Nutrient Media When unsealing an ampoule with a culture obtained from the All-Russian Collection of Microorgan- isms (VKM) and for initial reinoculations, it is recommended to use the medium specified for the strain in the Catalogue. General comments to the descriptions of the media 1. In all cases when the composition of a medium is given as the description of particular solutions, this implies that the given solutions should be sterilized separately and mixed upon sterilization. 2. In all cases, besides when specified otherwise, a given mode of sterilization assumes autoclaving. If the sterilization conditions of a medium are not specified, they can be set arbitrarily within the limits accepted in the general microbiological practice. 3. The purity of the reagents is not specified in the descriptions. It is assumed that the respective re- agents will be used, specifically, peptone, triptone, yeast extract, etc. marked 'Bacto' or 'For microbi- ological work'. For some media, descriptions for preparing the components are given instead of using commercial concentrates. 4. The pH values given indicate the magnitudes, which a medium shall have prior to inoculation. In the cases when some solutions are described and errors are possible, it is specified: pH of the medium. An insufficiently alkaline medium shall be alkalized, usually with a sterile solution of NaOH or NaHCO3; an insufficiently acidic one should be acidified, usually with a sterile solution of HCl. 5. Some media differ in a few components or their concentrations. When a medium similar to the al- ready mentioned one is given, the reader is referred to the latter. The references can (i) specify a com- pound added and its concentration; (ii) specify the set of trace elements or vitamins; (iii) specify the differences in the concentrations of particular compounds. In the latter case, the zero concentration of a substrate implies that it is not to be added. 1. ALLOMONAS ENTERICA MEDIUM Peptone 10.0 g NaCl 20.0 g Meat extract 5.0 g or yeast extract 3.0 g Distilled water 1000.0 ml 2. YEAST WATER Pressed yeast 200.0 g Tap water 1000.0 ml Twice filter hot through a paper filter or centrifuge. Sterilize at 121°C for 15 min. 3. POTATO AGAR Potato 200.0 g Agar 20.0 g Tap water 1000.0 ml Boil potatoes for 1 h, filter cold through a cotton wool-gauze filter. Sterilize at 121°C for 30 min. 4. WORT AGAR Wort extract (malt extract) 20.0 g Agar 20.0 g Water 1000.0 ml Sterilize at 121°C for 30 min. 5. PEPTONE MEAT AGAR Peptone 10.0 g NaCl 5.0 g Agar 20.0 g Meat water 1000.0 ml Preparation of meat water: comminute 500 g of meat free of bones, fat and tendons, add 1000.0 ml of tap water and leave for 12 h at room temperature or in a thermostat at 30°C, or for 2 h at 37°C. Then squeeze the meat through gauze or cloth and boil the filtrate for 5 min. The proteins are denatured. Fil- ter the cooled down mass through a cotton-wool filter and add water to the initial volume. Sterilize at 121°C for 30 min. 6. PEPTONE MEAT BROTH Peptone 10.0 g NaCl 5.0 g Meat water 1000.0 ml pH 7.2 - 7.4. Sterilize at 121°C for 30 min. Preparation of meat water: comminute 500 g of meat free of bones, fat and tendons, add 1000.0 ml of tap water and leave for 12 h at room temperature or in a thermostat at 30°C, or for 2 h at 37°C. Then squeeze the meat through gauze or cloth and boil the filtrate for 5 min. The proteins are denatured. Fil- ter the cooled down mass through a cotton-wool filter and add water to the initial volume. Sterilize at 121°C for 30 min. 7. OATMEAL AGAR Oats 30.0 g Agar 15.0 g Tap water 1000.0 ml Keep oats on a water bath at 58°C for 1 h, filter through 2 layers of gauze, dilute to 1000.0 ml and add 15.0 g agar. Sterilize at 121°C for 30 min. 8. PEPTONE MAIZE AGAR Peptone 5.0 g Maize extract 5.0 g Starch (soluble) 10.0 g NaCl 5.0 g CaCl2 0.5 g Agar 20.0 g Tap water 1000.0 ml pH 7.2-7.4. Sterilize at 121°C for 30 min. 9. WORT AGAR 7 B Malt wort 7 B 1000.0 ml Agar 20.0 g Sterilize at 105°C for 30 min. Preparation of malt wort: mix 250 g of ground malt in 1000.0 ml of tap water, heat to 55-60°C and keep at this temperature for 1.5-2 h at periodic stirring. Then increase the temperature to 80°C and keep for 10 min. Then cool the wort, squeeze through a linen bag, adjust the concentration of sugars to 7 B. 10. WORT AGAR 2-3 B Malt wort 2-3 B 1000.0 ml Agar 20.0 g Sterilize at 105°C for 30 min. Preparation of malt wort: mix 250 g of ground malt in 1000.0 ml of tap water, heat to 55-60°C and keep at this temperature for 1.5-2 h at periodic stirring. Then increase the temperature to 80°C and keep for 10 min. Then cool the wort, squeeze through a linen bag, adjust the concentration of sugars to 2-3 B. 11. WORT AGAR 3.5 B (MALT AGAR) Malt wort 3.5 B 1000.0 ml Agar 20.0 g Sterilize at 105°C for 30 min. Preparation of malt wort: mix 250 g of ground malt in 1000.0 ml of tap water, heat to 55-60°C and keep at this temperature for 1.5-2 h at periodic stirring. Then increase the temperature to 80°C and keep for 10 min. Then cool the wort, squeeze through a linen bag, adjust the concentration of sugars to 3.5B. 12. CZAPEK DOX MEDIUM NaNO2 3.0 g K2HPO4 1.0 g KCl 0.5 g MgSO4 x 7H2O 0.5 g FeSO4 x 7H2O 0.01 g Sucrose 30.0 g Agar 20.0 g Distilled water 1000.0 ml pH 6.0. Sterilize at 121°C for 30 min. 13. POTATO-GLUCOSE AGAR 1 Grated potato 200.0 g Glucose 20.0 g Agar 20.0 g Tap water 1000.0 ml Boil potatoes for 1 h in 1000.0 ml of water, filter through gauze, add water to the initial volume, add glucose and agar. Sterilize at 105°C for 30 min. 14. POTATO-CARROT AGAR Grated potato 20.0 g Grated carrot 20.0 g Agar 20.0 g Tap water 1000.0 ml Boil potato and carrot in 1000.0 ml of water for 1 h, filter, add water to the initial volume, adjust pH to 7.0 - 7.1 and add agar. Sterilize at 121°C for 30 min. 15. LB MEDIUM Tryptone 10.0 g Yeast extract 5.0 g NaCl 10.0 g Tap water 1000.0 ml 16. YT MEDIUM Tryptone 8.0 g Yeast extact 5.0 g NaCl 5.0 g Tap water 1000.0 ml 17. GLUCOSE PEPTONE AGAR WITH YEAST WATER Glucose 40.0 g Peptone 5.0 g Agar 20.0 g 10% yeast water 1000.0 ml Sterilize by steam. Preparation of yeast water: suspend 200.0 g of pressed yeast in 1000.0 ml of tap water. Twice filter hot through a paper filter or centrifuge. Sterilize at 121°C for 15 min. 18. GLUCOSE PEPTONE AGAR WITH 5% NaCl Glucose 40.0 g Peptone 5.0 g NaCl 50.0 g Agar 20.0 g 10% yeast water 1000.0 ml Sterilize by steam. Preparation of yeast water: suspend 200.0 g of pressed yeast in 1000.0 ml of tap water. Twice filter hot through a paper filter or centrifuge. Sterilize at 121°C for 15 min. 19. WORT AGAR 7 B WITH 5% NaCl Malt wort 7 B 1000.0 ml NaCl 50.0 g Agar 20.0 g Sterilize at 105°C for 30 min. Preparation of malt wort: mix 250 g of ground malt in 1000.0 ml of tap water, heat to 55-60°C and keep at this temperature for 1.5-2 h at periodic stirring. Then increase the temperature to 80°C and keep for 10 min. Then cool the wort, squeeze through a linen bag, adjust the concentration of sugars to 7 B. 20. LIESKE MEDIUM Mg-acetate 0.1 g Agar 15.0 g Distilled water 1000.0 ml 21. MANNITOL AGAR WITH YEAST WATER Mannitol 10.0 g Agar 15.0 g 10% yeast water 100.0 ml Tap water 900.0 ml Preparation of yeast water: suspend 200.0 g of pressed yeast in 1000.0 ml of tap water. Twice filter hot through a paper filter or centrifuge. Sterilize at 121°C for 15 min. 22. MALT AGAR WITH 60% SUCROSE Malt wort 3.5 B 1000.0 ml Sucrose 600.0 g Agar 20.0 g Sterilize at 121°C for 20 min. Preparation of malt wort: mix 250 g of ground malt in 1000.0 ml of tap water, heat to 55-60°C and keep at this temperature for 1.5-2 h at periodic stirring. Then increase the temperature to 80°C and keep for 10 min. Then cool the wort, squeeze through a linen bag, adjust the concentration of sugars to 3.5B. 23. MALT AGAR WITH 40% SUCROSE Malt wort 3.5 B 1000.0 ml Sucrose 400.0 g Agar 20.0 g Sterilize at 121°C for 20 min. Preparation of malt wort: mix 250 g of ground malt in 1000.0 ml of tap water, heat to 55-60°C and keep at this temperature for 1.5-2 h at periodic stirring. Then increase the temperature to 80°C and keep for 10 min. Then cool the wort, squeeze through a linen bag, adjust the concentration of sugars to 3.5B. 24. MALT AGAR WITH 20% SUCROSE Malt wort 3.5 B 1000.0 ml Sucrose 200.0 g Agar 20.0 g Sterilize at 121°C for 20 min. Preparation of malt wort: mix 250 g of ground malt in 1000.0 ml of tap water, heat to 55-60°C and keep at this temperature for 1.5-2 h at periodic stirring. Then increase the temperature to 80°C and keep for 10 min. Then cool the wort, squeeze through a linen bag, adjust the concentration of sugars to 3.5B. 25. MALT AGAR WITH FILTER PAPER Malt wort 3.5 B 1000.0 ml Agar 20.0 g Sterilize at 105°C for 30 min. Preparation of malt wort: mix 250 g of ground malt in 1000.0 ml of tap water, heat to 55-60°C and keep at this temperature for 1.5-2 h at periodic stirring. Then increase the temperature to 80°C and keep for 10 min. Then cool the wort, squeeze through a linen bag, adjust the concentration of sugars to 3.5B. Sterilize filter paper strips with dry heat and soak it with sterile medium. 26. MANURE AGAR Horse manure 100-125 g Agar 25.0 g Distilled water 1000.0 ml Boil manure in 1000.0 ml of water for 10 min, then keep for 16-20 h, filter through 1-2 layers of filter paper, adjust to the initial volume, add agar. Sterilize at 121°C for 15 min. 27. PEPTONE LACTOSE AGAR Peptone 10.0 g Lactose 10.0 g Agar 15.0 g Tap water 1000.0 ml Sterilize at 105°C for 15 min. 28. TRYPTOSE AGAR Tryptose 20.0 g Dextrose 1.0 g NaCl 5.0 g Agar 15.0 g Thiamine-HCl 0.005 g Distilled water 1000.0 ml Sterilize at 105°C for 15 min. 29. PEA AGAR Yellow peas 100.0 g K2HPO4 0.5 g Sucrose 10.0 g Agar 20.0 g Tap water 1000.0 ml Boil peas in 1000.0 ml of water, filter through gauze, add water to the initial volume; add phosphate, sucrose and agar. Sterilize at 121°C for 30 min. 30. AZOTOBACTER MEDIUM 1 Glucose 5.0 g Mannitol 5.0 g CaCl2 x 2 H2O 0.1 g MgSO4 x 7 H2O 0.1 g Na2MoO4 x 2 H2O 5.0 mg K2HPO4 0.9 g KH2PO4 0.1 g FeSO4 x 7 H2O 0.01 g CaCO3 5.0 g Agar 15.0 g Distilled water 950.0 ml pH 7.3. Sterilize glucose and mannitol separately (in 50.0 ml H2O) and add to the medium after auto- claving. 31. CABBAGE AGAR Cabbage 50.0 g Glucose 20.0 g Peptone 10.0 g Agar 20.0 g Tap water 1000.0 ml Boil 50.0 g of cabbage in 1000.0 ml of water, filter cabbage and adjust the volume of broth to the ini- tial value. 32. CURD DECOCTION Pour 9.0 l of distilled water into 3.0 kg of curd and add 150.0 of dry Aspergillus terricola mycelium, shake, pour chloroform, seal with a stopper. Decoct at 37°C for 10 days, adjust pH to 7.0 with 1 N NaOH. The decoction shall contain 400 mg% amine nitrogen and 300 mg% tryptophan. 33. PEPTONE MEAT AGAR WITH 0.2% UREA Peptone 10.0 g NaCl 5.0 g Urea 2.0 g Agar 20.0 g Meat water 1000.0 ml Sterilize urea at 105°C for 30 min to 1 h. Preparation of meat water: comminute 500 g of meat free of bones, fat and tendons, add 1000.0 ml of tap water and leave for 12 h at room temperature or in a thermostat at 30°C, or for 2 h at 37°C. Then squeeze the meat through gauze or cloth and boil the filtrate for 5 min. The proteins are denatured. Fil- ter the cooled down mass through a cotton-wool filter and add water to the initial volume. Sterilize at 121°C for 30 min. 34. SOIL EXTRACT Dry garden soil, rich in organic material, in the air by spreading in a thin layer, comminuting and stir- ring. Then sieve through a rough sieve, and mix 400 g of soil with 960 ml of tap water. Autoclave at 121°C for 1 h at the end of the day but leave the autoclave open overnight. Filter the cooled extract through filter paper, autoclave 300 ml of filtrate at 121°C for 20 min and allow to stay for 2 weeks or longer to settle the sediment. Decant the clear supernatant liquid and use to prepare the medium. 35. NITROBACTER MEDIUM 1 Solution 1 (see below) 0.5 ml Solution 2 (see below) 0.5 ml Solution 3 (see below) 1.0 ml Solution 4 (see below) 0.5 ml Solution 5 (see below) 0.5 ml Solution 6 (see below) 0.1 ml Distilled water to 1000.0 ml Solution 1: CaCl2 2.0 g Distilled water 100.0 ml Solution 2: MgSO4 x 7 H2O 20.0 g Distilled water 100.0 ml Solution 3: Chelated iron 0.1 g Distilled water 100.0 ml Solution 4: Na2MoO4 0.1 g MnCl2 x 4 H2O 0.2 g CoCl2 x 6 H2O 0.002 g ZnSO4 x 7 H2O 0.1 g CuSO4 x 5 H2O 0.02 g Distilled water 1000.0 ml Solution 5: NaNO3 41.4 g Distilled water 100.0 ml Solution 6: K2HPO4 1.74 g Distilled water 100.0 ml 36. PROPIONIBACTERIUM MEDIUM Yeast extract 10.0 g KH2PO4 1.0 g Na2HPO4 x 2 H2O 3.0 g Na-lactate (70%) 40.0 ml Distilled water 1000.0 ml Dissolve all ingredients and add lactate. pH 7.0. 37. KNOP MEDIUM WITH FILTER PAPER Ca(NO3)2 1.0 g KH2PO4 0.25 g MgSO4 0.25 g FeCl3 Traces Agar 20.0 g Distilled water 1000.0 ml Sterilize at 121°C for 30 min. Sterilize filter paper strips by dry heat and soak with sterile medium. 38. WORT AGAR WITH 12% NaCl Malt wort 7 B 1000.0 ml NaCl 120.0 g Agar 20.0 g Sterilize at 105°C for 30 min. Preparation of malt wort: mix 250 g of ground malt in 1000.0 ml of tap water, heat to 55-60°C and keep at this temperature for 1.5-2 h at periodic stirring. Then increase the temperature to 80°C and keep for 10 min. Then cool the wort, squeeze through a linen bag, adjust the concentration of sugars to 7 B. 39. WORT AGAR WITH 1% NaCl Malt wort 7 B 1000.0 ml NaCl 10.0 g Agar 20.0 g Sterilize at 105°C for 30 min. Preparation of malt wort: mix 250 g of ground malt in 1000.0 ml of tap water, heat to 55-60°C and keep at this temperature for 1.5-2 h at periodic stirring. Then increase the temperature to 80°C and keep for 10 min. Then cool the wort, squeeze through a linen bag, adjust the concentration of sugars to 7 B. 40. AZOTOBACTER MEDIUM 2 KH2PO4 0.2 g K2HPO4 0.8 g MgSO4 x 7 H2O 0.2 g CaSO4 x 2 H2O 0.1 g FeCl3 Traces Na2MoO4 Traces Yeast extract 0.5 g Sucrose 20.0 g Agar 15.0-20.0 g Distilled water 1000.0 ml pH 7.2. Sterilization at 105°C for 30 min. 41. FLAVOBACTERIUM MEDIUM Na-caseinate 2.0 g Yeast extract 0.5 g Peptone 1.0 g K2HPO4 0.5 g Agar 12.0 g Distilled water 1000.0 ml pH 7.4. 