Strongyloides stercoralis larvae are usu-ally the only larvae found in stool speci-mens. Depending on bowel transit time and the condition of the patient, rhabditi-form and, rarely, filarirhabditi-form larvae may be present. If there is delay in examination of the stool, then embryonated ova and larvae of hookworm may be present. Culture of feces for larvae is useful to (i) reveal the presence of larvae when they are too scanty to be detected by concentration methods, (ii) distinguish whether the
in-fection is due to Strongyloides or hook-worm on the basis of rhabditiform larval morphology by allowing hookworm eggs to hatch and release first-stage larvae, and (iii) allow development of larvae into the filariform stage for further differentiation.
The use of certain fecal-culture meth-ods (sometimes referred to as coprocul-ture) is especially helpful in detecting light infections of hookworm, Strongyloides spp., and Trichostrongylus spp. and for specifically identifying parasites.
Addi-tionally, such techniques are useful for ob-taining a large number of infective-stage larvae for research purposes. Three culture techniques and one enhanced-recovery method are described in this section.
Occasionally, it is necessary to exam-ine stool specimens for scolices and pro-glottids of cestodes and for adult nema-todes and tremanema-todes to confirm the diagnosis and/or to identify a species. A method for the recovery of these stages is also described in procedure 9.5.6.
P R E A N A L Y T I C A L C O N S I D E R A T I O N S
I. PRINCIPLE The Baermann technique uses a special apparatus and relies on the principle that active larvae will migrate out of a fecal specimen that has been placed on a wire mesh covered with several layers of gauze (1, 2). Larvae migrate through the gauze into the water and settle to the bottom of the funnel, where they can be collected
and examined. Modifications to simplify the procedure have been reported else-where (5). Besides being useful for diag-nosis from stool specimens directly or af-ter enhancement by culture, this technique can be used by epidemiologists to examine soil specimens for larvae.
II. SPECIMEN The specimen must be fresh stool that has not been refrigerated.
III. MATERIALS A. Reagent
䊓 Indicate the expiration date on the label and in the work record or on the manufacturer’s label.
Bleach (full strength) B. Supplies
1. Glass funnel (6 in. across at the mouth)
2. Rubber tubing to fit end of funnel 3. Clamp
4. Wire gauze screen or nylon filter 5. Centrifuge tubes (15-ml capacity) 6. Glass slides (1 by 3 in. or larger) 7. Disposable glass or plastic
pi-pettes
8. Wooden applicator sticks (non-sterile)
9. Gauze
10. Ring stand and ring for holding funnel
11. Glass beaker (500 ml) C. Equipment
1. Binocular microscope with 10⳯, 40⳯, and 100⳯ objectives (or the approximate magnifications for low-power, high dry power, and oil immersion examination)
2. Oculars should be 10⳯. Some workers prefer 5⳯; however, over-all smover-aller magnification may make final organism identifications more
A N A L Y T I C A L C O N S I D E R A T I O N S
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 confirmation of larval iden-tification.
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 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. If possible, use a fresh fecal specimen that has been obtained after administration of a mild saline cathartic, not a stool softener. Soft stool is recommended, but any fresh fecal specimen is acceptable.
C. Set up a clamp supporting a 6-in.-mouth glass funnel. Attach rubber tubing and a pinch clamp to the bottom of the funnel. Place a collection beaker underneath (see Fig. 9.5.1–1).
D. Place wire gauze or a nylon filter over the top of the funnel (or resting within the funnel), and then place a pad consisting of two layers of gauze over that.
E. Close the pinch clamp at the bottom of the tubing, and fill the funnel with tap water until it just soaks the gauze padding.
F. Spread a large amount of fecal material on the gauze padding so the specimen is in contact with water. If the fecal material is very firm, emulsify in water.
G. Allow the apparatus to stand for 2 or more hours, draw off 10 ml of fluid into the beaker by releasing the pinch clamp, centrifuge for 2 min at 500⳯ g, and examine the sediment under the microscope (100⳯ and 400⳯) for the presence of motile larvae. Make sure that the end of the tubing is well inside the beaker before slowly releasing the pinch clamp. Infective larvae may be present; wear gloves when performing this procedure.
H. If there are no larvae seen, allow the apparatus to stand at room temperature for 8 to 12 h and examine additional fluid (after centrifugation). Discard after 12 h.
VI. RESULTS A. Larval nematodes (hookworm, Strongyloides spp., or Trichostrongylus spp.) may be recovered.
B. Both infective and noninfective Strongyloides larvae may be recovered, par-ticularly in a heavy infection.
IV. QUALITY CONTROL
Larval-Stage Nematodes: Baermann Technique 9.5.1.3
Figure 9.5.1–1 Diagram of the Baermann apparatus used for recovery of larval-stage nematodes (from reference 2).
P O S T A N A L Y T I C A L C O N S I D E R A T I O N S
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 stercoralis larvae detected by fecal culture.
VIII. PROCEDURE NOTES 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. Preserved fecal specimens or specimens obtained after a barium meal are not suitable for processing by this method.
C. Wear gloves when you perform this procedure.
D. When you release the pinch clamp, do it slowly to prevent splashing.
E. For the same reason, hold the end of the tubing toward the bottom of the beaker.
F. Infective larvae may be found any time after the fourth day and occasionally after the first day in heavy infections. Caution must be exercised in handling the fluid, gauze pad, and beaker to prevent accidental infection. Always remem-ber to wear gloves.
VII. REPORTING RESULTS
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 (2–4). 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.
REFERENCES 1. Baermann, G. 1917. Eine einfache Methode zur Auffindung vor Ankylostomum (Nemato-den) Larven in Erdproben. Meded. Geneesk Laborat. Weltever Feestbundel, p. 41.
2. Garcia, L. S. 2001. Diagnostic Medical Par-asitology, 4th ed., p. 786–795. ASM Press, Washington, D.C.
3. Melyin, D. M., and M. M. Brooke. 1985.
Laboratory Procedures for the Diagnosis of Intestinal Parasites, p. 163–189. U.S. Depart-ment of Health, Education, and Welfare pub-lication no. (CDC) 85-8282. U.S. Government Printing Office, Washington, D.C.
4. 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.
5. Watson, J. M., and R. Al-Hafidh. 1957. A modification of the Baermann funnel tech-nique and its use in establishing the infection potential of human hookworm carriers. Ann.
Trop. Med. Parasitol. 51:15–16.
Ash, L. R., and T. C. Origel. 1987. Parasites: a Guide to Laboratory Procedures and Identifica-tion, 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.
SUPPLEMENTAL READING
9.5.2.1