Day 0
Plate pouring:
Autoclave the B and D medium and pour sterile glass plates (18 cm of diameter). Let them dry under bacterial hood and store them in the growth chamber (25°C, 80% humidity, dark).

Day 1
Start Bradyrhizobium japonicum culture:

In 200 ml of HM medium + chloramphenicol (20 μg/ml final concentration), 2 ml of a bacteria pre-culture are added.

The flask is shaking during 3 days at 30°C, 180 rpm.

Glycine max (Williams 82) seeds sterilization and sowing:
20% bleach 10’ (2 times)
Wash 3 times with sterile water
0.01M HCl (1 time)
Wash 3 times with sterile water
Let the seeds dry few minutes before sowing. 

The seeds are germinated on agar B and D medium. Each seed is separated from its
neighbors by around 2 centimeters. The plates are stored for 3 days in the growth
chamber (25°C, 80% humidity, dark). After 3 days germination occurs. Roots are around
1 to 2 cm long. 

Day 4
Bacterial inoculation:
After 3 days, the bacteria are washed in sterile water three times by centrifugation (4000 rpm, 10 min, room temperature) with gentle resuspension. After the last washing, the O.D.600nm is measured and sterile water is added to obtain an O.D.600nm of 0.8. The bacteria are sprayed on the 3 days old seedlings. As control, other plates are sprayed with water. After spray, the plates go back in the growth chamber for incubation (25°C, 80% humidity, dark) before to be harvested. We use a perfume sprayer for inoculation but any type of sprayer should work as long as it uniformly wets the plants.

Root Hair isolation:
The roots are cut from the seedlings and dropped directly into a liquid nitrogen container. The liquid nitrogen is stirred using a glass rod for 20 minutes. The stirring allows the separation of the roots hair cells from the roots. The stirring must not be too strong to avoid breaking the root tips. To isolate the root hairs, the liquid nitrogen is poured in a beaker through a sieve to retain roots. We use a metal strainer. The type you can buy at any store that sells kitchen supplies. To harvest the maximum amount of root hair, the liquid nitrogen container is rinse with liquid nitrogen 5 to7 times. Usually, from 1000 seedlings, 0.5 to 1 gram of root hair cells are harvested.


B and D medium:
Solution A (CaCl2 2 M) (per 100 ml)
22.2 g CaCl2
Solution B1 (KH2PO4 1 M) (per 100 ml)
13.6 g KH2PO4
Solution B2 (K2HPO4 1 M) (per 100 ml)
17.4 g K2HPO4
Solution C (Fe-citrate 0.02 M) (per 100 ml)
0.495 g Fe-citrate
Solution D (per 100 ml)
12.33 g MgSO4, 7 H2O (0.5 M)
8.7 g K2SO4 (0.5 M)
0.0338 g MnSO4.H2O (0.002 M)
0.0247 g H3BO3 (0.004 M)
0.0288 g ZnSO4.7H2O (0.001 M)
0.01 g CuSO4.5H2O (0.004 M)
0.0056 g CoSO4.7H2O (0.0002 M)
0.0048 g Na2MoO4.2H2O (0.0002 M)

Autoclave the 5 solutions.
In a 6L flask, add 2.25 ml of each solution (A, B1, B2, C and D) to 3 liters of water.
Complete the volume to 4.5 liters with water. Add 60 g of agar for 4.5 liters of medium. 

HM medium (per liter):
0.125 g Na2HPO4
0.25 g Na2SO4
0.32 g NH4Cl
0.18 g MgSO4.7H2O
0.004 g FeCl3
0.013 g CaCl2.2H2O
1.3 g HEPES
1.1 g MES
0.25 g Yeast extract
1 g D-Arabinose or L-Arabinose






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