Secondary antibiotic exposure
This protocol is extracted from research article:
Slow growth determines nonheritable antibiotic resistance in Salmonella enterica
Sci Signal, Jul 30, 2019; DOI: 10.1126/scisignal.aax3938

Stationary-phase bacteria from overnight LB cultures were collected by centrifugation (14,000 rpm for 1 min at room temperature), resuspended in fresh LB broth, and incubated at 37°C for 30 min. Cells were then diluted 1:200 into fresh LB broth in the presence or absence of antibiotics. After 24 hours of antibiotic exposure, cells were collected by centrifugation (14,000 rpm for 20 min at 4°C). Cells were washed with 1.2 ml of ice-cold phosphate-buffered saline, transferred to 1.5-ml microcentrifuge tubes, and collected by centrifugation (14,000 rpm for 20 min at 4°C). Last, cells were resuspended in 500 μl of LB medium, diluted, and plated onto LB agar (1.5%, w/v) plates. Cells were diluted 1:100 into fresh LB medium and allowed to grow overnight. Stationary-phase bacteria from overnight cultures were subjected to a second round of antibiotic exposure as described above.

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