ParaCon™

Cat. no. PK48 Fecal Parasite Concentration Kit 48 tests/kit
Cat. no. PK96 Fecal Parasite Concentration Filter-Funnels 96 filter-funnels/box

INTENDED USE

ParaCon™ is a test kit designed for concentrating protozoan cysts, helminth eggs and larvae, coccidian oocysts, and microsporidian spores from fecal specimens. This methodology is similar to the Ritchie formalin ether sedimentation concentration procedure. The ParaCon™ filter-funnel device provides a standard mesh size and allows filtration of fecal material without clogging.

SUMMARY

Diagnosis of intestinal parasite infections requires appropriate collection, processing, and identification of protozoan cysts, helminth eggs and larvae, coccidian oocysts, and microsporidian spores in fecal material.(1-11) ParaCon™ uses existing methodology and standardized components to accommodate specimen preparation for subsequent examination.

This kit utilizes the following components to achieve recovery of parasites from preserved fecal material:

KIT CONTENTS

PK48 contains the following components:

PK96 contains:

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 15-30ºC. Do not use if there are signs of deterioration or if the expiration date has passed.

PRECAUTIONS

PROCEDURE

Specimen Collection: Stool specimens, fresh or preserved in 5 or 10% formalin, sodium acetate-acetic acid formalin (SAF), merthiolate-iodine-formalin (MIF), or any of the single vial collection systems may be used. The ratio of stool specimen to preservative must be in the ratio of 1:3 to 1:5.

Specimen Preparation

1. The stool specimen should be thoroughly mixed in the preservative in order to break up any lumps. Vigorous shaking of the stool/preservative mixture is sufficient for adequate mixing (except for hard-formed stools - stir using applicator sticks).

2. Let the stool preservative mixture stand for at least 30 minutes at 15-30ºC. to allow adequate fixation.

3. Observe the stool/preservative mixture for mucus and/or fecal lumps. If present, add 2 drops of Triton® X-100 to the stool/preservative vial and shake vigorously. The Triton® X-100 will free additional parasites by reducing the adhesive forces in mucus and fecal lumps.

Concentration Procedure

1. Place ParaCon™ filter-funnel assembly into one of the 15ml centrifuge tubes. Pour sufficient volume of the stool/preservative mixture through the funnel assembly such that 1ml of sediment will be deposited at the base of the centrifuge tube after initial centrifugation. Approximately 3ml stool/preservative mixture should be filtered through the funnel device for a heavy stool suspension, and up to 10ml is required for a light stool suspension (as would be obtained from a watery stool specimen). Do not force fecal material through the ParaCon™ funnel assembly.

2. Discard the ParaCon™ filter-funnel assembly. Add physiological saline (0.85% NaCl) or 5-10% formalin to the 14ml line of the centrifuge tube.

3. Centrifuge at 500xG (2,000rpm) for 10 minutes.

4. Decant the supernatant fluid, retaining fecal sediment.

5. Suspend the fecal sediment in 8ml of 10% formalin or SAF. Add 4ml of ethyl acetate (Cat. no. ET105) and place cap on the centrifuge tube. If the amount of sediment left in the bottom of the tube is very small or the original specimen contained a lot of mucus, do not add ethyl acetate; merely add the formalin, spin, decant and examine the remaining sediment.(4)

6. Invert the centrifuge tube and shake for 30 seconds.

7. With the tube directed away from your face, slowly remove cap to release pressure built up after shaking. Replace cap and tighten.

8. Centrifuge at 500xG (2,000rpm) for 10 minutes.

9. Layers seen in the centrifuge tube after centrifugation will be as follows from the top down:

a.
Ethyl acetate (approximately 3ml)
b.
Fecal debris and fat (approximately 0.5ml)
c.
Discolored formalin or SAF (approximately 9ml)
d.
Fecal sediment (approximately 0.5ml)

10. Holding the centrifuge tube vertically, free the layer of fecal debris and fat by circling the rim of the layer with a wood applicator stick (Cat. no. 807).

11. Decant the layers of ethyl acetate, fecal debris and fat, and discolored formalin. Continue to hold the centrifuge tube in the decanting position while thoroughly cleaning the inside of the tube of remaining ethyl acetate and debris with a cotton swab (Cat. no. 958702). If the sediment is too dry after swabbing the tube, add several drops of saline before preparing the wet smear for examination.

12. Transfer a portion of the fecal sediment to a clean microscope slide and add a 22x22mm coverslip and examine microscopically. Examination of the slide should be performed before the smear dries.

