325. COPROBACTER MEDIUM

Ground beef (fat free) 500.0 g

Distilled water 1000.0 ml

NaOH 1 N 25.0 ml

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 1000.0 ml, and then add:

Casitone 30.0 g

Yeast extract 5.0 g

K₂HPO₄ 5.0 g

Resazurin 1.0 mg

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. Autoclave at 121°C for 15 min. Add to 1000.0 ml of medium after autoclaving: Haemin solution (see below) 10.0 ml Vitamin K₁ or Vitamin K₃ solution (see below) 0.2 ml.

Haemin solution: Dissolve 50 mg haemin in 1.0 ml 1 N NaOH; make up to 100.0 ml with distilled water and filter sterilize. Store refrigerated.

Vitamin K₁ solution: Dissolve 0.1 ml of vitamin K₁/K₃ in 20.0 ml 95% ethanol and filter sterilize. Store refrigerated in a brown bottle.

Vitamin K₃ solution: Dissolve 5 mg/ml of vitamin K₃ in 10.0 ml 95% ethanol and filter sterilize. Store refrigerated in a brown bottle.

 

Updated 1.02.2024