# 1494: . OMIZ-PAT Medium (modified) ## Main sol. 1494 100 ml Distilled water 10 ml **Solution A** 10 ml **Solution B** 10 ml **Solution C** 10 ml **Solution D** 10 ml **Solution E** 10 ml **Solution F** 10 ml **Solution G** 200 µl **Solution H** 8 µl **Solution I** 2 µl **Solution J** 40 µl **Solution K** 2 ml **Solution L** 2 ml **Trace elements I** (pre-dilute 1:1000; use) 2 ml **Trace elements II** (pre-dilute 1:1000; use) 200 µl **Trace elements III** (pre-dilute 1:1000; use) 200 ml Distilled water (add up to 200 ml) 200 µl **Supplement 1** 2 ml **Supplement 2** 2 ml **Supplement 3** 200 µl **Supplement 4** 2 ml **Supplement 5** 1. Add 10 ml rabbit serum, flush the medium with N2CO2, adjust the pH to 6.9 and filter-sterilize the completed medium into tubes. 10 ml Rabbit serum 2. This recipe describes the preparation of 200 ml medium, which can be used for about 4 weeks when kept at 4°C. ## Solution A 90 mg L-Alanine 350 mg L-Arginine 300 mg L-Asparagine 236 mg L-Aspartic acid 700 mg L-Cysteine HCl 1360 mg L-Glutamine 600 mg L-Glutamic acid 150 mg Glycine 1240 mg L-Histidine 262 mg L-Isoleucine 262 mg L-Leucine 364 mg L-Lysine HCl 300 mg L-Methionine 340 mg L-Ornithine HCl 330 mg L-Phenylalanine 230 mg L-Proline 1050 mg L-Serine 240 mg L-Threonine 204 mg L-Tryptophan 180 mg L-Tyrosine 234 mg L-Valine 100 ml Distilled water 1. Prepare Solutions A, flush with nitrogen to make them anoxic, filter-sterilize and store at -20°C ## Solution B 1936 mg KCl 500 mg MgSO4 x 7 H2O 280 mg NaH2PO4 x H2O 3200 mg NH4Cl 400 mg Citric acid, trisodium salt 100 ml Distilled water 1. Prepare Solutions B, flush with nitrogen to make them anoxic, filter-sterilize and store at -20°C ## Solution C 10 mg Calcium D-(+)-pantothenate 100 mg Choline chloride 100 mg Myo-inositol 10 mg Thiamine HCl 10 mg Thiamin pyrophosphate 2 mg Coenzym A, sodium salt 2 mg Flavin adenine dinucleotide (FAD) 2 mg ß-nicotinamide adenine dinucleotide (NAD) 20 mg 2-Mercaptoethanesulfonic acid (MES), sodium salt 50 mg N-acetylmuramic acid 400 mg N-Acetylglucosamine 1600 mg D-glucuronic acid, sodium salt 1600 mg D-galacturonic acid 1100 mg Pyruvic acid, sodium salt 1000 mg Fumaric acid, disodium salt 600 mg Formic acid, sodium salt 100 ml Distilled water 1. Prepare Solutions C, flush with nitrogen to make them anoxic, filter-sterilize and store at -20°C ## Solution D 10 mg Pyridoxal hydrochloride 10 mg Pyridoxal phosphate 1120 mg D, L-lactic acid, sodium salt 2.8 mg Hypoxanthine 2.2 mg Uracil 4.8 mg Thymidine 3.6 mg N-(2-acetamido)-2-aminoethanesulfonic acid 400 mg DL-carnitine 10 mg Putrescine 2 HCl 20 mg Phenol red, sodium salt 100 ml Distilled water 1. Prepare Solutions D, flush with nitrogen to make them anoxic, filter-sterilize and store at -20°C ## Solution E 4 g D-Glucose 1.6 g D-Fructose 1.6 g D-Maltose 1.6 g D-Mannitol 4 g D-Mannose 4 g D-Arabinose 4 g