**1. Introduction**

A vast majority of microorganisms in the world exist within biofilms, which are weak hydrogels that often form at various interfaces [1]. The biofilms consist of up to 98% water, and they are typically composed of polymicrobial aggregates that are encased in extrapolymeric substances (EPS) [2–5]. Besides acting as a protective barrier, the EPS, which is made of DNA, proteins, and polysaccharides, aid in adhesion and water retention [4]*. Pseudomonas aeruginosa* is a well-known opportunistic human pathogen that is a common cause of hospital-acquired infections in burn wounds and eyes [6–8], and it is known to create persistent infection in cystic fibrosis (CF) patients [8–15], having resistance to many classes of antibiotics

**4**

*Pseudomonas aeruginosa - An Armory Within*

[1] Winstanley C, O'Brien S, Brockhurst

diversification in cystic fibrosis chronic lung infections. Trends in Microbiology.

[2] Moradali MF, Ghods S, Rehm BH. Pseudomonas aeruginosa lifestyle: A paradigm for adaptation, survival, and persistence. Frontiers in Cellular and Infection Microbiology. 2017;**7**:39

[3] Stefani S, Campana S, Cariani L, et al. Relevance of multidrug-resistant Pseudomonas aeruginosa infections in cystic fibrosis. International Journal of Medical Microbiology.

MA. Pseudomonas aeruginosa evolutionary adaptation and

2016;**24**(5):327-337

**References**

2017;**307**(6):353-362

2010;**5**:e11044

2019;**54**(4):497-506

[4] Cox MJ, Allgaier M, Taylor B, et al. Airway microbiota and pathogen abundance in age-stratified cystic fibrosis patients. PLoS One.

[5] Rossi GA, Morelli P, Galietta LJ, Colin AA. Airway microenvironment alterations and pathogen growth in cystic fibrosis. Pediatric Pulmonology.

[6] Lobo LJ, Noone PG. Respiratory infections in patients with cystic fibrosis undergoing lung transplantation. Lancet Respiratory Medicine. 2014;**2**:73-82

[7] Chalermskulrat W, Sood N, Neuringer IP, Hecker TM, Chang L, Rivera MP, et al. Non-tuberculous mycobacteria in end stage cystic fibrosis: implications for lung transplantation.

[8] Jones AM. Which pathogens should we worry about? Paediatric Respiratory Reviews. 2019 [epub ahead of print]

Thorax. 2006;**61**:507-513

[16–18]. PAO1, a medically-relevant strain of *P. aeruginosa* that is used in this study, acts as the model for biofilm-forming bacteria. To grow, bacterial cultures need water, a source of carbon, a source of nitrogen, and trace amounts of salts. The lysogeny broth (LB) is a complex and non-selective medium; many different types of bacteria can grow on non-selective medium. Lysogeny broth was formulated by Giusseppe Bertani in 1951 to optimize *Shigella* growth, but it has since become the standard for growing many bacterial cultures [19]. Lysogeny broth is composed of: 1% tryptone (source of amino acids); 0.5% yeast extract (source of vitamins, amino acids, nitrogen, and carbon); [20] and 1% NaCl (provides osmotic balance) [21]. Yeast extract is made from baker's yeast (*Saccharomyces cerevisiae*) grown to a high concentration and then exposed to high temperature or osmotic shock, killing the yeast and starting autolysis of the cells through the yeast's own enzymes [20, 22, 23]. The resulting extract solution is further filtered and spray-dried into a powder [20]. Proteins make up the most significant component of the powdered yeast extract at 62.5–73.8 wt% [20]. The average molecular weight of the yeast extract is 438 Da with 59.1% of the total under 300 Da [23]. Using additions of glycerol, glucose, sucrose, sodium chloride (NaCl), and silver nitrate (AgNO3), this paper investigates various modifications of the LB medium for their effects on the biofilm.

Both the biofilm's structure and the cell-to-cell communication mechanism of the bacteria, known as quorum sensing (QS), are affected by their environment and the medium composition [24]. Quorum sensing controls additional properties that influence biofilm structures of bacteria, such as the production of extracellular DNA, proteins, mucus, and lipids [24–26]. When the growth environment becomes more viscous through the addition of glycerol, strains of *Pseudomonas* produced highmolecular-weight EPS and developed more robust biofilms [27]. The nutritional condition, such as the carbon source, influences the QS-associated swarming motility of *P. aeruginosa* [25]. While glucose supplementation limits bacterial motility, producing scattered, mushroom-like microcolonies, increasing the concentration of glucose from 0 to 2.7% caused an increase in the overall formation of biofilm [24, 25, 28, 29].