42. PSEUDOMONAS SACCHAROPHILA MEDIUM Solution 1: KH2PO4 4.4 g Na2HPO4 4.8 g NH4Cl 1.0 g MgSO4 x 7 H2O 0.5 g Agar (if needed) 20.0 g Distilled water 1000.0 ml Solution 2: Ferric ammonium citrate 50.0 mg CaCl2 5.0 mg Distilled water 5.0 ml Solution 3: Sucrose 1.0 g Distilled water 10.0 ml 43. SEA WATER MEDIUM WITH YEAST EXTRACT Sea salt 37.9 g Yeast extract 3.0 g Peptone 10.0 g Agar 20.0 g Distilled water to 1000.0 ml pH 7.2-7.4. 44. HALOBACTERIUM MEDIUM 1 Solution 1: NaCl 250.0 g MgSO4 10.0 g KCl 5.0 g CaCl2 x 6 H2O 0.2 g Tap water 800.0 ml Solution 2: Yeast extract 10.0 g Tryptone 2.5 g Agar 20.0 g Tap water 200.0 ml 45. STARVED AGAR Agar 20.0 g Distilled water 1000.0 ml Sterilize at 121°C for 15 min. 46. SP MEDIUM FOR STIGMATELLA AURANTIACA Raffinose 1.0 g Sucrose 1.0 g Galactose 1.0 g Starch (soluble) 5.0 g Casitone 2.5 g MgSO4 x 7 H2O 0.5 g K2HPO4 0.25 g Agar 15.0 g Distilled water 1000.0 ml 47. CM + YE MEDIUM Casamino acids 7.5 g Yeast extract 10.0 g MgSO4 x 7 H2O 20.0 g Na-citrate 3.0 g KCl 2.0 g NaCl 200.0 g Agar 15.0 g FeSO4 x 7H2O in 0.01N HCl (see below) 1.0 ml Distilled water 1000.0 ml Solution of FeSO4 x 7 H2O: 0.01 N HCl 100.0 ml FeSO4 x 7 H2O 4.98 g Adjust pH of medium to 7.4 with 1N NaOH. 48. CASEIN MEDIUM NaCl 250.0 g Casein hydrolysate 5.0 g Yeast extract 5.0 g MgCl2 x 6 H2O 20.0 g KCl 2.0 g CaCl2 x 2 H2O 0.2 g Agar 20.0 g Distilled water 1000.0 ml Adjust pH to 7.4 with NaOH. 49. HALOBACTERIUM MEDIUM 2 Solution 1: NaCl 120.0 g MgCl2 x 6 H2O 50.0 g K2SO4 5.0 g CaCl2 x 6 H2O 0.2 g Distilled water 500.0 ml Solution 2: Tryptone 5.0 g Yeast extract 5.0 g Agar 20.0 g Distilled water 500.0 ml pH 6.8. 50. YEAST GLUCOSE AGAR Yeast extract 5.0 g Peptone 5.0 g Glucose 10.0 g Agar 20.0 g Distilled water 1000.0 ml pH 7.2. Sterilize at 121°C for 15 min. 51. CASITONE AGAR Casitone 3.0 g CaCl2 x 2 H2O 1.36 g Agar 15.0 g Distilled water 1000.0 ml pH 7.2. 52. CASITONE YEAST MEDIUM Casitone 3.0 g CaCl2 x 2 H2O 1.36 g Yeast extract 1.0 g Agar 15.0 g Distilled water 1000.0 ml pH 7.2. 53. THERMUS THERMOPHILUS MEDIUM Yeast extract 4.0 g Polypeptone 8.0 g NaCl 2.0 g Agar 20.0 g Distilled water 1000.0 ml pH 7.0. 54. DESULFOVIBRIO MEDIUM WITH 1% NaCl K2HPO4 0.01 g NaCl 10.0 g MgSO4 0.2 g Na-lactate (40%) 4.0 ml Solution of More salt (see below) 1.0 ml Yeast extract 1.0 g Ascorbic acid 0.1 g Agar 6.0 g Distilled water 1000.0 ml Solution of More salt: Fe(NH4)2(SO4)2 x 6 H2O 1.0 g Distilled water 5.0 ml 55. CAULOBACTER MEDIUM Peptone 2.0 g Yeast extract 1.0 g MgSO4 x 7 H2O 0.2 g Agar 10.0 g Tap water 1000.0 ml 56. CAULOBACTER MEDIUM WITH GLUCOSE Peptone 2.0 g Yeast extract 1.0 g Glucose 2.0 g Riboflavin 1.0 mg MgSO4 x 7 H2O 0.2 g Agar 10.0 g Tap water 1000.0 ml pH 7.0. 57. PEPTONE MEAT AGAR WITH 1% UREA Peptone 10.0 g NaCl 5.0 g Urea 10.0 g Agar 20.0 g Meat water 1000.0 ml pH 8.0. Preparation of meat water: comminute 500 g of meat free of bones, fat and tendons, add 1000.0 ml of tap water and leave for 12 h at room temperature or in a thermostat at 30°C, or for 2 h at 37°C. Then squeeze the meat through gauze or cloth and boil the filtrate for 5 min. The proteins are denatured. Fil- ter the cooled down mass through a cotton-wool filter and add water to the initial volume. Sterilize at 121°C for 30 min. 58. HALOBACTERIUM MEDIUM 3 Solution 1: Casamino acids 7.5 g Yeast extract 10.0 g Na-citrate 3.0 g KCl 2.0 g MgSO4 x 7 H2O 20.0 g FeCl3 traces NaCl 250.0 g Distilled water 750.0 ml Solution 2: Agar 25.0 g Distilled water 250.0 ml pH 7.4. Sterilize at 121°C for 15 min. 59. Peptone 2.5 g Meat extract 2.5 g NaCl 2.5 g Yeast extract 0.1 g Glucose 2.5 g Sucrose 5.0 g Casein acidic hydrolysate 0.1 g Agar 15.0 g Distilled water 1000.0 ml 60. ALFALFA AGAR K2HPO4 0.5 g MgSO4 x 7 H2O 0.2 g KH2PO4 0.5 g NaCl 0.2 g CaSO4 0.1 g (NH4)2MoO4 Traces Mannitol 20.0 g Alfalfa meal 10.0 ml Agar 20.0 g Distilled water 1000.0 ml pH 7.0-7.2. Sterilize at 105°C for 20 min. 61. MEDIUM FOR NITROGEN-FIXING SPIRILLUM K2HPO4 0.1 g KH2PO4 0.4 g MgSO4 x 7 H2O 0.2 g NaCl 0.1 g CaCl2 0.02 g FeCl3 0.01 g Na2MoO4 x 2 H2O 0.002 g Na-malate 5.0 g Yeast extract 50.0 mg Distilled water 1000.0 ml pH 7.2-7.4. 62. MANURE TINCTURE Cow manure (fresh) 1.0 kg Distilled water 3000.0 ml Boil, squeeze through gauze into a bottle and dilute to 3.0 l. 63. MEDIUM WITH CURD DECOCTION Curd decoction 61.0 ml Manure tincture 184.0 ml Na-acetate 1.0 g Agar 20.0 g Tap water 735.0 ml pH 7.8. Preparation of curd decoction: pour 9.0 l of distilled water into 3.0 kg of curd and add 150.0 of dry Aspergillus terricola mycelium, shake, pour chloroform, seal with a stopper. Decoct at 37°C for 10 days, adjust pH to 7.0 with 1 N NaOH. The decoction shall contain 400 mg% amine nitrogen and 300 mg% tryptophan. Preparation of manure tincture: mix cow manure (fresh) 1.0 kg and distilled water 3000.0 ml. Boil, squeeze through gauze into a bottle and dilute to 3.0 l. 64. PEPTONE YEAST AGAR Peptone 5.0 g Yeast extract 3.0 g KH2PO4 0.2 g Glucose 5.0 g Agar 20.0 g Tap water 1000.0 ml pH 7.0-7.2. 65. OATMEAL AGAR FOR BACTERIA Oatmeal 2.0 g Peptone 0.5 g NaCl 1.0 g Galactose 0.5 g Agar 12.0 g Distilled water 1000.0 ml pH 7.5. 66. MEDIUM WITH SOIL EXTRACT Peptone 5.0 g Meat extract 3.0 g Agar 15.0 g Soil extract 250.0 ml Tap water 750.0 ml pH 7.0. Preparation of soil extract: dry garden soil, rich in organic material, in the air by spreading in a thin layer, comminuting and stirring. Then sieve through a rough sieve, and mix 400 g of soil with 960 ml of tap water. Autoclave at 121°C for 1 h at the end of the day but leave the autoclave open overnight. Filter the cooled extract through filter paper, autoclave 300 ml of filtrate at 121°C for 20 min and al- low to stay for 2 weeks or longer to settle the sediment. Decant the clear supernatant liquid and use to prepare the medium. 67. PEPTONE MEAT AGAR WITH TRACE ELEMENTS Yeast autolysate 2.0 g Trace element solution (see below) 1.0 ml Agar 20.0 g Peptone meat broth (see below) 1000.0 ml Trace element solution: H3BO3 5.0 g (NH4)2MoO4 5.0 g KI 0.5 g NaBr 0.5 g ZnSO4 0.2 g Al2(SO4)3 0.3 g Distilled water 1000.0 ml Peptone meet broth: Peptone 10.0 g NaCl 5.0 g Meat water 1000.0 ml pH 7.2 - 7.4. Sterilize at 121°C for 30 min. Preparation of meat water: comminute 500 g of meat free of bones, fat and tendons, add 1000.0 ml of tap water and leave for 12 h at room temperature or in a thermostat at 30°C, or for 2 h at 37°C. Then squeeze the meat through gauze or cloth and boil the filtrate for 5 min. The proteins are denatured. Fil- ter the cooled down mass through a cotton-wool filter and add water to the initial volume. Sterilize at 121°C for 30 min. 68. PEPTONE MEAT AGAR WITH 3% SEA SALT Peptone 10.0 g NaCl 5.0 g Sea salt 30.0 g Agar 20.0 g Meat water 1000.0 ml Preparation of meat water: comminute 500 g of meat free of bones, fat and tendons, add 1000.0 ml of tap water and leave for 12 h at room temperature or in a thermostat at 30°C, or for 2 h at 37°C. Then squeeze the meat through gauze or cloth and boil the filtrate for 5 min. The proteins are denatured. Fil- ter the cooled down mass through a cotton-wool filter and add water to the initial volume. Sterilize at 121°C for 30 min. 69. DAVIS SUPPLEMENTED MINIMAL MEDIUM Solution 1: Yeast extract 2.0 g Casein hydrolysate 2.0 g K2HPO4 7.0 g KH2PO4 3.0 g Na-citrate x 3 H2O 0.5 g MgSO4 x 7 H2O 0.1 g (NH4)2SO4 1.0 g Agar 15.0 g Distilled water 1000.0 ml Solution 2: Glucose 2.0 g Distilled water 20.0 ml pH 7.0. 70. MEDIUM VY/2 FOR STIGMATELLA AURANTIACA Baker's yeast 5.0 g CaCl2 x 2 H2O 1.36 g Vitamin B12 (cyanocobalamin) 0.5 mg Agar 15.0 g Distilled water 1000.0 ml Sterilize at 105°C for 20 min. Vitamin B12 sterilize separately with filtration. pH of the medium 7.2 (adjust with KOH before adding agar). 71. NITROSOCOCCUS MEDIUM 1 (NH4)2SO4 1.32 g MgSO4 x 7 H2O 380.0 mg CaCl2 x 2 H2O 20.0 mg Chelated iron (13% iron) 1.0 mg Na2MoO4 x 2 H2O 100.0 ?g MnCl2 x 4 H2O 200.0 ?g CoCl2 x 6 H2O 2.0 ?g ZnSO4 x 7 H2O 100.0 ?g K2HPO4 8.7 mg Phenol red (0.04%) 3.25 ml Sea water 1000.0 ml Adjust final pH to 7.5 - 7.8 with 1 N HCl. 72. BEAN AGAR Beans (peas or pulse) 100.0 g K2HPO4 0.5 g Sucrose 10.0 g Agar 20.0 g Water 1000.0 ml Boil beans in 1000.0 ml of water, filter through gauze, add water to the initial volume; add phosphate, sucrose and agar. Sterilize at 121°C for 30 min. 73. GYT-AGAR Glucose 10.0 g Yeast extract 1.0 g Tryptose 2.0 g FeSO4 x 7 H2O 1.0 mg Agar 20.0 g Distilled water 1000.0 ml pH 7.2. 74. HOTTINGER BROTH Boil meat (1-2 cm pieces) (without fat or tendons) in 2.0 l of water, then mince. Adjust pH of the de- coction to 8.0, mix with minced meat and cool down to 40°C. Then add 1.0 g of dry pancreatin, mix and again alkalinize to pH 7.8-8.0. Pour the mixture into a bottle with the rubber stopper (1/3 of the bottle to remain free), add chloroform (20 ml), mix and open the bottle for 1 min to remove the excess chloroform vapors. 2 h after pancreatin was added, adjust pH to 7.4-7.6 and leave the mixture for 2 weeks at 18-20°C. The first 4 days adjust pH of the medium; shake and mix 3 times a day, then stir once a day. Two days before the end of the procedure stop mixing to allow the decoction to settle. The liquid shall be of straw color, the reaction with tryptophan with bromine water shall be positive; in the decoction hydrolysate the total nitrogen shall be no less than 1100 mg%. Filter the decoction through the linen, pour into flasks and sterilize in autoclave at 121°C for 30 min. Filter prior to use. 75. MODIFICATION OF TWEEN-80 MEDIUM FOR MILK-ACID BACTERIA Yeast extract 5.0 g Glucose 2.5 g Tween-80 1.0 ml K2HPO4 2.0 g Na-acetate 5.0 g NH4-citrate 2.0 g MgSO4 x 7 H2O 0.2 g MnSO4 x 4 H2O 0.05 g Agar 5.0 g Meat water 400.0 ml Distilled water 600.0 ml pH 6.0-6.5. Preparation of meat water: comminute 500 g of meat free of bones, fat and tendons, add 1000.0 ml of tap water and leave for 12 h at room temperature or in a thermostat at 30°C, or for 2 h at 37°C. Then squeeze the meat through gauze or cloth and boil the filtrate for 5 min. The proteins are denatured. Fil- ter the cooled down mass through a cotton-wool filter and add water to the initial volume. Sterilize at 121°C for 30 min. 76. POTATO-PEPTONE MEDIUM Potato decoction 200.0 ml Yeast extract 1.0 g Peptone 5.0 g Agar 30.0 g Distilled water 800.0 ml Preparation of potato decoction: boil 200.0 g potatoes in 1.0 l of tap water for 1 h, filter cold through a cotton wool-gauze filter. Sterilize at 121°C for 30 min. 77. PEPTONE MEAT AGAR WITH VITAMINS Peptone 10.0 g NaCl 5.0 g Agar 20.0 g Yeast extract 1.0 g Glucose 1.0 g B12 2.0 mg B1 2.0 mg Meat water 1000.0 ml Sterilize at 105°C for 30 min. Preparation of meat water: comminute 500 g of meat free of bones, fat and tendons, add 1000.0 ml of tap water and leave for 12 h at room temperature or in a thermostat at 30°C, or for 2 h at 37°C. Then squeeze the meat through gauze or cloth and boil the filtrate for 5 min. The proteins are denatured. Fil- ter the cooled down mass through a cotton-wool filter and add water to the initial volume. Sterilize at 121°C for 30 min. 78. PEPTONE MEAT AGAR WITH 2% SOLUBLE STARCH Peptone 10.0 g NaCl 5.0 g Soluble starch 20.0 g Agar 20.0 g Meat water 1000.0 ml Preparation of meat water: comminute 500 g of meat free of bones, fat and tendons, add 1000.0 ml of tap water and leave for 12 h at room temperature or in a thermostat at 30°C, or for 2 h at 37°C. Then squeeze the meat through gauze or cloth and boil the filtrate for 5 min. The proteins are denatured. Fil- ter the cooled down mass through a cotton-wool filter and add water to the initial volume. Sterilize at 121°C for 30 min. 79. PEPTONE MEAT AGAR WITH 1% SOLUBLE STARCH Peptone 10.0 g NaCl 5.0 g Soluble starch 10.0 g Agar 20.0 g Meat water 1000.0 ml pH 7.2 Preparation of meat water: comminute 500 g of meat free of bones, fat and tendons, add 1000.0 ml of tap water and leave for 12 h at room temperature or in a thermostat at 30°C, or for 2 h at 37°C. Then squeeze the meat through gauze or cloth and boil the filtrate for 5 min. The proteins are denatured. Fil- ter the cooled down mass through a cotton-wool filter and add water to the initial volume. Sterilize at 121°C for 30 min. 80. PEPTONE MEAT AGAR WITH 6% NaCl Peptone 10.0 g NaCl 60.0 g Agar 20.0 g Meat water 1000.0 ml Preparation of meat water: comminute 500 g of meat free of bones, fat and tendons, add 1000.0 ml of tap water and leave for 12 h at room temperature or in a thermostat at 30°C, or for 2 h at 37°C. Then squeeze the meat through gauze or cloth and boil the filtrate for 5 min. The proteins are denatured. Fil- ter the cooled down mass through a cotton-wool filter and add water to the initial volume. Sterilize at 121°C for 30 min. 81. PEPTONE MEAT AGAR WITH 1.8% SEA SALT Peptone 10.0 g NaCl 5.0 g Sea salt 18.0 g Agar 20.0 g Meat water 1000.0 ml Preparation of meat water: comminute 500 g of meat free of bones, fat and tendons, add 1000.0 ml of tap water and leave for 12 h at room temperature or in a thermostat at 30°C, or for 2 h at 37°C. Then squeeze the meat through gauze or cloth and boil the filtrate for 5 min. The proteins are denatured. Fil- ter the cooled down mass through a cotton-wool filter and add water to the initial volume. Sterilize at 121°C for 30 min. 82. POTATO AGAR WITH 2% GLUCOSE Potato 200.0 g Agar 20.0 g Glucose 20.0 g Tap water 1000.0 ml Boil potatoes for 1 h, filter cold through a cotton wool-gauze filter. Add glucose. Sterilize at 105°C for 30 min. 83. LOPATINA MEDIUM Glucose 10.0 g KH2PO4 0.2 g MgSO4 x 7 H2O 0.2 g Thyrosin 1.0 g NaCl 0.2 g CaSO4 0.1 g Agar 20.0 g Water 1000.0 ml 84. MEDIUM WITH CASEIN HYDROLYSATE Casein hydrolysate 10.0 g Glucose 5.0 g p-Aminobenzoic acid 5.0 ?g Agar 20.0 g Water 1000.0 ml 85. MEDIUM WITH HOTTINGER BROTH Peptone 10.0 g Yeast autolysate 10.0 g Hottinger broth 10.0 ml Phosphate solution (see below) 0.5 ml Salt solution (see below) 0.5 ml Glucose 5.0 g Water 950.0 ml Phosphate solution: KH2PO4 100.0 mg K2HPO4 100.0 mg Distilled water 25.0 ml Salt solution: MgSO4 x 7 H2O 40.0 mg NaCl 2.0 mg FeSO4 2.0 mg Distilled water 25.0 ml Preparation of Hottinger broth: boil meat (1 - 2 cm pieces) (without fat or tendons) in 2.0 l of water, then mince. Adjust pH of the decoction to 8.0, mix with minced meat and cool down to 40°C. Then add 1.0 g of dry pancreatin, mix and again alkalinize to pH 7.8-8.0. Pour the mixture into a bottle with the rubber stopper (1/3 of the bottle to remain free), add chloroform (20 ml), mix and open the bottle for 1 min to remove the excess chloroform vapors. 