viii. list of media939 viii. list of media 1 potato sucrose agar (psa) potato 200 g sucrose 20 g...

939

VIII. List of Media

1 Potato Sucrose Agar (PSA) Potato 200 g

Sucrose 20 g Distilled water 1 L 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 Distilled water 1 L 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.

3 Potato 200 g

Glucose 120 g NaCl 50 g Distilled water 1 L 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.

4 Potato 200 g

Yeast extract 2 g Glucose 20 g Distilled water 1 L 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.

5 Mal t Ext ract Agar Malt extract 20 g

Glucose 20 g Peptone 1 g Distilled water 1 L Agar 20 g

pH 6.0

6 Sabouraud 's Agar Peptone 10 g

Glucose 40 g Distilled water 1 L Agar 20 g

pH 6.0

7 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.

8 Oatmeal Agar Oatmeal 50 g

Distilled water 1 L 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.

9 NaNO3 3 g

K2HPO4 1 g MgSO4·7H2O 0.5 g KCl 0.5 g FeSO4·7H2O 0.01 g Sucrose 200 g Distilled water 1 L Agar 20 g

pH 6.0

10 NaNO3 3 g

K2HPO4 1 g MgSO4·7H2O 0.5 g KCl 0.5 g FeSO4·7H2O 0.01 g Sucrose 400 g Distilled water 1 L Agar 20 g

pH 6.0

Media

940

13 Seawater Starch Agar (SWS) Soytone 1 g

Soluble starch 10 g Seawater (2% Salinity)* 1 L Agar 15 g

pH 8.2

* Dilute artificial seawater or natural seawater with distilled water.

14 Corn and F ish Meal Agar Corn and fish meal mixture (feed for birds) 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 Ju ice Seawater Agar (V-8 SWA) Vegetable juice* 200 ml

CaCO3* 3 g Seawater (2% salinity) 800 ml Agar 15 g

pH 7.0

*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 7.0.

16 Cornmeal Seawater Agar (CMSWA) Cornmeal extract 2 g

Seawater (2% Salinity) 1 L Agar 15 g

pH 7.0 - 7.5

17 Cornmeal Agar Cornmeal extract 2 g


pH 6.0

18 Pet r i -Cel lu lose Medium Cellulose* 10 g

Ca(NO3)2·4H2O 0.4 g K2HPO4 0.15 g MgSO4·7H2O 0.15 g KCl 0.06 g Distilled water 1 L

pH 7.0

* 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 5.8

* Wako Pure Chemicals Ind. Ltd., Osaka, Japan.

20 1/5 Mycorrhiza Medium Yeast extract 0.4 g


pH 5.0

22 Trypt icase Yeast Ext ract 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 Ext ract Seawater Agar (CMYSWA)

Cornmeal extract 2 g Yeast extract 1 g Seawater (2% salinity) 1 L Agar 15 g

pH 7.0 - 7.5

25 PYG Agar Proteose peptone 1 g


26 Mycorrh iza Medium Yeast extract 2 g


pH 5.0

103 Glucose 10 g

Peptone 5 g Yeast extract 3 g Malt extract 3 g Olive oil 10 g Distilled water 1 L Agar 15 g

pH 5.6

104 Glucose 10 g

Peptone 5 g Yeast extract 3 g Malt extract 3 g CaCO3 10 g Distilled water 1 L Agar 15 g

pH 5.6

Media

941

107 Glucose 10 g

Peptone 5 g Yeast extract 3 g Malt extract 3 g NaCl 100 g Distilled water 1 L Agar 15 g

pH 5.6 - 6.0

108 YM Agar Glucose 10 g

Peptone 5 g Yeast extract 3 g Malt extract 3 g Distilled water 1 L Agar 15 g

109 YPAD Medium Yeast extract 10 g

Peptone 20 g Glucose 20 g Adenine sulfate 40 mg Distilled water 1 L Agar 15 g

111 Bacto-Yeast Nitrogen Base w/o Amino acids (Difco) 6.7 g

Glucose 20 g L-Tryptophan 20 mg L-Histidine 20 mg Distilled water 1 L Agar 15 g

112 YPD Medium Yeast extract 10 g

Peptone 20 g Glucose 20 g Distilled water 1 L Agar 15 g

113 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 Distilled water 1 L Agar 15 g

114 Medium for the 2μ Mapping St ra ins 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 Distilled water 1 L Agar 15 g

116 25% Glucose-Yeast Ext ract -Peptone Glucose 250 g

Yeast extract 10 g Peptone 20 g Distilled water 1 L

117 MA2 Malt extract 20 g


118 MA4 Malt extract 40 g


119 YEP Alcohol Yeast extract 3 g

Peptone 10 g Distilled water 1 L Agar 15 g Ethyl alcohol 10 ml

201 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 Agar (if needed) 15 g

pH 7.0

* 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.

202 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 Agar (if needed) 15 g

pH 7.0

* 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.


Media

942

207 Mannitol 15 g

K2HPO4 0.2 g MgSO4·7H2O 0.2 g NaCl 0.2 g CaSO4·2H2O 0.1 g CaCO3 5 g Distilled water 1 L Agar (if needed) 15 g

pH 7.2

209 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.

210 Glucose 30 g

Yeast extract 5 g Peptone 5 g CaCO3 30 g Distilled water 1 L Agar (if needed) 15 g

pH 6.8

213 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, pH 7.0) 980 ml Agar (if needed) 15 g

* Sterilize separately by filtration.

214 Skim milk 100 g

Distilled water 1 L

216 Hear t In fus ion Agar Bacto Heart Infusion Agar (Difco) 40 g

Distilled water 1 L

219 Bacto Casitone (Difco) 20 g

MgSO4·7H2O 1 g Potassium phosphate buffer (0.01 M, pH 7.2) 1 L Agar (if needed) 15 g


Bacto Tryptone (Difco) 1 g Sodium acetate 1 g Soil extract* 50 ml Distilled water 950 ml Agar (if needed) 15 g

pH 7.4

* Autoclave 400 g of farm soil suspended in 1 L of distilled water for 30 min, and remove solids by filtration.

221 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 Distilled water 1 L Agar (if needed) 15 g

pH 7.0

222 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 Distilled water 1 L

pH 7.2 - 7.4

223 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 Distilled water 1 L

pH 7.0 - 7.2

224 (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 Na2MoO4·2H2O 0.3 mg Yeast extract 0.1 g Sulfur (powder)* 1 g Bromophenol blue 10 mg Distilled water 1 L

pH 4.0 - 4.6

* Sterilize separately by autoclaving for 60 min at 105℃ on each of 3 successive days and add after inoculation.

Media

943

225 Table wine 500 ml

Yeast extract 20 g Calcium lactate 10 g CaCO3 20 g Distilled water 500 ml Agar (if needed) 15 g

pH 6.6 - 6.8


Glucose 20 g Distilled water 1 L

pH 6.8 - 7.0

227 Yeast Ext ract -Mal t Ext rac t Agar ( ISP Medium No.2)

Yeast extract 4 g Malt extract 10 g Glucose 4 g Distilled water 1 L Agar 20 g

pH 7.3

228 Bennet t 's Agar Yeast extract 1 g

Beef extract 1 g NZ Amine, Type A* 2 g Glucose 10 g Distilled water 1 L 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 Distilled water 1 L Agar (if needed) 20 g

pH 7.3

230 Trypt icase Soy Agar Trypticase Soy (BBL) 30 g


pH 7.3

231 Mal tose-Bennet t 's Agar Yeast extract 1 g

Beef extract 1 g NZ Amine, Type A* 2 g Maltose 10 g Distilled water 1 L Agar 20 g

pH 7.3


232 Halophi l ic Act inomycetes Bacto Yeast Extract (Difco) 4 g

Bacto Malt Extract (Difco) 10 g Glucose 4 g NaCl 200 g Distilled water 1 L Agar (if needed) 20 g

pH 7.3

233 Methanol* 10 ml

(NH4)2HPO4 3 g NaCl 1 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

pH 7.0

* 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 Distilled water 1 L

*** Sterilize separately.

234 (NH4)2SO4 2 g

KCl 0.1 g K2HPO4 0.5 g MgSO4·7H2O 0.5 g Polypepton* 0.1 g Glucose** 1 g Distilled water 1 L Agar (if needed) 15 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.


** Sterilize separately.

235 (NH4)2SO4 2 g

K2HPO4 0.5 g MgSO4·7H2O 0.5 g KCl 0.1 g Ca(NO3)2 0.01 g FeSO4·7H2O 40 g Distilled water 1 L

Adjust pH to 3.0 with 6 N H2SO4 and sterilize by filtration.

Media

944

239 NaNO2 1 g

NaCl 0.3 g K2HPO4 0.5 g MgSO4·7H2O 0.5 g MnSO4·nH2O 0.002 g Fe2(SO4)3 0.005 g Distilled water 1 L

pH 7.5

240 (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 Distilled water 1 L

pH 8.0

* CaCO3 is practically insoluble.

243 Oatmeal 3 g

KNO3 0.2 g K2HPO4 0.5 g MgSO4·7H2O 0.2 g Distilled water 1 L Agar (if needed) 20 g

pH 7.0


Malt extract 10 g Glucose 4 g Seawater 750 ml Distilled water 250 ml Agar (if needed) 20 g

pH 7.6

245 Oatmeal Agar ( ISP Medium No.3) Oatmeal 20 g

Trace salts solution* 1 ml Distilled water 1 L Agar 18 g

pH 7.2

* Trace salts solution

FeSO4·7H2O 0.1 g MnCl2·4H2O 0.1 g ZnSO4·7H2O 0.1 g Distilled water 100 ml

246 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.

* 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.


Malt extract 10 g Glucose 4 g Na2CO3* 10 g Distilled water 1 L Agar (if needed) 20 g

pH 9.0 - 10.0

* Na2CO3 is sterilized separately.

249 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 Agar (if needed) 15 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 NaCl 1 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 Na2MoO4·2H2O 0.01 g Na2SeO3 0.001 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

Media

945

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


**** 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 Distilled water 1 L Agar (if needed) 15 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 Distilled water 1 L Agar (if needed) 15 g

pH 7.5

253 Bra in Hear t In fus ion Agar Bacto Brain Heart Infusion (Difco) 37 g

Distilled water 1 L Agar (if needed) 15 g

254 Blood Agar Bacto Blood Agar Base (Difco) 40 g

Distilled water 1 L

Sterilize by autoclaving and cool to 45-50℃ and add to it 50 ml of sterile defibrillated blood.

255 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 9.0 - 9.5.


257 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 Yeast extract 5 g Glucose 1 g Distilled water 1 L Agar (if needed) 20 g

pH 7.0

258 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 Distilled water 1 L Agar (if needed) 15 g

pH 7.0


Media

946


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 Distilled water 1 L Agar (if needed) 15 g

pH 6.0


Malt extract 5 g K2HPO4 5.74 g NH4H2PO4 1.15 g MgSO4·7H2O 0.06 g Catalase* 60 mg Distilled water 1 L Agar (if needed) 15 g

pH 7.0


262 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 Distilled water 1 L Agar (if needed) 15 g

The final pH of the freshly prepared medium is between 10.5 and 11.0.


** Na2CO3 is sterilized separately by autoclaving.

263 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 Agar (if needed) 15 g

pH 8.0

* HMSS solution

Nitrilotriacetic acid 10 g MgSO4·7H2O 29.7 g CaCl2·2H2O 3.3 g Na2MoO4·2H2O 12.7 mg FeSO4·7H2O 99 mg Metal 44 solution**** 50 ml Distilled water 1 L

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 Distilled water 1 L

Sterilize separately, and add after autoclaving.

*** Vitamin mixture


**** Metal 44 solution

2Na-EDTA 2.5 g ZnSO4·7H2O 10.95 g FeSO4·7H2O 5 g MnSO4·nH2O 1.54 g CuSO4·5H2O 0.392 g CoCl2·6H2O 0.203 g Na2B4O7·10H2O 0.177 g Distilled water 1 L

264 Peptone 10 g

Yeast extract 5 g Malt extract 5 g Bacto Casamino Acids (Difco) 5 g Beef extract 2 g Glycerol 2 g Tween 80 0.05 g MgSO4·7H2O 1 g Distilled water 1 L Agar (if needed) 15 g

pH 7.0

265 Glycero l -Asparagine Agar ( ISP Medium No.5)

L-Asparagine 1 g Glycerol 10 g K2HPO4 1 g Distilled water 1 L Trace salts solution* 1 ml Agar 20 g

pH 7.0 - 7.4



Media

947

266 Yeast Ext ract -Starch Agar Yeast extract 2 g

Soluble starch 10 g Distilled water 1 L Agar 15 g

pH 7.3

267 Modi f ied Hayf l ick 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 Sal ts Starch Agar ( ISP Medium No.4)

Soluble starch 10 g K2HPO4 (anhydrous base) 1 g MgSO4·7H2O 1 g NaCl 1 g (NH4)2SO4 2 g CaCO3 2 g Trace salts solution* 1 ml Bacto Agar (Difco) 20 g Distilled water 1 L

The unadjusted pH should be 7.0 - 7.4.



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 Distilled water 1 L Agar 20 g

pH 7.3


271 Yeast -Kra insky 's Agar Glucose 10 g

L-Asparagine 1 g K2HPO4 0.5 g Yeast extract 2 g Distilled water 1 L Agar 17 g

pH 7.3


Yeast extract 1 g CaCl2·2H2O 1.36 g Distilled water 1 L Agar (if needed) 15 g

pH 7.2

273 Peptone 5 g

Meat extract 3 g NaCl 60 g Distilled water 1 L Agar (if needed) 15 g

pH 7.0

274 Bacto Tryptone (Difco) 0.5 g

Yeast extract 0.5 g Beef extract 0.2 g Sodium acetate 0.2 g Distilled water 1 L Agar (if needed) 15 g

pH 7.2

275 LB Medium Peptone 10 g

Yeast extract 5 g NaCl 5 g Distilled water 1 L Agar (if needed) 15 g

pH 7.0


CaCl2·2H2O 1.36 g Yeast extract 1 g Cellobiose 5 g Distilled water 1 L Agar (if needed) 15 g

pH 7.2


Yeast extract 5 g MgSO4·7H2O 1 g Distilled water 1 L Agar (if needed) 15 g

pH 7.2

Media

948

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 CaCl2·2H2O 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 Na2MoO4·2H2O 0.1 mg Vitamin mixture* 5 ml Distilled water 1 L

Adjust pH to 7.5 with 1 N HCl

* Vitamin mixture


279 Tryptone Yeast Ext ract Agar (TYG) Bacto Tryptone (Difco) 3 g


pH 7.0

280 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 Distilled water 1 L


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℃.


(NH4)2SO4 1.3 g CaCl2·2H2O 0.08 g MgSO4·7H2O 0.25 g KH2PO4 0.28 g Na2SiO3·9H2O 0.3 g Na2MoO4·2H2O 0.03 g FeSO4·7H2O 0.02 g Distilled water 1 L

Adjust pH to 3.0 with 10N H2SO4

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℃.

282 Glucose 10 g

K2HPO4 1 g NH4NO3 2 g MgSO4·7H2O 0.2 g MnSO4·7H2O 0.005 g FeSO4·7H2O 0.005 g CaCl2·2H2O 0.1 g Yeast extract 3 g Polypepton* 0.3 g Resazurin 0.001 g Distilled water 1 L Agar (if needed) 15 g

After autoclaving, adjust pH to 10.0 with sterilized 10% Na2CO3 solution.


283 Sucrose 1 g

Bacto Casitone (Difco) 0.75 g Yeast extract 0.25 g Polypepton* 0.25 g Vitamin mixture** 10 ml Distilled water 1 L Agar (if needed) 15 g

pH 7.1


** Vitamin mixture


284 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 Distilled water 1 L Agar (if needed) 15 g

pH 7.4

Media

949

286 (NH4)2SO4 3 g

CaCl2·2H2O 0.08 g MgSO4·7H2O 0.25 g KH2PO4 0.28 g KCl 0.1 g Ca(NO3)2·4H2O 0.01 g Yeast extract* 0.2 g Sulfur (powder)** 10 g Distilled water 1 L

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.

287 (NH4)2SO4 1.3 g

CaCl2·2H2O 0.08 g MgSO4·7H2O 0.25 g KH2PO4 0.28 g Na2SiO3·9H2O 0.1 g Na2MoO4·2H2O 0.03 g FeSO4·7H2O 0.02 g Yeast extract* 1 g Sulfur (powder)** 10 g Distilled water 1 L


* 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.

288 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 0.006 g Distilled water 1 L

pH 6.8

Good growth can be obtained in semi-solid medium (1 g of agar in 1 liter).

289 KH2PO4 0.28 g

NaNO3 0.12 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 Distilled water 1 L

Adjust pH to 6.75 with 1 N NaOH

* Vitamin mixture


** Ferric quinate solution

FeCl3·6H2O 0.45 g Quinic acid 0.19 g Distilled water 1 L

*** HMSS solution

Nitrilotriacetic acid 10 g MgSO4·7H2O 29.7 g CaCl2·2H2O 3.3 g Na2MoO4·2H2O 12.7 mg FeSO4·7H2O 99 mg Metal 44 solution**** 50 ml Distilled water 1 L

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 10.95 g FeSO4·7H2O 5 g MnSO4·nH2O 1.54 g CuSO4·5H2O 0.392 g CoCl2·6H2O 0.203 g Na2B4O7·10H2O 0.177 g Distilled water 1 L

Good growth can be obtained in semi-solid medium (1 g of agar in 1 liter).

294 Bacto Yeast Extract (Difco) 5 g

Distilled water 1 L Adjust pH to 5.0 with 1 N HCl

296 V-8 Ju ice Tap Water Agar V-8 Juice* 200 ml

CaCO3* 3 g Tap water 800 ml Agar 15 g

* 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.

Media

950

297 Beef extract 1 g

Yeast extract 2 g Peptone 5 g NaCl 5 g Distilled water 1 L Agar (if needed) 15 g

pH 7.0

298 OA-Y Agar Oatmeal 20 g

Trace salts solution* 1 ml Bacto Yeast Extract (Difco) 1 g Distilled water 1 L Agar 18 g

pH 7.2



299 Gauze 's Synthet ic Medium No.1 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 Agar (if needed) 15 g

pH 7.2 - 7.4

302 Bacto Yeast Extract (Difco)* 1 g

(NH4)2SO4 1.3 g KH2PO4 0.28 g MgSO4·7H2O 0.25 g CaCl2·2H2O 0.07 g FeCl3·6H2O 0.02 g MnCl2·4H2O 0.018 g Na2B4O7 4.5 mg ZnSO4·7H2O 0.22 mg Na2MoO3·2H2O 0.05 mg Distilled water 1 L


* Sterilize by filtration as 5% solution, and add to other ingredients sterilized by autoclaving.


304 Oatmeal Agar (B) Oatmeal (powder, Quaker White Oats) 3 g

KNO3 0.2 g K2HPO4 0.5 g MgSO4·7H2O 0.2 g Distilled water 1 L Agar 15 g

pH 7.0

305 Sulfur (powder)* 0.5 g

(NH4)2SO4 1.3 g KH2PO4 0.28 g MgSO4·7H2O 0.25 g CaCl2·2H2O 0.07 g FeCl3·6H2O 0.02 g MnCl2·4H2O 0.018 g Na2B4O7 4.5 mg ZnSO4·7H2O 0.22 mg Na2MoO3·2H2O 0.05 mg Distilled water 1 L


* Sulfur is sterilized separately by autoclaving for 60 min at 105℃ on each of 3 successive days and added after inoculation.


306 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 Distilled water 1 L Agar (if needed) 15 g

pH 7.0


308 Glucose Nutr ient Agar Beef extract 1 g

Yeast extract 2 g Peptone 5 g NaCl 5 g Glucose 10 g Distilled water 1 L Agar 15 g

pH 7.0

309 Rhamnose Sal t 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 Distilled water 1 L

pH 7.0

Media

951

310 MRS Medium Peptone 10 g

Meat extract 10 g Yeast extract 5 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 Distilled water 1 L Agar (if needed) 15 g

pH 6.0 - 6.5

311 Todd Hewi t t Medium Bacto Todd Hewitt Broth (Difco) 30 g

Distilled water 1 L pH 7.3

312 GAM Medium Nissui GAM Broth* 59 g

Distilled water 1 L pH 7.3

* Nissui Pharmaceutical Co. Ltd., Tokyo, Japan.

313 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 0.335 g NH4Cl 0.25 g CaCl2·2H2O 0.14 g K2HPO4 0.14 g Fe(NH4)2(SO4)2·6H2O 0.002 g Resazurin 0.001 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.

314 Bacto Brain Heart Infusion (Difco) 37 g

Horse serum* 50 ml Distilled water 950 ml


315 Bacto Todd Hewitt Broth (Difco) 30 g

Horse serum* 5 ml Distilled water 1 L

pH 7.3


316 Polypepton* 10 g

Yeast extract 2 g MgSO4·7H2O 0.5 g Na2CO3** 10 g Distilled water 1 L Agar (if needed) 15 g


** Sterilize separately by autoclaving and add to other ingredients. The final pH is between 9.0 and 10.0.

317 Peptone 10 g

Yeast extract 10 g L-Cysteine 1 g Distilled water 1 L

pH 6.8

319 NZ Amine wi th Solub le Starch and Glucose Agar Medium

Glucose 10 g Soluble starch 20 g Yeast extract 5 g NZ Amine, Type A* 5 g CaCO3 (reagent grade) 1 g Distilled water 1 L Agar 15 g

pH 7.3


323 Solution A* 500 ml

Solution B** 500 ml

* Solution A

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 3.0 - 4.0

** Solution B

Glucose 1 g Agar (if needed) 20 g Distilled water 500 ml

Solutions A and B are separately sterilized by autoclaving and mixed.

324 (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 Distilled water 1 L


* Autoclave separately as 20% solution, and added to other autoclaved ingredients.

Media

952

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℃.


Yeast extract 2 g MgSO4·7H2O 0.5 g Seawater** 750 ml Distilled water 250 ml Agar (if needed) 15 g

pH 7.2 - 7.4


** Filtered, aged seawater or artificial seawater (Daigo's Artificial Seawater SP*).

326 Peptone 10 g

Yeast extract 2 g MgSO4·7H2O 1 g NaCl 30 g Distilled water 1 L Agar (if needed) 15 g

pH 7.0

328 Glycerol 30 g

Keratin 10 g Soil extract* 100 ml CaCO3 1 g Yeast extract 5 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.


Soluble starch 10 g NaCl 50 g Agar (if needed) 15 g Distilled water 1 L

pH 7.3

331 SP5 Medium Bacto Casitone (Difco) 9 g

Yeast extract 1 g Agar (if needed) 15 g Seawater* 1 L

pH 7.2

* Filtered, aged seawater or artificial seawater (Daigo's Artificial Seawater SP**).