High osmolarity had a detrimental effect on biofilm of *P. fluorescens*, at roughly 0.4 Osm L<sup>−</sup><sup>1</sup> of either NaCl or sucrose, and the formation of biofilm decreased by four-fold as compared to lower concentrations of each component [30]. Similarly, mutant strains of *P. aeruginosa* that are found in CF patients transition from a nonmucoid to an alginate-overproducing state under osmotic stress that is induced by concentrations of 0.2–0.5 M NaCl (~1.2–3%) or 10% sucrose [31]. Silver has broad-spectrum antimicrobial effects on gram-negative bacteria that are welldocumented [32, 33]. For instance, for concentrations of silver sulfadiazine that are lower than 0.16 μg mL<sup>−</sup><sup>1</sup> , planktonic growth of *P. aeruginosa* was unchanged; however, at or above this threshold amount, the concentration of the planktonic bacteria was reduced by five orders of magnitude [34]. Silver sulfadiazine was even effective against mature biofilms above a threshold dose of 1 μg mL<sup>−</sup><sup>1</sup> , and at concentrations of 10 μg mL<sup>−</sup><sup>1</sup> , it can completely eradicate a pre-established biofilm of *P. aeruginosa* [34].

The following sections of this study cover three different methods of characterizing biofilms: (i) rheology to quantify the impact of the modified medium on the mechanical strength of the biofilm; (ii) ferning to characterize the mass transport of the salts through the polymer matrix of the biofilm during desiccation; and (iii) birefringence to observe self-assembly behavior of the solute in the biofilm.

#### **1.1 Rheology of biofilms**

The study of flow and deformation of matter (rheology) enables characterization of its structure and mechanical properties. Rheology is an especially valuable

**7**

*liquid-like.*

**Figure 1.**

in **Figure 1b**–**d**.

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

tool for understanding a vast range of "soft matter" that falls between liquid and solid phases [35]. Soft matter can be divided into four classes: (1) polymers, a long repeating chain of monomers which for biological samples include proteins, DNA, and cellulose; (2) colloids, a large category of materials that describe a suspension of one material into another medium such as aerosols, foams, emulsions, suspensions, and pastes; (3) amphiphiles, molecules with dual characteristics where one end of the molecule likes the solvent (hydrophilic), while the other end does not (hydrophobic) include surfactants that are amphiphiles at the air-water interface; and (4) liquid crystals, rod or disk shaped molecules that self-assemble to form orientation order but not positional order, resulting in an anisotropic fluid [35, 36]. Rheological techniques can characterize the strength and behavior of clinically relevant biological fluids such as mucus, blood plasma, and bacterial biofilm. More importantly, we can also use rheological measurements to drive the treatment of the biofluids toward a favorable clinical outcome. A rheological testing can quantify the viscous and elastic properties of a material. Two main modes of testing exist on a rotational rheometer: (1) steady-shear testing mode (**Figure 1a**–**d**), where the material is sheared between a stationary bottom plate and rotating top plate at a given stress or strain; and (2) the oscillation mode (**Figure 1e**–**g**), where the top

plate oscillates back and forth at a set frequency and amplitude.

From the shear-flow sweep test, the stress (τ) versus shear rate (γ̇) curve gives important information on the flow properties of the material (**Figure 1b**). The flow behavior can be modeled by Herschel-Bulkley function for yield stress fluids (Eq. (1): τy, yield stress; c [Pa s], flow coefficient or consistency index; p, power-law index). Yield stress materials have a minimum stress that must be surpassed before the material starts flowing. Different types of material flow behavior are described