2 h after pancreatin was added, adjust pH to 7.4-7.6 and leave the mixture for 2 weeks at 18-20°C. The first 4 days adjust pH of the medium; shake and mix 3 times a day, then stir once a day. Two days before the end of the procedure stop mixing to allow the decoction to settle. The liquid shall be of straw color, the reaction with tryptophan with bromine water shall be positive; in the decoction hydrolysate the total nitrogen shall be no less than 1100 mg%. Filter the decoction through the linen, pour into flasks and sterilize in autoclave at 121°C for 30 min. Filter prior to use. 86. NITROSOLOBUS MEDIUM 1 CaCl2 x 2 H2O 0.02 g MgSO4 x 7 H2O 0.2 g Chelated iron 1.0 mg Na2MoO4 x 2 H2O 0.1 mg MnCl2 x 4 H2O 0.2 mg CoCl2 x 6 H2O 2.0 ?g CuSO4 x 5 H2O 0.02 mg ZnSO4 x 7 H2O 0.1 mg (NH4)2SO4 1.65 g K2HPO4 0.087 g Phenol red 5.0 mg Distilled water 1000.0 ml Adjust pH to 7.5 with 0.1 M Na2CO3. 87. NITROSOCOCCUS MEDIUM 2 NH4Cl 0.5 g KH2PO4 0.05 g CaCO3 5.0 g Chelated iron 1.0 mg Phenol red (0.04%) 3.25 ml Sea water 1000.0 ml Adjust pH to 7.5 - 7.8 with 1 N HCl. 88. SPIRILLUM MEDIUM Peptone 10.0 g Succinic acid 1.0 g (NH4)2SO4 1.0 g MgSO4 x 7 H2O 1.0 g FeCl3 x 6 H2O 2.0 mg MnSO4 x H2O 2.0 mg Distilled water 1000.0 ml pH 6.8. 89. MILK MEDIUM FOR HALOPHILS Solution 1: Milk 500.0 ml Solution 2: MgSO4 x 7 H2O 10.0 g KNO3 2.0 g NaCl 200.0 g Distilled water 100.0 ml Solution 3: Peptone 5.0 g Glycerol 10.0 g Agar 25.0 g Distilled water 400.0 ml pH 8.4. Sterilize at 121°C for 20 min. Sequence of mixing: add warm skim milk to a hot mixture of solutions 1 and 2. 90. DESULFOVIBRIO GIGAS MEDIUM Solution 1: KH2PO4 1.0 g NH4Cl 0.5 g MgSO4 x 7 H2O 0.4 g Na2SO4 2.0 g CaCl2 x 2 H2O 0.1 g Trace element solution (see below) 1.0 ml 2 M H2SO4 1.0 ml Na L-lactate 2.0 g Distilled water 950.0 ml Solution 2: NaHCO3 2.0 g Distilled water 40.0 ml Solution 3: Na2S x 9 H2O 300.0 mg Distilled water 6.0 ml Vitamine solution: Biotin 2.5 mg Nicotinic acid 25.0 mg Thiamine-HCl 12.5 mg p-Aminobenzoic acid 12.5 mg Calcium pantothenate 6.5 mg Pyridoxine-HCl 62.5 mg Distilled water 1000.0 ml Trace element solution SL-6: ZnSO4 x 7 H2O 0.1 g MnCl2 x 4 H2O 0.03 g H3BO3 0.3 g CoCl2 x 6 H2O 0.2 g CuCl2 x 2 H2O 0.01 g NiCl2 x 6 H2O 0.02 g Na2MoO4 0.03 g Distilled water 1000.0 ml Sterilize solutions 1 and 3 under nitrogen. Filter sterilize vitamin solution and add 5 ml of the solution to 1 l of basal medium. Solution 2 is not to be kept for long. pH of the medium 7.2. 91. THERMODESULFOBACTERIUM MEDIUM Solution 1: Na2SO4 3.0 g NH4Cl 1.0 g MgCl2 x 6 H2O 0.2 g KH2PO4 0.3 g Na2HPO4 x 12 H2O 2.0 g FeSO4 x 7 H2O 1.5 mg Resazurin 1.0 mg Distilled water 930.0 ml Solution 2: Trace element solution (see below) 10.0 ml Solution 3: Yeast extract 1.0 g Distilled water 25.0 ml Solution 4: Na-lactate 4.0 g Distilled water 25.0 ml Solution 5: Na2S x 9 H2O 0.5 g Distilled water 6.0 ml Solution 6: Vitamin solution (see below) 5.0 ml Trace element solution: Nitrilotriacetic acid 12.8 g FeCl3 x 4 H2O 0.2 g MnCl2 x 4 H2O 0.1 g CoCl2 x 6 H2O 0.17 g CaCl2 x 2 H2O 0.1 g ZnCl2 0.1 g CuCl2 0.02 g Na2MoO4 x 2 H2O 0.01 g NiCl2 x 6 H2O 0.026 g NaCl 1.0 g Na2SeO4 x 5 H2O 0.02 g Distilled water 1000.0 ml Vitamin solution: Biotin 2.0 mg Folic acid 2.0 mg Pyridoxine-HCl 10.0 mg Thiamine-HCl 5.0 mg Riboflavin 5.0 mg Nicotinic acid 5.0 mg Ca DL-pantothenate 5.0 mg Vitamin B12 0.1 mg p-Aminobenzoic acid 5.0 mg Lipoic acid 5.0 mg Distilled water 1000.0 ml Solutions 1, 3, 4 and 5 sterilize under N2. Solution 6 is filter sterilized. Before use, neutralize solution 5 by dropwise of 1 N HCl. pH of the medium 6.8 - 7.0. 92. DESULFOVIBRIO MEDIUM WITH LACTATE Solution 1: K2HPO4 0.5 g NH4Cl 1.0 g CaCl2 x 6 H2O 0.1 g MgSO4 x 7 H2O 2.0 g Na2SO4 1.0 g Na-lactate 5.0 g Yeast extract 1.0 g Resazurin 0.001 g Cysteine 0.5 g Distilled water 950.0 ml Solution 2: NaHCO3 4.0 g Distilled water 40.0 ml Solution 3: Na2S x 9 H2O 300.0 mg Distilled water 6.0 ml Solution 4: FeSO4 x 7 H2O 0.4 g Distilled water 10.0 ml Solution 1 bring to boil while simultaneously bubbling a mixture of oxygen-free gas composed of 97% N2 and 3% H2 through the mixture. Sterilize in atmosphere of this gas mixture. Solution 3 sterilize in atmosphere of N2. pH of the medium 6.8. 93. AZOSPIRILLUM BRASILIENSE MEDIUM 1 Ca-malate 10.0 g K2HPO4 1.0 g MgSO4 x 7 H2O 0.5 g CaCl2 x 2 H2O 0.02 g Distilled water 1000.0 ml pH 6.5. 94. MEDIUM FOR CARBON MONOOXIDE OXIDIZERS Na2HPO4 x 12 H2O 4.5 g KH2PO4 0.75 g NH4Cl 1.5 g MgSO4 x 7 H2O 0.2 g CaCl2 x 2 H2O 0.03 g Fe(NH4)-citrate 0.018 g Agar (if necessary) 1.2 g Trace element solution SL6 (see below) 1.0 ml Distilled water 1000.0 ml Trace element solution SL-6: ZnSO4 x 7 H2O 0.1 g MnCl2 x 4 H2O 0.03 g H3BO3 0.3 g CoCl2 x 6 H2O 0.2 g CuCl2 x 2 H2O 0.01 g NiCl2 x 6 H2O 0.02 g Na2MoO4 0.03 g Distilled water 1000.0 ml pH 7.0. For chemoautotrophic growth incubate under gas atmosphere of a) 20-80% CO2 + 10% O2 + 0-70% N2 or b) 70% H2 + 20% O2 + 10% CO2 adding 2.5 g NaHCO3 per liter of medium. For chemoorganotrophic growth add 3.0 g sodium acetate and incubate under air. 95. DESULFOTOMACULUM ACETOXIDANS MEDIUM Solution 1: NaCl 1.17 g MgCl2 x 6 H2O 0.4 g KCl 0.3 g CaCl2 x 2 H2O 0.15 g NH4Cl 0.27 g KH2PO4 0.2 g Na2SO4 2.84 g Na-acetate 1.4 g Na-butyrate 1.4 g Yeast extract 1.0 g Vitamin solution (see below) 1.0 ml Trace element solution 1.0 ml (see below) Distilled water 1000.0 ml Solution 2: Na2S x 9 H2O 0.36 g Distilled water 10.0 ml Sodium bicarbonate for alkalization: NaHCO3 4.5 g Distilled water 50.0 g Vitamin solution: p-Aminobenzoic acid 4.0 mg D(+)-Biotin 1.0 mg Thiamine-HCl 10.0 mg Distilled water 100.0 ml Trace element solution: FeCl3 x 4 H2O 1.5 g ZnCl2 68.0 mg MnCl2 x 4 H2O 100.0 ml H3BO3 62.0 mg CoCl2 x 6 H2O 120.0 ml Na2MoO4 x 2 H2O 24.0 mg HCl, 0.05 M 1000.0 ml Solution 1 is boiled before sterilization for a few minutes being flushed with gas mixture of 80% N2 and 20% CO2 with sodium bicarbonate added until an equilibrium pH of 6.9 - 7.1. Solution 1 is steril- ized under this gas mixture. Solution 2 is sterilized under 100% N2. Vitamin solution is filter sterilized. Final pH of the complete medium 7.0-7.2. 96. DESULFONEMA LIMICOLA MEDIUM Solution 1: Na2SO4 3.0 g NaCl 13.0 g MgCl2 x 6 H2O 2.2 g CaCl2 x 2 H2O 0.15 g KCl 0.5 g KH2PO4 0.2 g NH4Cl 0.3 g Distilled water 850.0 ml Solution 2: Trace element solution (see below) 1.0 ml Solution 3: NaHCO3 5.0 g Distilled water 100.0 ml Solution 4: Na-acetate x 3 H2O 2.5 g Distilled water 100.0 ml Solution 5: Disodium succinate 0.1 g Distilled water 1.0 ml Solution 6: Vitamin solution (see below) 5.0 ml Solution 7: AlCl3 x 6 H2O 245.0 mg Distilled water 5.0 ml Solution 8: Na2CO3 170.0 mg Distilled water 1.6 ml Solution 9: Rumen fluid, clarified 20.0 ml Solution 10: Na2S x 9 H2O 0.4 mg Distilled water 10.0 ml Trace element solution SL-10: HCl (25%; 7.7 M) 10.0 ml FeCl3 x 4 H2O 1.5 g ZnCl2 70.0 mg MnCl2 x 4 H2O 100.0 mg H3BO3 6.0 mg CoCl2 x 6 H2O 190.0 mg CuCl2 x 2 H2O 2.0 mg NiCl2 x 6 H2O 24.0 mg Na2MoO4 x 2 H2O 36.0 mg Distilled water 990.0 ml Vitamin solution: Biotin 2.0 mg Folic acid 2.0 mg Pyridoxine-HCl 10.0 mg Thiamine-HCl 5.0 mg Riboflavin 5.0 mg Nicotinic acid 5.0 mg Ca DL-pantothenate 5.0 mg Vitamin B12 0.1 mg p-Aminobenzoic acid 5.0 mg Lipoic acid 5.0 mg Distilled water 1000.0 ml The trace element solution preparation: FeCl3 x 4 H2O is dissolved firstly in HCl, then is mixed with water and other salts are dissolved in the sequence indicated. For the preparation and sterilization of the medium see medium 95. Solutions 7 and 8 are combined before sterilization. pH of the medium 7.6. 97. DESULFONEMA MAGNUM MEDIUM Solution 1: Na2SO4 3.0 g NaCl 21.0 g/l MgCl2 x 6 H2O 5.5 g/l CaCl2 x 2 H2O 1.35 g/l KCl 0.5 g KH2PO4 0.2 g NH4Cl 0.3 g Solution 2: Trace element solution (see below) 1.0 ml Solution 3: NaHCO3 2.5 g/l Distilled water 100.0 ml Solution 4: Na-benzoate 0.6 g Distilled water 10.0 ml Solution 5: Disodium succinate 0.1 g Distilled water 1.0 ml Solution 6: Vitamin solution (see below) 5.0 ml Solution 7: AlCl3 x 6 H2O 245.0 mg Distilled water 5.0 ml Solution 8: Na2CO3 170.0 mg Distilled water 1.6 ml Solution 9: Rumen fluid, clarified 20.0 ml Solution 10: Na2S x 9 H2O 0.4 mg Distilled water 10.0 ml Trace element solution: HCl (25%; 7.7 M) 10.0 ml FeCl3 x 4 H2O 1.5 g ZnCl2 70.0 mg MnCl2 x 4 H2O 100.0 mg H3BO3 6.0 mg CoCl2 x 6 H2O 190.0 mg CuCl2 x 2 H2O 2.0 mg NiCl2 x 6 H2O 24.0 mg Na2MoO4 x 2 H2O 36.0 mg Na2SeO4 3.0 mg Distilled water 990.0 ml Vitamin solution: Biotin 2.0 mg Folic acid 2.0 mg Pyridoxine-HCl 10.0 mg Thiamine-HCl 5.0 mg Riboflavin 5.0 mg Nicotinic acid 5.0 mg Ca DL-pantothenate 5.0 mg Vitamin B12 0.1 mg p-Aminobenzoic acid 5.0 mg Lipoic acid 5.0 mg Vitamin B12 50.0 mg Distilled water 1000.0 ml The trace element solution preparation: FeCl3 x 4 H2O is dissolved firstly in HCl, then is mixed with water and other salts are dissolved in the sequence indicated. For the preparation and sterilization of the medium see medium 95. Solutions 7 and 8 are combined before sterilization. pH of the medium 7.6. 98. WORT AGAR 7 B WITH 2% CaCO3 Malt wort 7 B 1000.0 ml CaCO3 20 g Agar 20.0 g Sterilize at 105°C for 30 min. Preparation of malt wort: mix 250 g of ground malt in 1000.0 ml of tap water, heat to 55-60°C and keep at this temperature for 1.5-2 h at periodic stirring. Then increase the temperature to 80°C and keep for 10 min. Then cool the wort, squeeze through a linen bag, adjust the concentration of sugars to 7 B. 99. MEDIUN YE Yeast extract 30.0 g Ethanol 20.0 ml Agar 20.0 g Distilled water 1000.0 ml pH 5.0-6.0. Apply sterile ethanol (0.1 ml/test tube) onto the surface of agar slants. 100. SAP-2 AGAR Tryptone 1.0 g Yeast extract 1.0 g Agar 20.0 g Sea water 1000.0 g pH 7.4. 101. SOIL AGAR Yeast extract 2.0 g Tryptone 1.0 g Na-acetate 1.0 g Soil extract 50.0 ml Agar 20.0 g Distilled water add to 1000.0 ml pH 7.4. Preparation of soil extract: dry garden soil, rich in organic material, in the air by spreading in a thin layer, comminuting and stirring. Then sieve through a rough sieve, and mix 400 g of soil with 960 ml of tap water. Autoclave at 121°C for 1 h at the end of the day but leave the autoclave open overnight. Filter the cooled extract through filter paper, autoclave 300 ml of filtrate at 121°C for 20 min and al- low to stay for 2 weeks or longer to settle the sediment. Decant the clear supernatant liquid and use to prepare the medium. 102. DESULFOBACTER POSTGATEI MEDIUM Solution 1: Na2SO4 3.0 g KH2PO4 0.2 g NH4Cl 0.3 g NaCl 7.0 g MgCl2 x 6 H2O 1.3 g KCl 0.5 g CaCl2 x 2 H2O 0.15 g Distilled water 870.0 ml Solution 2: Trace element solution (see below) 1.0 ml Solution 3: NaHCO3 5.0 g Distilled water 100.0 ml Solution 4: Na-acetate x 3 H2O 2.5 g Distilled water 10.0 ml Solution 5: Vitamin solution (see below) 10.0 ml Solutin 6: Na2S x 9 H2O 0.4 g Distilled water 10.0 ml Trace element solution SL-10: HCl (25%; 7.7 M) 10.0 ml FeCl3 x 4 H2O 1.5 g ZnCl2 70.0 mg MnCl2 x 4 H2O 100.0 mg H3BO3 6.0 mg CoCl2 x 6 H2O 190.0 mg CuCl2 x 2 H2O 2.0 mg NiCl2 x 6 H2O 24.0 mg Na2MoO4 x 2 H2O 36.0 mg Distilled water 990.0 ml Vitamin solution: Biotin 2.0 mg Folic acid 2.0 mg Pyridoxine-HCl 10.0 mg Thiamine-HCl 5.0 mg Riboflavin 5.0 mg Nicotinic acid 5.0 mg Ca DL-pantothenate 5.0 mg Vitamin B12 0.1 mg p-Aminobenzoic acid 5.0 mg Lipoic acid 5.0 mg Distilled water 1000.0 ml Solution 1 is boiled before sterilization for a few minutes being flushed with gas mixture of 80% N2 and 20% CO2 with sodium bicarbonate added until an equilibrium pH of 6.9-7.1. Solution 1 is steril- ized under this gas mixture. Solution 2 is sterilized under 100% N2. Vitamin solution is filter sterilized. Final pH of the complete medium 7.1-7.4. 103. DESULFOBULBUS MEDIUM Solution 1: Na2SO4 3.0 g KH2PO4 0.2 g NH4Cl 0.3 g NaCl 1.0 g MgCl2 x 6 H2O 0.4 g KCl 0.5 g CaCl2 x 2 H2O 0.15 g Distilled water 870.0 ml Solution 2: Trace element solution (see below) 1.0 ml Solution 3: NaHCO3 5.0 g Distilled water 100.0 ml Solution 4: Na-propionate 1.5 g Distilled water 10.0 ml Solution 5: Vitamin solution (see below) 10.0 ml Solutin 6: Na2S x 9 H2O 0.4 g Distilled water 10.0 ml Trace element solution SL-10: HCl (25%; 7.7 M) 10.0 ml FeCl3 x 4 H2O 1.5 g ZnCl2 70.0 mg MnCl2 x 4 H2O 100.0 mg H3BO3 6.0 mg CoCl2 x 6 H2O 190.0 mg CuCl2 x 2 H2O 2.0 mg NiCl2 x 6 H2O 24.0 mg Na2MoO4 x 2 H2O 36.0 mg Distilled water 990.0 ml Vitamin solution: Biotin 2.0 mg Folic acid 2.0 mg Pyridoxine-HCl 10.0 mg Thiamine-HCl 5.0 mg Riboflavin 5.0 mg Nicotinic acid 5.0 mg Ca DL-pantothenate 5.0 mg Vitamin B12 0.1 mg p-Aminobenzoic acid 5.0 mg Lipoic acid 5.0 mg Distilled water 1000.0 ml Solution 1 is boiled before sterilization for a few minutes being flushed with gas mixture of 80% N2 and 20% CO2 with sodium bicarbonate added until an equilibrium pH of 6.9-7.1. Solution 1 is steril- ized under this gas mixture. Solution 2 is sterilized under 100% N2. Vitamin solution is filter sterilized. Final pH of the complete medium 7.1-7.4. 