**3.3 Birefringence of bacterial biofilms**

Analyzing the pre-desiccation biofilm that was grown in 5% NaCl under polarized filters showed the entire sample sample lit with birefringent strands (**Figure 6**). The Pf bacteriophages produced by *P. aeruginosa* are known to self-assemble into liquid crystals that exhibit birefringence [64]. The birefringent strands (black arrows) are 50–100 μm in length, and they are evenly distributed throughout the sample (**Figure 6a** and **b**). A magnified view of the strands revealed that each strand was a bundle of smaller strands that were surrounded by cell clusters (yellow arrows) and biofilm (**Figure 6c**). The bacteriophages are about 6 μm in length, so the bundle was likely composed of hundreds of individual Pf phages that were assembled into one strand [64]. These birefringent strand clusters did not exist in samples lacking biofilm. A plate with 10% NaCl LB medium formed sheets of salt crystals, no visible biofilm, and no birefringent strands (Figure S6, https://ir.library.oregonstate.edu/ concern/defaults/g158bp85b).

The 5% NaCl sample that was previously analyzed (**Figure 6**) was desiccated (**Figure 7a**) and examined with and without a polarized filter (**Figure 7b**–**k**). The red lines outlined the specific regions of the fern under the microscope. The region outlined in the circle (**Figure 7b**–**e**) was the square crystalline structure on the plate. Without a filter this region showed a cubic structure with a length of 4–5 mm per side with a nucleation point in the center and thin diagonal lines running through it with cavernous voids coming from the sides of the structure (**Figure 7b**). The center of the ferning structure showed cuboid lattice-like patterns of growth emerging from the seed point (**Figure 7e**). Similarly, the previous study of the ferning pattern in a gelatin-salt mix revealed interlocking salt blocks with a length of 10–30 μm on a side that formed the backbone of the ferning structure [47].

#### **Figure 6.**

*Polarized microscopic images of inoculated samples of PAO1 in modified LB medium with a concentration of 5% NaCl. (a and b) Birefringent fragments exist throughout the liquid medium. (c) The birefringent threadlike fragments (black arrows) of about 50–100 μm in length were dispersed within medium that was full of bacterial clusters (yellow arrows).*

**21**

**Figure 7.**

*polarization.*

existed with only one color.

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

With a polarized filter, bright birefringent regions lit throughout the square (**Figure 7c**). A closer look at the center of the square revealed two forms of birefringent structures, large red and gold star formations (~0.5 mm in diameter) and red and gold strands that were about 50 μm in diameter (**Figure 7d**). In contrast to the birefringent strands that were scattered throughout the sample (black arrows), the birefringent stars (white arrows) were only in the crystallized ferns. Each birefringent strand that was visible in **Figure 7c** was a bundle of even smaller birefringent strands (**Figure 6c**). Therefore, the formation of the birefringent star demonstrated that having an even higher order of self-assembly during desiccation was possible such that the bundled strands further merged into a star formation. The alternating red and gold coloring indicated that the strands with matching orientations cluster together, but they must not have formed the entire cluster, as no star formation

*Desiccated biofilm that was grown in LB medium with 5% NaCl. (a) Ferning pattern from the bottom of the petri dish. The regions that are outlined in red were inspected under the microscope. (b–e) The cubic piece at the top right corner of the plate (red circle): (b) seen under normal light; (c and d) seen through a polarized filter at different magnifications; (e) focused on the seed point of crystallization. (f and k) The ferning region of the plate (red square): (f–h) different regions of the fern under normal light at different magnifications; (i and j) seen through a polarized filter at different magnifications; (k) viewed at a different angle of* 

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

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

#### **Figure 7.**

*Pseudomonas aeruginosa - An Armory Within*

**3.3 Birefringence of bacterial biofilms**

concern/defaults/g158bp85b).

that formed the backbone of the ferning structure [47].

salt ratio.

