The Standardization and Utilization of a Spot Plate Assay to Help Define the Mechanism of Lactoferrin-mediated Killing of S. mutans

Abstract

Lactoferrin(LF) is an iron-binding glycoprotein common to exocrine secretions and specific granules of neutrophils. Studies have shown apo-LF to possess bactericidal properties against numerous mircorganisms, including Streptococcus mutans, a cariogenic bacteria. The initial aspect of this study was to develop a new bactericidal assay that would allow for numerous parameters to be run quickly and simultaneously, while at the same time providing well-defined kinetics. A Spot Plate assay was standardized against the assay system, Spiral Plater, which is presently used to measure apo-LF mediated killing of S. mutans. The outcome of the comparison showed that the two assays exhibited analogous results. The Spot Plate assay killed the bacterial cells in a shorter time span. These results can be explained by a dilution of inoculant over the plate area. This new assay can be utilized in trying to prove the hypothesized killing mechanism, a reactive OH· generated from oxidation of Fe 2+ --> Fe 3+ by a LF molecule. The Spot Plate assay was used to test the bactericidal effects of numerous parameters carried out in different conditions, such as temperature, varying concentrations of apo-LF and bacterial cells. These particular tests provided some insight on the mechanism, as these results will used to further examine the bactericidal mechanism of apo-LF.