Soil Collection

diversity. To achieve this, one must sample a representative portion of the microhabitats in the area under investigation. Once sampled, conditions in the soil sample begin to change. Species may become inactive or may be stimulated to grow after sampling. Therefore, one tries to differ- entiate between active and inactive species at the time of sampling. In this chapter, we discuss approaches for: (i) estimating the total number of species in samples; (ii) extracting and enumerating active species at the time of sampling; and (iii) obtaining enriched samples and cultures for certain organisms. The chapter is not intended to be a description of methods, but a discussion of common approaches. Useful practical guides to methodology and laboratory procedures are provided at the end of the chapter and referenced in the text.

Soil can be collected with a variety of hand-held shovels, soil core sam- plers and augers, spoons or spatulas. The preferred tool will depend on the quantity of soil required and the type of soil being sampled. To pre-

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vent cross-contamination of samples with bacteria, protist cysts, spores or active cells between samples, it is necessary to clean sampling tools with a volatile alcohol which disrupts cell membranes (such as 5%

ethanol or 5% methanol). A small number of active cells is sufficient to colonize an entire sample in 2 or 3 days, if cells disperse through the sample or grow through several divisions. Therefore, adequate sampling protocols require attention to clean field techniques. Soil augers with an inner sleeve are commercially available and reduce the risk of cross-contamination. The collected soil sample must also be protected from crushing and desiccation. Once a soil sample is removed from the field, it will undergo some mixing or crushing, aeration, desiccation and temperature change. One should strive to minimize the changes before samples are analysed. Several precautionary measures are necessary. Excessive friction of soil particles will damage or kill protists, including hyphae, and the microinvertebrates such as nematodes, enchytraeids and microarthropods. A decrease in moisture content will initiate encystment or inactivation of many species. Thus, soil samples normally are placed in zip-lock plastic bags with care taken to avoid crushing the soil. This permits some gas exchange with air in the bag and reduces loss of moisture. A safer method, especially with protists, is to use screw-cap plastic tubes that protect the sample from desiccation, crushing and friction. Only a few centigrams of soil are required to extract and enumerate for protists and bacteria, but up to 100 g may be necessary for microarthropods. It is necessary to compromise between spatial distribution heterogeneity and the number of samples to analyse in the laboratory. For efficiency, it is best to obtain several small samples that can be fully analysed rather than fewer larger samples. For transport and temporary storage, soil samples are kept away from direct heat or cold in a cooler, at about the soil temperature.

The distribution of organisms varies with depth through the litter, organic horizon and B horizon, and along the surface. The diversities of conditions and food types available over several micrometres to several metres are called microhabitats. Surface soil microhabitats that influ- ence species distribution can be found inside and under a fallen tree trunk or coarse woody debris, in decomposing leaves, or animal debris and carcasses. Litter organisms require sampling of coarse woody debris (twigs, branches and bark), fallen leaves and other debris that may be present. Falling fruit, nuts, catkins or pollen deposition can also impact the litter and soil organisms. To determine species stratification with depth, it is more important to preserve the profile. A soil corer, large diameter cork borer or curved spatula are useful because the soil can be subsampled or the profile collected intact. Alternatively, one can dig to expose the profile before sampling at various depth intervals. With depth, the quality of the organic nutrients decreases, soil bulk density or compaction increases, and oxygen availability becomes limiting. These

determine species composition. To preserve the anaerobic species, it is necessary to fill the container completely with soil (without crushing) and to use a tight lid. Alternatively, anaerobic cores can be sealed in plastic, or commercially available sampling sleeves.

While in the field, it is important to remember the following points before samples are collected: (i) whether the soil profile needs to be preserved or subsampled, or if mixed bulk soil will do; (ii) whether the microhabitats in the litter can be amalgamated or if they need to be kept separate; (iii) how deep in the soil horizons one needs to sample; and (iv) how many replicates per sample are required. Once the soil is collected, the heterogeneity of the study site needs to have been sampled adequately for the project. The number of samplescollected needs to be manageable in the laboratory, by the number of people and amount of time dedicated to sample analysis. The idealized number of samples required statistically can be impractical or simply superfluous once in the field. The correct balance between site heterogeneity and number of samples must be adjusted to what can be analysed (see below, this chapter). This may require narrowing the scope of the project. It is more informative to have fewer well-analysed samples than too many poorly studied samples.

