Whatman grade no 1

Mycobacterium smegmatis cells were fixed with 2% glutaraldehyde in 0.1 M cacodylate at room temperature for 2 h, and then incubated overnight in 4% formaldehyde, 1% glutaraldehyde, and 0.1% PBS. For negative staining of the samples, a drop of the fixed bacterial suspension was applied directly onto a glow-discharged EM grid (QUANTIFOIL. Formvar/Carbon. Cu 400 mesh grids). The sample was allowed to be adsorbed and then blotted with filter paper (Whatman grade No. 1). The grid was washed by touching the surface with two consecutive drops of 0.75% (w/v) uranyl formate, blotting each time, and stained for 1 min with one more drop of the same staining agent. Negative stained samples were examined in a JEOL JEM-1230 (accelerating voltage 100 kV) electron microscope, and images were recorded with a CCD camera ORIUS SC100 (4 × 2.7 k pixel).

The powder from L. monopetalum was soaked in distilled water for 48 h at 60 °C for sample preparation to LC–QTOF–MS analysis for metabolite detection. Filter paper, Whatman Grade No. 1, was used for mixture filtration, then evaporated. Methanol (1 mL) was used for dissolving the aqueous extract (1 mg). An Agilent Extend-C18 column (2.1 mm × 50 mm, 1.8 μm) was used for the separation process aided with elution gradient 0–1 min, 5% B; 1–11 min, 5%–100% B; 11–13 min, 95% B; 13–15 min, 5% B; 15–16 min, 5% B, applying 0.1% HCOOH in water (mobile phase A and 0.1% HCOOH in methanol (mobile phase B), and 10 μL and 300 μL/min were the injection volume and sample flow rate, respectively. The MS1 acquisition approach was obtained using positive mode 100 to 600 m/z as mass range. Conditions of the mass spectrometer were designed at 300 °C = gas temperature; 8 I/min = gas flow; 35 psig = nebulizer; 350 °C = sheath gas temperature, and 11. MS1 = sheath gas flow. Data were obtained by a quantitative and qualitative analysis software (Agilent MassHunter, Agilent Technologies). A mass assessment of the spectrum was detected, and LC-MS data were used for extracting the chemical features by the recursive analysis workflow and molecular feature extraction (MFE) algorithm [22 (link)].

For negative staining (NS) of the samples, a drop of the protein solution was applied directly onto a glow-discharged EM grid (QUANTIFOIL. Formvar/Carbon. Cu 400 mesh grids), and allowed to be adsorbed on the grid surface (for few seconds or minutes, depending on protein concentration); then the drop was blotted with filter paper (Whatman grade No. 1) and the grid was washed by touching the surface with two consecutive drops of 0.75% (w/v) uranyl formate, blotting each time, and stained for 1 min with one more drop of the same staining agent. Finally, the grid was blotted again and allowed to air dry before observation.