A skim milk-supplemented axenic medium to support development and reproduction of C. elegans

The Reproductive Hazards Laboratory is engaged in efforts to assess effects of environmental chemicals on C. elegans. Incubations using liquid culture media containing E. coli have shown that the bacteria alone are capable of catabolizing organic compounds rapidly. Also, previously-available axenic media for liquid culture of C. elegans, as described by Sayre, Hansen, Vanfleteren and others, have produced relatively long generation intervals and have been found in our laboratory to contain enzymatic activity that can produce breakdown of chemicals. Either approach produces unacceptable confounding effects when applied to evaluation of toxicologic effects on worms from exposures to organic compounds. To circumvent this problem, a new axenic medium has been developed. This medium, the C. elegans Habitation and Reproduction (CeHR) medium, uses a base medium similar to previously published axenic media such as CbMM, but substitutes ultrahigh-temperature pasteurized skim milk at 20% of the medium volume for ingredients such as heated liver extract. The skim milk is obtained from grocery stores and is handled using sterile technique. Each batch of milk is checked thoroughly for sterility prior to use in the medium. The completed medium also is tested for sterility prior to use, and the worms added to the medium are either from existing microbiologically sterile cultures or from bleached embryos. The formula and directions for preparing CeHR medium may be accessed at http://usacehr.detrick.army.mil/clegglab.html. This medium has been used very successfully for liquid culture of N2 C. elegans. Beginning with synchronized cultures of L1 larvae, the interval to production of the next generation of L1 larvae is approximately 3.5 days. Worm motion is vigorous, and the medium supports high densities of worms. The CeHR medium is being used in the Reproductive Hazards Laboratory for multigenerational exposure experiments.