78c .
CHOPPED MEAT MEDIUM FOR FRETIBACTERIUM SP.
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Equipment needed:
Autoclave
Filter
Gassing station
Hungate tubes
Final pH:
7.0
Select a strain (optional):
ml
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Ground beef
(fat free) |
500.0 | g | ||
|
|
NaOH
(1 N) |
25.0 | ml | ||
|
|
Casein peptone
(BD BACTO) |
30.0 | g | ||
|
|
Yeast extract | 5.0 | g | ||
|
|
K2HPO4 | 5.0 | g | ||
|
|
Sodium resazurin
(0.1% w/v) |
0.5 | ml | ||
|
|
L-Cysteine HCl x H2O | 0.5 | g | ||
|
|
Na-fumarate | 6.0 | g | ||
|
|
Na-formate | 6.0 | g | ||
|
|
Clarified rumen fluid , clarified | 50.0 | ml | ||
|
|
Horse serum
(inactivated, INVITROGEN) |
50.0 | ml | ||
|
|
Fatty acid mixture | 20.0 | ml | ||
|
|
Wolin's vitamin solution (10x) | 1.0 | ml | ||
|
|
Haemin solution
(0.05% w/v) |
5.0 | ml | ||
|
|
Vitamin K1 solution
(0.05% w/v) |
2.0 | ml | ||
|
|
DL-Dithiothreitol
(DTT) |
0.3 | g | ||
|
|
Distilled water | 700.0 | ml | ||
| 1 Use lean beef or horse meat. Remove fat and connective tissue before grinding. Mix meat, water and NaOH, then boil for 15 min with stirring. Cool to room temperature, skim fat off surface, and filter, retaining both meat particles and filtrate. To the filtrate add Casitone, yeast extract, hydrogen phosphate and resazurin, then add water to a final volume of 700 ml. Sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic, then add cysteine and adjust pH to 7.0. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes containing meat particles (use ca. 7 ml liquid medium for 1 - 2 g meat particles), then autoclave at 121°C for 20 min. After autoclaving add fumarate, formate, rumen fluid, horse serum, fatty acids, vitamins, haemin, vitamin K1 and DTT rom sterile anoxic stock solutions prepared under 100% N2 gas atmosphere and sterilized by filtration. | |||||
Clarified rumen fluid #
1000
| 1 Rumen fluid from cow or sheep (obtained from fistulated animals or abattoir refuse) is filtered through muslin, autoclaved at 121°C for 15 min and then centrifuged at 27,000 g for 20 min. The supernatant is made anoxic by sparging with 100% N2 gas for 15 min, dispensed under same gas atmosphere into anoxic serum vials to 30% of volume and then stored frozen at -20°C. |
Fatty acid mixture #
1000
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Isobutyric acid | 23 | ml | ||
|
|
DL-2-Methylbutyric acid | 27 | ml | ||
|
|
Valeric acid | 27 | ml | ||
|
|
Isovaleric acid | 27 | ml | ||
|
|
Distilled water | 896 | ml | ||
| 1 Adjust pH to 7.5 with concentrated NaOH. | |||||
Haemin solution #
100
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Haemin | 50 | mg | ||
|
|
NaOH
(1 N) |
1 | ml | ||
|
|
Distilled water | 100 | ml | ||
| 1 Dissolve 50 mg haemin in 1 ml 1 N NaOH; make up to 100 ml with distilled water and filter sterilize. Store refrigerated. | |||||
Vitamin K1 solution #
20
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Vitamin K1 | 0.1 | ml | ||
|
|
Ethanol
(95 %) |
20.0 | ml | ||
| 1 Dissolve 0.1 ml of vitamin K1 in 20 ml 95% ethanol and filter sterilize. Store refrigerated in a brown bottle. | |||||
Wolin's vitamin solution (10x) #
1000
| Compound | Amount | Unit | |
|---|---|---|---|
|
|
Biotin | 20 | mg |
|
|
Folic acid | 20 | mg |
|
|
Pyridoxine hydrochloride | 100 | mg |
|
|
Thiamine HCl | 50 | mg |
|
|
Riboflavin | 50 | mg |
|
|
Nicotinic acid | 50 | mg |
|
|
Calcium D-(+)-pantothenate | 50 | mg |
|
|
Vitamin B12 | 1 | mg |
|
|
p-Aminobenzoic acid | 50 | mg |
|
|
(DL)-alpha-Lipoic acid | 50 | mg |
|
|
Distilled water | 1000 | ml |