332 Enr iched Cytophaga Agar Bacto Tryptone (Difco) 2 g

Beef extract 0.5 g Yeast extract 0.5 g Sodium acetate 0.2 g Agar (if needed) 15 g Distilled water 1 L

pH 7.2 - 7.4

333 Mar ine Cytophaga Agar Bacto Tryptone (Difco) 2 g

Beef extract 0.5 g Yeast extract 0.5 g Sodium acetate 0.2 g Agar (if needed) 15 g Seawater* 1 L

pH 7.2 - 7.4



334 F lavobacter ium co lumnare 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 Distilled water 1 L

pH 7.2 - 7.4

335 Medium for Mar ine F lex ibacter ia KNO3 0.5 g

Sodium glycerophosphate 0.1 g Trace element solution* 1 ml Tris (hydroxymethyl) aminomethane 1 g Bacto Tryptone (Difco) 5 g Yeast extract 5 g Agar (if needed) 15 g Seawater** 1 L

pH 7.0

* Trace element solution

H3BO4 2.85 g MnCl2·4H2O 1.8 g FeSO4·7H2O 1.36 g CuCl2·2H2O 26.9 mg ZnCl2 20.8 mg CoCl2·6H2O 40.4 mg Na2MoO4·2H2O 25.2 mg Sodium tartrate 1.77 g Distilled water 1 L

** Filtered, aged seawater or artificial seawater (Daigo's Artificial Seawater SP***).

*** Wako Pure Chemical Ind., Ltd., Osaka, Japan.

336 Cytophaga (Mar ine) Medium Bacto Tryptone (Difco) 1 g

Yeast extract 1 g Agar (if needed) 15 g Seawater* 1 L

pH 7.2

* Filtered, aged seawater or artificial seawater (Daigo's Artificial Seawater SP**)

Media

953


338 Caulobacter Medium Peptone 2 g

Yeast extract 1 g MgSO4·7H2O 0.2 g Agar (if needed) 10 g Distilled water 1 L

pH 7.0

339 Mar ine Broth 2216 Bacto Marine Broth 2216 (Difco) 37.4 g

Distilled water 1 L pH unadjusted

340 Mar ine Agar 2216 Bacto Marine Agar 2216 (Difco) 55.1 g


341 Medium for Freshwater F lex ibacter ia MgSO4·7H2O 0.1 g

KNO3 0.1 g CaCl2·2H2O 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 Agar (if needed) 15 g Distilled water 1 L

pH 7.5


H3BO4 2.85 g MnCl2·4H2O 1.8 g FeSO4·7H2O 1.36 g CuCl2·2H2O 26.9 mg ZnCl2 20.8 mg CoCl2·6H2O 40.4 mg Na2MoO4·2H2O 25.2 mg Sodium tartrate 1.77 g Distilled water 1 L

342 Nissu i P la te Sheep Blood Agar G* * Nissui Pharmaceutical Co. Ltd., Tokyo, Japan.

343 Sucrose-Bennet t 's Agar Yeast extract 1 g

Beef extract 1 g NZ Amine, Type A* 2 g Sucrose 10 g Distilled water 1 L Agar 20 g

pH 7.3


347 Nissu i P la te Sheep Blood Agar* * Nissui Pharmaceutical Co. Ltd., Tokyo, Japan.

350 Acetobacter xy l inus Medium Polypepton* 5 g

Yeast extract 5 g Glucose 5 g Mannitol 5 g MgSO4·7H2O 1 g Ethanol** 5 ml Agar (if needed) 15 g Distilled water 1 L

pH 6.6 - 7.0


** Sterilize separately by filtration.


Yeast extract 2 g N-Acetyl glucosamine** 30 mg MgSO4·7H2O 1 g Agar (if needed) 15 g Distilled water 1 L

pH 7.0



352 Methy lobacter ium Medium Polypepton* 10 g

Yeast extract 2 g Methanol** 5 ml MgSO4·7H2O 1 g Agar (if needed) 15 g Distilled water 1 L

pH 7.0



355 R-CW Medium Peptone 5 g

Bacto Tryptone (Difco) 5 g Yeast extract 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.


Yeast extract 2 g MgSO4·7H2O 1 g N-Acetyl-D-glucosamine 50 mg Distilled water 1 L Agar (if needed) 15 g

pH 7.0


Media

954


Yeast extract 2 g MgSO4·7H2O 1 g D-Alanine 50 mg Distilled water 1 L Agar (if needed) 15 g

pH 7.0



Yeast extract 2 g MgSO4·7H2O 1 g Thymine 100 mg Distilled water 1 L Agar (if needed) 15 g

pH 7.0



Yeast extract 2 g MgSO4·7H2O 1 g Cytidine 50 mg Distilled water 1 L Agar (if needed) 15 g

pH 7.0


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 Distilled water 1 L Agar (if needed) 15 g

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 Na2MoO4·2H2O 20 mg CuCl2·2H2O 10 mg NiCl2·6H2O 10 mg Na2SeO3 5 mg Distilled water 1 L

362 Polypepton* 5 g

Yeast extract 5 g Glucose 5 g MgSO4·7H2O 1 g NaCl 80 g Distilled water 1 L

pH 7.5


364 Polypepton* 5 g

Yeast extract 5 g Glucose 5 g MgSO4·7H2O 1 g Distilled water 1 L Agar (if needed) 15 g

pH 8.0


365 Polypepton* 5 g

Yeast extract 5 g Glucose 5 g MgSO4·7H2O 1 g Distilled water 1 L

pH 5.5


366 Peptone 10 g


pH 5.5

Media

955

367 mjYPGS Medium NaCl 3 g

K2HPO4 14 mg CaCl2·2H2O 14 mg NH4Cl 12.5 mg NaNO3 12.5 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 Distilled water 1 L

Adjust pH 7.5 - 8.0 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 0.01 mg KAl(SO4)2·12H2O 0.02 g H3BO3 0.01 g Na2MoO4·2H2O 0.01 mg Distilled water 1 L


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


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 Distilled water 1 L

pH 6.8

370 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 Distilled water 1 L

pH 6.8

* 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 Na2MoO4·2H2O 36 mg CuCl2·2H2O 17 mg NiCl2·6H2O 24 mg Distilled water 1 L

** Separately sterilize by filtration and aseptically add to the medium.

372 MMYS-I I Medium Solution I* 500 ml

Solution II** 500 ml pH 7.8

Separately autoclave Solutions I and II. Mix aseptically and adjust final pH to 7.7 - 7.8.

* Solution I

(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

** Solution II

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 Na2MoO4·2H2O 36 mg CuCl2·2H2O 17 mg NiCl2·6H2O 24 mg Distilled water 1 L

373 Nutrient Broth (OXOID) 6.5 g


pH unadjusted

374 PPES-I I 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 Agar 15 g Artificial sea water* 1 L

pH 7.8

Media

956

* Artificial sea water

NaCl 30 g KCl 0.7 g MgCl2·6H2O 10.8 g MgSO4·7H2O 5.4 g CaCl2·2H2O 1 g Distilled water 1 L

376 Ekho Lake St ra ins 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 Na2MoO4·2H2O 12.67 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 B12 0.2 mg p-Aminobenzoic acid 10 mg Distilled water 1 L

*** Artificial sea water

NaCl 23.477 g Na2SO4 3.917 g MgCl2·6H2O 4.981 g CaCl2 1.102 g NaHCO3 192 mg KCl 664 mg KBr 6 mg H3BO3 26 mg SrCl2 24 mg NaF 3 mg Distilled water 1 L

**** 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 Oceani thermus Medium KH2PO4 0.75 g

K2HPO4 0.78 g MgCl2·6H2O 0.36 g CaCl2·2H2O 0.1 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 Distilled water 1 L

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 NaCl 1 g FeSO4·7H2O 0.1 g CoCl2·6H2O 0.17 g CaCl2·2H2O 0.13 g ZnSO4·7H2O 0.18 g CuSO4 0.05 g KAl(SO4)2·12H2O 0.018 g H3BO3 0.01 g Na2MoO4·2H2O 0.011 g NiCl2·6H2O 0.025 g

Dissolve the 2Na-EDTA in 500 ml of distilled water, and after adding the salts, make up to 1 L.

** Vitamin solution

Biotin 2 mg Folic acid 2 mg Pyridoxine-HCl 10 mg Thiamine-HCl 5 mg Riboflavin 5 mg Nicotinic acid 5 mg Ca-pantothenate 5 mg p-Aminobenzoic acid 1 mg Vitamin B12 0.01 mg Distilled water 1 L

Media

957

380 NA + 0 .5% Yeast Ext ract Pepton 5 g

Beef extract 3 g Yeast extract 5 g NaCl 3 g Agar (if needed) 15 g Distilled water 1 L

pH 7.0

382 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 Na2MoO4·2H2O 0.03 g Distilled water 1 L

** 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.

383 2.5% NaCl GYPFK Agar /Broth Glucose 10 g

Polypepton* 5 g Yeast extract 5 g Extract Bonito* 5 g NaCl 25 g K2HPO4 10 g Salt solution** 5 ml Agar (if needed) 15 g Distilled water 1 L

pH 8.5

Sterilize by filtration.


** Salt solution

MgSO4·7H2O 40 g MnSO4·4H2O 2 g FeSO4·7H2O 2 g Distilled water 1 L

384 F lex i l ig lad ius Medium Bacto Heart Infusion Broth (Difco) 2.5 g


pH unadjusted

385 Bi f idobacter ium Medium Casein peptone, tryptic digest 10 g

Meat extract 5 g Yeast 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 Distilled water 1 L

pH 7.5

Add the vitamin solution aseptically to the cooled autoclaved medium.

Media

958

* Basal salts solution

3Na-EDTA 4.53 g FeSO4·7H2O 1.11 g MgSO4·7H2O 24.65 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 Na2MoO4·2H2O 24 mg H3BO3 31 mg Distilled water 1 L

** Phosphate solution

KH2PO4 75 g K2HPO4 78 g Distilled water 1 L

*** 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 Distilled water 1 L

pH 7.0

389 Medium for Tepidanaerobacter Glucose 2.2 g

Yeast extract 2.3 g KH2PO4 0.14 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 Distilled water 1 L

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 B12 2.7 mg p-Aminobenzoic acid 0.3 mg Lipoic acid 0.4 mg Distilled water 1 L


Nitrilotriacetic acid (NTA) 12.8 g FeCl3·6H2O 1.35 g MnCl2·4H2O 0.1 g CoCl2·6H2O 24 mg CaCl2·2H2O 0.1 g ZnCl2 0.1 g CuCl2·2H2O 25 mg H3BO3 10 mg Na2MoO4·2H2O 24 mg NiCl2·6H2O 0.12 g Distilled water 1 L

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 Distilled water 1 L

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 Modi f ied 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 K2HPO4 0.25 g Agar (if needed) 15 g Distilled water 1 L

pH 7.4

Media

959

391 Thermodesul fob ium narugense Medium K2HPO4 0.78 g

KH2PO4 0.75 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 Distilled water 1 L

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

Biotin 2 mg Folic acid 2 mg Pyridoxine-HCl 10 mg Thiamine-HCl 5 mg Riboflavin 5 mg Nicotinic acid 5 mg Ca-pantothenate 5 mg Vitamin B12 0.1 mg p-Aminobenzoic acid 5 mg Lipoic acid 5 mg Distilled water 1 L


Nitrilotriacetic acid (NTA) 12.8 g FeCl3·6H2O 1.35 g MnCl2·4H2O 0.1 g CoCl2·6H2O 0.024 g CaCl2·2H2O 0.1 g ZnCl2 0.1 g CuCl2·2H2O 0.025 g H3BO3 0.01 g Na2MoO4·2H2O 0.024 g NaCl 1 g NiCl2·6H2O 0.12 g Na2SeO3·5H2O 0.026 g Distilled water 1 L

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 K2HPO4 0.14 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 Resazurin 1 mg Na2S·9H2O 0.5 g Distilled water 1 L

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.


Nitrilotriacetic acid 1.5 g MgSO4·7H2O 3 g MnSO4·2H2O 0.5 g NaCl 1 g FeSO4·7H2O 0.1 g CoSO4·7H2O 0.18 g CaCl2·2H2O 0.1 g ZnSO4·7H2O 0.18 g CuSO4·5H2O 0.01 g KAl(SO4)2·12H2O 4 mg H3BO3 0.01 g Na2MoO4·2H2O 0.01 g NiCl2·6H2O 0.025 g Na2SeO3·5H2O 0.8 mg Na2WO4·H2O 0.8 mg Distilled water 1 L

First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Final pH 7.0 (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 K2HPO4 0.14 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 Resazurin 1 mg Na2S·9H2O 0.5 g Distilled water 1 L

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.

Media

960


Nitrilotriacetic acid 1.5 g MgSO4·7H2O 3 g MnSO4·2H2O 0.5 g NaCl 1 g FeSO4·7H2O 0.1 g CoSO4·7H2O 0.18 g CaCl2·2H2O 0.1 g ZnSO4·7H2O 0.18 g CuSO4·5H2O 0.01 g KAl(SO4)2·12H2O 0.02 g H3BO3 0.01 g Na2MoO4·2H2O 0.01 g NiCl2·6H2O 0.025 g Na2SeO3·5H2O 0.3 mg Distilled water 1 L


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 Resazurin 1 mg

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 13.85 g MgSO4·7H2O 3.5 g MgCl2·6H2O 2.75 g KCl 0.325 g NaBr 0.05 g H3BO3 15 mg SrCl2·6H2O 7.5 mg KI 0.05 mg CaCl2 0.75 g KH2PO4 0.5 g (NH4)2Ni(SO4)2·6H2O 2 mg Distilled water 1 L

** Trace minerals

Nitrilotriacetic acid 1.5 g MgSO4·7H2O 3 g MnSO4·xH2O 0.5 g NaCl 1 g FeSO4·7H2O 0.1 g CoSO4·7H2O 0.1 g CaCl2·2H2O 0.1 g ZnSO4·7H2O 0.1 g CuSO4·5H2O 0.01 g AlK(SO4)2 0.01 g H3BO3 0.01 g Na2MoO4·2H2O 0.01 g Distilled water 1 L

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

pH 7.0 - 7.2

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 Sul fo lobus tokodai i 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

(NH4)2SO4 1.3 g KH2PO4 0.28 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 Na2MoO4·2H2O 0.03 mg VOSO4·xH2O 0.03 mg CoSO4·7H2O 0.01 mg Distilled water 1 L


397 VY/2 Agar Baker's yeast 5 g

CaCl2·2H2O 1.36 g Vitamin B12 0.5 mg Bacto Agar (Difco) 15 g Distilled water 1 L

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.

Media

961

398 Methanothermobacter Medium KH2PO4 0.136 g

NH4Cl 0.54 g MgCl2·6H2O 0.2 g CaCl2·2H2O 0.147 g NaHCO3 2.5 g Bacto Yeast Extract (Difco) 0.2 g Vitamin solution* 10 ml Trace element solution** 10 ml Resazurin 1 mg Cysteine-HCl 0.5 g Na2S·9H2O 0.5 g Distilled water 1 L

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


** Trace elements solution



399 Methanobacter ium 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 0.002 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 0.001 g Cysteine-HCl 0.5 g Na2S·9H2O 0.5 g Distilled water 940 ml

Adjust pH to 6.7-7.0

Prepare the medium anaerobically under a atmosphere H2/CO2 (80/20, 150 kPa).

* Trace element solution SL-10

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 Na2MoO4·2H2O 36 mg Distilled water 990 ml

** Sludge fluid

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

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.

Media

962

402 CYC Agar 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 Distilled water 1 L

Adjust pH to 7.2

403 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 Agar (if needed) 15 g Distilled water 1 L

pH 6.7±0.2

404 Oceani thermus profundus Medium NH4Cl 0.33 g

MgCl2·6H2O 0.33 g CaCl2·2H2O 0.33 g KCl 0.33 g KNO3 0.33 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 Distilled water 1 L

Prepare the medium anaerobically, under N2, omitting the CaCl2, MgCl2, KNO3, tryptone, yeast extract, vitamins and sucrose. The pH should be 7.0-7.5. 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





405 Methanosarc ina Medium KH2PO4 0.227 g

K2HPO4 0.348 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 NaHCO3 0.85 g Methanol 10 ml Cysteine-HCl 0.3 g Na2S·9H2O 0.3 g Resazurin 1 mg 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


Media

963

** FeCl2 solution

25% HCl (7.7 M) 10 ml FeCl2·4H2O 1.5 g Distilled water 990 ml

*** Trace element solution

ZnCl2 70 mg MnCl2·4H2O 100 mg H3BO3 6 mg CoCl2·6H2O 190 mg CuCl2·2H2O 2 mg NiCl2·6H2O 24 mg Na2MoO4·2H2O 36 mg Distilled water 1 L


K2HPO4 0.3 g (NH4)2SO4 0.3 g NaCl 0.6 g MgSO2·7H2O 0.13 g FeSO2·7H2O 2 mg CaCl2·2H2O 8 mg Bacto Yeast Extract (Difco) 2 g Trypticase Peptone (BBL) 2 g Sodium acetate 2.5 g Sodium formate 2.5 g Trace minerals* 10 ml Trace vitamins** 10 ml NaHCO3 5 g Na2S·9H2O 0.5 g Cysteine-HCl 0.5 g Resazurin 1 mg Distilled water 1 L

Mix ingredients except NaHCO3, cysteine-HCl and Na2S·9H2O, and bring to a boil for 5-10 sec. Cool down under a stream of H2/CO2 (80/20), and add NaHCO3 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 H2/CO2 (80/20) and seal with butyl rubber stoppers. 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. Pressurize the inoculated vessels to 200 kPa with H2/CO2 (80/20).

* Trace minerals

Nitrilotriacetic acid 1.5 g MgSO4·7H2O 3 g MnSO4·xH2O 0.5 g NaCl 1 g FeSO4·7H2O 0.1 g CoSO4·7H20 0.1 g CaCl2·2H2O 0.1 g ZnSO4·7H2O 0.1 g CuSO4·5H2O 0.01 g AlK(SO4)2 0.01 g H3BO3 0.01 g Na2MoO4·2H2O 0.01 g Distilled water 1 L


** Trace vitamins

Biotin 2 mg Folic acid 2 mg Pyridoxine-HCl 10 mg Thiamine-HCl 5 mg Riboflavin 5 mg Nicotinic acid 5 mg Calcium pantothenate 5 mg Vitamin B12 0.1 mg p-Aminobenzoic acid 5 mg Lipoic acid 5 mg Distilled water 1 L


Yeast extract 2 g MgSO4·7H2O 1 g Distilled water 1 L

pH 7.0


Prepared medium is available from Wako Pure Chemical Ind., Ltd.

703 YM L iqu id Medium Glucose 10 g

Peptone 5 g Yeast extract 3 g Malt extract 3 g Distilled water 1 L

704 YPD L iqu id Medium Yeast extract 10 g

Peptone 20 g Glucose 20 g Distilled water 1 L

pH unadjusted

707 Peptone 5 g

Yeast extract 3 g MgSO4·7H2O 1 g Distilled water 1 L

pH 7.0

708 Polypepton* 5 g

Yeast extract 2 g MgSO4·7H2O 1 g Seawater** 750 ml Distilled water 250 ml

pH 7.0


** Filtered, aged seawater or artificial seawater (Daigo's Artificial Seawater SP*).


Yeast extract 2 g MgSO4·7H2O 1 g Distilled water 1 L Agar (if needed) 15 g

pH 7.0

Media

964


Prepared medium (Code No. 398-01671) is available from Wako Pure Chemical Ind., Ltd.

803 Polypepton* 5 g

Yeast extract 5 g Glucose 5 g Lactose 2 g Tween 80 0.5 g MgSO4·7H2O 1 g Distilled water 1 L Agar (if needed) 15 g

pH 6.5 - 6.8


804 Polypepton* 5 g


pH 6.6 - 7.0



Mannitol 5 g K2HPO4 0.7 g KH2PO4 0.1 g MgSO4·7H2O 1 g Distilled water 1 L Agar (if needed) 15 g

pH 7.0 - 7.2

807 Thiog lyco la te Medium* Polypepton** 15 g

Yeast extract 5 g Glucose 5 g NaCl 2.5 g L-Cystine 0.5 g Sodium thioglycolate 0.5 g Resazurin 1 mg Distilled water 1 L Agar (if needed) 15 g

pH 7.0 - 7.2

* Thioglycolate Medium I for JP general test, Wako Pure Chemicals Ind. Ltd., Osaka, Japan.


808 Methanospi r i l lum Medium KH2PO4 0.3 g

K2HPO4 0.3 g (NH4)2SO4 0.3 g NaCl 0.6 g MgSO2·7H2O 0.13 g FeSO2·7H2O 2 mg CaCl2·2H2O 8 mg Bacto Yeast Extract (Difco) 2 g Trypticase peptone (BBL) 2 g Sodium acetate 2.5 g Sodium formate 2.5 g Trace minerals* 10 ml Trace vitamins** 10 ml NaHCO3 5 g Na2S·9H2O 0.5 g Cysteine-HCl 0.5 g Resazurin 1 mg Distilled water 1 L

Mix ingredients except NaHCO3, cysteine-HCl and Na2S·9H2O, and bring to a boil for 5-10 sec. Cool down under a stream of H2/CO2 (80/20), and add NaHCO3 to the medium. 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. Pressurize the inoculated vessels to 200 kPa with H2/CO2 (80/20).

* Trace minerals



** Trace vitamins


Media

965

809 Glucose 2.2 g

Bacto Yeast Extract (Difco) 2.3 g KH2PO4 0.15 g NH4Cl 0.5 g MgCl2·6H2O 0.2 g CaCl2·2H2O 0.15 g NaHCO3 2.5 g Na2S·9H2O 0.3 g Cysteine-HCl 0.3 g Trace elements solution* 1 ml Vitamin solution** 1 ml Resazurin solution (1 mg/ml) 1 ml Distilled water 1 L

Adjust pH to 7.0 at 25℃

Prepare the medium anaerobically under a N2/CO2 (80/20) atmosphere. Neutralized substrates were added to the vials from stock solutions prior to inoculation.


Nitrilotriacetic acid (NTA) 12.8 g FeCl3·6H2O 1.35 g MnCl2·4H2O 0.1 g CoCl2·6H2O 0.024 g CaCl2·2H2O 0.1 g ZnCl2 0.1 g CuCl2·2H2O 0.025 g H3BO3 0.01 g Na2MoO4·2H2O 0.024 g NiCl2·6H2O 0.12 g Na2SeO3·5H2O 0.026 g Na2WO4·H2O 0.003 g Distilled water 1 L

** Vitamin solution


810 Methanocaldococcus jannaschi i Medium K2HPO4 0.14 g

CaCl2·2H2O 0.14 g NH4Cl 0.25 g MgSO4·7H2O 3.4 g MgCl2·2H2O 2.7 g KCl 0.33 g NiCl2·6H2O 0.75 mg Na2SeO3·5H2O 0.5 mg NaCl 30 g Fe(NH4)2(SO4)2·6H2O 1 mg Trace element solution* 10 ml Vitamin solution** 10 ml Resazurin 1 mg NaHCO3 1 g Cysteine-HCl 0.5 g Na2S·9H2O 0.5 g Distilled water 1 L

Mix ingredients except vitamin solution, cysteine-HCl and Na2S·9H2O, and autoclave under a H2/CO2 (80/20) atmosphere. Separately autoclave cysteine-HCl and Na2S·9H2O as 5% solutions, respectively, under a N2 atmosphere. Prior to inoculation, aseptically and anaerobically add filter-sterilized vitamin, cysteine-HCl and Na2S·9H2O. Pressurize the inoculated bottles to 200 kPa with H2/CO2 (80/20).


