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.