Formation of Clear Zones with 'Sensitive' and 'Resistant' Staphylococcus aureus in Penicillin Plate Assays

Abstract

The high-power microscopical examination of stained specimens of portions of agar plate cultures of bacteria allows bacterial cells and colonies to be studied in their original positions. The disposition and morphology of bacteria from the edges of clear zones on penicillin assay plates under conditions sufficiently standardized permitted a distinction between sensitive and resistant strains of Staphylococcus aureus. With a sensitive strain 'ghost zones ' were formed of partially lysed, distorted and abnormally staining cells ; with a resistant strain a ' scatter zone ' was formed of apparently normal cells in isolated colonies.For plate assay work, a sensitive strain may be used when only low concentrations of the drug are available ; a resistant strain may be expected to give a sharper zone edge, but will require higher concentrations to provide an adequate response.Examinations of assay plates have been made by a number of workers in studying the antibacterial properties of penicillin. The wide use of Staph. aureus for penicillin assay has prompted the inclusion of penicillin-sensitive strains in such investigations, but a detailed comparative study of assay plates seeded with sensitive and resistant forms does not appear to have been reported.From direct examination of assay plates, numerous observations on the structure of inhibition zones have been reported. Abraham, Chain, Fletcher, Gardner, Heatley, Jennings & Florey (1941) mentioned that indefinite zones might occur between the clear circle and the opaque normal growth in the cylinder-plate method. Foster & Woodruff (1943) noticed an indistinctness of the clear-zone edge, attributed to lysis of the staphylococci; the variable nature of intermediate zones was also noted. Knox (1945) used a 5 yo horseblood agar medium and a single central cylinder of penicillin solution to each Petri dish, multiple inocula being made in radial streaks. With sensitive staphylococci examination under 10 x magnification confirmed the naked-eye impression that lysis of colonies occurred in the periphery of the cleared part of the streaks. Haemolysis in the region of bacteriolysis suggested that bacterial growth here had previously been vigorous, although only the debris of colonies remained at the end of incubation. With a resistant staphylococcus, lysis was not seen, and the zone of haemolysis did not extend inwards from the region of normal growth. Vesterdal(1946), using an all-over surface-seeding technique with a sensitive staphylococcus, also noted the extension of clear-zone edges by lysis of marginal colonies, and recorded the diameters of outer and intermediate zone edges. With the lowest concentrations of penicillin used the intermediate zones were narrow and opaque. With greater concentrations these zones widened, and an inner, translucent zone appeared, which gradually displaced the opaque zone, and became still wider as the series was continued to the highest concentrations

Mecillinam susceptibility as an indicator of β-lactamase production in Staphylococcus aureus

“…Inhibition zones were measured in millimeters. A note was made as to whether bacterial growth tapered out (‘ghost zones’[5]) or was clear‐cut at the inhibition zone edge. A zone of 28 mm is the breakpoint between penicillin‐susceptible and β ‐lactamase‐producing strains [2].…”

Correlation of phenotypic tests with the presence of the blaZ gene for detection of beta-lactamase

“…However, little information about the reliability of the latter phenomenon is available,21, 27 especially for coagulase-negative staphylococci.…”

Classification of Staphylococci by Penicillin Lysis

“…The light streaks were l / l O O to l / l O O O dilutions of 24 hr. broth cultures, and the heavy streaks the centrifuged deposit from similar cultures suspended in a few drops of broth (Ingram, 1951).…”