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Mutagenized phages must be screened on mixed lawns because they do not express their mutant phenotypes until after they replicate once in E. coli.
We screened mutagenized phages for the ability to infect S. typhimurium by plating on mixed lawns of E. coli and S. typhimurium, and looking for clear plaques.
Young adult worms exposed to mixed lawns of E. coli/Verde2 at 25°C mostly died within 36 hr, and larvae were almost wholly unable to survive.
Worms grown on mixed lawns of E. coli and Verde1 (∼10:1) became impaired in movement and grew more slowly (generation time increased by 27%; Table S2), but they were able to grow and reproduce.
When the common competitor was a host-range mutant, the ratios of competing genotypes in the starting mixture (R0) and in the harvested lysate (R1) were obtained by plating on mixed lawns of P. phaseolicola and P. pseudoalcaligenes (200 1 mixture), where ordinary and host-range genotypes form turbid and clear plaques, respectively.
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φX174 forms clear plaques on this mixed lawn.
Initially (t0) and after 45 minutes (t45), phage concentrations were measured by plating on a mixed lawn consisting of 200 microliters of an overnight E. coli culture and 60 microliters of an onvernight S. typhimurium culture.
200 µL of E. coli ensures that both phages produce clearly visible plaques on the mixed lawn, and 60 µL of S. typhimurium is enough to make G4 plaques appear turbid, but not so much that G4 plaques are no longer clearly visible.
To verify the ingestion of E. coli, S. mitis and E. faecalis strains and the proliferation in the C. elegans intestine, L4 C. elegans were fed on a mixed lawn of E. faecalis and E. coli, E. faecalis and S. mitis or S. mitis and E. coli at a same ratio 1 1.
When these Verde2-resistant glycosylation mutants were exposed to Verde1, it was found that the Verde1 pathogen efficiently killed them, so populations of Bus or Srf worms were completely unable to survive on mixed E. coli/Verde1 lawns.
About 200 F2 progeny from each plate were then transferred to mixed E. coli/Verde1 (10:1) lawns and incubated at RT for a further 7 10 days, after which most (273/300) plates contained only dead or dying worms.
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