Media. 20 1/5 Mycorrhiza Medium. 13 Seawater Starch Agar (SWS) 22 Trypticase Yeast Extract Agar (TYS) 14 Corn and Fish Meal Agar

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1 VIII. List of Media 1 Potato Sucrose Agar (PSA) Potato 200 g Sucrose 20 g Agar 20 g ph 5.6 Wash potatoes with tap water, peel and cut into 1 cm cubes. Rinse with tap water quickly and boil 200 g of potato cubes with 1 L of distilled water for 20 min. Mash and squeeze through a muslin bag. Add sucrose and stir till dissolved. Adjust ph. Add agar. Make up to 1 L. Autoclave to sterilize. 2 Potato Carrot Agar (PCA) Potato 20 g Carrot 20 g Agar 20 g ph 6.0 Peel and slice potatoes and carrots, boil for 20 min with 1 L of distilled water. Mash and strain through a muslin bag. Adjust ph. Add agar. Make up to 1 L. Autoclave to sterilize. 5 Malt Extract Agar Malt extract 20 g Glucose 20 g Peptone 1 g Agar 20 g ph Sabouraud's Agar Peptone 10 g Glucose 40 g Agar 20 g ph Matsutake Medium Ebios (tablets)* 5 g Glucose 20 g Tap water 1 L Agar 20 g * Manufactured by Asahi Food & Health Care Co. Ltd., Tokyo, Japan. Distributed by Mitsubishi Tanabe Pharma Co., Osaka, Japan. 3 4 Potato 200 g Glucose 120 g NaCl 50 g Agar 20 g ph 5.6 Wash potatoes with tap water, peel and cut into 1 cm cubes. Rinse with tap water quickly and boil 200 g of potato cubes with 1 L of distilled water for 20 min. Mash and squeeze through a muslin bag. Add agar and boil till melted. Add glucose and NaCl, and stir till dissolved. Make up to 1 L. Adjust ph. Autoclave to sterilize. Potato 200 g Yeast extract 2 g Glucose 20 g Agar 20 g ph 5.6 Wash potatoes with tap water, peel and cut into 1 cm cubes. Rinse with tap water quickly and boil 200 g of potato cubes with 1 L of distilled water for 20 min. Mash and squeeze through a muslin bag. Add agar and boil till melted. Add yeast extract and glucose, and stir till dissolved. Make up to 1 L. Adjust ph. Autoclave to sterilize. 8 Oatmeal Agar 9 10 Oatmeal 50 g Agar 20 g ph unadjusted Boil powdered oatmeal with distilled water on a water bath at 80, stirring occasionally, for 1 h. Squeeze through a muslin bag, then make up to 1 L. Add agar and boil till melted. Autoclave to sterilize. NaNO3 3 g K2HPO4 1 g MgSO4 7H2O 0.5 g KCl 0.5 g FeSO4 7H2O 0.01 g Sucrose 200 g Agar 20 g ph 6.0 NaNO3 3 g K2HPO4 1 g MgSO4 7H2O 0.5 g KCl 0.5 g FeSO4 7H2O 0.01 g Sucrose 400 g Agar 20 g ph

2 13 Seawater Starch Agar (SWS) Soytone 1 g Soluble starch 10 g Seawater (2% Salinity)* 1 L ph 8.2 * Dilute artificial seawater or natural seawater with distilled water. 14 Corn and Fish Meal Agar Corn and fish meal mixture (feed for birds) 20 g Agar 20 g ph 6.5 Boil 20 g of the mixture in ca. 1 L water for 1 h. After filtering the solution, adjust it to 1 L with water and add 20 g of agar. 15 Vegetable Juice Seawater Agar (V-8 SWA) Vegetable juice* 200 ml CaCO3* 3 g Seawater (2% salinity) 800 ml *Add CaCO3 (3 g) to V-8 juice (ca. 300 ml) and stir it for 2 h. After centrifuging, add seawater and agar to 200 ml of the supernatant. Adjust ph to Cornmeal Seawater Agar (CMSWA) Cornmeal extract 2 g Seawater (2% Salinity) 1 L Cornmeal Agar Cornmeal extract 2 g Agar 20 g ph Petri-Cellulose Medium Cellulose* 10 g Ca(NO3)2 4H2O 0.4 g K2HPO g MgSO4 7H2O 0.15 g KCl 0.06 g * Whatman No. 1 filter paper pieces, 5 mm square. 19 Rust Fungi Medium Murashige & Skoog's plant salt mixture* 1.15 g Peptic Pepton (USB) 2 g Lab-Lemco Broth (OXOID) 2 g Sucrose 40 g Distilled water make up to 1 L Bacto Agar (Difco) 20 g ph /5 Mycorrhiza Medium Yeast extract 0.4 g Glucose 4 g ph Trypticase Yeast Extract Agar (TYS) Trypticase Peptone (BBL) or Polypepton* 0.1 g Yeast extract 1 g Agar 12 g Seawater (2% salinity) 1 L * Wako Pure Chemical Ind., Ltd., Osaka, Japan. 24 Cornmeal Yeast Extract Seawater Agar (CMYSWA) Cornmeal extract 2 g Yeast extract 1 g Seawater (2% salinity) 1 L PYG Agar Proteose peptone 1 g Yeast extract 1 g Glucose 1 g 26 Mycorrhiza Medium 103 Yeast extract 2 g Glucose 20 g ph 5.0 Glucose 10 g Peptone 5 g Yeast extract 3 g Malt extract 3 g Olive oil 10 g ph Glucose 10 g Peptone 5 g Yeast extract 3 g Malt extract 3 g CaCO3 10 g ph 5.6 * Wako Pure Chemicals Ind. Ltd., Osaka, Japan. 940

3 107 Glucose 10 g Peptone 5 g Yeast extract 3 g Malt extract 3 g NaCl 100 g ph YM Agar Glucose 10 g Peptone 5 g Yeast extract 3 g Malt extract 3 g 109 YPAD Medium 111 Yeast extract 10 g Peptone 20 g Glucose 20 g Adenine sulfate 40 mg Bacto-Yeast Nitrogen Base w/o Amino acids (Difco) 6.7 g Glucose 20 g L-Tryptophan 20 mg L-Histidine 20 mg 112 YPD Medium 113 Yeast extract 10 g Peptone 20 g Glucose 20 g Bacto-Yeast Nitrogen Base w/o Amino Acids and Ammonium Sulfate (Difco) 1.7 g Methanol 10 ml NH4Cl 2 g L-Histidine 10 mg DL-Methionine 20 mg DL-Tryptophan 20 mg 114 Medium for the 2μ Mapping Strains Bacto-Yeast Nitrogen Base w/o Amino acids (Difco) 6.7 g Glucose 20 g L-Leucine 60 mg L-Histidine 20 mg L-Tryptophan 20 mg L-Methionine 20 mg % Glucose-Yeast Extract-Peptone Glucose 250 g Yeast extract 10 g Peptone 20 g 117 MA2 Malt extract 20 g 118 MA4 Malt extract 40 g 119 YEP Alcohol Yeast extract 3 g Peptone 10 g Ethyl alcohol 10 ml Potato* 200 g Press yeast 30 g Liver infusion from* 25 g Meat extract 5 g Thioglycolate Medium Dehydrated** 10 g Glucose 5 g Glycerol 15 g Distilled water make up to 1 L * Gently boil sliced potatoes in 500 ml of water (or sliced liver in 150 ml of water) for 30 min, and remove solids by filtration through cloth. ** Wako Pure Chemical Ind., Ltd., Osaka, Japan. Potato* 200 g Press yeast 30 g Liver infusion from* 25 g Meat extract 5 g Thioglycolate Medium Dehydrated** 10 g Glucose 5 g Glycerol 15 g CaCO3 15 g Distilled water, make up to 1 L * Gently boil sliced potatoes in 500 ml of water (or sliced liver in 150 ml of water) for 30 min and remove solids by filtration through cloth. ** Wako Pure Chemical Ind., Ltd., Osaka, Japan. 941

