Standard Operating Procedure
TO BE PERFORMED FIRST IN THE SERIES OF ANALYSES
Title: Neutral Detergent Fiber Analysis via Filter Bag Technique for samples
containing < 5% fat (no soybean products)
Department: Agronomy
Created by: Virginia Nichols
Laboratory: Crop Production & Physiology Lab Suite
Supervisor: Dr. Fernando Miguez
Lab Supervisor: Whitney Bouma
Date approved:
Procedure Overview: This procedure determines Neutral Detergent Fiber, which is the
residue remaining after digesting material in a detergent solution. The remaining fiber
residues are predominantly hemicellulose, cellulose, and lignin.
Equipment and reagents necessary:
Reagents:
Neutral Detergent (ND) Solution (purchase pre-mixed from ANKOM Technology)
3.0% Sodium lauryl sulphate
1.86% EDTA disodium, dehydrate
0.68% Sodium borate, decahydrate
0.46% Sodium phosphate, dibasic, anhydrous
1.0% water
Alpha-amylase, activity 17,400 Liquefon Units / ml
Sodium sulfite (Na2SO3), anhydrous
Acetone
Miscellaneous:
Analytical Balance (0.1 mg accuracy)
Weigh spatula
Oven (capable of maintaining a temperature of 102±2⁰C)
ANKOM 200 Digestion instrument
Chemically inert filter bags (purchased from ANKOM Technology)
Heat sealer (purchased from ANKOM)
Desiccator pouch
Solvent and acid resistant marking pen
Gloves
Lab coat
Procedure:
1. Use marker to label all filter bags. Weigh filter bag (W 1), record weight, zero balance.
2. Weigh 0.45 – 0.55 g of prepared sample (W 2) directly into filter bag. Make sure there is
no sample in the top portion of the filter bag.
3. Using the heat sealer, completely seal filter bag within 4mm of the top. Heat sealer
should be set to 4-5. Allow 2 sec cool time before removing from heat sealer.
4. Spread sample uniformly inside filter bag, removing clumps (shake, flick).
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5. Weigh one blank bag, include in run to determine blank bag correction (C1).
Note: When doing more than one run, make sure to have reps of the same
sample straddled across runs. This will serve as a ‘standard’.
6. Place no more than 24 bags into the Bag Suspender. 8 trays should be used regardless
of the number of bags being processed – the 9th tray is used as a cover and should NOT
have bags in it. Place no more than 3 bags per tray then stack trays on center post with
each level rotated 120 degrees.
7. Insert the Bag Suspender with bags into the fiber analyzer vessel and place the weight
on top to keep it submerged.
8. The amount of ND solution added will depend on the number of bags you use.
9.

When processing 20-24 bags, add 1900 – 2000 mL of ambient ND solution to the
fiber analyzer vessel.

When processing less than 20 bags, add 100 mL/bag of ND solution, with a
minimum of 1500 mL to ensure the bag suspender is covered.
Add 0.5 g/50mL of sodium sulfite to the solution. For 24 bags, use 20 g of sodium
sulfite.
10. Regardless of the number of bags, add 4.0 mL of alpha-amylase to the solution in the
vessel.
11. Make sure all switches are ‘off’ before plugging in in the instrument. Once plugged in,
turn Agitate and Heat on and confirm agitation. Set timer for 75 min and close lid. Vessel
will maintain a solution temperature of 100⁰C.
12. At the end of the extraction, turn Heat and Agitate off. Open the drain valve (slowly!) and
drain the ND solution into a waste vessel before on before opening lid. Note that the
exhaust hose must be secured to the vessel, as the contents are under pressure.
13. (Rinse 1) After the solution has been drained, close the valve and open the lid. Add
1900 mL of (70-90°C) tap water and 4.0 mL of alpha-amylase. Turn Agitate on and rinse
for 5 min. The lid may be sealed with Heat on or left open with Heat off. Exhaust and
drain the ANKOM as in step 12. (NOTE: I am not sure if this should be drained into a
waste container or if it can go down the drain).
14. (Rinse 2) Repeat step 13.
15. (Rinse 3) Close the valve and add 1900 mL of (70-90°C) rinse water (no alphaamylase!). Turn Agitate on and rinse for 3-5 min. The lid may be sealed with Heat on or
left open with Heat off. Exhaust the vessel; the ANKOM may be drained into the sink or
waste container.
