SEM Observation of Bacterial Presence in Cut Tree Peony

The presence of bacteria in the xylem vessel at the stems end of the cut tree peony ‘Luoyang Hong’ flowers was observed by SEM (JSM-IT200(LA), JEOL, Tokyo, Japan) as described by Li et al. (2017) (link) and Thakur et al. (2022) (link), with some modifications. In brief, Cross-sections of stems (~2 mm length) were excised from the base of each stem using fresh surgical blades every other day. The stem segments were immediately immersed in 0.1 M phosphate buffer (pH 7.2, PBS), and then transferred to 2.5% glutaraldehyde for fixation 6 h at room temperature. After fixation, the stem segments were washed three times with PBS followed by dehydration using graded ethanol series (30%, 50%, 70%, 90% and 100%) of 15 min for each ethanol concentration. The stem segments were eventually dried in a critical point dryer (K850, Quorum Technologies, UK) with liquid CO₂. Then, the stem segments were mounted with double-sided carbon tape on SEM stub and coated with platinum using a sputter-coater (JFC-1600, JEOL Japan) for 30 s. The images of sample surfaces were observed at 20 kV accelerating voltage for capturing the digital micrographs.