4 Mannitol 15 g K2HPO4 0.2 g MgSO4 7H2O 0.2 g NaCl 0.2 g CaSO4 2H2O 0.1 g CaCO3 5 g ph 7.2 SI Medium Dehydrated* 50 g Ethyl alcohol** 100 ml Distilled water 900 ml * Nippon Jozo Kyokai, 6-30, Takinogawa 2 chome, Kita-ku, Tokyo 114. ** Sterilize by filtration and add to SI medium sterilized by autoclaving. Glucose 30 g Peptone 5 g CaCO3 30 g ph 6.8 Yeast extract 10 g Ethyl alcohol (99.5%)* 15 ml Sodium acetate 7.5 g (NH4)2SO4 2.6 g MgSO4 7H2O 0.25 g CaCl2 2H2O 0.01 g Sodium thioglycolate 0.5 g FeSO4 (0.02 M) 2.5 ml Na2MoO4 (0.01 M) 1 ml MnSO4 (0.01 M) 1 ml Potassium phosphate buffer (0.02 M, ) 980 ml * Sterilize separately by filtration. 214 Skim milk 100 g 216 Heart Infusion Agar Bacto Heart Infusion Agar (Difco) 40 g 219 Bacto Casitone (Difco) 20 g MgSO4 7H2O 1 g Potassium phosphate buffer (0.01 M, ph 7.2) 1 L Yeast extract 2 g Bacto Tryptone (Difco) 1 g Sodium acetate 1 g Soil extract* 50 ml Distilled water 950 ml ph 7.4 * Autoclave 400 g of farm soil suspended in 1 L of distilled water for 30 min, and remove solids by filtration. Uric acid 2 g Yeast extract 1 g K2HPO4 1 g MgSO4 7H2O 35 mg FeSO4 7H2O 35 mg CaCl2 2H2O 4.2 mg Mercaptoacetic acid (80% solution) 2 ml Sodium lactate 3.5 g Yeast extract 1 g NH4Cl 0.5 g K2HPO4 1 g MgSO4 7H2O 2 g CaSO4 2H2O 1 g Fe2(SO4)3 (NH4)2SO4 24H2O 0.5 g ph Peptone 5 g Beef extract 3 g Yeast extract 0.2 g Glucose 5 g MgSO4 7H2O 1.5 g Na2SO4 1.5 g Fe2(SO4)3 (NH4)2SO4 24H2O 0.1 g (NH4)2SO4 2 g KNO3 3 g KH2PO4 3 g MgCl2 6H2O 0.5 g CaCl2 2H2O 0.25 g FeSO4 7H2O 0.01 g Na2S2O3 5H2O 5 g 0.3 mg Yeast extract 0.1 g Sulfur (powder)* 1 g Bromophenol blue 10 mg ph * Sterilize separately by autoclaving for 60 min at 105 on each of 3 successive days and add after inoculation. 942

5 Table wine 500 ml Yeast extract 20 g Calcium lactate 10 g CaCO3 20 g Distilled water 500 ml ph Glucose 20 g ph Yeast Extract-Malt Extract Agar (ISP Medium No.2) Yeast extract 4 g Malt extract 10 g Glucose 4 g Agar 20 g ph Bennett's Agar 229 Yeast extract 1 g Beef extract 1 g NZ Amine, Type A* 2 g Glucose 10 g Agar 20 g ph 7.3 * Sheffield Chemical Co., San Ramon, CA 94583, USA or Wako Pure Chemical Ind., Ltd., Osaka, Japan. Glycerol 50 g CaCO3 1 g Agar (if needed) 20 g ph Trypticase Soy Agar Trypticase Soy (BBL) 30 g Agar 20 g ph Maltose-Bennett's Agar Yeast extract 1 g Beef extract 1 g NZ Amine, Type A* 2 g Maltose 10 g Agar 20 g ph 7.3 * Sheffield Chemical Co., San Ramon, CA 94583, USA or Wako Pure Chemical Ind., Ltd., Osaka, Japan. 232 Halophilic Actinomycetes Bacto Yeast Extract (Difco) 4 g Bacto Malt Extract (Difco) 10 g Glucose 4 g NaCl 200 g Agar (if needed) 20 g ph 7.3 Methanol* 10 ml (NH4)2HPO4 3 g MgSO4 7H2O 0.2 g FeSO4 7H2O 10 mg MnSO4 nh2o 5 mg Vitamin mixture** 5 ml Tap water 1 L Agar (if needed)*** 20 g * Sterilize by filtration and add to other autoclaved ingredients after cooling. ** Vitamin mixture Biotin 0.2 mg Ca-pantothenate 40 mg Folic acid 0.2 mg myo-inositol 200 mg Niacin 40 mg p-aminobenzoic acid 20 mg Pyridoxine-HCl 40 mg Riboflavin 40 mg Thiamin-HCl 20 mg Choline chloride 100 mg Cyanocobalamin 2 μg *** Sterilize separately. (NH4)2SO4 2 g KCl 0.1 g K2HPO4 0.5 g MgSO4 7H2O 0.5 g Polypepton* 0.1 g Glucose** 1 g Adjust ph to 3.0 with 6 N H2SO4 Glucose and Polypepton are autoclaved separately as 10% solution, and add to other autoclaved ingredients after cooling. * Wako Pure Chemical Ind., Ltd., Osaka, Japan. ** Sterilize separately. (NH4)2SO4 2 g K2HPO4 0.5 g MgSO4 7H2O 0.5 g KCl 0.1 g Ca(NO3) g FeSO4 7H2O 40 g 943 Adjust ph to 3.0 with 6 N H2SO4 and sterilize by filtration.

6 NaNO2 1 g NaCl 0.3 g K2HPO4 0.5 g MgSO4 7H2O 0.5 g MnSO4 nh2o g Fe2(SO4) g ph 7.5 (NH4)2SO4 0.5 g NaCl 0.3 g K2HPO4 1 g MgSO4 7H2O 0.3 g FeSO4 7H2O 0.03 g CaCO3* 7.5 g ph 8.0 * CaCO3 is practically insoluble. Oatmeal 3 g KNO3 0.2 g K2HPO4 0.5 g MgSO4 7H2O 0.2 g Agar (if needed) 20 g Yeast extract 4 g Malt extract 10 g Glucose 4 g Seawater 750 ml Distilled water 250 ml Agar (if needed) 20 g ph Oatmeal Agar (ISP Medium No.3) 246 Oatmeal 20 g Trace salts solution* 1 ml Agar 18 g ph 7.2 * Trace salts solution FeSO4 7H2O 0.1 g ZnSO4 7H2O 0.1 g Distilled water 100 ml Bacto PPLO Broth (Difco) 21 g Active horse serum 200 ml Fresh baker's yeast extract (25% solution)* 100 ml Distilled water 700 ml Horse serum and yeast extract are sterilized by filtration and added to PPLO broth autoclaved and cooled Yeast extract 4 g Malt extract 10 g Glucose 4 g Na2CO3* 10 g Agar (if needed) 20 g ph * Na2CO3 is sterilized separately. Na2HPO4 12H2O 4.2 g KH2PO4 1.5 g Na2CO3 4 g MgCl2 6H2O 0.4 g NH4Cl 0.6 g Yeast extract 1 g Bacto Tryptone (Difco) 1 g Na2WO4 2H2O 0.4 mg Wolfe's modified mineral solution* 50 ml VFA** 1 ml Distilled water 738 ml ph 6.5 Adjust ph to 6.5 with 10 N KOH and add the following medium. 0.1% Resazurin solution 1 ml myo-inositol 1 mg Vitamin mixture*** 10 ml Cellobiose 0.2 g Cellulose**** 200 ml Heat the above mixed solution at 60 for 5 to 10 min and then bubble with N2 gas. And then add the following ingredients. Cysteine 0.5 g Na2S 9H2O 0.5 g Under anaerobic condition, the medium is autoclaved at 100 for 10 min. * Wolfe's modified mineral solution Nitrilotriacetic acid (NTA) 1.5 g MgSO4 7H2O 3 g MnSO4 H2O 0.5 g FeSO4 7H2O 0.1 g Co(NO3)2 6H2O 0.1 g CaCl2 0.1 g ZnSO4 7H2O 0.1 g CuSO4 5H2O 0.01 g Boric acid 0.01 g 0.01 g Na2SeO g Nitrilotriacetic acid is suspended in 500 ml water, and dissolved by titrating with 2 N KOH until its ph stabilize at 6.5. The rest of the ingredients are added and dissolved. Finally total volume is adjusted to 1 L. ** VFA * Boil 250 g of dried baker's yeast suspended in 1 L of distilled water for 30 min, remove yeast cells by centrifugation, and sterilize by filtration. 944

7 Acetic acid 17 ml Propionic acid 6 ml Butyric acid 4 ml iso-butyric acid 1 ml n-valeric acid 1 ml iso-valeric acid 1 ml DL-α-Methylbutyric acid 1 ml *** Vitamin mixture Biotin 0.2 mg Ca-pantothenate 40 mg Folic acid 0.2 mg myo-inositol 200 mg Niacin 40 mg p-aminobenzoic acid 20 mg Pyridoxine-HCl 40 mg Riboflavin 40 mg Thiamin-HCl 20 mg Choline chloride 100 mg Cyanocobalamin 2 μg **** 3% Cellulose suspension which is ball-milled for 3 days. Na2S2O3 5H2O 5 g KH2PO4 1.5 g Na2HPO4 4.5 g MgSO4 7H2O 0.1 g NH4Cl 0.3 g ph 6.0 Na2S2O3 5H2O 5 g KH2PO4 1.5 g Na2HPO4 4.5 g MgSO4 7H2O 0.1 g NH4Cl 0.3 g ph Brain Heart Infusion Agar Bacto Brain Heart Infusion (Difco) 37 g 254 Blood Agar Bacto Blood Agar Base (Difco) 40 g Sterilize by autoclaving and cool to and add to it 50 ml of sterile defibrillated blood Bacto Casamino Acids (Difco) 7.5 g Yeast extract 10 g Trisodium citrate 3 g KCl 2 g MgSO4 7H2O 20 g FeSO4 7H2O 0.05 g MnSO4 nh2o 0.2 mg NaCl 250 g Distilled water, make up to 1 L Agar (if needed) * 20 g ph 7.4 * Dissolve agar by heating before adding NaCl. Bacto Casamino Acids (Difco) 5 g KH2PO4 1 g KCl 1 g NH4Cl 1 g MgSO4 7H2O 0.24 g CaSO4 2H2O 0.17 g Sodium glutamate 1 g Na2CO3 5 g NaCl 250 g Distilled water, make up to 1 L Agar (if needed)* 20 g Adjust ph to 6.5 before autoclaving. Sterilize Na2CO3 in solution separately, and add to other autoclaved ingredients. Check the final ph is between * Dissolve agar by heating before adding NaCl. NaCl 156 g MgCl2 6H2O 13 g MgSO4 7H2O 20 g CaCl2 2H2O 1 g KCl 4 g NaHCO3 0.2 g KBr 0.5 g Glucose 1 g Agar (if needed) 20 g NaCl 125 g MgCl2 6H2O 160 g CaCl2 2H2O 0.13 g K2SO4 5 g Polypepton* 1 g Yeast extract 1 g Soluble starch 2 g * Wako Pure Chemical Ind., Ltd., Osaka, Japan. 945

