*3.1.6 Modification with sodium chloride*

Unmodified LB medium already consists of sodium chloride (NaCl) at a concentration of 1%, and the modified concentration varied from 1 to 5% of NaCl. The complex modulus of biofilm remained constant for concentrations below 2.5% and increased by one order of magnitude for concentrations between 2.5 and 5%, while the modulus of the sterile modified LB medium was not affected by the concentration of NaCl (**Figure 2e**). Similarly, the yield stress increased as the concentration of NaCl was increased greater than 2.5% (**Figure 3e**). NaCl is already required for bacterial growth to provide osmotic balance, but a larger amount of salts appeared to promote stronger biofilm. The change in the biofilm could be caused by the higher salinity or osmolarity, making the environment hostile, triggering a higher level of production of alginate and other types of EPS as a countermeasure. Previous studies found that concentrations of NaCl between about 1 and 3% increased production of biofilms in *S. aureus* and *P. aeruginosa*, while concentrations of about 6% of NaCl prevented growth of biofilm in *S. aureus* [29, 31]. At concentrations of NaCl above 10%, no biofilm growth was observed, and the plate quickly crystalized to cubes of salt (Figure S6, https://ir.library.oregonstate.edu/concern/ defaults/g158bp85b).

### *3.1.7 Modification with silver nitrate*

Silver has antimicrobial properties that can inhibit bacterial growth and development of biofilm. Supplementation of silver nitrate (AgNO3) to the modified LB medium has no impact on the complex modulus (**Figure 2f**) or the yield stress of the biofilm for concentrations below 0.1 mM (**Figure 3f**). Past this concentration, the modulus instantly reduced to the same level as the sterile modified LB medium, and the yield stress disappeared. Correspondingly, the plates of biofilm at the higher silver concentrations appeared clear and less viscous, resembling sterile modified LB medium (Figure S1, https://ir.library. oregonstate.edu/concern/defaults/g158bp85b). Therefore, the antimicrobial activity of the silver appeared to be strongly dependent on concentration, with little to no effect on bacterial growth at concentrations lower than the threshold (0.1 mM) and deadly at higher concentrations. These results are consistent with previous studies where the inhibitory threshold for *S. aureus* was over 0.033 mM, while the inhibitory threshold for *P. aeruginosa* was over 0.16 μg mL<sup>−</sup><sup>1</sup> (0.45 mM) [34, 71].

#### *3.1.8 Summary of rheological characterization*

The rheological parameters of elastic modulus and yield stress are useful measures of the strength of a biofilm. The complex modulus and the yield stress of the biofilms increased with the addition of glucose, which served as an additional source of carbon, but they were unaffected by addition of sucrose, which is a complex sugar that the bacteria could not utilize. The strength increased to an extent with osmolarity (glycerol and NaCl) and dramatically reduced to their sterile baseline at concentrations that were higher than the inhibitory threshold of an antimicrobial agent (AgNO3). Samples with higher rheological properties correlated with a biofilm that appeared more viscous than the unmodified LB biofilm, while samples with lower modulus, and lacking a yield stress, such as high concentrations of glycerol and silver ions, appeared less viscous and free of biofilm. The values of modulus and yield stress for the samples of biofilm displayed the same medium dependent response; therefore, either measurement would be a useful metric of the strength of biofilm. Out of the five chemical modifications, three modifications increased the strength of the biofilm when compared to the unmodified LB biofilm: glycerol for concentrations up to 10%, glucose for concentrations at least up to 4.5%, and NaCl for concentrations higher than 2.5%. One of the chemicals, sucrose, had no measurable effect on the strength of the biofilm for concentrations at least up to 4.5%, while another modifier, AgNO3, inhibited bacterial growth at a concentration above 0.1 mM.

#### **3.2 Development of ferning patterns on dried plates of biofilm**

After the plates were dried over a span of weeks in the incubator, they were photographed, and the photographs were converted to black and white images and cropped (Table S1, https://ir.library.oregonstate.edu/concern/defaults/g158bp85b). The patterns of crystallization on the plates were analyzed based on the ferning coverage of the plate and the complexity of the pattern (**Figure 4**). The complexity of the ferning was scored qualitatively by the degree of branching of the pattern (**Figure 4**): 0, empty plate, no ferning; 1, seed or nucleation points; 2, lines without branching; 3, orthogonal pattern with 1° of branching; 4, orthogonal pattern with 2° of branching; 5, orthogonal pattern with 3° of branching; 6, orthogonal pattern with 4° of branching; 7, orthogonal pattern with 5° of branching; and 8, dendritic pattern with branching at acute angles. The branches at acute angles were distinctly smaller, and they were irregularly branched when compared to the orthogonal ferns that were scored 3–7 on the complexity scale.

