Critical point dryer

The same S. aureus and E. coli strains were grown for 7 h in tryptone soy yeast extract broth (TSYEB) at 37 °C. A total of 75 µL of concentrated green plum flesh water extract in 20% ethanol (from approximately 130 mg DW of flesh powder) was added to 1 mL bacteria and broth samples, and 75 µL of 20% ethanol was added to the controls, and incubated for 24 h at 37 °C. The samples and controls were washed three times in sterile phosphate buffered saline and fixed in 3% glutaraldehyde. They were adhered to poly-l-lysine-coated (1 mg/mL) coverslips and dehydrated in ethanol before being dried in a critical point dryer (Tousimis Research Corporation, Rockville, MD, USA). Coverslips were attached to stubs with double-sided carbon tabs and coated with gold before samples were imaged using scanning electron microscopy (SEM) on a Jeol Neoscope JCM 5000 (Jeol Ltd., Tokyo, Japan) at an accelerating voltage of 10 kV.

Infected coral fragments, and also cultured R. reptotaenium (clumps), were fixed in 2.5% glutaraldehyde and maintained at 4 °C until processing. After fixation, fragments were dehydrated through a graded series of alcohol by placing them for 10 min in ethanol at concentrations of 20, 40, 60, 70, 90 (one wash each) and 100% (three washes). Fragments were then freeze fractured in liquid nitrogen and dried in a critical point dryer (Tousimis, Rockville, MD, USA), after which they were affixed to an aluminum stub using carbon adhesive tape and coated with palladium/gold using a sputter coater (SPI-Module). Fragments were viewed using a JOEL JSM-5900 LV Scanning Electron Microscope (JOEL USA, Peabody, MA, USA) at Florida International University.

dtm-1, dtm-cr5, and their corresponding wild type SAMs were dissected from seedlings at different developmental stages by removing any visible leaves and examined with a stereomicroscope (M125/DFC420, Leica). For SEM and histological analysis, the dissected meristems were fixed in formalin-acetic acid-alcohol (10% formaldehyde, 5% acetic acid, and 50% alcohol by volume). The meristems were gradually dehydrated by incubation in an ethanol series (30–100%). Half of these meristem samples were used for SEM analysis and so further dried using a critical-point dryer (Tousimis, USA), before being sputter coated in gold particles. SEM images were collected using a high-resolution field emission SEM (Zeiss Merlin Compact, Zeiss or JSM-6360LV, JEOL). The other half were embedded in Paraplast (Sigma-Aldrich) for histological analysis and 8 μm sections made using a Leica microtome (Leica). The sections were briefly stained with 0.05% toluidine blue.

To investigate the ultrastructural changes of bacterial biofilms caused by oregano oil, scanning electron microscopy (SEM) was performed using the representative strains of P. aeruginosa IQ0042 and MRSA IQ0064. Briefly, biofilms of IQ0042 and IQ0064 were grown for 24 h on sterilized squares of ACLAR 33C (Electron Microscopy Sciences, Hatfield, PA, United States), and treated for 1 h with oregano oil at 0.75 mg/ml or 0.3 mg/ml, respectively. Untreated and oregano-treated biofilms were fixed at 4°C for 24 h in 0.1 M sodium cacodylate buffer containing 2.5% glutaraldehyde, 0.15% alcian blue, and 0.15% safranin O. The fixed biofilms were washed with 0.1 M sodium cacodylate buffer, infiltrated with 2% osmium tetroxide for 2 h, and dehydrated to 100% ethanol. The biofilms were dried using a critical-point dryer (Tousimis Research Corporation, Rockville, MD, United States), mounted on specimen stubs, sputter-coated with 10 nm Cressington 208 platinum (Cressington Scientific Instruments, Watford, United Kingdom), and examined on a S4800 SEM (Hitachi Ltd., Tokyo, Japan). Micrographs were acquired under high vacuum using an accelerating voltage of 3.0 kV.

The methicillin resistant S. aureus, clinical isolates of P. aeruginosa, L. monocytogenes, and B. Cereus strains were grown for 7 h in tryptone soya yeast extract broth (TSYEB) at 37 °C. Methanolic ASE of T. ferdinandiana fruits and leaves were reconstituted in 75 µL 20% v/v ethanol, added to 1 mL bacteria and broth samples, and incubated for 24 h at 37 °C. The negative control was comprised of 75 µL 20% v/v ethanol. The samples and controls were washed three times in sterile phosphate buffered saline and fixed in 3% v/v glutaraldehyde [27 (link)]. Glutaraldehyde-fixed samples were fixed again in 1% v/v osmium tetroxide and dehydrated with ethanol. Samples were adhered to coverslips coated with poly-L-lysine (1 mg/mL) and dehydrated in the same manner, before being dried in a critical point dryer (Tousimis Research Corporation, Rockville, MD, USA) according to manufacturer’s instructions. Coverslips were attached to stubs with double-sided carbon tabs and coated with gold using a sputter coater (Agar Scientific Ltd, Essex, UK), following the manufacturer’s instructions. Samples were imaged in a Jeol Neoscope JCM 5000 (Jeol Ltd., Tokyo, Japan) at an accelerating voltage of 10 kV and for high resolution images in a Jeol JSM 7100F (Jeol Ltd., Tokyo, Japan) field emission scanning electron microscopy (SEM) at an accelerating voltage of 1 kV.