plant surface microbiology.pdf

25 Analysing Interactions between Microorganisms and Cuticles 483
that after the inoculation period of 6 h bacterial cells are solely present on the
inoculated outer cuticle surface.
4.7 Determination of the Viable Cell Number on the Cuticle Surface
In order to document the microbial development on isolated cuticular membranes,
the cfu is determined. The initial cfu on isolated cuticles is determined
directly after inoculation of membranes with microorganisms. As an example,
the initial cfu of P. fluorescens attached to cuticles of V. major was
2.85x10 5 ±0.98x10 5 cfu/CM. Then cfu measurements are done in daily intervals
during the incubation period. First, the nutrition solution inside the
chamber is totally removed with a sterile syringe and kept in sterile glass
tubes to check for microbial growth (see below). After having removed the
nutrition solution, the membrane is cut out of the chamber with a sterile
scalpel blade and transferred in a 1.5-ml tube containing 0.05 g of sterile sand.
The cuticle is ground in 100 ml PBS with a micropestle for 2 min. After
homogenisation of the cuticle, 900 ml PBS is added and the tube contents
mixed. Serial dilutions of 100 µl are incubated on glucose yeast extract agar
plates at 25 °C for 2 days before colonies have been counted.
In order to determine the microbial cfu exclusively on the outer cuticle surface,
it is very important to check the nutrition solution inside the chamber
for microbial growth. Therefore, the nutrition solution removed from the
inner chamber volume is simply incubated at 25 °C for 2 days. Only if there is
no microbial growth detectable, is the cfu determined for that cuticle considered
to describe the microbial development on the outer cuticle surface.
4.8 Microscope Visualisation of Microorganisms on the Cuticle
Microscopic detection of microbial cells on isolated cuticles gives information
about the colonisation pattern and development. The fluorescent dyes acridine
orange and DAPI are used to stain bacteria. Both dyes are polar substances
with a very high affinity to bind nucleic acids. Thus, microbial cells
adhering to the cuticle surface are specifically stained, whereas the hydrophobic
cuticle surface itself is not stained. 0.02 % (w/v) acridine orange and
0.001 % (w/v) DAPI are dissolved in deionised water and filtered through
0.2 mm membrane filters to remove dye crystals and dust particles. Care is
taken that staining solutions are protected from daylight. For better handling
cuticles are left mounted in the chambers for staining of bacterial cells. Staining
solution (200 µl) is evenly distributed over the outer cuticle surface.
Chambers are incubated in the dark at room temperature on a horizontal
shaker (30 rpm). After different staining times of 5, 20, 40 and 60 min, respectively,
the cuticle surfaces are washed twice with 200 ml of sterile-filtered