The ferning coverage was calculated quantitatively based on the percent of white pixels in the black and white images that were converted from its original photograph (**Figure 5a**). A photograph of the ferning on a plate of unmodified LB biofilm showed high ferning coverage (top photos), while the plate of sterile unmodified LB medium was noticeably absent of ferning with low calculated coverage (bottom photos). Even without biofilm growth, the sterile coverage values were not zero because the lighting and the glare of the plate surface produced some pixel artifacts. **Figure 5b**–**f** shows the mean coverage of the plates of unmodified LB biofilm (red filled square, n = 12) and of the plates of sterile unmodified LB medium (red unfilled circle, n = 7) plotted with the data of the modified LB medium. The left black y-axis is the ferning coverage, while the right gray y-axis is the qualitative ferning complexity score for biofilm (gray filled square) and the sterile LB medium (gray unfilled circle).

#### *3.2.1 Modifications with glycerol*

With the addition of glycerol, the ferning pattern of the samples of biofilm initially changed from a complexity score of 5 and a coverage of 47% (unmodified LB

**17**

of about zero.

**Figure 4.**

*3.2.2 Modification with glucose*

*3.2.3 Modification with sucrose*

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

biofilm) to a complexity score of 8 (**Figure 5b**). The change in ferning morphology from the orthogonal form to the acute branching form occurred at the lowest tested concentration of glycerol (**Figure 5b**: 2% plate, top left). However, as the concentration of glycerol increased, the ferning coverage dropped dramatically, reaching zero at around 8%. From both the visual inspection and the rheological measurement, samples below 10% had strong biofilm. However, a large amount of glycerol prevented the sample from completely drying, leaving the surface of the sample looking shiny and wet, causing both the ferning coverage and the complexity score to drop between 4 and 10% glycerol (**Figure 5b**: 10% plate, top right). The plates with concentrations of glycerol above 10% never dried, so no photographs were taken, and values of the coverage and complexity score were assumed to be zero. Sterile LB medium coverage and complexity score did not change from the unmodified values

*A guide for the ferning complexity score of the dried biofilm ferning pattern.*

In samples that were modified with the addition of glucose, the ferning coverage remained consistent, while the complexity score changed from 5 to 8 at 2.5% and then held steady at higher concentrations of glucose (**Figure 5c**). The pattern on the plates transitioned from standard orthogonal ferning at low concentrations of glucose (**Figure 5c**: 0.5%, top left) to acute branching at high concentrations of glucose (**Figure 5c**: 4.5%, top right). The coverage and the complexity score on the

The desiccated plates of medium modified with sucrose had the most unusual patterns (**Figure 5d**). Both the coverage (47–30%) and the complexity score (5–3) dropped when the concentration of sucrose increased from 0 to 2%. However, with further increase from 2 to 4% sucrose, the coverage increased to 50%, while the complexity score continued to show less degrees of branching. Similar to glycerol, sucrose is hygroscopic, so plates appeared shinier and somewhat wet with increasing concentration of sucrose. At the same time, the ferning on the surface evolved (Table S1, https://ir.library.oregonstate.edu/concern/defaults/g158bp85b) from the orthogonal pattern (**Figure 5d**: 0.5%, top left) to fine strands to finally no ferning at high concentrations of sucrose (**Figure 5d**: 4.5%, top right). The glare in the photos from the shiny surface along with the packing of the fine crystalline strands produced high estimations of surface coverage until both the coverage and

sterile plate remained unchanged from the standard values.

