1. Add the following to 1 L of water
   • 500g fat-free ground beef
   • 25 ml 1N NaOH
2. Mix meat, water and NaOH and bring to a boil with stirring
3. Cool to room temperature
4. Skim fat from surface
5. Filter the solution and retain both meat particles and filtrate
6. To filtrate, add sufficient distilled water to restore volume to 1 L.
7. To the filtrate add:
   • 30 g trypticase peptone (BD 211921)
   • 5 g yYeast extract
   • 5 g KHPO₄
   • 4 ml resazurin solution (see below)
8. Boil and cool medium under 80% N₂, 10% H₂, 10% CO₂
9. To the solution add:
   • 0.5 g L-Cysteine-HCl
   • 10 ml Hemin solution (see below)
   • 0.2 ml Vitamin K1 Solution (see below)
10. Adjust medium for final pH 7.0
11. 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

1. Add 0.15 ml of vitamin K1 to 30 ml of 95% ethanol
2. Store solution in brown bottle under refrigeration.
3. Discard after one month.
   

Hemin Solution

1. Add the following to 99 ml of distilled water
   • 50 mg hemin
   • 1 ml of 1N NaOH
2. Autoclave to sterilize.