AFIA - Laboratory Methods Manual - Australian Fodder Industry ...

Reagents:
1. Hydrochloric Acid, 32% (w/v), AR grade
2. Pepsin powder (derived from porcine source) 469 units/mg or greater.
3. Sodium Acetate, anhydrous AR grade
4. Sodium Carbonate, AR grade
5. Acetic Acid, glacial AR grade
6. Cellulase, ‘Onozuka FA’
7. Heat stable α-amylase solution (NOVO Termamyl; Sigma Cat No A5426 or similar) , 18,000
units/mL or greater.
8. Acidified Pepsin Solution (0.3% w/v in 0.125 M HCl) - to prepare:
a) Add 11.00 ± 0.05 mL HCl to 500 mL dH20 and dilute to 1.0 L. Mix thoroughly and dissolve
3.00 ± 0.01 g pepsin in the solution.
b) Prepare this solution just before use. Adjust these quantities as required, depending on
the number of samples in the batch (15 mL required per tube).
9. Sodium Carbonate Solution (1 M):
a) Dissolve 106.0 ± 0.2 g sodium carbonate in distilled water and dilute to 1.0 L. Mix well.
10. Buffered Cellulase Solution:
a) Dissolve 20.4 g sodium acetate and 8.7 mL acetic acid in dH20 and dilute to 1.0 L.
b) Dissolve 12.5 g cellulase in the solution and mix thoroughly.
c) Prepare this solution just before use. Adjust these quantities as required, depending on
the number of samples in the batch (10 mL required per tube).
11. α-amylase, 2% (v/v): -
α-amylase stock reagent should be standardised so that 2 additions of 2 mL of α-amylase
solution will remove 0.5 g of starch as per Appendix 1.8A(b)(1).
a) Dilute an appropriate aliquot of heat stable α-amylase (NOVO Termamyl; Sigma Cat No
A5426 or similar, 18,000 units/mL or greater) in dH20.
b) Dilute 2.0mL α-amylase per 100mL dH20, to give a final activity of 360U/mL or greater.
c) Allow 4.0 ± 0.1 mL of the dilute enzyme preparation per tube.
d) Prepare diluted solution fresh daily.
12. In vivo fodder standards (WWAI)
Procedure:
Note: Analyses are performed only on samples previously dried at 55 to 60 o C (maximum) and
ground to pass a 1 mm screen.
Day 1
1. Weigh 0.25 g (± 0.005 g) well-mixed laboratory dry sample onto previously tared scoop, record
the weight to at least 0.01 mg (0.0001 g) accuracy (WS) along with the sample number and
number of the test tube.
2. Carefully transfer weighed sample into numbered test tube.
3. Weigh a second sub-sample for laboratory dry matter determination and ash determination.
AFIA Laboratory Methods Manual – v7 September 2011 Page 32 of 103