aid visualisation, samples were subjected to cell elongation, carried out using a modification of the method by Juhna et al (). The 1 ml sample was added to 4 ml ddH2O and 5 ml R2 broth (0.1% w/v peptone, 0.05% w/v yeast extract, 0.05% w/v glucose, 0.05% w/v starch, 0.03% w/v potassium dihydrogen phosphate, 0.03% w/v sodium pyruvate and 0.0024% w/v magnesium sulphate), with 10 µl pipemidic acid at a concentration of 10 µg/ml. The suspension was incubated for 18 hours at 22oC in darkness. The suspension was concentrated prior to DVC in the same manner as before. All samples were imaged using episcopic differential interference contrast (EDIC) and epifluorescent (EF) microscopy () and a QImaging Retiga EXi camera. Bacteria were quantified by counting visible cells across at least 30 fields of view per sample. Images were merged using ImageJ. Caenorhabditis elegans killing C. elegans were maintained on 5 cm nematode growth medium (NGM) agar plates, prepared according to standard methods () with a lawn of E. coli OP50. To prepare experimental plates, 50 µl of E. coli OP50, L. monocytogenes, or Salmonella Thompson cultures were added to the centre of the plates and incubated at 22oC for 24 hours. To produce VBNC cells, cultures of L. monocytogenes and Salmonella Thompson were pelleted by centrifugation and resuspended in 10 ml 200 ppm chlorine solution for 30 minutes. Chlorinated water was removed by vacuum filtration as described above, and bacteria were removed from the membrane by vortexing in 1 ml PBS for 2 minutes (), concentrating the sample to compensate for the growth of their culturable counterparts on the NGM plate.
aid visualisation, samples were subjected to cell elongation, carried out using a modification of the method by Juhna et al (). The 1 ml sample was added to 4 ml ddH2O and 5 ml R2 broth (0.1% w/v peptone, 0.05% w/v yeast extract, 0.05% w/v glucose, 0.05% w/v starch, 0.03% w/v potassium dihydrogen phosphate, 0.03% w/v sodium pyruvate and 0.0024% w/v magnesium sulphate), with 10 µl pipemidic acid at a concentration of 10 µg/ml. The suspension was incubated for 18 hours at 22oC in darkness. The suspension was concentrated prior to DVC in the same manner as before. All samples were imaged using episcopic differential interference contrast (EDIC) and epifluorescent (EF) microscopy () and a QImaging Retiga EXi camera. Bacteria were quantified by counting visible cells across at least 30 fields of view per sample. Images were merged using ImageJ. Caenorhabditis elegans killing C. elegans were maintained on 5 cm nematode growth medium (NGM) agar plates, prepared according to standard methods () with a lawn of E. coli OP50. To prepare experimental plates, 50 µl of E. coli OP50, L. monocytogenes, or Salmonella Thompson cultures were added to the centre of the plates and incubated at 22oC for 24 hours. To produce VBNC cells, cultures of L. monocytogenes and Salmonella Thompson were pelleted by centrifugation and resuspended in 10 ml 200 ppm chlorine solution for 30 minutes. Chlorinated water was removed by vacuum filtration as described above, and bacteria were removed from the membrane by vortexing in 1 ml PBS for 2 minutes (), concentrating the sample to compensate for the growth of their culturable counterparts on the NGM plate.
VBNC_phylloplane_paper_non_GFP_Salmonella_mBio_v3_CLEAN.docx