9.5.2
“Culture” of Larval-Stage Nematodes: Harada-Mori Technique
PREANALYTICAL CONSIDERATIONS
I.
PRIN PR INCI CIPL PLE E
This filter filter paper paper test tube cultu culture re techn technique ique was ini initia tially lly int introd roduce uced d by Har Harada ada and Mori in 1955 (2) and was later modified by others (3, 5). The technique employs a filte fil terr pa pape perr to wh whic ich h fe feca call ma mate teri rial al is added and a test tube into which the filter
paper is inserted. Moisture is provided by adding water to the tube. The water continuously soaks the filter paper by capillary action. Incubation under suitable condition dit ionss fav favors ors hat hatchi ching ng of ova and and/or /or
II.. II
The specimen must be fresh stool that has not been refrigerated.
SPEC SP ECIM IMEN EN
development of larvae. larvae. Fecal specimens specimens to be cul cultur tured ed sho should uld not be ref refri riger gerate ated, d, since some parasites (especially Necator americanus) are susceptible to cold and may fail to develop after refrigeration.
Observe standard precautions.
III.. III
MATERI MAT ERIALS ALS
A. Reagents Reagents None B. Suppl Supplies ies 1. Disposable glass or plastic pipettes 2. Glass slides (1 by 3 in. or larger) 3. Coverslips (22 by 22 mm; no. 1 or larger) Filter er paper paper (40 or 42 is is fine; weigh weightt 4. Filt is not critical) 5. Wooden applicator sticks (nonsterile) 6. 15-ml-capacity conical tube 7. Forceps
C. Equipment Equipment 1. Binocula Binocularr micr microscope oscope with 10, 40, and 100 objectives (or the approx app roxima imate te mag magnifi nificat cation ionss for low-power, high dry power, and oil immersion examination) Ocul ular arss sh shou ould ld be 10. So Some me 2. Oc workers prefer 5; however, overall smaller smaller magnification magnification may make final organism identifications more difficult.
A N A L Y T I C A L C O N S I D E R A T I ON S
IV.
QUALI QU ALITY TY CO CONTR NTROL OL
A. To ensure reliable results, follow routine procedures for optimal collection and handling of specimens for parasitologic examination. B. If available, examine known positive and negative samples of stools (from laboratory animals) to make sure that the procedure is precise. C. Review larval diagrams (any parasitology text) for confirmation of larval identification. D. The microscope should be calibrated, and the objectives and oculars used for the calibration procedure should be used for all measurements on the microscope. Post the calibration factors for all objectives on the microscope for easy access (multiplication factors can be pasted right on the body of the microscope) (see procedures 9.1 and 9.3.2). Although there is not universal agreement, the 9.5.2.1
9.5.2.2
Parasitology
IV. QU QUALI ALITY TY CON CONTRO TROL L (continued)
microscope should probably be recalibrated once each year. This recommendation dat ion sho should uld be con consid sidere ered d wit with h hea heavy vy use or if the microsc microscope ope has bee been n bumped or moved multiple times. If the microscope does not receive heavy use, then recalibration is not required on a yearly basis. E. Record all QC results.
V.
A. Wear gloves when performing this procedure. B. Cut a narr narrow ow (3/8 by 5 in.) strip of filter paper, paper, and taper it slightly at one end. Smear 0.5 to 1 g of feces in the center of the strip. C. Add 3 to 4 ml of distilled water to a 15-ml conical centrifuge tube. filter paper strip into into the tube so that the tapered end end is near the bottom D. Insert the filter of the tube. The water level should be slightly (0.5 in.) below the fecal spot. It is not necessary to cap the tube. However, a cork stopper or a cotton plug may be used (see Fig. 9.5.2–1). E. Allow the tube to stand upright in a rack at 25 to 28 C. Add distilled water to maintain the original level (usually evaporation takes place over the first 2 days, but then the culture becomes stabilized). F. Keep the tube for 10 days, and check daily by withdrawing a small amount of fluid from the bottom of the tube. Prepare a smear on a glass slide, cover with a coverslip, and examine with the 10 objective. G. Examine the larvae for motility and typical morphological features to reveal Trichostrongyluss larvae are present. whether hookworm, Strongyloides, or Trichostrongylu
PROCED PRO CEDUR URE E
Figure 9.5.2–1 Diagram of Harada-Mori culture system (from reference 1).
