Histological approach to Bacillus subtilis colony-biofilm: evolving internal architecture and sporulation dynamics.

Bacillus subtilis has been used as a classic model to study biofilm formation and sporulation process. Colonies of wild-type strains usually have a complex external morphology, but the details of their internal architecture are still undisclosed. Since bacterial biofilms fulfill the criteria to be considered tissues, the aim of this work was to analyse B. subtilis colony-biofilm internal architecture evolution and sporulation dynamics using histological techniques. Transversal sections of colony-biofilms incubated from 24 hours up to 20 days were stained using histochemical techniques to analyse the internal structure by light and electron microscopy. A morphometric study of the different structural biofilm components was performed by image analysis, and an application to quantify spores was developed. Internal biofilm architecture was characterised by a stratified pattern, which evolved from 3 strata at 24 hours, up to 5 strata at 20 days. At 48 hours, strata at the central area of the biofilm was folded, resulting in elevated structures (vein-like structures) that could reach up to 465 mu m in height. Sporulation started at 48 hours, at the top of the vein-like structures, at the interface between the two uppermost strata. At 20 days spores formed a continuous central layer, representing 7.5% of the total biofilm. In summary, our results demonstrate that B. subtilis colony-biofilm has a complex and organized internal architecture, evolving over time, and taking place in different cell subpopulations with different functionalities. Furthermore, in situ spore quantification described in this work could be a good alternative to the classical chamber counting.