# Electrochemical Control of Biofilm Formation and Approaches to Biofilm Removal
## 2 Biofilms
### Biofilm Structure in Biofilm Systems  
The formation of bacterial biofilms on solid surfaces within a fluid starts when bacteria attach to the substrate. Understanding the environmental factors affecting the attachment and early stages of biofilm development will help to develop methods of controlling biofilm growth. Here, we show that biofilm formation is strongly affected by the flows in thin layers of bacterial suspensions controlled by surface waves. Deterministic wave patterns promote the growth of patterned biofilms, while wave-driven turbulent motion discourages patterned attachment of bacteria. Strong biofilms form under wave antinodes, while inactive bacteria and passive particles settle under the nodal points. By controlling thewavelength, its amplitude, and horizontal mobility of the wave patterns, one can shape the biofilm and either enhance the growth or discourage the formation of the biofilm. The results suggest that deterministic wave-driven transport channels, rather than hydrodynamic forces acting on microorganisms, determine the preferred location for bacterial attachment [80]. Depending on the interaction between the surface and the constituent cells, the biofilms could be either of a monolayered or multilayered nature. Monolayer biofilm has prominent interactions between the cell and the surface, rather than the interaction between the constituent cells. Different classes of adhesive structures, such as flagellum and pilus, are helpful to accelerate and increase the formation of monolayer biofilm. On the other hand, microbes often develop multilayer biofilms when they adhere to a surface, as well as to each other.  
Numerous techniques have been developed for the detection of biofilms [81], as demonstrated in Figure 1. These include tube culture, Congo red agar, microtiter plate assay and confocal laser scanning microscopy, atomic force microscopy (AFM), quartz crystal microbalance (QCM), surface plasmon resonance (SPR), fluorescence in situ hybridization (FISH), peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH), locked nucleic acid-fluorescence in situ hybridization (LNA-FISH), catalyzed reported deposition-fluorescence in situ hybridization (CARD-FISH), double labeling of oligonucleotide probes-fluorescence in situ hybridization (DOPE-FISH), combinatorial labeling and spectral paging-fluorescence in situ hybridization (CLASI-FISH), fluorescence in situ hybridization/microautoradoactivity (FISH/MAR), fluorescence in situ hybridization-Raman spectroscopy (FISH-Raman), fluorescence in situ hybridization-nanometer-scale secondary-ion mass spectrometry (FISH-NanoSIM), confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), cryo-scanning electron microscopy (CryoSEM), environmental scanning electron microscopy (ESEM), focused ion-beam scanning electron microscopy (Fib-SEM), colony forming units (CFU), propidium monoazide quantitative real-time PCR (PMA-qPCR), quantitative polymerase chain reaction (qPCR), tetrazolium salt reduction (XTT), triphenyltetrazolzolium chloride (TTC), crystal violet (CV), ultrasonic time domain reflectometry (UTDR), fluorescence correlation spectroscopy (FCS), and yet some others [81].  
Figure 1: Overview of methods enabling the growth and characterization of biofilms [81], which includes different biofilm devices, methods to assess adhesion extent and strength, and techniques to measure biofilm biomass, viability, and matrix composition.  
Biofilm architecture has been extensively studied using optical sectioning, confocal laser scanning microscopy, scanning electron microscopy, and three-dimensional imaging [16; 82]. The combination of flow-cell technology and fluorescence in situ hybridization (FISH) with confocal laser scanning microscopy is the most favored tool to obtain quantifiable evidence on both the overall biomass and the individual strains [8].