Sampling

Sampling

Soil is sampled for two metagenomic communities from each of 72 unique sampling sites e.g 1 sampling site = 2 sampling locations reaching to a total 144 sampling locations.

Soil is inserted in 50 ml Falcon tubes, stored in dry ice containing coolers during the sampling event, stored at -20 0C at HCMR and shipped via courier to the US. Three Falcon tubes of soil at each sampling location are collected:

Tube 1: (DNA) for metagenomic analysis, Tube 2: (CHEM) for soil chemistry analysis, Tube 3: (HCMR) for storage at HCMR)

Leading to a total of 3 * 144 = 432 falcon tubes.

Additionally one plastic bag is used per location to store the plant specimen collected.

Metadata

Each team fills the following metadata form:

Sampling Team No:

*Sampling Site ID:

Team Members:

City (nearest town):

Plant type collected:

Land use: e.g agricultural, botanical garden, cul1vated, pris1ne, beach

Place name:

Environment feature (habitat): agricultural land, alpine, beach, botanical garden, boulder field, cave, crevice, cultivated habitat, desert, farm, field, forest (e.g., oak forest), gravel field, grassland, greenhouse, lacustrine beach, meadow, national forest, nature reserve, olive grove, orchard, park, pasture, planted forest, plateau, sandy beach, valley, vineyard

Soil temperature (°C):

Soil type: according to the Soil Identification Guide

Litter type: leaves, pine needles, seeds, debris, animal feces, unknown

Litter depth (approximation (cm):

Materials

Each team is provided with the following materials

1. Logsheets (1/site)
2. 200 ml 15% hydrogen peroxide for sterilization (1 bottle). This sterilizer is used because alchool with fire on field is dangerous to start a fire.
3. 50 ml Falcon tubes (6/site)
4. Plastic bags, to store falcon tubes (with soil) inside (2/site)
5. Plastic bag for plant collection (2/site)
6. Dry ice (1 steryfoam box) ~10 kg for long distances
7. Plastic Clear Tape
8. Cooler with thick walls
9. Water for cleaning (1 spray bottle)
10. Paper towels (1 pack)
11. Gloves 2 pairs/site
12. Permanent pens (1 piece)
13. Spoon (1 piece)

14. Thermometer
15. Soil borer (3 parts)
    1. an aluminum tube ~15cm long, 2cm wide
    2. a ram
    3. a narrow tube with a plastic head for pushing the ram
16. Hammer

The navigation to the sampling locations is suggested to be with the GAIA gps smartphone application available in Android and IOS operating systems.

Sampling procedure

1. Wear gloves to avoid contamination and because hydrogen peroxide is corrosive.
2. First wash the tube, the ram and the spoon with water, then dry with 1ssue paper to remove any dirt.
3. Spray the tube and the ram with hydrogen peroxide solution and leave for 3 minutes before using it.
4. Put the tube on the ground and the ram inside the tube and by using a stone or the plastic head or the hammer to push the tube in the soil.
5. There are two marks (rings) on the tube (5cm-8cm) - A depth between these two marks is enough for collecting 40 ml soil
6. If sampling sand or dry and loose soil, scoop horizontally or use the spoon
7. Carefully take out the tube from the ground and put the lower edge inside the falcon tube (if the soil is dry, the soil could fall out)
8. Use the narrow tube to push the ram down
9. After sampling, place the 3 replicate tubes of each location in the respectively prelabelled plastic bag.
10. Zip the bag and place it inside the dry ice container.
    1. In case of long distance between sampling sites, seal the container with tape