Materials and Reagents

1. Fresh activated sludge from the aeration basin of a wastewater treatment plant
2. 0.5-5 ml pipette with tips (Thermo Fisher Scientific, Finnpipette^TM F1, catalog number: 4641110N ; 5 ml Finntip^TM, catalog number: 9402030 )
3. Grid for aligning and spacing vials on scanner surface. Custom-made dark gray metal grid measuring 30.4 × 22.2 × 0.2 cm (L × W × thickness) (Figures 2A-2B)
   The grid should be sufficiently low so that it does not shadow the biomass from the side, e.g., it should not be much higher than the thickness of the vial bottom (Figure 3A-3B). The required cut-out where to place the vials can be produced using computer numerical control (CNC) metal milling or 3D printing. You can also make the grid out of other materials, such as paper.
4. Light-impermeable box for covering distance between vials and light source to prevent loss of light and uncontrolled illumination. Home-made cardboard box of the approximate dimensions of 65 × 35 × 65 cm (L × W × H), enclosing the scanner and the lighting device.
   
   
   Figure 2. Grid. A. Grid design; B. Custom-made dark gray metal grid positioned on top of the scanner.
   

Equipment

1. 1 L break resistant bottle with a wide neck for sampling (Fisher Scientific, Gosselin, catalog number: 11728643 )
2. 2 L polypropylene beaker for mixing (Thermo Fisher Scientific, Nalgene, catalog number: 1201-2000 )
3. 4 ml clear glass vials measuring 15 mm × 45 mm × 8 mm (outer diameter × height × inner diameter) with a screw top for cultivations (Sigma-Aldrich, Supelco, catalog number: 27111) (Figure 3A)
   A clear, smooth and flat bottom is critical for the success of this experiment (Figure 3C). We used glass vials here, and successfully used polystyrene vials previously.
4. Screw caps with a contrasting color to the biomass for image treatment (Agilent Technologies, catalog number: 5183-4305 )
5. Magnetic stirrer and stir bar (Bioblock Scientific, AM AMC BBS 3000 ) to homogenize activated sludge so that differences in sample composition between vials are minimized
6. Holding device for mounting a light source, e.g., copy stand with camera arm (Kaiser, RS1, RA1, catalog number: 205510 )
7. Flat light source, e.g., light emitting diode (LED) panel measuring 59.5 × 59.5 (cropped to 30) × 1.06 cm (L × W × H) (Rexel, LEDVANCE, PANEL LED 600, 40W, 6500K, 4000 lm, catalog number: 4058075000582 )
   We used cool white light (6,500 K) for this experiment, but other light temperatures were also successfully tested (e.g., 5,600 and 6,000 K)
8. Photoactive synthetically radiation (PAR) quantum light sensor and display meter system (Skye Instruments Ltd, SKP 215/S 39520 , SKP 200 39521 )
9. Desktop scanner (Epson, Perfection V500 Photo, model: J251A)
   
   
   Figure 3. Vials. A. Glass vial with screw cap; B. Vial bottom thickness; C. Clear, smooth and flat vial bottom.
   

Software

1. Scanner driver allowing time-lapse acquisition of images using a desktop scanner, e.g., VueScan version 9.5.51 (Hamrick Software, https://www.hamrick.com/)
2. Tool to read, write and edit meta information in batches of images, e.g., ExifTool version 11.78 (Phil Harvey, https://exiftool.org/)
3. ImageJ version 1.52a (image processing program) (National Insitutes of Health (NIH), https://imagej.nih.gov/ij/) (Schneider et al., 2012)
   MorphoLibJ plugin to ImageJ version 1.4.0 (collection of mathematical morphology methods and plugins) (INRA-IJPB Modeling and Digital Imaging lab, https://imagej.net/MorphoLibJ) (Legland et al., 2016).
4. R version 3.6.0 (software environment for statistical computing and graphics) (R Core Team, https://www.r-project.org/) (R Core Team, 2019)
   exiftoolr package version 0.1.3 (O’Brien, 2020)
   ggplot2 package version 3.2.0 (Wickham, 2016)
   plyr package version 1.8.4 (Wickham, 2011)
   readr package version 1.3.1 (Wickham et al., 2018)
   

Procedure