Bacterial Growth in Oxalate Assays

Bacillus subtilis strain 168 (ATCC 23857) and Escherichia coli (UTI89) were routinely cultured at 37°C in Luria-Bertani (LB) broth (61 (link)). Growth in oxalate was assayed in 96-well plates prepared as dilutions of sodium oxalate (NaOx) in LB broth with stationary-phase bacteria added at a final dilution of 1/100. Plates were incubated for 24 h at 37°C with optical density (OD) readings every 30 min using an Eon microplate spectrophotometer (BioTek, Winooski, VT, USA). NaOx concentrations were selected based on physiologic intestinal oxalate concentrations reported in the literature (62 (link)). Growth curves were analyzed with the R package Growthcurver and GraphPad Prism (version 8.1.2) (63 (link)).
D. melanogaster flies were cultured following homogenization until day 10 of the survival assay to assess BS168 loads. D. melanogaster flies were surface sterilized with 70% ethanol and homogenized in 0.01 M phosphate-buffered saline (PBS) by using a motorized pestle. Homogenates were diluted up to 100-fold, plated onto LB agar, and then incubated aerobically at 37°C for 24 h. The characteristic colony morphology of BS168 was easily differentiated on LB, which is not a growth medium amenable to culturing the typical D. melanogaster microbiota (64 (link)).