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

Note: Samples normally foam vigorously for 1 to 2 minutes. Do not reduce temperature of heating
unit during this time.
9. After 5 minutes, remove beaker from the reflux unit and add 2ml of the standardised amylase
solution.
10. Swirl beaker to thoroughly mix the amylase in the neutral detergent solution and resuspend
any particles that have crept up the sides of the beaker. Detach any sample attached to the
sides or bottom of the beaker using a rubber policeman. Rinse off policeman with aNDF
solution.
11. Return beaker to the reflux unit and allow to come to a boil. Reflux for 60 minutes.
12. Five to 10 minutes after adding amylase, rinse down the sides of the beaker with neutral
detergent solution. Continue with refluxing until 60 minutes have elapsed.
13. Remove sample from heating unit and allow to settle for 30 to 60 seconds before filtering.
14. Preheat the fritted glass crucible for filtering by adding 40ml of boiling water. Remove water
with vacuum.
15. Carefully decant the first 30 to 40 ml of solution from the Berzelius beaker into the crucible.
Rinse lip of beaker to prevent solution with particles from running down outside of beaker.
Keep beaker in "decant" position while emptying.
16. Remove the solution with a minimum amount of vacuum.
17. Close vacuum and rinse the remaining residue from the beaker into the crucible using a fine
stream of boiling water. Be certain that no particles remain in the beaker or on the lip to run
down the outside as the beaker is turned upright.
18. Apply minimum vacuum to filter.
19. Immediately fill crucible half full of hot water and add 2ml of standardised amylase solution.
20. Allow to react for approximately 45 to 60 seconds, while policing particles from sides of
Berzelius beaker.
21. Rinse Berzelius beaker with boiling water while inverted over the crucible until all residue is
transferred.
22. Filter and wash twice by adding 30 to 40ml boiling water to residue in fritted glass crucible and
allowing to soak for 2 minutes each time.
23. Rinse sample twice with 30ml of acetone, allowing at least 2 minutes soaking time between
rinses.
24. Rinse policeman, vacuum sample dry, and remove sample from manifold.
25. Dry crucibles at 100 o C for 8 hours or overnight and hot weigh recording weight (W3) to nearest
0.1mg.
AFIA Laboratory Methods Manual – v7 September 2011 Page 38 of 103