*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 4.** *A guide for the ferning complexity score of the dried biofilm ferning pattern.*

biofilm) to a complexity score of 8 (**Figure 5b**). The change in ferning morphology from the orthogonal form to the acute branching form occurred at the lowest tested concentration of glycerol (**Figure 5b**: 2% plate, top left). However, as the concentration of glycerol increased, the ferning coverage dropped dramatically, reaching zero at around 8%. From both the visual inspection and the rheological measurement, samples below 10% had strong biofilm. However, a large amount of glycerol prevented the sample from completely drying, leaving the surface of the sample looking shiny and wet, causing both the ferning coverage and the complexity score to drop between 4 and 10% glycerol (**Figure 5b**: 10% plate, top right). The plates with concentrations of glycerol above 10% never dried, so no photographs were taken, and values of the coverage and complexity score were assumed to be zero. Sterile LB medium coverage and complexity score did not change from the unmodified values of about zero.

### *3.2.2 Modification with glucose*

*Pseudomonas aeruginosa - An Armory Within*

*3.1.8 Summary of rheological characterization*

The rheological parameters of elastic modulus and yield stress are useful measures of the strength of a biofilm. The complex modulus and the yield stress of the biofilms increased with the addition of glucose, which served as an additional source of carbon, but they were unaffected by addition of sucrose, which is a complex sugar that the bacteria could not utilize. The strength increased to an extent with osmolarity (glycerol and NaCl) and dramatically reduced to their sterile baseline at concentrations that were higher than the inhibitory threshold of an antimicrobial agent (AgNO3). Samples with higher rheological properties correlated with a biofilm that appeared more viscous than the unmodified LB biofilm, while samples with lower modulus, and lacking a yield stress, such as high concentrations of glycerol and silver ions, appeared less viscous and free of biofilm. The values of modulus and yield stress for the samples of biofilm displayed the same medium dependent response; therefore, either measurement would be a useful metric of the strength of biofilm. Out of the five chemical modifications, three modifications increased the strength of the biofilm when compared to the unmodified LB biofilm: glycerol for concentrations up to 10%, glucose for concentrations at least up to 4.5%, and NaCl for concentrations higher than 2.5%. One of the chemicals, sucrose, had no measurable effect on the strength of the biofilm for concentrations at least up to 4.5%, while another modifier, AgNO3, inhibited bacterial growth at a concentration above 0.1 mM.

**3.2 Development of ferning patterns on dried plates of biofilm**

(gray filled square) and the sterile LB medium (gray unfilled circle).

With the addition of glycerol, the ferning pattern of the samples of biofilm initially changed from a complexity score of 5 and a coverage of 47% (unmodified LB

After the plates were dried over a span of weeks in the incubator, they were photographed, and the photographs were converted to black and white images and cropped (Table S1, https://ir.library.oregonstate.edu/concern/defaults/g158bp85b). The patterns of crystallization on the plates were analyzed based on the ferning coverage of the plate and the complexity of the pattern (**Figure 4**). The complexity of the ferning was scored qualitatively by the degree of branching of the pattern (**Figure 4**): 0, empty plate, no ferning; 1, seed or nucleation points; 2, lines without branching; 3, orthogonal pattern with 1° of branching; 4, orthogonal pattern with 2° of branching; 5, orthogonal pattern with 3° of branching; 6, orthogonal pattern with 4° of branching; 7, orthogonal pattern with 5° of branching; and 8, dendritic pattern with branching at acute angles. The branches at acute angles were distinctly smaller, and they were irregularly branched when compared to the orthogonal ferns that were scored 3–7 on the complexity scale. The ferning coverage was calculated quantitatively based on the percent of white pixels in the black and white images that were converted from its original photograph (**Figure 5a**). A photograph of the ferning on a plate of unmodified LB biofilm showed high ferning coverage (top photos), while the plate of sterile unmodified LB medium was noticeably absent of ferning with low calculated coverage (bottom photos). Even without biofilm growth, the sterile coverage values were not zero because the lighting and the glare of the plate surface produced some pixel artifacts. **Figure 5b**–**f** shows the mean coverage of the plates of unmodified LB biofilm (red filled square, n = 12) and of the plates of sterile unmodified LB medium (red unfilled circle, n = 7) plotted with the data of the modified LB medium. The left black y-axis is the ferning coverage, while the right gray y-axis is the qualitative ferning complexity score for biofilm

**16**

*3.2.1 Modifications with glycerol*

In samples that were modified with the addition of glucose, the ferning coverage remained consistent, while the complexity score changed from 5 to 8 at 2.5% and then held steady at higher concentrations of glucose (**Figure 5c**). The pattern on the plates transitioned from standard orthogonal ferning at low concentrations of glucose (**Figure 5c**: 0.5%, top left) to acute branching at high concentrations of glucose (**Figure 5c**: 4.5%, top right). The coverage and the complexity score on the sterile plate remained unchanged from the standard values.

