Development of Caenorhabditis briggsae and Hymenolepis nana in Interchanged Media

Abstract

Chemically defined media supplemented with proteinaceous growth factors have been developed independently for axenic culture of Caenorhabditis briggsae and Hymenolepis nana. The chemical compositions of the media and characteristics of the growth factors are compared. Either growth factor was found to be an effective supplement for each organism. The chemically defined portions of the media can be interchanged if suitable adjustments are made in the salts and buffers. It therefore appears possible to develop a single basal medium for both types of organism, and to use the convenience of the C. briggsae bioassay for further isolation and the characterization of growth factors for helminths. Media have been developed independently to meet the culture requirements of free-living and of parasitic helminths. These media consist of chemically defined components supplemented with partially characterized proteinaceous materials. The chemically defined medium designed for culture of Caenorhabditis briggsae has been used with or without minor modifications for the continuous culture of several species of free-living nematodes (Hansen and Cryan, 1966), in culture of Pelodera strongyloides (Yarwood and Hansen, 1968), and for the insect parasite Neoaplectana carpocapsae (Hansen et al., 1968) and the plant parasite Aphelenchoides sp. (Myers, 1968). It seemed possible that it could be used for an even wider range of species. It was, for example, used as a supporting medium for Haemonchus contortus for production of antigen (Silverman, Alger, and Hansen, 1966). The chemically defined medium designed for culture of Hymenolepis nana has been used for two other species of Hymenolepis, and for Spirometra mansonoides, Trichinella spiralis (Bemtzen, 1966), and Mesocestoides corti (Voge and Coulombe, 1966; Hart, 1968). The objective of this work was to arrive at a chemically defined medium which would be suitable for the culture of both parasitic and free-living organisms. For the comparisons described in this paper the species used were those for which the respective media were designed, i.e., C. briggsae and H. nana. It might be possible to use the simpler assay Received for publication 10 January 1969. system with a free-living organism to assess the effectiveness of supplements used in the more complex parasitic system. MATERIALS AND METHODS The methods used for cultivation of C. briggsae and H. nana under axenic conditions have been described in detail (Dougherty et al., 1959; Berntzen, 1966). For assay with C. briggsae, 3 newly hatched larvae were inoclulated into duplicate aliquots of 0.25 ml of medium, incubated at 20 C under air, and the Fi time, required for new larvae to appear was determined (Lower, Hansen, and Yarwood, 1966). For H. nana, evaluation of media was carried out under experimental conditions designed to achieve optimum comparison with control medium in each test. Portions of 4, 10, or 100 ml of medium were used. These were gently agitated by aspiration or rotation to facilitate gas exchange. Some experiments were carried out with continuous flow or periodic flow of medium (Berntzen, 1965, 1966). The gaseous environment was 5 or 10% carbon dioxide, and 5, 10, or 15% oxygen in nitrogen; the ratio 10:5:85 was used in most experiments. Axenized cysticercoids were inoculated, 2 per ml of medium. Response was determined by their increase in length and accompanying degree of differentiation of the proglottids and formation of ova. The time of appearance of the internal structures in the proglottids was determined as previously described (Berntzen, 1966). Worms in some culture media showed comparable organ formation even though they did not achieve the same length. Therefore, relative length was used to compare the media of differing composition shown in Table III. In all these cultures developed eggs were observed. The 2 chemically defined media used in this study were C. briggsae Maintenance Medium, designated as CbMM (Buecher et al., 1966) and medium 115, designated as M-115 (Berntzen and Mueller, 1964). Their components are listed in Table I.