Nitrilotriacetic acid 1.5 g MgSO4·7H2O 3 g MnSO4·2H2O 0.5 g NaCl 1 g FeSO4·7H2O 0.1 g CoSO4·7H2O 0.1 g CaCl2·2H2O 0.1 g ZnSO4·7H2O 0.1 g CuSO4·5H2O 0.01 g AlK(SO4)2 0.01 g H3BO3 0.01 g Na2MoO4·2H2O 0.01 g Distilled water 1 L


** Vitamin solution


811 Yeast extract 0.3 g

Ethanol 0.5 ml Disodium succinate 1 g Ammonium acetate 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 Neutralized sulfide solution*** 5 ml Resazurin 1 mg Distilled water 1 L

Mix ingredients except ethanol, vitamin B12 solution and neutralized sulfide solution, and adjust pH to 6.8 with 6 N H2SO4. Dispense the medium into suitable culture vessels under a stream of nitrogen gas, seal with butyl rubber stoppers, and autoclave at 121°C for 15 min. Separately autoclave neutralized sulfide solution under a N2 atmosphere. Sterilize ethanol and vitamin B12 by filtration. Sterile syringes are used to inoculate and remove samples. Prior to inoculation, aseptically and anaerobically add ethanol, vitamin B12 solution and neutralized sulfide solution.

Media

966

* Soluble in hot water.

** Trace element solution SL-6


*** Neutralized sulfide solution


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 2 N 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.

812 Rhodomicrob ium Medium Yeast extract 0.2 g

Disodium succinate 1 g Ammonium acetate 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 Neutralized sulfide solution*** 5 ml Distilled water 1 L

pH 5.7

Mix ingredients except vitamin B12 solution and neutralized sulfide solution, and adjust pH to 5.7 with 6 N H2SO4. Dispense the medium into suitable culture vessels (e.g. 20 ml of the medium in 120 ml serum bottles) under a stream of nitrogen gas, seal with butyl rubber stoppers, and autoclave at 121°C for 15 min. Separately autoclave neutralized sulfide solution under a N2 atmosphere. Sterilize vitamin B12 by filtration. Sterile syringes are used to inoculate and remove samples. Prior to inoculation, aseptically and anaerobically add vitamin B12 solution and neutralized sulfide solution.


** Trace element solution SL-6





813 NaCl 50 g

KCl 2 g MgSO4·7H2O 1 g CaCl2·2H2O 0.36 g NaBr 0.23 g NaHCO3 0.06 g Proteose-Peptone No.3 (Difco) 5 g Bacto Yeast Extract (Difco) 10 g Glucose 1 g Distilled water 1 L Agar (if needed) 15 g

pH 7.2 - 7.4

Trace amount of FeCl3 is added to the medium.

814 Trypt icase Soy Yeast Ext ract Medium Trypticase Soy Broth (Difco) 30 g

Yeast extract 3 g Agar (if needed) 15 g Distilled water 1 L

pH 7.0 - 7.2

815 1/10 Trypt ic Soy Medium Bacto Tryptic Soy Broth (Difco) 3 g


816 Tet ragenococcus ha lophi lus Medium (To MRS Medium add 6.5% NaCl)

Peptone 10 g Meat extract 10 g Yeast extract 5 g Glucose 20 g Tween 80 1 g NaCl 65 g K2HPO4 2 g Sodium acetate 5 g Diammonium hydrogencitrate 2 g MgSO4·7H2O 0.2 g MnSO4·nH2O 0.05 g Distilled water 1 L Agar (if needed) 15 g

pH 6.0 - 6.5

Media

967

817 Tet ragenococcus mur ia t icus Medium (To MRS Medium add 10% NaCl)

Peptone 10 g Meat extract 10 g Yeast extract 5 g Glucose 20 g Tween 80 1 g NaCl 100 g K2HPO4 2 g Sodium acetate 5 g Diammonium hydrogencitrate 2 g MgSO4·7H2O 0.2 g MnSO4·nH2O 0.05 g Distilled water 1 L Agar (if needed) 15 g

pH 6.0 - 6.5

818 GYP-Sodium Acetate-Minera l Sa l ts Broth Glucose 10 g

Bacto Yeast Extract (Difco) 10 g Bacto Peptone (Difco) 10 g Sodium acetate 10 g MgSO4·7H2O 0.2 g MnSO4·xH2O 10 mg FeSO4·7H2O 10 mg NaCl 10 mg Distilled water 1 L

pH 6.8

819 Glucose 40 g

Yeast extract 20 g Peptone 20 g Sodium acetate 20 g Solution B* 10 ml Agar (if needed) 20 g Distilled water 1 L

pH 6.8

* Solution B

MgSO4·7H2O 4 g MnSO4·xH2O 0.2 g FeSO4·7H2O 0.2 g NaCl 0.2 g Distilled water 100 ml

820 Leuconostoc oenos Medium Casein peptone, tryptic digest 10 g

Yeast extract 5 g Glucose 10 g Fructose 5 g MgSO4·7H2O 0.2 g MnSO4·H2O 0.05 g Diammonium hydrogencitrate 3.5 g Tween 80 1 ml Tomato juice, filtered 100 ml Cysteine-HCl 0.5 g Distilled water 900 ml

pH 4.8

821 R Agar Bacto Peptone (Difco) 10 g

Bacto Yeast Extract (Difco) 5 g Bacto Malt Extract (Difco) 5 g Bacto Casamino Acids (Difco) 5 g Bacto Beef Extract (Difco) 2 g Glycerol 2 g Tween 80 50 mg MgSO4·7H2O 1 g Agar 20 g Distilled water 1 L

pH 7.2

822 Micro lunatus Medium Glucose 0.5 g

Peptone 0.5 g Yeast extract 0.5 g Sodium glutamate 0.5 g KH2PO4 0.5 g (NH4)2SO4 0.1 g MgSO4·7H2O 0.1 g Distilled water 1 L

pH 7.0

823 Bacto PPLO Broth (Difco) 21 g

Horse serum* 100 ml L-Arginine 3 g Phenol red 0.02 g Penicillin G** (20,000 U/ml) 5 ml Distilled water 900 ml

pH 6.8-7.0

* Heat inactivated at 56℃ for 30 min.

** Sterilized by filtration.

Dissolve 21 g of PPLO broth, 3 g of L-arginine, and 0.02 g of phenol red in 900 ml of water (adjusted pH to 6.8-7.0. sterilized by autoclave at 121℃ for 15 min). Aseptically add 100 ml of horse serum and 5 ml of Penicillin G (200,000 U/ml, sterilized by filtration) to the cooled broth.

824 Medium for Co-cu l ture of St ra in JT wi th Methanospi r i l lum hungate i NBRC 100397

Na-terephthalate 0.42 g Yeast extract 0.2 g KH2PO4 0.14 g MgCl2·H2O 0.2 g CaCl2·2H2O 0.15 g NH4Cl 0.54 g Vitamin solution* 3 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 Distilled water 1 L

This medium is for co-culture of strain JT with Methanospirillum hungatei strain NBRC 100397.

Media

968

For vial cultures, dissolve all ingredients except for Na-terephthalate, Na2S·9H2O and cysteine-HCl (up to 900ml), flush N2/CO2 (80/20), and autoclave. Add 0.5 ml of each of filter-sterilized Na2S·9H2O solution (0.6g Na2S·9H2O/50 ml) and cysteine-HCl solution (0.6g cysteine-HCl/ 50ml) per each vial separately. Add 0.2 ml of neutralized and filter-sterilized 0.2 M Na-terephthalate solution. Yeast extract is also added at final concentration about 0.02%. Inoculum size 10-20%. Growth was observed after 3-4 weeks of incubation.

* Vitamin solution





*** Se/W solution

Na2SeO3 4 mg Na2WO4 4 mg



825 Ac id ic MRS Medium (pH 4.8) Peptone 10 g


pH 4.8

828 YCA Medium Dry yeast 10 g

CaCl2·2H2O 1 g Agar (if needed) 15 g Distilled water 1 L

pH 7.2

829 HEPES Medium (NH4)2SO4 2.5 g

KH2PO4 0.5 g HEPES* 11.92 g NaHCO3 0.5 g MgSO4·7H2O 100 mg CaCl2·2H2O 5 mg Fe-EDTA 75 mg Distilled water 1 L

pH 7.8 - 8.0

* N-2-Hydroxyethyl piperazine-N'-2-ethane sulfonic acid

830 Ac id ianus manzaensis Medium for Autot rophic Growth

(NH4)2SO4·7H2O 0.132 g KCl 0.052 g KH2PO4 0.041 g MgSO4·7H2O 0.49 g CaCl2·2H2O 0.009 g Fe2(SO4)3·nH2O 14.7 g ZnSO4·7H2O 1 mg CuSO4·5H2O 2 mg MnSO4·5H2O 1 mg NaMoO4·2H2O 0.5 mg CoCl2·6H2O 0.5 mg NiCl2·6H2O 1 mg FeSO4·7H2O 5 mg Distilled water 1 L

Adjust pH to 1.3 with H2SO4.

In aerobic condition, H2 is necessary for autotrophic growth as an electron donor. In anaerobic condition, H2 and Fe3+ are necessary for autotrophic growth as an electron donor and acceptor, respectively.

831 Ac id ianus manzaensis Medium for Heterot rophic Growth

Yeast extract 1 g (NH4)2SO4·7H2O 0.132 g KCl 0.052 g KH2PO4 0.041 g MgSO4·7H2O 0.49 g CaCl2·2H2O 0.009 g ZnSO4·7H2O 1 mg CuSO4·5H2O 2 mg MnSO4·5H2O 1 mg NaMoO4·2H2O 0.5 mg CoCl2·6H2O 0.5 mg NiCl2·6H2O 1 mg FeSO4·7H2O 5 mg Distilled water 1 L


Media

969

832 Pyrod ic t ium Medium NaCl 13.85 g

MgSO4·7H2O 3.5 g MgCl2·6H2O 2.75 g KCl 0.325 g NaBr 0.05 g H3BO3 15 mg SrCl2·6H2O 7.5 mg KI 0.05 mg CaCl2 0.75 g KH2PO4 0.5 g (NH4)2Ni(SO4)2·6H2O 2 mg Trace minerals* 10 ml Bacto Yeast Extract (Difco) 2 g Citric acid 5 mg Sulfur (powder) 30 g Resazurin 1 mg Na2S·9H2O 0.3 g Distilled water 1 L

Mix ingredients except sulfur and Na2S·9H2O, adjust pH to 5.5 with H2SO4, and autoclave under H2/CO2 (80/20) conditions. Separately autoclave neutralize Na2S·9H2O solution. Sulfur is sterilized separately by autoclaving for 60 min at 105℃ on each of 3 successive days. Aseptically and anaerobically add sterile sulfur to the medium under a H2/CO2 (80/20) atmosphere. Prior to inoculation, add Na2S·9H2O solution, and readjust pH to 5.5 if necessary. Pressurize inoculated bottles to 200 kPa with H2/CO2 (80/20).

* Trace minerals



833 Pyrod ic t ium occul tum Medium NaCl 13.85 g

MgSO4·7H2O 3.5 g MgCl2·6H2O 2.75 g KCl 0.325 g NaBr 0.05 g H3BO3 15 mg SrCl2·6H2O 7.5 mg KI 0.05 mg CaCl2 0.75 g KH2PO4 0.5 g (NH4)2Ni(SO4)2·6H2O 2 mg Trace minerals* 10 ml Bacto Yeast Extract (Difco) 2 g Citric acid 5 mg Na2S2O3·5H2O 2.5 g Sulfur (powder) 30 g Resazurin 1 mg Na2S·9H2O 0.3 g Distilled water 1 L

Mix ingredients except sulfur and Na2S·9H2O, adjust pH to 5.5 with H2SO4, and autoclave under H2/CO2 (80/20) conditions. Separately autoclave neutralize Na2S·9H2O solution. Sulfur is sterilized separately by autoclaving for 60 min at 105℃ on each of 3 successive days. Aseptically and anaerobically add sterile sulfur to the medium under a H2/CO2 (80/20) atmosphere. Prior to inoculation, add Na2S·9H2O solution, and readjust pH to 5.5 if necessary. Pressurize inoculated bottles to 200 kPa with H2/CO2 (80/20).

* Trace minerals



Media

970

835 Thermoproteus tenax Medium (NH4)2SO4 0.264 g

FeSO4·7H2O 0.556 g MgSO4·7H2O 0.492 g CaSO4·2H2O 0.344 g KH2PO4 0.014 g NaF 0.84 mg MnCl2·4H2O 0.18 mg Na2B4O7·10H2O 0.45 mg ZnSO4·7H2O 0.22 mg CuCl2·2H2O 0.05 mg Na2MoO7·2H2O 0.03 mg CoSO4·7H2O 0.01 mg Bacto Yeast Extract (Difco) 0.2 g Soluble starch 5 g Sulfur (powder) 10 g Na2S·9H2O 0.5 g Resazurin 1 mg Distilled water 1 L

Mix ingredients except yeast extract, starch, sulfur and Na2S·9H2O, and filter-sterilize while gassing with N2. Autoclave yeast extract and starch as 10% solutions and Na2S·9H2O as a 5% solution under a N2 atmosphere. Sulfur is sterilized separately by autoclaving for 60 min at 105℃ on each of 3 successive days. Aseptically and anaerobically add the yeast extract and starch solutions, and sulfur to the medium. Prior to inoculation, add the sterile Na2S·9H2O solution, and adjust pH to 5.5 with sterile 1 N H2SO4.

836 Thermoproteus neut rophi lus Medium (NH4)2SO4 1.3 g

KH2PO4 0.28 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 Na2MoO4·2H2O 0.03 mg VOSO4·xH2O 0.03 mg CoSO4·7H2O 0.01 mg NaHCO3 0.85 g Bacto Yeast Extract (Difco) 0.2 g Na2S2O4 25 mg Sulfur (powder) 8 g Resazurin 0.5 mg Distilled water 1 L

Mix ingredients except yeast extract, NaHCO3, Na2S2O4 and sulfur, and adjust pH to 6.5. Autoclave, then cool under a H2/CO2 (80/20) atmosphere. Separately autoclave yeast extract as a 10% solution under a N2 atmosphere. Filter-sterilize NaHCO3 as a 8% solution while gassing with N2. Sulfur is sterilized separately by autoclaving for 60 min at 105℃ on each of 3 successive days. Aseptically and anaerobically add the yeast extract and NaHCO3 solutions, and sulfur to the medium under a H2/CO2 (80/20) atmosphere. Readjust pH to 6.5 by further gassing with H2/CO2 (80/20) gas mixture, if necessary. Prior to inoculation, add filter-sterilized 2.5% Na2S2O4 solution. Pressurize inoculated bottles to 150 kPa with H2/CO2 (80/20).

837 Methanosarc ina Medium KH2PO4 0.75 g

K2HPO4 0.75 g MgCl2·6H2O 0.36 g NH4Cl 1 g Bacto Yeast Extract (Difco) 0.1 g Polypepton* 0.1 g Sodium acetate 2.5 g Trimethylamine-HCl 8 g Trace mineral solution** 9 ml Trace vitamins*** 10 ml NaHCO3 0.8 g Na2S·9H2O 0.5 g Cysteine-HCl 0.5 g Resazurin 1 mg Distilled water 981 ml

Mix ingredients except trace vitamins, Na2S·9H2O and cysteine-HCl, and autoclave under a N2/CO2 (80/20) atmosphere. Filter-sterilize vitamin solution while gassing with N2. Separately autoclave Na2S·9H2O and cysteine-HCl as 5% solutions under a N2 atmosphere. Aseptically and anaerobically add vitamin solution to the medium under a N2/CO2 (80/20) atmosphere. Prior to inoculation, add the sterile Na2S·9H2O and cysteine-HCl solutions, and adjust pH to 6.8.


** Trace mineral solution

Nitrilotriacetic acid 4.5 g FeCl2·4H2O 0.4 g MnCl2·4H2O 0.1 g CoCl2·6H2O 0.17 g ZnCl2 0.1 g CaCl2·2H2O 0.02 g H3BO3 0.019 g Na2MoO4·2H2O 0.01 g NiCl2·6H2O 0.03 g Distilled water 1 L

Adjust pH to 7.0 with 1 N KOH

*** Trace vitamins


Media

971

838 Thermoproteus tenax Medium 2 (NH4)2SO4 1.3 g

KH2PO4 0.28 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 Na2MoO4·2H2O 0.03 mg VOSO4·xH2O 0.03 mg CoSO4·7H2O 0.01 mg NaHCO3 0.85 g Bacto Yeast Extract (Difco) 0.2 g Soluble starch 5 g Na2S·9H2O 0.5 g Sulfur (powder) 8 g Resazurin 0.5 mg Distilled water 1 L

Mix ingredients except NaHCO3, yeast extract, soluble starch, Na2S·9H2O and sulfur, and adjust pH to 5.5. Autoclave, then cool under N2/CO2 (80/20) gas mixture. Under a N2 atmosphere, separately autoclave yeast extract, soluble starch and Na2S·9H2O as 2%, 10% and 5% solutions, respectively. Filter-sterilize NaHCO3 as a 8% solution while gassing with N2. Sulfur is sterilized separately by autoclaving for 60 min at 105℃ on each of 3 successive days. Aseptically and anaerobically add the yeast extract, soluble starch and NaHCO3 solutions, and sulfur to the medium under a N2/CO2 (80/20) atmosphere. Readjust pH to 5.5, if necessary. Prior to inoculation, add Na2S·9H2O.

839 SP Medium Raffinose 1 g

Sucrose 1 g Galactose 1 g Soluble starch 5 g Casitone 2.5 g MgSO4·7H2O 0.5 g K2HPO4 0.25 g Agar (if needed) 15 g Distilled water 1 L

pH 7.4

840 Archaeoglobus Medium KP buffer* 5 ml

MgCl2·6H2O 0.75 g CaCl2·2H2O 0.15 g NH4Cl 0.54 g NaCl 30 g Na2S2O3 3.2 g Sodium acetate 1.6 g Trace elements solution** 2 ml Vitamin solution*** 2 ml Coenzyme M 0.14 g Resazurin 1 mg Na2CO3 1 g Na2S·9H2O 0.36 g Distilled water 1 L

pH unadjusted

Mix ingredients except KP buffer, vitamin solution, Na2CO3 and Na2S·9H2O. Dispense the medium into suitable culture vessels under a stream of H2/CO2 (80/20) and seal with butyl rubber stoppers. Separately autoclave KP buffer and Na2S·9H2O (5% solution) under a N2 atmosphere. Sterilize vitamin and 10% Na2CO3 solutions by filtration. Prior to inoculation, aseptically and anaerobically add KP buffer, vitamin, Na2CO3 and Na2S·9H2O solutions. Pressurize the inoculated vessels to 150 kPa with H2/CO2 (80/20).

* KP buffer


Autoclaved under N2.


Nitrilotriacetic acid (NTA) 12.8 g FeCl3·6H2O 1.35 g MnCl2·4H2O 0.1 g CoCl2·6H2O 0.024 g CaCl2·2H2O 0.1 g ZnCl2 0.1 g CuCl2·2H2O 0.025 g H3BO3 0.01 g Na2MoO4·2H2O 0.024 g NaCl 1 g NiCl2·6H2O 0.12 g Na2SeO4 0.004 g Na2WO4 0.004 g Distilled water 1 L

First dissolve nitrilotriacetic acid and adjust pH to 6.5 with NaOH, and then add minerals. Final pH 7.0.




Yeast extract 2 g MgSO4·7H2O 1 g Kanamycin sulfate solution (250 μg/ml)** 100 ml Distilled water 900 ml Agar (if needed) 15 g

pH 7.0



Media

972

842 Mycoplasma Medium wi th Sucrose Bacto Heart Infusion Broth (Difco) 17.5 g

Horse serum* 200 ml 15% Yeast Extract Solution (Gibco 0689) 150 ml Sucrose 40 g Phenol red 40 mg Distilled water 650 ml

* Heat-inactivated at 56℃ for 30 min.

Dissolve 17.5 g of heart infusion broth and 40 mg of phenol red in 450 ml of water (sterilized by autoclaving at 121℃ for 15 min). Dissolve sucrose in 200 ml of water (sterilized by filtration). Aseptically add the horse serum, yeast extract solution, sterile sucrose, and 1 ml of Penicillin G (200,000 U/ml, sterilized by filtration) to the cooled heart infusion broth. Final pH 7.4 +/- 0.2.

843 L Medium Bacto Tryptone (Difco) 10 g

Bacto Yeast Extract (Difco) 5 g NaCl 5 g Glucose 1 g Agar (if needed) 15 g Distilled water 1 L

pH 7.0

844 Pyrococcus Medium NaCl 13.5 g

Na2SO4 4 g MgCl2·6H2O 10.8 g CaCl2·2H2O 1.5 g Yeast extract 1 g Tryptone 5 g Resazurin 1 mg Solution 1* 10 ml Solution 2** 1 ml Sulfur (powder) 30 g Na2S·9H2O 0.25 g Distilled water 1 L

Mix ingredients except solutions 1 and 2, sulfur, and Na2S·9H2O, and autoclave under a N2 atmosphere. Sulfur is sterilized separately by autoclaving for 60 min at 105℃ on each of 3 successive days. Neutralize Na2S·9H2O as a 5% solution, and autoclave under a N2 atmosphere. Aseptically and anaerobically add sterile sulfur and filter-sterile solutions 1 and 2 to the medium. Prior to inoculation, add the sterile Na2S·9H2O solution.

* Solution 1

KCl 7 g NaHCO3 2 g NaBr 1 g Distilled water 1 L

** Solution 2

H3BO3 3 g SrCl2·6H2O 2.5 g Distilled water 1 L

845 Ac id ic Glycero l -Asparagine Agar ( ISP Medium No.5)

L-Asparagine 1 g Glycerol 10 g K2HPO4 1 g Distilled water 1 L Trace salts solution* 1 ml Agar 20 g

pH 5.0



846 BYG Medium ( for Ac id iph i l ium mul t ivorum) Solution A* 500 ml

Solution B** 500 ml

Agar solid medium of BYG contained 2% agar. In order to prevent the hydrolysis of agar and glucose by autoclaving at low pH condition, Solutions A and B are autoclaved separately and combined before solidified (50-60℃).

* Solution A

(NH4)2SO4 2 g KCl 0.1 g KH2PO4 0.5 g MgSO4·7H2O 0.5 g Ca(NO3)2·4H2O 0.001 g Yeast extract 0.1 g Distilled water 500 ml


** Solution B


847 Thermoproteus and Vulcanisaeta Medium (NH4)2SO4 1.3 g

KH2PO4 0.28 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 Na2MoO4·2H2O 0.03 mg VOSO4·nH2O 0.03 mg CoSO4·7H2O 0.01 mg Bacto Yeast Extract (Difco) 1 g Sulfur (powder) 5 g Na2S·9H2O 0.5 g Resazurin 1 mg Distilled water 1 L

Media

973

Mix ingredients except yeast extract, sulfur and Na2S·9H2O, and autoclave under a N2 atmosphere. Separately autoclave yeast extract as a 10% solution and Na2S·9H2O as a 5% solution under a N2 atmosphere. Sulfur is autoclaved for 60 min at 105℃ on each of 3 successive days. Aseptically and anaerobically add yeast extract and sulfur to the medium. Prior to inoculation, add sterile Na2S·9H2O solution, and adjust pH to 4.5-5.0 with sterile 1 N H2SO4.

848 BL Agar BL Agar* 58 g

Defibrinated horse blood 50 ml Distilled water 950 ml

Autoclave except horse blood and cool to about 50℃. Aseptically add 5 % (final) sterile defibrinated horse blood. Mix and quickly dispense into sterile test tubes or petri dishes.

* Nissui Pharmaceutical Co. Ltd., Tokyo, Japan.

