Scd 004 sputter coater

Samples were transferred to a Petri dish to be observed under an inverted microscope (Leica DMI8), up to 400x magnification DIC. A Leica MC170 HD camera with the Leica application suite (v. 4.12.0) software was used to take photographs.
Observed individuals were isolated using a small diameter pipette and transferred to a drop of sterile water, then further rinsed into another drop in order to get rid of other eukaryotic contaminants. Living cells were stored, individually or in groups of up to four cells, in Eppendorf tubes containing 100 µL of guanidine thiocyanate-based nucleic acids extraction buffer (Chomczynski and Sacchi 1987 (link)) for later DNA extraction.
Some individuals or empty tests were deposited, after washing in distilled water, on stubs for scanning electron microscopy. Stubs were desiccated in a box with silica gel at least one day before metallization and observation. Then they were coated with 8-nm gold using a Balzers SCD 004 sputter coater and a tension of 15 kV. They were observed with a Hitachi S-3000 N and a JEOL JSM-5510 (operating at 10 kV) scanning electron microscope.

All the reagents were of the best grade available, and were used without any further purification. The sole exception was fungal laccase (LC), and LC was produced and purified as already described [51 (link)] UV–Vis spectra and absorbance readings were recorded using an Ultrospec 2100pro UV–VIS spectrophotometer (Amersham Biosciences, Milan, Italy).
FT-IR spectra were recorded after the samples were prepared as KBr pellets, with a KBr beam-splitter and KBr windows using a Thermo Nicolet 5700 (Waltham, MA, USA) spectrometer at room temperature.
Sample morphology was observed using scanning electron microscopy (SEM) (S-4100, Hitachi, Tokyo, Japan). The samples were fixed on a brass stub using double-sided carbon coated with gold blazers on a SCD 004 sputter coater (Hitachi, Tokyo, Japan) for 2 min and observed under an excitation voltage of 5 kV.