ANKOM RF - Manual (Page 42)

Operator’s Manual
pg. 42
Rev F 8/29/14
Appendix B – Buffer, Inoculum, & Sample prep for Rumen studies
Buffer: Goering - Van Soest (see ref. 3)
The information contained herein comes from the following paper:
Goering, H.K. and Van Soest, P.J., 1970, Forage fiber analysis (apparatus, reagents, procedures and
some applications), Agricultural Handbook No. 379 ARS-USDA, Washington, DC.
To prepare for the study of apparent digestibility within ruminant animals using the Goering – Van Soest buffer,
follow the procedure below.
(1) Maintain all glassware at 39°C.
(2) Prepare the following solutions using Distilled Water throughout.
Resaruzin 0.1% (w/v) solution
Dissolve 0.1 g resaruzin into 100 ml H
2
0
In vitro buffer solution In vitro macromineral solution
NH
4
HCO
3
4 g Na
2
HPO
4
anhydrous 5.7 g
NaHCO
3
35 g KH
2
PO
4
anhydrous 6.2 g
Bring volume to 1 L using Distilled Water MgSO
4
·7 H
2
O 0.6 g
Bring volume to 1 L using Distilled Water
In vitro micromineral solution Reducing solution
CaCl
2
·2 H
2
O 13.2 g Cysteine·HCl 625.0 mg
MnCl
2
·4H
2
0 10.0 g 1N NaOH 4.0 ml
CoCl
2
·6 H
2
O 1.0 g Na
2
S·9H
2
O 625.0 mg
FeCl
3
·6 H
2
O 8.0 g Bring volume to 100 ml using Distilled Water
Bring volume to 100 ml using Distilled Water
(3) Mix 2 g trypticase with 400 ml of water and 0.1 ml micromineral solution. Agitate to dissolve.
(4) Add 200 ml of buffer solution, 200 ml of macromineral solution, and 1 ml of resaruzin solution to the
solution in step 3. Mix together to create your final buffer solution.
(5) Prepare enough buffer solution to support the planned number of Gas Production Modules (80 ml buffer
per 250 ml bottle – adjust the amount according to this ratio if using different size bottles).).
(6) Add sample to each Glass Bottle (1 g per 250 ml bottle – adjust the amount according to this ratio if
using different size bottles).
(7) Add buffer to each Glass Bottle used in the run (80 ml per 250 ml bottle – adjust the amount according
to this ratio if using different size bottles).
(8) Allow the temperature of the Glass Bottle, buffer, and sample to equilibrate for 20 to 30 minutes at
39°C.
(9) Prepare the rumen inoculum while the buffer and sample are equilibrating.
(10) To remove O
2
from the buffer solution, add 2 ml of reducing solution. The buffer solution color should
change from a red to colorless.
(11) Add rumen inoculum to each bottle (20 ml per 250 ml bottle – adjust the amount according to this ratio
if using different size bottles).