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Two separate microscope filters were prepared for each sample, and evaluated by 2 independent, experienced microscopists.

The red channel, used for mOrange, tdTomato, TagRFP and mRFP1.2, used a polychromator set at 550 nm, a 570DRLP beamsplitter, and a 595/40 nm emission filter placed in the filterwheel under the microscope (filters were obtained from Omega Optical).

To directly compare the two filter configurations in the microscope, both filters are mounted onto a small optical bread board, which slides on rails on the optical table.

Both filters were mounted on microscope slides with non-fluorescent oil, and abundance was determined by epifluorescence microscopy equipped with a Hg 100-W lamp, blue-light excitation filter cube (exciter BP460 to 490 nm, dichroic mirror 505 nm, barrier 515 nm) and bright line multiband filter cube (exciter BP360 to 730 nm, dichroic mirror 400 nm, barrier 420 nm).

Filters were mounted on microscope slides with Vectashield (Vector Labs, Burlingame, California).

Visual monitoring using strain PAO1 tagged with the green fluorescent protein (GFP) and confocal scanning laser microscope (CSLM) imaging demonstrated that after filters were inoculated with single P. aeruginosa cells, large microcolonies had formed after 8 hours which then further developed into confluent biofilms after 14 hrs [ 5].

The filters were mounted onto glass microscope slides in 50% glycerol and coverslipped.

Cell nuclei on the filters were visualized under a fluorescent microscope.

Cells on undersides of filters were examined and counted under microscope.

Filters were examined with an epifluorescence microscope (Eclipse 90i, Nikon Corp., Japan).

Whole filters were manually counted under the inverted microscope.