There are two “secret weapons” behind the scientific success of C. elegans: NGM media and OP50 bacteria. The Heterorhabditis bacteriophora/Photorhabdus luminescens entomopathogenic nematode/bacterium (EPN/EPB) complexes are widely used in biological control. Therefore elaboration of genetic toolkits for EPN species would have a practical impact. C. elegans is considered as a model for genetic analysis of EPN species (Fodor et al., 1990). The genome sequence of H. bacteriophora (H. b.) is almost complete. The “secret tools” (the proper NGM-like media and OP50-like bacteria) for genetics of H. b. have, however, been missing. H. b. can only be fed on its own symbiont in lab media, which is rich in N sources and contains oil. Neither H. b. nor its bacterial symbiont could grow in NGM, even if it was supplemented with oil and an abundant peptone N-source. The results were hardly better with supplemented TSY or TSA. In a rich oily media (Woots, NA) the bacteria overgrow the nematodes and the animals cannot be observed individually. In Woots agar media the growth was remarkable, but the visibility of the nematodes was poor. In Woots agar C. elegans could propagate, but “feeding RNAi” (Fire et al., 1998) could not be induced. We elaborated a novel media, called ENGM (Entomopathogenic Nematode Growth Media) in which H. b., C. elegans, as well as their food-source bacteria (P. luminescens, E. coli TT01, OP50) could properly grow. The visibility of the nematodes on ENGM is almost as good as that on NGM. Feeding RNAi can be induced in C. elegansby feeding in both NGM and ENGM with similar frequencies, suggesting that RNAi in H. b. might also be studied in ENGM. Since then we successfully induced heterolog RNAi in H. b. (in preparation). The food source for H. b. is the moderately growing Tn-10-induced NS107 rifR, kmR, apR mutant (isolated by us) of P. luminescens TT01 (Duchaud E. et al., 2003) (kindly provided by Dr. T. Ciche). The recipe of ENGM is as follows: 2.5 g bacto-peptone; 1.5 g beef extract; 2.3 g brain-heart infusion; 15 g agar to 1 liter of deionized water. After autoclaving: 5 g vegetable oil; 1 ml of 5 mg/ml cholesterol (dissolved in EtOH); 2 ml of 0.5M MgSO4, rifampicin100, kanamycin30) are to be added. ENGM plates should be seeded with moderately growing symbiotic bacteria, such as NS107. (For details on the isolation of NS107, please contact the corresponding author).
Duchaud E, Rusniok C, Frangeul L, Buchrieser C, Givaudan A, Taourit S, Bocs S, Boursaux-Eude C, Chandler M, Charles JF, et al. (2003). The genome sequence of the entomopathogenic bacterium Photorhabdus luminescens Nat. Biotechnol. 21, 1307 – 1313.
Fodor A, Vecseri G, Farkas T. (1990). C. elegans, as a model for studying entomopathogenic nematodes. In Entomopathogenic Nematodes in Biological Control, R. Gaugler and H. Kaya eds. (Boca Raton, FL, USA: CRC Press), pp. 285-300.
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