### *3.2.3 Modification with sucrose*

The desiccated plates of medium modified with sucrose had the most unusual patterns (**Figure 5d**). Both the coverage (47–30%) and the complexity score (5–3) dropped when the concentration of sucrose increased from 0 to 2%. However, with further increase from 2 to 4% sucrose, the coverage increased to 50%, while the complexity score continued to show less degrees of branching. Similar to glycerol, sucrose is hygroscopic, so plates appeared shinier and somewhat wet with increasing concentration of sucrose. At the same time, the ferning on the surface evolved (Table S1, https://ir.library.oregonstate.edu/concern/defaults/g158bp85b) from the orthogonal pattern (**Figure 5d**: 0.5%, top left) to fine strands to finally no ferning at high concentrations of sucrose (**Figure 5d**: 4.5%, top right). The glare in the photos from the shiny surface along with the packing of the fine crystalline strands produced high estimations of surface coverage until both the coverage and

#### **Figure 5.**

*(a) The original photograph was converted to a black and white image before the ferning coverage was calculated from the processed image. An example of ferns from biofilm that was grown in unmodified LB medium with 46.2% ferning coverage and from a plate of sterile unmodified LB medium showing zero ferning coverage. (b–f) Ferning coverage (left y-axis in black) and complexity score (right y-axis in gray) of biofilm that was grown in modified LB medium (coverage: black filled squares; complexity: Gray filled squares) and of sterile plates (coverage: black unfilled circles; complexity: Gray unfilled circles). The mean coverage and standard deviation of biofilm that was grown in unmodified LB medium (n = 12) and in sterile unmodified LB medium (n = 7) are plotted in red across figures (b–f). The results of the biofilm that was grown in modified LB medium with (b) glycerol, (c) glucose, (d) sucrose, (e) NaCl and (f) AgNO3 are shown. The change in morphology of the biofilm ferning pattern with (b) glycerol at concentrations of 2% (top left) and 10% (top right); (c) glucose at concentrations of 0.5% (top left) and 4.5% (top right); (d) sucrose at concentrations of 0.5% (top left) and 4.5% (top right); (e) NaCl at concentrations of 1.5% (top left) and 5% (top right); and (f) AgNO3 at concentrations of 0.001 mM (top left) and 1 mM (top right) are shown. The gray regions in (b) and (f) represent inhibiting concentrations that had no biofilm growth.*

the complexity score suddenly dropped to zero at the maximum concentration of sucrose that was tested (4.5%). The complexity score of the pattern steadily dropped from 5 to 0 with sucrose, while the calculation of coverage resulted in scattered results, as the surface glare and the interaction of the densely packed linear striations with light affected the procedure.

#### *3.2.4 Modification with sodium chloride*

With further addition of sodium chloride (NaCl) to the modified LB medium, the ferning coverage remained consistently around 50%, and the complexity score remained around 5 (**Figure 5e**). Still, a change to the pattern exists, as the ferns evolved from thin branches (**Figure 5e**: 1.5%, top left) to a more pronounced branching with large crystalline formations with increasing concentration of NaCl (**Figure 5e**: 5%, top right). While the ferning branches became wider with the further addition of NaCl, the complexity score did not change. The orthogonal morphology and the non-overlapping crystallization appear to naturally limit the maximum coverage of the ferning pattern, resulting in a consistent 40–50% coverage. The sterile dishes with modified LB medium that contained the same amount

**19**

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

The complexity score for the plates that were treated with silver nitrate (AgNO3) remained 5 for concentrations below 0.1 mM, but at higher concentrations, no biofilm growth occurred, resulting in a complexity score of 1 (**Figure 5f**). Even with no measurable biofilm, the plates contained clusters of dried materials that resembled nucleation points (**Figure 5f**: 1 mM, top right). For concentration below 0.1 mM of silver nitrate, the ferning patterns were orthogonal (**Figure 5f**: 0.001 mM, top left), and the coverage was in the range of 30–40%. Finally, the coverage dropped below 10% at concentrations that were greater than the inhibiting concentration of 0.1 mM. The coverage did not completely drop to 0, as beige clusters were left behind on the plate, which was the same reason that the complexity score was 1 for