VI.. VI
RESU RE SULT LTS S
P O S T A N A L Y T I CA L
A. Larval nematodes (hookworm, Strongyloides spp., or Trichostrongylus spp.) may be recovered. B. If Strongyloides organisms are present, free-living stages and larvae may be found after several days in culture.
C O N S I D E R A T I O NS
VII.
REPORTIN REPO RTING G RESU RESULTS LTS
A. Report your findings as “No larvae detected” if no larvae could be detected at the end of incubation. B. Report larvae detected by fecal culture. Example: Strongyloides Example: Strongyloides stercoralis larvae detected by fecal culture.
VIII.. VIII
PROCEDU PROC EDURE RE NOTE NOTES S
A. It is often difficult to observe details in rapidly moving larvae. If desired, use slight heating or a drop of iodine or formalin to kill the larvae. B. Infective larvae may be found anytime after day 4 or even on day 1 in a heavy infection. Since infective larvae may migrate upward as well as downward on the filter paper strip, be careful when handling the fluid and the paper strip itself to prevent infection. Handle the filter paper with forceps. C. It is important to maintain the original water level to keep optimum humidity. D. Preserved fecal specimens or specimens obtained after a barium meal are not suitable for processing by this method. E. Wear gloves when you perform this procedure.
Larval-Stage Nematodes: Harada-Mori Technique
9.5.2.3
IX. LIM IX. LIMITA ITATIO TIONS NS OF THE PROCEDURE
A. This technique allows both parasitic and free-living forms of nematodes to develop. If specimens have been contaminated with soil or water containing these forms, it may be necessary to distinguish parasitic from free-living forms. This distinction is possible since parasitic forms are more resistant to slight acidity than are free-living forms. Proceed as follows (4, 6). Add 0.3 ml of concentrated hydrochloric acid per 10 ml of water containing the larvae (adjust the volume to achi achieve eve a 1:30 dilution dilution of acid acid). ). FreeFree-livi living ng nema nematodes todes are killed, while parasitic species live for about 24 h. B. Specimens that have been refrigerated are not suitable for culture. Larvae of certain species are susceptible to cold. C. This method requires too much time to be clinically useful, but it can be used for field or survey studies where rapid results are not that important. Due to the time factor, this method, as well as the petri dish-filter paper slant method (procedure 9.5.3), may be replaced by the agar plate method (procedure 9.5.4), which is not only more rapid but also more sensitive.
REFERENCES
Garcia, L. S. 2001. Diagnostic Medical Par1. Garcia, asitology, 4th ed., p. 786– 786–795. 795. ASM Press, Washington,, D.C. Washington Harada, U., and O. Mor Mori. i. 1955 2. Harada, 1955.. A ne new w method for culturing hookworm. Yonago Acta 1: 177–179. Med. 1: Med. Hsieh, eh, H. C. 1962. A test 3. Hsi test-tub -tubee filter filter-pape -paperr method for the diagnosis of Ancylostoma duodenale, dena le, Neca Necator tor ameri americanus canus,, and Strongyloides stercoralis. W. H. O. Tech. Rep. Ser. 255: 27–30. 4. Melvin, D. M., and M. M. Brooke. 1985. Laboratory Procedures for the Diagnosis of Intestinal Parasites, p. 163–189. U.S. Department of Health, Education and Welfare publication no. (CDC) 85-8282. U.S. Government Printing Office, Washington, D.C.
SUPPLEMENTAL SUPPLEMENTA L READING
Ash, L. R., and T. C. Orihel. 1987. Parasites: a Guide to Laboratory Procedures and Identification, p. 59–66. ASCP Press, Chicago, Ill. Markell, E. K., M. Voge, and D. T. John. 1986. Medical Parasitology, 6th ed., p. 348. The W. B. Saunders Co., Philadelphia, Pa.
Hayashi, H. Tanak Tanaka, a, and R. 5. Sasa, M., S. Hayashi, Shirasaka. 1958. Application of test-tube cultivation method on the survey of hookworm and related human nematode infection. Jpn. J. Exp. Med. Med. 28: 28: 129–137. 6. Shorb, D. A. 1937. A method of separating Haemonchus contortus infect inf ective ive lar larvae vae of Haemonchus (Trichostrongylidae) (Trichostrong ylidae) from free living nematodes. Proc. Helminthol. Soc. Wash. 4: 52.