or oblique branching seemed to be the most common type of ferning with the examples of branches with acute angles being rare [47]. The cause of the change in the morphology of the fern in biofilms from 90° angles to acute angles is not immediately clear. However, other studies have reported that the gelatin-to-salt ratio was the key factor controlling the ferning morphology of salt-gelatin mixtures [47]. Therefore, the samples with higher rheological properties (glycerol and glucose samples), which arguably has a higher amount of EPS, may have produced branching with acute angles due to the increased EPS-to-salt ratio. Thus, the acute-angle morphology dominated when the biofilms had larger rheological values, indicating higher EPS-to-salt ratio, while orthogonal-branching morphology dominated at intermediate ratios with no ferning at extremely high or low values of the EPS-to-

Analyzing the pre-desiccation biofilm that was grown in 5% NaCl under polarized filters showed the entire sample sample lit with birefringent strands (**Figure 6**). The Pf bacteriophages produced by *P. aeruginosa* are known to self-assemble into liquid crystals that exhibit birefringence [64]. The birefringent strands (black arrows) are 50–100 μm in length, and they are evenly distributed throughout the sample (**Figure 6a** and **b**). A magnified view of the strands revealed that each strand was a bundle of smaller strands that were surrounded by cell clusters (yellow arrows) and biofilm (**Figure 6c**). The bacteriophages are about 6 μm in length, so the bundle was likely composed of hundreds of individual Pf phages that were assembled into one strand [64]. These birefringent strand clusters did not exist in samples lacking biofilm. A plate with 10% NaCl LB medium formed sheets of salt crystals, no visible biofilm, and no birefringent strands (Figure S6, https://ir.library.oregonstate.edu/

The 5% NaCl sample that was previously analyzed (**Figure 6**) was desiccated (**Figure 7a**) and examined with and without a polarized filter (**Figure 7b**–**k**). The red lines outlined the specific regions of the fern under the microscope. The region outlined in the circle (**Figure 7b**–**e**) was the square crystalline structure on the plate. Without a filter this region showed a cubic structure with a length of 4–5 mm per side with a nucleation point in the center and thin diagonal lines running through it with cavernous voids coming from the sides of the structure (**Figure 7b**). The center of the ferning structure showed cuboid lattice-like patterns of growth emerging from the seed point (**Figure 7e**). Similarly, the previous study of the ferning pattern in a gelatin-salt mix revealed interlocking salt blocks with a length of 10–30 μm on a side

*Polarized microscopic images of inoculated samples of PAO1 in modified LB medium with a concentration of 5% NaCl. (a and b) Birefringent fragments exist throughout the liquid medium. (c) The birefringent threadlike fragments (black arrows) of about 50–100 μm in length were dispersed within medium that was full of* 

**20**

**Figure 6.**

*bacterial clusters (yellow arrows).*

*Desiccated biofilm that was grown in LB medium with 5% NaCl. (a) Ferning pattern from the bottom of the petri dish. The regions that are outlined in red were inspected under the microscope. (b–e) The cubic piece at the top right corner of the plate (red circle): (b) seen under normal light; (c and d) seen through a polarized filter at different magnifications; (e) focused on the seed point of crystallization. (f and k) The ferning region of the plate (red square): (f–h) different regions of the fern under normal light at different magnifications; (i and j) seen through a polarized filter at different magnifications; (k) viewed at a different angle of polarization.*

With a polarized filter, bright birefringent regions lit throughout the square (**Figure 7c**). A closer look at the center of the square revealed two forms of birefringent structures, large red and gold star formations (~0.5 mm in diameter) and red and gold strands that were about 50 μm in diameter (**Figure 7d**). In contrast to the birefringent strands that were scattered throughout the sample (black arrows), the birefringent stars (white arrows) were only in the crystallized ferns. Each birefringent strand that was visible in **Figure 7c** was a bundle of even smaller birefringent strands (**Figure 6c**). Therefore, the formation of the birefringent star demonstrated that having an even higher order of self-assembly during desiccation was possible such that the bundled strands further merged into a star formation. The alternating red and gold coloring indicated that the strands with matching orientations cluster together, but they must not have formed the entire cluster, as no star formation existed with only one color.

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 was not tested during this study.
