Evaluation of difference imagery for visualizing and quantitating microbial growth

Abstract

Online computer image enhancement and analysis were used to visualize and quantitate microbial growth in slide cultures. Images of a single field were collected, enhanced, and stored in random access memory at 15-min time intervals. The image obtained at time 0 was subtracted from each of the subsequent images. This produced a series of difference images showing microbial growth as a black object on a grey background and eliminating debris. Data obtained using difference imaging were compared to data obtained by computer analysis of the original images. The initial rate of growth (2.8 μm3∙field−1∙h−1) was determined within an error of 2% and continuously increased during the first 2 h of incubation. The images of seven soil fractions and of stationary phase cells were superimposed on both the initial and final images before subtraction to evaluate difference imaging as an aid in visually discriminating growing microorganisms from simulated artifacts. The coarse clay fraction (0.2–2.0 μm) was the most difficult to visually distinguish from microbial cells, but was easily eliminated in difference images. The potential for application of difference imaging in the direct measurement of in situ metabolic activity is also discussed.