- Add the following to 1 L of water
- 500g fat-free ground beef
- 25 ml 1N NaOH
- Mix meat, water and NaOH and bring to a boil with stirring
- Cool to room temperature
- Skim fat from surface
- Filter the solution and retain both meat particles and filtrate
- To filtrate, add sufficient distilled water to restore volume to 1 L.
- To the filtrate add:
- 30 g trypticase peptone (BD 211921)
- 5 g yYeast extract
- 5 g KHPO4
- 4 ml resazurin solution (see below)
- Boil and cool medium under 80% N2, 10% H2, 10% CO2
- To the solution add:
- 0.5 g L-Cysteine-HCl
- 10 ml Hemin solution (see below)
- 0.2 ml Vitamin K1 Solution (see below)
- Adjust medium for final pH 7.0
- Anaerobically dispense 7 ml into tubes containing meat particles (1 part meat to 5 parts fluid) under the gas phase noted above.
Vitamin K1 Solution
- Add 0.15 ml of vitamin K1 to 30 ml of 95% ethanol
- Store solution in brown bottle under refrigeration.
- Discard after one month.
- Add the following to 99 ml of distilled water
- 50 mg hemin
- 1 ml of 1N NaOH
- Autoclave to sterilize.