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*This amount has been used traditionally, however more recent findings (H. Keyser, unpublished) show that 1 g l-1 is sufficient for most rhizobia.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 121C for 15 min.

Yeast Mannitol Agar (YMA)

Constituents:

Yeast Mannitol Broth Agar

1 liter 15 g

Preparation: - Prepare YMB - Add agar, shake to suspend evenly, autoclave. - After autoclaving, shake flask to ensure even mixing of melted agar with medium.

Glucose Peptone Agar

Ingredients per liter: Glucose Peptone Agar Preparation: - Dissolve glucose and peptone in 1 liter distilled water - Add 10 ml BCP stock solution* to achieve a BCP concentration of 100g ml l-1 (Prepare BCP stock solution by dissolving 1 g BCP in 100 ml ethanol) - Add agar and suspend evenly - Autoclave at 121C for 15 minutes 5 g 10 g 15 g

Fermentor Broth (Burton, 1967) Constituents per liter: Mannitol 2.0 g

Sucrose Tripotassium phosphate (K3PO4) Monopotassium phosphate (KH2PO4) Magnesium sulphate (MgSO4.7H2O) Sodium chloride (NaCl) Calcium carbonate (CaCO3) Calcium sulphate (CaSO4.H2O) Yeast Extract Ammonium phosphate [(NH4)2HPO4] Water

10.0 0.2 0.4 0.2

g g g g

0.06 g 0.2 g

0.04 g 0.5 0.1 1000 g g ml

Micronutrient Stock Solution (Burton)

Constituents: Boric Acid (H3BO3) Manganese sulphate (MnSO4.7H2O) Zinc sulphate (ZnSO4.7H2O) Sodium molybdate (Na2MoO4) Ferric chloride (FeCl3.6H2O) Cobalt sulphate (CoSO4.6H2O) Lactic acid (88%) Distilled water 2.78 g 1.54 g 0.21 g 4.36 g 5.00 g 0.004 g 580 420 ml ml

*Addition of 1.0 ml per liter of medium gives: boron 0.5 g; manganese 0.5 g; zinc 0.05 g; molybdenum 1.0 g; iron 100 g and cobalt 0.0005 g per liter (or parts per million).

- Dissolve mannitol, sucrose, yeast extract and salts in 1 liter distilled water - Add 1 ml of micronutrient stock solution - Autoclave at 121C for 15 min.

Bergersens defined medium for preparation of Rhizobium for antiserum production

Constituents: K2HPO4 KH2PO4 MgSO47H2O CaCl26H2O FeCl36H2O Sodium glutamate Mannitol Agar Water 1.0 g 1.0 g 0.25 g 0.1 g 0.01 g 1.10 g 10.00 g 15.00 g 1 liter

Dispense known volumes into bottles, autoclave and add 1 ml of Biotin-thiamine solution per liter.

a. Dissolve 0.1 g thiamine and 0.025 g biotin in 1 liter distilled water.

b. Dispense 2 ml quantities via sterile Seitz or Millipore filter into small bottles (dispense 50 and discard remainder of

solution).

c. Store in freezer and dispense aseptically into autoclaved medium at 1 ml/liter.

DYES INCORPORATED IN MEDIA

Bromthymol Blue (BTB)

Stock solution: 0.5 g/100 ml ethanol Add 5 ml stock/liter YMA Final concentration of BTB: 25 ppm.

Congo Red (CR)

Stock solution: 0.25 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. Final concentration: 100 ppm.

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: 1.25 ppm.

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. 10 ml str stock/500 ml YMA for plates containing 80 g str/ml.

Spectinomycin (spc)

Stock solution: 1.25 g spc/50 ml water (250 mg spc/ml) Add 5 ml spc stock to 500 ml YMA for plates with 250 g spc/ml. Add 10 ml spc stock to 500 ml YMA for plates with 500 mg spc/ml.