849 R2A Medium Bacto Yeast Extract (Difco) 0.5 g

Bacto Proteose Peptone No. 3 (Difco) 0.5 g Bacto Casamino Acids (Difco) 0.5 g Glucose 0.5 g Soluble starch 0.5 g Sodium pyruvate 0.3 g K2HPO4 0.3 g MgSO4·7H2O 0.05 g Agar (if needed) 15 g Distilled water 1 L

pH 7.2+/-0.2

850 Modi f ied MB Medium NH4Cl 1 g

K2HPO4 0.3 g KH2PO4 0.3 g MgCl2·6H2O 0.5 g NaCl 2 g KCl 0.2 g CaCl2·2H2O 0.05 g Yeast extract 1 g Bacto Tryptone (Difco) 2 g Resazurin 0.5 mg Soluble starch 10 g Na2S2O3·5H2O 5 g Cysteine-HCl 0.5 g Trace element solution* 10 ml Distilled water 1 L

Adjust pH to 7.5 with 2 M NaOH

Dissolve ingredients except thiosulfate and cysteine-HCl, boil medium for 3 min, then cool to room temperature under a N2 atmosphere, and add cysteine-HCl. Adjust pH to 7.5, dispense under same gas atmosphere in culture vessels and autoclave. Before inoculation, add thiosulfate from a sterile, anaerobic stock solutions prepared under N2.




851 Thermotoga mar i t ima Medium Soluble starch 5 g

KH2PO4 0.5 g Trace minerals* 10 ml NaCl 20 g Bacto Yeast Extract (Difco) 0.5 g Resazurin 1 mg Na2S·9H2O 0.5 g Artificial sea water** 250 ml Distilled water 750 ml

Mix ingredients except soluble starch and Na2S·9H2O, and adjust pH to 6.5 with NaOH. Sterilize by filtration under a N2 atmosphere. Soluble starch and neutralize Na2S·9H2O are prepared as 10 and 5% solutions, respectively, and autoclaved under a N2 atmosphere. Prior to inoculation, add the sterile soluble starch and Na2S·9H2O solutions.

* Trace minerals

Nitrilotriacetic acid 1.5 g MgSO4·7H2O 3 g MnSO4·2H2O 0.5 g NaCl 1 g FeSO4·7H2O 0.1 g CoSO4·7H2O 0.18 g CaCl2·2H2O 0.1 g ZnSO4·7H2O 0.18 g CuSO4·5H2O 0.01 g H3BO3 0.01 g Na2MoO4·2H2O 0.01 g NiCl2·6H2O 0.025 g Na2SeO3·5H2O 0.8 mg Na2WO4 0.8 mg KAl(SO4)2·12H2O 4 mg Distilled water 1 L


** Artificial sea water

Media

974

NaCl 27.7 g MgSO4·7H2O 7 g MgCl2·6H2O 5.5 g CaCl2·2H2O 2.25 g KCl 0.65 g NaBr 0.1 g H3BO3 30 mg SrCl2·6H2O 15 mg Citric acid 10 mg KI 0.05 mg Distilled water 1 L

852 Pyrobaculum aerophi lum Medium Marine medium/synthetic seawater mix solution* 125 ml

Trace elements** 10 ml NH4Cl 0.25 g NaHCO3 2.2 g KH2PO4 0.07 g Fe(NH4)2(SO4)2·6H2O 2 mg (NH4)2Ni(SO4)2·6H2O 2 mg Bacto Yeast Extract (Difco) 0.5 g Na2S2O3·5H2O 1 g Distilled water 865 ml

After flushing with N2/CO2, adjust pH to 7.0 with 1 N H2SO4. Sterilize by filtration. Dispense the medium into vessels under a N2/CO2/O2 (79/20/1) atmosphere. Pressurize the inoculated vessels to 200 kPa with N2/CO2/O2.

* Marine medium/synthetic seawater mix solution

NaCl 47 g MgCl2·6H2O 18 g MgSO4·7H2O 7 g CaCl2·2H2O 3 g Na2SO4 3.13 g Na2CO3 0.1 g NaBr 0.2 g SrCl2·6H2O 72 mg H3BO3 52 mg Na2HPO4 8.1 mg NaF 2.4 mg KI 0.05 mg Sodium silicate 0.4 g Distilled water 1 L

** Trace elements

Nitrilotriacetic acid 1.5 g MgSO4·7H2O 3 g MnSO4·2H2O 0.5 g NaCl 1 g FeSO4·7H2O 0.1 g CoSO4·7H2O 0.18 g CaCl2·2H2O 0.1 g ZnSO4·7H2O 0.18 g CuSO4·5H2O 0.01 g H3BO3 0.01 g Na2MoO4·2H2O 0.01 g NiCl2·6H2O 0.025 g Na2SeO3·5H2O 0.4 mg Na2WO4 0.4 mg KAl(SO4)2·12H2O 2 mg Distilled water 1 L


853 Picrophi lus tor r idus Medium KH2PO4 3 g

MgSO4·7H2O 0.5 g CaCl2·2H2O 0.25 g Bacto Yeast Extract (Difco) 2 g Glucose 10 g (NH4)2SO4 0.2 g Distilled water 1 L

Mix ingredients except glucose, and adjust pH to 1.0 with 10 N H2SO4. After autoclaving, add filter-sterilized 40% glucose solution.

854 Shr imp Agar Dried shrimp 3 g

Agar 15 g Distilled water 1 L

855 Green Sul fur Bacter ium Medium Solution I* 45 ml

Solution II** 2.5 ml Neutralized sulfide solution*** 2 ml Acetate solution**** 0.5 ml

pH 6.8

After aseptically inject solution II, sulfide solution and acetate solution into vessel of solution I, the pH is adjusted to 6.8 by adding of sterile 0.6 N H2SO4 with a syringe without opening the vessel. If it is necessary, 0.5 ml thiosulfate solution***** is injected.

* Solution I

KH2PO4 0.5 g MgSO4·7H2O 0.5 g CaCl2·2H2O 0.05 g KCl 0.025 g NH4Cl 0.5 g Ammonium acetate 0.5 g Ferric citrate solution (0.1%) 5 ml Vitamin B12 solution (0.001%) 2 ml Trace element solution SL-6 2 ml Resazurin 1 mg Distilled water 950 ml

pH 5.0

Mix ingredients except vitamin solution, and distribute the medium into culture vessels under a stream of nitrogen gas and seal with butyl rubber stoppers. After autoclaving (121°C, 15 min), aseptically inject vitamin solution.

** Solution II

NaHCO3 1.5 g Distilled water 50 ml

pH 8.2, Filter sterile




Media

975

**** Acetate solution

Ammonium acetate 2.5 g Magnesium acetate 2.5 g Fructose 2.5 g Distilled water 100 ml

pH 7.2

***** Thiosulfate solution

Na2S2O3·5H2O 5 g Distilled water 100 ml

pH 7.1

Solution is prepared in a vessel sealed with a butyl rubber stopper. Autoclave at 121℃ for 15 min under a N2 atmosphere.

856 Potato Dext rose Agar (PDA) Potato 200 g

Glucose 10 g Agar 15 g Distilled water 1 L

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 stir till dissolved. Make up to 1 L. Adjust pH to 5.4 - 5.6. Autoclave to sterilize.

857 Sul fa te Reducer Medium KP buffer* 10 ml

MgCl2·6H2O 0.75 g CaCl2·2H2O 0.15 g NH4Cl 0.54 g Na2S2O3 1.6 g Na2SO4 1.4 g Trace elements solution** 2 ml Vitamin solution*** 2 ml Resazurin 1 mg Na2CO3 0.5 g Na2S·9H2O 0.36 g Distilled water 1 L

pH unadjusted


* KP buffer




Nitrilotriacetic acid (NTA) 12.8 g FeCl3·6H2O 1.35 g MnCl2·4H2O 0.1 g CoCl2·6H2O 0.024 g CaCl2·2H2O 0.1 g ZnCl2 0.1 g CuCl2·2H2O 0.025 g H3BO3 0.01 g Na2MoO4·2H2O 0.024 g NaCl 1 g NiCl2·6H2O 0.12 g Na2SeO4 0.004 g Na2WO4 0.004 g KAl(SO4)2·12H2O 0.02 g Distilled water 1 L




859 Methanopyrus Medium NH4Cl 0.25 g

K2HPO4 0.05 g KH2PO4 0.09 g NaCl 11.8 g MgSO4·7H2O 1.75 g MgCl2·6H2O 4.5 g Na2SO4 0.81 g CaCl2·2H2O 0.78 g KCl 0.3 g NaHCO3 1 g Fe(NH4)2(SO4)2·6H2O 2 mg (NH4)2Ni(SO4)2·6H2O 2 mg NaBr 0.04 g SrCl2·6H2O 0.018 g H3BO3 0.013 g KI 0.0125mg Sodium silicate 1 mg NaF 0.6 mg KNO3 0.4 mg Na2HPO4·12H2O 4.6 mg Bacto Yeast Extract (Difco) 0.2 g Coenzyme M 0.14 g Trace elements solution* 10 ml Vitamin solution** 10 ml Resazurin 1 mg Na2S·9H2O 0.5 g Distilled water 1 L

Mix ingredients except Na2S·9H2O, and adjust pH to 6.0 with H2SO4. Sterilize by filtration while gassing H2/CO2 (80/20). Separately autoclave Na2S·9H2O (5% solution) under a N2 atmosphere. Prior to inoculation, aseptically and anaerobically add Na2S·9H2O solution. Pressurize the inoculated vessels to 150 kPa with H2/CO2 (80/20).

Media

976




** Vitamin solution


860 Methanosarc ina acet ivorans Medium NaCl 23.4 mg

MgSO4·7H2O 9.45 g Na2CO3 5 g NH4Cl 1 g KCl 0.8 g CaCl2·2H2O 0.14 g Na2HPO4 0.6 g Bacto Yeast Extract (Difco) 1 g Trace elements solution* 10 ml Vitamin solution** 10 ml Methanol 6.5 ml Resazurin 1 mg Cysteine-HCl 0.3 g Na2S·9H2O 0.3 g Distilled water 1 L

Mix ingredients except vitamin solution, cysteine-HCl, and Na2S·9H2O. Flush with N2/CO2, and adjust pH to 7.0 with HCl. Dispense the medium into suitable culture vessels under a stream of N2/CO2 (80/20) and seal with butyl rubber stoppers. Separately autoclave cysteine-HCl and Na2S·9H2O (5% solution) under a N2 atmosphere. Sterilize vitamin solution by filtration. Prior to inoculation, aseptically and anaerobically add vitamin, cysteine-HCl and Na2S·9H2O solutions.




** Vitamin solution


861 Sul fur isphaera Medium (pH 2.5) (NH4)2SO4 1.3 g

KH2PO4 0.28 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 Na2MoO4·2H2O 0.03 mg VOSO4·nH2O 0.03 mg CoSO4·7H2O 0.01 mg Bacto Yeast Extract (Difco) 1 g Distilled water 1 L

pH 2.5

Mix ingredients except Bacto Yeast Extract, adjust pH to 2.5 with H2SO4, and autoclave at 121°C for 15 min. Separately autoclave Bacto Yeast Extract as a 10% solution. Prior to inoculation, add Bacto Yeast Extract solution.

Media

977

862 Sul fur isphaera Medium (pH 5.0) (NH4)2SO4 1.3 g

KH2PO4 0.28 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 Na2MoO4·2H2O 0.03 mg VOSO4·nH2O 0.03 mg CoSO4·7H2O 0.01 mg Bacto Yeast Extract (Difco) 1 g Distilled water 1 L

pH 5.0

Mix ingredients except Bacto Yeast Extract, adjust pH to 5.0 with H2SO4, and autoclave at 121°C for 15 min. Separately autoclave Bacto Yeast Extract as a 10% solution. Prior to inoculation, add Bacto Yeast Extract solution.

863 Modi f ied MMJY Medium NaCl 20 g

K2HPO4 0.14 g CaCl2·2H2O 1 g NaNO3 1 g MgSO4·7H2O 3.4 g MgCl2·6H2O 4.2 g KCl 0.33 g Bacto Yeast Extract (Difco) 0.1 g Sodium pyruvate 0.2 g Sodium lactate 0.2 g Sodium acetate 0.2 g Trace elements solution* 10 ml Vitamin solution** 10 ml NaHCO3 0.1 g Resazurin 1 mg Na2S·9H2O 0.5 g Distilled water 1 L

Mix ingredients except vitamin solution, NaHCO3 and Na2S·9H2O, and adjust pH to 7.5 - 8.0 with HCl. Dispense the medium into suitable culture vessels under a stream of H2/CO2 (80/20) and seal with butyl rubber stoppers. Separately autoclave Na2S·9H2O (5% solution) under a N2 atmosphere. Sterilize vitamin and NaHCO3 solutions by filtration. Prior to inoculation, aseptically and anaerobically add vitamin, NaHCO3 and Na2S·9H2O solutions, and pressurize the inoculated vessels to 150 kPa with H2/CO2 (80/20).


Nitrilotriacetic acid (NTA) 12.8 g FeCl3·6H2O 1.35 g MnCl2·4H2O 0.1 g CoCl2·6H2O 0.024 g CaCl2·2H2O 0.1 g ZnCl2 0.1 g CuCl2·2H2O 0.025 g H3BO3 0.01 g Na2MoO4·2H2O 0.024 g NaCl 1 g NiCl2·6H2O 0.12 g Na2SeO4 0.8 mg Na2WO4 0.8 mg KAl(SO4)2·12H2O 4 mg Distilled water 1 L

First dissolve nitrilotriacetic acid and adjust pH to 6.5 with NaOH, and then add minerals. Final pH is 7.0.

** Vitamin solution


864 Medium for A l icyc lobac i l lus ac id iph i lus Solution A* 500 ml

Solution B** 500 ml

* Solution A

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 2.5 - 3.0

** Solution B


pH 5.5 - 6.0


865 Haroarcu la Medium Casamino Acids (Difco) 5 g

Bacto Yeast Extract (Difco) 5 g Sodium glutamate 1 g Trisodium citrate 3 g MgSO4·7H2O 20 g KCl 2 g NaCl 200 g FeCl2·4H2O 36 mg MnCl2·4H2O 0.26 mg Distilled water 1 L Agar (if needed) 20 g

pH 7.0-7.2

866 Thermus Nutr ient Medium Polypeptone 8 g

Bacto Yeast Extract (Difco) 4 g NaCl 2 g CaCl2·2H2O 0.05 g MgCl2·6H2O 0.08 g Distilled water 1 L

Adjust pH to 7.5 with NaOH

867 Thermus Medium Polypeptone 8 g

Bacto Yeast Extract (Difco) 4 g NaCl 2 g Distilled water 1 L


Media

978

For solid medium, equal volumes of separately autoclaved 1.6% gelrite and double-strength liquid medium containing 2 g/L MgSO4·7H2O are mixed.

868 1/5 Mar ine Broth + 0 .5% Sodium Pyruvate Bacto Marine Broth 2216 (Difco) 7.5 g

Sodium pyruvate 5 g Agar (if needed) 15 g Sea water* 800 ml Distilled water 200 ml

pH 7.6

* Filtered, aged seawater or artificial seawater (Daigo's Artificial Seawater SP**)


869 Medium for St ra in C/G2 KH2PO4 0.2 g

NH4Cl 0.25 g NaCl 20 g MgCl2·6H2O 3 g KCl 0.5 g CaCl2·2H2O 0.15 g Trace element mixture* 1 ml Selenite-tungstate solution** 1 ml Vitamin mixture*** 1 ml Vitamin B12 solution**** 1 ml Bicarbonate solution (1.0 M)***** 30 ml Dithionite (ca.) 20 mg Distilled water 1 L

Mix ingredients except vitamin mixture, vitamin B12 solution, bicarbonate solution and dithionite, and adjust pH to about 7.5. Dispense the medium into suitable culture vessels under a stream of nitrogen gas, seal with butyl rubber stoppers, and autoclave at 121°C for 15 min. Aseptically and anaerobically add vitamin mixture, vitamin B12 solution, bicarbonate solution and dithionite solution using sterile syringes, and the N2/CO2 (80/20) mixed gas is enclosed with the vessels. Adjust pH to 7.2- 7.8 using compact pH meter. An aqueous solution of sodium glutamate (final concentration, 10 mM) and sodium thiosulfate (final concentration, 20 mM) are added before inoculation. Addition of yeast extract (0.5 - 1 g/L) greatly improves the growth of the strain.

* Trace element mixture

HCl (25%) 12.5 ml FeSO4·7H2O 2100 mg H3BO3 30 mg MnCl2·4H2O 100 mg CoCl2·6H2O 190 mg NiCl2·6H2O 24 mg CuCl2·2H2O 2 mg ZnSO4·7H2O 144 mg Na2MoO4·2H2O 36 mg Distilled water 987 ml

Sterilized by autoclaving.

** Selenite-tungstate solution

NaOH 0.4 g Na2SeO·5H2O 6 mg Na2WO4·2H2O 8 mg Distilled water 1 L


*** Vitamin mixture

4-Aminobenzoic acid 4 mg D(+)-Biotin 1 mg Nicotinic acid 10 mg Calcium D(+)-panthothenate 5 mg Pyridoxine dihydrochloride 15 mg Thiamine-HCl 10 mg Distilled water 100 ml


**** Vitamin B12 solution

Cyanocobalamin 5 mg Distilled water 100 ml


***** Bicarbonate solution (1.0 M)

NaHCO3 84 g Distilled water 1 L


870 Medium for St ra in C/L2 Na2SO4 4 g

KH2PO4 0.2 g NH4Cl 0.25 g NaCl 20 g MgCl2·6H2O 3 g KCl 0.5 g CaCl2·2H2O 0.15 g Trace element mixture* 1 ml Selenite-tungstate solution** 1 ml Vitamin mixture*** 1 ml Vitamin B12 solution**** 1 ml Bicarbonate solution (1.0 M)***** 30 ml Sulfide solution****** 24 ml Distilled water 1 L

Mix ingredients except vitamin mixture, vitamin B12 solution, bicarbonate solution and sulfide solution, and adjust pH to about 7.5. Dispense the medium into suitable culture vessels under a stream of nitrogen gas, seal with butyl rubber stoppers, and autoclave at 121°C for 15 min. Aseptically and anaerobically add vitamin mixture, vitamin B12 solution, bicarbonate solution and sulfide solution using sterile syringes, and the N2/CO2 (80/20) mixed gas is enclosed with the vessels. Adjust pH to 7.2- 7.8 using compact pH meter. An aqueous solution of sodium lactate (final concentration, 20 mM) is added as the electron donor and carbon source before inoculation. Addition of yeast extract (0.5 - 1 g/L) greatly improves the growth of the strain.

* The trace element mixture

HCl (25%) 12.5 ml FeSO4·7H2O 2100 mg H3BO3 30 mg MnCl2·4H2O 100 mg CoCl2·6H2O 190 mg NiCl2·6H2O 24 mg CuCl2·2H2O 2 mg ZnSO4·7H2O 144 mg Na2MoO4·2H2O 36 mg Distilled water 987 ml


Media

979

** The selenite-tungstate solution

NaOH 0.4 g Na2SeO·5H2O 6 mg Na2WO4·2H2O 8 mg distilled water 1 L


*** The vitamin mixture

4-Aminobenzoic acid 4 mg D(+)-Biotin 1 mg Nicotinic acid 10 mg Calcium D(+)-panthothenate 5 mg Pyridoxine dihydrochloride 15 mg Thiamine-HCl 10 mg Distilled water 100 ml


**** The vitamin B12 solution

Cyanocobalamin 5 mg Distilled water 100 ml


***** The bicarbonate solution (1.0 M)

NaHCO3 84 g Distilled water 1 L


******Neutralized sulfide solution



871 Trypt ic Soy Broth Bacto Tryptic Soy Broth (Difco) 30 g


872 Trypt ic Soy Agar Bacto Tryptic Soy Agar (Difco) 40 g


873 Skim milk 100 g

Tomato juice* 100 ml Yeast extract 5 g Distilled water, make up to 1 L

Autoclave at 121℃ for 15 minutes.

* Filter canned tomatoes through paper. Leave overnight at 10℃. Adjust pH to 7.0.

874 Mar ine Ac idoph i l ic Sul fur -ox id iz ing Bacter ia Medium

(NH4)2SO4 3 g K2HPO4 5 g MgSO4·7H2O 5 g KCl 1 g Ca(NO3)2 0.3 g NaCl 20 g Sulfur (powder)* 10 g Distilled water 1 L

pH 4

* Sterilize separately by autoclaving for 60 min at 105℃ on each of 3 successive days and add after inoculation.

875 M17 Broth M17 Broth (OXOID) 37.25 g

Distilled water 950 ml

After sterilization by autoclaving at 121°C for 15 min, aseptically add 50 ml of sterile lactose solution (10% w/v).

876 Yeast -Starch Agar (pH 5) Yeast extract 2 g


Adjust pH 5 after autoclaving.

877 Archaeoglobus profundus Medium KP buffer* 5 ml

MgCl2·6H2O 0.75 g CaCl2·2H2O 0.15 g NH4Cl 0.54 g NaCl 30 g Na2SO4 2.4 g Na2S2O3·5H2O 2.4 g Sodium acetate 0.8 g Sodium pyruvate 1.1 g Bacto Yeast Extract (Difco) 0.5 g Fe(NH4)2(SO4)2·7H2O 2 mg Trace elements solution** 2 ml Vitamin solution*** 2 ml Resazurin 1 mg Na2CO3 1 g Na2S·9H2O 0.36 g Distilled water 1 L

pH unadjusted


* KP buffer



Media

980







K2HPO4 0.75 g NH4Cl 1 g MgCl2·6H2O 0.36 g Bacto Yeast Extract (Difco) 0.1 g Polypeptone 0.1 g Trace elements solution* 10 ml Vitamin solution 10 ml Resazurin 1 mg NaHCO3 4.8 g Cysteine-HCl 0.5 g Na2S·9H2O 0.5 g Distilled water 1 L

Mix ingredients except for vitamin solution, cysteine-HCl and Na2S·9H2O, and autoclave under H2/CO2 (80/20) condition. Separately autoclave cysteine-HCl and Na2S·9H2O as a 5% solution under a N2 atmosphere. Aseptically and anaerobically add filter-sterile vitamin solution, and cysteine-HCl and Na2S·9H2O solutions, and pressurize inoculated bottles to 150 kPa with H2/CO2.


Nitrilotriacetic acid (NTA) 4.1 g FeCl3·6H2O 0.36 g MnCl2·4H2O 0.09 g CoCl2·6H2O 0.15 g CaCl2·2H2O 0.018 g ZnCl2 0.09 g H3BO3 0.017 g Na2MoO4·2H2O 0.009 g NiCl2·6H2O 0.018 g Distilled water 1 L


** Vitamin solution


880 Methanocul leus Medium NH4Cl 0.25 g

KCl 0.34 g MgCl2·6H2O 4 g MgSO4·7H2O 3.5 g CaCl2·2H2O 0.14 g K2HPO4 0.14 g NaCl 6 g Trace elements solution* 10 ml Vitamin solution** 10 ml Bacto Yeast Extract (Difco) 2 g Polypeptone 2 g Sodium acetate 1 g Resazurin 1 mg NaHCO3 5 g Cysteine-HCl 0.5 g Na2S·9H2O 0.5 g Distilled water 1 L

Mix ingredients except for vitamin solution, NaHCO3, cysteine-HCl and Na2S·9H2O, and autoclave under H2/CO2 (80/20) condition. Separately autoclave cysteine-HCl and Na2S·9H2O as a 5% solution under a N2 atmosphere. Aseptically and anaerobically add filter-sterile vitamin and NaHCO3 solutions, and cysteine-HCl and Na2S·9H2O solutions, and pressurize inoculated bottles to 150 kPa with H2/CO2.