16. Remove the samples, and gently press out excess water from bags.
17. Place bags in a 250 mL beaker, add enough acetone to cover bags and soak for 3-5
min.
18. Remove bags from acetone and place on wire screen to air dry. Completely dry in oven
at 102 ± 2°C. Most ovens will complete drying within 2-4 hours. Do NOT place bags in
oven until acetone has completely evaporated.
19. Remove bags from oven, place directly into a collapsible desiccant pouch and flatten to
remove air. Do NOT use a conventional desiccator container. Cool to ambient
temperature.
20. Weigh bags (W 3). Blank bag correction (C1) is the final oven-dried weight divided by the
original blank bag weight.
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21. If vessel will not be used for >1 month, add 1900 mL of water, agitate 10 min, drain. This
will ensure there is no acid remaining to corrode the machine.
Calculations
% NDF (as received basis) =[ [(W3 – (W1 x C1))] / W2 ] x 100
W1 = bag tare weight
W2 = sample weight
W3 = dried weight of bag with fiber after extraction process
C1 = (final oven weight of blank bag) / (original blank bag weight)
Personal Protective Equipment / Engineering Controls:
Nitrile gloves
Safety glasses
Face shield
Dust mask
Latex gloves
Splash goggles
Lab coat
Fume hood
Neoprene gloves
Vented goggles
Apron
Biosafety cabinet
Insulated gloves
Eye wash station
Safety shower
Respirator
Note: Open-toed and heeled shoes are NOT allowed.
Other Control Measures:
Handling & Storage Precautions:
Sodium Sulfite (anhydrous): Air and moisture sensitive. Keep container tightly closed. Keep container in
a cool, well-ventilated area.
Alpha-amylase: Protect from contamination. Keep container tightly closed in a cool, well-ventilated
area.
Neutral Detergent (ND) Solution: Keep container tightly closed in a cool, dry, well-ventilated area away
from ignition sources. Keep from freezing.
Acetone -
Waste Disposal Procedures:
Unless EH&S specifically instructs otherwise, all chemical/reagent waste (including excess solutions)
must be placed in an appropriately labeled hazardous waste container for EH&S disposal.
Compatible substances may be combined into one waste container.
Spill/Release Containment and Clean Up/Decontamination Procedures:
Sodium sulfite: use appropriate tools to put spilled solid in a convenient waste disposal container. Rinse
area with water.
Alpha-amylase: absorb material with suitable absorbent and containerize for disposal.
Neutral Detergent (ND) Solution: absorb material with suitable absorbent and containerize for disposal.
Acetone: Use with adequate ventilation, wash hands and clothing thoroughly after handling. Store in a
cool, dry, well-ventilated area away from incompatible substances and away from ignition sources.
Health & Safety Summary for Required Reagents:
Sodium sulfite is a stable, non-flammable, low-reactivity substance. It is a skin and eye irritant, and
should not be ingested or inhaled. When heated to >500°C it emits toxic Na2O and Sox fumes.
However, these conditions are unlikely to be met when using this protocol, as the maximum
exposure temperature is ~100°C.
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Alpha-amylase is a stable, non-flammable, low-reactivity substance. It is non-hazardous to the skin, but
may cause irritation to eyes, gastro-intestinal tract, and lungs.
ND solution is a stable, non-flammable, low-reactivity substance. It may cause skin and eye irriation,
and it may be harmful if swallowed. If excessively heated outside the temperature ranges of this
protocol, thermal decomposition may produce oxides of carbon, sulfur, sodium, boron,
phosphorous, and nitrogen.
Acetone: Remove all sources of ignition and provide adequate ventilation. Soak up with an inert
absorbent material.
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Target Organ(s)
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Sodium sulfite
Incompatibilities
Alpha-amylase
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None
None.
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Neutral Detergent Sltn
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Eyes, skin, and
digestive tract.
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Acetone
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Central
system
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reducing agents, iron,
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compounds, acids, strong
oxidizers, alkalies.
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nervous
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 The above summary consists of guidelines for proper handling & disposal of
chemicals used in this procedure. You must read and understand the
contents of the entire MSDS(s) before starting this procedure.
References:
ANKOM Procedures, available through ANKOM website.
Sodium Sulfite MSDS, available through ANKOM website.
Alpha Amylase MSDS, available through ANKOM website.
Neutral Detergent Solution MSDS, available through ANKOM website.
Acetone MSDS, available through ANKOM website
Dec 2012 VNichols