Operator’s manual, Buffer / mineral solution: cone (see ref. 4), Buffer: kansas state (see ref. 5) – ANKOM RF User Manual
Page 43: Inoculum preparation procedure
Operator’s Manual
Rev F 8/29/14
pg. 43
Buffer / Mineral Solution: Cone (see ref. 4)
Solution
grams / liter
NaHCO
3
8.75
NH
4
HCO
3
1.00
Na
2
HPO
4
1.43
KH
2
PO
4
1.55
MgSO
4
·7H
2
O
0.15
Na
2
S
0.52
CaCl
2
·2 H
2
O
0.017
MnCl
2
·4 H
2
O
0.015
CoCl
2
·6 H
2
O
0.002
FeCl
3
·6 H
2
O
0.012
Resazurin (optional)
0.125
Buffer: Kansas State (see ref. 5)
Mix 20 ml of Solution B with 1000 ml of Solution A and adjust pH to 6.8 by adding Solution B before each use.
Solution A
grams / liter
Solution B
grams / 100 ml
KH
2
PO
4
10.0
Na
2
CO
3
15.0
MgSO
4
·7 H
2
O
0.5
Na
2
S·9 H
2
O
1.0
NaCl
0.5
CaCl
2
·2 H
2
O
0.1
Urea (optional)
0.5
Inoculum Preparation Procedure
(1)
Preheat two 2 L thermos bottles by filling with 39°C water.
(2)
Empty heated water just prior to collection of rumen inoculum.
(3)
Using the appropriate collection procedure, remove 600 to 1,000 ml of rumen inoculum and place in
thermos. Include approximately two "fistfuls" of the fibrous mat from the rumen with your collection in
one thermos.
(4)
Empty the rumen inoculum and fibrous mat from the thermoses into a pre-warmed blender.
(5)
Purge the blender container with CO
2
gas and blend at a high speed for 30 seconds. The blending action
serves to dislodge microbes that are attached to the mat and assure a representative microbial population
for the fermentation.
(6)
Filter the blended digesta through 4 layers of cheesecloth into a pre-heated (39°C) flask. NOTE: Allow
for extra cheesecloth around the edges to facilitate squeezing contents of filtered mat.
(7)
The flask should be continually purged with CO
2
before and after the transfer of the inoculum.
(8)
Be careful to maintain temperature to minimize cold shock of the microorganisms.
(9)
Add the 20 ml of inoculum to the equilibrated buffer solution and sample in each Module’s Glass Bottle.
(10) Purge the Glass Bottle with CO
2
gas for 30 seconds. A purge system can be purchased from ANKOM
Technology which allows the purging to be accomplished in a closed Module. If you don’t have an
ANKOM purge system then remove the Glass Bottle and allow CO
2
to flow into it.
(11) Repeat process for all Glass Bottles to be used.
Do not allow CO
2
gas to bubble through the buffered inoculum. Instead, use the
CO
2
to form a gaseous blanket over the contents of the Glass Bottle.