the date of manufacture. The user should verify this examining the concentrator unit for cracks, and the surfactant for bacterial or fungal contamination.

Bibliography

1. ASMT, 1978. Recommended Procedures for the Examination of Clinical Specimens Submitted for the Diagnosis of Parasitic Infections. Am. J. Med. Technol., 44:1101-1106.
2. Brooke, M.M., 1974 “Intestinal and Urogenital Protozoa”, Manual of Clinical Microbiology, ASM Washington D.C., 2nd Edition, 582-601
3. Garcia, L.S., 2001. Diagnostic Medical Parasitology, 4th ed.; ASM Press: Washington D.C.
4. Garcia, L.S. and R. Shimizu, 1981. Comparison of Clinical Results for the use of Ethyl Acetate and Diethyl Ether in the Formalin-Ether Sedimentation Technique Performed on Polyvinyl Alcohol Preserved Specimens. J. Clin. Microbiol., 13:709-713
5. Melvin,D.M. and M.M. Brooke, 1980. “Laboratory Procedures for the Diagnosis of Intestinal Parasites”, U.S. D.S.E.W., 79:8282, CDC Atlanta, GA, 23-65
6. Yang, J., and Scholten, T., 1977. A Fixative for Intestinal Parasites Permitting the Use of Concentration and Permanent Staining Procedures. Am. J. Clin. Pathol., 67:300-304
7. Young, Kirk H., et al, 1979. Ethyl Acetate as a Substitute for Diethyl Ether in the Formalin-Ether Sedimentation Technique. J. Clin. Microbiol., 10:852-853.