104. MACROMONAS MEDIUM 1 Na-acetate 1.0 g CaCl2 x 2 H2O 0.1 g Casein acidic hydrolysate 0.1 g Yeast extract 0.1 g FeS or CaS 0.2 g Agar 1.0 g Distilled water 1000.0 ml pH 7.2-7.4. Prepare suspension of FeS separately from the equimolar solutions of Na2S x 9 H2O and FeSO4, wash with freshly boiled distilled water under the flow of inert gas, sterilize separately from the main medium under inert gas at 105°C. 105. MACROMONAS MEDIUM 2 (NH4)2SO4 0.5 g MgSO4 x 7 H2O 0.2 g Casein acidic hydrolysate 1.0 g Na-acetate 1.0 g or succinate, or benzoate 0.5 g Na2S2O3 x 5 H2O 0.5 g Catalase 2.0 mg Vitamin solution (see below) 1.0 ml Distilled water 1000.0 ml Vitamin solution: Biotin 200.0 mg Folic acid 20.0 mg Pyridoxine-HCl 100.0 mg Thiamine-HCl 50.0 mg Riboflavin 100.0 mg Nicotinic acid 50.0 mg DL-Pantothenic acid 50.0 mg Vitamin B12 1.0 mg p-Aminobenzoic acid 50.0 mg Distilled water 1000.0 ml Sterilize catalase and vitamins separately from the main medium by filtration. Thiosulfate should also better be sterilized separately - at 105°C for 30 min. 106. BEGGIATOA MEDIUM 1 (NH4)2SO4 500.0 mg MgSO4 x 7 H2O 100.0 mg CaCl2 x 2 H2O 50.0 mg Na-lactate 500.0 mg Na2S2O3 x 5 H2O 500.0 mg K2HPO4 110.0 mg KH2PO4 85.0 mg FeCl3 x 6 H2O 2.0 mg EDTA 3.0 mg Vitamin solution (see below) 1.0 ml Buffer HEPES 0.01 m Distilled water 1000.0 ml Vitamin solution: Biotin 200.0 mg Folic acid 20.0 mg Pyridoxine-HCl 100.0 mg Thiamine-HCl 50.0 mg Riboflavin 100.0 mg Nicotinic acid 50.0 mg DL-Pantothenic acid 50.0 mg Vitamin B12 1.0 mg p-Aminobenzoic acid 50.0 mg Distilled water 1000.0 ml Sterilize thiosulfate, lactate and vitamins each separately and add into the main medium prior to inocu- lation. Sterilize the vitamin solution by filtration. pH of the medium 7.2-7.5 (adjust with NaOH). 107. DESULFOSARCINA MEDIUM Solution 1: Na2SO4 3.0 g KH2PO4 0.2 g NH4Cl 0.3 g NaCl 13.5 g MgCl2 x 6 H2O 2.0 g KCl 0.5 g CaCl2 x 2 H2O 0.15 g Distilled water 870.0 ml Solution 2: Trace element solution (see below) 1.0 ml Solution 3: NaHCO3 5.0 g Distilled water 100.0 ml Solution 4: Na-benzoate 0.6 g Na-lactate 1.0 g Distilled water 10.0 ml Solution 5: Vitamin solution (see below) 10.0 ml Solutin 6: Na2S x 9 H2O 0.4 g Distilled water 10.0 ml Trace element solution: HCl (25%; 7.7 M) 10.0 ml FeCl3 x 4 H2O 1.5 g ZnCl2 70.0 mg MnCl2 x 4 H2O 100.0 mg H3BO3 6.0 mg CoCl2 x 6 H2O 190.0 mg CuCl2 x 2 H2O 2.0 mg NiCl2 x 6 H2O 24.0 mg Na2MoO4 x 2 H2O 36.0 mg Na2SeO4 x 5 H2O 3.0 mg Distilled water 990.0 ml Vitamin solution: Biotin 2.0 mg Folic acid 2.0 mg Pyridoxine-HCl 10.0 mg Thiamine-HCl 5.0 mg Riboflavin 5.0 mg Nicotinic acid 5.0 mg Ca DL-pantothenate 5.0 mg Vitamin B12 0.1 mg p-Aminobenzoic acid 5.0 mg Lipoic acid 5.0 mg Distilled water 1000.0 ml Solution 1 is boiled before sterilization for a few minutes being flushed with gas mixture of 80% N2 and 20% CO2 with sodium bicarbonate added until an equilibrium pH of 6.9-7.1. Solution 1 is steril- ized under this gas mixture. Solution 2 is sterilized under 100% N2. Vitamin solution is filter sterilized. Final pH of the complete medium 7.1-7.4. 108. BEGGIATOA MEDIUM 2 MgCl2 x 7 H2O 50.0 mg CaCl2 x 2 H2O 30.0 mg Na-lactate 500.0 mg Na2S2O3 x 5 H2O 500.0 mg NaH2PO4 125.0 mg KCl 125.0 mg NaHCO3 125.0 mg Na2SO4 500.0 mg NaNO3 620.0 mg Vitamin solution (see below) 1.0 ml Trace element solution (see below) 1.0 ml Distilled water 1000.0 ml Vitamin solution: Biotin 200.0 mg Folic acid 20.0 mg Pyridoxine-HCl 100.0 mg Thiamine-HCl 50.0 mg Riboflavin 100.0 mg Nicotinic acid 50.0 mg DL-Pantothenic acid 50.0 mg Vitamin B12 1.0 mg p-Aminobenzoic acid 50.0 mg Distilled water 1000.0 ml Trace element solution according to Hogland: EDTA 5.0 g FeSO4 x 7 H2O 2.0 g ZnSO4 x 7 H2O 100.0 mg MnCl2 x 4 H2O 30.0 mg H3BO3 300.0 mg CoCl2 x 6 H2O 200.0 mg CuCl2 10.0 mg NiCl2 x 6 H2O 20.0 mg Na2MoO4 x 2 H2O 20.0 mg Distilled water 1000.0 ml To prepare the trace element solution, preliminarily acidify water to pH 3.0-4.0 with HCl. Sterilize thi- osulfate, lactate, bicarbonate, trace elements and vitamins separately and add to the main medium prior to inoculation. Lactate and thiosulfate can be more conveniently prepared as 10% solutions; bicarbon- ate, as 5% solution. Sterilize the vitamin solution by filtration. pH of the medium 7.5 (adjust with 1% HCl). 109. DESULFOVIBRIO MEDIUM Solution 1: K2HPO4 0.5 g NH4Cl 1.0 g Na2SO4 1.0 g CaCl2 x 2 H2O 0.1 g MgSO4 x 7 H2O 2.0 g Na DL-lactate 2.0 g Yeast extract 1.0 g Resazurin 1.0 g Distilled water 980.0 ml Solution 2: FeSO4 x 7 H2O 0.5 g Distilled water 10.0 ml Solution 3: Na-thioglycollate 0.1 g Ascorbic acid 0.1 g Distilled water 10.0 ml Solution 4: Trace element solution (see below) 1.0 ml Trace element solution: FeCl3 x 4 H2O 1.5 g ZnCl2 68.0 mg MnCl2 x 4 H2O 100.0 ml H3BO3 62.0 mg CoCl2 x 6 H2O 120.0 ml Na2MoO4 x 2 H2O 24.0 mg HCl, 0.05 M 1000.0 ml pH 7.4-7.8. Sterilization at 121°C for 15 min under an atmosphere of N2. Solution 1 is boiled before sterilization, being blown with N2. 110. DESULFOVIBRIO MEDIUM WITH 2% NaCl Solution 1: K2HPO4 0.5 g NH4Cl 1.0 g NaCl 20 g Na2SO4 1.0 g CaCl2 x 2 H2O 0.1 g MgSO4 x 7 H2O 2.0 g Na DL-lactate 2.0 g Yeast extract 1.0 g Resazurin 1.0 g Distilled water 980.0 ml Solution 2: FeSO4 x 7 H2O 0.5 g Distilled water 10.0 ml Solution 3: Na-thioglycollate 0.1 g Ascorbic acid 0.1 g Distilled water 10.0 ml Solution 4: Trace element solution (see below) 1.0 ml Trace element solution: FeCl3 x 4 H2O 1.5 g ZnCl2 68.0 mg MnCl2 x 4 H2O 100.0 ml H3BO3 62.0 mg CoCl2 x 6 H2O 120.0 ml Na2MoO4 x 2 H2O 24.0 mg HCl, 0.05 M 1000.0 ml pH 7.4-7.8. Sterilization at 121°C for 15 min under an atmosphere of N2. Solution 1 is boiled before sterilization, being blown with N2. 111. DESULFOVIBRIO MEDIUM WITH 3% NaCl Solution 1: K2HPO4 0.5 g NH4Cl 1.0 g NaCl 30 g Na2SO4 1.0 g CaCl2 x 2 H2O 0.1 g MgSO4 x 7 H2O 2.0 g Na DL-Lactate 2.0 g Yeast extract 1.0 g Resazurin 1.0 g Distilled water 980.0 ml Solution 2: FeSO4 x 7 H2O 0.5 g Distilled water 10.0 ml Solution 3: Na-thioglycollate 0.1 g Ascorbic acid 0.1 g Distilled water 10.0 ml Solution 4: Trace element solution (see below) 1.0 ml Trace element solution: FeCl3 x 4 H2O 1.5 g ZnCl2 68.0 mg MnCl2 x 4 H2O 100.0 ml H3BO3 62.0 mg CoCl2 x 6 H2O 120.0 ml Na2MoO4 x 2 H2O 24.0 mg HCl, 0.05 M 1000.0 ml pH 7.4-7.8. Sterilization at 121°C for 15 min under an atmosphere of N2. Solution 1 is boiled before sterilization, being blown with N2. 112. GLUCOSE YEAST EXTRACT AGAR Glucose 20.0 g Yeast extract 10.0 g CaCO3 20.0 g Agar 17.0 g Distilled water 1000.0 ml 113. DESULFOVIBRIO BAARSII MEDIUM Solution 1: Na2SO4 3.0 g KH2PO4 0.2 g NH4Cl 0.3 g NaCl 1.0 g MgCl2 x 6 H2O 0.4 g KCl 0.5 g CaCl2 x 2 H2O 0.15 g Distilled water 870.0 ml Solution 2: Trace element solution (see below) 1.0 ml Solution 3: NaHCO3 5.0 g Distilled water 100.0 ml Solution 4: Na-butyrate 0.7 g Na-caproate 0.3 g Na-octanoate 0.15 g Distilled water 10.0 ml Solution 5: Vitamin solution (see below) 10.0 ml Solutin 6: Na2S x 9 H2O 0.4 g Distilled water 10.0 ml Trace element solution SL-10: HCl (25%; 7.7 M) 10.0 ml FeCl3 x 4 H2O 1.5 g ZnCl2 70.0 mg MnCl2 x 4 H2O 100.0 mg H3BO3 6.0 mg CoCl2 x 6 H2O 190.0 mg CuCl2 x 2 H2O 2.0 mg NiCl2 x 6 H2O 24.0 mg Na2MoO4 x 2 H2O 36.0 mg Distilled water 990.0 ml Vitamin solution: Biotin 2.0 mg Folic acid 2.0 mg Pyridoxine-HCl 10.0 mg Thiamine-HCl 5.0 mg Riboflavin 5.0 mg Nicotinic acid 5.0 mg Ca DL-pantothenate 5.0 mg Vitamin B12 0.1 mg p-Aminobenzoic acid 5.0 mg Lipoic acid 5.0 mg Distilled water 1000.0 ml Solution 1 is boiled before sterilization for a few minutes being flushed with gas mixture of 80% N2 and 20% CO2 with sodium bicarbonate added until an equilibrium pH of 6.9-7.1. Solution 1 is steril- ized under this gas mixture. Solution 2 is sterilized under 100% N2. Vitamin solution is filter sterilized. Final pH of the complete medium 6.8-7.0. 114. AZOSPIRILLUM BRASILIENSE MEDIUM 2 Ca-malate 10.0 g or glucose 20.0 g K2HPO4 0.1 g KH2PO4 0.2 g MgSO4 x 7 H2O 0.5 g FeCl3 x 6 H2O 0.1 g CaCl2 x 2 H2O 0.02 g Na2MoO4 x 2 H2O 0.02 mg Yeast extract 0.1 g Agar 15.0 g Distilled water 1000.0 ml pH 6.9. 115. METHANOBACTERIUM MEDIUM KH2PO4 0.5 g MgSO4 x 7 H2O 0.4 g NaCl 0.4 g NH4Cl 0.4 g CaCl2 x 2 H2O 0.05 g Trace element solution (see below) 1.0 ml Vitamin solution (see below) 5.0 ml Yeast extract 1.0 g Na-acetate 1.0 g Na-formate 2.0 g NaHCO3 4.0 g Resazurin 1.0 mg Cystein-HCl 0.5 g Na2S x 9 H2O 0.5 g Fatty acid mixture (see below) 20.0 ml Sludge fluid (see below) 50.0 ml Distilled water 940.0 ml Trace element solution SL-10: HCl (25%; 7.7 M) 10.0 ml FeCl3 x 4 H2O 1.5 g ZnCl2 70.0 mg MnCl2 x 4 H2O 100.0 mg H3BO3 6.0 mg CoCl2 x 6 H2O 190.0 mg CuCl2 x 2 H2O 2.0 mg NiCl2 x 6 H2O 24.0 mg Na2MoO4 x 2 H2O 36.0 mg Distilled water 990.0 ml Vitamin solution: Biotin 2.0 mg Folic acid 2.0 mg Pyridoxine (B2) 0.1 mg Riboflavin (B1) 5.0 mg Pantotenoic acid 5.0 mg p-Aminobenzoic acid 5.0 mg Thiamine-HCl 5.0 mg Nicotinic acid 5.0 mg Cyanocobalamin (B12) 0.1 mg Lipoic (tioctoic) acid 5.0 mg Distilled water 1000.0 ml Fatty acid mixture: Valeric acid 0.5 g Isovaleric acid 0.5 g p-Methylbutyric acid 0.5 g Isobutyric acid 0.5 g Distilled water 20.0 ml Adjust pH to 7.5 with conc. NaOH. Sludge water: to sludge from an anaerobic digester add 0.4% yeast extract and after gassing with ni- trogen 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 under nitrogen gas. pH of the medium, 6.7-7.0. Prepare the medium anaerobically under a gas atmosphere of 80% H2 and 20% CO2. 116. THERMUS RUBER MEDIUM Pepton 5.0 g Yeast extract 1.0 g Starch (soluble) 1.0 g Agar 12.0 g Distilled water 1000.0 ml pH 8.0. 117. METHANOSARCINA MEDIUM Solution 1: NaCl 0.9 g MgCl2 x 7 H2O 0.2 g CaCl2 x 2 H2O 0.1 g NH4Cl 1.0 g Yeast extract 2.0 g Resazurin 0.001 g Methanol 10% 10.0 ml Trace element solution (see below) 10.0 ml Vitamin solution (see below) 5.0 ml Distilled water 965.0 ml Solution 2 (reducing agents): Cystein-HCl 0.5 g Na2S x 9 H2O 0. 5 g Distilled water 10.0 ml Buffer solutions: a) K2HPO4 29.0 g Distilled water 100.0 ml b) KH2PO4 15.0 g Distilled water 100.0 ml Trace element solution: Nitrilotriacetic acid 12.8 mg FeSO4 x 7 H2O 0.1 mg MnCl2 x 6 H2O 0.1 mg CoCl2 x 2 H2O 0.17 mg CaCl2 x 2 H2O 0.1 mg ZnCl2 0.1 mg CuCl2 0.02 mg H3BO3 0.01 mg Na2MoO4 x 2 H2O 0.01 mg NaCl 1.0 mg Na2SeO4 0.017 mg Distilled water 1000.0 ml Vitamin solution: Biotin 2.0 mg Folic acid 2.0 mg Pyridoxine (B2) 0.1 mg Riboflavin (B1) 5.0 mg Pantotenoic acid 5.0 mg p-Aminobenzoic acid 5.0 mg Thiamine-HCl 5.0 mg Nicotinic acid 5.0 mg Cyanocobalamin (B12) 0.1 mg Lipoic (tioctoic) acid 5.0 mg Distilled water 1000.0 ml Prepare medium in anaerobic conditions, blowing through with N2 without O2 up to sterilization. Solu- tions of reducing agents (10 ml) and of buffer (per 1 ml) add to base medium after separate steriliza- tion. pH 7.2-7.4. 118. CLARK AGARIZED MEDIUM Peptone 5.0 g Glucose 5.0 g K2HPO4 5.0 g Agar 2.0 g Distilled water 1000.0 ml pH 6.9-7.0. Sterilize at 112°C for 20 min. Reagents for VP test: 6% alcoholic solution of p-naphthol and 40% aqueous solution of KOH. Reagent for MR test: 0.02% alcoholic-aqueous solution of methyl red. 119. NITROSOLOBUS MEDIUM 2 (NH4)2SO4 0.5 g KH2PO4 0.2 g CaCl2 x 2 H2O 0.02 g MgSO4 x 7 H2O 0.04 g EDTA FeNa 3.8% 0.1 ml Phenol red 0.05% 2.0 ml Distilled water 1000.0 ml pH 8.0 (adjust with 6% Na2CO3). 120. MANNITOL AGAR WITH PEPTONE Yeast extract 5.0 g Mannitol 25.0 g Peptone 3.0 g Agar 15.0 g Distilled water 1000.0 ml pH 5.0-6.0. 121. THIOBACILLUS THIOOXIDANS MEDIUM (WAKSMAN MEDIUM) (NH4)2SO4 300.0 mg MgSO4 x 7 H2O 500.0 mg KH2PO4 3.5 g CaCl2 x 2 H2O 250.0 mg FeSO4 x 7 H2O 18.0 mg Finely dispersed sulfur 5.0 g Distilled water 1000.0 ml 122. THIOBACILLUS FERROOXIDANS MEDIUM (LEATHEN MEDIUM) (NH4)2SO4 150.0 mg KCl 50.0 mg MgSO4 x 7 H2O 500.0 mg KH2PO4 100.0 mg Ca(NO3)2 x 4 H2O 10.0 mg Distilled water 1000.0 ml After sterilization of the medium, add 10 ml of 10% FeSO4 x 7 H2O preliminarily acidified to pH 3.5 and sterilized separately. Sterilize this solution of iron in sealed ampoules under nitrogen or with minimal content of air by boiling on a water bath. pH of the medium 4.0 (adjust after sterilization and addition of iron solution). 123. MEDIUM FOR MARINE NITROBACTER Solution 1 (see below) 0.5 ml Solution 2 (see below) 0.5 ml Solution 3 (see below) 1.0 ml Solution 4 (see below) 0.5 ml Solution 5 (see below) 0.5 ml Solution 6 (see below) 0.1 ml Distilled water 300.0 ml Sea water 700.0 ml Solution 1: CaCl2 2.0 g Distilled water 100.0 ml Solution 2: MgSO4 x 7 H2O 20.0 g Distilled water 100.0 ml Solution 3: Chelated iron 0.1 g Distilled water 100.0 ml Solution 4: Na2MoO4 0.1 g MnCl2 x 4 H2O 0.2 g CoCl2 x 6 H2O 0.002 g ZnSO4 x 7 H2O 0.1 g CuSO4 x 5 H2O 0.02 g Distilled water 1000.0 ml Solution 5: NaNO3 41.4 g Distilled water 100.0 ml Solution 6: K2HPO4 1.74 g Distilled water 100.0 ml 124. AZOSPIRILLUM MEDIUM Solution 1: Yeast extract 0.05 g FeSO4 x 7 H2O 0.01 g K2HPO4 0.25 g Na2MoO4 x 2 H2O 1.0 mg MnSO4 x H2O 2.0 mg MgSO4 x 7 H2O 0.2 g NaCl 0.1 g CaCl2 x 2 H2O 0.02 g (NH4)2SO4 1.0 g Biotin 0.1 mg Bromothymol blue 25.0 mg Distilled water 950.0 ml Solution 2: Glucose 20% 25.0 ml Solution 3: Na-malate 20% 25.0 ml Dissolve bromothymol blue in diluted KOH before adding into the medium. pH of the medium 7.1. 125. NITROSOLOBUS MEDIUM 3 (NH4)2SO4 2.0 g CaCl2 x 2 H2O 20.0 mg MgSO4 x 7 H2O 200.0 mg Chelated iron 1.0 mg Na2MoO4 x 2 H2O 100.0 ?g MnCl2 x 4 H2O 200.0 ?g CoCl2 x 6 H2O 2.0 ?g ZnSO4 x 7 H2O 100.0 ?g K2HPO4 15.9 mg CuSO4 x 5 H2O 20.0 ?g Distilled water 1000.0 ml pH 7.5-7.8. 