13. Systematically scan with the 10X objective. The entire coverslip area should be examined under low power (total magnification of X100). If something suspicious is seen, the 40X objective can be used for more detailed study. At least one-third of the coverslip should be examined under high dry power (total magnification of X400) even if nothing suspicious has been seen. Iodine may be added to enhance morphological detail. It is not practical to examine these wet mounts using oil immersion objectives.

14. Portions of the sediment can also be used for the preparation of smears for staining (modified acid-fast stains for coccidia or modified trichrome stains for microsporidia). Sediment is also recommended as the specimen of choice for the Direct Fluorescent Antibody (DFA) and fecal immunoassay procedures (to enhance the sensitivity of organism detection).

INTERPRETATION OF RESULTS

Protozoan trophozoites and/or cysts and helminth eggs and larvae may be seen and identified. Protozoan trophozoites are less likely to be seen. Consult listed references for information regarding identification of intestinal parasites, including the coccidia and microsporidia.(4-5)

LIMITATIONS

Do not use if product performance does not meet specified standards. See Quality Control section for specified standards.

If there is excessive ethyl acetate in the smear of the sediment prepared for examination, there will be bubbles present which will obscure the material that you are trying to see.(4)

Too much or too little sediment will result in an ineffective concentration.(4)

If the centrifugation time at the proper speed is reduced, some of the organisms (coccidian oocysts or microsporidian spores) may not be recovered in the sediment.(1,3-5)

Results obtained with wet smears should usually be confirmed by permanent stained smears. Some protozoa are very small and difficult to identify as to the species with just the direct wet smear. Also, special stains are sometimes necessary for organism identification.(4)

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies such as cotton swabs, glass microscope slides, coverslips, microscopes, centrifuge, pasteur pipets, collection container with preservative, iodine (Cat. no. Z66), ethyl acetate (Cat. no. ET105), and formalin (Cat. no. 575A128), etc., are not provided.

QUALITY CONTROL

User Quality Control

Visibly examine funnel assembly for presence of plastic filter. Do not use if filter is absent or if filter spaces are blocked. Examine reagent bottle of 10% Triton® X-100 for cloudiness. Do not use if reagent appears cloudy. Check reagent bottle of 10% Triton® X-100 for expiration date. Do not use if expiration date has passed.

The microscope should be calibrated (within the last 12 months), and the objectives and oculars used for the calibration procedure should be in place on the microscope when objects are measured.

ParaCon™

ParaCon™ (Cat. no. PK48).

REFERENCES

1. Clinical and Laboratory Standards Institute. 2005. Procedures for the Recovery and Identification of Parasites from the Intestinal Tract, Approved Guideline, M28-2A. Clinical and Laboratory Standards Institute, Villanova, PA.

2. Deplazes, P., L.S. Garcia and R.Y. Shimizu. 2007. Diagnostic Parasitology: Specimen Collection, Transport, and Processing, p. 1995-2012. Manual of Clinical Microbiology, 9th ed. ASM Press, Washington D.C.

3. Garcia, L.S. (Coordinating Editor). 2003. Selection and Use of Laboratory Procedures for Diagnosis of Parasitic Infections of the Gastrointestinal Tract, Cumitech 30A, ASM Press, Washington, D.C.

4. Garcia, L.S. 2009. Practical Guide to Diagnostic Parasitology, 2nd ed. ASM Press, Washington, D.C.

5. Garcia, L.S. 2007. Diagnostic Medical Parasitology, 5th ed. ASM Press, Washington D.C.

6. 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.

7. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

8. Melvin, D.M. and M.M. Brooke. 1980. Laboratory Procedures for the Diagnosis of Intestinal Parasites. U.S.D.H.E.W., CDC, Atlanta, GA.; 80:8282.

9. Perry, J.L., J.S. Mathews and G.R. Miller. 1990. Parasite Detection Efficiencies of Five Stool Concentration Systems. J. Clin. Microbiol.; 28:1094-1097.

10. Ritchie, L.S. 1948. An Ether Sedimentation Technique for Routine Stool Examination. Bull. U.S. Army Med. Dept.; 8:326.

11. Young, K.H., S. Bullock, C.M. Melvin and C.L. Sprull. 1979. Ethyl Acetate as a Substitute for Diethyl Ether in the Ether-Formalin Sedimentation Technique. J. Clin. Microbiol. ; 10:852-853.

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