Richard Lab:ADL

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Contents

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°C
  • dessicator
  • zip-loc bags with dessicant pouches (recently recharged at 105°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°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.

Notes

  • ALWAYS add acid to water and work in fume hood when making 72% sulfuric acid

References

  1. Van Soest, P. J., Robertson, J. B., and Lewis, B. A. (1991). "Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition." Journal of Dairy Science, 74(10), 3583-3597.
  2. See ANKOM Technology website for procedures (http://www.ankom.com/09_procedures/procedures.shtml)

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