*Shear and oscillatory rheological techniques. (a) In a two-plate steady-shear system where the top plate is moving, the velocity (v) of the fluid is dependent on the gap height (h). (b) Samples can exhibit several different flow behaviors of stress versus strain rate, including (1) ideally viscous Newtonian fluid; (2) shearthinning fluid; (3) shear-thickening fluid; and (4) yield stress fluid. Yield stress materials have a minimum stress (τy) that must be overcome before flow starts. (c) The stress-strain curve demonstrates a material with (2) no yield stress and (4) a material with a clear yield stress calculated using the tangent crossover point method. (d) The viscosity-shear rate plot shows (1) Newtonian fluid; (2) shear-thinning fluid with no yield stress reaching zero-shear viscosity (η0); (3) shear-thickening fluid; and (4) shear-thinning fluid with yield stress. (e) In a two-plate system the top plate can oscillate back and forth at set amplitude or frequency for oscillatory rheology tests. The amplitude sweep test has constant frequency (ω0) with changing strain amplitude while the frequency sweep test has constant strain amplitude (γ0) with changing frequency. (f) During the amplitude sweep test, the strain values up to the limit of the strain where the G***′** *and G*″ *values are constant (γL) are called the linear viscoelastic region (LVR). The point where G*″ *crosses over G***′** *is called the flow point (γf) [72]. (g) In a frequency sweep, when G***′** *> G*″ *materials are said to be solid-like and when G*″ *> G***′***, materials are said to be* 

*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*

tool for understanding a vast range of "soft matter" that falls between liquid and solid phases [35]. Soft matter can be divided into four classes: (1) polymers, a long repeating chain of monomers which for biological samples include proteins, DNA, and cellulose; (2) colloids, a large category of materials that describe a suspension of one material into another medium such as aerosols, foams, emulsions, suspensions, and pastes; (3) amphiphiles, molecules with dual characteristics where one end of the molecule likes the solvent (hydrophilic), while the other end does not (hydrophobic) include surfactants that are amphiphiles at the air-water interface; and (4) liquid crystals, rod or disk shaped molecules that self-assemble to form orientation order but not positional order, resulting in an anisotropic fluid [35, 36].

Rheological techniques can characterize the strength and behavior of clinically relevant biological fluids such as mucus, blood plasma, and bacterial biofilm. More importantly, we can also use rheological measurements to drive the treatment of the biofluids toward a favorable clinical outcome. A rheological testing can quantify the viscous and elastic properties of a material. Two main modes of testing exist on a rotational rheometer: (1) steady-shear testing mode (**Figure 1a**–**d**), where the material is sheared between a stationary bottom plate and rotating top plate at a given stress or strain; and (2) the oscillation mode (**Figure 1e**–**g**), where the top plate oscillates back and forth at a set frequency and amplitude.

From the shear-flow sweep test, the stress (τ) versus shear rate (γ̇) curve gives important information on the flow properties of the material (**Figure 1b**). The flow behavior can be modeled by Herschel-Bulkley function for yield stress fluids (Eq. (1): τy, yield stress; c [Pa s], flow coefficient or consistency index; p, power-law index). Yield stress materials have a minimum stress that must be surpassed before the material starts flowing. Different types of material flow behavior are described in **Figure 1b**–**d**.

#### **Figure 1.**

*Pseudomonas aeruginosa - An Armory Within*

[16–18]. PAO1, a medically-relevant strain of *P. aeruginosa* that is used in this study, acts as the model for biofilm-forming bacteria. To grow, bacterial cultures need water, a source of carbon, a source of nitrogen, and trace amounts of salts. The lysogeny broth (LB) is a complex and non-selective medium; many different types of bacteria can grow on non-selective medium. Lysogeny broth was formulated by Giusseppe Bertani in 1951 to optimize *Shigella* growth, but it has since become the standard for growing many bacterial cultures [19]. Lysogeny broth is composed of: 1% tryptone (source of amino acids); 0.5% yeast extract (source of vitamins, amino acids, nitrogen, and carbon); [20] and 1% NaCl (provides osmotic balance) [21]. Yeast extract is made from baker's yeast (*Saccharomyces cerevisiae*) grown to a high concentration and then exposed to high temperature or osmotic shock, killing the yeast and starting autolysis of the cells through the yeast's own enzymes [20, 22, 23]. The resulting extract solution is further filtered and spray-dried into a powder [20]. Proteins make up the most significant component of the powdered yeast extract at 62.5–73.8 wt% [20]. The average molecular weight of the yeast extract is 438 Da with 59.1% of the total under 300 Da [23]. Using additions of glycerol, glucose, sucrose, sodium chloride (NaCl), and silver nitrate (AgNO3), this paper investigates various modifications of the LB medium for their effects on the biofilm. Both the biofilm's structure and the cell-to-cell communication mechanism of the bacteria, known as quorum sensing (QS), are affected by their environment and the medium composition [24]. Quorum sensing controls additional properties that influence biofilm structures of bacteria, such as the production of extracellular DNA, proteins, mucus, and lipids [24–26]. When the growth environment becomes more viscous through the addition of glycerol, strains of *Pseudomonas* produced highmolecular-weight EPS and developed more robust biofilms [27]. The nutritional condition, such as the carbon source, influences the QS-associated swarming motility of *P. aeruginosa* [25]. While glucose supplementation limits bacterial motility, producing scattered, mushroom-like microcolonies, increasing the concentration of glucose from 0 to 2.7% caused an increase in the overall formation of biofilm [24, 25, 28, 29]. High osmolarity had a detrimental effect on biofilm of *P. fluorescens*, at roughly

