plant surface microbiology.pdf

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Daniel Knoll and Lukas Schreiber
deionised water to remove unbound dye molecules. Cuticles are left over silica
gel until dry. The dried cuticle surfaces are excised from the chambers
with a scalpel blade and cut into four parts. Cuticle pieces are transferred onto
a thin hydrophobic layer of silicon grease on a microscopic slide. A cover slip
together with one drop of immersion oil is put on the top of the cuticle prior
to microscopic examination. Due to the hydrophobic layer of silicon grease
and the immersion oil, the entire surfaces of the cuticle pieces are spread
totally flat minimising problems with depth focus. Furthermore, the refraction
of light is markedly reduced allowing fluorescence microscopy with isolated
cuticles. Samples can be viewed with a Zeiss Axioplan microscope
(Zeiss, Oberkochen, Germany) equipped with a 50 W mercury high pressure
bulb, a 40x objective (Zeiss, Plan-Neofluar) and a Zeiss filter set No. 09 (excitation:
450–490 nm; dichroic beamsplitter ≥510 nm; emission ≥520 nm). One
examples of a fluorescence microscopy micrograph of a colonised cuticle surface
is shown in Fig. 8. The surface coverage of the cuticle colonised by bacte-
Fig. 8. Epifluorescent microscope image of an isolated cuticular membrane of Prunus
laurocerasus artificially colonised with Pseudomonas fluorescens (magnification ¥400).
Bacterial cells are stained with acridine orange and viewed at an excitation of
450–490 nm.Approximately 27.4 % of the cuticle surface is covered by bacteria. Bacterial
cells are accumulated in small clusters over the entire cuticle surface