Division 5.3.1

Observe the slides immediately with a microscope fitted for epifluorescence microscopy at 630 or 1000x magnification under immersions oil. With a filter suitable for fluorescein isothiocyanate (FITC) eubacterial cells (including most gram negative cells) in the sample are stained fluorescent green. Using a filter for tetramethylrhodamine-5-isothiocyanate, Cy3-stained cells of C. m. subsp. sepedonicus appear fluorescent red. Compare cell morphology with that of the positive controls. Cells must be bright fluorescent and completely stained The FISH test (section 9.4) must be repeated if the staining is aberrant. Scan windows across two diameters at right angles and around the perimeter. For samples showing no or low number of cells observe at least 40 microscope fields.