Nitrilotriacetic acid (NTA) 4.1 g FeCl3·6H2O 0.36 g MnCl2·4H2O 0.09 g CoCl2·6H2O 0.15 g CaCl2·2H2O 0.018 g ZnCl2 0.09 g H3BO3 0.017 g Na2MoO4·2H2O 0.009 g NiCl2·6H2O 0.018 g Distilled water 1 L


** Vitamin solution


Media

981

881 GYP Medium (pH 6) Glucose 10 g

Bacto Yeast Extract (Difco) 10 g Polypepton* 5 g Sodium acetate 2 g Tween 80 solution** 10 ml Salts solution*** 5 ml Distilled water 1 L

pH 6.0

Add 5 g/L CaCO3 and 15 g/L agar after the pH is adjusted for the solid medium.


** Tween 80 solution

Tween 80 50 mg Distilled water 1 ml

*** Salts solution

MgSO4·7H2O 40 mg MnSO4·4H2O 2 mg FeSO4·7H2O 2 mg NaCl 2 mg Distilled water 1 ml

Add a drop of 12 N HCl to 500 ml of the salts solution.

882 Defer r ibacter Medium NH4Cl 0.535 g

KH2PO4 0.136 g MgCl2·6H2O 0.204 g CaCl2·2H2O 0.147 g Trace elements solution* 1 ml Vitamin solution** 1 ml Sodium acetate 0.82 g Sodium nitrate 0.85 g NaHCO3 2.52 g Resazurin 1 mg Na2S·9H2O 0.5 g Distilled water 1 L

Mix ingredients except for vitamin solution and Na2S·9H2O, and autoclave under H2/CO2 (80/20) condition. Separately autoclave Na2S·9H2O as a 5% solution under a N2 atmosphere. Aseptically and anaerobically add filter-sterile vitamin solution and Na2S·9H2O solution, and pressurize inoculated bottles to 150 kPa with H2/CO2.




** Vitamin solution


883 TSB + 0.2%(w/v) Na 2 CO3 Bacto Tryptic Soy Broth* 30 g

Na2CO3** 2 g Agar (if needed) 15 g Distilled water 1 L

* Becton Dickinson and Company, Cockeysville, MD USA.

** Sterilize separately by autoclaving and add to other ingredients. The final pH is around 8.5.

884 LB Broth + 0.05%(w/v) Na 2 CO3 Bacto LB Broth, Lennox (Difco) 20 g

Na2CO3* 0.5 g Agar (if needed) 15 g Distilled water 1 L

* Sterilize separately by autoclaving and add to other ingredients. The final pH is around 8.0.

885 Nut r ient Broth + 0.5%(w/v) Na 2 CO 3 Bacto Nutrient Broth (Difco) 8 g

Na2CO3* 5 g Agar (if needed) 15 g Distilled water 1 L

* Sterilize separately by autoclaving and add to other ingredients. The final pH is around 10.5.

886 Al lanto in Minera l Medium K2HPO4 0.8 g

KH2PO4 0.2 g MgSO4·7H2O 0.5 g CaCl2·2H2O 0.05 g FeSO4·7H2O 0.01 g MnSO4·H2O 1 mg Allantoin 20 g Agar 15 g Distilled water 1 L

pH 7.2

887 Guaiaco l -CMC Agar Difco Yeast Carbon Base 11.7 g

Ammonium tartrate 0.2 g CM-Cellulose 0.1 g Agar 15 g Guaiacol 0.1 g Distilled water 1 L

pH 4.5

Media

982

889 Tre-P-Y Agar Trehalose 20 g

Peptone 3 g Bacto Yeast Extract (Difco) 1 g Distilled water 1 L Agar 15 g

pH 6.7

890 LB + Ampic i l l in Medium Bacto Tyrptone (Difco) 10 g

Yeast extract 5 g NaCl 10 g Sodium ampicillin solution (750 μg/ml)* 100 ml Distilled water 900 ml Agar (if needed) 15 g

pH 7.0


891 Soybean Casein Digest Agar Casein peptone 15 g

Soybean peptone 5 g NaCl 5 g Agar 15 g Distilled water 1 L

pH 7.1-7.5

892 Soybean Casein Digest Broth Casein peptone 17 g

Soybean peptone 3 g K2HPO4 2.5 g Glucose 2.5 g NaCl 5 g Distilled water 1 L

pH 7.1-7.5

893 1/5 LBM Medium Bacto Tryptone (Difco) 2 g

Bacto Yeast Extract (Difco) 1 g Agar (if needed) 15 g Seawater* 1 L

pH 7.0



895 Solution A* 500 ml

Solution B** 500 ml

* Solution A

Yeast extract 1 g Malt extract 2.5 g Glucose 1 g Distilled water 500 ml

pH 4 (by H2SO4)

** Solution II

Agar 18 g Distilled water 500 ml


897 Methanothr ix Medium Sodium acetate 6.56 g

NH4Cl 1 g KH2PO4 0.3 g NaCl 0.6 g MgCl2·6H2O 0.1 g CaCl2·2H2O 0.08 g Trace elements solution* 5 ml Vitamin solution** 10 ml Resazurin 1 mg NaHCO3 3.5 g Cysteine-HCl 0.5 g Na2S·9H2O 0.5 g Distilled water 1 L

Mix ingredients except for vitamin solution, cysteine-HCl and Na2S·9H2O, and autoclave under N2/CO2 (80/20) condition. Separately autoclave cysteine-HCl and Na2S·9H2O as a 5% solution under a N2 atmosphere. Aseptically and anaerobically add filter-sterile vitamin, cysteine-HCl and Na2S·9H2O solutions.


Nitrilotriacetic acid (NTA) 4.1 g FeCl3·6H2O 0.36 g MnCl2·4H2O 0.09 g CoCl2·6H2O 0.15 g CaCl2·2H2O 0.018 g ZnCl2 0.09 g H3BO3 0.017 g KAl(SO4)2·12H2O 0.02 g Na2MoO4·2H2O 0.009 g NiCl2·6H2O 0.018 g Na2SeO3 1.6 mg Na2WO4 1.6 mg KAl(SO4)2·12H2O 8 mg Distilled water 1 L


** Vitamin solution


898 1/5 Nutr ient Agar Bacto Nutrient Agar (Difco) 4.6 g

Agar 14 g Distilled water 1 L

899 GYP Medium Glucose 10 g

Bacto Yeast Extract (Difco) 10 g Polypepton* 5 g Sodium acetate 2 g Tween 80 solution** 10 ml Salts solution*** 5 ml Distilled water 1 L

pH 6.8

Media

983

Add 5 g/L CaCO3 and 15 g/L agar after the pH is adjusted for the solid medium.


** Tween 80 solution

Tween 80 50 mg Distilled Water 1 ml

*** Salts solution

MgSO4·7H2O 40 mg MnSO4·4H2O 2 mg FeSO4·7H2O 2 mg NaCl 2 mg Distilled Water 1 ml

Add a drop of 12 N HCl to 500 ml of the salts solution.

900 Thermanaeromonas Medium K2HPO4 0.78 g

KH2PO4 0.75 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 NaHCO3 5 g Na2S2O3 0.8 g Glucose 0.5 g Bacto Yeast Extract (Difco) 0.5 g Vitamin solution* 10 ml Trace element solution** 10 ml Cysteine-HCl 0.5 g Distilled water 1 L

Mix ingredients except vitamin solution and cysteine-HCl. Dispense the medium into suitable culture vessels under a stream of N2/CO2 (80/20), seal with butyl rubber stoppers and autoclave. Separately autoclave 5% cysteine-HCl solution under a N2 atmosphere. Sterilize vitamin solution by filtration. Prior to inoculation, aseptically add vitamin and cysteine-HCl solutions.

* Vitamin solution



Nitrilotriacetic acid (NTA) 12.8 g FeCl3·6H2O 1.35 g MnCl2·4H2O 0.1 g CoCl2·6H2O 0.024 g CaCl2·2H2O 0.1 g ZnCl2 0.1 g CuCl2·2H2O 0.025 g H3BO3 0.01 g Na2MoO4·2H2O 0.024 g NaCl 1 g NiCl2·6H2O 0.12 g Na2SeO3·5H2O 0.026 g Distilled water 1 L

901 Sul fur -ox id iz ing Bacter ia Medium KP buffer* 10 ml

MgCl2·6H2O 0.75 g CaCl2·2H2O 0.15 g NH4Cl 0.54 g Na2S2O3 3.2 g Bacto Yeast Extract (Difco) 0.1 g Trace element solution** 2 ml Vitamin solution*** 2 ml Na2CO3 0.5 g Distilled water 1 L

Mix ingredients except KP buffer and vitamin solution. Dispense the medium into suitable culture vessels under a stream of N2/CO2/O2 (75/20/5) and seal with butyl rubber stoppers. Separately autoclave KP buffer under a N2 atmosphere. Sterilize vitamin solution by filtration. Prior to inoculation, aseptically add KP buffer and vitamin solution. Pressurize the inoculated vessels to 150 kPa with N2/CO2/O2.

* KP buffer






Media

984



902 Reinforced Clost r id ia l Medium CM0149 Broth (OXOID) 38 g


Autoclave at 121℃ for 15 minutes.

903 TcP Medium NaCl 30 g

KCl 0.33 g NH4Cl 0.25 g MgCl2·6H2O 2.8 g MgSO4·7H2O 3.4 g FeSO4·7H2O 0.025 g K2HPO4 0.3 g NaBr 0.01 g Trace minerals* 10 ml Vitamin solution** 10 ml Bacto Yeast Extract (Difco) 3 g Bacto Tryptone (Difco) 3 g PIPES 6 g Sulfur (powder) 10 g Resazurin 1 mg Na2S·9H2O 0.5 g Distilled water 1 L

Mix ingredients except vitamin solution, sulfur and Na2S·9H2O, and adjust pH to 7. Autoclave under a N2 atmosphere. Filter-sterilize vitamin solution. Sulfur is sterilized separately by autoclaving for 60 min at 105℃ on each successive days. Neutralize Na2S·9H2O as 5% solution and autoclave under a N2 atmosphere. Prior to inoculation, aseptically and anaerobically add vitamin, sulfur and Na2S·9H2O.

* Trace minerals



** Vitamin solution


904 Polypepton* 5 g

Yeast extract 5 g Glucose 5 g MgSO4·7H2O 1 g NaCl 10 g Agar (if needed) 15 g Distilled water 1 L

pH 7.0 - 7.5


905 M Medium (EBR45) KH2PO4 0.4 g

K2HPO4·3H2O 0.4 g NH4Cl 1 g MgCl2·6H2O 0.1 g Yeast extract 0.2 g NaHCO3 6 g Cysteine-HCl 0.5 g Na2S·9H2O 0.25 g Resazurin 1 mg Cellulose (or Cellobiose) 10 g Vitamin solution* 10 ml Mineral solution** 10 ml Distilled water 1 L

pH 7.5

Mix ingredients except NaHCO3, cysteine-HCl, Na2S·9H2O and vitamin solution, distribute the medium into culture vessels under a stream of nitrogen gas, and seal with butyl rubber stoppers. After autoclaving (121°C, 15 min), aseptically inject other ingredients. The pH is adjusted to 7.5 by adding of sterile 0.6 N H2SO4 with a syringe without opening the vessel.

* Vitamin solution

Biotin 2 mg Folic acid 2 mg Pyridoxine-HCl 10 mg Thiamine-HCl 5 mg Riboflavin 5 mg Nicotinic acid 5 mg Ca-pantothenate 5 mg Vitamin B12 1 mg p-Aminobenzoic acid 5 mg Lipoic acid 5 mg Distilled water 1 L

Media

985

** Mineral solution

Nitrilotriacetic acid (NTA) 4.5 g FeCl2·4H2O 0.4 g CoCl2·6H2O 0.12 g AlK(SO4)2 0.01 g NaCl 1 g CaCl2 0.02 g Na2MoO4·2H2O 0.01 g MnCl2·4H2O 0.1 g ZnCl2 0.1 g H3BO3 0.01 g CuSO4·5H2O 0.01 g NiCl2·6H2O 0.02 g Distilled water 1 L

906 MRS+8% NaCl Peptone 10 g

Lab-Lemco powder 8 g Yeast extract 4 g Glucose 20 g Sorbitan mono-oleate 1 ml K2HPO4 2 g Sodium acetate 5 g Triammonium citrate 2 g MgSO4·7H2O 0.2 g MnSO4·4H2O 0.05 g NaCl 80 g Distilled water 1 L Agar (if needed) 15 g

pH 6.0 - 6.4

907 ChrysPYG Chrysalis extract 1 g

Proteose peptone 1 g Yeast extract 1 g Glucose 1 g Distilled water 1 L Agar 15 g

908 Medium for St ra in SANAE KH2PO4 0.14 g

NH4Cl 0.54 g MgCl2·6H2O 0.2 g CaCl2·2H2O 0.15 g Bacto Yeast Extract (Difco) 0.1 g Sodium acetate 1 g Trace elements solution* 1 ml Vitamin solution 2 ml Resazurin 1 mg NaHCO3 2.5 g Cysteine-HCl 0.3 g Na2S·9H2O 0.3 g Distilled water 1 L

Mix ingredients except for vitamin solution, cysteine-HCl and Na2S·9H2O, and autoclave under H2/CO2 (80/20) condition. Separately autoclave cysteine-HCl and Na2S·9H2O as a 6% solution under a N2 atmosphere. Aseptically and anaerobically add filter-sterile vitamin solution, and cysteine-HCl and Na2S·9H2O solutions, and pressurize inoculated bottles to 150 kPa with H2/CO2.


Nitrilotriacetic acid (NTA) 4.1 g FeCl3·6H2O 0.36 g MnCl2·4H2O 0.09 g CoCl2·6H2O 0.15 g CaCl2·2H2O 0.018 g ZnCl2 0.09 g H3BO3 0.017 g Na2MoO4·2H2O 0.009 g NiCl2·6H2O 0.018 g Na2SeO4 0.004 g Na2WO4 0.004 g Distilled water 1 L


** Vitamin solution


909 Micro lunatus Medium Glucose 0.5 g

Peptone 0.5 g Yeast extract 0.5 g Monosodium glutamate 0.5 g KH2PO4 0.44 g (NH4)2SO4 0.1 g MgSO4·7H2O 0.1 g Distilled water 1 L Agar 15 g

Adjust pH to 7.0

910 GS Medium KCl 0.355 g

KH2PO4 0.14 g MgCl2·6H2O 4 g MgSO4·7H2O 3.45 g NH4Cl 0.25 g CaCl2·2H2O 0.14 g NaCl 6 g Fe(NH4)2(SO4)2·6H2O 2 mg S medium trace mineral solution* 10 ml Trace vitamins** 10 ml NaHCO3 5 g Bacto Yeast Extract (Difco) 2 g Trypticase Peptone (BBL) 2 g Glucose 1.8 g Na2S-cysteine solution*** 10 ml Resazurin 1 mg Distilled water 970 ml

pH 7.0

Mix ingredients except for trace vitamins and Na2S-cysteine solution, and autoclave under N2/CO2 (80/20) condition. Separately autoclave Na2S-cysteine solution under a N2 atmosphere. Aseptically and anaerobically add filter-sterile trace vitamins and Na2S-cysteine solution.

Media

986

* S medium trace mineral solution

Nitrilotriacetic acid 1.5 g MgSO4·7H2O 3 g MnSO4·2H2O 0.5 g NaCl 1 g FeSO4·7H2O 0.1 g CoSO4·7H2O 0.1 g CaCl2·2H2O 0.1 g ZnSO4·7H2O 0.1 g CuSO4·5H2O 0.01 g AlK(SO4)2 0.01 g H3BO3 0.01 g Na2MoO4·2H2O 0.01 g NiCl2·6H2O 0.025 g Na2SeO3·5H2O 0.3 g (NH4)2Ni(SO4)2·6H2O 2 g Na2WO4·2H2O 10 mg Distilled water 1 L

First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add other minerals. Final pH 7.0 (with KOH). Sterilize by filtration.

** Trace vitamins


*** Na2S-cysteine solution

Cysteine-HCl 5 g Na2S·9H2O 5 g Distilled water 100 ml

The gas phase is replaced with N2.

911 Mar ine H2-us ing Methanogen Medium KP buffer* 5 ml

MgCl2·6H2O 3.05 g CaCl2·2H2O 0.15 g NH4Cl 0.54 g Na2SO4 0.3 g NaCl 30 g Bacto Yeast Extract (Difco) 0.02 g Sodium acetate 0.8 g Trace elements solution** 2 ml Vitamin solution*** 2 ml Resazurin 1 mg Na2CO3 1.5 g Na2S·9H2O 0.5 g Cysteine-HCl 0.5 g Distilled water 1 L

pH unadjusted

Mix ingredients except KP buffer, vitamin solution, Na2CO3, Na2S·9H2O and cysteine-HCl. Dispense the medium into suitable culture vessels under a stream of H2/CO2 (80/20) and seal with butyl rubber stoppers. Separately autoclave KP buffer, Na2S·9H2O (5% solution) and cysteine-HCl (5% solution) under a N2 atmosphere. Sterilize vitamin and 10% Na2CO3 solutions by filtration. Prior to inoculation, aseptically and anaerobically add KP buffer, vitamin, Na2CO3 and Na2S·9H2O solutions. Pressurize the inoculated vessels to 150 kPa with H2/CO2.

* KP buffer








Media

987

912 Methanogenium Medium KCl 0.355 g

MgCl2·6H2O 4 g MgSO4·7H2O 3.45 g NH4Cl 0.25 g CaCl2·2H2O 0.14 g KH2PO4 0.14 g NaCl 18 g Trace element solution* 10 ml Vitamin solution** 10 ml Fe(NH4)2(SO4)2·6H2O 2 mg NaHCO3 5 g Sodium acetate 1 g Bacto Yeast Extract (Difco) 2 g Trypticase Peptone (BBL) 2 g Na2S·9H2O 0.5 g Cysteine-HCl 0.5 g Distilled water 1 L

Mix ingredients except NaHCO3, Na2S·9H2O and cysteine-HCl, and adjust pH to 6.5. Bring to a boil for 5-10 seconds, then cool under a stream of H2/CO2 (80/20) and add NaHCO3 to the medium. After autoclaving under a H2/CO2 atmosphere, stand the medium overnight. Separately autoclave cysteine-HCl and Na2S·9H2O as 5% solutions under a N2 atmosphere. Prior to inoculation, aseptically and anaerobically add the cysteine-HCl and Na2S·9H2O solutions. Pressurize the inoculated bottles to 200 kPa with H2/CO2.


Nitrilotriacetic acid 1.5 g MgSO4·7H2O 3 g MnSO4·xH2O 0.5 g NaCl 1 g FeSO4·7H2O 0.1 g CoSO4·7H2O 0.1 g CaCl2·2H2O 0.1 g ZnSO4·7H2O 0.1 g CuSO4·5H2O 0.01 g AlK(SO4)2 0.01 g H3BO3 0.01 g Na2MoO4·2H2O 0.01 g NiCl2·6H2O 0.025 g Na2SeO3·5H2O 0.3 g (NH4)2Ni(SO4)2·6H2O 2 g Na2WO4·2H2O 0.01 g Distilled water 1 L

Dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH. Then add minerals. Adjust final pH to 7.0.

** Vitamin solution


913 Casamino Ac ids-Peptone-Czapek’s Agar Casamino Acids (Difco) 1 g

Bacto Peptone (Difco) 2 g K2HPO4 1 g KCl 0.5 g MgSO4·7H2O 0.5 g FeSO4·7H2O 10 mg Sucrose 30 g Agar 15 g Distilled water 1 L

914 Bacto Tryptone (Difco) 5 g

Yeast extract 3 g CaCl2·2H2O 0.87 g Distilled water 1 L Agar (if needed) 15 g

pH 7.0


Yeast extract 3 g CaCl2·2H2O 0.87 g Rifampicin solution (500 μg/ml)* 100 ml Spectinomycin solution (500 μg/ml)* 100 ml Distilled water 800 ml Agar (if needed) 15 g

pH 7.0



Yeast extract 3 g CaCl2·2H2O 0.87 g Spectinomycin solution (500 μg/ml)* 100 ml Distilled water 900 ml Agar (if needed) 15 g

pH 7.0



Yeast extract 3 g CaCl2·2H2O 0.87 g Kanamycin solution (500 μg/ml)* 100 ml Rifampicin solution (500 μg/ml)* 100 ml Distilled water 800 ml Agar (if needed) 15 g

pH 7.0



Yeast extract 3 g CaCl2·2H2O 0.87 g Kanamycin solution (500 μg/ml)* 100 ml Rifampicin solution (500 μg/ml)* 100 ml Spectinomycin solution (500 μg/ml)* 100 ml Distilled water 700 ml Agar (if needed) 15 g

pH 7.0


Media

988


Yeast extract 3 g CaCl2·2H2O 0.87 g Rifampicin solution (500 μg/ml)* 100 ml Distilled water 900 ml Agar (if needed) 15 g

pH 7.0



Yeast extract 3 g CaCl2·2H2O 0.87 g Kanamycin solution (500 μg/ml)* 100 ml Distilled water 900 ml Agar (if needed) 15 g

pH 7.0


922 Oceani thermus profundus Medium KH2PO4 0.75 g

K2HPO4 0.78 g MgCl2·6H2O 0.36 g CaCl2·2H2O 0.1 g NH4Cl 0.54 g NaHCO3 5 g Trace elements solution* 10 ml Vitamin solution** 10 ml Yeast extract 2 g Sucrose 6.8 g NaNO3 1.7 g NaCl 20 g Distilled water 1 L

pH unadjusted

Mix ingredients except sucrose, 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 sucrose, NaHCO3 and vitamin solutions.




** Vitamin solution


923 Hydrogenobacter Medium KP buffer* 10 ml

MgCl2·6H2O 0.75 g CaCl2·2H2O 0.15 g NH4Cl 0.54 g Na2S2O3 3.2 g Bacto Yeast Extract (Difco) 0.1 g Peptone (BBL) 1 g Glucose 0.2 g Sodium succinate 0.2 g Trace elements solution** 2 ml Vitamin solution*** 2 ml Na2CO3 0.5 g Distilled water 1 L

Mix ingredients except KP buffer and vitamin solution. Dispense the medium into suitable culture vessels under a stream of H2/CO2/O2 (75/20/5) and seal with butyl rubber stoppers. Separately autoclave KP buffer under a N2 atmosphere. Sterilize vitamin solution by filtration. Prior to inoculation, aseptically add KP buffer and vitamin solution. Pressurize the inoculated vessels to 150 kPa with H2/CO2/O2.

* KP buffer






Media

989



924 1/4 YpSs Soluble starch 3.75 g

Yeast extract 1 g K2HPO4 0.25 g MgSO4·7H2O 0.125 g Agar 15 g Distilled water 1 L

925 Methanosaeta harundinacea Medium Sodium acetate 1.7 g

NH4Cl 1 g KH2PO4 0.3 g NaCl 0.6 g MgCl2·6H2O 0.1 g CaCl2·2H2O 0.08 g Trace elements solution* 10 ml Vitamin solution** 10 ml Bacto Yeast Extract (Difco) 0.5 g Polypepton 0.5 g Resazurin 1 mg KHCO3 4.5 g Cysteine-HCl 0.3 g Na2S·9H2O 0.3 g Distilled water 1 L

Mix ingredients except for vitamin solution, cysteine-HCl and Na2S·9H2O, and autoclave under N2/CO2 (80/20) condition. Separately autoclave cysteine-HCl and Na2S·9H2O as a 3% solution under a N2 atmosphere. Aseptically and anaerobically add filter-sterile vitamin, cysteine-HCl and Na2S·9H2O solutions. Final pH is 7.2-7.5.


