# The Viable but Non-Culturable (VBNC) State, a Poorly Explored Aspect of Beneficial Bacteria
## 4 Techniques to Evaluate Bacterial Viability in the VBNC State  
Currently, several methodologies have been reported that can be used to determine the viability of a bacterium under the VBNC state (Figure 3). The methodologies are divided into three groups: molecular techniques, techniques focused on metabolism, and staining techniques. The molecular techniques are based on detecting individual and global gene expression in non-culturable cells. According to the literature, the use of transcripts is an excellent alternative, given that mRNA half-lives are typically in the range of seconds to minutes [104; 105] and the identification of the mRNA is evidence that cells remain metabolically active. In addition, their detection provides an essential insight into the factors that may be regulated in the VBNC sate. The techniques focused on metabolism search for metabolites or enzymatic activity, generally using methodologies with colorimetric results, biosensors, or matrices. The detection of metabolic activity in bacteria in a viable but non-culturable state serves as an indicator of cell viability. This detection signifies that despite the bacteria's inability to be cultured under standard laboratory conditions, there is biological activity. Evidence indicates that bacteria in the VBNC state retain the ability to conduct fundamental metabolic processes, including respiration, nutrient assimilation, and gene expression (see Sections 5 and 6). The staining techniques mainly are based on detecting activity in the electron transport system and the integrity of the cytoplasmic membrane. Cellular respiration allows the rapid identification of metabolically active cells, only using a compound fluorescent or a compound that reacts and forms a fluorescent compound. For example, CTC is reduced to a red fluorescent compound called formazan. The BacLight(r) Live/Dead Kit is a tool that is frequently used, in which, by differential staining and fluorescence microscopy, bacteria in the VBNC state stain green, indicating that they have largely intact membranes and thus can be considered to be alive [16]. However, in the case of bacteria under desiccation stress, the membranes suffer apparent sublethal damage during the VBNC state and they stain red [4]. On the other hand, it has been reported that some dead bacteria are observed as empty particles lacking cytosol that do not allow propidium iodide entry, but their membranes remain intact. This type of situation can lead to the erroneous interpretation of the results [106]. It is important to note that there is not a decisive test for bacterial viability in the VBNC state, and it is usually recommended that, when trying to prove this fact, two or more methodologies should be performed. [PERSON] and colleagues [107] have raised concerns that the employed method could potentially result in a misinterpretation of the VBNC state. The integration of various approaches enables the assessment of multiple parameters, such as metabolic activity and membrane integrity, ensuring more reliable determination. Interestingly, although several techniques have been developed to elucidate the VBNC state, their application in beneficial bacteria is limited. The most commonly used techniques include Live/Dead Baclight staining, CTC, DFA-DVC, RT-qPCR, and qPCR (Figure 3).  
Figure 3: Methodologies for determination of bacterial viability in the VBNC state. Molecular biology-based techniques are highlighted in blue. Methods reliant on identifying metabolic activity are represented in green. Techniques employing dyes for cell staining are depicted in orange. The orange and green methodologies involve techniques for staining and the identification of metabolic activity. The blue text shows beneficial microorganisms and the techniques that have been used to study their VBNC state.