Abstract:
Microbes and their exopolysaccharides (EPS) can block xylem vessels, thereby increasing the hydraulic
resistance and decreasing the vase life of cut flowers and foliage. Scanning electron microscopy (SEM)
provides a powerful tool for investigation of bacteria-induced xylem occlusion. However, conventional
preparation protocols for SEM involving chemicals can cause loss of hydrated EPS material, and thereby
damage the bacterial biofilms during dehydration. A modified chemical fixation protocol involving prefixation
with 75 mM lysine plus 2.5% glutaraldehyde followed by the normal fixation in 3% glutaraldehyde
was, therefore, tested for improved preservation of bacterial biofilm at the stem-ends of cut Acacia
holosericea foliage stems. Stem-end segments with different stages of bacterial growth were obtained
from stems stood into water. The lysine-based protocol was compared with four other processing protocols
of critical point drying (CPD) without fixation (control), freeze-drying (FD), conventional chemical
fixation followed by drying with hexamethyldisilazane (HMDS), and conventional chemical fixation with
CPD. The non-fixed control, FD and the glutaraldehyde fixation with HMDS drying gave poor preservation
of hydrated material, including bacterial EPS. Conventional glutaraldehyde fixation followed by CPD was
superior to these three methods in terms of better preserving the EPS. However, this fourth method gave
condensation of biofilms during dehydration. In contrast, the modified lysine-based protocol resulted in
superior preservation of EPS and biofilm structure. Thus, this fifth method was the most appropriate for
examination of bacterial stem-end blockage in cut ornamentals.
