Nuclepore track etch polycarbonate membranes

Nuclepore Track-Etch polycarbonate membranes (Whatman; 0.4-μM pore size, 90-mm diameter) were used for assessing biosurfactant production. As previously described (23 (link)), one side of the membrane is shiny and the other is dull due to the manufacturing process. The dull side’s surface contains gaps and ridges that physically trap cells, but the biosurfactant permeates to produce a visible ring around colonies. The shiny side’s surface is smooth, which allows biosurfactant-producing cells to spread out through growth. Sterile forceps were used to overlay the membrane on the PAF agar surface, and 20 μL of overnight culture was spotted directly on the membrane and allowed to fully dry before the plates were inverted and incubated overnight at room temperature.

Water samples (50–150 mL) were fixed with formaldehyde (2% v/v) and stored at 4°C for 48 h. PA and FL bacteria were separated via sequential filtration through 3.0 and 0.2 μm (Kegler et al., 2017 (link)) Nuclepore TrackEtch polycarbonate membranes (Whatman, Dassel, Germany), respectively. Filters were air-dried and frozen until microscopic analysis (Rieck et al., 2015 (link)). 4′,6-Diamidino-2-phenylindole (DAPI; Thermofisher Scientific Inc., Waltham, MA, United States) was diluted in a 3:1 mounting solution (made of Citifluor AF mounting medium; Citifluor Ltd., London, United Kingdom) and Vecta shield (Vector Laboratories Inc., Burlingame, CA, United States) to a concentration of 1 μg mL^-1. The DAPI/mounting medium solution was directly added onto the filter. FL bacteria were enumerated by using an automatic microbial cell enumeration system. A multipurpose fully automated microscope imaging system (MPISYS) was used for the refined image acquisition. Image selection, cell determination, and enumeration were carried out using the ACMEtool2.0 (Bennke et al., 2016 (link)). PA bacteria were manually enumerated with an epifluorescence microscope “Axioskop 40” (Zeiss, Jena, Germany) at 1000× magnifications (Grossart et al., 2005 (link)). A minimum of 40 grids (grid size: 15,625 μm²) was counted.