of either NaCl or sucrose, and the formation of biofilm decreased by

, planktonic growth of *P. aeruginosa* was unchanged;

, it can completely eradicate a pre-established biofilm

, and at

four-fold as compared to lower concentrations of each component [30]. Similarly, mutant strains of *P. aeruginosa* that are found in CF patients transition from a nonmucoid to an alginate-overproducing state under osmotic stress that is induced by concentrations of 0.2–0.5 M NaCl (~1.2–3%) or 10% sucrose [31]. Silver has broad-spectrum antimicrobial effects on gram-negative bacteria that are welldocumented [32, 33]. For instance, for concentrations of silver sulfadiazine that

however, at or above this threshold amount, the concentration of the planktonic bacteria was reduced by five orders of magnitude [34]. Silver sulfadiazine was even effective against mature biofilms above a threshold dose of 1 μg mL<sup>−</sup><sup>1</sup>

to observe self-assembly behavior of the solute in the biofilm.

The following sections of this study cover three different methods of characterizing biofilms: (i) rheology to quantify the impact of the modified medium on the mechanical strength of the biofilm; (ii) ferning to characterize the mass transport of the salts through the polymer matrix of the biofilm during desiccation; and (iii) birefringence

The study of flow and deformation of matter (rheology) enables characterization of its structure and mechanical properties. Rheology is an especially valuable

**6**

0.4 Osm L<sup>−</sup><sup>1</sup>

are lower than 0.16 μg mL<sup>−</sup><sup>1</sup>

concentrations of 10 μg mL<sup>−</sup><sup>1</sup>

of *P. aeruginosa* [34].

**1.1 Rheology of biofilms**

*Shear and oscillatory rheological techniques. (a) In a two-plate steady-shear system where the top plate is moving, the velocity (v) of the fluid is dependent on the gap height (h). (b) Samples can exhibit several different flow behaviors of stress versus strain rate, including (1) ideally viscous Newtonian fluid; (2) shearthinning fluid; (3) shear-thickening fluid; and (4) yield stress fluid. Yield stress materials have a minimum stress (τy) that must be overcome before flow starts. (c) The stress-strain curve demonstrates a material with (2) no yield stress and (4) a material with a clear yield stress calculated using the tangent crossover point method. (d) The viscosity-shear rate plot shows (1) Newtonian fluid; (2) shear-thinning fluid with no yield stress reaching zero-shear viscosity (η0); (3) shear-thickening fluid; and (4) shear-thinning fluid with yield stress. (e) In a two-plate system the top plate can oscillate back and forth at set amplitude or frequency for oscillatory rheology tests. The amplitude sweep test has constant frequency (ω0) with changing strain amplitude while the frequency sweep test has constant strain amplitude (γ0) with changing frequency. (f) During the amplitude sweep test, the strain values up to the limit of the strain where the G***′** *and G*″ *values are constant (γL) are called the linear viscoelastic region (LVR). The point where G*″ *crosses over G***′** *is called the flow point (γf) [72]. (g) In a frequency sweep, when G***′** *> G*″ *materials are said to be solid-like and when G*″ *> G***′***, materials are said to be liquid-like.*