126. DESULFOCOCCUS NIACINI MEDIUM Solution 1: Na2SO4 3.0 g KH2PO4 0.2 g NH4Cl 0.3 g NaCl 13.5 g MgCl2 x 6 H2O 2.2 g KCl 0.5 g CaCl2 x 2 H2O 0.15 g Distilled water 870.0 ml Solution 2: Trace element solution (see below) 1.0 ml Solution 3: NaHCO3 5.0 g Distilled water 100.0 ml Solution 4: Na-nicotinate 5.0 mM Solution 5: Vitamin solution (see below) 10.0 ml Solutin 6: Na2S x 9 H2O 0.4 g Distilled water 10.0 ml Trace element solution SL-10: HCl (25%; 7.7 M) 10.0 ml FeCl3 x 4 H2O 1.5 g ZnCl2 70.0 mg MnCl2 x 4 H2O 100.0 mg H3BO3 6.0 mg CoCl2 x 6 H2O 190.0 mg CuCl2 x 2 H2O 2.0 mg NiCl2 x 6 H2O 24.0 mg Na2MoO4 x 2 H2O 36.0 mg Na2SeO4 x 5 H2O 40.0 mg Distilled water 990.0 ml Vitamin solution: Biotin 2.0 mg Folic acid 2.0 mg Pyridoxine-HCl 10.0 mg Thiamine-HCl 5.0 mg Riboflavin 5.0 mg Nicotinic acid 5.0 mg Ca DL-pantothenate 5.0 mg Vitamin B12 0.1 mg p-Aminobenzoic acid 5.0 mg Lipoic acid 5.0 mg Distilled water 1000.0 ml Solution 1 is boiled before sterilization for a few minutes being flushed with gas mixture of 80% N2 and 20% CO2 with sodium bicarbonate added until an equilibrium pH of 6.9-7.1. Solution 1 is steril- ized under this gas mixture. Solution 2 is sterilized under 100% N2. Vitamin solution is filter sterilized. Final pH of the complete medium - 7.4. 127. SELENITE CONTROL MEDIUM Solution 1: Peptone 5.0 g Na2HPO4 7.0 g NaH2PO4 3.0 g Lactose 4.0 g Distilled water 960.0 ml pH 6.9-7.1. Solution 2: 10% Na2SeO4 x 5 H2O 40.0 ml Sterilize at 112°C for 30 min. 128. HIRSCH MEDIUM KH2PO4 1.36 g Na2HPO4 2.15 g MgSO4 x 7 H2O 0.2 g MnSO4 x 5 H2O 1.05 g (NH4)2SO4 0.5 g CaCl2 x 2 H2O 5.97 mg Na2MoO4 x 2 H2O 1.5 mg Methanol 5.0 ml Distilled water to 1000.0 ml pH 7.0. 129. CONTROL MEDIUM C-1 KH2PO4 2.0 g K2HPO4 2.0 g MgSO4 x 7 H2O 0.2 g NaCl 5.0 g Raffinose 2.0 g Bromothymol blue (1.6% alkaline) 2.0 ml Crystal violet (0.01%) 20.0 ml 50% Urea 4.0 ml Distilled water 1000.0 ml 130. GLUCOSE POTATO AGAR Potato 200.0 g Agar 20.0 g Glucose 10.0 g Tap water 1000.0 ml Boil potatoes for 1 h, filter cold through a cotton wool-gauze filter. Add agar and glucose. Sterilize at 121°C for 30 min. pH 7.0. 131. MEDIUM Q MOD FOR FRANKIA K2HPO4 300.0 mg NaH2PO4 200.0 mg MgSO4 x 7 H2O 200.0 mg KCl 200.0 mg Yeast extract 500.0 mg Peptone 5.0 g Glucose 10.0 g Fe-citrate 1.0 ml Trace element solution (see below) 1.0 ml CaCO3 100.0 mg Tween-80 2.0 g Agar 15.0 g Distilled water 1000.0 ml Trace element solution: H3BO3 1.5 g MnSO4 x 7 H2O 0.8 g ZnSO4 x 7 H2O 0.6 g CuSO4 x 7 H2O 0.1 g (NH4)6Mo7O24 x 4 H2O 0.2 g CoSO4 0.001 g Distilled water 1000.0 ml Sterilize at 105°C for 30 min. 132. THIOBACILLUS FERROOXIDANS MEDIUM 9K Solution 1: KCl 100.0 mg MgSO4 x 7 H2O 500.0 mg K2HPO4 500.0 mg Ca(NO3)2 x 4 H2O 10.0 mg Distilled water 700.0 ml Solution 2: (NH4)2Fe(SO4)2 63.0 g H2SO4 (10 N) 1.0 ml Distilled water 300.0 ml Sterilize solution 1 at 121°C for 15 min, solution 2, at 105°C. Mix the solutions before inoculation. pH of the medium 3.5. 133. COLBY AND ZATMAN MEDIUM WITH METHANOL K2HPO4 1.2 g KH2PO4 0.62 g CaCl2 x 6 H2O 0.05 g MgSO4 x 7 H2O 0.2 g NaCl 0.1 g FeCl3 x 6 H2O 1.0 mg (NH4)2SO4 0.5 g CuSO4 x 5 H2O 5.0 ?g MnSO4 x 5 H2O 10.0 ?g Na2MoO4 x 2 H2O 10.0 ?g H3BO3 10.0 ?g ZnSO4 x 7 H2O 70.0 ?g CoCl2 x 6 H2O 5.0 ?g Purified agar 15.0 g Methanol 1.0 ml Distilled water 1000.0 ml pH 6.8. 134. METHYLOTROPH MEDIUM 1 KH2PO4 2.0 g (NH4)2SO4 2.0 g MgSO4 x 7 H2O 0.025 g NaCl 0.5 g FeSO4 x 7 H2O 0.002 g Methanol 5.0 ml or methylamine 3.0 g Distilled water 1000.0 ml pH 7.0. 135. METHYLOTROPH MEDIUM 2 KH2PO4 0.8 g Na2HPO4 x 12 H2O 3.0 g (NH4)2SO4 0.8 g MgSO4 x 7 H2O 0.1 g FeSO4 x 7 H2O 10.0 mg Trace element solution (see below) 1.0 ml Distilled water 1000.0 ml Trace element solution: CaCl2 x 2 H2O 1.25 g CoCl2 x 6 H2O 1.25 g MnSO4 x 4 H2O 0.1 g ZnSO4 x 7 H2O 1.25 g Na2MoO4 x 2 H2O 50.0 mg CuCl2 x 2 H2O 250.0 mg Distilled water 250.0 ml pH of the medium, 7.0-7.2. Sterilize at 121°C for 30 min. 136. MEDIUM FOR PERCHLORATE-REDUCING BACTERIA Solution 1: NH4Cl 0.1 g NaCl 0.02 g KH2PO4 0.1 g K2HPO4 0.4 g MgSO4 x 7 H2O 0.1 g Tap water 1000.0 ml Solution 2: HCl, to dissolve the precipitate in solution 1 Solution 3: 5% NaOH, to adjust pH 6.9-7.2 Solution 4: 5% NH4ClO4 5.0 ml Solution 5: 5% Na-acetate 5.0 ml Solution 6: Trace element solution (see below) 0.5 ml Solution 7: Vitamin B12 (dispensarymade solution) 0.2 ml Solution 8: 96° ethanol 0.5 ml Trace element solution according to Hogland: EDTA 5.0 g FeSO4 x 7 H2O 2.0 g ZnSO4 x 7 H2O 100.0 mg MnCl2 x 4 H2O 30.0 mg H3BO3 300.0 mg CoCl2 x 6 H2O 200.0 mg CuCl2 10.0 mg NiCl2 x 6 H2O 20.0 mg Na2MoO4 x 2 H2O 20.0 mg Distilled water 1000.0 ml Add solutions and additions to the main medium in the order of their enumeration. 137. MEDIUM FOR CHROMATE-REDUCING BACTERIA Solution 1: NH4Cl 0.3 g NaCl 0.1 g KH2PO4 0.5 g K2HPO4 0.3 g MgSO4 x 7 H2O 0.1 g CaCO3 0.05 g FeCl3 x 6 H2O 0.05 g K2CrO4 0.1 g Paper-filtered pond water 900.0 ml Solution 2: Peptone meat broth 100.0 ml Solution 3: 5% Na-acetate 5.0 ml Solution 4: Trace element solution (see below) 0.5 ml Solution 5: Vitamin B12 (dispensarymade solution) 0.2 ml Trace element solution according to Hogland: EDTA 5.0 g FeSO4 x 7 H2O 2.0 g ZnSO4 x 7 H2O 100.0 mg MnCl2 x 4 H2O 30.0 mg H3BO3 300.0 mg CoCl2 x 6 H2O 200.0 mg CuCl2 10.0 mg NiCl2 x 6 H2O 20.0 mg Na2MoO4 x 2 H2O 20.0 mg Distilled water 1000.0 ml Add solutions and additions to the main medium in the order of their enumeration. pH 7.0-7.2. 138. LARSEN PHOTOTROPH MEDIUM NH4Cl 0.5 g KH2PO4 1.0 g MgCl2 0.5 g CaCl2 0.1 g Trace element solution (see below) 1.0 ml NaHCO3 5.0 g Na-acetate 2.0 g Na2S2O3 0.4-0.6 g Na2S 0.1 g Fe-citrate Traces Distilled water 1000.0 ml Trace element solution SL-12B: Ethylenediaminetetraacetate (EDTA) Na 3.0 g FeSO4 x 7 H2O 1.1 g CoCl2 x 6 H2O 190.0 mg MnCl2 x 2 H2O 50.0 mg ZnCl2 42.0 mg NiCl2 x 6 H2O 24.0 mg Na2MoO4 x 2 H2O 18.0 mg H3BO3 300.0 mg CuCl2 x 2 H2O 2.0 mg Distilled water 1000.0 ml pH of the trace element solution, 6.0. pH of the medium 8.4. 139. POSTGATE MEDIUM B FOR SULFATE REDUCERS NaCl 1.0 g KH2PO4 0.5 g NH4Cl 1.0 g CaSO4 x 2 H2O 1.0 g MgSO4 x 7 H2O 2.0 g Na-lactate 3.5 g Yeast extract 1.0 g Ascorbic acid 1.0 g Thioglycolic acid 1.0 g FeSO4 x 7 H2O 0.5 g Tap water 1000.0 ml 140. GLYCEROL-FUCHSIN BROTH Solution 1: Hottinger broth (see below) 1000.0 ml Solution 2: Basic fuchsin, 10% alcoholic saturated solution 2.5 ml Solution 3: 10% Na2SO4 16.6 ml Solution 4: Glycerol 10.0 ml Sterilize at 112°C for 15 min. Preparation of Hottinger broth: boil meat (1-2 cm pieces) (without fat or tendons) in 2.0 l of water, then mince. Adjust pH of the decoction to 8.0, mix with minced meat and cool down to 40°C. Then add 1.0 g of dry pancreatin, mix and again alkalinize to pH 7.8-8.0. Pour the mixture into a bottle with the rubber stopper (1/3 of the bottle to remain free), add chloroform (20 ml), mix and open the bottle for 1 min to remove the excess chloroform vapors. 2 h after pancreatin was added, adjust pH to 7.4-7.6 and leave the mixture for 2 weeks at 18-20°C. The first 4 days adjust pH of the medium; shake and mix 3 times a day, then stir once a day. Two days before the end of the procedure stop mixing to allow the decoction to settle. The liquid shall be of straw color, the reaction with tryptophan with bromine water shall be positive; in the decoction hydrolysate the total nitrogen shall be no less than 1100 mg%. Filter the decoction through the linen, pour into flasks and sterilize in autoclave at 121°C for 30 min. Filter prior to use. 141. CITRATE AGAR NaCl 5.0 g MgSO4 x 7 H2O 0.2 g NH4H2PO4 1.0 g K2HPO4 1.0 g Na-citrate 3.0 g Bromothymol blue 0.08 g Agar 20.0 g Distilled water 1000.0 ml pH 7.2. Sterilize at 121°C for 15 min or at 112°C for 30 min. 142. PPYA Potato decoction (see below) 200.0 ml Peptone 5.0 g Yeast extract 1.0 g Agar 25.0 g Distilled water to 1000.0 ml pH 8.0. Preparation of potato decoction: boil 200.0 g potatoes in 1.0 l of tap water for 1 h, filter cold through a cotton wool-gauze filter. Sterilize at 121°C for 30 min. 143. PEPTONE MEAT AGAR WITH 1% GLYCEROL Peptone 10.0 g NaCl 5.0 g Glycerol 10.0 ml Agar 20.0 g Meat water 1000.0 ml Preparation of meat water: comminute 500 g of meat free of bones, fat and tendons, add 1000.0 ml of tap water and leave for 12 h at room temperature or in a thermostat at 30°C, or for 2 h at 37°C. Then squeeze the meat through gauze or cloth and boil the filtrate for 5 min. The proteins are denatured. Fil- ter the cooled down mass through a cotton-wool filter and add water to the initial volume. Sterilize at 121°C for 30 min. 144. PEPTONE MEAT AGAR WITH 0.5% GLUCOSE Peptone 10.0 g NaCl 5.0 g Glucose 5.0 g Agar 20.0 g Meat water 1000.0 ml Preparation of meat water: comminute 500 g of meat free of bones, fat and tendons, add 1000.0 ml of tap water and leave for 12 h at room temperature or in a thermostat at 30°C, or for 2 h at 37°C. Then squeeze the meat through gauze or cloth and boil the filtrate for 5 min. The proteins are denatured. Fil- ter the cooled down mass through a cotton-wool filter and add water to the initial volume. Sterilize at 121°C for 30 min. 145. PSEUDOMONAS SP. (ARTHROBACTER GLOBIFORMIS) MEDIUM Glucose 10.0 g Maize extract 10.0 g Tap water 1000.0 ml pH 7.8 (adjust with NH4OH); paper filter. Sterilize at 105°C for 30 min. 146. FUCHSIN-SULFITE AGAR ENDO Meat extract 5.0 g Peptone 10.0 g NaCl 5.0 g Lactose 5.0 g Na2SO4 5.0 g Basic fuchsin 0.4 g Agar 20.0 g Tap water 1000.0 ml Prepare the basic medium without fuchsin and sulfite, pH of the medium 7.4-7.6. Prepare separately 10% fuchsin solution in 90° ethanol, filter, add sulfite (possible as 10-20% aqueous solution) to bright green staining. Mix the melted medium with fuchsin and sulfite immediately before pouring into Petri dishes. 147. HYPHOMICROBIUM MEDIUM NaNO3 1.0 g NaCl 0.5 g K2HPO4 1.0 g MgSO4 x 7 H2O 0.2 g Methanol 5.0-10.0 ml Distilled water 1000.0 ml Grow in an exsiccator in the presence of ethanol vapors. 148. UROBACTERIA MEDIUM Urea 20.0 g Gelatine 150.0 g or agar 15.0 g Meat broth 1000.0 ml 149. CLAVIBACTER XYLI MEDIUM Solution 1: Flour agar 5.0 g Papaya hydrolysate of soybean meal 8.0 g K2HPO4 1.0 g KH2PO4 1.0 g MgSO4 x 7 H2O 0.2 g Distilled water 965.0 ml Solution 2: Bovine hemine chloride (0.1% in 0.05 N NaOH) 15.0 ml Solution 3: Bovine serum albumin, fraction 5 (20%) 10.0 ml Solution 4: Glucose (50%) 1.0 ml Solution 5: Cysteine (10%) 10.0 ml Sterilize solutions 2-5 by filtration. 150. GETCHINSON MEDIUM WITH FILTER PAPER K2HPO4 1.3 g MgSO4 x 7 H2O 0.3 g CaCl2 x 6 H2O 0.1 g FeCl3 x 6 H2O 0.01 g NaNO3 2.5 g Distilled water 1000.0 ml pH 7.2-7.3. Cut filter paper into strips, sterilize by dry heat and immerse into the medium so that they are not completely in the liquid medium. 151. MUNZ MEDIUM FOR METHANE-OXIDIZING BACTERIA K2HPO4 0.5 g KH2PO4 0.5 g MgSO4 x 7 H2O 0.5 g NH4Cl 1.0 g Tap water 1000.0 ml Cultivate in the mixed atmosphere of air and methane (2:1). 152. TRYPTONE THIOGLYCOLLATE MEDIUM Solution 1: K2HPO4 5.45 g KH2PO4 1.20 g MgSO4 x 7 H2O 0.025 g CaCl2 x 2 H2O 0.015 g FeSO4 x 7 H2O 0.01 g MnCl2 x 4 H2O 2.0 mg CoCl2 x 6 H2O 2.5 mg Na2MoO4 x 2 H2O 2.5 mg Peptone 2.0 g Tryptone 2.0 g Yeast extract 6.0 g Na-thioglycollate 0.5 g Distilled water 950.0 ml Solution 2: Glucose 20.0 g Distilled water 50.0 ml pH 7.5. Solution 2 sterilize separately. 153. MEDIUM P-2 FOR THERMOPHILIC ANAEROBIC BACTERIA K2HPO4 3.0 g KH2PO4 2.0 g NH4Cl 2.0 g MgCl2 x 6 H2O 0.2 g CaCl2 x 6 H2O 0.05 g Tryptone 10.0 g Glucose 5.0 g Yeast extract 5.0 g Resazurin 0.001 g Distilled water 1000.0 ml 154. DESULFOVIBRIO SULFODISMUTANS MEDIUM Solution 1: KH2PO4 0.2 g NH4Cl 0.3 g NaCl 1.0 g MgCl2 x 6 H2O 0.4 g KCl 0.5 g CaCl2 x 2 H2O 0.15 g Distilled water 920.0 ml Solution 2: Trace element solution (see below) 1.0 ml Solution 3: NaHCO3 2.5 g Distilled water 50.0 ml Solution 4: Na-acetate x 3 H2O 0.3 g Distilled water 10.0 ml Solution 5: D(+)-Biotin 10.0 ?g Ca-D(+)-Pantothenate 50.0 ?g Distilled water 1.0 ml Solution 6: Na2S x 9 H2O 0.4 g Distilled water 10.0 ml Solution 7: Na2S2O3 (0.5 M) 10.0 ml pH 7.5-8.0, adjust with NaOH Trace element solution SL-10: HCl (25%; 7.7 M) 10.0 ml FeCl3 x 4 H2O 1.5 g ZnCl2 70.0 mg MnCl2 x 4 H2O 100.0 mg H3BO3 6.0 mg CoCl2 x 6 H2O 190.0 mg CuCl2 x 2 H2O 2.0 mg NiCl2 x 6 H2O 24.0 mg Na2MoO4 x 2 H2O 36.0 mg Distilled water 990.0 ml Solution 1 is prepared and autoclaved anaerobically under 80% N2 + 20% CO2. Solutions 2, 4, 5 and 6 are gassed with N2 and sterilized separately. Solution 3 (gassed with N2 + CO2) and solution 7 (gassed with N2) are filter-sterilized. pH of the medium 7.1-7.4. 155. SCHATZ AND BOVELL MEDIUM FOR HYDROGEN-OXIDIZING BACTERIA KH2PO4 1.0 g NH4NO2 1.0 g MgSO4 x 7 H2O 0.2 g FeSO4 x 7 H2O 0.01 g CaCl2 x 2 H2O 0.01 g NaHCO3 0.5 g Agar 15.0 g Distilled water 1000.0 ml pH 6.8-7.2. Cultivate in a gas mixture of carbon dioxide, air and hydrogen (1:3:6). 156. RHIZOBIUM MEDIUM Yeast extract 1.0 g Mannitol 10.0 g Agar 15.0 g Soil extract (see below) 200.0 ml Distilled water 800.0 ml Soil extract: Air-dried garden soil 80.0 g Na2CO3 0.2 g Distilled water 200.0 ml pH 7.2. Sterilize at 121°C for 1 h. 157. EMERSON STARCH YEAST EXTRACT AGAR Yeast extract 4.0 g Starch (soluble) 15.0 g KH2PO4 1.0 g MgSO4 x 7 H2O 0.5 g Agar 20.0 g Distilled water 1000.0 ml 158. GLYCEROL YEAST AGAR Yeast extract 5.0 g Glycerol 50.0 g CaCO3 1.0 g Agar 20.0 g Distilled water 1000.0 ml 159. AGAR WITH GLUCOSE AND YEAST EXTRACT Glucose 20.0 g Yeast extract 10.0 g CaCO3 20.0 g Agar 17.0 g Distilled water 1000.0 ml 160. MEAT GLUCOSE MEDIUM Peptone 10.0 g Glucose 10.0 g NaCl 5.0 g Meat water 1000.0 ml pH 7.2-7.4. Sterilize at 121°C for 15 min. Preparation of meat water: comminute 500 g of meat free of bones, fat and tendons, add 1000.0 ml of tap water and leave for 12 h at room temperature or in a thermostat at 30°C, or for 2 h at 37°C. Then squeeze the meat through gauze or cloth and boil the filtrate for 5 min. The proteins are denatured. Fil- ter the cooled down mass through a cotton-wool filter and add water to the initial volume. Sterilize at 121°C for 30 min. 161. MEDIUM FOR MIXOBACTERIA Casein hydrolysate 2.5 g Asparagin 2.5 g K2HPO4 2.0 g NaCl 1.0 g MgSO4 x 7 H2O 0.1 g FeSO4 x 7 H2O 0.003 g CaCl2 x 2 H2O 0.01 g Distilled water 1000.0 ml 162. ANCYLOBACTER-SPIROSOMA MEDIUM Glucose 1.0 g Peptone 1.0 g Yeast extract 1.0 g Agar 15.0 g Distilled water 1000 0 ml 163. MICROCYCLUS MEDIUM Glucose 5.0 g Peptone 5.0 g Yeast extract 5.0 g Agar 15.0 g Distilled water 1000.0 ml pH 6.8. 