8 Yeast extract 1 g Na2S2O3 5H2O 5 g KH2PO4 1.5 g Na2HPO4 4.5 g MgSO4 7H2O 0.1 g NH4Cl 0.3 g Sodium aspartate 1 g ph 6.0 Malt extract 5 g K2HPO g NH4H2PO g MgSO4 7H2O 0.06 g Catalase* 60 mg * Sterilize separately by filtration. Polypepton* 5 g Yeast extract 1.5 g Glucose 5 g Na2HPO4 12H2O 1.5 g NaCl 1.5 g MgCl2 6H2O 0.1 g Na2CO3** 5.03 g The final ph of the freshly prepared medium is between 10.5 and * Wako Pure Chemical Ind., Ltd., Osaka, Japan. ** Na2CO3 is sterilized separately by autoclaving. NaCl 100 g MgCl2 6H2O 5 g MgSO4 7H2O 9.5 g KCl 5 g CaCl2 2H2O 0.2 g (NH4)2SO4 0.1 g KNO3 0.1 g Bacto Casamino Acids (Difco) 5 g Yeast extract 1 g HMSS solution* 20 ml PS solution** 20 ml Vitamin mixture*** 10 ml Distilled water 960 ml ph 8.0 * HMSS solution Nitrilotriacetic acid 10 g MgSO4 7H2O 29.7 g CaCl2 2H2O 3.3 g 12.7 mg FeSO4 7H2O 99 mg Metal 44 solution**** 50 ml Neutralize the nitrilotriacetic acid with KOH. Dissolve the remaining ingredients and adjust the ph to 7.2 with KOH. ** PS solution K2HPO4 2.5 g KH2PO4 2.5 g Sterilize separately, and add after autoclaving. *** Vitamin mixture Biotin 0.2 mg Ca-pantothenate 40 mg Folic acid 0.2 mg myo-inositol 200 mg Niacin 40 mg p-aminobenzoic acid 20 mg Pyridoxine-HCl 40 mg Riboflavin 40 mg Thiamin-HCl 20 mg Choline chloride 100 mg Cyanocobalamin 2 μg **** Metal 44 solution 2Na-EDTA 2.5 g ZnSO4 7H2O g FeSO4 7H2O 5 g MnSO4 nh2o 1.54 g CuSO4 5H2O g CoCl2 6H2O g Na2B4O7 10H2O g Peptone 10 g Malt extract 5 g Bacto Casamino Acids (Difco) 5 g Beef extract 2 g Glycerol 2 g Tween g MgSO4 7H2O 1 g 265 Glycerol-Asparagine Agar (ISP Medium No.5) L-Asparagine 1 g Glycerol 10 g K2HPO4 1 g Trace salts solution* 1 ml Agar 20 g * Trace salts solution FeSO4 7H2O 0.1 g ZnSO4 7H2O 0.1 g Distilled water 100 ml

9 266 Yeast Extract-Starch Agar Yeast extract 2 g Soluble starch 10 g ph Modified Hayflick Medium Bacto Heart Infusion Broth (Difco) 25 g Distilled water 700 ml Bacto Agar (Difco, if needed) 15 g ph 7.8 Sterilized by autoclaving (121 for 20 min). Aseptically add 200 ml of horse serum (heat inactivated, 56 for 30 minutes), 100 ml of fresh yeast extract (25% solution)*, 12 ml of calf thymus DNA (Sigma, Type I) (0.2% (w/v) solution), 10 ml of thallium acetate (1% (w/v) solution), 25 ml of Penicillin G (20,000 IU/ml). * Boil 250 g of dried baker's yeast suspended in 1 L of distilled water for 30 min, remove yeast cells by centrifugation, and sterilize by filtration. 268 Inorganic Salts Starch Agar (ISP Medium No.4) Soluble starch 10 g K2HPO4 (anhydrous base) 1 g MgSO4 7H2O 1 g (NH4)2SO4 2 g CaCO3 2 g Trace salts solution* 1 ml Bacto Agar (Difco) 20 g The unadjusted ph should be * Trace salts solution FeSO4 7H2O 0.1 g ZnSO4 7H2O 0.1 g Distilled water 100 ml 270 Hickey & Tresner Agar Yeast extract 1 g Beef extract 1 g NZ Amine, Type A* 2 g Dextrin 10 g CoCl2 6H2O 0.02 g Agar 20 g ph Bacto Casitone (Difco) 3 g Yeast extract 1 g CaCl2 2H2O 1.36 g ph 7.2 Peptone 5 g Meat extract 3 g NaCl 60 g Bacto Tryptone (Difco) 0.5 g Yeast extract 0.5 g Beef extract 0.2 g Sodium acetate 0.2 g ph LB Medium 276 Peptone 10 g NaCl 5 g Bacto Casitone (Difco) 3 g CaCl2 2H2O 1.36 g Yeast extract 1 g Cellobiose 5 g ph Bacto Casitone (Difco) 5 g MgSO4 7H2O 1 g ph 7.2 * Sheffield Chemical Co., San Ramon, CA 94583, USA or Wako Pure Chemical Ind., Ltd., Osaka, Japan. 271 Yeast-Krainsky's Agar Glucose 10 g L-Asparagine 1 g K2HPO4 0.5 g Yeast extract 2 g Agar 17 g ph

10 278 Tris (hydroxymethyl) aminomethane 1.2 g Bacto Casamino Acids (Difco) 1 g Glucose 1 g MgSO4 7H2O 0.1 g KNO3 0.1 g Sodium glycerophosphate 0.1 g MnCl2 4H2O 1.8 mg FeSO4 7H2O 1.4 mg H3BO3 2.8 mg ZnCl2 2H2O 0.1 mg 0.1 mg Vitamin mixture* 5 ml Adjust ph to 7.5 with 1 N HCl * Vitamin mixture Biotin 0.2 mg Ca-pantothenate 40 mg Folic acid 0.2 mg myo-inositol 200 mg Niacin 40 mg p-aminobenzoic acid 20 mg Pyridoxine-HCl 40 mg Riboflavin 40 mg Thiamin-HCl 20 mg Choline chloride 100 mg Cyanocobalamin 2 μg 279 Tryptone Yeast Extract Agar (TYG) 280 Bacto Tryptone (Difco) 3 g Glucose 5 g Agar 20 g KH2PO4 3 g MgSO4 7H2O 0.5 g CaCl2 2H2O 0.25 g Bacto Yeast Extract (Difco) 1 g Glucose 10 g (NH4)2SO4 2 g Adjust ph to 2.0 with 10 N H2SO For solid medium, equal volumes of separately autoclaved double-strength liquid medium (ph 2.0) and 1.6% Gelrite (Kelco, Division of Merck & Co, Inc.) are mixed, after cooling to 45. Glucose 10 g K2HPO4 1 g NH4NO3 2 g MgSO4 7H2O 0.2 g MnSO4 7H2O g FeSO4 7H2O g Yeast extract 3 g Polypepton* 0.3 g Resazurin g After autoclaving, adjust ph to 10.0 with sterilized 10% Na2CO3 solution. * Wako Pure Chemical Ind., Ltd., Osaka, Japan. Sucrose 1 g Bacto Casitone (Difco) 0.75 g Yeast extract 0.25 g Polypepton* 0.25 g Vitamin mixture** 10 ml ph 7.1 * Wako Pure Chemical Ind., Ltd., Osaka, Japan. ** Vitamin mixture Biotin 0.2 mg Ca-pantothenate 40 mg Folic acid 0.2 mg myo-inositol 200 mg Niacin 40 mg p-aminobenzoic acid 20 mg Pyridoxine-HCl 40 mg Riboflavin 40 mg Thiamin-HCl 20 mg Choline chloride 100 mg Cyanocobalamin 2 μg 281 Glucose is autoclaved separately as 40% solution, and added to other autoclaved ingredients after cooling. For solid medium, equal volumes of separately autoclaved double-strength liquid medium (ph 2.0) and 1.6% Gelrite (Kelco, Division of Merck & Co, Inc.) are mixed, after cooling to 45. Yeast extract 1 g (NH4)2SO4 1.3 g CaCl2 2H2O 0.08 g MgSO4 7H2O 0.25 g KH2PO g Na2SiO3 9H2O 0.3 g 0.03 g FeSO4 7H2O 0.02 g Adjust ph to 3.0 with 10N H2SO Bacto Vitamin Assay Casamino Acids (Difco) 7.5 g Bacto Yeast Extract (Difco) 10 g MgSO4 7H2O 20 g Trisodium citrate 2H2O 3 g KCl 2 g NaCl 150 g FeSO4 7H2O (4.98% solution) 1 ml ph 7.4

