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APPENDIX 3

MEDIA AND STAINING SOLUTIONS

Yeast Mannitol Broth (YMB) Constituents: Mannitol K2HPO4 MgSO4.7H2O NaCl Yeast Extract Distilled Water 10.0 g* 0.5 g 0.2 g 0.1 g 0.5 g 1.0 liter

*This amount has been used traditionally, however more recent findings (H. Keyser, unpublished) show that 1 g l-1 is sufficient for most rhizobia.

Preparations: - Add mannitol and salts to 1 l distilled water - Dissolve under continuous stirring - Adjust pH to 6.8 with 0.1 N NaOH - Autoclave at 121°C for 15 min.

Yeast Mannitol Agar (YMA)

Constituents:

Add 10 ml BCP stock solution* to achieve a BCP concentration of 100μg ml l-1 (Prepare BCP stock solution by dissolving 1 g BCP in 100 ml ethanol) .Dissolve glucose and peptone in 1 liter distilled water .After autoclaving. shake flask to ensure even mixing of melted agar with medium.Add agar.Prepare YMB . . shake to suspend evenly.Autoclave at 121°C for 15 minutes 5 g 10 g 15 g Fermentor Broth (Burton. 1967) Constituents per liter: Mannitol 2. autoclave.Yeast Mannitol Broth Agar 1 liter 15 g Preparation: . Glucose Peptone Agar Ingredients per liter: Glucose Peptone Agar Preparation: .Add agar and suspend evenly .0 g .

54 g 0.H2O) Yeast Extract Ammonium phosphate [(NH4)2HPO4] Water 10.0 ml per liter of medium gives: boron 0.6H2O) Lactic acid (88%) Distilled water 2. iron 100 μg and cobalt 0. molybdenum 1.2 0.00 g 0.5 0. manganese 0.7H2O) Sodium chloride (NaCl) Calcium carbonate (CaCO3) Calcium sulphate (CaSO4.7H2O) Zinc sulphate (ZnSO4.0 μg. zinc 0. .5 μg.05 μg.36 g 5.0 0.004 g 580 420 ml ml *Addition of 1.21 g 4.0005 μg per liter (or parts per million).Sucrose Tripotassium phosphate (K3PO4) Monopotassium phosphate (KH2PO4) Magnesium sulphate (MgSO4.7H2O) Sodium molybdate (Na2MoO4) Ferric chloride (FeCl3.04 g 0.06 g 0.2 g g g g 0.6H2O) Cobalt sulphate (CoSO4.4 0.5 μg.1 1000 g g ml Micronutrient – Stock Solution (Burton) Constituents: Boric Acid (H3BO3) Manganese sulphate (MnSO4.2 g 0.78 g 1.

sucrose.Autoclave at 121°C for 15 min. Dispense 2 ml quantities via sterile Seitz or Millipore filter into small bottles (dispense 50 and discard remainder of .Add 1 ml of micronutrient stock solution . a. b. yeast extract and salts in 1 liter distilled water . Dissolve 0. Bergersen’s defined medium for preparation of Rhizobium for antiserum production Constituents: K2HPO4 KH2PO4 MgSO4·7H2O CaCl2·6H2O FeCl3·6H2O Sodium glutamate Mannitol Agar Water 1.00 g 1 liter Dispense known volumes into bottles.025 g biotin in 1 liter distilled water.25 g 0. autoclave and add 1 ml of Biotin-thiamine solution per liter.10 g 10.00 g 15.01 g 1.1 g 0.Dissolve mannitol.0 g 0.0 g 1..1 g thiamine and 0.

solution). DYES INCORPORATED IN MEDIA Bromthymol Blue (BTB) Stock solution: 0. Final concentration: 100 ppm. Congo Red (CR) Stock solution: 0.5 g/100 ml ethanol Add 5 ml stock/liter YMA Final concentration of BTB: 25 ppm. c. Store in freezer and dispense aseptically into autoclaved medium at 1 ml/liter.25 g/100 ml Add 10 ml stock/liter YMA Final concentration of CR: 25 ppm. Bromcresol Purple (BCP) Stock solution: 1 g/100 ml ethanol Add 10 ml stock per liter peptone glucose agar. Brilliant Green (BG) .

Stock solution: 125 mg/100 ml ethanol Add 1 ml stock to 1 liter of YMA before autoclaving Final concentration of BG: 1. YMA with antibiotics Streptomycin (str) Stock solution: 400 mg str/100 ml water (4 mg str/ml) Add 5 ml str stock/500 ml YMA to make plates containing 40 g str/ml. Add 10 ml spc stock to 500 ml YMA for plates with 500 mg spc/ml.25 g spc/50 ml water (250 mg spc/ml) Add 5 ml spc stock to 500 ml YMA for plates with 250 µg spc/ml. Autoclave YMA together with magnetic stirring bar in an Erlenmeyer flask.25 ppm. 10 ml str stock/500 ml YMA for plates containing 80 µg str/ml. . Add filter sterilized antibiotics after the Mix well and pour after bubbles agar has cooled below 80oC. Spectinomycin (spc) Stock solution: 1.

