Table 1.
Methods to study VBNC Lm.
| Acronym | Full name | Mechanism of action | References |
|---|---|---|---|
| CTC–DAPI | 5- cyano-2,3-ditolyltetrazolium chloride– 4',6-diamidino-2-phenylindole staining | CTC, a redox dye, is reduced to an insoluble fluorescent red CTC-formazan salt by the electron transport chain in actively respiring bacterial cells. The presence of red CTC crystals in bacterial cells is visualized by epifluorescence microscopy. Counterstaining with DAPI, which fluoresces blue, provides contrast and allows enumeration of total bacteria. | Bremer et al., 1998; Besnard et al., 2000; Cappelier et al., 2005; Dreux et al., 2007 |
| DVC | Direct Viable Count | Incubation of the samples in a medium containing a limited level of nutrients and an antibiotic acting as an inhibitor of DNA replication, allows the viable cells to metabolize nutrients and elongate, while blocking their division. After staining with a fluorescent dye, elongated cells are counted by fluorescence microscopy. | Besnard et al., 2000; Besnard et al., 2002; Dreux et al., 2007; Giao and Keevil, 2014; Highmore et al., 2018 |
| DVC –FISH | Direct Viable Count –Fluorescence In Situ Hybridization | This method combines DVC and FISH with a Lm fluorescent oligonucleotide probe (usually 16S rRNA) used to visualize bacteria by microscopy. | Moreno et al., 2012 |
| Live/Dead BacLight |
Live/Dead BacLight bacterial viability assay |
This method discriminates between live and dead bacteria based on membrane integrity, using a dual staining with DNA-intercalating dyes, SYTO 9 green-fluorescent stain and Propidium Iodide (PI) red-fluorescent stain. These dyes differ in their ability to enter bacteria. SYTO 9 enters in all cells, whereas PI only enters bacteria with compromised membranes, causing reduced SYTO 9 fluorescence. With an optimized mixture, dead bacteria will fluoresce red, while live bacteria will fluoresce green. | Dreux et al., 2007; Lindback et al., 2010; Giao and Keevil, 2014; Kortebi et al., 2017; Afari and Hung, 2018; Robben et al., 2019; Gu et al., 2020 |
| Flow cytometry | Flow cytometry | This method estimates the number of viable cells in a heterogeneous population using a flow cytometer. Viable cells are discriminated by fluorescent dyes that either penetrate all cells (e.g., SYTO 9 or SYTO 24) or penetrate only cells with compromised membranes (e.g., PI). | Truchado et al., 2020; Chiara et al., 2021 |
| CFDA | Carboxy-fluorescein diacetate staining | CFDA is a colorless fluorogenic ester that enters bacterial cells through diffusion. CFDA is enzymatically cleaved by esterase enzymes of viable cells yielding a fluorescent probe, which accumulates exclusively in bacteria with intact membranes. This labeling can be used for monitoring viable cells by fluorescence microscopy or flow cytometry. | Olszewska et al., 2015; Arvaniti et al., 2021 |
| ATP | BacTiter-Glo Microbial Cell Viability assay | The method is based on the quantification of ATP. A sample is mixed with a luciferase reagent and the luminescence is recorded in relative light units (RLU) by a luminometer. Data are converted to ATP concentration per cell using a standard curve, and is proportional to the number of viable cells. | Lindback et al., 2010; Robben et al., 2019 |
| qPCR | Quantitative polymerase chain reaction | Amplification of a bacterial DNA fragment by real time polymerase chain reaction. This method indicates presence of bacterial DNA but does not assess viability. | Wery et al., 2006; Klein et al., 2011; Maynaud et al., 2016; Afari and Hung, 2018 |
| RT-PCR | Reverse transcription polymerase chain reaction | Detection of a messenger RNA (mRNA), by PCR amplification of a cDNA sequence synthesized from the mRNA template. | Lindback et al., 2010; Zolfaghari et al., 2020 |
| v-PCR (EMA- or PMA-qPCR) |
viability-PCR | Ethidium monoazide (EMA) and propidium monoazide (PMA) are photoreactive DNA-intercalating dyes that, when used in combination with polymerase chain reaction, prevent the DNA of dead bacteria from being amplified. The dyes selectively enter only the compromised cells (PMA being more selective for dead cells than EMA). Exposure to light crosslinks the dye to the DNA and blocks the PCR reaction (generally in qPCR). | Desneux et al., 2016; Overney et al., 2016; Overney et al., 2017; Brauge et al., 2020; Gu et al., 2020; Truchado et al., 2020; Bernardo et al., 2021 |
| Metataxonomics 16S-rRNA |
16S rRNA gene sequencing | This method is based on the sequencing of the 16S rRNA gene, which is found in all bacteria and archaea. 16S rRNA gene sequencing is commonly used for identification, classification and quantification of microbes within microbial communities. This method indicates presence of bacterial DNA but does not assess viability. | Pang et al., 2018 |
| Metagenomics | Whole shotgun metagenomic sequencing | Direct sequencing of all the DNA present in a sample. Shotgun sequencing has more power to identify less abundant taxa than 16S rRNA sequencing, but requires in-depth bioinformatics analysis. This method indicates presence of bacterial DNA but does not assess viability. | De Goffau et al., 2019 |
Only methods cited in this review are listed and ordered as follows: fluorescence-based methods, biochemical methods and molecular biology methods. The reference list provides examples, but is not exhaustive.