Similar to the dependence of rheological properties of the biofilm on the nutri-

The complexity score and the ferning coverage was higher for stronger biofilms (higher G\* and τy) that caused more limitations in mass transfer, and both values dropped to nearly zero when no biofilm was present. The morphology of ferns with a complexity score of 8 that were produced by the biofilms with higher elasticity was similar to random/acute-angled branching ferns that were produced under conditions of increased diffusion limitation in a previous study [47]. Exceptions were the plates that appeared to never fully dry due to the high concentration of hygroscopic materials like sucrose or glycerol. So, even as the rheological properties of the biofilm increased (glycerol) or stayed constant (sucrose), the complexity score and coverage dropped with increasing amounts of the modifying chemical. The ferning coverage never exceeded 60%, indicating a natural growth limitation that was based on the available space and the morphology of the ferns. The videos of the ferning process demonstrated how these branches quickly started and stopped growing without any of the branches overlapping (Video S1, https://

The ferning patterns of the biofilms were large (visible without microscopy on the order of centimeters) with most of the patterns consisting of orthogonal branches, and the ferns were reproducible in coverage and complexity score. The ferning patterns that were formed by the biofilm had the same morphology as ferning patterns of saliva, cervical mucus, *E. coli*, salt-gel, or salt-protein [44, 47, 49, 50]. From reports in the literature and the results in this work, the orthogonal

ent environment, the ferning pattern was dependent on the properties of the bacterial biofilm, so it changed with the composition of the medium. No ferning existed on plates that lacked biofilm. The presence of biofilm, confirmed rheologically and visually, correlated with robust ferning patterns. Using the same boxcounting method, ferning patterns on the samples of unmodified LB biofilm had a fractal dimension of 1.8 (Figure S5, https://ir.library.oregonstate.edu/concern/ defaults/g158bp85b), which is consistent with samples of mucus that had a fractal dimension of 1.7 [44]. The plates of sterile medium that were incubated and dried under the same conditions as the plates of biofilm had no visible ferning pattern, no yield stress, and little to no elastic modulus. So, the medium composition not only affected the growth of the biofilm and its rheological properties but also, by exten-

of salt did not produce ferning patterns, so the coverage and complexity score

the highest concentration even though no biofilm was present.

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

*3.2.5 Modification with silver nitrate*

*3.2.6 Summary of ferning patterns*

sion, affected the ferning pattern.

ir.library.oregonstate.edu/concern/defaults/g158bp85b).

remained zero.

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

of salt did not produce ferning patterns, so the coverage and complexity score remained zero.

## *3.2.5 Modification with silver nitrate*

*Pseudomonas aeruginosa - An Armory Within*

the complexity score suddenly dropped to zero at the maximum concentration of sucrose that was tested (4.5%). The complexity score of the pattern steadily dropped from 5 to 0 with sucrose, while the calculation of coverage resulted in scattered results, as the surface glare and the interaction of the densely packed linear

*(a) The original photograph was converted to a black and white image before the ferning coverage was calculated from the processed image. An example of ferns from biofilm that was grown in unmodified LB medium with 46.2% ferning coverage and from a plate of sterile unmodified LB medium showing zero ferning coverage. (b–f) Ferning coverage (left y-axis in black) and complexity score (right y-axis in gray) of biofilm that was grown in modified LB medium (coverage: black filled squares; complexity: Gray filled squares) and of sterile plates (coverage: black unfilled circles; complexity: Gray unfilled circles). The mean coverage and standard deviation of biofilm that was grown in unmodified LB medium (n = 12) and in sterile unmodified LB medium (n = 7) are plotted in red across figures (b–f). The results of the biofilm that was grown in modified LB medium with (b) glycerol, (c) glucose, (d) sucrose, (e) NaCl and (f) AgNO3 are shown. The change in morphology of the biofilm ferning pattern with (b) glycerol at concentrations of 2% (top left) and 10% (top right); (c) glucose at concentrations of 0.5% (top left) and 4.5% (top right); (d) sucrose at concentrations of 0.5% (top left) and 4.5% (top right); (e) NaCl at concentrations of 1.5% (top left) and 5% (top right); and (f) AgNO3 at concentrations of 0.001 mM (top left) and 1 mM (top right) are shown. The gray regions in (b)* 

