Quantitative Imaging of Nitrogen Fixation by Individual Bacteria Within Animal Cells

C. P. Lechene, Y. Luyten, G. McMahon, D. L. Distel
2007 Science  
Materials and Methods Sample Preparation: Cells of T. turnerae T7902 were grown as previously described (S1). The headspace gas was replaced with 0.5 ml of 15 N 2 (98%). The cells were incubated at room temperature for up to 96 hr (approx. eight doublings). Cells of E. faecalis were also incubated in 15 N 2 as above for 2.5 hr (> 4 doublings) at 37°C in tryptic soy broth medium (Bacto). Specimens of L. pedicellatus were raised as previously described (S2). Specimens residing in wood were
more » ... rred to sealed 500 ml flasks containing 15 N 2 -enriched seawater medium buffered with 20mM Hepes (pH 8), stripped of dissolved atmospheric gas by bubbling with 99.99% He for 1 hr at room temperature, and equilibrated with a mixture of 10.1 ml of 15 N 2 (98%) and 3 ml of O 2 (99.5%) for 24 hrs at room temperature. The specimens were incubated at room temperature for eight days. Additional O 2 (2 ml, 99.5%) was added to the headspace daily. The T. turnerae (cells) and L. pedicellatus (whole animals with shells and pallets removed) were fixed after incubation as described (S3).
doi:10.1126/science.1145557 pmid:17872448 fatcat:wwwutlf6qffrjopgoh2i6tbivu