AOAC 2019 First Action Methods

B. Equipment

( a ) Ethanol Assay Kit.-- Cat. No. K-ETOH (Megazyme, Bray, Co. Wicklow, Ireland; www.megazyme.com); contains all components required for analysis. ( b ) Volumetric flasks .--50 and 100 mL.

( c ) Disposable plastic cuvettes .--1 cm light path, 3.0 mL. ( d ) Micro-pipettors .--e.g., Gilson Pipetman® P20 and P100. ( e ) Positive displacement pipettor .--e.g., Eppendorf Multipette® with 5.0 and 25.0 mL Combitip®. ( f ) Analytical balance .--e.g., Mettler Toledo XS204 ( g ) UV-Vis spectrophotometer .--Set at 340 nm (e.g., Amersham Biosciences Ultrospec 4300 pro spectrophotometer). ( h ) Vortex mixer .--e.g., IKA® Yellowline Test Tube Shaker TTS2. ( i ) Filter papers .--e.g., Whatman No. 1, 9 cm.

( j ) Disposable cuvette caps. C. Chemicals and Reagents

All reagents listed below are supplied in the Megazyme Ethanol Assay Kit.

( a ) Bottle 1 .--Buffer (15 mL, pH 9.0) plus sodium azide (0.02%, w/v) as a preservative. Use contents of bottle 1 as supplied. Stable for >2 years at 4°C. ( b ) Bottle 2 .--NAD + . Stable for >5 years at -20°C. Dissolve contents of bottle 2 in 12.4 mL distilled water. Stable for >1 year at 4°C or stable for >2 years at -20°C (to avoid repetitive freeze/thaw cycles, divide into appropriately sized aliquots and store in polypropylene tubes). ( c ) Bottle 3.-- Aldehyde dehydrogenase solution (3.25 mL). Stable for >2 years at -20°C. Use contents of bottle 3 as supplied. Before opening for the first time, shake the bottle to remove any enzyme that may have settled on the rubber stopper. Subsequently, store the bottle in an upright position. Swirl the bottle to mix contents before use. ( d ) Bottle 4.-- ADH suspension (1.3 mL). Stable for >2 years at 4°C. Use the contents of bottle 4 as supplied. Before opening for the first time, shake the bottle to remove any enzyme that may have settled on the rubber stopper. Subsequently, store the bottle in an upright position. Swirl the bottle to mix contents before use. ( e ) Bottle 5.-- Ethanol standard solution (5 mL, 5 mg/mL). Stable in a well-sealed container (as supplied) for >2 years at 4°C. Dilute 0.5 mL of the contents of bottle 5 to 50 mL with distilled water. Store in a well-sealed Duran® bottle. When diluted, this solution is stable for 2 days at 4°C.

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