183752.fig.004
Figure 4: The in situ hybridization process can be divided into four stages: (A) sampling and immediate fixation in formaldehyde to preserve the integrity of the cells, especially the ribosomes; (B) hybridization with a specific probe, labelled with a fluorescent dye at its 50-end and matched with a sequence of the 16S rRNA; (C) counterstaining with a universal marker (DAPI, which attaches nonspecifically to DNA molecules) or another more general probe labelled with a different fluorescent dye; (D) visualization via fluorescence microscopy. Direct quantification is possible by manual counting of hybridized cells (epifluorescence and laser confocal microscope) or by image analysis of digital photos (both microscopes) or automated counting with a flow cytometer.