Table 3.

A comparative analysis of the NanoLuc method under different experimental conditions

	Plate counting (CFU/mL) (Klančnik et al. 2017a)	NanoLuc bioluminescence assay (this manuscript)	Crystal violet assay (Kurinčič et al. 2016; Klančnik et al. 2017a)	Resazurin (Kovač et al. 2015)
Biofilm state (early/mature biofilm)	Less suitable for mature biofilm. Increase in damaged, VBNC and dead cells that cannot be cultured results in underestimation of cell number	Enables the detection of culturable, VBNC and damaged cells, and is therefore suitable for various biofilm conditions	Increase in biofilm biomass over time due to the accumulation of extracellular polymers and not due to an increase in the number of cells in mature biofilms; inaccurate for mature biofilms	Detects metabolically active cells. Suitable for various biofilm conditions
Mixed biofilms	Selection method, such as antibiotic resistance, is required for the identification of individual organisms	Suitable for mixed biofilms. Only organisms with the expressed NanoLuc protein can be detected	The distinction between organisms is not possible. Only the total biomass can be determined	The distinction between organisms is not possible. Only the general metabolic activity can be determined
Biofilm thickness	Problem with detachment and degradation of the biofilm, cells could be present in clusters	Limited substrate penetration into the thick biofilm and signal attenuation due to the multilayer biofilm	Limited dye penetration into the thick biofilm	Limited substrate penetration into the thick biofilm and signal attenuation due to the multilayer biofilm
Type of surface used for biofilm growth	Difficult removal of biofilms from rough surfaces	Risk of light diffusion on certain surfaces	The rinsing protocol must be adapted to the surface used	Risk of light diffusion on certain surfaces
Dynamic biofilm	Detachment of cells in the flow-through system results in the destruction of the dynamic biofilm	Retention of the substrate prevents continuous monitoring. Intervals between measurements should be considered	Rinsing results in the destruction of the dynamic biofilm	Retention of the substrate prevents continuous monitoring. Intervals between measurements should be considered