APPENDIX 3

MEDIA AND STAINING SOLUTIONS

Yeast Mannitol Broth (YMB)

Constituents:

Mannitol10.0 g*

K2HPO4 0.5 g

MgSO4.7H2O 0.2 g

NaCl 0.1 g

Yeast Extract 0.5 g

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*This amount has been used traditionally, however more recent findings (H. Keyser, unpublished) show that 1 g l-1 is sufficient for most rhizobia.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

Yeast Mannitol Broth 1 liter

Agar15 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 5 g

Peptone10 g

Agar15 g

Preparation:

- Dissolve glucose and peptone in 1 liter distilled water

- 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)

- Add agar and suspend evenly

- Autoclave at 121C for 15 minutes

Fermentor Broth (Burton, 1967)

Constituents per liter:

Mannitol 2.0 g

Sucrose 10.0 g

Tripotassium phosphate (K3PO4) 0.2 g

Monopotassium phosphate (KH2PO4) 0.4 g

Magnesium sulphate (MgSO4.7H2O) 0.2 g

Sodium chloride (NaCl) 0.06 g

Calcium carbonate (CaCO3) 0.2 g

Calcium sulphate (CaSO4.H2O) 0.04 g

Yeast Extract 0.5 g

Ammonium phosphate [(NH4)2HPO4] 0.1 g

Water 1000 ml

Micronutrient – Stock Solution (Burton)

Constituents:

Boric Acid (H3BO3) 2.78 g

Manganese sulphate (MnSO4.7H2O) 1.54 g

Zinc sulphate (ZnSO4.7H2O) 0.21 g

Sodium molybdate (Na2MoO4) 4.36 g

Ferric chloride (FeCl3.6H2O) 5.00 g

Cobalt sulphate (CoSO4.6H2O) 0.004 g

Lactic acid (88%) 580 ml

Distilled water 420 ml

*Addition of 1.0 ml per liter of medium gives: boron 0.5 μg; manganese 0.5 μg; zinc 0.05 μg; molybdenum 1.0 μg; iron 100 μg and cobalt 0.0005 μg 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 121C for 15 min.

Bergersen’s defined medium for preparation of Rhizobium for antiserum production

Constituents:

K2HPO41.0 g

KH2PO41.0 g

MgSO4·7H2O0.25 g

CaCl2·6H2O0.1 g

FeCl3·6H2O0.01 g

Sodium glutamate1.10 g

Mannitol 10.00 g

Agar 15.00 g

Water1 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 µg 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 µg 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 agar has cooled below 80oC. Mix well and pour after bubbles resulting from mixing have dispersed.

Fahraeus C- and N-free Madium*

CaCl20.1 g

MgSO4·7H2O0.12 g

KH2PO40.1 g

Na2HPO4·2H2O0.15 g

Ferric citrate0.005 g

*Mn, Cu, Zn, B, Motraces

Distilled water1000 ml

PH after autoclaving is 6.5

Sterilize at 121oC for 20 minutes.

Seedling Agar (Jensen, 1942)*

CaHPO41.0 g

K2HPO40.2 g

MgSO2·7H2O0.2 g

NaCl0.2 g

FeCl30.1 g

Water1.0 liter

Agar 15.0 g

Microelementsa1.0 ml (Gibson 1963)*

a 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

Crystal violet10 g

Ammonium oxalate 4 g

Ethanol 100 ml

Water (distilled) 400 ml

Solution II: Iodine solution

Iodine 1 g

Potassium iodide 2 g

Ethanol25 ml

Water (distilled) 100 ml

Solution III: 95% Ethanol

Solution IV: Counterstain

2.5% Safranin in ethanol10 ml

Water (distilled) 100 ml

Carbol Fuchsin Stain

Basic fuchsin 1 g

Ethanol10 ml

5% phenol solution 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 yeast-water. 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 121C. Following sterilization, the yeast water may be stored at room temperature.

Dried yeast may also be used in making yeast-water. One kg of dry yeast is equivalent to about 2.5 kg of wet yeast. Suspend 40 g of dry yeast in one liter of water. Boil, decant, bottle, and sterilize in the same way as described for fresh yeast. One 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. Dry preparations are convenient and usually satisfactory.

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. Boil slowly for 2 hours replacing the lost water regularly. Allow to cool and centrifuge at 5000 rpm. Remove the supernatant, autoclave, and store. For rhizobia media, use 100 ml per liter. 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 / Element / M / Form / MW / g/l / M
1 / Ca / 1000 / CaCl2•2H2O / 147.03 / 294.1 / 2.0
2 / P / 500 / KH2PO4 / 136.09 / 136.1 / 1.0
3 / Fe / 10 / Fe-citrate / 355.04 / 6.7 / 0.02
Mg / 250 / MgSo4•7H2O / 246.5 / 123.3 / 0.5
K / 250 / K2SO4 / 174.06 / 87.0 / 0.5
Mn / 1 / MnSO4•H2O / 169.02 / 0.338 / 0.002
4 / B / 2 / H3BO3 / 61.84 / 0.247 / 0.004
Zn / .5 / ZnSO4•7H2O / 287.56 / 0.288 / 0.001
Cu / .2 / CuSO4•5H2O / 249.69 / 0.100 / 0.0004
Co / .1 / CoSO4•7H2O / 281.12 / 0.056 / 0.0002
Mo / .1 / Na2MoO2•2H2O / 241.98 / 0.048 / 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. Use 1 N NaOH to adjust the pH to 6.6-6.8. For plus N control treatments, KNO3 (0.05%) is added giving an N concentration of 70 ppm.