11 (NH4)2SO4 3 g CaCl2 2H2O 0.08 g MgSO4 7H2O 0.25 g KH2PO g KCl 0.1 g Ca(NO3)2 4H2O 0.01 g Yeast extract* 0.2 g Sulfur (powder)** 10 g Adjust ph to 1.5 to 2.5 with 6 N H2SO4 * Yeast extract is autoclaved separately as 10% solution. ** Sulfur is sterilized separately by autoclaving for 60 min at 105 on each of 3 successive days and added after inoculation. (NH4)2SO4 1.3 g CaCl2 2H2O 0.08 g MgSO4 7H2O 0.25 g KH2PO g Na2SiO3 9H2O 0.1 g 0.03 g FeSO4 7H2O 0.02 g Yeast extract* 1 g Sulfur (powder)** 10 g Adjust ph to 2.5 with 6 N H2SO4 * Yeast extract is autoclaved separately as 10% solution. ** Sulfur is sterilized separately by autoclaving for 60 min at 105 on each of 3 successive days and added after inoculation. MgSO4 7H2O 0.1 g K2HPO4 0.5 g Sodium acetate 1 g NH4Cl 0.1 g Bacto Yeast Extract (Difco) 0.1 g Sodium thioglycolate 0.5 g Ferric citrate g ph 6.8 Good growth can be obtained in semi-solid medium (1 g of agar in 1 liter). KH2PO g NaNO g Sodium thioglycolate 0.05 g Sodium tartrate 0.37 g Sodium succinate 0.37 g Sodium acetate 0.05 g Vitamin mixture* 5 ml Ferric quinate solution** 2 ml HMSS solution*** 1 ml Adjust ph to 6.75 with 1 N NaOH 294 * Vitamin mixture Biotin 0.2 mg Ca-pantothenate 40 mg Folic acid 0.2 mg myo-inositol 200 mg Niacin 40 mg p-aminobenzoic acid 20 mg Pyridoxine-HCl 40 mg Riboflavin 40 mg Thiamin-HCl 20 mg Choline chloride 100 mg Cyanocobalamin 2 μg ** Ferric quinate solution FeCl3 6H2O 0.45 g Quinic acid 0.19 g *** HMSS solution Nitrilotriacetic acid 10 g MgSO4 7H2O 29.7 g CaCl2 2H2O 3.3 g 12.7 mg FeSO4 7H2O 99 mg Metal 44 solution**** 50 ml Neutralize the nitrilotriacetic acid with KOH. Dissolve the remaining ingredients and adjust the ph to 7.2 with KOH. **** Metal 44 solution EDTA 2.5 g ZnSO4 7H2O g FeSO4 7H2O 5 g MnSO4 nh2o 1.54 g CuSO4 5H2O g CoCl2 6H2O g Na2B4O7 10H2O g Good growth can be obtained in semi-solid medium (1 g of agar in 1 liter). Bacto Yeast Extract (Difco) 5 g Adjust ph to 5.0 with 1 N HCl 296 V-8 Juice Tap Water Agar V-8 Juice* 200 ml CaCO3* 3 g Tap water 800 ml * Add CaCO3 (3 g) to V-8 juice (ca 300 ml) and stir it for 2 h. After centrifuging, adjust the supernatant to 200 ml with tap water. 949

12 297 Beef extract 1 g Yeast extract 2 g Peptone 5 g NaCl 5 g 298 OA-Y Agar Oatmeal 20 g Trace salts solution* 1 ml Bacto Yeast Extract (Difco) 1 g Agar 18 g ph 7.2 * Trace salts solution FeSO4 7H2O 0.1 g ZnSO4 7H2O 0.1 g Distilled water 100 ml 299 Gauze's Synthetic Medium No Soluble starch 20 g KNO3 1 g NaCl 0.5 g MgSO4 7H2O 0.5 g K2HPO4 0.5 g FeSO4 7H2O 10 mg Tap water 1 L ph Bacto Yeast Extract (Difco)* 1 g (NH4)2SO4 1.3 g KH2PO g MgSO4 7H2O 0.25 g CaCl2 2H2O 0.07 g FeCl3 6H2O 0.02 g MnCl2 4H2O g Na2B4O7 4.5 mg ZnSO4 7H2O 0.22 mg Na2MoO3 2H2O 0.05 mg Adjust ph to 3.5 with 10 N H2SO4 * Sterilize by filtration as 5% solution, and add to other ingredients sterilized by autoclaving. For solid medium, equal volumes of separately autoclaved double-strength liquid medium (ph 2.0) and 1.6% Gelrite (Kelco, Division of Merck & Co, Inc.) are mixed, after cooling to Oatmeal Agar (B) Oatmeal (powder, Quaker White Oats) 3 g KNO3 0.2 g K2HPO4 0.5 g MgSO4 7H2O 0.2 g Sulfur (powder)* 0.5 g (NH4)2SO4 1.3 g KH2PO g MgSO4 7H2O 0.25 g CaCl2 2H2O 0.07 g FeCl3 6H2O 0.02 g MnCl2 4H2O g Na2B4O7 4.5 mg ZnSO4 7H2O 0.22 mg Na2MoO3 2H2O 0.05 mg Adjust ph to 3.0 with 10 N H2SO4 * Sulfur is sterilized separately by autoclaving for 60 min at 105 on each of 3 successive days and added after inoculation. For solid medium, equal volumes of separately autoclaved double-strength liquid medium (ph 2.0) and 1.6% Gelrite (Kelco, Division of Merck & Co, Inc.) are mixed, after cooling to 45. NaCl 80 g Bacto Casamino Acids (Difco) 7.5 g Polypepton* 5 g Yeast extract 1 g Sodium citrate 3 g MgSO4 7H2O 20 g K2HPO4 0.5 g Fe(NH4)2(SO4)2 6H2O 0.05 g * Wako Pure Chemical Ind., Ltd., Osaka, Japan. 308 Glucose Nutrient Agar Beef extract 1 g Yeast extract 2 g Peptone 5 g NaCl 5 g Glucose 10 g 309 Rhamnose Salt Medium K2HPO4 2.9 g KH2PO4 2.1 g NH4Cl 2 g MgSO4 7H2O 0.4 g NaCl 30 mg CaCl2 2H2O 3 mg FeSO4 7H2O 1 mg Yeast extract 3 g Rhamnose 10 g 950

13 310 MRS Medium Peptone 10 g Meat extract 10 g Glucose 20 g Tween 80 1 g K2HPO4 2 g Sodium acetate 5 g Diammonium hydrogencitrate 2 g MgSO4 7H2O 0.2 g MnSO4 nh2o 0.05 g ph Todd Hewitt Medium Bacto Todd Hewitt Broth (Difco) 30 g ph GAM Medium Nissui GAM Broth* 59 g ph 7.3 * Nissui Pharmaceutical Co. Ltd., Tokyo, Japan. NaCl 18 g MgCl2 6H2O 7 g Bacto Yeast Extract (Difco) 1 g Fructose (20% solution)* 50 ml Cysteine-HCl (2% solution)* 25 ml Na2S (2% solution)* 25 ml NaHCO3 5 g KCl g NH4Cl 0.25 g CaCl2 2H2O 0.14 g K2HPO g Fe(NH4)2(SO4)2 6H2O g Resazurin g Distilled water 900 ml The ph is not adjusted and usually a value of 7.4. * Sterilize separately and add to other autoclaved ingredients. Bacto Brain Heart Infusion (Difco) 37 g Horse serum* 50 ml Distilled water 950 ml * Sterilize separately by filtration. Bacto Todd Hewitt Broth (Difco) 30 g Horse serum* 5 ml ph 7.3 * Sterilize separately by filtration Polypepton* 10 g Yeast extract 2 g MgSO4 7H2O 0.5 g Na2CO3** 10 g * Wako Pure Chemical Ind., Ltd., Osaka, Japan. ** Sterilize separately by autoclaving and add to other ingredients. The final ph is between 9.0 and Peptone 10 g Yeast extract 10 g L-Cysteine 1 g ph NZ Amine with Soluble Starch and Glucose Agar Medium Glucose 10 g Soluble starch 20 g NZ Amine, Type A* 5 g CaCO3 (reagent grade) 1 g ph 7.3 * Sheffield Chemical Co., San Ramon, CA 94583, USA or Wako Pure Chemical Ind., Ltd., Osaka, Japan. Solution A* 500 ml Solution B** 500 ml * Solution A ** Solution B Yeast extract 1 g (NH4)2SO4 0.2 g MgSO4 7H2O 0.5 g CaCl2 2H2O 0.25 g KH2PO4 0.6 g Distilled water 500 ml ph Glucose 1 g Agar (if needed) 20 g Distilled water 500 ml Solutions A and B are separately sterilized by autoclaving and mixed. (NH4)2SO4 2 g KCl 0.1 g K2HPO4 0.5 g MgSO4 7H2O 0.5 g Glucose* 1 g Yeast extract * 0.1 g Adjust ph to 3.5 with 1 N H2SO4 * Autoclave separately as 20% solution, and added to other autoclaved ingredients. 951

