inoculation
Leishmania spp.
Trypanosoma spp.
Toxoplasma gondii
Xenodiagnosis
Very few clinical laboratories offer specific culture techniques for parasites. The methods for in vitro culture are often complex, while quality control is difficult and not really feasible for the routine diagnostic laboratory. In certain institutions, some techniques may be available, particularly when consultative services are provided (reference laboratory situation) and for research purposes. However, most laboratories do not offer these techniques.
Few parasites can be routinely cultured; however, methods are available for Entamoeba histolytica, Naegleria fowleri, Acanthamoeba spp., Trichomonas vaginalis, Dientamoeba fragilis, Toxoplasma gondii, Blastocystis spp., Trypanosoma cruzi, and the leishmanias. Often, when specimens are cultured for potential pathogens, nonpathogenic protozoa could also be recovered. These procedures are usually available only after consultation with the laboratory and on special request. For those who may be interested in trying these techniques, the several different media presented below are representative of those available. More extensive options can be found in the literature (1–13).
Cultures of parasites grown in association with an unknown microbiota are referred to as xenic cultures. A good example of this type of culture would be stool specimens cultured for E. histolytica. If the parasites are grown with a single known bacterium, the culture is referred to as monoxenic. An example of this type of culture would be clinical specimens (corneal biopsy specimens) cultured with Escherichia coli as a means of recovering species of Acanthamoeba and Naegleria. If parasites are grown as pure culture without any bacterial associate, the culture is referred to as axenic. An example of this type of culture would be the use of media for the isolation of Leishmania spp. or Trypanosoma cruzi. All three types of cultures are discussed in this chapter.
Pancreatic digests of casein are major ingredients of media used in the axenic cultivation of lumen-dwelling parasitic protozoa. Unfortunately, the digest used almost exclusively in the development of these media has not been available since the early 1980s. Many digest products have been tried in the interim with marginal results in supporting the growth of E. histolytica. Diamond et al. (14) have developed a casein-free medium, YI-S, consisting of a nutrient broth, vitamin mixture, and serum. This may serve as a replacement for TYI-S-33, widely used for the axenic culture of E. histolytica and other intestinal protozoa.
Specimens include stool material, mucus, or a combination of the two. The clinical specimen(s) should be no more than 24 h old; a maximum of 2 to 3 h is recommended.
Balamuth’s Aqueous Egg Yolk Infusion Medium for Amebae
Balamuth’s aqueous egg yolk infusion medium is used to detect the presence of amebae. The specific solutions required are phosphate buffer and whole-liver concentrate solution.
Balamuth’s Aqueous Egg Yolk Infusion Medium
Phosphate buffer
Mix the solution in the ratio of 3 parts tribasic (A) to 2 parts monobasic (B) to make 1 M phosphate buffer stock. Dilute the stock buffer to 0.067 M before use (add 492 ml of distilled H2O to 1 liter of 1 M phosphate buffer).
Whole-liver concentrate solution
Suspend the powder in cold water, and autoclave. Filter through a Büchner funnel to remove sediment, dispense in 10-ml quantities, and reautoclave.
Preparation of Complete Medium (15)
1. Using a blender, blend 12 fresh hard-boiled egg yolks with 375 ml of 0.8% sodium chloride.
2. Autoclave at 7 lb/in.2 pressure for 10 min, repressurize slowly, and stir.
3. Autoclave again for 45 min at 7 lb/in.2 pressure.
4. Allow to cool slightly, and add distilled water to replace evaporation loss. Transfer the material to a muslin bag, and express the liquid portion, saving all the fluid. Return the volume to 375 ml with 0.8% sodium chloride.
5. Autoclave for 20 min at 121°C; cool to 5°C. Do not agitate the fluid at this point or during filtration.
6. Decant the fluid carefully through gauze into a Büchner funnel with Whatman no. 3 filter paper. Filter papers can be replaced as necessary.
7. Measure the filtrate, add an equal volume of 0.067 M phosphate buffer, and autoclave for 20 min at 121°C. After cooling, add stock liver concentrate (1 part stock liver to 9 parts medium).
8. Material can then be decanted into sterile flasks, which are stored until the medium is dispensed.
Procedure
Tubes should contain 6 to 8 ml of fluid and should be incubated for 4 days at 37°C as a sterility check. Before inoculation, a loopful of sterile rice powder or starch (can be autoclaved in a screw-cap tube) is added to each tube. To each tube, add stool material, mucus, or a combination of the two (about the size of a small pea), break it up thoroughly in the medium, and incubate at 37°C. The cultures should be checked at 2, 3, and 4 days by examining 0.1 ml of sediment under the microscope (low-intensity light) for characteristic motility. Although the initial culture may appear to be negative, subcultures may reveal organisms (Fig. 8.1).
Figure 8.1 Protozoa from culture systems. (Upper left) Entamoeba histolytica/E. dispar trophozoite from liquid medium containing rice starch (note that there are no definitive erythrocytes within the cytoplasm, so that it is not possible to differentiate the true pathogen, E. histolytica, from the nonpathogen, E. dispar). (Upper right) Naegleria fowleri trophozoite from nonnutrient agar culture with bacterial overlay (note that this trophozoite has been stained). (Lower left) Acanthamoeba spp. trophozoite from nonnutrient agar culture with bacterial overlay (note the spiky acanthapodia). (Lower right) Acanthamoeba spp. cysts from nonnutrient agar culture with bacterial overlay (note the double hexagonal wall appearance. doi:10.1128/9781555819002.ch8.f1
According to Dolkart and Halpern (16), the addition of gastric mucin to the egg component is reported to improve the performance of Balamuth’s medium.
The addition of rice flour (prerinsed with distilled water) to Balamuth’s medium is reported