Zn.005 g traces 1000 ml PH after autoclaving is 6.0 g 1. 0.5 Sterilize at 121oC for 20 minutes.1 g 0. Fahraeus C. 0.1 g 0.005% Mo.and N-free Madium* CaCl2 MgSO4·7H2O KH2PO4 Na2HPO4·2H2O Ferric citrate *Mn. . Cu.15 g 0. B.005% Zn.1 g 1.2 g 0. and 0.002% Cu. Seedling Agar (Jensen.5% B.resulting from mixing have dispersed.0 g 0.2 g 0.2 g 0. Mo Distilled water 0. 1942)* CaHPO4 K2HPO4 MgSO2·7H2O NaCl FeCl3 Water Agar Microelementsa 1.12 g 0.05% Mn.0 ml (Gibson 1963)* a From stock containing: 0.0 liter 15. 0.

a 5-10 cm long agar face is presented for seedling growth.*Taken from Vincent 1970 Seedling Agar Slants Autoclave seedling agar at 121oC for 15 minutes and dispense equal volumes into tubes (tube size depends on plant species). An appropriate amount of molten agar is dispensed so that after solidifying in inclined tubes. 1970) Solution I: Crystal violet solution 10 g 4 g 100 ml 400 ml Crystal violet Ammonium oxalate Ethanol Water (distilled) Solution II: Iodine Iodine solution 1 g 2 g 25 ml 100 ml Potassium iodide Ethanol Water (distilled) Solution III: 95% Ethanol . SOLUTIONS FOR GRAM STAIN (Vincent.

straw-colored liquid. bottle and autoclave for 30 to 40 minutes at 121°C.6 to 6. Preparation of Yeast Water Fresh starch-free cakes of yeast are preferred in making yeastwater. Siphon off the clear. Suspend 100 g of yeast in 1. adjust the liquid to pH 6.5% Safranin in ethanol Water (distilled) Carbol Fuchsin Stain Basic fuchsin Ethanol 5% phenol solution 1 g 10 ml 100 ml The fuchsin stain should be diluted 5-10 times with distilled water before use. the yeast water may be stored at room temperature.000 ml of water and boil slowly or steam for 3 to 4 hours. Following sterilization.8 with sodium hydroxide. . replacing the water lost regularly. Allow the cooled suspension to stand until yeast cells have settled (usually 10 to 12 hours) to the bottom.Solution IV: Counterstain 10 ml 100 ml 2.

When these are available. bottle. dry yeast is equivalent to about 2. autoclave.Dried yeast may also be used in making yeast-water. For rhizobia media. regularly. about 0. 40 g of dry yeast in one liter of water. Boil slowly for 2 hours replacing the lost water Allow to cool and centrifuge at 5000 rpm. Yeast extract powders prepared by spray-drying aqueous autolyzed yeast preparations are available in many countries. use Nitrogen sources can also be prepared from other grain legume seeds in the same way. Preparation of Soybean Water Grind 100 g soybean seeds to a course flour and place in 1000 ml of water. and usually satisfactory. decant. Remove the supernatant. One and sterilize in the same way as described for fresh yeast. 100 ml per liter. .5 g per liter of the dried preparation is used to replace yeast-water. hundred ml of yeast-water should contain about 75 mg of nitrogen. and store. One kg of Suspend Boil. Foaming can be controlled by adding a small amount of sterile white mineral oil or silicone emulsion. Dry preparations are convenient The media containing yeast may foam excessively when aerated vigorously in fermentor vessels.5 kg of wet yeast.

2 .5 .04 246.1 136. N-free Nutrient Solution (Broughton and Dillworth.001 0.0 0.02 61.0002 0. KNO3 (0. then dilute to 10 liters. .98 g/l 294.0002 For each 10 liters of full strength culture solution.09 355. take 5.05%) is added giving an N concentration of 70 ppm.3 87.6-6.8.56 249.7 123.69 281.002 0.288 0.12 241.5 0.0 ml each of solutions 1 to 4. Use 1 N NaOH to adjust the For plus N control treatments.056 0.247 0.Table A.03 136.338 0. Stock Solutions 1 2 3 Element Ca P Fe Mg K Mn 4 B Zn Cu Co Mo M 1000 500 10 250 250 1 2 .02 0.84 287.004 0.0 liters of water.1.5 174.1 6. pH to 6.0 0. 1970).06 169.0 1.1 .1 Form CaCl2•2H2O KH2PO4 Fe-citrate MgSo4•7H2O K2SO4 MnSO4•H2O H3BO3 ZnSO4•7H2O CuSO4•5H2O CoSO4•7H2O Na2MoO2•2H2O MW 147. then add to 5.100 0.0004 0.048 M 2.5 0.