Nitrilotriacetic acid (NTA) 4.1 g FeCl3·6H2O 0.36 g MnCl2·4H2O 0.09 g CoCl2·6H2O 0.15 g CaCl2·2H2O 0.018 g ZnCl2 0.09 g H3BO3 0.017 g Na2MoO4·2H2O 0.009 g NiCl2·6H2O 0.018 g Na2SeO3 1.6 mg Na2WO4 1.6 mg Distilled water 1 L

First dissolve nitrilotriacetic acid and adjust pH to 6.5 with NaOH, and then add other minerals. Final pH 7.0.

** Vitamin solution


928 Yeast Ext ract -Mal t Ext rac t Agar ( ISP Medium No. 2 , pH 5.0)


pH 5.0

930 Potato Dext rose Agar Nissui Potato Dextrose Agar 39 g

Distilled water 1 L

931 MPA Medium NH4Cl 0.5 g

KH2PO4 0.15 g MgCl2·6H2O 0.2 g CaCl2·2H2O 0.15 g Yeast extract 0.5 g Crotonic acid 0.86 g NaHCO3 2.5 g Cysteine-HCl 0.5 g Na2S·9H2O 0.5 g Trace element solution* 1 ml Vitamin solution** 1 ml Resazurin 1 mg Distilled water 1 L

Mix ingredients except NaHCO3, cysteine-HCl, Na2S·9H2O and vitamin solution, and autoclave under a N2 atmosphere. Separately autoclave cysteine-HCl and Na2S·9H2O as 5% solutions under a N2 atmosphere. After adding filter-sterile NaHCO3 solution to the medium, adjust pH to 7.0. Prior to inoculation, aseptically and anaerobically add filter-sterile vitamin, cysteine-HCl and Na2S·9H2O solutions.


FeCl2 1.27 g CoCl2·6H2O 0.24 g MnCl2·4H2O 0.2 g ZnCl2 0.14 g H3BO3 6.2 mg NiCl2·6H2O 0.024 g AlCl3 0.013 g Na2MoO4·2H2O 0.024 g Na2SeO3·5H2O 2.6 mg Na2WO4·H2O 3 mg CuCl2·2H2O 1.7 mg Distilled water 1 L

Media

990

** Vitamin solution

Biotin 5 mg p-Aminobenzoic acid 5 mg Ca-pantothenate 5 mg Pyridoxine-HCl 5 mg Nicotinic acid 5 mg Thiamine-HCl 5 mg Lipoic acid 5 mg Folic acid 2 mg Vitamin B12 0.1 mg Riboflavin 5 mg Distilled water 1 L

932 Sul fo lobus meta l l icus Medium (NH4)2SO4 1.3 g

KH2PO4 0.28 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 Na2MoO4·2H2O 0.03 mg VOSO4·nH2O 0.03 mg CoSO4·7H2O 0.01 mg Bacto Yeast Extract (Difco) 1 g Sulfur (powder) 1 g Distilled water 1 L

pH 2.5

Mix ingredients except Bacto Yeast Extract and sulfur, adjust pH to 2.5 with H2SO4, and autoclave at 121°C for 15 min. Separately autoclave Bacto Yeast Extract as a 10% solution. Sulfur is sterilized separately by autoclaving for 60 min at 105°C on each of 3 successive days. Prior to inoculation, add Bacto Yeast Extract solution and sulfur powder. Cultivate under air containing 1-10% CO2.

933 M Medium (EBR46, EBR599) KH2PO4 0.4 g

K2HPO4·3H2O 0.4 g NH4Cl 1 g MgCl2·6H2O 0.1 g Yeast extract 0.2 g NaHCO3 6 g Cysteine-HCl 0.5 g Na2S·9H2O 0.25 g Resazurin 1 mg Bacto Tryptone (Difco) 10 g Vitamin solution* 10 ml Mineral solution** 10 ml Distilled water 1 L

pH 7.5

Mix ingredients except NaHCO3, cysteine-HCl, Na2S·9H2O and vitamin solution, distribute the medium into culture vessels under a stream of nitrogen gas, and seal with butyl rubber stoppers. After autoclaving (121°C, 15 min), aseptically inject other ingredients. The pH is adjusted to 7.5 by adding of sterile 0.6 N H2SO4 with a syringe without opening the vessel.

* Vitamin solution


** Mineral solution


934 Mar ine Broth 2216 + 0.5% Alg inate Bacto Marine Broth 2216 (Difco) 37.4 g

Sodium alginate 5 g Agar (if needed) 15 g Distilled water 1 L

pH unadjusted

935 Alka l ine Haloph i le Medium KH2PO4 1 g

KCl 1 g NH4Cl 1 g MgSO4·7H2O 0.24 g CaSO4·2H2O 0.17 g NaCl 200 g Sodium glutamate 1 g Bacto Yeast Extract (Difco) 5 g Casamino acids 5 g Trace elements solution 1 ml Na2CO3 5 g Agar (if necessary) 20 g Distilled water 1 L

Mix ingredients except Na2CO3 and adjust pH to 6.5 before autoclaving. Add Na2CO3 as a filter-sterile 10% solution after autoclaving. Check final pH to be 9.0-9.5. If agar medium is prepared, dissolve agar by heating before adding NaCl.

Media

991


2Na-EDTA 0.64 g MgSO4·7H2O 6.2 g MnSO4·4H2O 0.55 g NaCl 1 g FeSO4·7H2O 0.1 g CoCl2·6H2O 0.17 g CaCl2·2H2O 0.13 g ZnSO4·7H2O 0.18 g CuSO4 0.05 g H3BO3 0.01 g Na2MoO4·2H2O 0.011 g NiCl2·6H2O 0.025 g


936 Hydrogen-ox id iz ing Bacter ia Medium KP buffer* 10 ml

MgCl2·6H2O 0.75 g CaCl2·2H2O 0.15 g NH4Cl 0.54 g Na2S2O3 1.6 g Bacto Yeast Extract (Difco) 0.1 g Trace elements solution** 2 ml Vitamin solution*** 2 ml Na2CO3 0.5 g Distilled water 1 L

Mix ingredients except KP buffer and vitamin solution. Dispense the medium into suitable culture vessels under a stream of H2/CO2/O2 (75/20/5) and seal with butyl rubber stoppers. Separately autoclave KP buffer under a N2 atmosphere. Sterilize vitamin solution by filtration. Prior to inoculation, aseptically add KP buffer and vitamin solution. Pressurize the inoculated vessels to 150 kPa with H2/CO2/O2.

* KP buffer








937 KH2PO4 0.2 g

K2HPO4 0.8 g MgSO4·7H2O 0.2 g CaSO4·2H2O 0.1 g FeCl3·6H2O 10 mg Na2MoO4·2H2O 10 mg Yeast extract 0.5 g Sucrose 20 g Agar (if needed) 15 g Distilled water 1 L

pH 7.2

939 802 + 5% NaCl Polypepton* 10 g


pH 7.0


940 802 + 10% NaCl Polypepton* 10 g


pH 7.0


941 Methanol inea Medium KH2PO4 0.14 g

NH4Cl 0.54 g MgCl2·6H2O 0.2 g CaCl2·2H2O 0.15 g Bacto Yeast Extract (Difco) 0.1 g Sodium acetate 0.1 g Sodium formate 2.72 g Trace elements solution* 1 ml Vitamin solution 2 ml Resazurin 1 mg NaHCO3 2.5 g Cysteine-HCl 0.3 g Na2S·9H2O 0.3 g Distilled water 1 L

Media

992

Mix ingredients except for sodium formate, vitamin solution, cysteine-HCl and Na2S·9H2O, and autoclave under N2/CO2 (80/20) condition. Separately autoclave cysteine-HCl and Na2S·9H2O as a 6% solution under a N2 atmosphere. Aseptically and anaerobically add filter-sterile vitamin and neutralized 1.6 M formate solutions, and cysteine-HCl and Na2S·9H2O solutions.


Nitrilotriacetic acid (NTA) 4.1 g FeCl3·6H2O 0.36 g MnCl2·4H2O 0.09 g CoCl2·6H2O 0.15 g CaCl2·2H2O 0.018 g ZnCl2 0.09 g H3BO3 0.017 g Na2MoO4·2H2O 0.009 g NiCl2·6H2O 0.018 g Na2SeO4 0.004 g Na2WO4 0.004 g Distilled water 1 L


** Vitamin solution


942 Modi f ied 802 (pH 10.0) Polypepton* 10 g

Yeast extract 2 g MgSO4·7H2O 1 g K2HPO4 1 g Distilled water 900 ml Agar (if needed) 15 g


After autoclaving, aseptically add 100 ml of a 10% Na2CO3 solution to the medium. Check final pH to be about 10.0.

943 Modi f ied Prosthecomicrob ium and Ancalomicrobium Medium

Bacto Peptone (Difco) 0.15 g Yeast extract 0.15 g (NH4)2SO4 0.25 g Glucose 1 g Vitamin solution* 10 ml Modified Hutner's solution** 20 ml Agar (if needed) 15 g Distilled water 970 ml

pH unadjusted

* Vitamin solution

Biotin 2 mg Folic acid 2 mg Thiamine-HCl 5 mg Ca-pantothenate 5 mg Vitamin B12 0.1 mg Riboflavin 5 mg Distilled water 1 L

** Modified Hutner's solution

Nitrilotriacetic acid (NTA) 10 g MgSO4·7H2O 29.7 g CaCl2·2H2O 3.34 g (NH4)6Mo7O24·2H2O 9.25 mg FeSO4·7H2O 99 mg Metal salt solution 44*** 50 ml

Adjust volume to 1 L with distilled water.

Dissolve and neutralize the nitrilotriacetic acid with KOH (7.3 g); add the other ingredients and adjust the pH to 6.6-6.8 before bringing the volume to 1.0 L with distilled water.

*** Metal salt solution 44

Na-EDTA 250 mg ZnSO4·7H2O 1095 mg FeSO4·7H2O 0.5 g 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


944 Peptone Yeast Ext ract A lka l ine Medium Bacto Peptone (Difco) 8 g

Yeast extract 3 g K2HPO4 1 g EDTA-2Na 3.5 mg ZnSO4·7H2O 3 mg FeSO4·7H2O 10 mg MnSO4·nH2O 2 mg CuSO4·5H2O 1 mg Co(NO3)2·6H2O 2 mg H3BO3 1 mg Agar (if needed) 15 g 100 mM NaHCO3/100 mM Na2CO3 buffer (pH 10) 1 L

pH unadjusted

945 Mal tose-Yeast Ext ract Medium Maltose 20 g

Yeast extract 10 g KH2PO4 2 g NaCl 5 g Agar (if needed) 15 g Distilled water 1 L

pH 6.0

Media

993

946 Trypt ic Soy Medium wi th 10% Horse Serum Bacto Tryptic Soy Broth (Difco) 30 g

Horse serum* 100 ml Agar (if needed) 15 g Distilled water 900 ml

pH unadjusted

* Heat inactivated at 56°C for 30 min.

947 Mar ine Broth wi th 0.5% Methanol Bacto Marine Broth 2216 (Difco) 37.4 g

Methanol* 5 ml Agar (if needed) 15 g Distilled water 1 L

pH unadjusted


948 YSG Medium Solution A* 500 ml

Solution B** 500 ml

* Solution A

Yeast extract 2 g Distilled water 500 ml

Adjust pH to 3.7 with H2SO4

** Solution B

Soluble starch 2 g Glucose 1 g Agar (if needed) 15 g Distilled water 500 ml


949 TSA (pH 5.5) Bacto Tryptic Soy Agar (Difco) 40 g

Distilled water 1 L Adjust pH to 5.5

950 TSB (pH5.5) Bacto Tryptic Soy Broth (Difco) 30 g

Distilled water 1 L Adjust pH to 5.5

951 1/2MY Malt Extract Agar (Nissui) 2.7 g

Yeast extract 1.5 g Bacto Peptone (Difco) 2.5 g Glucose 5 g Agar 18.8 g Distilled water 1 L

pH unadjusted

952 LCA Glucose 1 g

KH2PO4 1 g MgSO4·7H2O 0.2 g KCl 0.2 g NaNO3 2 g Yeast extract 0.2 g Agar 13 g Distilled water 1 L

pH 6.5-7.0

954 Nut r ient Agar /Broth Nutrient Broth (OXOID) 13 g


pH unadjusted

955 Hydrogen-us ing Methanogen Medium KCl 0.355 g

MgCl2·6H2O 4 g MgSO4·7H2O 3.45 g NH4Cl 0.25 g CaCl2·2H2O 0.14 g KH2PO4 0.14 g Fe(NH4)2(SO4)2·6H2O 2 mg Sodium acetate 0.82 g Bacto Yeast Extract (Difco) 2 g Polypepton (Nihon seiyaku) 2 g Trace elements solution* 10 ml Vitamin solution** 10 ml NaHCO3 5 g Resazurin 1 mg Cysteine-HCl 0.5 g Na2S·9H2O 0.5 g Distilled water 1 L

Mix ingredients except vitamin solution, NaHCO3, cysteine-HCl and Na2S·9H2O, and adjust pH to 6.5 with NaOH. Dispense the medium into suitable culture vessels under a stream of H2/CO2 (80/20) and seal with butyl rubber stoppers. Separately autoclave cysteine-HCl and Na2S·9H2O as 5% solutions under a N2 atmosphere. Sterilize vitamin and 8% NaHCO3 solutions by filtration. Prior to inoculation, aseptically and anaerobically add vitamin, NaHCO3, cysteine-HCl and Na2S·9H2O solutions. Pressurize the inoculated vessels to 150 kPa with H2/CO2. Adjust pH to 6.8-7.0.




** Vitamin solution


Media

994

957 Trypt icase Soy Agar + 7% NaCl Trypticase Soy (BBL) 30 g

NaCl 70 g Distilled water 1 L Agar 20 g

pH 7.3

959 Medium-H Glucose 2 g

Yeast extract 0.2 g Monosodium glutamate 0.5 g Agar (if needed) 12 g Seawater 1 L

960 Agar 15 g

Distilled water 1 L pH around 5.5

961 TSB (pH 9.0 wi th K 2 CO3) Bacto Tryptic Soy Broth (Difco) 30 g

Distilled water 900 ml Agar (if needed) 15 g

After autoclaving, aseptically add 100 ml of a 10% K2CO3 solution to the medium. Check final pH to be about 9.0.

962 Matsuar ia Medium Chitosan 10 g

Yeast extract 0.25 g Peptone 0.25 g K2HPO4 0.25 g KH2PO4 0.7 g MgSO4·7H2O 0.3 g Distilled water 1 L

pH 7.0

963 IMK (SW) Daigo's IMK Medium* 0.253 g

Agar (if needed) 12 g Seawater 1 L

* Wako Pure Chemical Industries, Ltd. (Product Code: 398-01333).

964 AF6 NaNO3 140 mg

NH4NO3 22 mg MgSO4·7H2O 30 mg KH2PO4 10 mg K2HPO4 5 mg CaCl2·2H2O 10 mg CaCO3 10 mg Fe-citrate 2 mg Citric acid 2 mg Biotin 2 μg Thiamine-HCl 10 μg Vitamin B6 1 μg Vitamin B12 1 μg P IV Metals solution* 5 ml MES 400 mg Agar (if needed) 12 g Distilled water 1 L

pH 6.6

* P IV Metals solution

FeCl3·6H2O 19.6 mg MnCl2·4H2O 3.6 mg ZnSO4·7H2O 2.2 mg CoCl2·6H2O 0.4 mg Na2MoO4·2H2O 0.25 mg Na2EDTA·2H2O 100 mg Distilled water 1 L

965 KO2(NO3 ) NaNO3 1.7 g

KH2PO4 0.27 g MgSO4·7H2O 0.25 g CaCl2·6H2O 0.11 g FeSO4·7H2O 0.02 g EDTA-2Na 0.03 g N1 Metals solution* 1 ml Agar (if needed) 20 g Distilled water 1 L

pH 2

* N1 Metals solution

H3BO3 2.86 g MnCl2·4H2O 1.81 g ZnSO4·7H2O 0.22 g CuSO4·5H2O 0.08 g MoO3 15 mg Distilled water 1 L

968 KO2(NH4 ) (NH4)2SO4 1.32 g

KH2PO4 0.27 g MgSO4·7H2O 0.25 g CaCl2·6H2O 0.11 g FeSO4·7H2O 0.02 g EDTA-2Na 0.03 g N1 Metals solution* 1 ml Agar (if needed) 20 g Distilled water 1 L

pH 2

* N1 Metals solution

H3BO3 2.86 g MnCl2·4H2O 1.81 g ZnSO4·7H2O 0.22 g CuSO4·5H2O 0.08 g MoO3 15 mg Distilled water 1 L

970 M Medium (EBR596) KH2PO4 0.4 g

K2HPO4·3H2O 0.4 g NH4Cl 1 g MgCl2·6H2O 0.1 g Yeast extract 2 g NaHCO3 6 g Cysteine-HCl 0.5 g Na2S·9H2O 0.25 g Resazurin 1 mg Cellobiose 5 g NaCl 10 g Vitamin solution* 10 ml Mineral solution** 10 ml Distilled water 1 L

pH 6.5

Media

995

Mix ingredients except NaHCO3, cysteine-HCl, Na2S·9H2O and vitamin solution, distribute the medium into culture vessels under a stream of nitrogen gas and seal with butyl rubber stoppers. After autoclaving (121°C, 15 min), aseptically inject other ingredients. The pH is adjusted to 6.5 by adding of sterile 0.6 N H2SO4 with a syringe without opening the vessel.

* Vitamin solution


** Mineral solution


971 Pseudomonas saccharophi la Medium KH2PO4 4.4 g

Na2HPO4 4.8 g NH4Cl 1 g MgSO4·7H2O 0.5 g Solution A* 10 ml Solution B** 10 ml Distilled water 980 ml Agar (if needed) 20 g

pH unadjusted

Solution A and B are autoclaved separately and add to other autoclaved ingredients after cooling.

* Solution A.

Ferric ammonium citrate 5 g CaCl2 0.5 g Distilled water 1 L

** Solution B.

Sucrose 100 g Distilled water 1 L

972 Nutr ient Broth + 5% NaCl (pH 10.0) Nutrient Broth (OXOID) 13 g

NaCl 50 g Sodium-sesquicarbonate solution* 100 ml Distilled water 900 ml Agar (if needed) 15 g

* Sodium-sesquicarbonate solution

NaHCO3 4.2 g Na2CO3 (anhydrous) 5.3 g Distilled water 100 ml

Sterilize separately and add to other autoclaved ingredients. Check final pH to be about 10.0.

974 Medium for St ra in RASEN Bacto Yeast Extract (Difco) 1 g

NaCl 30 g K2HPO4 90 mg KH2PO4 70 mg CaCl2·2H2O 1.06 g MgCl2·6H2O 4.18 g KCl 0.33 g NH4Cl 0.535 g NaHCO3 2 g Na2S·9H2O 0.3 g Cysteine-HCl 0.3 g Trace element solution* 10 ml Vitamin solution** 10 ml Resazurin 1 mg Distilled water 1 L

pH 7

For vial cultures, dissolve all ingredients except for yeast extract, vitamin solution, Na2S·9H2O and cysteine-HCl (up to 900 ml), flush N2/CO2 (80/20) (pH 7), dispense 19 ml into 50 ml vials, seal with butyl rubber stoppers with flushing N2/CO2 (80/20) and autoclave. Add 0.5 ml of each of filter-sterilized Na2S·9H2O solution (0.3 g Na2S·9H2O/50 ml) and cysteine-HCl solution (0.3 g cysteine-HCl/50 ml) per each vial separately. Add 0.5 ml of filter-sterilized 40 g/l yeast extract solution. Inoculum size 5%. Growth was observed after 2 days of incubation.


Nitrilotriacetic acid 0.15 g MnCl2·4H2O 0.53 g CoCl2·6H2O 0.42 g ZnCl2 90 mg CuCl2·2H2O 7 mg AlCl3 6 mg H3BO3 10 mg Na2MoO4·2H2O 1 mg SrCl2·6H2O 10 mg NaBr 10 mg KI 10 mg FeCl2·4H2O 0.1 g NiCl2·6H2O 25 mg Na2SeO3 0.2 mg Na2WO4·2H2O 0.3 mg Distilled water 1 L

** Vitamin solution


Media

996

975 Thermus Medium Bacto Yeast Extract (Difco) 1 g

Bacto Tryptone (Difco) 1 g CaSO4·2H2O 0.06 g MgSO4·7H2O 0.1 g NaCl 0.008 g KNO3 0.1 g NaNO3 0.69 g Na2HPO4 0.11 g Trace elements solution* 10 ml Agar (if needed) 20 g Distilled water 1 L





976 Sul fur -ox id izer Medium KP buffer* 10 ml

MgCl2·6H2O 0.75 g CaCl2·2H2O 0.15 g NH4Cl 0.54 g Na2S2O3 0.8 g Trace elements solution** 2 ml Vitamin solution*** 2 ml Na2CO3 0.5 g Distilled water 1 L

pH unadjusted

Mix ingredients except KP buffer and vitamin solution. Dispense the medium into suitable culture vessels under a stream of N2/CO2/O2 (60/20/20) and seal with butyl rubber stoppers. Separately autoclave KP buffer under a N2 atmosphere. Sterilize vitamin solution by filtration. Prior to inoculation, aseptically and anaerobically add KP buffer and vitamin solution. Pressurize the inoculated vessels to 150 kPa with N2/CO2/O2 (60/20/20).

* KP buffer








978 2.5% NaCl GYPFK Agar /Broth for A lka l ibacter ium

Glucose 10 g Polypepton* 5 g Yeast extract 5 g Extract Bonito* 5 g NaCl 25 g K2HPO4 10 g Salt solution** 5 ml Agar (if needed) 15 g Distilled water 1 L

pH 8.5



** Salt solution

MgSO4·7H2O 40 g MnSO4·4H2O 2 g FeSO4·7H2O 2 g Distilled water 1 L

Media

997

979 Clost r id ium thermocel lum Medium (NH4)2SO4 1.3 g

MgCl2·6H2O 2.6 g KH2PO4 1.43 g K2HPO4·3H2O 7.2 g CaCl2·2H2O 0.13 g Sodium glycerophosphate 6 g FeSO4·7H2O 1.1 mg Glutathione 0.25 g Yeast extract 4.5 g Resazurin 1 mg Cellulose (Avicel or MN 300) 10 g or Cellobiose 5 g Distilled water 1 L

pH 7.0-7.2

Mix ingredients except for cellobiose and autoclave under H2/CO2 (95/5) condition. Aseptically and anaerobically add filter-sterile 10% cellobiose solution before inoculation.

980 S6 Medium KH2PO4 1.8 g

Na2HPO4 1.2 g (NH4)2SO4 0.1 g MgSO4·7H2O 0.1 g FeCl3·6H2O 30 mg MnSO4·xH2O 30 mg CaCl2·2H2O 40 mg Na2S2O3 10 g Distilled water 1 L

Adjust pH to 7

Separately sterilize 10% Na2S2O3 solution by filtration. Aseptically add sterile Na2S2O3 solution to the medium.

981 Verrucomicrob ium Medium Peptone 0.25 g

Yeast extract 0.25 g Glucose 0.25 g Vitamin solution* 10 ml Hutner's basal salts** 20 ml 0.1 M Tris-HCl (pH 7.5) 50 ml Artificial sea water*** 250 ml Distilled water 670 ml

Autoclave at 121°C for 15 min.