L-Fucose 4 g D-Trehalose 4 g D-Sucrose 4 g L-Rhamnose 100 ml Distilled water 1. Prepare Solutions E, flush with nitrogen to make them anoxic, filter-sterilize and store at -20°C ## Solution F 300 mg CaCl2 x H2O 100 ml Distilled water 1. Prepare Solution F, flush with nitrogen to make it anoxic, filter-sterilize and store at 4°C ## Solution G 1 mg/ml Asialofetuin 1. Prepare Solutions G, flush with nitrogen to make them anoxic, filter-sterilize and store at -20°C ## Solution H 5 mg Vitamine B12 5 mg D-(+)-biotin 5 mg Folic acid 10 mg Folinic acid, calcium salt 5 mg Nicotinamide 10 mg Nicotinic acid 1 mg Riboflavine 100 ml Distilled water 1. Prepare Solutions H, flush each with nitrogen to make them anoxic, filter-sterilize and store at -20°C ## Solution I 10 µl Isobutyric acid 10 µl 2-Methylbutyric acid 10 µl Valeric acid 10 µl Isovaleric acid 1. Prepare Solution I and store at -20°C ## Solution J 1 mg/ml DL-alpha-lipoic acid 0.1 ml 2-Mercaptoethanol 0.9 ml Ethanol 1. D,L-alpha-lipoic acid (1 mg/ml); in 2-mercaptoethanol-ethanol (1:10); store at -20°C ## Solution K 0.2 mM Hemin 10 mM NaOH 1. Hemin (0.2 mM) in NaOH (10 mM) ## Solution L 42 g Yeast extract (fresh) 140 ml Distilled water 1. Homogenize 42 g fresh baker yeast in 140 ml distilled water, incubate for 24h at 56°C and centrifuge to harvest the supernatant. Adjust pH to 7 with NaOH, make anoxic, filter-sterilize and store at -80°C ## Trace elements I 7.89 mg CuSO4 169 mg MnSO4 x H2O 2.87 g ZnSO4 x 7 H2O 100 ml HCl (10 mM) 1. Prepare Trace elements I, flush with nitrogen to make them anoxic, filter-sterilize and store at 4°C ## Trace elements II 173 mg Na2SeO3 100 ml HCl (10mM) 1. Prepare Trace elements II, flush with nitrogen to make them anoxic, filter-sterilize and store at 4°C ## Trace elements III 13.1 mg NiSO4 x 6 H2O 11.8 mg SnCl2 x 2 H2O 61 mg NaVO3 1240 mg (NH4)6Mo7O24 x 4 H2O 100 ml HCl (10 mM) 1. Prepare Trace elements III, flush with nitrogen to make them anoxic, filter-sterilize and store at 4°C ## Supplement 1 10 mM Fe SO4 x 7 H2O 10 mM HCl 1. FeSO4 x 7H2O (10 mM) in HCl (10 mM) 2. Prepare Supplements 1, flush with nitrogen to make them anoxic, filter-sterilize and store at -20°C ## Supplement 2 10 %(w/v) Ascorbic acid 2 ml Distilled water 1. Ascorbic acid (10%; w/v); in distilled water, neutralize with NaOH 2. Prepare Supplements 2, flush with nitrogen to make them anoxic, filter-sterilize and store at -20°C ## Supplement 3 0.5 M Glutathione 2 ml Distilled water 1. Glutathion (0.5 M); in distilled water, neutralize with NaOH 2. Prepare Supplements 3, flush with nitrogen to make them anoxic, filter-sterilize and store at -20°C ## Supplement 4 1 mg/ml Cholesterol 1 ml Ethanol (100 %) 1. Cholesterol (1 mg/ml); in Ethanol (100%); store at -20°C ## Supplement 5 10 %(w/v) NaHCO3 2 ml Distilled water 1. NaHCO3 (10%; w/v) in distilled water, store at 4°C