**2. Materials and methods**

*Pseudomonas aeruginosa - An Armory Within*

**1.3 Birefringence of** *P. aeruginosa*

salt ratio fluids.

Studying the ferning pattern and complexity of biofluids or biogels gives a simple and indirect measurement of the structures within the material that guide or hinder the movement of ions that ultimately form these distinct crystallization patterns. While much of the ferning patterns seen in biofluids are linear patterns with 90° branching angles, tightly packed and curved ferning patterns can be expected to develop in environments that induce fast evaporation such as in low-viscosity fluids or environments that are highly diffusion limited such as in high macromolecule to

One of the techniques of self-assembly for small particles is through depletion attraction in a solvent during solvent evaporation [59, 60]. Depletion attraction is an entropic force that becomes relevant when the particles in the solvent move close enough together that their excluded volumes overlap [61]. This overlap increases the osmotic pressure in the surrounding fluid and further pushes the particles together [59]. These highly ordered or anisotropic solution is described as having a liquid crystal phase and this phase is birefringent, which means that their ordered state will split light into two beams with perpendicular polarization [36, 60, 62]. Liquid crystal phases have been observed with many different types of biopolymers such as DNA, peptides, glycopolymers, proteoglycans, viruses, collagen, cellulose, phages, and chitin [60, 61, 63]. Liquid crystals form: (i) nematic phase where the molecules form directional order but no positional order; (ii) smectic phase with positional order; or (iii) chiral phase with twisting order [60]. Of these, biopolymers most commonly have nematic phase. *P. aeruginosa* that exists in a viscous or anaerobic environment is stimulated to transcribe filamentous Pf bacteriophages that are about 2 μm in length and 6 nm in diameter [64]. In *P. aeruginosa* biofilm, the filamentous phage self-assembles through depletion attraction, with the biopolymers exerting the osmotic force that bundles the phage strands. These highly ordered anisotropic regions of nematic phase liquid crystals are birefringent, possessing a large negative charge, and the anisotropy was shown to increase with the ionic strength and the molecular weight [64, 65]. Birefringence is not only a direct indicator of molecular order, but it is an indicator for *P. aeruginosa* biofilm strength, surface adhesivity, desiccation tolerance, and antibiotic resistance [62, 64]. The filamentous bacteriophages facilitate chronic infection of *P. aeruginosa* in the host by promoting a less invasive, less inflammatory but more resistant, more persistent form of *P. aeruginosa* [66]. In addition, Pf phages can bind iron to inhibit the metabolic activity of other pathogens such as *Aspergillus fumigatus* [67]. Liquid crystal methods provide the means to study the structure and behavior of filamentous bacteriophages without perturbation [65]. Moreover, liquid crystal analysis, specifically through detection of its birefringence, was used to detect analytes such as glucose, cholesterol, *E. coli*, and even viruses such as Ebola and HIV [68]. This detection method was made possible through enzymatic reaction in response to analytes within the mesophase of the normally optically isotropic lipidic cubic phases that results in the formation of strongly birefringent liquid crystal phases that are easily detected optically [68]. The exogenous and endogenous birefringence from various classes of analytes were exploited to make simple and cheap detection tool that was proposed as a new diagnostic tool that can be utilized

**10**

**1.4 Objective**

in industry or in the field to detect biothreats [68].

Biofilm is composed of motile bacterial cells, non-motile bacterial aggregates, and mucoid hydrogels of EPS that have a heterogeneous, highly-porous microstructure,

## **2.1 Bacterial strains, media, and growth conditions**

The strain of *Pseudomonas aeruginosa* that was used for the entire study was the laboratory-adapted wild-type strain, PAO1. Miller lysogeny broth (LB) was prepared from BD Difco dry powder and autoclaved. Five different types of chemical modifications were made to the lysogeny broth: (i) glycerol was added to form between 1 and 15 v/v% in LB medium; (ii) glucose was added to form concentrations between 0.5 and 4.5 w/v% in LB; (iii) sucrose was added to form concentrations between 0.5 and 4.5 w/v% in LB; (iv) NaCl added to form concentrations between 1.5 and 5 w/v% in LB; and (v) AgNO3 added to form concentrations between 0.001 and 1 mM in LB. Modified LB medium in a petri dish (3.6 mL) was inoculated with an overnight culture of PAO1 (0.4 mL) and was incubated for 6 days at 37°C. Some of the dishes of modified LB medium were kept sterile and incubated along with the biofilm samples to act as a negative control.

#### **2.2 Measuring bulk biofilm rheology**

The sample rheology was measured on the Discovery Hybrid Rheometer 3 (DHR3, TA Instruments, USA) using a 40-mm stainless steel plate geometry at 25°C. The measurements took place in the following order:


Detailed description of the sample inoculation, the incubation, and the rheological measurement methods are located in our previous work [43].

#### **2.3 Ferning properties of dried bacterial biofilms**

The biofilm samples were dried in the incubator, forming ferning patterns that were large enough to be easily seen by the unaided eye. In this paper, the previous qualitative method for ferning characterization was converted to a quantitative method of image analysis by calculating the coverage area, the fractal dimension, and the complexity score (degree of branching) of the ferning pattern. This analysis was completed by taking photographs of the surface of the petri dish, converting

the photographs to black and white image on MATLAB (Figure S4, https://ir.library. oregonstate.edu/concern/defaults/g158bp85b), and finally calculating the ferning coverage by determining the ratio between white and black pixel areas in the image. The fractal dimension was calculated using the box-counting method on MATLAB (Figure S4, https://ir.library.oregonstate.edu/concern/defaults/g158bp85b).

#### **2.4 Dried bacterial biofilms under the microscope**

Microscopic images of the biofilm in its liquid and its dried ferning state were taken using an Eclipse Ti-S inverted microscope (Nikon, Japan). The polarized images were produced with polarized filters.
