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Viability-PCR (vPCR) protocols are mainly based on photo-reactive dyes impermeant to intact cell membranes. The absence of cell barriers allows the reagent's interaction with the genetic material after a short incubation period. By light-induced reaction, DNA becomes the unsuitable mould for the polymerases and thus cannot be amplified and detected by PCR. General rules and consensus exist on critical aspects of successful vPCR protocol development. However, the understanding of the vPCR reaction concerning how much reagent is really effective or the proper amount of light has been poorly studied. The convenience of using 600 times more dye than bases pairs exist suggests that although these dyes are DNA intercalating reagents, many organic molecules can adsorb it. Concerning light, no exact references exist about how much energy is needed to activate the azide group of reagents such as propidium monoazide. Therefore, it cannot be calculated in terms of energy how much light needs a vPCR protocol. The general rule is to provide reagents and energy in excess. This work provides different responses (based on experimental results) to both questions, which can contribute to a better understanding of the theoretical basis of vPCR protocols. • The general rule for viability PCR (vPCR) protocols is to provide reagents and energy in excess. • The data suggest that although these reagents are DNA intercalating reagents, many organic molecules must be adsorbing them. • No exact references exist about how much energy is needed to activate the azide group of reagents such as PMA. • In this work, the light/energy balance allows a better understanding of the analytical ranges of the vPCR. • If a concentration of 50 μM of PMA is exceeded there may not be enough energy to activate all the reagent molecules. [ABSTRACT FROM AUTHOR]