University of Cape Town
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modified on 2019-04-04, 13:37

Live-cell imaging of M. smegmatis wild type and metH knockdown strains using time-lapse phase-contrast microscopy. To perform single cell analysis using microfluidics, a 100ml suspension of 2.0 x 106 bacterial cells/ml was prepared and loaded on the four-chambered CellASIC ONIX B04A-03 microfluidic platform (Merck) kept at 37⁰C throughout the experiment. Cells were trapped with the following pressure and flow time settings: channel A8 at 13.8 kPa for 15 seconds; channel A6 at 27.6kPa for 15 seconds. Channel A6 was then rinsed at 6.9kPa for 30 seconds. Untrapped cells were washed out by flowing inlet solution at 34.5kPa for 5 minutes. Prior to trapping, cells at OD600nm = 1 was diluted to OD600nm= 0.1 in 10ml 7H9-OADC with or without 100ng/ml of ATc and incubated in a shaking incubator for 6 h at 37⁰C. ATc-containing media was perfused continuously in channels loaded with cells that had been preexposed to ATc and for cells that were not preexposed to ATc, only 7H9-OADC was perfused. The experiment was run for 43 h. Live-cell imaging was performed on a Zeiss AxioObserver using a 100X, 1.4NA Objective with Phase Contrast and Colibri.7 fluorescent illumination system. Images were captured every 15 minutes using a Zeiss Axiocam 503 and processed using the FIJI software (https://fiji.sc/).