Laboratory of Ann Hirsch


Sweetclover Methods

Standard Media Used In Nodulation Studies

JENSEN'S AGAR SLANTS

Recipe for one liter:

TY FOR BACTERIA
(per liter)

CaHPO4....................................

1.0 g

Tryptone................................

5.0 g

K2HPO4....................................
(or 0.26 g K2HPO4.3H2O)

0.2 g

Yeast Extract.........................

3.0 g

MgSO4.7H2O.........................

0.2 g

CaCl2....................................

0.5 g

NaCl.........................................

0.2 g

pH to 7.2

FeCl3........................................

0.1 g

Hoagland's micronutrients...........

5 mL

YMB FOR RHIZOBIA
(per liter)

Sucrose..................................

0.5 g

pH to 6.8

K2HPO4.................................

5.0 g

MgSO4...................................

0.2 g

Add 12 g agar

NaCl......................................

0.1 g

Mannitol.................................

10.0 g

There will be a precipitate. Stir continuously while dispensing.

Yeast Extract..........................

0.4 g

pH to 7.0; add ~3 drops 2N HCl

(RDM) Rhizobium Defined Medium (a minimal medium for agrobacteria and rhizobia)
Reference: Vincent, 1970, IBP Handbook No. 15, Blackwell

Materials:

RDM A STOCK = 10 X PER LITER
6 g KNO3
1 g CaCl2.2H2O
2.5 g MgSO4.7H2O
0.1 g FeCl3.6H2O
RDM B STOCK = 10 X PER LITER
10 g K2HPO4
10 g KH2PO4

BIOTIN STOCK = 0.25 mg/mL
THIAMINE STOCK = 10 mg/mL

Methods:

To make RDM: add the following to 700 mL of distilled water
5 g sucrose, dissolved
100 mL RDM A
100 mL RDM B
4 mL Biotin Stock
1 mL Thiamine Stock
Then bring up the final solution to 1000 mL and add 15 g of agar if you are making plates.

Complete
FeEDTA

Complete
FeCl2

Ca

-S

-Mg

-K

-N

-P

-Fe

-Micro-
nutrients

(per liter)

mL

mL

mL

mL

mL

mL

mL

mL

mL

mL

1M Ca(NO3)2

5

5

-

5

5

5

-

5

5

5

1M KNO3

5

5

5

5

5

-

-

5

5

5

1M MgSO4

2

2

2

-

-

2

2

2

2

2

1M KH2PO4

1

1

1

1

1

-

1

-

1

1

FeEDTA stock

1

-

1

1

1

1

1

1

-

1

FeCl2 stock

-

1

-

-

-

-

-

-

-

-

Micronutrients stock

1

1

1

1

1

1

1

1

1

-

1M NaNO3

-

-

10

-

-

5

-

-

-

-

1M MgCl2

-

-

-

2

-

-

-

-

-

-

1M Na2SO4

-

-

-

-

2

-

-

-

-

-

1M NaH2PO4

-

-

-

-

-

1

-

-

-

-

1M CaCl2

-

-

-

-

-

-

5

-

-

-

1M KCl

-

-

-

-

-

-

5

1

-

-

pH at 5.7-5.8

FeEDTA is a solution of an iron complex of ethylenediaminetetraacetic  acid. Each mL of the stock solution contains 5 mg of Fe. Each mL of the  FeCl3 stock solution also contains 5 mg of Fe. To make FeEDTA,  dissolve 7.45 g Na2EDTA in 1 L H2O, then add 5.57  g FeSO4.7H2O.

The micronutrients stock solution contains 2.86 g of H2 BO3 (boric acid), 1.81 g of MnCl2.4H20 (manganese  chloride), 0.11 g of ZnCl2 (zinc chloride), 0.05 g of CuCl2.2H2O  (copper chloride), and 0.025 g of Na2 MoO4.2H2O  (sodium molybdate) per liter.