164. PD BROTH FOR FLEXIBACTER Peptone 1.0 g KNO3 100.0 mg Yeast extract 100.0 mg K2HPO4 66.7 mg MgSO4 x 7 H2O 33.3 mg Trace element solution (see below) 1.0 ml Distilled water 1000.0 ml Trace element solution: Zn SO4 x 7 H2O 22.0 mg MnSO4 x 7 H2O 1.81 g CuSO4 x 5 H2O 79.0 mg Na3BO3 x 4 H2O 1.0 g (NH4)6Mo7O24 x 4 H2O 9.3 g FeSO4 x 7 H2O 20.0 mg Co(NO3)2 x H2O 20.0 mg Trilon B 10.0 g Distilled water 1000.0 ml pH 7.0. Sterilize at 105°C for 30 min. 165. INDICATOR MEDIUM WITH MALONATE Yeast extract 1.0 g (NH4)2SO4 2.0 g KH2PO4 0.4 g K2HPO4 0.6 g NaCl 2.0 g Na-malonate 3.0 g Glucose 0.25 g Bromothymol blue (0.2%) 12.0 ml Distilled water 1000.0 ml Dissolve components of the medium in boiling water in the specified sequence (except the indicator). Then filter through a cotton wool-gauze filter to remove the possible precipitate, bring to the initial volume, cool, adjust pH 6.7, add the indicator. Sterilize at 112°C for 30 min or at 121°C for 15 min. 166. CORYNEBACTERIUM AGAR Casein peptone, tryptic digest 10.0 g Yeast extract 5.0 g Glucose 5.0 g NaCl 5.0 g Agar 15.0 g Distilled water 1000.0 ml pH 7.2-7.4. 167. PEPTONE MEAT AGAR WITH 3% NaCl Peptone 10.0 g NaCl 30.0 g Agar 20.0 g Meat water 1000.0 ml Preparation of meat water: comminute 500 g of meat free of bones, fat and tendons, add 1000.0 ml of tap water and leave for 12 h at room temperature or in a thermostat at 30°C, or for 2 h at 37°C. Then squeeze the meat through gauze or cloth and boil the filtrate for 5 min. The proteins are denatured. Fil- ter the cooled down mass through a cotton-wool filter and add water to the initial volume. Sterilize at 121°C for 30 min. 168. CORYNEBACTERIUM MEDIUM WITH SALT Casein peptone, tryptic digest 10.0 g Yeast extract 5.0 g Glucose 5.0 g NaCl 60.0 g Agar 15.0 g Distilled water 1000.0 ml pH 7.2-7.4. 169. ALCALIGENES PARADOXUS MEDIUM Solution 1: KH2PO4 2.3 g Na2HPO4 x 2 H2O 2.9 g NH4Cl 1.0 g MgSO4 x 7 H2O 0.5 g NaHCO3 0.5 g CaCl2 x 2 H2O 0.5 g Trace element solution (see below) 5.0 ml Agar (if necessary) 15.0 g Distilled water 980.0 ml Solution 2: Fe(NH4)-citrate 0.05 g Distilled water 20.0 ml Trace element solution SL-6: ZnSO4 x 7 H2O 0.1 g MnCl2 x 4 H2O 0.03 g H3BO3 0.3 g CoCl2 x 6 H2O 0.2 g CuCl2 x 2 H2O 0.01 g NiCl2 x 6 H2O 0.02 g Na2MoO4 0.03 g pH 6.8. Sterilize at 121°C for 15 min. 170. CYTOPHAGA MEDIUM Yeast extract 10.0 g NH4Cl 1.0 g MgSO4 x 7 H2O 1.0 g K2HPO4 0.2 g NaCl 20.0 g FeCl3 x 6 H2O Traces Agar (if necessary) 2.0-3.0 g Distilled water 1000.0 ml pH 7.5. 171. YEAST AGAR K2HPO4 1.0 g MgSO4 x 7 H2O 0.5 g Yeast extract 10.0 g Agar 20.0 g Tap water 1000.0 ml pH 7.0-7.2. 172. PEPTONE SUCCINATE AGAR (NH4)2SO4 1.0 g MgSO4 x 7 H2O 1.0 g MnSO4 x 5 H2O 2.0 mg FeCl3 x 6 H2O 2.0 mg Succinic acid 1.68 g Peptone 5.0 g Agar 1.5 g Distilled water 1000.0 ml pH 7.0. 173. SPIRILLUM GRACILLE MEDIUM Peptone 5.0 g Yeast extract 0.5 g Tween 80 0.02 g K2HPO4 0.1 g Agar (if needed) 15.0 g Tap water 1000.0 ml Adjust pH to 7.2. 174. MEDIUM FOR DENTRIFYING BACTERIA (GILTAY MEDIUM) Solution 1: KNO3 1.0 g Asparagin 1.0 g Distilled water 250.0 ml Solution 2: Ca-citrate 8.5 g KH2PO4 1.0 g Distilled water 500.0 ml Solution 3: MgSO4 x 7 H2O 1.0 g FeCl3 x 4 H2O traces CaCl2 x 2 H2O 0.2 g Distilled water 250.0 ml 175. PEPTONE MEAT AGAR WITH 1% GLUCOSE Peptone 10.0 g NaCl 5.0 g Glucose 10.0 g Agar 20.0 g Meat water 1000.0 ml Preparation of meat water: comminute 500 g of meat free of bones, fat and tendons, add 1000.0 ml of tap water and leave for 12 h at room temperature or in a thermostat at 30°C, or for 2 h at 37°C. Then squeeze the meat through gauze or cloth and boil the filtrate for 5 min. The proteins are denatured. Fil- ter the cooled down mass through a cotton-wool filter and add water to the initial volume. Sterilize at 121°C for 30 min. 176. METHANOBACTERIUM MEDIUM Solution 1: NaCl 0.9 g MgCl2 x 7 H2O 0.2 g CaCl2 x 2 H2O 0.1 g NH4Cl 1.0 g Yeast extract 2.0 g Resazurin 0.001 g Trace element solution (see below) 10.0 ml Vitamin solution (see below) 5.0 ml Distilled water 965.0 ml Solution 2 (reducing agents): Cystein-HCl 0.5 g Na2S x 9 H2O 0. 5 g Distilled water 10.0 ml Buffer solutions: a) K2HPO4 29.0 g Distilled water 100.0 ml b) KH2PO4 15.0 g Distilled water 100.0 ml Trace element solution: Nitrilotriacetic acid 12.8 mg FeSO4 x 7 H2O 0.1 mg MnCl2 x 6 H2O 0.1 mg CoCl2 x 2 H2O 0.17 mg CaCl2 x 2 H2O 0.1 mg ZnCl2 0.1 mg CuCl2 0.02 mg H3BO3 0.01 mg Na2MoO4 x 2 H2O 0.01 mg NaCl 1.0 mg Na2SeO4 0.017 mg Distilled water 1000.0 ml Vitamin solution: Biotin 2.0 mg Folic acid 2.0 mg Pyridoxine (B2) 0.1 mg Riboflavin (B1) 5.0 mg Pantotenoic acid 5.0 mg p-Aminobenzoic acid 5.0 mg Thiamine-HCl 5.0 mg Nicotinic acid 5.0 mg Cyanocobalamin (B12) 0.1 mg Lipoic (tioctoic) acid 5.0 mg Distilled water 1000.0 ml Prepare medium in anaerobic conditions, blowing through with N2 without O2 up to sterilization. Solu- tions of reducing agents (10 ml) and of buffer (per 1 ml) add to base medium after separate steriliza- tion. pH 7.2-7.4. Cultivate in a gas mixture of 80% H2 and 20% CO2. 177. Starch (soluble) 10.0 g Yeast extract 1.0 g Meat extract 1.0 g Pancreatic casein hydrolysate 2.0 g CoCl2 x 7 H2O 0.02 g Agar 20.0 g Distilled water 1000.0 ml pH 7.3. 178. MEDIUM FOR MARINE CYTOPHAGA Solution 1: Yeast extract 1.0 g Tryptone 1.0 g KCl 0.7 g NaCl 24.7 g MgSO4 x 7 H2O 6.3 g MgCl2 x 6 H2O 4.6 g Agar 15.0 g Distilled water 950.0 ml Solution 2: CaCl2 x 2 H2O 1.2 g Distilled water 25.0 ml Solution 3: NaHCO3 0.2 g Distilled water 25.0 ml pH 7.2. 179. Glucose 1.0 g Trypticase 5.0 g KH2PO4 1.0 g Na-acetate 4.0 g Yeast extract 2.0 g n-Valeric acid 0.1 ml Resazurin 1.0 mg Na2CO3 4.0 g Cystein-HCl 0.5 g Distilled water 1000.0 ml pH 7.0. Gas atmosphere: 100% CO2. 180. DESULFOVIBRIO CARBINOLICUS MEDIUM Solution 1: Na2SO4 3.0 g KH2PO4 0.2 g NH4Cl 0.3 g NaCl 1.0 g MgCl2 x 6 H2O 0.4 g KCl 0.5 g CaCl2 x 2 H2O 0.15 g Distilled water 870.0 ml Solution 2: Trace element solution (see below) 1.0 ml Solution 3: NaHCO3 5.0 g Distilled water 100.0 ml Solution 4: Ethanol 0.7 g Casamino acids 0.1 g Yeast extract 0.1 g Distilled water 10.0 ml Solution 5: Vitamin solution (see below) 10.0 ml Solutin 6: Na2S x 9 H2O 0.4 g Distilled water 10.0 ml Trace element solution SL-10: HCl (25%; 7.7 M) 10.0 ml FeCl3 x 4 H2O 1.5 g ZnCl2 70.0 mg MnCl2 x 4 H2O 100.0 mg H3BO3 6.0 mg CoCl2 x 6 H2O 190.0 mg CuCl2 x 2 H2O 2.0 mg NiCl2 x 6 H2O 24.0 mg Na2MoO4 x 2 H2O 36.0 mg Distilled water 990.0 ml Vitamin solution: Biotin 2.0 mg Folic acid 2.0 mg Pyridoxine-HCl 10.0 mg Thiamine-HCl 5.0 mg Riboflavin 5.0 mg Nicotinic acid 5.0 mg Ca DL-pantothenate 5.0 mg Vitamin B12 0.1 mg p-Aminobenzoic acid 5.0 mg Lipoic acid 5.0 mg Distilled water 1000.0 ml Solution 1 is boiled before sterilization for a few minutes being flushed with gas mixture of 80% N2 and 20% CO2 with sodium bicarbonate added until an equilibrium pH of 6.9-7.1. Solution 1 is steril- ized under this gas mixture. Solution 2 is sterilized under 100% N2. Vitamin solution is filter sterilized. Final pH of the complete medium 7.1-7.4. 181. CASEIN-CITRATE AGAR Casein hydrolysate 7.5 g Yeast extract 10.0 g MgSO4 x 7 H2O 20.0 g Na-citrate 3.0 g KCl 2.0 g NaCl 200.0 g 4.98% FeSO4 in 0.001 N HCl 1.0 ml Agar 20.0 g Distilled water 1000.0 ml pH 7.4. 182. DESULFOBACTERIUM MEDIUM Solution 1: Na2SO4 3.0 g KH2PO4 0.2 g NH4Cl 0.3 g NaCl 21.0 g MgCl2 x 6 H2O 3.0 g KCl 0.5 g CaCl2 x 2 H2O 0.15 g Resazurin 1.0 mg Distilled water 930.0 ml Solution 2: Trace element solution SL-10 (see below) 1.0 ml Solution 3: Vitamin solution (see below) 10.0 ml Solution 4: NaHCO3 2.5 g Distilled water 50.0 ml Solution 5: Na2SeO4 x 5 H2O (3 mg in 1 l 0.01 M NaOH) 1.0 ml Solution 6: Na2S x 9 H2O 0.4 g Distilled water 10.0 ml Solition 7: Substrate - depending on the species of bacteria: 25% Na-acetate 10.0 ml or indole 0.3 g NaCl 2.1 g MgCl2 x 6 H2O 0.3 g Distilled water 100.0 ml Phenol 40.0 mg or Na-benzoate 400.0 mg Distilled water 4.0 ml Trace element solution SL-10: HCl (25%; 7.7 M) 10.0 ml FeCl3 x 4 H2O 1.5 g ZnCl2 70.0 mg MnCl2 x 4 H2O 100.0 mg H3BO3 6.0 mg CoCl2 x 6 H2O 190.0 mg CuCl2 x 2 H2O 2.0 mg NiCl2 x 6 H2O 24.0 mg Na2MoO4 x 2 H2O 36.0 mg Distilled water 990.0 ml Vitamin solution: p-Aminobenzoic acid 4.0 mg D(+)-Biotin 1.0 mg Thiamine-HCl 10.0 mg Distilled water 100.0 ml Solution 1 is prepared and autoclaved anaerobically under 80% N2 + 20% CO2. Solutions 2, 4, 5 and 6 are gassed with N2 and sterilized separately. Solution 3 (gassed with N2 + CO2) and solution 7 (gassed with N2) are filter-sterilized. pH of the medium 7.1-7.4. 183. BENETT MEDIUM Yeast extract 1.0 g Meat extract 1.0 g Fermentative casein hydrolysate 2.0 g Glucose 10.0 g Agar 15.0 g Distilled water 1000.0 ml pH 7.2. 184. ISP 2 MEDIUM Glucose 4.0 g Yeast extract 4.0 g Malt extract 10.0 g Agar 15.0 g Distilled water 1000.0 ml pH 7.2. 185. HALOBACTERIUM MEDIUM 4 Yeast extract 5.0 g Casamino acids 5.0 g Na-glutamate 1.0 g KCl 2.0 g Na-citrate 3.0 g MgSO4 x 7 H2O 20.0 g NaCl 200.0 g FeCl3 x 4 H2O 36.0 mg MnCl2 x 4 H2O 0.36 mg Agar 20.0 g Distilled water to 1000.0 ml pH 7.0-7.2. 186. HALOBACTERIUM MEDIUM 5 Solution 1: Casamino acids 7.5 g Yeast extract 10.0 g Na-citrate 3.0 g KCl 2.0 g MgSO4 x 7 H2O 20.0 g FeSO4 x 7 H2O 0.05 g MnSO4 x H2O 0.2 g NaCl 250.0 g Distilled water 750.0 ml Solution 2: Agar 20.0 g Distilled water 250.0 ml pH 7.4. Sterilize at 121°C for 15 min. 187. HALOCOCCUS MEDIUM Solution 1: Skim milk 50.0 g Distilled water 500.0 ml Solution 2: MgSO4 x 7 H2O 10.0 g KNO3 2.0 g NaCl 200.0 g Fe-citrate traces Distilled water 100.0 ml Solution 3: Neopeptone 5.0 g Glycerol 10.0 g Agar 25.0 g Distilled water 400.0 ml Sterilization of solution 1 at 112°C for 15 min. Mix together heated solutions 2 and 3, adjust pH of the mixture to 8.4 and sterilize at 121°C for 20 min. 188. NATRONOBACTERIA MEDIUM Solution 1: KH2PO4 1.0 g KCl 1.0 g NH4Cl 1.0 g MgSO4 x 7 H2O 0.24 g CaSO4 x 2 H2O 0.17 g Trace element solution (see below) 1.0 ml Agar, if necessary (heat and dissolve it before adding sodium chloride) 20.0 g NaCl 200.0 g Glutamate 1.0 g Yeast extract 5.0 g Casamini acids 5.0 g Distilled water to 1000.0 ml pH of solution 1 before sterilization, 6.5. Solution 2: Na2CO3 5.0 g Distilled water 50.0 ml Trace element solution SL-10: HCl (25%; 7.7 M) 10.0 ml FeCl3 x 4 H2O 1.5 g ZnCl2 70.0 mg MnCl2 x 4 H2O 100.0 mg H3BO3 6.0 mg CoCl2 x 6 H2O 190.0 mg CuCl2 x 2 H2O 2.0 mg NiCl2 x 6 H2O 24.0 mg Na2MoO4 x 2 H2O 36.0 mg Distilled water 990.0 ml pH of the medium, 9.0 - 9.5. 189. HALOBACTERIUM MEDIUM 6 NaCl 156.0 g MgCl2 x 6 H2O 13.0 g MgSO4 x 7 H2O 20.0 g CaCl2 x 6 H2O 1.0 g KCl 4.0 g NaHCO3 0.2 g NaBr 0.5 g Yeast extract 5.0 g Glucose 1.0 g Distilled water 1000.0 ml pH 7.0. 190. PRAUSER MEDIUM 79 Glucose 10.0 g Peptone 10.0 g Yeast extract 2.0 g Casamino acids 2.0 g NaCl 6.0 g Agar 20.0 g Tap water 1000.0 ml pH 7.5. 191. STARCH-YEAST AGAR Yeast extract 2.0 g Starch (soluble) 10.0 g Agar 20.0 g Tap water 1000.0 ml pH 7.3. 192. MYA-AGAR Glucose 2.0 g L-Asparagin 1.0 g K2HPO4 0.5 g MgSO4 x 7 H2O 0.5 g Trace element solution (see below) 1.0 ml Agar 20.0 g Distilled water 1000.0 ml Trace element solution: FeSO4 x 7 H2O 0.1 g CuSO4 x 5 H2O 0.1 g ZnSO4 x 7 H2O 0.1 g Distilled water 100.0 ml pH 7.4. 193. ACETATE AGAR NaCl 5.0 g K2HPO4 1.0 g NH4H2PO4 1.0 g MgSO4 x 7 H2O 0.2 g Na-acetate 2.0 g Bromothymol blue (0.2%) 40.0 ml Agar 20.0 g Distilled water 1000.0 ml pH 7.2. Add the indicator last, after pH is set and the possible precipitate is separated by filtration through a cotton wool-gauze filter. Sterilize at 112°C for 15 min. 194. INMI MEDIUM 3 NaCl 250.0 g KCl 2.0 g Na-citrate 3.0 g MgSO4 x 7 H2O 20.0 g Casamino acids 5.0 g Yeast extract 2.5 g Agar 20.0 g Distilled water 1000.0 ml pH 7.2. 195. INMI MEDIUM 4 Yeast extract 2.5 g Casamino acids 5.0 g pH 9.5. 196. MEDIUM FOR PURPLE BACTERIA (VAN NIEL MEDIUM) NH4Cl 1.0 g K2HPO4 0.5 g MgCl2 x 6 H2O 0.2 g NaHCO3 1.0 g Na2S x 9 H2O 1.0 g Tap water 1000.0 ml Sterilize sulfide separately. pH of the medium 7.6. 197. MEDIUM FOR RHODOSPIRILLUM (PFENNIG MEDIUM) NH4Cl 0.4 g KH2PO4 0.5 g MgSO4 x 7 H2O 0.2 g NaCl 0.4 g CaCl2 x 2 H2O 0.05 g Acetate, or butyrate, or propionate, or succinate 1.0 g Yeast extract 0.2 g Fe-citrate (0.1%) 5.0 ml Trace element solution (see below) 1.0 ml Vitamin B12 (1.0%) 1.0 ml Distilled water 1000.0 ml Trace element solution according to Pfennig: EDTA 1.5 g Trace element solution according to Hogland (see below): 6.0 ml FeSO4 x 7 H2O 0.2 g MnCl2 x 4 H2O 0.02 g ZnSO4 x 7 H2O 0.1 g Distilled water 1000.0 ml Trace element solution according to Hogland: EDTA 5.0 g FeSO4 x 7 H2O 2.0 g ZnSO4 x 7 H2O 100.0 mg MnCl2 x 4 H2O 30.0 mg H3BO3 300.0 mg CoCl2 x 6 H2O 200.0 mg CuCl2 10.0 mg NiCl2 x 6 H2O 20.0 mg Na2MoO4 x 2 H2O 20.0 mg Distilled water 1000.0 ml 198. THIOBACILLUS DENITRIFICANS MEDIUM (TAYLOR MEDIUM) KNO3 2.0 g NH4Cl 1.0 g KH2PO4 2.0 g NaHCO3 2.0 g MgSO4 x 7 H2O 0.8 g Na2S2O3 x 5 H2O 5.0 g Trace element solution (see below) 1.0 ml Distilled water 1000.0 ml Trace element solution according to Pfennig: EDTA 1.5 g Trace element solution according to Hogland (see below): 6.0 ml FeSO4 x 7 H2O 0.2 g MnCl2 x 4 H2O 0.02 g ZnSO4 x 7 H2O 0.1 g Distilled water 1000.0 ml Trace element solution according to Hogland: EDTA 5.0 g FeSO4 x 7 H2O 2.0 g ZnSO4 x 7 H2O 100.0 mg MnCl2 x 4 H2O 30.0 mg H3BO3 300.0 mg CoCl2 x 6 H2O 200.0 mg CuCl2 10.0 mg NiCl2 x 6 H2O 20.0 mg Na2MoO4 x 2 H2O 20.0 mg Distilled water 1000.0 ml 199. THIOBACILLUS DENITRIFICANS MEDIUM (BAALSRUD MEDIUM) KNO3 2.0 g NH4Cl 0.5 g KH2PO4 2.0 g NaHCO3 1.0 g MgCl2 x 6 H2O 0.5 g Na2S2O3 x 5 H2O 5.0 g FeSO4 x 7 H2O 0.01 g Distilled water 1000.0 ml Sterilize iron, phosphorus and bicarbonate salts separately. pH 7.0. 