Autoclave YMA together with magnetic stirring bar in an Erlenmeyer flask. Add filter sterilized antibiotics after the Mix well and pour after bubbles

agar has cooled below 80oC.

resulting from mixing have dispersed. Fahraeus C- and N-free Madium*

CaCl2 MgSO47H2O KH2PO4 Na2HPO42H2O Ferric citrate *Mn, Cu, Zn, B, Mo Distilled water

0.1 g 0.12 g 0.1 g 0.15 g 0.005 g traces 1000 ml

PH after autoclaving is 6.5 Sterilize at 121oC for 20 minutes.

Seedling Agar (Jensen, 1942)*

CaHPO4 K2HPO4 MgSO27H2O NaCl FeCl3 Water Agar Microelementsa

1.0 g 0.2 g 0.2 g 0.2 g 0.1 g 1.0 liter 15.0 g 1.0 ml (Gibson 1963)*

From stock containing: 0.5% B; 0.05% Mn; 0.005% Zn; 0.005% Mo;

and 0.002% Cu.

*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, a 5-10 cm long agar face is presented for seedling growth.

SOLUTIONS FOR GRAM STAIN (Vincent, 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

Solution IV:

Counterstain 10 ml 100 ml

2.5% 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.

Preparation of Yeast Water

Fresh starch-free cakes of yeast are preferred in making yeastwater. Suspend 100 g of yeast in 1,000 ml of water and boil

slowly or steam for 3 to 4 hours, replacing the water lost regularly. Allow the cooled suspension to stand until yeast

cells have settled (usually 10 to 12 hours) to the bottom. Siphon off the clear, straw-colored liquid; adjust the liquid to pH 6.6 to 6.8 with sodium hydroxide; bottle and autoclave for 30 to 40 minutes at 121C. Following sterilization, the yeast water

may be stored at room temperature.

Dried yeast may also be used in making yeast-water. dry yeast is equivalent to about 2.5 kg of wet yeast. 40 g of dry yeast in one liter of water.

One kg of Suspend

Boil, decant, bottle, One

and sterilize in the same way as described for fresh yeast.

hundred ml of yeast-water should contain about 75 mg of nitrogen.

Yeast extract powders prepared by spray-drying aqueous autolyzed yeast preparations are available in many countries. When these

are available, about 0.5 g per liter of the dried preparation is used to replace yeast-water. and usually satisfactory. Dry preparations are convenient

The media containing yeast may foam excessively when aerated vigorously in fermentor vessels. Foaming can be controlled by

adding a small amount of sterile white mineral oil or silicone emulsion.

Preparation of Soybean Water

Grind 100 g soybean seeds to a course flour and place in 1000 ml of water. regularly. Boil slowly for 2 hours replacing the lost water Allow to cool and centrifuge at 5000 rpm. Remove

the supernatant, autoclave, and store. 100 ml per liter.

For rhizobia media, use

Nitrogen sources can also be prepared from

other grain legume seeds in the same way.

Table A.1. N-free Nutrient Solution (Broughton and Dillworth, 1970). 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 .5 .2 .1 .1 Form CaCl22H2O KH2PO4 Fe-citrate MgSo47H2O K2SO4 MnSO4H2O H3BO3 ZnSO47H2O CuSO45H2O CoSO47H2O Na2MoO22H2O MW 147.03 136.09 355.04 246.5 174.06 169.02 61.84 287.56 249.69 281.12 241.98 g/l 294.1 136.1 6.7 123.3 87.0 0.338 0.247 0.288 0.100 0.056 0.048 M 2.0 1.0 0.02 0.5 0.5 0.002 0.004 0.001 0.0004 0.0002 0.0002

For each 10 liters of full strength culture solution, take 5.0 ml each of solutions 1 to 4, then add to 5.0 liters of water, then dilute to 10 liters. pH to 6.6-6.8. Use 1 N NaOH to adjust the

For plus N control treatments, KNO3 (0.05%) is added giving an N

concentration of 70 ppm.