14 For solid medium, equal volumes of separately autoclaved double-strength liquid medium (ph 3.5) and 1.6% Gelrite (Kelco, Division of Merck & Co, Inc.) were mixed, after cooling to 50. Polypepton* 10 g Yeast extract 2 g MgSO4 7H2O 0.5 g Seawater** 750 ml Distilled water 250 ml ph * Wako Pure Chemical Ind., Ltd., Osaka, Japan. ** Filtered, aged seawater or artificial seawater (Daigo's Artificial Seawater SP*). Peptone 10 g Yeast extract 2 g MgSO4 7H2O 1 g NaCl 30 g Glycerol 30 g Keratin 10 g Soil extract * 100 ml CaCO3 1 g Distilled water 900 ml ph 7.2 * 500 g of soil in 500 ml of water was autoclaved at 121 for 30 min and filtered. Yeast extract 2 g Soluble starch 10 g NaCl 50 g ph SP5 Medium Bacto Casitone (Difco) 9 g Yeast extract 1 g Seawater* 1 L ph 7.2 * Filtered, aged seawater or artificial seawater (Daigo's Artificial Seawater SP**). ** Wako Pure Chemical Ind., Ltd., Osaka, Japan. 332 Enriched Cytophaga Agar Bacto Tryptone (Difco) 2 g Beef extract 0.5 g Yeast extract 0.5 g Sodium acetate 0.2 g ph Marine Cytophaga Agar Bacto Tryptone (Difco) 2 g Beef extract 0.5 g Yeast extract 0.5 g Sodium acetate 0.2 g Seawater* 1 L ph * Filtered, aged seawater or artificial seawater (Daigo's Artificial Seawater SP**). ** Wako Pure Chemical Ind., Ltd., Osaka, Japan. 334 Flavobacterium columnare Agar Bacto Tryptone (Difco) 2 g Beef extract 0.5 g Yeast extract 0.5 g Sodium acetate 0.2 g Agar (if needed) 10 g ph Medium for Marine Flexibacteria KNO3 0.5 g Sodium glycerophosphate 0.1 g Trace element solution* 1 ml Tris (hydroxymethyl) aminomethane 1 g Bacto Tryptone (Difco) 5 g Seawater** 1 L * Trace element solution H3BO g MnCl2 4H2O 1.8 g FeSO4 7H2O 1.36 g CuCl2 2H2O 26.9 mg ZnCl mg CoCl2 6H2O 40.4 mg 25.2 mg Sodium tartrate 1.77 g ** Filtered, aged seawater or artificial seawater (Daigo's Artificial Seawater SP***). *** Wako Pure Chemical Ind., Ltd., Osaka, Japan. 336 Cytophaga (Marine) Medium Bacto Tryptone (Difco) 1 g Yeast extract 1 g Seawater* 1 L ph * Filtered, aged seawater or artificial seawater (Daigo's Artificial Seawater SP**)

15 ** Wako Pure Chemical Ind., Ltd., Osaka, Japan. 338 Caulobacter Medium Peptone 2 g Yeast extract 1 g MgSO4 7H2O 0.2 g Agar (if needed) 10 g 339 Marine Broth 2216 Bacto Marine Broth 2216 (Difco) 37.4 g ph unadjusted 340 Marine Agar 2216 Bacto Marine Agar 2216 (Difco) 55.1 g ph unadjusted 341 Medium for Freshwater Flexibacteria MgSO4 7H2O 0.1 g KNO3 0.1 g Sodium glycerophosphate 0.1 g Trace element solution* 1 ml Tris (hydroxymethyl) aminomethane 1 g Thiamine 1 mg Bacto Casamino Acids (Difco) 1 g Glucose 1 g Cobalamine 1 μg ph 7.5 * Trace element solution H3BO g MnCl2 4H2O 1.8 g FeSO4 7H2O 1.36 g CuCl2 2H2O 26.9 mg ZnCl mg CoCl2 6H2O 40.4 mg 25.2 mg Sodium tartrate 1.77 g 342 Nissui Plate Sheep Blood Agar G* * Nissui Pharmaceutical Co. Ltd., Tokyo, Japan. 343 Sucrose-Bennett's Agar Yeast extract 1 g Beef extract 1 g NZ Amine, Type A* 2 g Sucrose 10 g Agar 20 g ph 7.3 * Sheffield Chemical Co., San Ramon, CA 94583, USA or Wako Pure Chemical Ind., Ltd., Osaka, Japan. 347 Nissui Plate Sheep Blood Agar* * Nissui Pharmaceutical Co. Ltd., Tokyo, Japan. 350 Acetobacter xylinus Medium 351 Polypepton* 5 g Glucose 5 g Mannitol 5 g MgSO4 7H2O 1 g Ethanol** 5 ml ph * Wako Pure Chemical Ind., Ltd., Osaka, Japan. ** Sterilize separately by filtration. Polypepton* 10 g Yeast extract 2 g N-Acetyl glucosamine** 30 mg MgSO4 7H2O 1 g * Wako Pure Chemical Ind., Ltd., Osaka, Japan. ** Sterilize separately by filtration. 352 Methylobacterium Medium Polypepton* 10 g Yeast extract 2 g Methanol** 5 ml MgSO4 7H2O 1 g * Wako Pure Chemical Ind., Ltd., Osaka, Japan. ** Sterilize separately by filtration. 355 R-CW Medium 356 Peptone 5 g Bacto Tryptone (Difco) 5 g KH2PO4 5 g Sodium acetate trihydrate 5 g Diammonium hydrogencitrate 2 g MgSO4 7H2O 0.5 g MnSO4 nh2o 0.5 g Tween 80 1 ml Cheese whey* 1 L ph 6.0 * Add HCl to warmed milk and coagulate. After centrifuging, use the supernatant. Polypepton* 10 g Yeast extract 2 g MgSO4 7H2O 1 g N-Acetyl-D-glucosamine 50 mg * Wako Pure Chemical Ind., Ltd., Osaka, Japan. 953

16 Polypepton* 10 g Yeast extract 2 g MgSO4 7H2O 1 g D-Alanine 50 mg * Wako Pure Chemical Ind., Ltd., Osaka, Japan. Polypepton* 10 g Yeast extract 2 g MgSO4 7H2O 1 g Thymine 100 mg * Wako Pure Chemical Ind., Ltd., Osaka, Japan. Polypepton* 10 g Yeast extract 2 g MgSO4 7H2O 1 g Cytidine 50 mg * Wako Pure Chemical Ind., Ltd., Osaka, Japan. 360 SA Medium Sodium acetate 1 g Sodium succinate 1 g KH2PO4 0.5 g K2HPO4 0.6 g (NH4)2SO4 1 g MgSO4 7H2O 0.2 g NaCl 0.2 g CaCl2 2H2O 0.05 g Na2S2O3 5H2O 0.1 g Yeast extract 0.1 g Vitamin mixture* 1 ml Trace element solution** 1 ml ph 6.7 * Vitamin mixture Thiamine-HCl 50 mg Niacine 50 mg p-aminobenzoic acid 30 mg Pyridoxal-HCl 10 mg Biotin 5 mg Vitamin B12 5 mg Distilled water 100 ml ** Trace element solution 2Na-EDTA 1 g FeCl3 6H2O 2 g ZnCl2 0.1 g MnCl2 2H2O 0.1 g H3BO3 0.1 g CoCl2 6H2O 0.1 g 20 mg CuCl2 2H2O 10 mg NiCl2 6H2O 10 mg Na2SeO3 5 mg Polypepton* 5 g Glucose 5 g MgSO4 7H2O 1 g NaCl 80 g ph 7.5 * Wako Pure Chemical Ind., Ltd., Osaka, Japan. Polypepton* 5 g Glucose 5 g MgSO4 7H2O 1 g ph 8.0 * Wako Pure Chemical Ind., Ltd., Osaka, Japan. Polypepton* 5 g Glucose 5 g MgSO4 7H2O 1 g ph 5.5 * Wako Pure Chemical Ind., Ltd., Osaka, Japan. 366 Peptone 10 g Meat extract 10 g Glucose 20 g Tween 80 1 g K2HPO4 2 g Sodium acetate 5 g Diammonium hydrogencitrate 2 g MgSO4 7H2O 0.2 g MnSO4 nh2o 0.05 g ph