200. THIOBACILLUS DENITRIFICANS MEDIUM (LIESKE MEDIUM) KNO3 5.0 g KH2PO4 0.2 g NaHCO3 1.0 g MgCl2 x 6 H2O 0.1 g Na2S2O3 x 5 H2O 5.0 g CaCl2 x 6 H2O Traces FeCl3 x 6 H2O Traces Distilled water 1000.0 ml 201. MODIFIED BROCK MEDIUM FOR SULFUROXIDIZING BACTERIA (NH4)2SO4 1.3 g KH2PO4 0.37 g MgSO4 x 7 H2O 0.25 g CaCl2 x 6 H2O 0.07 g Distilled water 1000.0 ml pH 2.4 (range: 2.3 - 3.0; adjust with H2SO4). Additions to the medium (sterilize separately each): Trace element solution (see below) 1.0 ml Yeast extract 0.2 g Element sulfur 10.0 g Chalk (CaCO3) 10.0 g For cultivation of heterotrophic representatives of the group the medium after sterilization is to be also supplemented with sterilized as separate solutions at 105°C: Peptone 1.75 g Sucrose 0.25 g Trace element solution according to Pfennig: EDTA 1.5 g Trace element solution according to Hogland (see below): 6.0 ml FeSO4 x 7 H2O 0.2 g MnCl2 x 4 H2O 0.02 g ZnSO4 x 7 H2O 0.1 g Distilled water 1000.0 ml Trace element solution according to Hogland: EDTA 5.0 g FeSO4 x 7 H2O 2.0 g ZnSO4 x 7 H2O 100.0 mg MnCl2 x 4 H2O 30.0 mg H3BO3 300.0 mg CoCl2 x 6 H2O 200.0 mg CuCl2 10.0 mg NiCl2 x 6 H2O 20.0 mg Na2MoO4 x 2 H2O 20.0 mg Distilled water 1000.0 ml 202. MEDIUM FOR MAGNETIC BACTERIA Tartaric acid 0.37 g Succinic acid 0.37 g Na-acetate 0.05 g KH2PO4 0.68 g NaNO3 0.12 g Agar 1.3 g Vitamin solution (see below) 10.0 ml Trace element solution (see below) 5.0 ml Fe-quinate 2.0 ml Na-thioglycolate 0.05 g Resazurin 0.5 mg Bidistilled water 1000.0 ml Vitamin solution: Biotin 200.0 mg Folic acid 20.0 mg Pyridoxine-HCl 100.0 mg Thiamine-HCl 50.0 mg Riboflavin 100.0 mg Nicotinic acid 50.0 mg DL-Pantothenic acid 50.0 mg Vitamin B12 1.0 mg p-Aminobenzoic acid 50.0 mg Distilled water 1000.0 ml Trace element solution: Nitrilotriacetic acid 12.8 g FeCl3 x 4 H2O 0.2 g MnCl2 x 4 H2O 0.1 g CoCl2 x 6 H2O 0.17 g CaCl2 x 2 H2O 0.1 g ZnCl2 0.1 g CuCl2 0.02 g Na2MoO4 x 2 H2O 0.01 g NiCl2 x 6 H2O 0.026 g NaCl 1.0 g Na2SeO4 x 5 H2O 0.02 g Distilled water 1000.0 ml pH 6.75 (adjust with NaOH). Prior to sterilization the medium is to be blown down by the flow of N2 and sterilized in the nitrogen atmosphere at 105°C for 20 min. 203. MEDIUM FOR FLEXIBACTER (LEWIN MEDIUM) Na-glycerophosphate 0.1 g KNO3 0.1 g MgSO4 x 7 H2O 0.1 g Trace element solution (see below) 1.0 ml Vitamin B12 1.0 ?g CaCl2 x 6 H2O 0.1 g Tris buffer 1.0 g Thiamine 1.0 mg Casamino acids 1.0 g Distilled water 1000.0 ml Trace element solution: Nitrilotriacetic acid 12.8 g FeCl3 x 4 H2O 0.2 g MnCl2 x 4 H2O 0.1 g CoCl2 x 6 H2O 0.17 g CaCl2 x 2 H2O 0.1 g ZnCl2 0.1 g CuCl2 0.02 g Na2MoO4 x 2 H2O 0.01 g NiCl2 x 6 H2O 0.026 g NaCl 1.0 g Na2SeO4 x 5 H2O 0.02 g Distilled water 1000.0 ml pH 7.5. 204. MEDIUM FOR CLOSTRIDIUM Glucose 10.0 g Peptone 12.0 g NaCl 2.0 g Agar 16.0 g Distilled water 1000.0 ml The medium can be used with chalk addition as buffer against acidification of the medium during cul- tivation. 205. OATMEAL AGAR A Oatmeal 20.0 g Agar 20.0 g Tap water 1000.0 ml pH 7.2. 206. ISP MEDIUM 3 Oatmeal 20.0 g Salt solution A (see below) 1.0 ml Agar 20.0 g Distilled water 1000.0 ml Salt solution A: FeSO4 0.1 g MnCl2 0.1 g ZnSO4 0.1 g Distilled water 100.0 ml pH 7.2. 207. MINERAL AGAR 1 Starch (soluble) 20.0 g K2HPO4 0.5 g MgSO4 x 7 H2O 0.5 g KNO3 1.0 g NaCl 0.5 g FeSO4 0.01 g Agar 30.0 g Distilled water 1000.0 ml pH 7.2 - 7.4. 208. GLUCOSE ASPARAGIN AGAR Glucose 10.0 g L-Asparagin 0.5 g K2HPO4 0.5 g Agar 20.0 g Distilled water 1000.0 ml pH 6.8. Sterilize at 105°C for 30 min. 209. MODIFICATION OF THE CZAPEK MEDIUM WITH STARCH Starch (soluble) 20.0 g K2HPO4 0.5 g KNO3 1.0 g NaCl 0.2 g MgSO4 x 7 H2O 0.3 g CaCO3 0.5 g FeSO4 0.001 g Agar 20.0 g Distilled water 1000.0 ml pH 7.2-7.4. Sterilize at 105°C for 30 min. 210. GLYCEROL-ASPARAGIN AGAR L-Asparagin 1.0 g Glycerol 10.0 g K2HPO4 1.0 g Salt solution A (see below) 1.0 ml Agar 20.0 g Distilled water 1000.0 ml Salt solution A: FeSO4 0.1 g MnCl2 0.1 g ZnSO4 0.1 g Distilled water 100.0 ml pH 7.0-7.4. 211. OATMEAL AGAR WITH 0.1% YEAST EXTRACT Oatmeal 20.0 g Yeast extract 1.0 g Agar 20.0 g Tap water 1000.0 ml pH 7.2. 212. MODIFICATION OF CZAPEK MEDIUM WITH GLUCOSE Glucose 20.0 g K2HPO4 0.5 g KNO3 1.0 g NaCl 0.2 g MgSO4 x 7 H2O 0.3 g CaCO3 0.5 g FeSO4 0.001 g Agar 20.0 g Distilled water 1000.0 ml pH 7.2-7.4. 213. ORGANIC AGAR 2 Hottinger broth (see below) 30.0 ml Peptone 5.0 g NaCl 5.0 g Glucose 10.0 g Agar 30.0 g Tap water 1000.0 ml pH 7.0-7.2. Preparation of Hottinger broth: boil meat (1-2 cm pieces) (without fat or tendons) in 2.0 l of water, then mince. Adjust pH of the decoction to 8.0, mix with minced meat and cool down to 40°C. Then add 1.0 g of dry pancreatin, mix and again alkalinize to pH 7.8-8.0. Pour the mixture into a bottle with the rubber stopper (1/3 of the bottle to remain free), add chloroform (20 ml), mix and open the bottle for 1 min to remove the excess chloroform vapors. 2 h after pancreatin was added, adjust pH to 7.4-7.6 and leave the mixture for 2 weeks at 18-20°C. The first 4 days adjust pH of the medium; shake and mix 3 times a day, then stir once a day. Two days before the end of the procedure stop mixing to allow the decoction to settle. The liquid shall be of straw color, the reaction with tryptophan with bromine water shall be positive; in the decoction hydrolysate the total nitrogen shall be no less than 1100 mg%. Filter the decoction through the linen, pour into flasks and sterilize in autoclave at 121°C for 30 min. Filter prior to use. 214. STARCH AMMONIA AGAR Starch (soluble) 10.0 g K2HPO4 1.0 g MgSO4 x 7 H2O 1.0 g NaCl 1.0 g (NH4)2SO4 2.0 g CaCO3 2.0 g Salt solution A (see below) 1.0 ml Agar 20.0 g Distilled water 1000.0 ml Salt solution A: FeSO4 0.1 g MnCl2 0.1 g ZnSO4 0.1 g Distilled water 100.0 ml pH 7.0-7.4. 215. GLYCEROL NITRATE AGAR Glycerol 30.0 g NaNO3 2.0 g K2HPO4 1.0 g MgSO4 x 7 H2O 0.5 g KCl 0.5 g FeSO4 0.01 g Agar 20.0 g Distilled water 1000.0 ml pH 7.0-7.2. Sterilize at 121°C for 30 min. 216. TETRATHIONATE BROTH (MULLER MEDIUM) Solution 1: Hottinger broth (see below) 900.0 ml Solution 2: Lugol solution (see below) 20.0 ml Solution 3: 50% Na2S2O3 x 5 H2O 100.0 ml Lugol solution: KJ 20.0 g J2 25.0 g Distilled water 100.0 ml pH 7.2 - 7.4. Sterilize solution 3 with steam for 30 min. Pour the medium into sterile vials with CaCO3 (25 g CaCO3 per 1 liter of medium). Sterilize the flasks with CaCO3 with dry heat. Preparation of Hottinger broth: boil meat (1-2 cm pieces) (without fat or tendons) in 2.0 l of water, then mince. Adjust pH of the decoction to 8.0, mix with minced meat and cool down to 40°C. Then add 1.0 g of dry pancreatin, mix and again alkalinize to pH 7.8-8.0. Pour the mixture into a bottle with the rubber stopper (1/3 of the bottle to remain free), add chloroform (20 ml), mix and open the bottle for 1 min to remove the excess chloroform vapors. 2 h after pancreatin was added, adjust pH to 7.4-7.6 and leave the mixture for 2 weeks at 18-20°C. The first 4 days adjust pH of the medium; shake and mix 3 times a day, then stir once a day. Two days before the end of the procedure stop mixing to allow the decoction to settle. The liquid shall be of straw color, the reaction with tryptophan with bromine water shall be positive; in the decoction hydrolysate the total nitrogen shall be no less than 1100 mg%. Filter the decoction through the linen, pour into flasks and sterilize in autoclave at 121°C for 30 min. Filter prior to use. 217. MEDIUM FOR MICROMONOSPORES Glucose 10.0 g Starch (soluble) 20.0 g Yeast extract 5.0 g Fermentative casein hydrolysate 5.0 g CaCO3 1.0 g Agar 20.0 g Distilled water 1000.0 ml 218. PYA WITH MARINE WATER Peptone 5.0 g Yeast extract 3.0 g Agar 12.0 g Distilled water 250.0 ml Aged filtered sea water 750.0 ml pH 7.5-7.6. 219. MEDIUM FOR HALOPHILIC BACILLI NaCl 100.0 g NaHCO3 10.0 g Na2CO3 10.0 g Nutrient broth 1000.0 ml pH 9.5 220. PYEA MEDIUM Peptone 10.0 g Yeast extract 10.0 g NaCl 5.0 g Agar 15.0 g Distilled water 1000.0 ml pH 7.2 221. MEDIUM FOR METHANOTROPHIC BACTERIA KNO3 1.0 g KH2PO4 0.7 g MgSO4 x 7 H2O 0.2 g CaCl2 0.02 g Na2HPO4 x 5 H2O 1.5 g Trilon B 5.0 mg FeSO4 x 7 H2O 2.0 mg ZnSO4 x 7 H2O 0.1 mg MnCl2 x 4 H2O 0.03 mg CoCl2 x 6 H2O 0.2 mg CuCl2 x 5 H2O 0.1 mg NiCl2 x 6 H2O 0.02 mg Na2MoO4 0.03 mg Distilled water 1000.0 ml pH 6.7-7.1. Cultivation under mixture of methane and air (1:1). 222. MEDIUM FOR MARINE METHYLOTROPHIC BACTERIA KH2PO4 2.0 g (NH4)2SO4 2.0 g NaCl 30.0 g MgSO4 x 7 H2O 0.025 g FeSO4 x 7 H2O 0.002 g Yeast extract 0.1 g Methanol 5.0 ml Biotine 0.01 mg Distilled water 1000.0 ml pH 7.0 223. MEDIUM FOR OLIGOCARBOPHILIC BACTERIA. Solution 1 (basic solution): Peptone 0.25 g Yeast extract 0.25 g Agar 5.0 g Solution 2 20.0 ml Distilled water 965.0 ml Sterilize 20 min at 121°C. After cooling add the following solutions: Glucose (2.5 %) 10.0 ml Solution 3 5.0 ml Adjust pH to 7.5. Solution 2 (trace elements): Nitrilotriacetate 10.0 g MgSO4 x 7 H2O 29.7 g CaCl2 x 2 H2O 3.34 g Na2MoO4 x 2 H2O 12.67 mg FeSO4 x 7 H2O 350.0 mg Na-EDTA 125.0 mg ZnSO4 x 7 H2O 548.0 mg MnSO4 x H2O 77.0 mg CuSO4 x 5 H2O 20.0 mg Co(NO3)2 x 6 H2O 12.4 mg Na2B4O7 x 10 H2O 8.8 mg Distilled water 950.0 ml Dissolve nitriltriacetate first by neutralizing with KOH, then add other salts. Adjust pH to 7.2. Adjust volume to 1000 ml. Solution 3 (vitamin solution): Biotin 4.0 mg Folic acid 4.0 mg Pyridoxine-HCl 20.0 mg Riboflavine 10.0 mg Thiamine-HCl 10.0 mg Nicotin amide 10.0 mg Calcium D-pantothenate 10.0 mg Vitamin B12 0.2 mg p-aminobenzoic acid 10.0 mg Distilled water 1000.0 ml Store in refrigerator at +5°C. 224. MEDIUM FOR DESULFOTOMACULUM ALKALIPHILUM Solution 1 (basic solution): Solution 2 10.0 ml Na2CO3 0.5 g Na2SO4 5.0 g NaCl 5.0 g Na formate 5.0 g Yeast extract 1.0 g Solution 3 2.0 ml Solution 4 1.0 ml Rezazurine traces Distilled water 1000.0 ml Sterilize 20 min at 121°C. After cooling add the following solutions: NaHCO3 final concentration 8.0 g/l Na2S x 9 H2O final concentration 0.5 g/l Solution 2: KH2PO4 0.2 g MgCl2 x 6 H2O 0.1 g NH4Cl 1.0 g KCl 0.2 g Distilled water 1000.0 ml Solution 3 (vitamin solution): Biotin 10.0 mg Folic acid 10.0 mg Pyridoxine-HCl 50.0 mg Riboflavine 25.0 mg Thiamine-HBr 25.0 mg Nicotin amide 25.0 mg D-pantothenate 25.0 mg Vitamin B12 0.5 mg p-aminobenzoic acid 25.0 mg Thioctoic acid 25.0 mg Distilled water 500.0 ml Store in refrigerator at +5°C. Solution 4 (trace elements): Na2MoO4 x 2 H2O 20.0 mg FeSO4(NH4)2SO4 x 6 H2O 400.0 mg FeSO4 x 7 H2O 200.0 mg ZnSO4 x 7 H2O 200.0 mg MnCl2 x 4 H2O 720.0 mg NiCl2 100.0 mg CuSO4 x 5 H2O 20.0 mg AlK(SO4)2 x 12 H2O 20.0 mg CoCl2 x 6 H2O 200.0 mg H3BO3 20.0 mg HCl 5.0 ml Distilled water 200.0 ml 225. 1/5 STARCH-YEAST AGAR Yeast extract 0.4 g Soluble starch 2.0 g Agar 20.0 g Distilled water 1000.0 ml pH 7.3. 226. MEDIUM FOR ACTINOPOLYSPORA MORTIVALLIS Bacto vitamin assay casamino acids 7.5 g Yeast extract 10.0 g MgSO4x7 H2O 20.0 g Trisodium citrate x 2 H2O 3.0 g KCl 2.0 g NaCl 150.0 g 4.98% FeSO4 in 0.001 N HCl 1.0 ml Agar 20.0 g Distilled water 1000.0 ml pH 7.4. 227. MICROLUNATUS 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 x 7H2O 0.1 g Distilled water 1000.0 ml pH 7.0. 228. MICROCOCCUS HALOPHILUS MEDIUM Peptone 10.0 g Yeast extract 5.0 g Malt extract 5.0 g Casamino acids 5.0 g Meat extract 2.0 g Glycerol 2.0 g Tween-80 50.0 mg MgSO4 x 7 H2O 1.0 g NaCl 50.0 g Agar 20.0 g Distilled water 1000.0 ml pH 7.2. 229. ALKALIBACTER MEDIUM (NH4)2SO4 1.00 g NH4Cl 0.40 g Na2S2O3 x 5 H2O 0.10 g K2HPO4 0.50 g MgSO4 x 7 H2O 0.10 g CaCl2 x 2 H2O 0.05 g NaCl 10.00 g FeSO4 x 7 H2O 2.00 mg Trace element solution (see below) 10.00 ml Resazurin 0.001 g Yeast extract 0.25 g Tryptone 2.0 g Glucose 5.0 g Vitamin solution (see below) 10.00 ml L-cysteine 0.50 g Distilled water 950.00 ml Trace element solution: Nitrilotriacetic acid 12.8 mg FeSO4 x 7 H2O 0.1 mg MnCl2 x 6 H2O 0.1 mg CoCl2 x 2 H2O 0.17 mg CaCl2 x 2 H2O 0.1 mg ZnCl2 0.1 mg CuCl2 0.02 mg H3BO3 0.01 mg Na2MoO4 x 2 H2O 0.01 mg NaCl 1.0 mg Na2SeO4 0.017 mg Distilled water 1000.0 ml Vitamin solution: Biotin 2.0 mg Folic acid 2.0 mg Pyridoxine (B2) 0.1 mg Riboflavin (B1) 5.0 mg Pantotenoic acid 5.0 mg p-Aminobenzoic acid 5.0 mg Thiamine-HCl 5.0 mg Nicotinic acid 5.0 mg Cyanocobalamin (B12) 0.1 mg Lipoic (tioctoic) acid 5.0 mg Distilled water 1000.0 ml After autoclaving, add from sterile anaerobic solution (per liter medium): 50 ml of 5% w/v Na2CO3 Adjust final pH of medium to 9.0. 230. CALDITHRIX ABYSSI MEDIUM Sea salt 37.9 g NH4Cl 0.33 g KH2PO4 0.33 g Resazurin 0.50 mg Distilled water 900.00 ml Prepare medium anaerobically under 80% N2 + 20% CO2 gas mixture. After autoclaving add from separately prepared, sterile anaerobic stock solutions NaHCO3 (5% w/v) 50.00 ml Yeast extract (20% w/v) 15 ml Trace element solution (see below) 10.00 ml Vitamin solution (see below) 10.00 ml Na2S x 9 H2O (3% w/v) 20.00 ml Selenite-tungstate solution (0.5 g NaOH, 3 mgNa2SeO3 x 5 H2O, 4 mgNa2WO4 x 2 H2O, 1 l distilled water) 1.00 ml Trace element solution: Nitrilotriacetic acid 12.8 mg FeSO4 x 7 H2O 0.1 mg MnCl2 x 6 H2O 0.1 mg CoCl2 x 2 H2O 0.17 mg CaCl2 x 2 H2O 0.1 mg ZnCl2 0.1 mg CuCl2 0.02 mg H3BO3 0.01 mg Na2MoO4 x 2 H2O 0.01 mg NaCl 1.0 mg Na2SeO4 0.017 mg Distilled water 1000.0 ml Vitamin solution: Biotin 2.0 mg Folic acid 2.0 mg Pyridoxine-HCl 10.0 mg Thiamine-HCl x 2 H2O 5.0 mg Riboflavin 5.0 mg Nicotinic acid 5.0 mg D-Ca-pantothenate 5.0 mg Vitamin B12 0.1 mg p-Aminobenzoic acid 5.0 mg Lipoic acid 5.0 mg Distilled water 1000.0 ml Adjust final pH of the medium to 6.8. 