$$
\tau = \tau\_{\mathcal{V}} + c \ast \dot{\mathcal{V}}^p \tag{1}
$$

Two main types of oscillatory testing exist (**Figure 1e**–**g**). An amplitude sweep test oscillates the upper plate back and forth at a set frequency (ω0) at increasing strains (**Figure 1e**). On the modulus (G′—elastic; G″—viscous) versus strain plot (**Figure 1f**), the plateau is the linear viscoelastic region (LVR), and the strain limit of the region is γL. A frequency sweep test oscillates at a set amplitude (γ0), which has been determined previously from the amplitude sweep test to be within the LVR, at increasing frequency (**Figure 1e**). The frequency sweep describes how the material acts when the material is stressed for different periods. For example, when Silly Putty is stressed quickly by throwing it on the floor, it bounces back, acting like a rigid solid. However, when the Silly Putty sits at rest and experiences low stress over a long period of time, it spreads out, acting like a viscous fluid. The frequency sweep (**Figure 1g**) reveals if the material is solid-like (G′ > G″) or liquid-like (G″ > G′) and if the behavior is frequency dependent (G′(ω), G″(ω)) or independent. Stable gels and suspensions are typically solid-like and frequencyindependent, so these types of materials are called "gel-like."

Previous studies on biofilm rheology using various techniques of rheological measurement have found the elastic modulus (G′) to range in order of magnitude from 10<sup>−</sup><sup>2</sup> to 104 Pa for bulk biofilms at solid-liquid interfaces using plate-on-plate methods, while the values of yield stress (τy) range in order of magnitude from 10<sup>−</sup><sup>1</sup> to 105 Pa [2, 37–42]. The wide-ranging values of G′ and τy in the literature reflect the variability in the compositions of the biofilms, diversity of growth mediums, variability of growth conditions, and most importantly, natural variability of response of the microorganisms, even to the same medium and growth conditions. This chapter uses the techniques established in our previously published work on the nondestructive development and characterization of rheological properties of biofilms [43]. Using this non-destructive method, the measured values of elastic modulus and yield stress of PAO1 that were grown in standard LB medium were both between 0.1 and 10 Pa [43].

#### **1.2 Crystallization of biological materials**

Biological fluids like tears, cervical mucus, and saliva are all shown to self-assemble into fractal-like patterns of crystallization when they are dried [44, 45]. A fractal is a structure that is made of smaller parts that resemble the bigger parts, with a high degree of organization and self-similarity. This structure can be characterized with a specific fractal dimension [44]. Fractal dimension is a measure of complexity of the fractal pattern [46]. Random nucleations of salts initiate the process of crystallization, where its growth is limited by the diffusion of salt through the polymer matrix (proteins or macromolecules) [47]. Therefore, the combined effects of ionic strength, osmolarity, and the size and concentration of macromolecules control the behavior of crystallization, where too little or too much of one factor can dramatically alter the pattern of crystallization [48, 49]. A typical crystallization of biosaline proceeds in the following manner: (i) salt nucleation initiates the process of crystallization; (ii) the nucleation point grows with some symmetry into a highly-branched structure whose growth is modified by the interaction of the salt with the biological matter; (iii) the branches do not overlap or merge [47, 50, 51]. The process of crystallization of biofluids is called "arborization," "ferning," or "dendritic growth" in various literature [45]. In this paper, the general formation of salt crystals will continue to be called crystallization, while the specific crystallization of the biofluids that result in fractal patterns will be called ferns.

