serum or bovine serum albumin is required for T. vaginalis grown under axenic conditions. However, these components inhibit several biological properties of these parasites. PACSR is a serum replacement, free of bovine serum albumin, which is used for axenic cultivation of E. histolytica. Studies indicate that PACSR added to several of the standard media used for the cultivation of T. vaginalis can support growth in the absence of serum (29).
Dientamoeba fragilis
Dientamoeba fragilis is a protozoan pathogen of the human gastrointestinal tract that was originally classified as an amoeba. Later studies confirmed its relationship with trichomonads. The approach to in vitro culture is not new; however, some excellent improvements have recently been developed (30, 31). Currently, no axenic cultures of D. fragilis exist. D. fragilis will grow happily with a support flora consisting mostly of E. coli. Slight variations in the species of prokaryotic support flora present within D. fragilis cultures are unlikely to exhibit any significant effect on growth. A modified Earle’s Balanced Salt Solution (EBSS) containing cholesterol, ferric ammonium citrate, and rice starch can be regarded as a superior liquid overlay that can be used along with the Loeffler’s serum slope for culture of D. fragilis under anaerobic conditions.
Loeffler’s Serum Slants (30)
1. Aliquot 5 ml into tubes, slant, and inspissate in 80°C drying oven.
Note If the slants are left too long at 80°C, Dientamoeba will not grow. Slants should be allowed to cool IMMEDIATELY after they solidify.
2. Add approximately 2 mg of rice starch into the bottom of each slant.
3. Overlay each slant with 5 ml of PBS.
4. After inoculation with fresh stool (pea size), incubate at 42°C under microaerophilic conditions (6% O2, 7.2% CO2, 3.6% H2, 83.3% N2).
Earle’s Balanced Salt Solution Overlay
Another overlay that has proven to be very effective is EBSS, pH 7.8. EBSS provides the highest trophozoite numbers and contains a variety of inorganic salts, as well as sodium hydrogen carbonate (NaHCO3) and glucose. The inorganic salts in the medium help to maintain the osmolarity as well as providing a range of divalent cations needed for cell metabolism. NaHCO3 maintains the buffering capacity in the anaerobic in vitro culture and glucose provides an additional energy source in addition to the rice starch. This saline solution has also been used with good results when supplemented with 40 mg/liter ferric ammonium citrate and 50 mg/liter cholesterol (31).
Autoclave and store at room temperature.
Cryoprxrvation (30)
1. Pool four culture tubes of Loeffler’s medium containing viable organisms.
2. Centrifuge at 500 × g for 5 min; discard supernatant fluid.
3. Add 1 ml of PBS to the pellet and invert tube to obtain even cell suspension.
4. Count the cells present.
5. Dilute quantified cell suspensions in PBS to a final concentration of 106 trophozoites/µl.
6. Plate tubes overnight at −80°C freezer.
7. Remove tubes from the −80°C freezer the following morning and place in a liquid nitrogen freezer.
Flagellates of Blood and Tissue
The first types of media used to culture the blood and tissue flagellates, which are still useful for establishment of cultures, were undefined and contained a complex mixture of ingredients. Improvements led to semidefined formulations that included tissue culture media as a base and, as a next step, addition of tissue culture cells as a feeder layer to promote parasite growth. Newer developed media are completely defined, having replaced the feeder cells with various supplements. Serum, a variable component of the media, can now be replaced by various serum substitutes. Fully defined formulations are available for the cultivation of many of these organisms (8).
For the hemoflagellates found in the bloodstream, probably the most direct means of diagnosis is by examination of a stained blood smear under the microscope (any blood stain is acceptable). However, if isolation of the parasite is required for confirmation, culture can be used. The easiest approach involves the use of Novy-MacNeal-Nicolle (NNN) medium. Because of its relatively short shelf life (2 to 4 weeks with refrigeration) and the infrequent need for culture, most clinical laboratories would not routinely stock NNN medium. However, the Centers for Disease Control and Prevention (Division of Parasitic Diseases, Atlanta, GA) can provide culture services, and in other parts of the world, major reference laboratories may provide a similar service. The procedure for isolating Leishmania spp. from patients with suspected cases of cutaneous, mucocutaneous, and visceral leishmaniases is basically similar to that for isolating Trypanosome spp. The amastigote found within host cells, or the promastigote that develops from the amastigote in culture, would be confirmatory. Punch biopsy specimens or needle aspirates serve as the inocula for NNN medium or Schneider’s Drosophila medium with 30% fetal bovine serum. Other procedures such as splenic puncture and liver biopsy can also yield material but are invasive and less likely to be performed (8).
Specimens for culturing Leishmania spp. may consist of aspirates, scrapings, or biopsy material from skin lesions of patients with cutaneous leishmaniasis; bone marrow aspirates or, more rarely, splenic aspirates from visceral leishmaniasis patients; or normal skin biopsy specimens, lymph node aspirates, or pieces of liver and spleen from suspected or potential wild- or domestic-animal reservoirs. Ensure that the skin surrounding the ulcer is thoroughly cleaned and swabbed with 70% alcohol (sterile saline is not acceptable as a cleansing agent) and allowed to dry before the sample is removed. Also ensure that alcohol does not get into the ulcerated area or broken skin. A punch biopsy specimen taken from the advancing margin of the lesion is often recommended. If skin biopsy fragments are placed in transport medium (RPMI medium supplemented with 10% fetal calf serum) maintained at ambient temperature, delays in transit through the mail will still not prevent recovery of the organisms. In one study, after being received in the laboratory (transit time as long as 3 to 17 days), the biopsy specimens were ground in sterile saline and inoculated into NNN culture tubes. The tubes were incubated at 25°C and subcultured every week until the fifth week. Cultures were positive in 9 of 16 cases in all seasons and for three different Leishmania species. Thus, delayed culture can still yield valuable results from biopsy specimens obtained under field conditions (32).
It is imperative that only a few drops of bone marrow juice or spleen aspirate be inoculated into tubes. Inoculate several tubes with a few drops each rather than inoculating a single
