9.5.2.

1
9.5.2 Culture of Larval-Stage
Nematodes: Harada-Mori Technique
P R E ANAL Y T I CAL CONS I DE R AT I ONS
I. PRINCIPLE
This lter paper test tube culture technique
was initially introduced by Harada and
Mori in 1955 (2) and was later modied
by others (3, 5). The technique employs a
lter paper to which fecal material is
added and a test tube into which the lter
paper is inserted. Moisture is provided by
adding water to the tube. The water con-
tinuously soaks the lter paper by capil-
lary action. Incubation under suitable con-
ditions favors hatching of ova and/or
development of larvae. Fecal specimens to
be cultured should not be refrigerated,
since some parasites (especially Necator
americanus) are susceptible to cold and
may fail to develop after refrigeration.
The specimen must be fresh stool that has not been refrigerated.
A. Reagents
None
B. Supplies
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)
4. Filter paper (40 or 42 is ne; weight
is not critical)
5. Wooden applicator sticks (nonster-
ile)
6. 15-ml-capacity conical tube
7. Forceps
C. Equipment
1. Binocular microscope with 10,
40, and 100 objectives (or the
approximate magnications for
low-power, high dry power, and oil
immersion examination)
2. Oculars should be 10. Some
workers prefer 5; however, over-
all smaller magnication may make
nal organism identications more
difcult.
ANAL Y T I CAL CONS I DE R AT I ONS
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 lab-
oratory animals) to make sure that the procedure is precise.
C. Review larval diagrams (any parasitology text) for conrmation of larval iden-
tication.
D. The microscope should be calibrated, and the objectives and oculars used for
the calibration procedure should be used for all measurements on the micro-
scope. 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
Observe standard precautions.
IV. QUALITY CONTROL
II. SPECIMEN
III. MATERIALS
9.5.2.2 Parasitology
microscope should probably be recalibrated once each year. This recommen-
dation should be considered with heavy use or if the microscope has been
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. PROCEDURE
A. Wear gloves when performing this procedure.
B. Cut a narrow (3/8 by 5 in.) strip of lter 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.
D. Insert the lter paper strip into the tube so that the tapered end is near the bottom
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. 9.5.21).
E. Allow the tube to stand upright in a rack at 25 to 28C. Add distilled water to
maintain the original level (usually evaporation takes place over the rst 2 days,
but then the culture becomes stabilized).
F. Keep the tube for 10 days, and check daily by withdrawing a small amount of
uid 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
whether hookworm, Strongyloides, or Trichostrongylus larvae are present.
Figure 9.5.21 Diagram of Harada-Mori
culture system (from reference 1).
VI. RESULTS
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.
P OS T ANAL Y T I CAL CONS I DE R AT I ONS
A. Report your ndings as No larvae detected if no larvae could be detected at
the end of incubation.
B. Report larvae detected by fecal culture.
Example: Strongyloides stercoralis larvae detected by fecal culture.
A. It is often difcult 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 lter paper strip, be careful when handling the uid and the paper strip itself
to prevent infection. Handle the lter 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.
IV. QUALITY CONTROL
(continued)
VII. REPORTING RESULTS
VIII. PROCEDURE NOTES
IX. LIMITATIONS OF THE
PROCEDURE
A. This technique allows both parasitic and free-living forms of nematodes to de-
velop. 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 achieve a 1:30 dilution of acid). Free-living nematodes 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 eld or survey studies where rapid results are not that important. Due to the
time factor, this method, as well as the petri dish-lter 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
1. Garcia, L. S. 2001. Diagnostic Medical Par-
asitology, 4th ed., p. 786795. ASM Press,
Washington, D.C.
2. Harada, U., and O. Mori. 1955. A new
method for culturing hookworm. Yonago Acta
Med. 1:177179.
3. Hsieh, H. C. 1962. A test-tube lter-paper
method for the diagnosis of Ancylostoma duo-
denale, Necator americanus, and Strongy-
loides stercoralis. W. H. O. Tech. Rep. Ser.
255:2730.
4. Melvin, D. M., and M. M. Brooke. 1985.
Laboratory Procedures for the Diagnosis of
Intestinal Parasites, p. 163189. U.S. Depart-
ment of Health, Education and Welfare pub-
lication no. (CDC) 85-8282. U.S. Government
Printing Ofce, Washington, D.C.
5. Sasa, M., S. Hayashi, H. Tanaka, and R.
Shirasaka. 1958. Application of test-tube cul-
tivation method on the survey of hookworm
and related human nematode infection. Jpn. J.
Exp. Med. 28:129137.
6. Shorb, D. A. 1937. A method of separating
infective larvae of Haemonchus contortus
(Trichostrongylidae) from free living nema-
todes. Proc. Helminthol. Soc. Wash. 4:52.
SUPPLEMENTAL READING
Ash, L. R., and T. C. Orihel. 1987. Parasites: a
Guide to Laboratory Procedures and Identica-
tion, p. 5966. 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.
Larval-Stage Nematodes: Harada-Mori Technique 9.5.2.3