**9**

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

The ferning patterns of the dried samples of tears and saliva have been used for years as a supplementary diagnostic tool [49, 52]. The ferning patterns in saliva and in tears exhibit different morphologies; saliva produces linear ferns with branching angles of 90°, while ferns from tears have more curvature with tightly packed branches with acute angles. Ferning patterns from tears and saliva are traditionally classified in a qualitative manner according to Rolando's system as Type I to Type IV [53]. Type I has the most ferning and the highest concentration of protein, while Type IV has no ferning and the lowest amount of protein [49]. Samples of tears from healthy individuals typically exhibit robust, highly-branched ferning patterns (Type I and II), while samples from patients with eye or immune diseases show little to no ferning (Type III and IV) [48, 49, 52]. Through analysis of X-ray microscopy and scanning electron microscopy (SEM), the molecular structure of the ferns from tears is revealed to be composed of NaCl, KCl, and proteins [48]. In addition to helping detect infection, saliva ferning pattern has been shown to be useful for tracking ovulation cycle from highest fertility level during estrus to lowest fertility level during diestrus [54]. Cervical mucus is a heterogeneous hydrogel that changes over the course of an animal's reproductive cycle [44, 45, 55]. Regardless of the source, human or otherwise, high levels of estrogen are produced during ovulation or peak fertility, resulting in linear ferns with branching angles of 90°, while no ferning is found during the period of low fertility when progesterone is dominant [44, 45]. During ovulation, the cervical mucus is over 98% water with its highest level of salt while both water content and salt content drops during low fertility period [45]. The low salt content during low fertility period is the cause of the lack of ferning pattern. SEM analysis of ferns from gelatin-NaCl mixture revealed that the backbone of the ferning pattern was a series of interlocking crystalline blocks that were 10–30 μm in size [47]. When the fractal dimension of bovine cervical mucus (BCM) that was taken during ovulation was determined using the box-counting method, it was about 1.7, characteristic of diffusion-limited growth processes [44, 46]. Box counting method is based on counting non-empty boxes making up a fractal pattern on a grid [46]. A diffusion limitation was observed with the gelatin-NaCl mix as well. The ferning pattern became much less geometric and increasingly random at higher gelatin-to-NaCl ratios, where more diffusion limitation occurs due to the crosslinking of the gelatin, and the ferning ceased at extremely high gelatin-to-NaCl ratios [47]. Furthermore, the ferning pattern developed curvatures at evaporation

 [47]. Bacterial biofilms produce ferning patterns that are similar to gelatin and mucus samples [50, 56]. Upon evaporation of droplets of solutions of various salts with cells of *E. coli* and *Bacillus subtilis*, ferning patterns emerged where the crystallized top layer covered a base layer that consisted of bacterial cells. The structure of the *E. coli* ferns was linear with branching angles of 90°, similar to cervical mucus. Neither sterile saline solutions nor *E. coli* in pure water produced ferning, confirming the previous findings that ferning results from balanced proportions of salts and macromolecules [50, 56]. Bacteria inside the crystalline structure were effectively in a state of suspended animation that was capable of reanimation after rehydration, even a week later [50]. This crystallization was hypothesized to be a form of biomineralization [50], which occurs when biological organisms produce organo-mineral hybrids that give the organism mechanical strength and hardness. Examples of biomineralization that are found in nature include bones, teeth, shells, corals, and algal silica [57]. Previous studies on strain PAO1 of *P. aeruginosa* in flow cell reactors have shown biomineralization of calcium carbonate within the EPS of the biofilm [58]. SEM of the ferning sample of *E. coli* revealed a 3D structure that was composed of dried EPS, bacteria, and salts, with the salts concentrated in the

crystalline region, consistent with the previously mentioned studies [50].

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

rates above a threshold value of 11 μm s<sup>−</sup><sup>1</sup>

#### *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 ferning patterns of the dried samples of tears and saliva have been used for years as a supplementary diagnostic tool [49, 52]. The ferning patterns in saliva and in tears exhibit different morphologies; saliva produces linear ferns with branching angles of 90°, while ferns from tears have more curvature with tightly packed branches with acute angles. Ferning patterns from tears and saliva are traditionally classified in a qualitative manner according to Rolando's system as Type I to Type IV [53]. Type I has the most ferning and the highest concentration of protein, while Type IV has no ferning and the lowest amount of protein [49]. Samples of tears from healthy individuals typically exhibit robust, highly-branched ferning patterns (Type I and II), while samples from patients with eye or immune diseases show little to no ferning (Type III and IV) [48, 49, 52]. Through analysis of X-ray microscopy and scanning electron microscopy (SEM), the molecular structure of the ferns from tears is revealed to be composed of NaCl, KCl, and proteins [48]. In addition to helping detect infection, saliva ferning pattern has been shown to be useful for tracking ovulation cycle from highest fertility level during estrus to lowest fertility level during diestrus [54].

