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The RS and FRS samples were cut into 1 mm and put in separated vials in triplicate and fixed in fixative solution (4% glutaraldehyde) for 2 days at 4°C.
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To test the reproducibility of the analysis method, this sample was divided between six vials and analyzed in triplicate as a randomized sample list.
To calculate the actual challenge dose given, direct plating of aliquots from the challenge inoculum and remaining working cell bank vials was performed in triplicate using tryptone soya agar (Oxoid) and culture for 24 hours before colony counting.
Each vial was analyzed in triplicate.
A no-template control was included for all assays, and all reactions were run in triplicate vials.
Cellobiohydrolase assays were carried out in triplicate vials at 40°C, pH 5.0 in 20 mM azide-containing acetate buffer, with continuous mixing by inversion at 10 rpm while immersed in a water bath.
Single-vial enzyme assays were completed in triplicate in 2 mL amber glass GC sample vials as previously described [ 51] in three different buffer/substrate combinations with approximately 60 μg of purified protein per 500 μL assay.
Blank wheat samples (0.500 ± 0.002 g) were weighed into an 8-mL vial and spiked with 500 μg kg−1 D3G in triplicate.
All reactions were run in triplicate and for two different vials per sampling date 6 qPCR reactions per sampling date.
Experiments with glucose as carbon source were carried out in triplicate in 117-ml serum vials sealed with rubber stoppers and aluminium crimps.
Biomass from each sample tube was dispensed in triplicate into three independent output vials, and each digestion assay was quantified for glucose and pentose in quadruplicate.
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