## 3 Biofilm Characterization
### Quantitative Assessment Methods
#### 3.1.2 Indirect Methods
In metabolically active cells, these salts are reduced to colored formazan products, which can be quantified by measuring the absorbance. These assays specifically assess the metabolic activity of cells within the biofilm, providing an indirect measure of viable cell biomass. They are useful for distinguishing between live and dead cells in biofilms [28]. Total protein determination involves quantifying the amount of protein within a biofilm using colorimetric assays like the Bradford or BCA assay. The biofilm is lysed to release proteins, which are then quantified. This method provides an estimate of the total cellular content within the biofilm as protein content is directly proportional to the number of cells. It is useful for comparing the cellular density of different biofilms [28]. The QCM is a highly sensitive technique that measures changes in mass on a quartz crystal surface. As biofilm forms on the crystal, the increase in mass causes a change in the frequency of the crystal's oscillation, which can be precisely measured. The QCM allows for real-time monitoring of biofilm formation and growth. It is particularly valuable for studying the dynamics of biofilm development and the effects of various treatments on biofilm mass [40; 41; 42]. Total Organic Carbon (TOC) analysis measures the total amount of carbon in the biofilm, representing the organic matter present. The biofilm is oxidized to convert all carbon into CO\({}_{2}\), which is then quantified. TOC provides a measure of the total organic content of the biofilm, including both cellular material and extracellular polymeric substances (EPS). This method is useful for assessing the overall organic load of biofilms [28]. The Resazurin assay, also known as Alamar Blue, is a redox-based assay that measures cell viability. Resazurin is a non-toxic blue dye that is reduced to the pink fluorescent compound resoruffin in metabolically active cells. The fluorescence intensity or absorbance is then measured. This assay is a sensitive method for determining the viability and metabolic activity of cells within a biofilm. It is particularly useful for assessing the effects of antimicrobial agents on biofilm-embedded cells [43]. Each of these methods provides valuable information about different aspects of the biofilm's structure and function, allowing researchers to assess biofilm formation, viability, and response to treatments from multiple perspectives. In Table 2, the advantages and disadvantages of different indirect methods to assess biofilm quantity are presented. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Method** & **Advantages** & **Disadvantages** & **References** \\ \hline Dry mass & Rapid technique. & Heat or precipitation is required to fully eliminate water molecules. & [28] \\ \hline Crystal Violet staining & Enables the measurement of total biomass. & \begin{tabular}{c} \begin{tabular}{c} \begin{tabular}{c} \begin{tabular}{c} \begin{tabular}{c} \begin{tabular}{c} \begin{tabular}{c} \begin{tabular}{c} \begin{tabular}{c} \begin{tabular}{c} \begin{tabular}{c} \begin{tabular}{c} \begin{tabular}{c} \
\begin{tabular}{c}
\begin{tabular}{c} \end{tabular} \\ \end{tabular} \\ \end{tabular} \\ \end{tabular} \\ \end{tabular} \\ \end{tabular} \\ \end{tabular} \\ \end{tabular}
\end{table} 
Table 2: Indirect methods to quantify biofilms and their advantages and disadvantages.