Cervical mucus is a heterogeneous hydrogel that changes over the course of an animal's reproductive cycle [44, 45, 55]. Regardless of the source, human or otherwise, high levels of estrogen are produced during ovulation or peak fertility, resulting in linear ferns with branching angles of 90°, while no ferning is found during the period of low fertility when progesterone is dominant [44, 45]. During ovulation, the cervical mucus is over 98% water with its highest level of salt while both water content and salt content drops during low fertility period [45]. The low salt content during low fertility period is the cause of the lack of ferning pattern.

SEM analysis of ferns from gelatin-NaCl mixture revealed that the backbone of the ferning pattern was a series of interlocking crystalline blocks that were 10–30 μm in size [47]. When the fractal dimension of bovine cervical mucus (BCM) that was taken during ovulation was determined using the box-counting method, it was about 1.7, characteristic of diffusion-limited growth processes [44, 46]. Box counting method is based on counting non-empty boxes making up a fractal pattern on a grid [46]. A diffusion limitation was observed with the gelatin-NaCl mix as well. The ferning pattern became much less geometric and increasingly random at higher gelatin-to-NaCl ratios, where more diffusion limitation occurs due to the crosslinking of the gelatin, and the ferning ceased at extremely high gelatin-to-NaCl ratios [47]. Furthermore, the ferning pattern developed curvatures at evaporation rates above a threshold value of 11 μm s<sup>−</sup><sup>1</sup> [47].

Bacterial biofilms produce ferning patterns that are similar to gelatin and mucus samples [50, 56]. Upon evaporation of droplets of solutions of various salts with cells of *E. coli* and *Bacillus subtilis*, ferning patterns emerged where the crystallized top layer covered a base layer that consisted of bacterial cells. The structure of the *E. coli* ferns was linear with branching angles of 90°, similar to cervical mucus. Neither sterile saline solutions nor *E. coli* in pure water produced ferning, confirming the previous findings that ferning results from balanced proportions of salts and macromolecules [50, 56]. Bacteria inside the crystalline structure were effectively in a state of suspended animation that was capable of reanimation after rehydration, even a week later [50]. This crystallization was hypothesized to be a form of biomineralization [50], which occurs when biological organisms produce organo-mineral hybrids that give the organism mechanical strength and hardness. Examples of biomineralization that are found in nature include bones, teeth, shells, corals, and algal silica [57]. Previous studies on strain PAO1 of *P. aeruginosa* in flow cell reactors have shown biomineralization of calcium carbonate within the EPS of the biofilm [58]. SEM of the ferning sample of *E. coli* revealed a 3D structure that was composed of dried EPS, bacteria, and salts, with the salts concentrated in the crystalline region, consistent with the previously mentioned studies [50].

*Pseudomonas aeruginosa - An Armory Within*

*τ = τ<sup>y</sup> + c* ∗ *γ*̇

independent, so these types of materials are called "gel-like."

Two main types of oscillatory testing exist (**Figure 1e**–**g**). An amplitude sweep test oscillates the upper plate back and forth at a set frequency (ω0) at increasing strains (**Figure 1e**). On the modulus (G′—elastic; G″—viscous) versus strain plot (**Figure 1f**), the plateau is the linear viscoelastic region (LVR), and the strain limit of the region is γL. A frequency sweep test oscillates at a set amplitude (γ0), which has been determined previously from the amplitude sweep test to be within the LVR, at increasing frequency (**Figure 1e**). The frequency sweep describes how the material acts when the material is stressed for different periods. For example, when Silly Putty is stressed quickly by throwing it on the floor, it bounces back, acting like a rigid solid. However, when the Silly Putty sits at rest and experiences low stress over a long period of time, it spreads out, acting like a viscous fluid. The frequency sweep (**Figure 1g**) reveals if the material is solid-like (G′ > G″) or liquid-like (G″ > G′) and if the behavior is frequency dependent (G′(ω), G″(ω)) or independent. Stable gels and suspensions are typically solid-like and frequency-

Previous studies on biofilm rheology using various techniques of rheological measurement have found the elastic modulus (G′) to range in order of magnitude

methods, while the values of yield stress (τy) range in order of magnitude from 10<sup>−</sup><sup>1</sup>

variability in the compositions of the biofilms, diversity of growth mediums, variability of growth conditions, and most importantly, natural variability of response of the microorganisms, even to the same medium and growth conditions. This chapter uses the techniques established in our previously published work on the nondestructive development and characterization of rheological properties of biofilms [43]. Using this non-destructive method, the measured values of elastic modulus and yield stress of PAO1 that were grown in standard LB medium were both between 0.1

