Soil collection, plant bioassays and endosphere sampling

For the soil collection and plant bioassays you could check any paper from Raaijmakers JM.

For the endosphere sampling, find attached two manuscripts and a more detailed protocol. 
 

Isolation of bacterial cells from sugarbeet plants through Nycodenz density centrifugation (VJC).

1/ Prepare a solution of Nycodenz at 0.5 g/mL (ND50) in the suspension buffer (Mgsol or Phosphate buffer…):

Add the powder progressively to 75% of the desired final volume, stir with heating to 50-60C (takes a while), adjust the volume once dissolved and autoclave. Before autoclaving the ND dilutions can be prepared:

ND35= 35 ml ND50 + 15 ml Mgsol ND20= 20 ml ND50 + 30 ml Mgsol ND10= 10 ml ND50 + 40 ml Mgsol

2/ Prepare the plant material:

• Weigh the root material before sterilization

• Harvest root systems with adhering soil in the suspension buffer (Mgsol)

• Shake the roots rinsed with 10 ml of Mgsol (repeat until the Mgsol is transparent, so most of the rhizosphere is gone, for sugar beet 5 times was enough depending of the moisture of the roots)

• Wash the roots with 10 ml of Mgsol + Tween20 (0.01%)
• Rinse with 10 ml of Mgsol.
• Wash the roots with 10 ml of bleach at 1% during 2 min under slow shaking (depends of type of roots, stage of growth…)
• Wash the roots with sterile water until the bleach smell is gone (for sugarbeet was 5 times)
• Controls of sterilization-plate 100ul of the last rinsed solution and roll the root in a rich media.

3/ Centrifugation:

• Weigh the root material after sterilization
• Mix with Mgsol (20-100 ml depending on the size of the roots)
• Macerate the root tissues using a special blender, the maceration should be done in ice to avoid the warming of the sample.
• Filter the sample using miracloth filter paper (better to use double layer)

• Centrifuge at 9500 rpm during 10-20 min (this has to be a big centrifuge because you will end up with 50-100 ml of sample), keep the pellet… contain bacteria, fungi, plant material and soil particles…

• Suspend in 3.5 ml of ND50 by pipetting (has to be in special glass tubes, depends also of the centrifuge)
• Overlay carefully (very carefully, don’t mix the layers) with 3 ml of ND35, 2ml ND20 and 2 ml ND10.

• Equilibrate the tubes
• Centrifuge 45 min at 8500 rpm
• After centrifugation the bacterial cells form a whitish ring floating between ND50 and ND35.

• Harvest the bacterial layer with a Pasteur pipet and pour by 0.5 mL aliquots in 1.5 mL Ep’s.

• To rinse the Nycodenz add 1 mL suspension buffer in each tube, mix well and spin 5 min at max speed in a bench centrifuge to pellet the cells.

• Remove supernatant, pool pellets and rinse again with 1 Ml

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