Lactophenol blue

Light microscopy analyses of C. acutatum development were performed on strawberry leaf discs (10 mm diameter) randomly excised from infected leaflet at 1, 3, 5, 7, 9 dpi, using a method modified from Debode et al. (2009 (link)). In breaf, leaf discs were cleared in 0.15% trichloroacetic acid (TCA) in a 3:1 (v/v) mixture of ethanol and chloroform for 48 h, with at least three changes of the bleaching solution, rinsed briefly in lactoglycerol and incubated at room temperature for 1 h in lactophenol blue (Sigma), and washed 3 times in lactoglycerol. For ROS detection, leaf discs were infiltrated in a 10 mg/ml DAB (3,3′ -diaminobenzidine, Sigma) solution for 10 min and subsequently incubated overnight at room temperature in dark, and cleared for 24 h in 3:1 (v/v) ethanol: glacial acetic acid with three changes. Treated leaf disc was mounted in 50% fresh glycerol on glass slides and examined using a Leica DM5000B microscope. Images were captured with a Leica DC500 digital camera. Three leaftlets were sampled from each plant, and 3 plants were observed at each time point. The overall number of conidia (germinated and non-germinated) and appresoria were counted per leaf disc taken up to 9 dpi.

The alteration in P. digitatum mycelial morphology, after treatment with MFC from NCT/PPE/SeNPs, was microscopically detected; with digital optical microscope “Labomed Lx400; Labo America Inc., Fremont, CA”, after incubation of fungal mycelia with the nanocomposite for 12 and 24 h under stirring and staining of treated mycelia with lactophenol-blue (Sigma-Aldrich, MO).

A direct examination of collected hairs, skin and nail scrapings, and phenotypic characteristics was performed based on comparative analyses of macro- and micromorphology of cultivated fungi and molecular biology methods. The last-mentioned factor allowing for unique molecular identification to the species level was based on sequencing of the PCR product obtained with ITS1 and ITS4 pair of primers complementary to the rDNA gene cluster. For direct microscopical examination of the clinical material collected from the patients’ clearing fluid, a solution comprising dimethyl sulphoxide (DMSO) and 10% KOH was used. For better visualisation, additional staining with lactophenol blue (Sigma-Aldrich, Saint Louis, MO, USA) or calcofluor white (Sigma-Aldrich, Saint Louis, MO, USA) was performed. The preparations were examined in the presence of any fungal elements under a light or fluorescence microscope (Olympus BX51, Tokyo, Japan). In each microscopic preparation, 10 visual fields were examined under a magnification of 400×.