17 367 mjypgs Medium 368 NaCl 3 g K2HPO4 14 mg CaCl2 2H2O 14 mg NH4Cl 12.5 mg NaNO mg MgSO4 7H2O 0.34 g MgCl2 6H2O 41.8 mg KCl 33 mg NiCl2 6H2O 0.05 mg Na2SeO3 5H2O 0.05 mg Ferric citrate 5 mg Trace mineral solution* 1 ml Na2S2O3 5H2O 2 g Na2SiO3 9H2O 1 g Bacto Yeast Extract (Difco) 1 g Peptone (BBL) 1 g Glucose 0.2 g Sodium succinate 0.2 g Adjust ph with H2SO4 * Trace mineral solution Nitlirotriacetic acid 1.5 g MnSO4 2H2O 0.5 g CoSO4 7H2O 0.5 g ZnSO4 7H2O 0.18 g CuSO4 5H2O KAl(SO4)2 12H2O 0.02 g Yeast extract 10 g Casamino acids 7.5 g NaCl 250 g MgSO4 7H2O 40 g KCl 2 g Trisodium citrate 3 g FeCl3 4H2O 36 mg MnCl2 4H2O 0.36 mg Distilled water, make up to 1 L Agar* (if needed) 20 g ph 7.2 * Dissolve agar by heating before adding NaCl. 369 MYCA Medium Sodium DL-malate 1 g Bacto Yeast Extract (Difco) 3 g Bacto Casamino Acids (Difco) 2 g (NH4)2SO4 0.5 g ph PYS Medium KH2PO4 1 g (NH4)2SO4 1 g NaCl 0.2 g MgCl2 6H2O 0.2 g CaCl2 2H2O 0.05 g Trace element solution SL8* 1 ml Sodium pyruvate** 2.2 g Yeast extract 1 g ph * Trace element solution SL8 2Na-EDTA 5.2 g FeCl2 4H2O 1.5 g ZnCl2 70 mg MnCl2 4H2O 100 mg H3BO3 62 mg CoCl2 6H2O 190 mg 36 mg CuCl2 2H2O 17 mg NiCl2 6H2O 24 mg ** Separately sterilize by filtration and aseptically add to the medium. 372 MMYS-II Medium 373 Solution I* 500 ml Solution II** 500 ml ph 7.8 Separately autoclave Solutions I and II. Mix aseptically and adjust final ph to * Solution I ** Solution II (NH4)2SO4 1 g NaCl 8.2 g MgCl2 6H2O 0.2 g CaCl2 2H2O 0.05 g Trace element solution SL8*** 1 ml Sodium DL-malate 3.6 g Yeast extract 1 g Na2S2O3 5H2O 0.5 g Distilled water 500 ml 20 mm Potassium phosphate buffer 500 ml ph 7.8 *** Trace element solution SL8 2Na-EDTA 5.2 g FeCl2 4H2O 1.5 g ZnCl2 70 mg MnCl2 4H2O 100 mg H3BO3 62 mg CoCl2 6H2O 190 mg 36 mg CuCl2 2H2O 17 mg NiCl2 6H2O 24 mg Nutrient Broth (OXOID) 6.5 g ph unadjusted 374 PPES-II Agar Medium Peptone 2 g Bacto Proteose Peptone No.3 (Difco) 1 g Soytone 1 g Yeast extract 1 g Fe(III)-EDTA 0.1 g Artificial sea water * 1 L ph 7.8

18 * Artificial sea water NaCl 30 g KCl 0.7 g MgCl2 6H2O 10.8 g MgSO4 7H2O 5.4 g CaCl2 2H2O 1 g 376 Ekho Lake Strains Medium Mineral salt solution* ("Hutner/Cohen-Bazire") 20 ml Bacto Peptone (Difco) 0.25 g Bacto Yeast Extract (Difco) 0.25 g Bacto Agar (Difco) 15 g Glucose solution (2.5%, filter-sterilized) 10 ml Vitamin solution** 5 ml Artificial sea water*** 250 ml Distilled water 715 ml ph 7.5 Mix ingredients (except glucose and vitamin solutions), and sterilize 20 min at 121. After cooling to 60, aseptically add glucose and vitamin solutions to the medium. The medium is only weakly buffered; one needs approximately 10 drops/1 medium of 6 N KOH. * Mineral salt solution Nitrilotriacetic acid (NTA) 10 g MgSO4 7H2O 29.7 g CaCl2 2H2O 3.34 g mg FeSO4 7H2O 99 mg Metal salt solution 44**** 50 ml Distilled water 900 ml Dissolve NTA first by neutralizing with KOH, then add other salts. Adjust ph to 7.2 with KOH or H2SO4. Adjust volume to 1 L with distilled water. ** Vitamin solution Biotin 4 mg Folic acid 4 mg Pyridoxine-HCl 20 mg Riboflavin 10 mg Thiamine-HCl 10 mg Nicotinamide 10 mg Ca-pantothenate 10 mg Vitamin B mg p-aminobenzoic acid 10 mg *** Artificial sea water NaCl g Na2SO g MgCl2 6H2O g CaCl g NaHCO3 192 mg KCl 664 mg KBr 6 mg H3BO3 26 mg SrCl2 24 mg NaF 3 mg **** Metal salt solution 44 Na-EDTA 250 mg ZnSO4 7H2O 1095 mg FeSO4 7H2O 500 mg MnSO4 H2O 154 mg CuSO4 5H2O 39.2 mg Co(NO3)2 6H2O 24.8 mg Na2B4O7 10H2O 17.7 mg Distilled water 100 ml Dissolve the EDTA and add a few drops of concentrated H2SO4 to retard precipitation of the heavy metal ions. Store in the dark and cold (5 ). 377 Oceanithermus Medium KH2PO g K2HPO g MgCl2 6H2O 0.36 g NH4Cl 0.54 g NaHCO3 5 g Trace element solution* 10 ml Vitamin solution** 10 ml Yeast extract 2 g NaNO3 1.7 g NaCl 20 g ph unadjusted Mix ingredients, except NaHCO3 and vitamin solution. Dispense the medium into suitable culture vessels under a stream of N2 and seal with butyl rubber stoppers. After autoclaving, add filter-sterile NaHCO3 and vitamin solutions. * Trace elements solution 2Na-EDTA 0.64 g MgSO4 7H2O 6.2 g MnSO4 4H2O 0.55 g FeSO4 7H2O 0.1 g CoCl2 6H2O 0.17 g CaCl2 2H2O 0.13 g ZnSO4 7H2O 0.18 g CuSO g KAl(SO4)2 12H2O g g NiCl2 6H2O Dissolve the 2Na-EDTA in 500 ml of distilled water, and after adding the salts, make up to 1 L. ** Vitamin solution p-aminobenzoic acid 1 mg Vitamin B12 956

19 380 NA + 0.5% Yeast Extract 382 Pepton 5 g Beef extract 3 g NaCl 3 g Yeast extract 0.3 g Ethanol 0.5 ml Sodium succinate 1 g Diammonium hydrogencitrate 0.5 g Ferric citrate solution (0.1%) 5 ml KH2PO4 0.5 g MgSO4 7H2O 0.4 g NaCl 0.4 g NH4Cl 0.4 g CaCl2 2H2O 0.05 g Vitamin B12 solution (0.01%) 0.4 ml Trace element solution SL-6* 1 ml Sodium malate 1 g Na2S2O3 0.5 g Distilled water 1.05 L ph 6.8 Boil the medium under a stream of nitrogen gas for few minutes and distribute 45 ml medium into 50 ml screw-capped bottles (already flushed with nitrogen gas). Bubble each bottle with nitrogen gas and close immediately with a rubber septum and screw tight. Autoclave at 121 for 15 min. Sterile syringes are used to inoculate and remove samples. After sterilization neutralized sterile sodium sulfide solution** is added to a final concentration of 0.03%. Incubate in the light using a tungsten lamp. * Trace element solution SL-6 ZnSO4 7H2O 0.1 g MnCl2 4H2O 0.03 g H3BO3 0.3 g CoCl2 6H2O 0.2 g CuCl2 2H2O 0.01 g NiCl2 6H2O 0.02 g 0.03 g ** Neutralized sulfide solution Na2S 9H2O 1.5 g Distilled water 100 ml The sulfide solution is prepared in a vessel sealed with a butyl rubber stopper. Autoclave at 121 for 15 min. under a N2 atmosphere. After cooling to room temperature the ph is adjusted to about 7.3 by adding of sterile 2N H2SO4 drop-wise with a syringe without opening the bottle. Appearance of yellow colour indicates the ph is about 8. The vessel should be shaken continuously to avoid precipitation of elemental sulfur. The final solution should be transparent yellow % NaCl GYPFK Agar/Broth Glucose 10 g Polypepton* 5 g Extract Bonito* 5 g NaCl 25 g K2HPO4 10 g Salt solution** 5 ml ph 8.5 Sterilize by filtration. * Wako Pure Chemical Ind., Ltd., Osaka, Japan. ** Salt solution MgSO4 7H2O 40 g MnSO4 4H2O 2 g FeSO4 7H2O 2 g 384 Flexiligladius Medium Bacto Heart Infusion Broth (Difco) 2.5 g ph unadjusted 385 Bifidobacterium Medium Casein peptone, tryptic digest 10 g Meat extract 5 g Glucose 10 g K2HPO4 3 g Tween 80 1 ml Tap water 1 L ph 6.8 After sterilization, aseptically add solutions of sodium ascorbate and cysteine-hcl to final concentration of 1.0% and 0.05%, respectively. 387 PE Medium Basal salts solution* 5 ml Phosphate solution** 5 ml Vitamin solution*** 1 ml (NH4)2SO4 0.5 g Sodium glutamate 0.5 g Sodium succinate 0.5 g Sodium acetate 0.5 g Yeast extract 0.5 g Casamino acids 0.5 g Na2S2O3 5H2O 0.5 g ph 7.5 Add the vitamin solution aseptically to the cooled autoclaved medium. 957

