# 645: MIDDLEBROOK MEDIUM ## Main sol. 645 19 g Bacto Middlebrook 7H10 agar (Difco 262710) 5 ml Glycerol 900 ml Distilled water 1. Sterilize for 15 min by 121°C cool to 50 - 55°C. Add 100 ml Middlebrook OADC enrichment (Becton Dickinson 212240) under aseptic conditions and mix thoroughly. 2. If you can´t order the commercial media then prepare the media in this way: ## Bacto Middlebrook 7H10 agar 0.5 g (NH4)2SO4 1.5 g KH2PO4 1.5 g Na2HPO4 0.4 g Sodium citrate 0.025 g MgSO4 0.5 mg CaCl2 1 mg ZnSO4 1 mg CuSO4 0.5 g L-Glutamic acid 0.04 g Ferric ammonium citrate 1 mg Pyridoxine 0.5 mg Biotin 0.25 mg Malachite green 15 g Agar (Bacto ) 1000 ml Distilled water 1. Final pH at 25°C 6.6 ± 0.2. ## Bacto Middlebrook OADC enrichment 0.05 g Oleic acid 5 g Bovine albumin (albumin fraction V) 2 g Dextrose 4 mg Catalase (beef) 0.85 g NaCl 100 ml Distilled water