Richard Lab:ADL

Overview
Fiber analysis is an established method of determining fiber contents and digestibility of animal feeds and forages. When the steps are performed in a sequential manner, fiber analysis also provides estimates of hemicellulose and cellulose contents. The steps are: (1) neutral detergent fiber (NDF), (2) acid detergent fiber (ADF), and acid detergent lignin (ADL).

Materials

 * 2000 mL flask
 * acetone (in flammables cabinet below fume hood in room 113B)
 * 5 glass jars
 * microwave
 * pH paper
 * concentrated sulfuric acid
 * top-loading balance (1 decimal place)
 * ANKOM Daisy incubator and jars
 * analytical balance
 * drying oven at 105&deg;C
 * dessicator
 * zip-loc bags with dessicant pouches (recently recharged at 105&deg;C)
 * hazardous waste containers

Procedure

 * 1) Prepare 72% (w/w) sulfuric acid the night before performing ADL. Wear appropriate safety equipment (lab coat, safety glasses, rubber gloves (over-top of blue nitrile gloves).  Move balance into fume hood.
 * 2) Weigh 440 g nanopure water into 2000 mL flask.
 * 3) Weigh 400 g concentrated sulfuric acid into 400 mL beaker. Slowly add to water in flask.
 * 4) Repeat sulfuric acid addition 2 more times for a total of 1200 g sulfuric acid. This makes ~1 L 72% sulfuric acid, enough for 2 ADL batches.
 * 5) Cover flask with foil and let cool overnight in the fume hood.
 * 6) Label 2 Daisy incubator jars with batch number.
 * 7) Add 1 batch of bags to each jar (~10-12 bags on each side of divider).
 * 8) Split 72% sulfuric acid evenly between two jars (~500 mL in each jar).
 * 9) Place lids on jars and put in Daisy incubator.
 * 10) Rotate for 3 hours with door to Daisy incubator open.
 * 11) After 2 h 45 min, fill 5 glass jars (to just below neck) with nanopure water. Microwave 1st jar for 11 min.
 * 12) After 3 h, pour acid into hazardous waste container using funnel.
 * 13) Add heated rinse water to jars (split evenly between jars) and rotate jars in Daisy incubator while heating next rinse in microwave.
 * 14) Repeat rinses until water is neutral pH. Check this using pH paper and compare to nanopure water for reference.  Should require 5 rinses.  After 1st rinse, water can be disposed of down drain with running water.
 * 15) After final rinse is complete, remove bags from jars (do 1 batch at a time so they do not get mixed together). Gently squeeze bags (4-6 at a time) to remove excess water.
 * 16) Place bags in 1 L beaker. Add acetone to cover (~400 mL) and let soak in fume hood for 3-5 min.  Gently squeeze bags (4-6 at a time) to remove acetone.  Let air-dry on clean tray in fume hood for 1 h (until acetone has evaporated).  Pour used acetone into hazardous waste container.
 * 17) Place tray in 105&deg;C oven to dry overnight.
 * 18) Remove bags from oven and place immediately in zip-loc bags (flatten to remove air) and into dessicator. After cool (~30 min), record ADL bag weight using appropriate analytical balance.  Proceed with ash content determination.

Contact

 * Megan
 * Jess

Back to Protocols