Soil Microbial Sampling Protocol

Preparatory Steps for Soil Sampling

Formulate a Sampling Plan:

Before initiating the sampling process, it is crucial to establish the purpose of the study, determine the sampling site's location, identify the required number of samples, decide on the sampling methodology, list the necessary tools, plan transportation routes, and consider prevailing weather conditions. It is customary to prepare a detailed sampling report that includes specifics such as the sampling date, time, precise GPS-tracked location of the sampling site, and weather conditions, encompassing temperature, precipitation, humidity, and cloud cover.

Data Collection at the Sampling Site:

After selecting the sampling site, collect and analyze data regarding the local soil type, weather conditions, and hydrological information. Conducting a field inspection before actual sampling helps pinpoint specific locations for sampling based on the collected data.

Sampling Equipment:

1 Sampling Tools:

A. Tool Types: Augers, shovels, sieves, tweezers, medicine spoons, brushes, and other specialized equipment based on specific sampling requirements.

B. Equipment Types: Sterile 50 mL tubes, 50 mL sterile bottles, Parafilm sealing film, measuring tape, GPS, inclinometer, soil color charts, sample boxes (with cooling functionality or containing dry ice), ice packs, sample bags, disposable gloves, face masks, 75% alcohol, sterilized cotton balls, sterile water, liquid nitrogen, dry ice, and more.

C. Stationery: Sample labels, marking pens, sampling recording sheets, and others.

D. Safety Equipment: Work clothes, work shoes, rain gear, safety helmets, medicine boxes, and more.

2 Note: A. All sampling tools and containers used for soil samples must be sterilized in advance (wrapped in tin foil, sterilized with high-pressure steam, and left overnight in a 120 ℃ oven) to prevent interference from exogenous substances. B. Avoid using tools or containers that absorb water, release solvents, or plasticizers to hold soil samples.

Soil Sampling Protocol

Location Selection:

The selection of sampling sites should align with research objectives and adhere to principles such as:

Sites with defined soil type features, relatively flat and stable terrain, and good vegetation.
Avoid sampling in subordinate landscapes like foot slopes and depressions.
Avoid sites with major human interference that could disrupt soil characteristics, such as towns, residences, fences, roads, ditches, ridges, manure pits, composting spots, graves, etc.
Sampling sites should be at least 300 meters away from railways and highways.
Sites should have well-developed soil profiles with clear class distinctions and no intrusive bodies. Avoid sampling in areas with severe soil erosion or damaged topsoil.
F. Avoid areas where multiple soil types and parent materials intermingle or where landscape coverage is small.

Important Note: Once sampling sites are confirmed, they can be located using a precision map or GPS or marked for ease in future repetitive sampling or comparative experimentation.

Setting a Sampling Quadrat:

To illustrate the direction of soil sampling, refer to the schematic diagram.

Soil Sampling Operations

Methods for Bulk Soil Sampling:

Remove surface soil litter, including plants, moss, visible roots, fallen leaves, and visible soil animals.
Wipe the sampler with an alcohol-soaked cotton ball and moisten it with soil from the sampling quadrat after alcohol has evaporated.
Select several sampling sites within each quadrat (4-5 recommended), mix equal amounts uniformly into one sample.
After removing plants, visible animals, and stones, sieve the soil based on its condition: 1. Use a 2 mm sieve; 2. For organic matters that cannot pass through a 2 mm sieve, use a 4-5 mm sieve; 3. If the soil is too sticky or has high moisture content, sieving can be avoided. E. Divide the mixed soil sample into several parts (3-5 backup samples), each weighing about 3-5 g. Label and freeze them quickly in liquid nitrogen or place them in a foam box with ice/dry ice and transport them back to the laboratory. Reserve a portion for physicochemical measurements.

Rhizosphere Soil Sampling Methods:

1. Crop-Class (and other low-lying plants) Rhizosphere Soil Sampling Method:

Collect plant samples, remove large soil chunks, and transport them back to the laboratory.
In a sterile workstation, shake plant roots to remove loose soil, use a sterile brush to collect residual soil, or wash the root system with sterile water (or PBS buffer). Centrifuge or filter the washing liquid.
Mix rhizosphere soil samples from multiple points within the same quadrat, label, freeze quickly in liquid nitrogen, and store in a -80°C freezer.

2. Forestry-Class Rhizosphere Soil Sampling Method:

Use a soil auger to collect rhizosphere soil 10-20 cm below ground level. Transport the soil back to the laboratory.
Select a sterile sifter with a pore size of 2-5mm, mix rhizosphere soil samples, label, freeze quickly in liquid nitrogen, and store in a -80°C freezer.

Soil Sampling Protocol (Nature, 2013)

Sample Storage:

Avoid repeated freeze-thaw processes to ensure sample integrity under constant low-temperature conditions.
Emphasize the importance of sample sealing during storage and analysis in the laboratory to prevent contamination from condensed water vapor or other pollutants.

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Sample Transportation:

For DNA: Total quantity should exceed 500 ng, concentration should be over 20 ng/µl, with no significant degradation. Soil: 2 g. B. For single sampling, the number of samples should be ≥ 20. Clearly label sample numbers on the tubes, seal with Parafilm, and send with a complete sample order form in a zip lock bag, along with dry ice for transportation.

References

Wang J, Qi J, Zhao H, et al. Metagenomic sequencing reveals microbiota and its functional potential associated with periodontal disease. Scientific reports, 2013, 3: 1843.
Shi B, Chang M, Martin J, et al. Dynamic changes in the subgingival microbiome and their potential for diagnosis and prognosis of periodontitis. MBio, 2015, 6(1): e01926-14.
Mendes L W, Kuramae E E, Navarrete A A, et al. Taxonomical and functional microbial community selection in soybean rhizosphere. The ISME journal, 2014, 8(8): 1577-1587.
Panke-Buisse K, Poole A C, Goodrich J K, et al. Selection on soil microbiomes reveals reproducible impacts on plant function. The ISME journal, 2015, 9(4): 980-989.
Li X, Zhu T, Peng F, et al. Inner Mongolian steppe arbuscular mycorrhizal fungal communities respond more strongly to water availability than to nitrogen fertilization. Environmental microbiology, 2015, 17(8): 3051-3068.
Zhou J, Deng Y, Shen L, et al. Temperature mediates continental-scale diversity of microbes in forest soils. Nature communications, 2016, 7.
Bulgarelli D, Rott M, Schlaeppi K, et al. Revealing structure and assembly cues for Arabidopsis root-inhabiting bacterial microbiota. Nature, 2013, 501(7468): S25.