20 * Basal salts solution 3Na-EDTA 4.53 g FeSO4 7H2O 1.11 g MgSO4 7H2O g CaCl2 2H2O 2.94 g NaCl 23.4 g MnSO4 4H2O 112 mg ZnSO4 7H2O 29 mg Co(NO3)2 6H2O 29 mg CuSO4 5H2O 25 mg 24 mg H3BO3 31 mg ** Phosphate solution KH2PO4 75 g K2HPO4 78 g *** Vitamin solution 3Na-EDTA 200 mg Nicotinic acid 100 mg Thiamine-HCl 100 mg Biotin 5 mg p-aminobenzoic acid 50 mg Vitamin B12 1 mg Ca-pantothenate 50 mg Pyridoxine-HCl 50 mg Folic acid 50 mg Distilled water 100 ml Sterilized by filtration. 388 NPB Medium Bacto Tryptone (Difco) 10 g Bacto Yeast Extract (Difco) 2 g MgSO4 7H2O 1 g K2HPO4 1 g KH2PO4 0.5 g D-Glucose 5 g 389 Medium for Tepidanaerobacter Glucose 2.2 g Yeast extract 2.3 g KH2PO g MgCl2 6H2O 0.2 g CaCl2 2H2O 0.15 g NH4Cl 0.54 g Vitamin solution* 2 ml Trace element solution** 1 ml Se/W solution*** 1 ml Resazurin solution**** 1 ml NaHCO3 2.5 g Na2S 9H2O 0.3 g Cysteine-HCl 0.3 g ph7.0 * Vitamin solution Biotin 0.5 mg Folic acid 0.9 mg Pyridoxine-HCl 0.4 mg Thiamine-HCl 0.7 mg Riboflavin 0.8 mg Nicotinic acid 0.2 mg Ca-pantothenate 0.5 mg Vitamin B mg p-aminobenzoic acid 0.3 mg Lipoic acid 0.4 mg ** Trace element solution Nitrilotriacetic acid (NTA) 12.8 g FeCl3 6H2O 1.35 g CoCl2 6H2O 24 mg ZnCl2 0.1 g CuCl2 2H2O 25 mg H3BO3 10 mg 24 mg NiCl2 6H2O 0.12 g First dissolve NTA in 200 ml of water and adjust the ph to 6.5 with KOH. *** Se/W solution Na2SeO3 4 mg Na2WO4 4 mg **** Resazurin solution Resazurin 1 g For vial cultures, dissolve all ingredients except for glucose, yeast extract, Na2S 9H2O and cysteine-hcl (up to 900 ml), flush N2/CO2 (80/20) (ph 7), dispense 18 ml into 50-ml vials, fix with flushing N2/CO2 (80/20) and autoclave. Add 0.5 ml of each of filter-sterilized Na2S 9H2O solution (0.6 g Na2S 9H2O/50 ml) and cysteine-hcl solution (0.6 g cysteine-hcl/ 50ml) separately. Add 0.5 ml of neutralized and filter sterilized 0.8 M glucose solution and 0.5 ml of autoclaved yeast extract solution (%). Inoculum size 10%. 390 Modified SP Medium Bacto Casitone (Difco) 2.5 g Yeast extract 1 g Raffinose 1 g Sucrose 1 g Galactose 1 g Soluble starch 5 g MgSO4 7H2O 0.5 g K2HPO g ph

21 391 Thermodesulfobium narugense Medium K2HPO g KH2PO g 3Na-EDTA 0.04 g FeSO4 7H2O 0.01 g MgSO4 7H2O 0.25 g CaCl2 2H2O 0.03 g NaCl 0.2 g NH4Cl 0.5 g NaSO4 20 mm Sodium acetate 2 mm Vitamin solution* 10 ml Trace element solution** 10 ml Cysteine-HCl 0.25 g For vial culture, dissolve all ingredients except cysteine-hcl, flush N2/CO2, dispense 20 ml/50-ml vials, fix with a stopper with flushing N2/CO2 and autoclave. After autoclaved, the gas phase is replaced with H2/CO2 (80/20, 150 kpa), and add 0.1 ml of autoclaved cysteine-hcl solution (50 g/l) per each vial separately. Inoculum size 5-10%. * Vitamin solution Vitamin B mg p-aminobenzoic acid 5 mg Lipoic acid 5 mg ** Trace element solution Nitrilotriacetic acid (NTA) 12.8 g FeCl3 6H2O 1.35 g CoCl2 6H2O ZnCl2 0.1 g CuCl2 2H2O NiCl2 6H2O 0.12 g Na2SeO3 5H2O g 392 Archaeoglobus Medium KCl 0.34 g MgCl2 6H2O 4 g MgSO4 7H2O 3.45 g NH4Cl 0.25 g CaCl2 2H2O 0.14 g K2HPO g NaCl 18 g NaHCO3 3 g Bacto Yeast Extract (Difco) 0.5 g Sodium lactate 1.5 g Fe(NH4)2(SO4)2 7H2O 2 mg Trace element solution* 10 ml Na2S 9H2O 0.5 g 959 Dissolve ingredients except bicarbonate and sulfide, bring to boil for few minutes, then cool quickly to room temperature while gassing with N2/CO2 (80/20), add sodium bicarbonate and adjust ph to 6.9. Distribute in serum bottles (20 ml per 100 ml bottle) under N2/CO2, seal, pressurize bottles up to 2 bar overpressure, then autoclave. Before use, reduce the medium with sodium sulfide from anaerobic sterile neutral stock solution. * Trace element solution Nitrilotriacetic acid 1.5 g MgSO4 7H2O 3 g MnSO4 2H2O 0.5 g FeSO4 7H2O 0.1 g CoSO4 7H2O 0.18 g ZnSO4 7H2O 0.18 g CuSO4 5H2O 0.01 g KAl(SO4)2 12H2O 4 mg 0.01 g NiCl2 6H2O Na2SeO3 5H2O 0.8 mg Na2WO4 H2O 0.8 mg KOH, then add minerals. Final (with KOH). 393 Archaeoglobus profundus Medium KCl 0.34 g MgCl2 6H2O 4 g MgSO4 7H2O 3.45 g NH4Cl 0.25 g CaCl2 2H2O 0.14 g K2HPO g NaCl 18 g NaHCO3 1 g Sodium acetate 1 g Na2SO4 2.7 g Bacto Yeast Extract (Difco) 0.5 g Fe(NH4)2(SO4)2 7H2O 2 mg Trace element solution* 10 ml Na2S 9H2O 0.5 g Dissolve ingredients except bicarbonate and sulfide, bring to boil for few minutes, then cool quickly to room temperature while gassing with H2/CO2 (80/20), add sodium bicarbonate and adjust ph to 6.5. Distribute in serum bottles (20 ml per 100 ml bottle) under H2/CO2, seal, then autoclave. Before use, reduce the medium with sodium sulfide from anaerobic sterile neutral stock solution, and pressurize bottles up to 2 bar overpressure.

