At different stages of fermentation (0, 15, and 90 days), the presence of biofilms on the epidermis of fruits was assessed by scanning electron microscopy (SEM), as described by Kroupitski et al. (2009) (link) with slight modifications. First, fruits were rinsed twice for 15 min in PBS to remove the non-adhered cells and then fixed in 2.5% glutaraldehyde (Sigma–Aldrich, St. Louis, MI, USA) in PBS for 2.5 h. Later, the olives were dehydrated through a graded ethanol series (50, 70, 80, 90, 95, and 100%, 5 min in each one). Finally, fruits were treated for 20 min in 2-methyl-2-propanol. For SEM observation, 2 mm × 2 mm slices of the skin of olives were taken, placed on glass slides, and coated with gold in a Scancoat Six SEM sputter coater (Edwards, Crawley, England). Pictures were taken with a JEOL JSM- 6460LV SEM model (JEOL USA, Inc., Peabody, MA) in the Technology and Innovation Research Center (CITIUS, University of Seville, Spain).
Scancoat six sem sputter coater
Manufactured by Edwards Lifesciences
Sourced in United Kingdom, Israel
The Scancoat Six SEM sputter coater is a laboratory equipment designed for the preparation of samples for scanning electron microscopy (SEM) analysis. Its core function is to apply a thin conductive coating on the surface of non-conductive samples, which is necessary for effective imaging and analysis using an SEM.
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2 protocols using scancoat six sem sputter coater
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Biofilm Formation on Fruit Epidermis
2
SEM Imaging of Olive Fruit Biofilms
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Scanning electron microscope (SEM) techniques were used to observe fruits (biofilms and flesh) at 14 days of fermentation. For this purpose, and after washing the olives with sterile tap water to remove non-adhering microorganisms, fruits were treated according to the protocol of Kubota et al. (2008) , using glutaraldehyde and increasing concentrations of ethanol to dehydrate the samples. Then, samples were placed in 2-methyl-2-propanol for 20 min, and slices (0.5 cm 2 ) of the fruit epidermis before GB treatment and after the first and fifth treatments were fixed onto glass slides. Samples of the olive pulp were also obtained after removing the olive epidermis with a scalpel under sterile conditions. Olive slices were sputtered with gold using a Scancoat Six SEM sputter coater (Edwards, Gat, Israel) for 180 s. Finally, the preparations were observed with an SEM model JSM-6460LV (Jeol Ltd, Tokyo, Japan) at CITIUS (University Seville, Spain) facilities.
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