Axenic culture of Brachionus plicatilis using antibiotics
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The rotifer Brachionus plicatilis culture is composed of complex microcosms including bacteria, protozoans, algae, and fungi. Previous studies reported methods to establish axenic rotifer cultures, but further refinement of these techniques is needed, for molecular biological research which requires pure culture to isolate nucleic acids from rotifers only. In order to render rotifer culture axenic, we tested five antibiotics: ampicillin (Amp), chloramphenicol (Cp), kanamycin (Km), nalidixic acid (Na), and streptomycin (Sm) at 30–100 μg/ml. Except for Cp, which reduces rotifer reproduction, all other antibiotics at the tested concentrations did not affect rotifer reproduction or show any toxic effects. A rotifer disinfection method was finally established by treating the resting eggs with 0.25% (w/v) sodium hypochlorite (NaOCl) for 3 min, washing with sterilized sea water, and then exposing the neonates to an Amp, Km, Na, and Sm mixture. Using four nutrient media, we confirmed that this protocol renders the rotifer culture bacterial and fungus free. The axenic rotifer culture generated here is useful not only for genetic analysis of Brachionus plicatilis, but for studying the rotifer life cycle without bacterial influence.
KeywordsBrachionus plicatilis Antibiotic Bacteria free Resting egg disinfection
A part of this research was supported by a Grant in Aid for Scientific Research (B), 2009–2011, No. 21380125, from the Ministry of Education, Culture, Sports, Science and Technology of Japan, to A. H. We would like to thank Dr. Helen S. Marcial for her review and comments.
- Aoki, S. & A. Hino, 1996. Nitrogen flow in a chemostat culture of the rotifer Brachionus plicatilis. Fisheries Science 62: 8–14.Google Scholar
- Hagiwara, A., 2002. Frist live food of marine fish: biological function of rotifers and application to larval rearing. Suisan Zoshoku 50: 473–478. (in Japanese).Google Scholar
- Hagiwara, A., A. Hino & R. Hirano, 1988. Comparison of resting egg formation among five Japanese stocks of the rotifer Brachionus plicatilis. Nippon Suisan Gakkaishi 54: 577–580.Google Scholar
- Hirata, H., O. Murata, S. Yamada, H. Ishitani & M. Wachi, 2004. Probiotic culture of the rotifer Brachionus plicatilis. Hydrobiologia 387(388): 495–498.Google Scholar
- Hirayama, K. & H. Funamoto, 1983. Supplementary effect of several nutrients on nutritive deficiency of baker’s yeast for population growth of the rotifer Brachionus plicatilis. Bulletin of the Japanese Society of Scientific Fisheries 49: 505–510.Google Scholar
- Hirayama, K., K. Takagi & H. Kimura, 1979. Nutritional effect of eight species of marine phytoplankton on population growth of the rotifer, Brachionus plicatilis. Nippon Suisan Gakkaishi 45: 11–16.Google Scholar
- Kitaori, N. & M. Takahashi, 2005. Improvements in the sterilization performance of sodium hypochlorite. Electrochemistry 73: 141–144.Google Scholar
- Maruyama, I., Y. Ando, T. Maeda & K. Hirayama, 1989. Uptake of vitamin B12 by various strains of unicellular algae Chlorella. Nippon Suisan Gakkaishi 55: 1785–1790.Google Scholar
- Miyakawa, M. & K. Muroga, 1988. Bacterial flora of cultured rotifer Brachionus plicatilis. Suisan Zoshoku 35: 237–243.Google Scholar
- Oo, A. K., G. Kaneko, M. Hirayama, S. Kinoshita & S. Watabe, 2009. Identification of genes differentially expressed by calorie restriction in the rotifer (Brachionus plicatilis). Journal of Comparative Physiology B: Biochemical, Systemic, and Environmental Physiology. doi: 10.1007/s00360-009-0389-6.
- Raleigh, E. A., K. Elbing & R. Brent, 2002. Selected topics from classical bacterial genetics. In Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Sediman, J. A. Smith & K. Struhl (eds), Current Protocol in Molecular Biology. Wiley, New York, NY (Chapter 1, Unit 1.4).Google Scholar
- Snell, T. W. & M. J. Carmona, 1995. Comparative toxicant sensitivity of sexual and asexual reproduction in the rotifer Brachionus calyciflorus. Environmental Toxicology and Chemistry 14: 415–420.Google Scholar
- ZoBell, C. E., 1941. Studies on marine bacteria. I. The cultural requirement of heterotrophic aerobes. Journal of Marine Research 4: 42–75.Google Scholar