Fig. 4.

Viability PCR workflow (e.g., using EMA, PMA, or similar dyes). The initial sample is divided in two. One sample (left side) remains untreated, leaving total DNA—including extracellular DNA (yellow) and DNA in living (blue DNA, blue membrane) and dead (red DNA, black membrane) cells—relatively intact and available for downstream applications. The other sample (right side) is stained with a viability dye that binds to free DNA and to DNA in cells with compromised membranes. Upon photoactivation in the treated sample, bound DNA is degraded, such that it is no longer a suitable template for amplification. After amplification, a comparison of treated versus untreated samples can reveal relative proportions and/or types of living and dead microorganisms (e.g., via qPCR and/or DNA sequencing, respectively)