231. CARBOXYDOCELLA SPOROPRODUCENS MEDIUM KCl 0.33 g MgCl2 x 6 H2O 0.52 g CaCl2 x 2 H2O 0.29 g NH4Cl 0.33 g KH2PO4 0.33 g NaHCO3 1.00 g Trace element solution (see below) 10.00 ml Resazurin 0.50 mg Vitamin solution (see below) 10.00 ml Pyruvate 2.5 g Yeast extract 0.05 g Na2S x 9 H2O 0.30 g Distilled water 1000.00 ml Trace element solution SL-4: EDTA 0.5 g FeSO4 x 7H2O 0.2 g Trace element solution SL-6 (see below) 100.0 ml Distilled water 900.0 ml Vitamin solution: Biotin 2.0 mg Folic acid 2.0 mg Pyridoxine (B2) 0.1 mg Riboflavin (B1) 5.0 mg Pantotenoic acid 5.0 mg p-Aminobenzoic acid 5.0 mg Thiamine-HCl 5.0 mg Nicotinic acid 5.0 mg Cyanocobalamin (B12) 0.1 mg Lipoic (tioctoic) acid 5.0 mg Distilled water 1000.0 ml Trace element solution SL-6: ZnSO4 x 7 H2O 0.1 g MnCl2 x 4 H2O 0.03 g H3BO3 0.3 g CoCl2 x 6 H2O 0.2 g CuCl2 x 2 H2O 0.01 g NiCl2 x 6 H2O 0.02 g Na2MoO4 0.03 g Distilled water 1000.0 ml Dissolve ingredients except bicarbonate, yeast extract, vitamins, pyruvate and sulfide, boil medium for 1 min., then cool to room temperature under N2 gas atmosphere. Dispense medium under same gas at- mosphere in tubes or serum bottles and autoclave. Add vitamins (sterilized by filtration), yeast extract, pyruvate and sulfide from sterile anoxic stock solutions prepared under N2 gas atmosphere and bicar- bonate from a sterile anoxic solution prepared under 80% N2 and 20% CO2 gas mixture. Adjust pH to 7.0 with a sterile anoxic solution of 10% (w/v) NaHCO3. Inoculated vessels are pressurized with car- bon monoxide gas to 2 bar overpressure. 232. CARBOXYDOTHERMUS FERRIREDUCENS MEDIUM KH2PO4 0.33 g NH4Cl 0.33 g KCl 0.33 g MgCl x 6 H2O 0.33 g CaCl2 x 2 H2O 0.33 g NaHCO3 2.00 g Glycerol (87%) 3.00 ml Vitamin solution (see below) 10.00 ml Trace element solution (see below) 1.00 ml NiCl2 x 6 H2O 200.00 ?g Na2SeO3 x 5 H2O 120.00 ?g Na2WO4 x 2 H2O 30.00 ?g Yeast extract 1.00 g Na2-9,10-anthraquinone-2,6-disulfonate (Sigma A9706) 8.25 g Distilled water 1000.00 ml Vitamin solution: Biotin 2.0 mg Folic acid 2.0 mg Pyridoxine (B2) 0.1 mg Riboflavin (B1) 5.0 mg Pantotenoic acid 5.0 mg p-Aminobenzoic acid 5.0 mg Thiamine-HCl 5.0 mg Nicotinic acid 5.0 mg Cyanocobalamin (B12) 0.1 mg Lipoic (tioctoic) acid 5.0 mg Distilled water 1000.0 ml Trace element solution SL-10: HCl (25%; 7.7 M) 10.0 ml FeCl3 x 4 H2O 1.5 g ZnCl2 70.0 mg MnCl2 x 4 H2O 100.0 mg H3BO3 6.0 mg CoCl2 x 6 H2O 190.0 mg CuCl2 x 2 H2O 2.0 mg NiCl2 x 6 H2O 24.0 mg Na2MoO4 x 2 H2O 36.0 mg Distilled water 990.0 ml Dissolve ingredients (except CaCl2 x 2 H2O, NaHCO3, and vitamins), boil medium for some minutes to dissolve the anthraquinone, then cool under 80% N2 and 20% CO2 gas atmosphere to room tempera- ture. Add solid NaHCO3 and adjust medium pH to 6.8 with NaOH. Dispense medium in tubes or bot- tles under same gas. Autoclave at 121°C for 20 min. Before use, add CaCl2 and vitamins from anoxic, sterile stock solutions. 233. CLOSTRIDIUM ALKALICELLULOSI MEDIUM NH4Cl 0.5 g KH2PO4 0.2 g MgCl2 x 6 H2O 0.1 g KCl 0.2 g Trace element solution (see below) 1.0 ml Selenite-tungstate solution (0.5 g NaOH, 3 mgNa2SeO3 x 5 H2O, 4 mgNa2WO4 x 2 H2O, 1 l distilled water) 1.0 ml Na2CO3 1.0 g NaHCO3 7.6 g NaCl 10.0 g Yeast extract 0.2 g Cellobiose 3.0 g Na2S x 9 H2O 0.5 g Trace element solution SL-10: HCl (25%; 7.7 M) 10.0 ml FeCl3 x 4 H2O 1.5 g ZnCl2 70.0 mg MnCl2 x 4 H2O 100.0 mg H3BO3 6.0 mg CoCl2 x 6 H2O 190.0 mg CuCl2 x 2 H2O 2.0 mg NiCl2 x 6 H2O 24.0 mg Na2MoO4 x 2 H2O 36.0 mg Distilled water 990.0 ml Dissolve ingredients except cellobiose and sulfide, flush medium with N2 gas for 30-60 min, dispense under N2 gas atmosphere and autoclave. Add cellobiose after autoclaving from an anoxic stock solu- tion sterilized by filtration and sulfide from a sterile, anoxic stock solution prepared under N2. Adjust final pH of the medium to pH 8.8-9.0. 234. DESULFOHALOBIUM UTAHENSE MEDIUM NaCl 100.0 g MgSO4 x 7 H2O 10.0 g KCl 6.0 g CaCl2 x 2 H2O 0.4 g NH4Cl 1.0 g KH2PO4 0.1 g Yeast extract 0.5 g Trace element solution (see below) 1.0 ml Selenite-tungstate solution (0.5 g NaOH, 3 mgNa2SeO3 x 5 H2O, 4 mgNa2WO4 x 2 H2O, 1 l distilled water) 1.0 ml NaHCO3 4.0 g Na-(L)-lactate 2.5 g Resazurin 0.5 mg Na2S x 9 H2O 0.3 g Distilled water 1000.0 ml Trace element solution SL-10: HCl (25%; 7.7 M) 10.0 ml FeCl3 x 4 H2O 1.5 g ZnCl2 70.0 mg MnCl2 x 4 H2O 100.0 mg H3BO3 6.0 mg CoCl2 x 6 H2O 190.0 mg CuCl2 x 2 H2O 2.0 mg NiCl2 x 6 H2O 24.0 mg Na2MoO4 x 2 H2O 36.0 mg Distilled water 990.0 ml Dissolve ingredients (except lactate, bicarbonate and sulfide), boil medium for 1 min., then cool to room temperature under 80% N2 and 20% CO2 gas mixture. Dispense under same gas atmosphere in culture vessels and autoclave. Add sodium lactate and sulfide from sterile anoxic stock solutions pre- pared under N2 and bicarbonate from a sterile stock solution prepared under 80% N2 and 20% CO2. Adjust the final pH of the medium to 7.0-7.2. 235. DESULFONATRONUM COOPERATIVUM MEDIUM KH2PO4 0.2 g MgCl2 x 6 H2O 0.1 g NH4Cl 1.0 g KCl 0.2 g Na2SO4 5.0 g NaCl 10.0 g Na2CO3 3.5 g Trace element solution (see below) 1.0 ml Vitamin solution (see below) 10.0 ml Yeast extract 1.0 g Resazurin 0.5 mg Na-formate 4.0 g Na2S x 9 H2O 0.5 g Distilled water 1000.0 ml Trace element solution SL-10: HCl (25%; 7.7 M) 10.0 ml FeCl3 x 4 H2O 1.5 g ZnCl2 70.0 mg MnCl2 x 4 H2O 100.0 mg H3BO3 6.0 mg CoCl2 x 6 H2O 190.0 mg CuCl2 x 2 H2O 2.0 mg NiCl2 x 6 H2O 24.0 mg Na2MoO4 x 2 H2O 36.0 mg Distilled water 990.0 ml Vitamin solution: Biotin 2.0 mg Folic acid 2.0 mg Pyridoxine (B2) 0.1 mg Riboflavin (B1) 5.0 mg Pantotenoic acid 5.0 mg p-Aminobenzoic acid 5.0 mg Thiamine-HCl 5.0 mg Nicotinic acid 5.0 mg Cyanocobalamin (B12) 0.1 mg Lipoic (tioctoic) acid 5.0 mg Distilled water 1000.0 ml Dissolve the ingredients (except formate and sulfide) and flush medium with 100 % N2 for 30 min. Add the sodium sulfide, adjust the pH to 8.8-9.0, dispense in Hungate tubes under N2, and autoclave. Before use add sodium formate from a sterile, anaerobic stock solution. 236. DESULFOTOMACULUM CARBOXYDIVORANS MEDIUM Use medium 95 but lower the amount of yeast extract to 0.5 g/l. Na-acetate and Na-butyrate is re- placed by 2.2 g/l Na-pyruvate added from an anoxic stock solution (sterilized by filtration) after auto- claving. 237. DESULFUROCOCCUS FERMENTANS MEDIUM NH4Cl 0.33 g KH2PO4 0.33 g KCl 0.33 g CaCl2 x 2 H2O 0.44 g MgCl2 x 6 H2O 0.7 g NaCl 0.50 g Trace elements (see below) 1.0 ml Vitamin solution (see below) 10.0 ml Yeast extract 0.2 g Starch 5.0 g Resazurin 1.0 mg NaHCO3 0.8 g Na2S x 9 H2O 0.5 g Distilled water 1000.0 ml Trace element solution SL-10: HCl (25%; 7.7 M) 10.0 ml FeCl3 x 4 H2O 1.5 g ZnCl2 70.0 mg MnCl2 x 4 H2O 100.0 mg H3BO3 6.0 mg CoCl2 x 6 H2O 190.0 mg CuCl2 x 2 H2O 2.0 mg NiCl2 x 6 H2O 24.0 mg Na2MoO4 x 2 H2O 36.0 mg Distilled water 990.0 ml Vitamin solution: Biotin 2.0 mg Folic acid 2.0 mg Pyridoxine (B2) 0.1 mg Riboflavin (B1) 5.0 mg Pantotenoic acid 5.0 mg p-Aminobenzoic acid 5.0 mg Thiamine-HCl 5.0 mg Nicotinic acid 5.0 mg Cyanocobalamin (B12) 0.1 mg Lipoic (tioctoic) acid 5.0 mg Distilled water 1000.0 ml Dissolve ingredients (except vitamins, bicarbonate and sulfide), boil medium for 1 min, then cool to room temperature under 80% N2 and 20% CO2 gas atmosphere. Adjust pH to 6.2-6.4 and autoclave. After autoclaving add vitamins from an anoxic stock solution sterilized by filtration and bicarbonate from a sterile stock solution prepared under 80% N2 and 20% CO2 gas mixture. Prior to inoculation reduce medium by adding sulfide from a sterile, anoxic stock solution prepared under N2. 238. AQUASPIRILLUM MEDIUM (NH4)2SO4 1.0 g MgSO4 x 7 H2O 1.0 g CaCl2 x 6 H2O 30.0 mg Na2HPO4 10.0 mg Casamino acids 1.5 g Agar 0.5 g Distilled water 1000.0 ml pH 7.5 Add after autoclaving sterile solutions: Sodium succinate (10% solution) 10.0 ml Na2S2O3 x 5 H2O (10% solution) 1.0 ml Vitamin solution (see below) 5.0 ml Trace element solution (see below) 1.0 ml Vitamin solution: Biotin 200.0 mg Folic acid 20.0 mg Pyridoxine-HCl 100.0 mg Thiamine-HCl 50.0 mg Riboflavin 100.0 mg Nicotinic acid 50.0 mg DL-Pantothenic acid 50.0 mg Vitamin B12 1.0 mg p-Aminobenzoic acid 50.0 mg Distilled water 1000.0 ml Trace element solution SL-10: HCl (25%; 7.7 M) 10.0 ml FeCl3 x 4 H2O 1.5 g ZnCl2 70.0 mg MnCl2 x 4 H2O 100.0 mg H3BO3 6.0 mg CoCl2 x 6 H2O 190.0 mg CuCl2 x 2 H2O 2.0 mg NiCl2 x 6 H2O 24.0 mg Na2MoO4 x 2 H2O 36.0 mg Distilled water 990.0 ml 239. METHYLOPHAGA ALCALICA MEDIUM KH2PO4 1.0 g KNO3 1.0 g MgSO4 x 7 H2O 0.22 g NaCl 30.0 g Na2CO3 5.0 g Trace element solution (se below) 1.0 ml Distilled water 1000.0 ml Final pH 9.5 Trace elements solution: Ferric citrate 30.0 mg CaCl2 x 2 H2O 30.0 mg MgCl2 x 4 H2O 5.0 mg ZnSO4 x 7 H2O 5.0 mg CuSO4 x 5 H2O 0.5 g Distilled water 1000.0 ml Prepare the medium without the Na2CO3, which can be sterilised separately by autoclaving. 10 ml/l sterile methanol is added to the cooled medium. When preparing liquid media cool the mineral salts solution and Na2CO3 to room temperature before mixing. When preparing agar add 2.0 % agar to the mineral salts solution and autoclave. Cool the Na2CO3 stock solution and agar to 50-55°C before mix- ing. 240. METHYLOTHERMUS THERMALIS MEDIUM KNO3 0.25 g NH4Cl 0.25 g KH2PO4 0.13 g Na2HPO4.x 12H2O 0.358 g MgSO4.x 7H2O 0.4 g CaCl2 0.1 g Agar 20.0 g Distilled water 1000.00 ml pH 6*8. The gas phase methane/air mixture (4:1) 241. OCEANITHERMUS PROFUNDUS MEDIUM NH4Cl 0.33 g MgCl2 x 6 H2O 0.33 g CaCl2 x 2 H2O 0.33 g KCl 0.33 g KNO3 0.33 g NaCl 30.00 g HEPES 2.38 g Yeast extract 0.2 g Tryptone 1.0 g Sucrose 2.0 g Vitamin solution (see below) 1.0 ml Trace elements (see below) 1.0 ml Distilled water 1000.0 ml Vitamin solution: Biotin 2.0 mg Folic acid 2.0 mg Pyridoxine (B2) 0.1 mg Riboflavin (B1) 5.0 mg Pantotenoic acid 5.0 mg p-Aminobenzoic acid 5.0 mg Thiamine-HCl 5.0 mg Nicotinic acid 5.0 mg Cyanocobalamin (B12) 0.1 mg Lipoic (tioctoic) acid 5.0 mg Distilled water 1000.0 ml Trace element solution: Nitrilotriacetic acid 12.8 mg FeSO4 x 7 H2O 0.1 mg MnCl2 x 6 H2O 0.1 mg CoCl2 x 2 H2O 0.17 mg CaCl2 x 2 H2O 0.1 mg ZnCl2 0.1 mg CuCl2 0.02 mg H3BO3 0.01 mg Na2MoO4 x 2 H2O 0.01 mg NaCl 1.0 mg Na2SeO4 0.017 mg Distilled water 1000.0 ml Prepare the medium anaerobically, under nitrogen, 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 so- lutions should be autoclaved, while the vitamin solution is sterile filtered. 242. PSYCHROBACTER MEDIUM Peptone 5.0 g Yeast extract 1.0 g Sea salts 17.0 g Distilled water 1000.0 ml pH 7.2 243. ROSEICYCLUS MEDIUM KH2PO4 0.3 g MgSO4 2.0 g NH4Cl 0.3 g KCl 0.3 g CaCl2 x 2H2O 0.05 g Na2SO4 15.0 g NaHCO3 0.5 g Na-acetate 1.0 g Na-malate 1.0 g Yeast extract 1.0 g Peptone 0.5 g Agar 20.0 g/l Distilled water 1000.0 ml pH 7.8-8.0. 244. SULFOBACILLUS MEDIUM Solution A: (NH4)2SO4 3.0 g KCl 0.1 g K2HPO4 0.5 g MgSO4 x 7 H2O 0.5 g Ca(NO3)2 0.01 g Distilled water 700.0 ml Adjust pH to 2.0-2.2 with sulfuric acid. Solution B: FeSO4 x 7 H2O 44.2 g Distilled water 300.0 ml H2SO4, 10 N 1.0 ml Solution C: Yeast extract (1% w/v in water) 20.0 ml After autoclaving, combine the three solutions. Medium pH 1.9-2.4. 245. THERMINCOLA MEDIUM NH4Cl 1.0 g MgCl2 x 6 H2O 0.33 g CaCl2 x 2 H2O 0.1 g KCl 0.33 g KH2PO4 0.5 g Resazurin 0.5 mg Wolfe's mineral elixir (see below) 1.0 ml Vitamin solution (see below) 20.0 ml NaHCO3 0.5 g Na2CO3 0.5 g Na-acetate 0.2 g Yeast extract 0.2 g Na2S x 9 H2O 1.0 g Distilled water 1000.0 ml Wolfe's mineral elixir: MgSO4 x 7 H2O 30.0 g MnSO4 x H2O 5.0 g NaCl 10.0 g FeSO4 x 7 H2O 1.0 g CoCl2 x 6 H2O 1.8 g CaCl2 x 2 H2O 1.0 g ZnSO4 x 7 H2O 1.8 g CuSO4 x 5 H2O 0.1 g KAl(SO4)2 x 12 H2O 0.18 g H3BO3 0.10 g Na2MoO4 x 2 H2O 0.1 g (NH4)2Ni(SO4)2 x 6 H2O 2.8 g Na2WO4 x 2 H2O 0.1 g Na2SeO4 0.1 g Distilled water 1000.0 ml Vitamin solution: Biotin 2.0 mg Folic acid 2.0 mg Pyridoxine (B2) 0.1 mg Riboflavin (B1) 5.0 mg Pantotenoic acid 5.0 mg p-Aminobenzoic acid 5.0 mg Thiamine-HCl 5.0 mg Nicotinic acid 5.0 mg Cyanocobalamin (B12) 0.1 mg Lipoic (tioctoic) acid 5.0 mg Distilled water 1000.0 ml First adjust pH to 1.0 with diluted H2SO4, then add and dissolve the salts. Dissolve ingredients except carbonates, yeast extract and sulfide, boil medium for 1 min., then cool to room temperature under N2 gas atmosphere. Add carbonates and sulfide to the medium, dispense under CO gas atmosphere in cul- ture vessels (e.g., 10 ml medium in 50 ml serum bottles) and autoclave. Prior to inoculation add yeast extract from a sterile, anoxic stock solution prepared under N2 and adjust pH of final medium to 8.0 with a sterile, anoxic solution of 1 N HCl. 246. VULCANITHERMUS MEDIUM NH4Cl 0.33 g MgCl2 x 6 H2O 0.33 g CaCl2 x 2 H2O 0.33 g KCl 0.33 g KNO3 0.33 g NaCl 25.0 g PIPES 3.6 g Yeast extract 0.5 g Tryptone 1.0 g Sucrose 1.0 g Vitamin (see below) 1.0 ml Trace elements (see below) 1.0 ml Distilled water 1000.0 ml Vitamin solution: Biotin 2.0 mg Folic acid 2.0 mg Pyridoxine (B2) 0.1 mg Riboflavin (B1) 5.0 mg Pantotenoic acid 5.0 mg p-Aminobenzoic acid 5.0 mg Thiamine-HCl 5.0 mg Nicotinic acid 5.0 mg Cyanocobalamin (B12) 0.1 mg Lipoic (tioctoic) acid 5.0 mg Distilled water 1000.0 ml Trace element solution: Nitrilotriacetic acid 12.8 mg FeSO4 x 7 H2O 0.1 mg MnCl2 x 6 H2O 0.1 mg CoCl2 x 2 H2O 0.17 mg CaCl2 x 2 H2O 0.1 mg ZnCl2 0.1 mg CuCl2 0.02 mg H3BO3 0.01 mg Na2MoO4 x 2 H2O 0.01 mg NaCl 1.0 mg Na2SeO4 0.017 mg Distilled water 1000.0 ml Prepare the medium anaerobically, under nitrogen, omitting the CaCl2, MgCl2, KNO3, tryptone, yeast extract, vitamins and sucrose. The pH should be 6.8. Dispense the medium into vessels suitable for an- aerobic 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 so- lutions should be autoclaved, while the vitamin solution is sterile filtered. 247. RHODOBLASTUS MEDIUM Yeast extract 0.1 g Na2-succinate 1.0 g KH2PO4 0.5 g MgSO4 x 7 H2O 0.4 g NaCl 0.4 g NH4Cl 0.4 g CaCl2 x 2 H2O 0.05 g Trace element solution (see below) 1.0 ml Distilled water 1000.0 ml Adjust pH to 5.7 Trace element solution SL-6: ZnSO4 x 7 H2O 0.1 g MnCl2 x 4 H2O 0.03 g H3BO3 0.3 g CoCl2 x 6 H2O 0.2 g CuCl2 x 2 H2O 0.01 g NiCl2 x 6 H2O 0.02 g Na2MoO4 x 2 H2O 0.03 g Distilled water 1000.0 ml 248. PFENNIG'S MEDIUM KH2PO4 0.34 g NH4Cl 0.34 g MgSO4 x 7 H2O 0.5 g CaCl2 x 2 H2O 0.05 g KCl 0.34 g Trace element solution SLA (see below) 1 ml Cyanocobalamin (B12) 20 µg NaHCO3 1.5 g Na2S x 7-9 H2O 0.4 g Na2S2O3 x 5 H2O 0.5 g NaCl 15.0 g MgCl2 x 6 H2O 2.5 g. The pH was adjusted to 7.5. Trace element solution SLA: FeCl2 x 4 H2O 1.8 mg CoCl2 x 6 H2O 250 mg NiCl2 x 6 H2O 10 mg CuCl2 x 2 H2O 10 mg MnCl2 x 4 H2O 70 mg ZnCl2 100 mg H3BO3 500 mg Na2MoO4 x 2 H2O 30 mg Na2SeO3 x 5 H2O 10 mg Distilled water 1000 ml