Quantification of the in situ distribution of soil bacteria by large-scale imaging of thin sections of undisturbed soil

A method for determining the number and in situ spatial distribution of bacterial cells over spatial scales ranging from micrometres to centimetres in mineral soils is described. Biological thin sections of undisturbed cores of soil were prepared in order to preserve the spatial distribution of bacterial cells. Composite (tessellated) images in which individual bacteria can be resolved within an area of 0.282 mm2 were acquired by means of a motorised scanning microscope stage. An image processing and analysis procedure was developed to determine the numbers and locations of bacterial cells in the composite images. The image processing procedure first homogenised the background of the images and then discriminated between bacteria and non-bacterial features using the colour and morphological properties of the images of the bacterial cells. Feature edges were detected in the green channel of colour (red, green, blue) images and bacterial cell edges were confirmed in the blue channel after elimination of autofluorescent features in the red channel. No significant difference was found between the number of bacteria or associated distributions determined automatically and control values derived interactively on individual fields of view. Data relating to total bacterial counts in thin sections and in paired dispersed samples suggested that all soil bacteria were being visualised in thin sections. Significant differences between samples taken from a depth profile of a fallow arable soil were found for both cell numbers and for cell distribution as measured by an index of dispersion.