Virtual colony count

Abstract

Virtual colony count (VCC) is a kinetic, 96-well microbiological assay originally developed to measure the activity of defensins.[1] It has since been applied to other antimicrobial peptides including LL-37.[2] It utilizes a method of enumerating bacteria called quantitative growth kinetics, which compares the time taken for a bacterial batch culture to reach a threshold optical density with that of a series of calibration curves. The name VCC has also been used to describe the application of quantitative growth kinetics to enumerate bacteria in cell culture infection models.[3] Antimicrobial susceptibility testing (AST) can be done on 96-well plates by diluting the antimicrobial agent at varying concentrations in broth inoculated with bacteria and measuring the minimum inhibitory concentration that results in no growth. However, these methods cannot be used to study some membrane-active antimicrobial peptides, which are inhibited by the broth itself. The virtual colony count procedure takes advantage of this fact by first exposing bacterial cells to the active antimicrobial agent in a low-salt buffer for two hours, then simultaneously inhibiting antimicrobial activity and inducing exponential growth by adding broth. The growth kinetics of surviving cells can then be monitored using a temperature-controlled plate reader. The time taken for each growth curve to reach a threshold change in optical density is then converted into virtual survival values, which serve as a measure of antimicrobial activity. This article presents a brief review of published VCC experiments, followed by the presentation of an example VCC experiment investigating the effect of varying the inoculum of Escherichia coli cells when assayed against the defensin HNP1. A Microsoft Excel file containing the macro used for the calculation of threshold times and data analysis is also presented. The experiment demonstrated a pronounced inoculum effect at high inocula. The results of five similar experiments are also reported, showing variation among replicate data. © 2020, WikiJournal User Group. All rights reserved.