Journal of Methods Microbiological Journal of Microbiological Methods 40 (2000) 199–206 www.elsevier.com / locate / jmicmeth Chromogenic plate assay distinguishing bacteriolytic from bacteriostatic activity of an antibiotic agent * Gonzalo Mardones, Alejandro Venegas ´ ´ ´ ´ Laboratorio de Bioquımica, Departamento de Genetica Molecular y Microbiologıa, Pontificia Universidad Catolica de Chile, Casilla 114-D, Santiago, Chile Received 26 April 1999; received in revised form 20 December 1999; accepted 5 January 2000 Abstract A solid agar plate assay was devised to discriminate bacteriolytic from bacteriostatic activity for a given antibacterial agent. The assay uses a bacterial culture harboring b-galactosidase enzyme as reporter of cellular lysis. When a drop of bacteriolytic compound is placed on the agar, b-galactosidase is released from the bacteria to the external solid medium where it hydrolyzes X-Gal substrate analogue, developing a blue halo at the edge of the inhibition growth zone. The assay was successfully evaluated against several antibiotics with well-known mechanism of action. It was found that bacteriostatic compounds consistently did not display blue halo at the inhibition zone.  2000 Elsevier Science B.V. All rights reserved. Keywords: Antibiotics; Apidaecin; Bacteriolytic assay; X-Gal plates 1. Introduction similar way to hormones by binding to specific cellular receptors which require specific peptide The knowledge about the mechanism of action of conformation. In contrast, the mechanisms of action a new antimicrobial agent is basic to understanding of other antibacterial agents is less dependent on the events occurring during bacterial growth inhibi- such stringent structural requirement. Among these tion. This issue is very important for the develop- compounds, cytolytic cationic peptides with a wide ment of any antibacterial compound for therapeutic spectrum of action have been isolated from mam- use. malian macrophages — the so called defensins Recently, several efforts have focused on studying (Ganz et al., 1990), from insects, — melittin (Haber- the mechanisms of a number of new antibacterial mann, 1972), cecropins (Steiner et al., 1981) and peptides. Surface active peptides which bind and sarcotoxins (Okada and Natori, 1985) and from alter amphipatic surfaces, including membranes and amphibians, — magainin (Zasloff, 1987). The target receptors, have been extensively studied (DeGrado et for these surface-active peptides seems to be the lipid al., 1981; Kaiser and Kezdy, 1983, 1984; Kaiser, bilayer of the cellular membrane. It has been re- 1988). Some of these antibacterial agents act in a ported that their activity is exclusively due to their unique structural features, which allow them to bind to the corresponding cells, modulating the membrane *Corresponding author. Tel.: 1 56-2-686-2661; fax: 1 56-2-222- voltage and affecting membrane permeability (Wes- 2810. E-mail address: avenegas@genes.bio.puc.cl (A. Venegas) terhoff et al., 1989; Ganz et al., 1990). Participation 0167-7012 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0167-7012(00)00125-1