1. Bacteria exposed to C14- or S35-labelled penicillin bound and concentrated the antibiotic. The amount bound from low concentrations (0.001 to 0.01 µg. per ml.) was related to the penicillin sensitivity of the strain; and highly sensitive organisms such as Streptococcus pyogenes concentrated the antibiotic as much as 200-fold. The material bound by highly sensitive strains from these low concentrations of penicillin was removed to only a minor extent by washing. Penicillin inactivated with penicillinase, by hot H2SO4 or by cold HCl was not similarly concentrated.
2. Despite wide differences in their sensitivity to penicillin, at equieffective (LD99.9) levels, four of the five strains here studied had bound comparable amounts of antibiotic. That lethal intracellular concentration averaged 1.7 to 4 µg. per gm., and 1600 to 3300 molecules per cell.
3. Bacteria in the logarithmic phase of growth, "resting" organisms, and cell-free sonic extracts, had roughly the same reactivity with penicillin. The differences in the amount of penicillin bound by bacteria of varying sensitivity therefore do not rest on differences in the permeability of the cell.
4. Escherichia coli (K12)-inactivated penicillin within the cell; and that inactivation adequately explains its low binding affinity, and its relative resistance. The other species here studied inactivated the diffusible cellular penicillin to only a minor degree, and not enough to explain the differences in their penicillin-sensitivity. The working hypothesis is suggested that their varying sensitivity is instead determined by differences in the reactivity of vital cell components with the antibiotic.
5. At high concentrations of antibiotic (1 to 1000 µg. per ml.), there was non-specific binding by all the cell strains and cell extracts here studied, unrelated to their sensitivity, and masking the specific differences in reactivity noted at low concentrations. At these high levels, penicilloic acid was bound to the same degree as the active antibiotic.