Herpetomonas megaseliae and Crithidia harmosa: growth on blood-agar plates.

A technique for culturing two species of trypanosomatid flagellates on blood-agar plates is described. Herpetomonas megaseliae and Crithidia harmosa were grown as discrete clone colonies on plates prepared in sterile petri dishes with a medium derived from that of Tobie et al. (1950, J Parasitol 36: 48-54). Plates were inoculated with 0.01 ml flagellate suspension and the inoculum was spread evenly over the surface of the agar with a sterile glass spreader. Plates were incubated in a high humidity chamber at 25 C and colonies of both species, easily visible to the unaided eye, appeared within 1 week. There was a virtual 1:1 ratio between calculated number of CFU (colony forming units) and actual CFU observed on the plates. Colony morphology differed between the two species. Herpetomonas megaseliae produced a translucent flattened colony with a raised center and raised spiral arms, while C. harmosa produced an opaque hemispherical colony. Applications of the culture technique include potential diagnosis of trypanosomatid infections and potential studies in trypanosomatid genetics. Agar plates have been used for many years for the culture of microorganisms, and agarbased media have been used for nearly as long for culture of protozoa not normally grown on plates (Noller, 1917). Modifications of plating techniques have enabled workers to study the genetics of not only the cultured microorganisms, but also their parasites (Franklin et al., 1965; Lederberg and Lederberg, 1953; Puck, 1959). In the present paper we extend the technique of agar plate culture to include two protozoan species of the family Trypanosomatidae and suggest possible applications of the technique to problem areas in the study of trypanosomatid biology. MATERIALS AND METHODS Laboratory stock cultures of Herpetomonas megaseliae Daggett, Dollahon, and Janovy 1972 (ATCC # 30209) and Crithidia harmosa McGhee, Hanson, and Schmittner 1969, were used. The C. harmosa stock was obtained from Dr. S. H. Hutner, Pace University, and is from the same stock as ATCC # 30256. Organisms were loop transferred to 5-ml Mansour's medium with human blood (Dollahon and Janovy, 1971) and incubated at 25 C for 72 hr before use. Cultures used in these experiments were in the exponential phase of growth. Culture medium was prepared from the bloodagar base of that of Tobie et al. (1950) with the Locke's solution overlay salts added directly to the agar. Medium composition was thus: 10 g Bacto agar (Difco), 1.5 g Bacto beef (Difco), 2.5 g Neopeptone (Difco), 4 g NaCl, 0.1 g CaC2, 0.1 g KC1, 0.15 g NaH2PO4, 1.25 g glucose, and Received for publication 14 December 1976. 500 ml distilled water. This solution was boiled and adjusted to pH 7.2, autoclaved in a 1 liter screw cap flask, and allowed to cool until the flask was just warm to the touch. Forty ml of outdated whole human blood, lysed and diluted 1: 1 with distilled water, was added per 200 ml medium. Blood-agar plates were prepared in sterile glass petri dishes (15 by 100 mm) poured with 20 ml complete medium and allowed to solidify. Stock cultures of C. harmosa and H. megaseliae were counted by hemocytometer and diluted to contain the desired theoretical number of colony forming units (CFU) per 0.01 ml dilution medium. The dilution medium was Mansour's base (Dollahon and Janovy, 1971). Plates were then inoculated with 0.01 ml flagellate suspension and Giemsastained slides were also made of 0.01 ml inoculum suspension. The droplet on the agar plate was spread with a sterile Pasteur pipet with the tip bent at a right angle and sealed in a flame. Plates were sealed with masking tape, labeled, and placed in a high humidity chamber at 25 C. After 5 days growth, the numbers of colonies were counted and checked against the theoretical number of CFU inoculated. Inoculum slides were checked for differentiation state (of H. megaseliae) according to the methods of Janovy et al. (1975). The high humidity chamber consisted of plastic shoe boxes (10 by 15 by 30 cm) or aquaria (16 by 16 by 20 cm) with covers, containing several cotton balls soaked in water. All inoculation procedures were carried out in an isolation hood sterilized by 24-hr continuous UV lighting. Smears of organisms were air dried, fixed in absolute methanol, and Giemsa-stained. Stain was diluted 1:50 with phosphate buffer at pH 7.2.