plant surface microbiology.pdf

25 Analysing Interactions between Microorganisms and Cuticles 477
sterilised by UV radiation for 30 min on each side. Sterilised cuticles are then
mounted in the chambers under sterile conditions. Care is taken that the
physiological outer side of the cuticles is orientated to the outside. The physiological
inner side of the cuticle faces 800 ml of a highly concentrated nutrition
solution consisting of 20 % (w/v) glucose and 5 % (w/v) yeast extract or
simply water. The inner volume of the chamber is accessible by sampling
ports that can be closed by metal stoppers. Using a sterile plastic syringe, the
solution inside the chamber can be replaced several times during the course
of the experiment. Chambers were incubated upside down on a metal grid in
a climate-controlled incubation box for some hours at 25 °C before the inoculation
with microbial cells. Incubation boxes are 10x20 cm in size and can be
closed with an air-tight lid. Boxes are sterilised with 70 % (v/v) ethanol and
with UV radiation. Sterile pressurised air is conducted through the incubation
box. Air humidity is set by simply changing the temperature of the water
reservoir. At a temperature of 25 °C, the air has a humidity of 100 %. Lower
moisture levels can be set in the incubation box by reducing the temperature
of the water reservoir under 25 °C as the saturation vapour pressure of water
in air is dependent on temperature (Nobel 1991). One incubation box is
equipped with a hygrometer and a temperature sensor in order to verify the
actual climate conditions inside the box.
4.4 Inoculation of Cuticular Membranes with Epiphytic Microorganisms
A cell culture of P. fluorescens is cultivated in glucose-yeast-medium overnight
at 25 °C. Cells are harvested by centrifugation (2120xg, 20 min), resuspended
and washed twice in 10 –2 M phosphate buffered saline (PBS, pH 7.4; Sigma
Chemicals). Prior to inoculation the cell suspension is adjusted to an optical
density of 1.0 that corresponds to 2.5◊10 8 cfu/ml. The outer cuticle surface is
inoculated with bacteria by spreading 200 ml of a washed cell suspension of P.
fluorescens evenly over the entire exposed cuticle surface (Fig. 5B). Chambers
are incubated for 6 h at 25 °C in a sterile glass Petri dish containing PBSbuffer-moistened
filter papers at the bottom in order to avoid evaporation of
water from the inoculation solution. During the inoculation period, bacterial
cells adhere to the cuticle. After 6 h the suspension is withdrawn and the surface
is carefully washed five times with 200 ml sterile deionised water to
remove unbound bacteria. Chambers are left in a laminar flow hood until dry.
Immediately after the drying of the washed cuticle surface, the chambers are
transferred upside down in the incubation box (Fig. 5C). Furthermore, two
control experiments are performed. One control is necessary for checking
sterile conditions during the course of experiment. Therefore, cuticles are
incubated with 200 ml of sterile PBS and treated in the same way as described
above.Another control is to verify that during the inoculation period bacteria
are not able to pass through the silicone grease from the outer cuticle surface