SEM Analysis of M. smegmatis Morphology

Scanning electron microscopy (SEM) of M. smegmatis culture was performed to observe their morphology under stress environment. Cultures from above experiments were taken for this study. The cells were fixed overnight at 4°C in a mixture of 4% (v/v) paraformaldehyde (Sigma-Aldrich) and 2.5% (v/v) glutaraldehyde (Sigma-Aldrich) in 0.1 M phosphate buffer (pH 7.4). The sample suspension was then centrifuged, the supernatant was discarded, and the pellet was washed with 0.1 M phosphate buffer twice; the specimens were then fixed with 1% (w/v) osmium tetraoxide (Sigma-Aldrich) in 0.1 M phosphate buffer for 1 h. The cells were then dehydrated in ascending concentration using molecular grade ethanol (Merck) from 50%, 70%, and 100% (10 min at room temperature). The cells were transferred on a glass coverslip and air dried. All samples were coated with gold using a sputter coater (Quorm, SC7620) and examined using a scanning electron microscope (THERMO FEISEM, Volumescope) (Kim et al., 2017 (link); Pawar et al., 2020 (link)). SEM was performed at the Advanced Technology Platform Centre (ATPC) facility, RCB, Faridabad, India.