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Unique mode of cell division by the mycobacterial genetic resister clones emerging de novo from the antibiotic surviving population

Citation

Ajitkumar, Parthasarathi (2020), Unique mode of cell division by the mycobacterial genetic resister clones emerging de novo from the antibiotic surviving population, Dryad, Dataset, https://doi.org/10.5061/dryad.9kd51c5fk

Abstract

Live cell and timelapse microscopic images of the cells taken from different time points post antibiotic (Rifampicin and Moxifloxacin) exposure. The cells post antibiotic exposure, during their regrowth, showed multiple constriction to divide and generate sister antibiotic resister daughter cells with abrupt increased cell number within less division time by multiple septation. The phenomena of  multiple septation can be seen in Miscellaneous Figures (MF. 1-4) and Miscellaneous Movies (MF. 1-4). 

Methods

Agarose pad method was used for the Live-cell time-lapse microscopy of Msm regrowth phase cells. In brief, Agarose pad was prepared by 1.75% low melting point agarose on a clean glass slide using spent media of 90 hr rifampicin (25 µg/ml); 60 hr and 54 hr of moxifloxacin (1.5 µg/ml) exposed cultures. After the solidification of the agarose, for the introduction of rifampicin into the agarose pad, a portion of the agarose (about 1/5th of the total agarose pad area) was cut out using a blade to make a well. A tip was attached towards one side of the well, which was connected to a syringe for the removal of rifampicin containing medium. On top of the agarose pad,10 µl of 90 hr rifampicin (25 µg/ml); 60 and 54 hr of moxifloxacin (1.5 µg/ml) exposed Msm cells were placed and by tilting the slide the cells were spread evenly. Leaving a portion of the well open for the introduction of rifampicin with a syringe, the slide was covered by a cover slip at 45º angle from the base of the glass slide. To facilitate cell adhesion, the agarose pad with the cells was kept at 37ºC for 1 hr. Under Carl Zeiss AxioVision 4 microscope this slide was observed. We have used a live cell imaging option, at 100x magnification (DIC), with 0.2 µm slice distance Z-stacking at 37ºC. DIC images were taken at every 10 min time interval. The parameters like cell length and cell constriction, data analysis of  the images was performed using AxioVision 4 and ImageJ software.

Funding

Department of Biotechnology-Indian Institute of Science Partnership Programme

Department of Biotechnology-Indian Institute of Science Partnership Programme