Whatman no 1 paper

Laccase activity in R. placenta 45 was measured after 16 weeks incubation in untreated, MCA_LTBA and MCA_HTBA-treated wood colonized by R. placenta 45 (see protective effectiveness of MCA active ingredients) and after 2, 4, and 9 weeks in liquid cultures. For detection of laccase in wood, the colonized samples were treated according to Wei et al. [30 (link)]. The grounded wood was stirred overnight at 4C° in dist. H₂O containing 1M NaCl to extract extracellular proteins. The liquid phase was separated from the solids by vacuum filtration through Whatman no 1 paper and concentrated in an ultrafiltration cell (Ultracel, Millipore) fitted with 10-kDa cutoff membrane.
Laccase activity was measured as initial velocity of the oxidation of ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), 3 mM) to its cation radical at room temperature (22–25°C) and at pH 4.5 (citrate buffer 100 mM). Changes in absorbance (ΔA) at 420 nm were recorded with UV-visible spectrophotometer (Genesys 10S UV-vis, Thermo Scientific Inc., Waltham, MA, USA). One volumetric activity unit (U) was defined as the amount of enzyme transforming 1 μmol of ABTS per min and the volumetric activities were calculated using an extinction coefficient (ε) of 36000 mol^-1 L cm^-1[38 , 39 ].

The composition and preparation of CLUPS medium are the same as those of PEHPS medium [14 (link)], except for the substitution of beef liver (PEHPS) by beef lung (CLUPS). The main components of CLUPS are casein, lung and pancreas extract, and bovine serum. Table 1 compares the composition of the three media, PEHPS, TYI-S-33, and CLUPS. Once prepared, the basal medium was sterilized by filtration over a 0.22-μm filter (Millipore, Millipore Corporation, Billerica, MA, USA) and stored in 5.5-mL aliquots at -20°C. Just before use, CLUPS is supplemented with 8.3% v/v bovine serum. Bovine serum was also produced in-house as follows: about 15 L fresh blood from cows for human consumption was collected in a sterile way at a local slaughterhouse. After blood coagulation, the serum was recovered, and the complement was heat-inactivated (56°C, 1 h). Next, the serum was prefiltered through Whatman No. 1 paper before being sterile-filtered through a series of sterile Millipore HAWP filters (Millipore, Millipore Corporation, Billerica, MA, USA) from 5 to 0.22 μm. Serum was frozen at -70°C until use.

To obtain the supernatant of Gl006, the fungus was grown in Erlenmeyer (300 mL) with 150 mL of sterile PDB (Difco®) for five days (125 rpm, 25 °C) with an initial concentration of 1 × 10⁵ conidia.mL⁻¹. After incubation, the fermentation broth was harvested filtering by Whatman No. 1 paper and subsequently by sterile 0.22 μm filters (Millipore^®) and stored at −20 °C until its use. To obtain the supernatant of Bs006, bacteria was grown in sterile LB broth for 48 hours and then was centrifuged. Supernatant was harvested and filtered with sterile 0.22 μm filters (Millipore^®). Biomass obtained from Bs006 culture was washed twice to eliminate waste of supernatant and resuspended in SDW, homogenizing by shaking in vortex at maximum speed for two minutes, to be used as inoculum in these experiments. On the other hand, conidia of Gl006 were harvested from a seven-day-old culture on PDA, flooding the medium with sterile distilled water and scraping with a sterile metal rake. The suspension obtained was shaken in a vortex at maximum speed for 30 seconds and then was filtered with sterile muslin (pore size 0,5 mm) to eliminate pieces of mycelium.