Adjust pH so that value after sterilization is 8.5 ± 0.2. Dispense into screw-cap tubes. Autoclave 10 min at 121°C.
M8. Anaerobe Agar Base Trypticase (tryptic) soy agar 40 g Agar 5 g Yeast extract 5 g L-Cysteine (dissolved in 5 ml 1 N NaOH) 0.4 g Distilled water l liter
Heat with agitation to dissolve agar. Adjust pH to 7.5 ± 0.2. Autoclave 15 min at 121°C. Cool to 50°C.
Hemin solution. Suspend 1 g hemin in 100 ml distilled water. Autoclave 15 min at 121°C. Refrigerate at 4°C.
Vitamin K1 solution. Dissolve 1 g vitamin K1 (Sigma Chemical Co., St. Louis, MO) in 100 ml 95% ethanol. Solution may require 2-3 days with intermittent shaking to dissolve. Refrigerate at 4°C.
Final medium. To 1 liter base add 0.5 ml hemin solution and 1 ml Vitamin K1 solution. Mix and pour 20 ml portions into 15 x 100 mm petri dishes. Medium must be reduced before inoculation by 24 h anaerobic incubation in anaerobic glove box or GasPak jar.
M9. Anaerobic Egg Yolk Agar Agar base Yeast extract 5 g