Quantifying Bacterial Colonization in C. elegans

Nematodes were age-synchronized by bleaching [78 ], and embryos were incubated at 25°C on mNGM agar plates containing E. coli OP50 or S. typhimurium SL1344, as above, to prepare for the bacterial colonization assays. Bacterial colonization of C. elegans was determined using a method adapted from Garsin et al. [64 (link)] and RA Alegado (personal communication and [89 (link)]). At each time point tested, 10 worms were picked and placed on an agar plate containing 100 μg/ml gentamicin to remove surface bacteria. They then were washed in 5 μl drops of 25 mM levamisole in M9 buffer (LM buffer) for paralysis and inhibition of pharyngeal pumping and expulsion, then were washed twice more with LM buffer containing 100 μg/ml gentamicin, and twice more with M9 buffer alone. The washed nematodes then were placed in a 1.5 ml Eppendorf tube containing 50 μl of PBS buffer with 1% Triton X-100 and mechanically disrupted using a motor pestle. Worm lysates were diluted in PBS buffer and incubated overnight at 37°C on MacConkey agar. Lactose-fermenting (E. coli) and non-fermenting (Salmonella) colonies were quantified, and used to calculate the number of bacteria per nematode.