it daily by withdrawing a small amount of fluid from the bottom of the tube. Prepare a smear on a glass slide, cover the slide with a coverslip, and examine the smear with the 10× objective.
6. Examine the larvae for motility and typical morphological features to reveal whether hookworm, Strongyloides, or Trichostrongylus larvae are present.
Results and Patient Reports from Harada-Mori Filter Paper Strip 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 the incubation.
2. Report larvae detected by fecal culture.
Example: Strongyloides stercoralis larvae detected by fecal culture
Procedure Notes for Harada-Mori Filter Paper Strip Culture
1. If the larvae are too active to observe under the microscope and morphologic details are difficult to see, the larvae can be heat killed within the tube or after removal to the slide; iodine can also be used to kill larvae.
2. Infective larvae may be found any time after the fourth day or even on the first day in a heavy infection. Since infective larvae may migrate upward as well as downward on the filter paper strip, caution must be exercised in handling the fluid and the paper strip itself to prevent infection. Handle the filter paper with forceps. Wear gloves when handling the cultures.
3. It is important to maintain the original water level to keep optimum humidity.
4. Fresh stool is required for this procedure; preserved fecal specimens or specimens obtained after a barium meal are not suitable.
Procedure Limitations for Harada-Mori Filter Paper Strip Culture
1. The Harada-Mori 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.
Filter Paper/Slant Culture Technique (Petri Dish)
An alternative technique for culturing Strongyloides larvae is a filter paper/slant culture on a microscope slide placed in a glass or plastic petri dish (Fig. 4.1), which was originally described by Little (11). As with previous techniques, sufficient moisture is provided by continuous soaking of the filter paper in water. Fresh stool material is placed on the filter paper, which is cut to fit the dimensions of a standard (1 by 3 in.) microscope slide. The filter paper is then placed on a slanted glass slide in a glass or plastic petri dish containing water. This technique allows direct examination of the culture system with a dissecting microscope to look for nematode larvae and free-living stages of S. stercoralis in the fecal mass or the surrounding water without having to sample the preparation. Always wear gloves when performing these procedures.
Quality Control for the Filter Paper/Slant Culture Technique (Petri Dish)
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 the Filter Paper/Slant Culture Technique (Petri Dish)
1. Cut a filter paper strip (1 by 3 in.), and smear a film of 1 to 2 g of fresh fecal material in the center of the strip.
2. Place the strip on a glass slide (1 by 3 in.). Place the slide inclined at one end of the petri dish by resting the slide on a piece of glass rod or glass tubing; identify the specimen on the dish (Fig. 4.1).
3. Add water to the petri dish so that at least the bottom one-fourth of the slide is immersed in water. The stool will be kept moist by capillary action. Cover the dish, and maintain it at 25 to 28°C. As needed, add water to maintain the original level.
4. Keep the dish for 10 days. Examine daily, either with the dissecting microscope or by withdrawing a small amount of fluid and placing it on a microscope slide. Cover with a coverslip, and examine microscopically with the 10× and 40× objectives.
5. Examine any larvae recovered for typical morphologic features.
Results and Patient Reports from the Filter Paper/Slant Culture Technique (Petri Dish)
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 the Filter Paper/Slant Culture Technique (Petri Dish)
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 migrate anywhere on the filter paper strip, caution must be exercised in handling the fluid and the paper strip itself to prevent infection. Wear gloves when handling the cultures.
3. There may be infective larvae in the moisture that accumulates under the petri dish lid, so be careful not to allow the water to touch the skin when raising the lid.
4. It is important to maintain the original water level to keep optimum humidity.
5. Preserved fecal specimens or specimens obtained after a barium meal are not suitable; fresh stool specimens
