Design a two-step laboratory assay that distinguishes whether tetracycline resistance in an isolate is due to an efflux pump versus target protection (ribosomal polypeptide preventing binding). Which approach best separates these mechanisms?

• A. Treat bacteria with a combination of high salt and low pH and observe growth; if growth persists, call it enzymatic inactivation because efflux pumps and target protection are both inhibited by pH shifts equally and thus indistinguishable.
• B. Only perform a disk diffusion test with a single tetracycline concentration; if a zone of inhibition is absent, conclude target protection because disk diffusion cannot identify efflux contributions reliably.
• C. Measure intracellular accumulation of radiolabeled tetracycline with and without an efflux pump inhibitor (e.g., CCCP); if accumulation increases with inhibitor, efflux is involved. Then perform a ribosomal protection assay or PCR to detect known protection genes; if pump inhibition doesn't restore sensitivity and protection gene is present, target protection is the mechanism.
• D. Sequence the whole genome and if any mutation is found anywhere, attribute resistance to efflux because genome sequencing is always sufficient to separate molecular mechanisms without functional assays.

Answer: (C)