**4. Conclusions**

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 its inhibition concentration.

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 analysis of ferning biofluids.

**23**

*Effects of Medium Components on the Bulk Rheology and on the Formation of Ferning Patterns…*

The birefringence that was produced by liquid crystals within the samples of biofilm had two different morphologies, bundled strands that were about 50 μm in length in hydrated biofilm and star-shaped bundle of strands that were almost ten times larger inside the crystalline region of the ferning pattern. So, in addition to the self-assembly of the phages to strands inside the biofilm, a more complex assembly took place during crystallization in the biofilm that produced this tertiary structure. During the ferning process, clusters of bacteria became entrapped within the crystalline phase. Other researchers have found that these entrapped bacteria are in suspended animation state and that they could be brought back to life upon rehydration. If PAO1 can also reanimate, then ferning is yet another mechanism that *P. aeruginosa* could utilize to survive extreme conditions, similar to how liquid crystals formed by phages enhanced the resistance and persistence of *P. aeruginosa*.

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,

This work would not be possible without funding from Medical Research

The supplemental documents for this section may be found at: https://ir.library.

and Dalton Myas for helping with experimental preparation.

We do not have any conflict of interests to declare.

*DOI: http://dx.doi.org/10.5772/intechopen.85240*

**Acknowledgements**

**Conflict of interest**

Foundation of Oregon.

**Supplemental materials**

oregonstate.edu/concern/defaults/g158bp85b.

**Funding**

*Effects of Medium Components on the Bulk Rheology and on the Formation of Ferning Patterns… DOI: http://dx.doi.org/10.5772/intechopen.85240*

The birefringence that was produced by liquid crystals within the samples of biofilm had two different morphologies, bundled strands that were about 50 μm in length in hydrated biofilm and star-shaped bundle of strands that were almost ten times larger inside the crystalline region of the ferning pattern. So, in addition to the self-assembly of the phages to strands inside the biofilm, a more complex assembly took place during crystallization in the biofilm that produced this tertiary structure. During the ferning process, clusters of bacteria became entrapped within the crystalline phase. Other researchers have found that these entrapped bacteria are in suspended animation state and that they could be brought back to life upon rehydration. If PAO1 can also reanimate, then ferning is yet another mechanism that *P. aeruginosa* could utilize to survive extreme conditions, similar to how liquid crystals formed by phages enhanced the resistance and persistence of *P. aeruginosa*.
