Dm500 light microscope

Euglena gracilis culture was obtained from the Canadian Phycological Culture Centre (CPCC; University of Waterloo, Waterloo, ON, Canada) and grown in modified acid medium (MAM) [39 (link)] at pH 3 and supplemented with one simple carbon source, i.e., glutamic acid, malic acid, glucose or reduced glutathione (GSH) at a concentration of 5 g L⁻¹. Cultures were grown in a pre-combusted 1L Erlenmeyer flask and maintained under photoautotrophic conditions with an alternating light:dark cycle of 16:8 h. The light intensity was fixed at 150 µmol photons m⁻² s⁻¹ and the temperature kept between 20–25 °C. Previous studies [40 (link),41 (link),42 (link)] have shown that the addition of simple carbon sources in photoautotrophic conditions allowed faster growth. Having a greater biomass was crucial to carry out an in-depth study of the molecular characterization of phenolic compounds produced by E. gracilis. The cell concentration was counted using a Leica DM500 light microscope and a hemacytometer. The initial cell density of each culture was 1.0 × 10⁶ cell mL⁻¹, and the biomass was harvested on day 1, 3, 5, 6, 8 and 10 of growth. The cell pellet was washed 3 times with milliQ water and stored freeze-dried until further analysis.

Bee pollen sample loads were collected in 2018 from Bayburt, Turkey, with bottom-fitted pollen traps. The pollen samples were stored at −18 °C until analysed. The botanical origin of the bee pollen loads was prepared according to Wodehouse [55 (link)]. A few drops of 96% ethyl alcohol were dropped on the pollen on the slide. The preparation was allowed to stand on the heater until the alcohol evaporated. A portion of basic fuchsine added glycerin-gelatin was placed on the pollen and mixed. Pollen grains were examined by a Leica DM500 light microscope and diagnosed using an immersion objective lens (×100) [17 ,56 ].

For toluidine blue staining, briefly, the slides were deparaffinized and hydrated with deionized H₂O, followed by staining with toluidine blue solution pH 2.3 (Merck) for three minutes. They were subsequently washed with deionized H₂O three times. Slides were dehydrated quickly using 95% and 100% ethanol and then cleared in xylene and mounted with permanent mounting media (Vector Lab). Stained slides were observed under a Leica DM500 light microscope, and images were taken at 10× and 40×magnifications.

Macro-morphological descriptions were based on fresh and dried herbarium specimens. Microstructures are described using dried herbarium specimens. Fine sections through the basidiomata were prepared for observation using a razor blade under a stereomicroscope Leica EZ4 and mounted in 5% aqueous solution of potassium hydroxide (KOH) mixed with 1% aqueous solution of Phloxine. Melzer’s reagent (to test for dextrinoid or amyloid reactions), Cotton Blue (to test for cyanophilic reaction) were used and then examined at a magnification of 1000× using a Leica DM500 light microscope. Measurements were done with the software “Makroaufmaßprogramm” from Jens Rüdigs (https://ruedig.de/tmp/messprogramm.htm) and analysed with the software “Smaff” version 3.2 (Wilk 2012 ). In total, 135 basidiospores were measured from the sequenced specimen OAB0022 and additional examined specimen OAB0268. The basidiospore size is given as length and width of the spore. As measurements we present the mean with standard deviation and minimum and maximum values in parentheses (see below). The length (L), arithmetic average of all spore lengths, and the width (W), arithmetic average of all spore widths, were calculated. In addition, the ratio of length/width (Q) was calculated.