## 3 Biofilm Characterization
### Quantitative Assessment Methods
#### 3.1.2 Indirect Methods  
Indirect assessment methods include (i) dry mass; (ii) Crystal Violet assay (CV); (iii) Tetrazolium salts (e.g., 2,3,5-triphenyl-tetrazolium chloride (TTC) and 2-(4,5-dimethyl-2-thiazol)-3,5-diphenyl-2H-tetrazolium bromide (MTT)); (iv) total protein determination; (v) Quartz Crystal Microbalance (QCM); (vi) Total Organic Carbon; (vii) Resazurin assay (or Alamar Blue) [28; 32]. Briefly, the dry mass method involves measuring the total biomass of the biofilm. The biofilm is first collected, typically by scraping it from a surface, and then dried to a constant weight. The dried sample is weighed to determine the total mass of the biofilm. This method provides a direct measure of the total biofilm mass, including both the cellular and extracellular matrix components. It is simple and straightforward but does not differentiate between living and dead cells [39]. The Crystal Violet assay is a widely used technique to quantify biofilm formation. Biofilms are stained with Crystal Violet dye, which binds to the biofilm matrix. The bound dye is then solubilized, and its absorbance is \begin{table}
\begin{tabular}{c c c} \hline \hline
**Method** & **Advantages** & **Disadvantages** & **References** \\ \hline \multirow{6}{*}{\begin{tabular}{c} Colony-forming unit \\ (CFU) counting \\ \end{tabular} } & Allows viable counting. & Lengthy and requires repetitive tasks. \\  & Special equipment or dyes & Involves complex and indirect \\  & & procedures that increase ambiguity \\  & & & and uncertainty. \\  & are not required. & Detects only viable bacteria. \\  & May be performed in most & Requires removal of bacteria \\  & laboratory scenarios. & through sonication or \\  & & matrix-degrading enzymes. \\ \hline \multirow{6}{*}{\begin{tabular}{c} 4\({}^{\prime}\),6\({}^{\prime}\)-diamino-2-fenil-indol \\ (DAPI) staining \\ \end{tabular} } & \begin{tabular}{c} Enables measurement of the total \\ cells on the surface. \\ Particularly effective for handling \\ a large number of samples. \\ \end{tabular} & \begin{tabular}{c} Imaging is restricted to two dimensions. \\ Needs the use of fluorochromes, \\ epifluorescence microscopy, and \\ confocal laser scanning \\ microscopy (CLSM). \\ Cannot distinguish between live and \\ dead bacterial cells. \\ \end{tabular} & \begin{tabular}{c} [28; 35] \\ \end{tabular} \\ \hline \multirow{6}{*}{\begin{tabular}{c} Live/Dead staining \\ \end{tabular} } & Enables counting and \\  & distinguishing between live \\  & and dead bacteria. \\  & Particularly suitable for handling \\  & a large number of samples. \\ \end{tabular} & \begin{tabular}{c} Inaging is restricted to two dimensions. \\ Fluorochromes are sensitive to light. \\ Requires epifluorescence and confocal \\ laser scanning microscopy (CLSM). \\ \end{tabular} & \
\begin{tabular}{c} [31; 36; 37] \\ \end{tabular} \\ \hline \multirow{6}{*}{
\begin{tabular}{c} Fluorescence in situ \\ hybridization (FISH) \\ \end{tabular} } & Enables distinguishing and \\  & quantifying the sessile population. \\ \cline{1-1}  & Analyzes the spatial distribution & Requires specific probes, \\ \cline{1-1}  & within biofilms without \\ \cline{1-1}  & damaging their structure. & Involves costly equipment. \\ \cline{1-1}  & Discriminates microorganism & Protocol optimization is necessary for \\ \cline{1-1}  & species using fluorescent \\ \cline{1-1}  & labeling with specific \\ \cline{1-1}  & oligonucleotide probes. \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Direct methods to quantify biofilms and their advantages and disadvantages. measured spectrophotometrically. The CV assay is a quick and easy method to assess the total biomass of the biofilm, including cells and extracellular polymeric substances (EPS). It is especially useful for high-throughput screening but does not provide information on cell viability [28]. Tetrazolium salts, such as 2,3,5-triphenyl-tetrazolium chloride (TTC) and MTT (2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide), are used in colorimetric assays to measure cell viability.