The recently developed transparent soil consists of particles of Nafion, a polymer with a low refractive index (RI), which is prepared by milling and chemical treatment for use as a soil analogue. After the addition of a RI-matched solution, confocal imaging can be carried out in vivo and without destructive sampling. In a previous study, we showed that the new substrate provides a good approximation of plant growth conditions found in natural soils. In this paper, we present further development of the techniques for detailed quantitative analysis of images of root-microbe interactions in situ. Using this system it was possible for the first time to analyse bacterial distribution along the roots and in the bulk substrate in vivo. These findings indicate that the coupling of transparent soil with light microscopy is an important advance towards the discovery of the mechanisms of microbial colonisation of the rhizosphere.