22 * Trace element solution Nitrilotriacetic acid 1.5 g MgSO4 7H2O 3 g MnSO4 2H2O 0.5 g FeSO4 7H2O 0.1 g CoSO4 7H2O 0.18 g ZnSO4 7H2O 0.18 g CuSO4 5H2O 0.01 g KAl(SO4)2 12H2O 0.02 g 0.01 g NiCl2 6H2O Na2SeO3 5H2O 0.3 mg KOH, then add minerals. Final (with KOH). 394 Pyrococcus Medium 2 Salt base solution* 1 L Trace minerals** 10 ml Citric acid 5 mg Bacto Yeast Extract (Difco) 1 g Bacto Peptone (Difco) 5 g Sulfur (powder)*** 30 g Na2S 9H2O 0.5 g Mix ingredients except sulfur and Na2S 9H2O, and adjust ph to 6.5 with H2SO4. Sterilize by filtration under a N2 atmosphere. Prepare Na2S 9H2O as a 5% solution, and autoclave under a N2 atmosphere. Aseptically and anaerobically add sterile sulfur to the medium. Prior to inoculation, add the sterile Na2S 9H2O solution, and readjust ph to 6.5, if necessary. * Salt base solution NaCl g MgSO4 7H2O 3.5 g MgCl2 6H2O 2.75 g KCl g NaBr 0.05 g H3BO3 15 mg SrCl2 6H2O 7.5 mg KI 0.05 mg CaCl g KH2PO4 0.5 g (NH4)2Ni(SO4)2 6H2O 2 mg ** Trace minerals Nitrilotriacetic acid 1.5 g MgSO4 7H2O 3 g MnSO4 xh2o 0.5 g FeSO4 7H2O 0.1 g CoSO4 7H2O 0.1 g ZnSO4 7H2O 0.1 g CuSO4 5H2O 0.01 g AlK(SO4) g 0.01 g Dissolve nitrilotriacetic acid and adjust ph to 6.5 with KOH solution. Then proceed to add minerals. Adjust final ph to 7.0. *** Sulfur powder Sulfur is sterilized separately by autoclaving for 60 min at 105 on each of 3 successive days. 395 JXT Medium Bacto Yeast Extract (Difco) 1 g Trypticase Peptone (BBL) 1 g Na2S2O3 5H2O 1 g Sea water 1 L Use filtrated natural seawater or artificial seawater (e.g. Aquamarine, Yashima Co.). Separately sterilize a solution of Na2S2O3 5H2O by filtration, and aseptically add to the medium. 396 Sulfolobus tokodaii Medium Salt base solution* 1 L Bacto Yeast Extract (Difco) 1 g Adjust ph to 3.5 with 10 N H2SO4 Add yeast extract solution just prior to inoculation. * Salt base solution 397 VY/2 Agar (NH4)2SO4 1.3 g KH2PO g MgSO4 7H2O 0.25 g CaCl2 2H2O 0.07 g FeCl3 6H2O 0.02 g MnCl2 4H2O 1.8 mg Na2B4O7 10H2O 4.5 mg ZnSO4 7H2O 0.22 mg CuCl2 2H2O 0.05 mg 0.03 mg VOSO4 xh2o 0.03 mg CoSO4 7H2O Adjust ph to 3.5 with 10 N H2SO4 Baker's yeast 5 g CaCl2 2H2O 1.36 g Vitamin B mg Bacto Agar (Difco) 15 g Sterilized vitamin B12 separately by filtration. Kill yeast cells by heating before adding other ingredients and ph adjustment. Adjust ph to 7.2 with KOH before adding agar. 960

23 398 Methanothermobacter Medium KH2PO g NH4Cl 0.54 g MgCl2 6H2O 0.2 g CaCl2 2H2O g NaHCO3 2.5 g Bacto Yeast Extract (Difco) 0.2 g Vitamin solution* 10 ml Trace element solution** 10 ml Cysteine-HCl 0.5 g Na2S 9H2O 0.5 g Mix ingredients except vitamin solution, cysteine-hcl and Na2S 9H2O, and autoclave under a H2/CO2 atmosphere (80/20). Concentrated cysteine-hcl and sodium sulfide solutions are sterilized separately under a N2 atmosphere in tightly closed vessels. Prior to inoculation, add the filter-sterile vitamin solution, Cysteine-HCl and Na2S 9H2O. Pressurize the inoculated vessels to 150 kpa with H2/CO2 (80/20). * Vitamin solution p-aminobenzoic acid 1 mg Vitamin B12 ** Trace elements solution 2Na-EDTA 0.64 g MgSO4 7H2O 6.2 g MnSO4 4H2O 0.55 g FeSO4 7H2O 0.1 g CoCl2 6H2O 0.17 g CaCl2 2H2O 0.13 g ZnSO4 7H2O 0.18 g CuSO g KAl(SO4)2 12H2O g g NiCl2 6H2O Dissolve the 2Na-EDTA in 500 ml of distilled water, and after adding the salts, make up to 1 L. 399 Methanobacterium Medium KH2PO4 0.5 g MgSO4 7H2O 0.4 g NaCl 0.4 g NH4Cl 0.4 g CaCl2 2H2O 0.05 g FeSO4 7H2O g Trace element solution SL-10* 1 ml Yeast extract 1 g Sodium acetate 1 g Sodium formate 2 g Sludge fluid** 50 ml NaHCO3 4 g Fatty acid mixture*** 20 ml Resazurin g Cysteine-HCl 0.5 g Na2S 9H2O 0.5 g Distilled water 940 ml Adjust ph to Prepare the medium anaerobically under a atmosphere H2/CO2 (80/20, 150 kpa). * Trace element solution SL-10 ** Sludge fluid HCl (25%; 7.7 M) 10 ml FeCl2 4H2O 1.5 g ZnCl2 7 mg MnCl2 4H2O 100 mg H3BO3 6 mg CoCl2 6H2O 190 mg CuCl2 2H2O 2 mg NiCl2 6H2O 24 mg 36 mg Distilled water 990 ml Add 0.4% yeast extract to sludge from an anaerobic digester, and after gassing with nitrogen gas for a few minutes incubate it at 37 C for 24 hours. Then centrifuge the sludge at 13,000 g and autoclave the resulting, clear supernatant in screw-capped vessels under nitrogen gas. The sludge fluid can be stored at room temperature in the dark. *** Fatty acid mixture Valeric acid 0.5 g Isovaleric acid 0.5 g alpha-methylbutyric acid 0.5 g Isobutyric acid 0.5 g Distilled water 20 ml Adjust ph to 7.5 with conc. NaOH 400 M1D Medium Bacto PPLO Broth w/o CV (Difco) 2 g Bacto Tryptone (Difco) 1 g Bacto Peptone (Difco) 0.8 g Glucose 0.1 g Fructose 0.1 g Sucrose 1 g Sorbitol 7.5 g Phenol red 6 mg 961 Dissolve above in 80 ml of water and adjust to ph 7.8. Bring to 90 ml with water. Autoclave and cool to room temperature. Add the following under sterile conditions: Schneider's Drosophila medium (Invitorogen, Gibco) 160 ml, Fetal bovine serum (heat-inactivated) 50 ml, Penicillin G (100,000 U/ml) 2.5 ml.

24 402 CYC Agar 403 Czapek-Dox Liquid Medium, Modified (OXOID) 33.4 g Bacto Yeast Extract (Difco) 2 g Bacto Vitamin Assay Casamino Acids (Difco) 6 g Agar 16 g Adjust ph to 7.2 K2HPO4 0.5 g KH2PO4 0.6 g MgSO4 7H2O 0.2 g NaCl 0.1 g CaCO3 3 g Yeast extract 0.6 g Arabinose 4 g Sodium glutamate 6 g ph 6.7± Oceanithermus profundus Medium NH4Cl 0.33 g MgCl2 6H2O 0.33 g CaCl2 2H2O 0.33 g KCl 0.33 g KNO g NaCl 30 g HEPES 2.38 g Yeast extract 0.2 g Tryptone 1 g Sucrose 2 g Vitamin solution* 1 ml Trace element solution** 1 ml Prepare the medium anaerobically, under N2, omitting the CaCl2, MgCl2, KNO3, tryptone, yeast extract, vitamins and sucrose. The ph should be Dispense the medium into vessels suitable for anaerobic growth (Hungate tubes or serum bottles) under an atmosphere of nitrogen and autoclave. To the sterile, cooled medium add, from sterile stock solutions the CaCl2, MgCl2, KNO3, tryptone, yeast extract, vitamins and sucrose. The CaCl2, MgCl2, KNO3, tryptone, yeast extract, and sucrose stock solutions should be autoclaved, while the vitamin solution is filter-sterilized. * Vitamin solution Vitamin B mg p-aminobenzoic acid 5 mg Lipoic acid 5 mg ** Trace element solution Nitrilotriacetic acid 1.5 g MgSO4 7H2O 3 g MnSO4 2H2O 0.5 g FeSO4 7H2O 0.1 g CoSO4 7H2O 0.18 g ZnSO4 7H2O 0.18 g CuSO4 5H2O 0.01 g KAl(SO4)2 12H2O 0.02 g 0.01 g NiCl2 6H2O Na2SeO3 5H2O 0.3 mg KOH, then add minerals. Final (with KOH). 405 Methanosarcina Medium KH2PO g K2HPO g NH4Cl 0.5 g MgSO4 7H2O 0.5 g CaCl2 2H2O 0.25 g NaCl 2.25 g FeSO4 7H2O 2 mg Bacto Yeast Extract (Difco) 2 g Bacto Casitone (Difco) 2 g Trace vitamins* 10 ml FeCl2 solution** 1 ml Trace element solution*** 1 ml NaHCO g Methanol 10 ml Cysteine-HCl 0.3 g Na2S 9H2O 0.3 g Distilled water 980 ml Mix ingredients except NaHCO3, methanol, cysteine-hcl and Na2S 9H2O, and bring to a boil for 5-10 sec. Cool down under a stream of N2/CO2 (80/20), and add NaHCO3 and methanol to the medium. Dispense the medium into suitable culture vessels (e.g. 20 ml of the medium in 120 ml serum bottles) under a stream of N2/CO2 (80/20) and seal with butyl rubber stoppers. After autoclaving, stand the medium overnight. Separately autoclave cysteine-hcl and Na2S 9H2O as 5% solutions, respectively, under a N2 atmosphere. Prior to inoculation, aseptically and anaerobically add the cysteine-hcl and Na2S 9H2O solutions. * Trace vitamins Vitamin B mg p-aminobenzoic acid 5 mg Lipoic acid 5 mg 962