* Vitamin solution

Vitamin B12 0.1 mg Biotin 2 mg Thiamine-HCl 5 mg Ca-pantothenate 5 mg Folic acid 2 mg Riboflavin 5 mg Nicotinamide 5 mg Distilled water 1 L

** Hutner's basal salts

Nitrilotriacetic acid (NTA) 10 g MgSO4·7H2O 29.7 g CaCl2·2H2O 3.34 g (NH4)6MoO7O24·4H2O 9.25 mg FeSO4·7H2O 99 mg Metal salt solution 44**** 50 ml Distilled water 950 ml

*** Artificial sea water

NaCl 23.477 g Na2SO4 3.917 g MgCl2·6H2O 4.981 g CaCl2 1.102 g NaHCO3 192 mg KCl 664 mg KBr 6 mg H3BO3 26 mg SrCl2 24 mg NaF 3 mg Distilled water 1 L

**** 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 1 L


988 TYG Medium Trypticase Peptone (BBL) 2 g

Bacto Yeast Extract (Difco) 2 g Glucose 5 g Sodium n-hexanoate 0.2 g K solution* 10 ml M solution** 2.5 ml C solution*** 2.5 ml Resazurin 1 mg 15% Na2CO3 solution 20 ml Cysteine-HCl 0.75 g Distilled water 1 L

pH 7

Mix ingredients except for Na2CO3 and cysteine-HCl, dispense the medium into suitable culture vessels under a stream of N2/CO2 (80/20), seal with butyl rubber stoppers, and autoclave at 115℃ for 25 min. Aseptically and anaerobically add filter-sterile Na2CO3 and cysteine-HCl solutions, and adjust pH to 7.0 with 3 N HCl.

* K solution

KH2PO4 90 g NaCl 90 g (NH4)2SO4 90 g Distilled water 1 L

** M solution

MnCl2·4H2O 4 g CoCl2·6H2O 4 g Distilled water 1 L

*** C solution

CaCl2·2H2O 10.6 g Distilled water 1 L

Media

998

989 TYL Medium Trypticase Peptone (BBL) 2 g

Bacto Yeast Extract (Difco) 2 g 50% Sodium lactate 25 ml Biotin solution* 2 ml K solution** 10 ml M solution*** 2.5 ml C solution**** 2.5 ml Resazurin 1 mg 15% Na2CO3 solution 20 ml Cysteine-HCl 0.75 g Distilled water 1 L

pH 7

Mix ingredients except for Na2CO3 and cysteine-HCl, dispense the medium into suitable culture vessels under a stream of N2/CO2 (80/20), seal with butyl rubber stoppers, and autoclave at 115℃ for 25 min. Aseptically and anaerobically add filter-sterile Na2CO3 and cysteine-HCl solutions, and adjust pH to 7.0 with 3 N HCl.

* Biotin solution

Biotin 5 mg Ethanol 100 ml

** K solution

KH2PO4 90 g NaCl 90 g (NH4)2SO4 90 g Distilled water 1 L

*** M solution

MnCl2·4H2O 4 g CoCl2·6H2O 4 g Distilled water 1 L

**** C solution

CaCl2·2H2O 10.6 g Distilled water 1 L

990 1/10 Mar ine Broth (Agar) Bacto Marine Broth 2216 (Difco) 3.7 g

Agar (if needed) 15 g Distilled water 1 L

pH unadjusted

991 1/2 R2A-75%ASW R2A Broth (DAIGO) 1.6 g

Agar (if needed) 15 g Artificial sea water* 750 ml Distilled water 250 ml

* Artificial sea water

NaCl 23.5 g MgCl2 4.9 g Na2SO4 3.9 g CaCl2·2H2O 1.1 g KCl 0.66 g NaHCO3 0.19 g KBr 96 mg H3BO3 26 mg SrCl2 24 mg NaF 3 mg Distilled water 1 L

992 M1 Medium Starch 10 g

Yeast extract 4 g Peptone 2 g Agar (if needed) 18 g Seawater* 1 L



993 MMJS Medium NaCl 30 g

K2HPO4 0.14 g CaCl2·2H2O 0.14 g NH4Cl 0.125 g NaNO3 0.125 g MgSO4·7H2O 3.4 g MgCl2·6H2O 4.2 g KCl 0.33 g Fe(NH4)2(SO4)2·6H2O 0.01 g Bacto Yeast Extract (Difco) 1 g Trace elements solution* 10 ml Vitamin solution** 2 ml NaHCO3 0.1 g Sulfur (powder) 30 g Resazurin 1 mg Na2S·9H2O 0.5 g Distilled water 1 L

Mix ingredients except for vitamin solution, sulfur, NaHCO3, and Na2S·9H2O. Dispense the medium into suitable culture vessels under a stream of H2/CO2 (80/20), seal with butyl rubber stoppers, and autoclave at 121°C for 15 min. Separately autoclave Na2S·9H2O (5% solution) under N2 atmosphere. Sterilize vitamin and NaHCO3 solutions by filtration. Sulfur is sterilized separately by autoclaving for 60 min at 105°C on each of 3 successive days. Prior to inoculation, aseptically and anaerobically add vitamin, sulfur, NaHCO3, and Na2S·9H2O, and pressurize the inoculated vessels to 150 kPa with H2/CO2 (80/20). Final pH is 5.5-6.0.


Nitrilotriacetic acid (NTA) 12.8 g FeCl3·6H2O 1.35 g MnCl2·4H2O 0.1 g CoCl2·6H2O 0.024 g CaCl2·2H2O 0.1 g ZnCl2 0.1 g CuCl2·2H2O 0.025 g H3BO3 0.01 g Na2MoO4·2H2O 0.024 g NaCl 1 g NiCl2·6H2O 0.12 g Na2SeO4 0.8 mg Na2WO4 0.8 mg KAl(SO4)2·12H2O 4 mg Distilled water 1 L

First dissolve nitrilotriacetic acid and adjust pH to 6.5 with NaOH, and then add minerals. Final pH is 7.0.

Media

999

** Vitamin solution


994 Methanosaeta conc i l i i Medium Sodium acetate 4.1 g

(NH4)2SO4 0.45 g K2HPO4 2.19 g KH2PO4 1.5 g NaCl 0.5 g MgSO4·7H2O 0.12 g CaCl2·2H2O 0.06 g Resazurin 1 mg Trace element solution* 10 ml Vitamin solution** 10 ml Rumen fluid*** 10 ml FeSO4·7H2O**** 0.01 g Na2CO3 3 g Cysteine-HCl 0.25 g Na2S·9H2O 0.25 g Distilled water 1 L

Mix ingredients except for vitamin solution, rumen fluid, FeSO4, Na2CO3, cysteine-HCl and Na2S·9H2O, and autoclave under N2/CO2 (80/20) condition. A white precipitate that appears in the medium after autoclaving should dissolve again in about 2 days. Separately autoclave cysteine-HCl and Na2S·9H2O as a 5% solution under a N2 atmosphere. Vitamin and Na2CO3 (as a 10% solution) are sterilized by filtration. Aseptically and anaerobically add vitamin, rumen fluid, FeSO4, Na2CO3, cysteine-HCl and Na2S·9H2O. Adjust the pH of the medium to 7.3-7.5. Use 20% culture as an inoculum.


Nitrilotriacetic acid (NTA) 4.1 g FeCl3·6H2O 0.36 g MnCl2·4H2O 0.09 g CoCl2·6H2O 0.15 g CaCl2·2H2O 0.018 g ZnCl2 0.09 g H3BO3 0.017 g Na2MoO4·2H2O 0.009 g NiCl2·6H2O 0.018 g Na2SeO3 1.6 mg Na2WO4 1.6 mg Distilled water 1 L


** Vitamin solution


*** Rumen fluid:

Remove solid particles from a fresh rumen fluid by filtration and centrifugation, and then autoclave under a N2 atmosphere. Stock at 4°C.

**** FeSO4·7H2O:

FeSO4·7H2O is dissolved in 0.1 N H2SO4 as a 0.2% solution and then autoclaved under a N2 atmosphere.

996 YSG Medium (pH 4.5) Solution A* 500 ml

Solution B** 500 ml

* Solution A

Yeast extract 2 g Distilled water 500 ml

Adjust pH to 4.5 with H2SO4

** Solution B

Soluble starch 2 g Glucose 1 g Agar (if needed) 15 g Distilled water 500 ml


Check the final pH around 4.5.

999 PmTG Agar Peptonized Milk (Oxoid) 1 g

Tryptone 1 g Glucose 5 g Agar 10 g Distilled water 1 L

1000 Xy Agar Xylan 5 g

NH4NO3 0.16 g K2HPO4 0.5 g MgSO4·7H2O 0.25 g Ca(NO3)2·4H2O 0.06 g KCl 0.06 g Thiamine 2 mg Agar 10 g Distilled water 1 L

Media

1000

1002 Ac id imicrob ium Medium MgSO4·7H2O 0.5 g

(NH4)2SO4 0.4 g K2HPO4 0.2 g KCl 0.1 g Distilled water 1 L


Heterotrophic growth on yeast extract:

Add 10 mg/l of FeSO4·7H2O to the medium. After autoclaving, add yeast extract from sterile stock solution to final concentration of 0.25 g/l.

Autotrophic growth on ferrous sulfate:

Add 13.9 g/l of FeSO4·7H2O to the medium, and adjust medium pH to 1.7 with H2SO4 prior to autoclaving.

1003 Glucose 10 g

Polypepton* 5 g Yeast extract 5 g NaCl 10 g K2HPO4 1 g MgSO4·7H2O 0.2 g Na2CO3 10 g Agar (if needed) 18 g Distilled water 1 L

pH 10


1004 Leeming & Notman Agar Modi f ied (MLNA) Bacteriological peptone 10 g

Glucose 10 g Yeast extract 2 g Ox bile, desiccated 8 g Glycerol 10 ml Glycerol monostearate 0.5 g Tween 60 5 ml Olive oil 20 ml Agar 15 g Distilled water 965 ml

1008 Pelobacter carb ino l icus Medium KH2PO4 0.2 g

NH4Cl 0.25 g NaCl 20 g MgCl2·6H2O 3 g KCl 0.5 g CaCl2·2H2O 0.15 g Trace element solution SL-10* 1 ml Resazurin 1 mg NaHCO3 2.5 g 2,3-Butanediol 0.9 g Na2S·9H2O 0.36 g Distilled water 1 L

pH to 7.2

Gas atmosphere: N2/CO2 (80/20). Solutions of sodium bicarbonate, trace elements, and 2,3-butanediol are filter-sterilized separately. Sodium sulfide (3.6% w/v in water) is autoclaved under nitrogen atmosphere.

* Trace element solution SL-10:


1010 Na2SO4 3 g

KH2PO4 0.2 g NH4Cl 0.3 g NaCl 21 g MgCl2·6H2O 3 g KCl 0.5 g CaCl2·2H2O 0.15 g Trace element solution SL-10* 1 ml NaHCO3 2.5 g Na-L-lactate 2.3 g Selenite solution** 1 ml Sulfide solution*** 10 ml Resazurin 1 mg Distilled water 988 ml

pH 7

Dissolve ingredients except bicarbonate, lactate, selenite and sulfide, dispense under a N2/CO2 atmosphere (80/20) in culture vessels, and autoclave. Add bicarbonate, lactate, vitamin and sulfide from sterile anaerobic stock solutions prepared under N2 or N2/CO2 (bicarbonate). The bicarbonate and vitamin solutions should be sterilized by filtration. Adjust the final pH of the medium to 7.0. Addition of 10 - 20 mg sodium dithionite per liter (e.g. from 5% (w/v) solution, freshly prepared under N2 and filter-sterilized) may stimulate growth at the beginning. For transfers use 5 - 10% inoculum.



** Selenite solution

NaOH 0.4 g Na2SeO3·5H2O 3 mg Distilled water 1 L


*** Sulfide solution


Media

1001

1011 Modi f ied Pfenn ig ’s Medium I (Purp le Sul fur Bacter ia)

CaCl2·2H2O 0.25 g KH2PO4 0.34 g NH4Cl 0.34 g KCl 0.34 g MgSO4 0.5 g Vitamin B12 (0.002%) solution 1 ml Trace element solution SL-12B* 1 ml NaHCO3 1.5 g Na2S·9H2O 0.4 g Resazurin 0.5 mg Distilled water 998 ml

pH 7.3

Dissolve ingredients except bicarbonate, Vitamin B12 and sulfide, dispense under a N2/CO2 atmosphere (80/20) in culture vessels, and autoclave. Add bicarbonate, vitamin and sulfide** from sterile anaerobic stock solutions prepared under N2 or N2/CO2 (bicarbonate). The bicarbonate and vitamin solutions should be sterilized by filtration. Adjust the pH of the medium with sterile HCl or Na2CO3 solutions (2 mol/L each) to pH 7.3.

* Trace element solution SL-12B

Na2-EDTA 3 g FeSO4·7H2O 1.1 g CoCl2·6H2O 190 mg MnCl2·2H2O 50 mg ZnCl2 42 mg NiCl2·6H2O 24 mg Na2MoO4·2H2O 18 mg H3BO3 300 mg CuCl2·2H2O 2 mg Distilled water 1 L

pH 6




1012 Medium for Ectoth iorhodospi ra CaCl2·2H2O 0.25 g

KH2PO4 0.34 g NH4Cl 0.34 g KCl 0.34 g MgSO4 0.5 g NaCl 30 g Vitamin B12 (0.002%) solution 1 ml Trace element solution SL-12B* 1 ml NaHCO3 1.5 g Na2S·9H2O 0.4 g Resazurin 0.5 mg Distilled water 998 ml

pH 7.3



Na2-EDTA 3 g FeSO4·7H2O 1.1 g CoCl2·6H2O 190 mg MnCl2·2H2O 50 mg ZnCl2 42 mg NiCl2·6H2O 24 mg Na2MoO4·2H2O 18 mg H3BO3 300 mg CuCl2·2H2O 2 mg Distilled water 1 L

pH 6




1013 Modi f ied Pfenn ig ’s Medium I I (Green Sul fur Bacter ia)

CaCl2·2H2O 0.25 g KH2PO4 0.34 g NH4Cl 0.34 g KCl 0.34 g MgSO4 0.5 g Vitamin B12 (0.002%) solution 1 ml Trace element solution SL-10B* 1 ml NaHCO3 1.5 g Na2S·9H2O 0.6 g Resazurin 0.5 mg Distilled water 998 ml

pH 6.8


Media

1002


HCl (25%) 7.7 ml FeSO4·7H2O 1.5 g ZnCl2 70 mg MnCl2·4H2O 100 mg H3BO3 300 mg CoCl2·6H2O 190 mg CuCl2·2H2O 2 mg NiCl2·6H2O 24 mg Na2MoO4·2H2O 36 mg Distilled water 1 L




1016 Pelobacter acety len icus Medium KH2PO4 0.2 g

NH4Cl 0.25 g NaCl 1 g MgCl2·6H2O 0.4 g KCl 0.5 g CaCl2·2H2O 0.15 g Trace element solution SL-10* 1 ml Resazurin 1 mg NaHCO3 2.5 g Acetoin 1 g Na2S·9H2O 0.36 g Distilled Water 1 L

pH to 7.2

Gas atmosphere: N2/CO2 (80/20). Solutions of sodium bicarbonate, trace elements and acetoin are filter-sterilized separately. Sodium sulfide (3.6% w/v in water) is autoclaved under nitrogen atmosphere.



1017 Pelobacter venet ianus Medium KH2PO4 0.2 g

NH4Cl 0.25 g NaCl 20 g MgCl2·6H2O 3 g KCl 0.5 g CaCl2·2H2O 0.15 g Trace element solution SL-10* 1 ml Resazurin 1 mg NaHCO3 2.5 g Polyethylene glycol (Mw 106-20000) 1 g Na2S·9H2O 0.36 g Distilled Water 1 L

pH to 7.2

Gas atmosphere: N2/CO2 (80/20). Solutions of sodium bicarbonate, trace elements and polyethylene glycol are filter-sterilized separately. Sodium sulfide (3.6% w/v in water) is autoclaved under nitrogen atmosphere.



1019 Archaeoglobus fu lg idus Medium KP buffer* 5 ml

MgCl2·6H2O 0.75 g CaCl2·2H2O 0.15 g NH4Cl 0.54 g NaCl 30 g Na2SO4 2.4 g Sodium lactate 2.2 g Bacto Yeast Extract (Difco) 0.5 g Fe(NH4)2(SO4)2·7H2O 2 mg Trace element solution** 2 ml Vitamin solution*** 2 ml Resazurin 1 mg Na2CO3 1 g Na2S·9H2O 0.36 g Distilled water 1 L

pH unadjusted

Mix ingredients except KP buffer, vitamin solution, Na2CO3 and Na2S·9H2O. Dispense the medium into suitable culture vessels under a stream of N2/CO2 (80/20) and seal with butyl rubber stoppers. Separately autoclave KP buffer and Na2S·9H2O (5% solution) under a N2 atmosphere. Sterilize vitamin and 10% Na2CO3 solutions by filtration. Prior to inoculation, aseptically and anaerobically add KP buffer, vitamin, Na2CO3 and Na2S·9H2O solutions.

* KP buffer



Media

1003






1020 Methanofo l l is e thanol icus Medium Ethanol 0.46 ml

Yeast extract 0.1 g KH2PO4 0.14 g MgCl2·6H2O 0.2 g CaCl2·2H2O 0.15 g NH4Cl 0.54 g Sodium acetate 0.82 g Vitamin solution* 2 ml Trace element solution** 2 ml Se/W solution*** 1 ml Resazurin solution**** 1 ml NaHCO3 2.5 g Na2S·9H2O 0.3 g Cysteine-HCl 0.3 g Distilled water 1 L

For vial cultures, dissolve all ingredients except for ethanol, Na2S·9H2O and cysteine-HCl, flush N2/CO2 (80/20) and autoclave. Add each of filter-sterilized Na2S·9H2O solution (0.6 g Na2S·9H2O/50 ml) and cysteine-HCl solution (0.6 g cysteine-HCl/50 ml) per each vial separately. Add filter-sterilized 0.8 M ethanol solution. Inoculum size 10-20%.

* Vitamin solution





*** Se/W solution

Na2SeO3 4 mg Na2WO4 4 mg Distilled water 1 L



1021 Desul fov ibr io Medium K2HPO4 0.5 g

NH4Cl 1 g Na2SO4 1 g CaCl2·2H2O 0.1 g MgSO4·7H2O 2 g Sodium lactate 2 g Yeast extract 1 g FeSO4·7H2O 0.5 g Resazurin 1 mg Thioglycolate solution* 10 ml Distilled water 990 ml

pH 7.8

Dissolve ingredients except thioglycolate solution, dispense under a N2 atmosphere in culture vessels, and autoclave. Adjust the final pH of the medium to 7.8.

* Thioglycolate solution

Sodium thioglycolate 0.1 g Ascorbic acid 0.1 g Distilled water 10 ml


Media

1004

1022 (NH4)2SO4 3 g

K2HPO4 5 g MgSO4·7H2O 5 g KCl 1 g Ca(NO3)2 0.3 g NaCl 20 g Sodium tetrathionate 2 g Distilled water 1 L

pH 4

1024 Alka l i A Medium Solution A* 500 ml

Solution B** 500 ml

* Solution A

Glucose 10 g Peptone 5 g Yeast extract 5 g K2HPO4 1 g MgSO4·7H2O 0.2 g Agar 12 g Distilled water 500 ml

** Solution B

Na2CO3 10 g Distilled water 500 ml

Solutions A and B are autoclaved separately and combined before solidified. Final pH 10.3.

1026 Medium for St ra ins JA121 and JA322 KH2PO4 0.5 g

MgSO4·7H2O 2 g NaCl 20 g NH4Cl 0.6 g CaCl2·2H2O 0.15 g Sodium pyruvate 3 g Yeast extract 0.5 g Ferric citrate solution* (0.1%) 5 ml Vitamin B12 (0.002% (w/v)) 1 ml Trace element solution SL7** 1 ml Na2S·9H2O 0.48 g Distilled water 1 L

pH 7

Mix ingredients except vitamin B12 and Na2S·9H2O, and autoclave under a N2 atmosphere. Separately autoclave Na2S·9H2O solution under a N2 atmosphere. Sterilize vitamin B12 solution by filtration. Prior to inoculation, aseptically and anaerobically add them.


** Trace element solution SL7


Initially, dissolve FeCl2·4H2O in 25% HCl, then add distilled water followed by the other components.

1028 Modi f ied WS Medium KH2PO4 0.136 g

NH4Cl 0.54 g MgCl2·6H2O 3.05 g CaCl2·2H2O 0.147 g NaCl 20 g Methanol 0.8 ml NaHCO3 2.52 g Vitamin solution* 2 ml Trace element solution** 1 ml Resazurin 1 mg Cysteine-HCl 0.3 g Na2S·9H2O 0.3 g Distilled water 1 L

Mix ingredients except for methanol, vitamin solution, cysteine-HCl and Na2S·9H2O, and autoclave under a N2/CO2 atmosphere (80/20). Separately autoclave cysteine-HCl and Na2S·9H2O solutions as 3% solutions under a N2 atmosphere. Sterilize methanol and vitamin solution by filtration. Prior to inoculation, add methanol, vitamin solution, cysteine-HCl and Na2S·9H2O.

* Vitamin solution





1029 Alka l ine Yeast Ext ract -Mal t Ext ract Agar Bacto Yeast Extract (Difco) 4 g

Bacto Malt Extract (Difco) 10 g Glucose 4 g Agar 15 g Artificial seawater 1 L

After autoclaving, adjust pH to 10.0 with sterilized 10% Na2CO3 solution.

Media

1005

1030 Yeast Ext ract -Mal t Ext ract Agar ( ISP-2) wi th Ar t i f ic ia l Seawater

Bacto Yeast Extract (Difco) 4 g Bacto Malt Extract (Difco) 10 g Glucose 4 g Agar 15 g Artificial seawater 1 L

Adjust pH to 7.3

1031 Sodium pyruvate 2 g

KH2PO4 1 g (NH4)2SO4 1 g Na2S2O3·5H2O 0.5 g Yeast extract 0.5 g MgCl2·6H2O 0.2 g CaCl2 45 mg NaCl 0.2 g Vitamin B12 20 μg Nicotinic acid 10 μg p-Aminobenzoic acid 3 μg Trace element solution* 1 ml Distilled water 1 L

pH 6.8


2Na-EDTA 5 g FeCl2·4H2O 1 g H3BO3 0.3 g CoCl2·6H2O 0.11 g ZnCl2 50 mg MnCl2·4H2O 30 mg Na2MoO4·2H2O 30 mg NiSO4·6H2O 30 mg CuCl2·2H2O 10 mg Distilled water 1 L

1038 1/2 ISP-2 + 10% NaCl Yeast extract 2 g

Malt extract 5 g Glucose 2 g NaCl 100 g Distilled water 1 L Agar 18 g

pH 7.3

1039 Thermoheterot roph Medium K2HPO4 0.05 g

KH2PO4 0.09 g NH4Cl 0.25 g MgSO4·7H2O 0.25 g CaCl2·2H2O 0.15 g Bacto Yeast Extract (Difco) 5 g Na2S2O3·5H2O 2.5 g Na2CO3 0.25 g Vitamin solution* 2 ml Trace element solution** 1 ml Resazurin 1 mg Cysteine-HCl 0.3 g Na2S·9H2O 0.3 g Distilled water 1 L

Mix ingredients except for Na2CO3, vitamin solution, cysteine-HCl and Na2S·9H2O, and autoclave under a N2/CO2 atmosphere (80/20). Separately autoclave cysteine-HCl and Na2S·9H2O solutions as 3% solutions under a N2 atmosphere. Prior to inoculation, add the filter-sterile 5% Na2CO3 and vitamin solutions, cysteine-HCl, and Na2S·9H2O.

