209 .
EUBACTERIUM LENTUM MEDIUM
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Equipment needed:
Autoclave
Filter
Gassing station
Hungate tubes
Final pH:
7.0
Select a strain (optional):
Main sol. 78 #
1025
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Ground beef
(fat free) |
500.0 | g | ||
|
|
Distilled water | 1000.0 | ml | ||
|
|
NaOH
(1 N) |
25.0 | 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 water to a final volume of 1000 ml, and then add: | |||||
|
|
Casitone | 30.0 | g | ||
|
|
Yeast extract | 5.0 | g | ||
|
|
K2HPO4 | 5.0 | g | ||
|
|
Resazurin | 1.0 | mg | ||
| 2 To make medium anoxic bring it to a boil, cool under 100% N2 gas atmosphere, add 0.5 g/l L-cysteine hydrochloride and adjust pH to 7.0. Dispense under 100% N2 gas atmosphere by filling 7 ml medium into anoxic Hungate-type tubes (for strains demanding meat particles put those first into the tube (use 1 part meat particles to 4 or 5 parts fluid)). Autoclave at 121°C for 30 min. For agar slants use 15.0 g agar per 1000.0 ml medium. | |||||
|
|
L-Cysteine HCl | 0.5 | g | ||
|
|
Agar , for solid medium | 15.0 | g | ||
| 3 In some cases (as indicated in the catalogue) the addition of Haemin and Vitamin K1 or Vitamin K3 is necessary. Add to 1000 ml of medium after autoclaving: | |||||
|
|
Haemin solution
optional |
10.0 | ml | ||
|
|
Vitamin K1 solution
, alternative
optional |
10.0 | ml | ||
|
|
Vitamin K3 solution
, alternative
optional |
10.0 | ml | ||
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 K3 solution #
100
| Compound | Amount | Unit | |||
|---|---|---|---|---|---|
|
|
Vitamin K3 | 5 | mg | ||
|
|
Ethanol
(95%) |
1 | ml | ||
|
|
Water | 100 | ml | ||
| 1 Dissolve 5 mg vitamin K3 in 1 ml 95% ethanol, dilute to 0.05 mg/ml in water and filter sterilize. Store refrigerated in a brown bottle. | |||||
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. | |||||