Pa [2, 37–42]. The wide-ranging values of G′ and τy in the literature reflect the

Biological fluids like tears, cervical mucus, and saliva are all shown to self-assemble into fractal-like patterns of crystallization when they are dried [44, 45]. A fractal is a structure that is made of smaller parts that resemble the bigger parts, with a high degree of organization and self-similarity. This structure can be characterized with a specific fractal dimension [44]. Fractal dimension is a measure of complexity of the fractal pattern [46]. Random nucleations of salts initiate the process of crystallization, where its growth is limited by the diffusion of salt through the polymer matrix (proteins or macromolecules) [47]. Therefore, the combined effects of ionic strength, osmolarity, and the size and concentration of macromolecules control the behavior of crystallization, where too little or too much of one factor can dramatically alter the pattern of crystallization [48, 49]. A typical crystallization of biosaline proceeds in the following manner: (i) salt nucleation initiates the process of crystallization; (ii) the nucleation point grows with some symmetry into a highly-branched structure whose growth is modified by the interaction of the salt with the biological matter; (iii) the branches do not overlap or merge [47, 50, 51]. The process of crystallization of biofluids is called "arborization," "ferning," or "dendritic growth" in various literature [45]. In this paper, the general formation of salt crystals will continue to be called crystallization, while the specific crystallization of the biofluids

Pa for bulk biofilms at solid-liquid interfaces using plate-on-plate

*<sup>p</sup>* (1)

**8**

from 10<sup>−</sup><sup>2</sup>

and 10 Pa [43].

**1.2 Crystallization of biological materials**

that result in fractal patterns will be called ferns.

to 105

to 104

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 salt ratio fluids.

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

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

#### **1.4 Objective**

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

**11**

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

allowing diffusion of water, nutrients, waste, and electrolytes [26, 69]. A complex set of interactions between the electrolytes, solutes, bacteria, and biopolymers dictate the strength, bacterial resistance, and infection persistence of the biofilm. The objective of this study is to characterize the behavior of the bacterial culture in the presence of various environmental conditions, including a highly viscous media, nutrient-enhanced media, high osmolarity media, and antimicrobial media. The interaction of the bacterial culture with its nutrient environment is measured as a function of the strength of its biofilm through rheological analysis, while its ferning pattern characterizes the mass transport through the environment in the biofilm. Additionally, birefringence inside a

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

biofilm provides insight into the solute interaction with the biofilm.

incubated along with the biofilm samples to act as a negative control.

25°C. The measurements took place in the following order:

The sample rheology was measured on the Discovery Hybrid Rheometer 3 (DHR3, TA Instruments, USA) using a 40-mm stainless steel plate geometry at

b.Frequency sweep: γ0 = 0.1 (biofilm), γ0 = 0.005 (sterile LB), ω ∈[0.01, 1] rad s<sup>−</sup><sup>1</sup>

c.Stress sweep: τ ∈ [0.01, 1] Pa (biofilm), τ ∈ [0.001, 0.1] Pa (sterile LB), termi-

Detailed description of the sample inoculation, the incubation, and the rheo-

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

.

logical measurement methods are located in our previous work [43].

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

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

**2. Materials and methods**

**2.2 Measuring bulk biofilm rheology**

a.Pre-stressed: 0.1 Pa, 2 minutes

nating if the shear rate γ̇ > 10 s<sup>−</sup><sup>1</sup>

**2.3 Ferning properties of dried bacterial biofilms**

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

allowing diffusion of water, nutrients, waste, and electrolytes [26, 69]. A complex set of interactions between the electrolytes, solutes, bacteria, and biopolymers dictate the strength, bacterial resistance, and infection persistence of the biofilm. The objective of this study is to characterize the behavior of the bacterial culture in the presence of various environmental conditions, including a highly viscous media, nutrient-enhanced media, high osmolarity media, and antimicrobial media. The interaction of the bacterial culture with its nutrient environment is measured as a function of the strength of its biofilm through rheological analysis, while its ferning pattern characterizes the mass transport through the environment in the biofilm. Additionally, birefringence inside a biofilm provides insight into the solute interaction with the biofilm.
