Cells were plated into 12 well plates at a density of 25,000 cells per well in 0.5 mL of medium described above and allowed to adhere for 2 – 3 hours before treatment.
Afterward, this solution was added to 1 mL of previously described PCPP solution.
In Europe, cases of ML were described in Italy, France, Spain, Malta, Portugal, Holland, the United Kingdom, and Austria [ 4, 12, 13].
Formation of the complex was obtained using 3 mL molybdovanadate solution instead of 2 mL as described in the original method.
All animals were euthanized 14 dpi and BALF was sampled from the exenterated lung in 6 fractions of 20 mL as described [ 31].
Unless other specified, enzyme hydrolysis reactions were performed in 96-well deep-well plates in a reaction volume of 0.5 ml, as described [ 20].
Glucose limited fed batch cultivations of the selected P. pastoris high producing strains were carried out in duplicate in 1.0 L bioreactors (SR0700ODLS, DASGIP, Germany) with a fed batch starting volume of 350 mL as described previously [ 17].
From the same animals, broncho-alveolar lavage fluid (BALF) was endoscopically sampled at 4 and 9 dpi under general anesthesia in 5 fractions of 20 mL as described elsewhere [ 32].
In 2008, a volume of 2.4 mL was described as sufficiently effective by the FDA's Tentative Final Monograph for Healthcare Antiseptic Products [ 9] for formulations based on 85% ethanol [ 10].
Partitioned analyses were conduced using MrBayes for BI and a pre-release version of RAxML for ML, as described above.
