used.
Procedure Limitations for the Filter Paper/Slant Culture Technique (Petri Dish)
1. The filter paper/slant culture 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 (9, 10).
Add 0.3 ml of concentrated hydrochloric acid per 10 ml of water containing the larvae (adjust the volume accordingly to achieve a 1:30 dilution of acid). Free-living nematodes are killed, while parasitic species live for about 24 h.
2. Specimens that have been refrigerated or preserved are not suitable for culture. Larvae of certain species are susceptible to cold environments.
Another way to culture hookworm, Strongyloides, and trichostrongyle larvae is by using a granulated charcoal culture. The conditions of this culture provide an environment for larval development that mimics conditions in nature. It provides an efficient way to harvest large numbers of infective-stage larvae for use in experimental infections.
Quality Control for Charcoal Culture
1. Follow routine procedures for optimal collection and handling of fresh fecal specimens for parasitologic examination.
2. Examine known positive and negative samples of stools (from laboratory animals), if available, to make sure that the procedure is precise.
3. Review larval diagrams and descriptions for confirmation of larval identification.
4. The microscope should be calibrated, and the objectives and oculars used for the calibration procedure should be used for all measurements on the microscope. The calibration factors for all objectives should be posted on the microscope for easy access (multiplication factors can be pasted on the body of the microscope).
5. Record all QC results.
Procedure for Charcoal Culture
1. Mix 20 to 40 g of fresh fecal material in tap water until a thick suspension is obtained.
2. Add this suspension to a storage dish (4 by 3 in.) that is slightly more than half filled with no. 10 granulated hardwood charcoal. Mix thoroughly with a wooden tongue depressor until the fecal suspension is evenly distributed throughout the moistened charcoal. Water can be added to ensure that there is adequate moisture, but do not add so much water than it forms a layer on the bottom of the dish. The surface of the charcoal should glisten.
3. Cover the dish, and place it in the dark (in a drawer or cabinet).
4. Check the dish the next day to make sure that there is still sufficient moisture (i.e., that the charcoal still glistens); if water is needed, sprinkle it on the surface without further mixing.
5. Check the dish each day for moisture content. Caution must be used because moisture will accumulate on the underface of the lid, and it may contain infective-stage larvae.
6. Approximately 5 or 6 days after the culture has been prepared, hookworm and Strongyloides larvae will have reached the infective stage. This can occur earlier if the patient has a heavy infection with numerous larvae in the stool.
7. To harvest the larvae, prepare a round gauze pad of 10 to 12 layer thickness stapled at the edges and cut to fit the dish. Moisten the pad (not dripping wet), and apply it carefully with forceps so that it snugly covers the surface of the charcoal. Do not allow your hands to touch the charcoal—the larvae will be infective!
8. Expose the dish, with lid off, to a light source such as a gooseneck lamp. The lamp should be 6 to 8 in. from the surface of the charcoal. Make sure the lamp is not too close; the larvae can be killed by the heat.
9. After approximately 1 h, the pad can be carefully removed with forceps and inverted onto the surface of water in a pilsner glass filled with water. The gauze pad will remain at the top, and the larvae will now make their way through the pad, enter the water, and fall to the bottom of the glass, where they can be harvested with a pipette after another 30 to 60 min. With care, there will be no charcoal at the bottom of the glass, and the larvae will form a clean sediment.
Results and Patient Reports from Charcoal Culture
Larval nematodes of hookworm, S. stercoralis, or Trichostrongylus spp. may be recovered. If Strongyloides organisms are present, free-living stages and larvae may be found after several days in culture.
1. Report “No larvae detected” if no larvae could be detected at the end of incubation.
2. Report larvae detected by fecal culture.
Example: Strongyloides stercoralis larvae detected by fecal culture
Procedure Notes for Charcoal Culture
1. It is often difficult to observe details in rapidly moving larvae; a drop of iodine or formalin or slight heating can be used to kill the larvae.
2. Infective larvae may be found any time after the fourth day and occasionally after the first day in heavy infections. Since infective larvae may be present, use caution when handling the fluid, gauze pad, and charcoal to prevent infection. Wear gloves when handling the cultures.
3. It is important to maintain the moisture on the charcoal to keep optimum humidity (the charcoal should glisten).
4. Preserved fecal specimens or specimens obtained after a barium meal are not suitable; fresh stool specimens must be obtained.
Procedure Limitations for Charcoal Culture
1. 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 (9, 10).
Add 0.3 ml of concentrated hydrochloric acid per 10 ml of water containing the larvae (adjust the volume accordingly to achieve a 1:30 dilution of acid). Free-living nematodes are killed, while parasitic species live for about 24 h.
2. Specimens that have been refrigerated or preserved are not suitable for culture. Larvae of certain species are susceptible to cold environments.
Another method of examining a stool specimen suspected of containing small numbers of Strongyloides larvae is the use of a modified Baermann apparatus (Fig. 4.2). The Baermann technique, in which a funnel apparatus is used, relies on the principle that active larvae will migrate from a fresh fecal specimen that has been placed on a wire mesh with several layers of gauze which are in contact with tap water (
