Computer-enhanced darkfield microscopy for the quantitative analysis of bacterial growth and behavior on surfaces

Abstract

Computer image analysis techniques were integrated with low magnification darkfield microscopy to quantify colonization kinetics, growth rates, motility, plasmolysis, behavior and interactions of microorganisms on surfaces. Darkfield provided the potential advantages of a large observation field, large sample size, increased depth of field and ease of automation. Plumbicon, SIT and ISIT imaging devices were evaluated for their suitability in darkfield applications. Computer image analysis programs (IBAS 2000) were developed utilizing single and multiple image inputs, image averaging, difference imagery, and size and shape factors to perform analyses on dark field images. These programs allowed quantitation of colonization kinetics, bacterial growth rates, enumeration of cells, determination of rates of motility, number of motility events and determination of the recolonization kinetics of bacteria growing in surface microenvironments. Growth parameters obtained using darkfield were compared with those obtained from the specific rate of increase in cell number, as well as the specific rate of increase in cell area, determined using high magnification phase contrast and image analysis. Darkfield growth rates were comparable to other measures of growth if cameras with manual gain and voltage control were used to optimize darkfield image input. Significant errors (average error 35%) ocurred when imaging devices with automatic controls were used to collect primary darkfield images. The inability of video cameras to process weak and intense signals in the same field (intrascene range) further contributed to the potential for errors in growth rate estimation when using darkfield microscopy. Errors were also introduced when the developing biofilm became three dimensional making the measurement of area and inadequate estimate of biomass. Use of light sensitive SIT or ISIT rather than plumbicon tubes was required to avoid significant impairment of microbial growth and behavior by microscope illumination.