With further addition of sodium chloride (NaCl) to the modified LB medium, the ferning coverage remained consistently around 50%, and the complexity score remained around 5 (**Figure 5e**). Still, a change to the pattern exists, as the ferns evolved from thin branches (**Figure 5e**: 1.5%, top left) to a more pronounced branching with large crystalline formations with increasing concentration of NaCl (**Figure 5e**: 5%, top right). While the ferning branches became wider with the further addition of NaCl, the complexity score did not change. The orthogonal morphology and the non-overlapping crystallization appear to naturally limit the maximum coverage of the ferning pattern, resulting in a consistent 40–50% coverage. The sterile dishes with modified LB medium that contained the same amount

striations with light affected the procedure.

*and (f) represent inhibiting concentrations that had no biofilm growth.*

*3.2.4 Modification with sodium chloride*

**18**

**Figure 5.**

The complexity score for the plates that were treated with silver nitrate (AgNO3) remained 5 for concentrations below 0.1 mM, but at higher concentrations, no biofilm growth occurred, resulting in a complexity score of 1 (**Figure 5f**). Even with no measurable biofilm, the plates contained clusters of dried materials that resembled nucleation points (**Figure 5f**: 1 mM, top right). For concentration below 0.1 mM of silver nitrate, the ferning patterns were orthogonal (**Figure 5f**: 0.001 mM, top left), and the coverage was in the range of 30–40%. Finally, the coverage dropped below 10% at concentrations that were greater than the inhibiting concentration of 0.1 mM. The coverage did not completely drop to 0, as beige clusters were left behind on the plate, which was the same reason that the complexity score was 1 for the highest concentration even though no biofilm was present.

#### *3.2.6 Summary of ferning patterns*

Similar to the dependence of rheological properties of the biofilm on the nutrient environment, the ferning pattern was dependent on the properties of the bacterial biofilm, so it changed with the composition of the medium. No ferning existed on plates that lacked biofilm. The presence of biofilm, confirmed rheologically and visually, correlated with robust ferning patterns. Using the same boxcounting method, ferning patterns on the samples of unmodified LB biofilm had a fractal dimension of 1.8 (Figure S5, https://ir.library.oregonstate.edu/concern/ defaults/g158bp85b), which is consistent with samples of mucus that had a fractal dimension of 1.7 [44]. The plates of sterile medium that were incubated and dried under the same conditions as the plates of biofilm had no visible ferning pattern, no yield stress, and little to no elastic modulus. So, the medium composition not only affected the growth of the biofilm and its rheological properties but also, by extension, affected the ferning pattern.

The complexity score and the ferning coverage was higher for stronger biofilms (higher G\* and τy) that caused more limitations in mass transfer, and both values dropped to nearly zero when no biofilm was present. The morphology of ferns with a complexity score of 8 that were produced by the biofilms with higher elasticity was similar to random/acute-angled branching ferns that were produced under conditions of increased diffusion limitation in a previous study [47]. Exceptions were the plates that appeared to never fully dry due to the high concentration of hygroscopic materials like sucrose or glycerol. So, even as the rheological properties of the biofilm increased (glycerol) or stayed constant (sucrose), the complexity score and coverage dropped with increasing amounts of the modifying chemical. The ferning coverage never exceeded 60%, indicating a natural growth limitation that was based on the available space and the morphology of the ferns. The videos of the ferning process demonstrated how these branches quickly started and stopped growing without any of the branches overlapping (Video S1, https:// ir.library.oregonstate.edu/concern/defaults/g158bp85b).

The ferning patterns of the biofilms were large (visible without microscopy on the order of centimeters) with most of the patterns consisting of orthogonal branches, and the ferns were reproducible in coverage and complexity score. The ferning patterns that were formed by the biofilm had the same morphology as ferning patterns of saliva, cervical mucus, *E. coli*, salt-gel, or salt-protein [44, 47, 49, 50]. From reports in the literature and the results in this work, the orthogonal 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-tosalt ratio.
