**Acknowledgements**

*Pseudomonas aeruginosa - An Armory Within*

was not tested during this study.

its inhibition concentration.

analysis of ferning biofluids.

**4. Conclusions**

From the linear ferning section that was outlined by the red square (**Figure 7f**–**k**), the branches appeared to be about 1 mm in width with a distinct centerline running through each branch (**Figure 7f**). A magnified view of one of these branches revealed latticed or layered networks emanating from this central line (blue arrows) and cavities (pink arrows) that were present throughout the structure (**Figure 7g**). Some of the cavities were large, tunneling deep into the ferning structure (**Figure 7h**). Under polarized light, the branch was shown to have dozens of the star-shaped red and gold birefringent bundles (**Figure 7i**). Changing the angle of the polarized filter changed the color of the birefringent region from red and gold to gold and green (**Figure 7k**). The star-shaped birefringent clusters only existed within the crystal regions of the fern pattern, while the strands were scattered throughout the plate regardless of the ferning pattern (**Figure 7j**). This localization of the morphologies implied that the birefringent strands were produced within the biofilm; thus, they could be found throughout the material, while the formations of the birefringent stars were created as a result of crystallization, so they were only found within the crystalline regions. Clusters of bacterial cells appeared to be entrapped within the crystallized fern (yellow arrows), especially around the extremities of the ferning structure (**Figure 7c** and **j**). Similarly entrapped bacterial clusters were capable of reanimation at least a week after desiccation within the ferning structure [50]. Therefore, clusters of *P. aeruginosa* that were seen in **Figure 7c** and **j** may be in a suspended animation state as well, though this hypothesis

In environments that contained high viscosity (glycerol), high osmolarity (glycerol, NaCl), and high concentrations of simple carbon (glucose), the elasticity and the yield stress of the biofilm increased. Silver nitrate had an inhibiting effect on the biofilm formation, but only at concentrations that were greater than 0.1 mM. Similarly, concentrations of glycerol greater than 10% completely inhibited biofilm growth. However, the complex carbon structure of sucrose meant that it could not be utilized as an additional carbon source by PAO1 in the same way that glucose was utilized. Therefore, sucrose did not change the rheological properties of the biofilm. So, *P. aeruginosa* developed stronger biofilm under nutrient-rich conditions, certain levels of osmotic stress, and certain levels of diffusion limitation. However, it would not develop biofilm when the osmotic stress or diffusion limitation exceeded an inhibition amount or when an antimicrobial agent exceeded

While the rheological properties of biofilm revealed information about the strength of the biofilm, the morphology of the ferning pattern best described the interactions between the electrolytes and the EPS in the biofilm. Typically, the biofilm had ferning coverage of about 50% and a ferning complexity score of 5. The ferning complexity increased with the strength of the biofilm (high complex modulus and yield stress), as stronger biofilm increased diffusion limitation that was experienced by the solutes within the matrix. The coverage and complexity score both dropped to zero when no biofilm formed, so the macromolecule-to-salt ratio was too low for ferning to occur, as with high concentrations of silver nitrate and glycerol. Many of the analysis methods of biofluid ferning patterns were qualitative and subjective, which is currently problematic considering its use as an indicator of certain medical symptoms. The image analysis and ferning classification method that was presented here could easily be applied to the other fields to give more quantitative values to the

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The authors would like to thank Dr. Skip Rochefort for consulting on the rheology tests and Kristin Marshall for her help with the image conversion work. Additionally, the authors would like to thank Marisa Thierheimer, Curran Gahan, and Dalton Myas for helping with experimental preparation.
