Design a basic in vitro experiment that would distinguish whether a new drug is bactericidal by inhibiting cell wall synthesis or merely bacteriostatic. Which experimental approach and observation pair would most directly support a bactericidal mechanism targeting peptidoglycan?

• A. Perform a time-kill assay measuring colony-forming units (CFU/mL) over time after drug exposure; a rapid, dose-dependent decline in CFU (≥3-log decrease) indicates bactericidal activity consistent with cell wall inhibition, whereas stable CFU with inhibited growth indicates bacteriostatic effect.
• B. Measure optical density (OD600) over time and conclude bactericidal activity if OD600 increases slowly, since any decrease in turbidity is always due to bacteriostatic effects rather than cell death, making OD an unambiguous measure of killing.
• C. Use a colorimetric metabolic assay to show that metabolic activity increases in the presence of drug and infer bactericidal activity because dead cells universally increase metabolic dye turnover before lysis, indicating that peptidoglycan synthesis is blocked.
• D. Test for inhibition of human cell culture proliferation in parallel and, if human cells are unaffected, directly conclude the drug is bactericidal against bacteria without measuring bacterial viability over time, because host toxicity rules out bacteriostatic action.

Answer: (A)