APPENDIX 3
MEDIA AND STAINING SOLUTIONS
Yeast Mannitol Broth (YMB)
Constituents:
Mannitol
10.0 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findings (H. Keyser, unpublished) show that 1 g l-1 is sufficient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
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
5 g
Peptone
10 g
Agar
15 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
Calcium sulphate (CaSO4.H2O)
0.04 g
Yeast Extract
0.5
Ammonium phosphate [(NH4)2HPO4]
Water
g
g
0.1
1000
g
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:
K2HPO4
1.0 g
KH2PO4
1.0 g
MgSO4·7H2O
0.25 g
CaCl2·6H2O
0.1 g
FeCl3·6H2O
0.01 g
Sodium glutamate
1.10 g
Mannitol
10.00 g
Agar
15.00 g
Water
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 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*
CaCl2
0.1 g
MgSO4·7H2O
0.12 g
KH2PO4
0.1 g
Na2HPO4·2H2O
0.15 g
Ferric citrate
0.005 g
*Mn, Cu, Zn, B, Mo
traces
Distilled water
1000 ml
PH after autoclaving is 6.5
Sterilize at 121oC for 20 minutes.
Seedling Agar (Jensen, 1942)*
CaHPO4
1.0 g
K2HPO4
0.2 g
MgSO2·7H2O
0.2 g
NaCl
0.2 g
FeCl3
0.1 g
Water
1.0 liter
Agar
Microelementsa
a
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
Crystal violet
Ammonium oxalate
10 g
4 g
Ethanol
100 ml
Water (distilled)
400 ml
Solution II:
Iodine solution
Iodine
1 g
Potassium iodide
2 g
Ethanol
25 ml
Water (distilled)
Solution III:
95% Ethanol
100 ml
Solution IV:
Counterstain
2.5% Safranin in ethanol
Water (distilled)
10 ml
100 ml
Carbol Fuchsin Stain
Basic fuchsin
1 g
Ethanol
10 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 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 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.
g of dry yeast in one liter of water.
Suspend 40
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.
regularly.
Boil slowly for 2 hours replacing the lost water
Allow to cool and centrifuge at 5000 rpm.
supernatant, autoclave, and store.
ml per liter.
Remove the
For rhizobia media, use 100
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
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
4
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.