* Vitamin solution





1040 1/2 ISP-2 + 15% NaCl Yeast extract 2 g

Malt extract 5 g Glucose 2 g NaCl 150 g Distilled water 1 L Agar 18 g

pH 7.3

1041 Medium for St ra in JA143 KH2PO4 0.5 g

MgSO4·7H2O 0.2 g NaCl 0.4 g NH4Cl 0.6 g CaCl2·2H2O 0.05 g Sodium pyruvate 3 g Yeast extract 0.5 g Na2S2O3·5H2O 5 g Ferric citrate solution* (0.1%) 5 ml Trace element solution SL7** 1 ml Na2S·9H2O 0.48 g Distilled water 1 L

pH 7

Mix ingredients except for Na2S·9H2O and autoclave under a N2 atmosphere. Separately autoclave Na2S·9H2O solution under a N2 atmosphere. Prior to inoculation, aseptically and anaerobically add it.

Media

1006





1044 Hagem Medium KH2PO4 0.5 g

MgSO4·7H2O 0.5 g NH4Cl 0.5 g Fe-citrate 1% solution 0.5 ml Malt extract 5 g Glucose 5 g Agar 15 g Distilled water 1 L

pH 5.6

1045 Trypt icase Soy Agar + 10% NaCl Trypticase Soy (BBL) 30 g

NaCl 100 g Distilled water 1 L Agar 20 g

pH 7.3

1046 Synthet ic Low-Nutr ient Agar (SNA) Glucose 0.2 g

Sucrose 0.2 g KH2PO4 1.0 g KNO3 1.0 g MgSO4·7H2O 0.5 g KCl 0.5 g 1 N NaOH solution 0.6 ml Agar 23 g Distilled water 1 L

pH unadjusted

1047 802 + 5% Glucose Polypepton* 5 g


pH 6.6 - 7.0


1049 AS Medium NaCl 20 g

Bacto Yeast Extract (Difco) 2 g MgCl2·6H2O 3 g MgSO4·7H2O 6 g (NH4)2SO4 1 g CaCl2·2H2O 0.3 g KH2PO4 0.2 g KCl 0.5 g NaBr 0.05 g H3BO3 0.025 g SrCl2·6H2O 0.02 g Fe(III) ammonium citrate 0.01 g Bis Tris Propane 2.25 g Trace elements solution 10 ml Vitamin solution 10 ml Resazurin 1 mg Sulfur (powder) 10 g Na2S·9H2O 0.3 g Distilled water 1 L

Mix ingredients except for vitamin, sulfur and Na2S·9H2O, adjust pH to 7 with H2SO4, and autoclave under a N2 atmosphere. Sulfur is separately sterilized by autoclaving for 60 min at 105°C on each of 3 successive days. Separately autoclave Na2S·9H2O as 5% solution under a N2 atmosphere. Anaerobically add filter-sterile vitamin, sulfur and Na2S·9H2O.


Nitrilotriacetic acid (NTA) 4.1 g FeCl3·6H2O 0.36 g MnCl2·4H2O 0.09 g CoCl2·6H2O 0.15 g CaCl2·2H2O 0.018 g ZnCl2 0.09 g H3BO3 0.017 g Na2MoO4·2H2O 0.009 g NiCl2·6H2O 0.018 g Na2SeO3 1.6 mg Na2WO4 1.6 mg AlK(SO4)2 5 mg Distilled water 1 L


** Vitamin solution


Media

1007

1050 A Medium NaCl 20 g

Bacto Yeast Extract (Difco) 2 g MgCl2·6H2O 3 g MgSO4·7H2O 6 g (NH4)2SO4 1 g CaCl2·2H2O 0.3 g KH2PO4 0.2 g KCl 0.5 g NaBr 0.05 g H3BO3 0.025 g SrCl2·6H2O 0.02 g Fe(III) ammonium citrate 0.01 g Bis Tris Propane 2.25 g Trace elements solution 10 ml Vitamin solution 10 ml Resazurin 1 mg Na2S·9H2O 0.3 g Distilled water 1 L

Mix ingredients except for vitamin and Na2S·9H2O, adjust pH to 7 with H2SO4, and autoclave under a N2 atmosphere. Separately autoclave Na2S·9H2O as 5% solution under a N2 atmosphere. Anaerobically add filter-sterile vitamin and Na2S·9H2O.


Nitrilotriacetic acid (NTA) 4.1 g FeCl3·6H2O 0.36 g MnCl2·4H2O 0.09 g CoCl2·6H2O 0.15 g CaCl2·2H2O 0.018 g ZnCl2 0.09 g H3BO3 0.017 g Na2MoO4·2H2O 0.009 g NiCl2·6H2O 0.018 g Na2SeO3 1.6 mg Na2WO4 1.6 mg AlK(SO4)2 5 mg Distilled water 1 L


** Vitamin solution


1051 Yeast Ext ract -Mal t Ext rac t Agar ( ISP Medium No. 2 , pH 5.5)


pH 5.5

1052 Oatmeal Agar ( ISP Medium No.3, pH 5.5) Oatmeal 20 g

Trace salts solution* 1 ml Distilled water 1 L Agar 18 g

pH 5.5



1053 Yeast Ext ract -Starch Agar (pH 5.5) Yeast extract 2 g


pH 5.5

1056 Desul fov ibr io magnet icus Medium for Magnetosome Format ion

KH2PO4 0.2 g NH4Cl 0.06 g Cysteine-HCl 0.05 g Ferric quinate solution* 2 ml Wolfe's vitamin solution** 4 ml Wolfe's mineral solution*** 2 ml Sodium fumarate 0.58 g Sodium pyruvate 0.44 g Distilled water 1 L

pH 7

Mix ingredients except for vitamin solution and cysteine-HCl. Dispense the medium into vials suitable for anaerobic cultures, sparge with argon gas to establish anoxic condition (at least 30 min), seal with butyl rubber stoppers, and autoclave at 121°C for 15 min. Separately autoclave cysteine-HCl (0.5% solution) under argon atmosphere. Sterilize vitamin solution by filtration. Prior to inoculation, aseptically and anaerobically add the vitamin and cysteine-HCl solutions to the medium, and sparge the vials with sterile argon gas for 5 min. The cysteine-HCl more than the above-mentioned amount (0.05 g/L) is unsuitable for growth of strain RS-1. And the strain produces only a small amount of magnetite in the presence of sulfate.

* Ferric quinate solution

FeCl3 0.27 g Quinate 0.19 g Distilled water 100 ml

** Wolfe's vitamin solution


Media

1008

*** Wolfe's mineral solution

Nitrilotriacetic acid (NTA) 1.5 g MgSO4·7H2O 3 g MnSO4·H2O 0.5 g NaCl 1 g FeSO4·7H2O 0.1 g CoCl2·6H2O 0.1 g CaCl2 0.1 g ZnSO4·7H2O 0.1 g CuSO4·5H2O 0.01 g KAl(SO4)2·12H2O 0.01 g H3BO3 0.01 g Na2MoO4·2H2O 0.01 g Distilled water 1 L

Add nitrilotriacetic acid to approximately 500 ml of water and adjust to pH 6.5 with KOH to dissolve the compound. Bring volume to 1 L with remaining water and add remaining compounds one at a time.

1061 Alka l ic MRS Medium (pH 8.0) Peptone 10 g


pH 8.0

1066 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 myo-Inositol 1 mg Vitamin mixture*** 10 ml Cellobiose 5 g Cysteine-HCl 0.5 g Na2S·9H2O 0.5 g Resazurin 1 mg Distilled water 939 ml

pH 6.5

Dissolve ingredients except for vitamin, cysteine-HCl and sulfide, dispense under a N2 atmosphere in culture vessels and autoclave. Add vitamin, cysteine-HCl and sulfide from sterile anaerobic stock solutions prepared under a N2 atmosphere. The vitamin and cysteine-HCl solutions should be sterilized by filtration. Adjust the pH of the medium with sterile 10 N KOH solution to pH 6.5.

* Wolfe's modified mineral solution

Nitrilotriacetic acid (NTA) 1.5 g MgSO4·7H2O 3 g NaCl 1 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 Na2MoO4·2H2O 0.01 g Na2SeO3 0.001 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

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 Thiamine-HCl 20 mg Choline chloride 100 mg Cyanocobalamin 2 μg Distilled water 1 L


NH4Cl 0.54 g MgCl2·6H2O 0.2 g CaCl2·2H2O 0.147 g NaHCO3 2.5 g Bacto Yeast Extract (Difco) 0.2 g Sodium acetate 0.8 g Vitamin solution* 10 ml Trace element solution** 10 ml Resazurin 1 mg Cysteine-HCl 0.5 g Na2S·9H2O 0.5 g Distilled water 1 L

Mix ingredients except for 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).

Media

1009

* Vitamin solution



2Na-EDTA 0.64 g MgSO4·7H2O 6.2 g MnSO4·4H2O 0.55 g NaCl 1 g FeSO4·7H2O 0.1 g CoCl2·6H2O 0.17 g CaCl2·2H2O 0.13 g ZnSO4·7H2O 0.18 g CuSO4 0.05 g KAl(SO4)2·12H2O 0.018 g H3BO3 0.01 g Na2MoO4·2H2O 0.011 g NiCl2·6H2O 0.025 g Na2SeO4 0.004 g Na2WO4 0.004 g Distilled water 1 L


1068 Medium for St ra ins JA145, JA193, JA310, JA334, JA415, JA430 and JA447

KH2PO4 0.5 g MgSO4·7H2O 0.2 g NaCl 0.4 g NH4Cl 0.6 g CaCl2·2H2O 0.05 g Sodium pyruvate 3 g Yeast extract 0.5 g Ferric citrate solution* (0.1%) 5 ml Vitamin B12 (0.002% (w/v)) 1 ml Trace element solution SL7** 1 ml Resazurin 1 mg Distilled water 1 L

pH 7

Mix ingredients except for vitamin B12 and autoclave under a N2 atmosphere. Prior to inoculation, aseptically and anaerobically add vitamin B12 solution sterilized by filtration.

For strain JA310, add 2 mM Na2S2O3 (separately sterilized).





1071 PYGSW Agar Peptone 1 g

Yeast extract 1 g Glucose 0.5 g Seawater (2% salinity) 1 L Agar 15 g

1072 1% Tryptone Agar Bacto Tryptone (Difco) 10 g

Bacto Agar 15 g Distilled water 1 L

pH 7.0

1074 Alka l ine Agar Glucose 10 g

Bacto Peptone (Difco) 5 g Bacto Yeast Extract (Difco) 5 g KH2PO4 1 g MgSO4·7H2O 0.2 g 20% NaCl solution 200 ml 10% Na2CO3 solution 100 ml Agar 15 g Distilled water 700 ml

Autoclave NaCl and Na2CO3 solutions separately and add the solutions to the medium aseptically.

1078 Desul fov ibr io magnet icus Medium for Sul fa te- reduc ing Growth Mode

KH2PO4 0.2 g NH4Cl 0.06 g Cysteine-HCl 0.05 g Ferric quinate solution* 2 ml Wolfe's vitamin solution** 4 ml Wolfe's mineral solution*** 2 ml Na2SO4 0.3 g Sodium lactate 0.45 g Distilled water 1 L

pH 7

Mix ingredients except for vitamin solution and cysteine-HCl. Dispense the medium into vials suitable for anaerobic cultures, sparge with argon gas to establish anoxic condition (at least 30 min), seal with butyl rubber stoppers, and autoclave at 121°C for 15 min. Separately autoclave cysteine-HCl (0.5% solution) under an argon atmosphere. Sterilize vitamin solution by filtration. Prior to inoculation, aseptically and anaerobically add the vitamin and cysteine-HCl solutions to the medium, and sparge the vials with sterile argon gas for 5 min. The cysteine-HCl more than the above-mentioned amount (0.05 g/L) is unsuitable for growth of strain RS-1.

Media

1010

* Ferric quinate solution

FeCl3 0.27 g Quinate 0.19 g Distilled water 100 ml

** Wolfe's vitamin solution


*** Wolfe's mineral solution

Nitrilotriacetic acid (NTA) 1.5 g MgSO4·7H2O 3 g MnSO4·H2O 0.5 g NaCl 1 g FeSO4·7H2O 0.1 g CoCl2·6H2O 0.1 g CaCl2 0.1 g ZnSO4·7H2O 0.1 g CuSO4·5H2O 0.01 g KAl(SO4)2·12H2O 0.01 g H3BO3 0.01 g Na2MoO4·2H2O 0.01 g Distilled water 1 L

Add nitrilotriacetic acid to approximately 500 ml of water and adjust to pH 6.5 with KOH to dissolve the compound. Bring volume to 1 L with remaining water and add remaining compounds one at a time.

1079 Mar ine Microaerob ic Thermus Medium Bacto Yeast Extract (Difco) 1 g

Bacto Tryptone (Difco) 1 g NaCl 30 g K2HPO4 0.14 g CaCl2·2H2O 0.14 g NH4Cl 0.25 g MgSO4·7H2O 3.4 g MgCl2·6H2O 4.18 g KCl 0.33 g Fe(NH4)2(SO4)2·6H2O 0.01 g Trace elements solution* 10 ml Distilled water 1 L

Mix ingredients, adjust pH to 7.0 and autoclave under a N2/O2 atmosphere (95/5). Pressurize the vessels to 150 kPa with N2/O2.


2Na-EDTA 0.64 g MgSO4·7H2O 6.2 g MnSO4·4H2O 0.55 g NaCl 1 g FeSO4·7H2O 0.1 g CoCl2·6H2O 0.17 g CaCl2·2H2O 0.13 g ZnSO4·7H2O 0.18 g CuSO4 0.05 g KAl(SO4)2·12H2O 0.018 g H3BO3 0.01 g Na2MoO4·2H2O 0.011 g NiCl2·6H2O 0.05 g NaSeO3·5H2O 0.05 g


1082 LB Medium wi th 50 ppm Naphthalene Peptone 10 g

Yeast extract 5 g NaCl 5 g Naphthalene solution (10,000 ppm)* 5 ml Distilled water 1 L Agar (if needed) 15 g

pH 7.0

* Naphthalene solution (10,000 ppm)

Naphthalene 50 mg Ethanol 5 ml

Sterilize separately by filtration.

1083 Methanobacter ium a lca l iph i lum Medium NH4Cl 1 g

MgCl2·6H2O 0.1 g CaCl2·2H2O 0.1 g K2HPO4 0.4 g Bacto Yeast Extract (Difco) 2 g Polypeptone 2 g Resazurin 1 mg Carbonate mixture* 210 ml Tris-HCl buffer** 10 ml Vitamin solution*** 10 ml Trace elements solution**** 10 ml Cysteine-HCl 0.3 g Na2S·9H2O 0.3 g Distilled water 760 ml

Mix ingredients except for carbonate mixture, Tris-HCl buffer, vitamin solution, cysteine-HCl and Na2S·9H2O, and autoclave under a H2 atmosphere. Carbonate mixture, Tris-HCl buffer, cysteine-HCl (3% solution) and Na2S·9H2O (3% solution) are separately autoclaved under a N2 atmosphere. Prior to inoculation, add filter-sterile vitamin solution, carbon mixture, Tris-HCl buffer, cysteine-HCl and Na2S·9H2O. Pressurize the inoculated vessels to 150 kPa with H2.

* Carbonate mixture

NaHCO3 10 g Na2CO3 0.5 g Distilled water 210 ml

Mix ingredients and autoclave under a N2 atmosphere.

Media

1011

** Tris-HCl buffer

Dissolve 2 M Tris, adjust pH to 8.4 with HCl, and autoclave under a N2 atmosphere.



**** Trace elements solution

2Na-EDTA 0.64 g MgSO4·7H2O 6.2 g MnSO4·4H2O 0.55 g NaCl 1 g FeSO4·7H2O 0.1 g CoCl2·6H2O 0.17 g CaCl2·2H2O 0.13 g ZnSO4·7H2O 0.18 g CuSO4 0.05 g KAl(SO4)2·12H2O 0.018 g H3BO3 0.01 g Na2MoO4·2H2O 0.011 g NiCl2·6H2O 0.025 g Na2SeO4 0.004 g Na2WO4 0.004 g Distilled water 1 L


1084 Act inota lea fermentas Medium K2HPO4 2.21 g

KH2PO4 1.5 g (NH4)2SO4 1.3 g MgCl2·6H2O 0.1 g CaCl2·2H2O 0.02 g FeSO4·7H2O 1.25 mg Yeast extract 5 g Cellobiose or Cellulose (MN 300) 5 g NaHCO3 0.8 g Resazurin 1 mg Cysteine-HC 0.5 g Distilled water 1 L

Adjust pH to 7.4 with 8 M NaOH

Prepare the medium anaerobically under 100% nitrogen. Cellobiose, NaHCO3, and cysteine-HCl are sterilized separately by autoclaving under N2. For aerobic growth, omit resazurin and cysteine-HCl.

1091 MRS + Fructose Peptone 10 g

Meat extract 10 g Yeast extract 5 g Glucose 20 g Fructose 10 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 Distilled water 1 L Agar (if needed) 15 g

pH 6.0 - 6.5

1095 LMG 157 Medium Beef extract 0.5 g

Yeast extract 0.5 g Tryptone 2 g Sodium acetate 0.2 g Glucose* 0.25 mg Distilled water 1 L Agar (if needed) 15 g

pH 7.2- 7.4

* Sterilize separately by autoclaving and add to other ingredients. The final concentration is 0.025%.


Bacto Tryptone (Difco) 1 g Sodium glutamate monohydrate 1 g CaSO4·2H2O 0.06 g MgSO4·7H2O 0.1 g NaCl 0.008 g KNO3 0.1 g NaNO3 0.69 g Na2HPO4 0.11 g Trace elements solution* 10 ml Agar (if needed) 20 g Distilled water 1 L





Media

1012

1109 Modi f ied Norkans ' C (MNC) KH2PO4 1 g

MgSO4 0.5 g ZnSO4 0.2 % solution 0.5 ml NH4-tartrate 0.5 g Fe-citrate 1.0 % solution 0.5 ml Thiamine-HCl 50 μg Casein hydrolysate 0.23 g Yeast extract 0.5 g Glucose 10 g Agar 15 g Distilled water 1 L

1110 Alka l ic MRS (pH 8.5) + 3% NaCl Solution A* 500 ml

Solution B** 100 ml Solution C*** 400 ml

Separately autoclave Solutions A, B and C. Mix aseptically. Final pH around 8.5.

* Solution A

Peptone 10 g Meat extract 10 g Yeast extract 5 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 NaCl 30 g Distilled water 500 ml

pH unadjusted

** Solution B

Na2CO3 5 g Distilled water 100 ml

pH unadjusted

*** Solution C

Agar 15 g Distilled water 400 ml

pH unadjusted

In the case of liquid medium, prepare Solution A with 900 ml of distilled water and mix only with Solution B.

1112 Anaero Columbia Agar wi th Rabbi t B lood Anaero Columbia agar with rabbit blood (BD-BBL)

1113 Sporu la t ion Medium Dissolve each regent in distilled water and sterilize them

individually. Cool them down to about 60℃, and mix all the solution together.

Solution A

Nutrient broth 8 g FeSO4·7H2O 0.278 mg MgSO4·7H2O 0.25 g KCl 1 g Agar (if needed) 15 g Distilled water 800 ml

pH unadjusted

Solution B

Ca(NO3)2·4H2O 236.15 mg Distilled water 100 ml

pH unadjusted

Solution C

MnCl2·4H2O 1.979 mg Distilled water 100 ml

pH unadjusted

1118 Thermotoga Medium K2HPO4 0.05 g

KH2PO4 0.09 g NH4Cl 0.25 g MgSO4·7H2O 0.25 g CaCl2·2H2O 0.15 g Bacto Yeast Extract (Difco) 1 g Glucose 1.8 g Na2CO3 0.25 g Vitamin solution* 2 ml Trace element solution** 1 ml Resazurin 1 mg Cysteine-HCl 0.3 g Na2S·9H2O 0.3 g Distilled water 1 L

Mix ingredients except for Na2CO3, vitamin solution, cysteine-HCl and Na2S·9H2O, and autoclave under a N2/CO2 atmosphere (80/20). Separately autoclave cysteine-HCl and Na2S·9H2O solutions as 3% solutions under a N2 atmosphere. Prior to inoculation, add the filter-sterile 5% Na2CO3 and vitamin solutions, cysteine-HCl, and Na2S·9H2O.

* Vitamin solution





Media

1013


Bacto Tryptone (Difco) 1 g Sodium glutamate monohydrate 1 g CaSO4·2H2O 0.06 g MgSO4·7H2O 0.1 g NaCl 0.008 g KNO3 0.1 g NaNO3 0.69 g Na2HPO4 0.11 g Trace elements solution* 10 ml Gelrite (if needed) 8 g Distilled water 1 L







Bacto Tryptone (Difco) 1 g Sodium glutamate monohydrate 1 g CaSO4·2H2O 0.06 g MgSO4·7H2O 0.1 g NaCl 0.008 g KNO3 0.1 g NaNO3 0.69 g Na2HPO4 0.11 g Na2S2O3·5H2O 1.24 g Trace elements solution* 10 ml Gelrite (if needed) 8 g Distilled water 1 L






1128 Ac id ianus Medium (NH4)2SO4 1.3 g

KH2PO4 0.28 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 Na2MoO4·2H2O 0.03 mg VOSO4·xH2O 0.03 mg CoSO4·7H2O 0.01 mg Bacto Yeast Extract (Difco) 0.5 g Sulfur (powder) 1 g Resazurin 1 mg Na2S·9H2O 0.5 g Distilled water 1 L

Mix ingredients except for Bacto Yeast Extract, sulfur and Na2S·9H2O, and adjust pH to 2.0 with H2SO4. Dispense the medium into suitable culture vessels under a stream of H2/CO2 (80/20) and seal with butyl rubber stoppers. Separately autoclave Bacto Yeast Extract and Na2S·9H2O as 5% solutions under a N2 atmosphere. Sulfur is sterilized separately by autoclaving for 60 min at 105°C on each of successive days. Prior to inoculation, add Bacto Yeast Extract, sulfur and Na2S·9H2O, and pressurize the inoculated vessels to 150 kPa with H2/CO2.


Bacto Tryptone (Difco) 1 g Sodium glutamate monohydrate 1 g CaSO4·2H2O 0.06 g MgSO4·7H2O 0.1 g NaCl 0.008 g KNO3 0.1 g NaNO3 0.69 g Na2HPO4 0.11 g Na2S2O3·5H2O 0.1 g Trace elements solution* 10 ml Agar (if needed) 20 g Distilled water 1 L


Media

1014




1130 Sazae YGS Turban shell (sazae) meat cut 75 g

Yeast extract 10 g Glucose 30 g Agar 12 g Seawater (3 % salinity) 1 L

1132 Mal t Ext ract Eb ios Agar (MEBA) Malt extract 20 g

Ebios (tablets)* 1 g Distilled water 1 L Agar 15 g

* Manufactured by Asahi Breweries, Ltd., Tokyo, Japan. Distributed by Tanabe Seiyaku Co. Ltd., Osaka, Japan.

1136 CHROMagerT M Malassez ia /Candida* * Kanto Chemical Co., Inc., Tokyo, Japan.

viii. list of media939 viii. list of media 1 potato sucrose agar (psa) potato 200 g sucrose 20 g...

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