Soil handling

The soil matrix contains decomposing litter and organic matter that are a source of nutrients to species of bacteria, protists and invertebrate animals. The source of some of the litter is animal dung and corpses, including those of mammals. Since animals carry internal and external parasites, they can release them to the environment through excretions, secretions and cadavers. Therefore, viral, bacterial, protist and invertebrate parasites of animals can be found in the soil, where some have intermediate hosts or life cycle stages. To avoid infections, it is necessary to avoid contact with potentially contaminated soil. Some species are not normally parasitic to humans, but can still cause a mild infection.

However, non-parasitic soil species can become invasive and infectious, under certain conditions. These are called opportunistic parasites. For example, Acanthamoeba castellaniiand similar species, which are common soil amoebae, can proliferate in the eye and cause blindness. Similarly, many species of soil bacteria are pathogenic, or can become pathogenic if in contact with a wound, or internalized. There are also fungi that are inedible and poisonous, so contact can be dangerous, especially if ingested. Lastly, tissues of certain plant species (roots or leaves) contain poisonous chemicals, poison glands or trichomes (Colgate and Darling, 1994). These may remain in the litter for some time even if partially decomposed. Skin irritations (contact dermatitis) can be caused by contact with these molecules in the soil and litter.

Another source of toxicity in soils is pollution caused by human activity. These anthropogenic sources can affect large areas (e.g. through atmospheric deposition) or be very localized (an accidental spillage).

Deposition of air pollutants from the atmosphere causes accumulation of heavy metals, radioactivity, acid rain and organic poisons. Accidental spillage of petroleum products and hazardous chemicals, at the site of manufacturing or in transport, are realistically unavoidable.

Accumulation of toxic chemicals occurs gradually and cumulatively, or in large discharges. Soil pollution can be legal, for instance at a mining site, or illegal, by undeclared and improper disposal of toxic materials.

Agricultural and urban applications of pesticides, herbicides and other poisonous chemicals further contribute to soil toxicity. The dispersal and range of a pollution event depend on regional and global biogeochemi- cal cycling, hydrology, soil composition and many other parameters.

Standards for collection, storage, handling and analysis of pollutants, and other chemical analysis of soils, are set by various national govern- mental agencies. These sources must be referred to for guidelines on methodological assays and protocols.

For biological analysis, collected soil must be handled on a clean bench space and with clean or sterile equipment. Cysts and spores of soil protists and some bacteria are resistant to desiccation, but also to a variety of detergents and disinfectants. Thus, contamination of samples from laboratory surfaces is easy without sufficient care. Moreover, cysts and spores in spilt air-dried soil can be disturbed and carried in the laboratory air. Once airborne, contamination of soil isolates and cultures is more frequent. Cysts of colpodid ciliates are notorious invasive coloniz- ers, and they can be isolated from sinks and floors of most soil laboratories. They are the most common protozoan culture contaminant in cell biology laboratories. Often, the laboratory space is shared with bacteriol- ogists who apply sterile techniques, or with invertebrate or plant biolo- gists who do not. Clean work habits are the best deterrent to permanent contamination problems.

Soil storage

Storageof soil samples can affect the biological and chemical properties of the soil. For example, the sorption and release of dissolved organic carbon (DOC) in soil samples varies with soil storage conditions, which is effectively a pre-treatment step (Kaiser et al., 2001). The least effect is observed in fresh samples, but with an increase in soil DOC release of 23–50% depending on the horizon after 1 month at 3°C. Freezing at –18°C more than doubled the DOC measured, and air drying the samples increased the measured DOC by 4.3–4.7 times. Variations in soil storage and handling are significant in altering the chemical release of

material from soils. In part this is due to killing and lysis of organisms and denaturing the chemical–physical properties of the organic matter.

For many soil organisms, biological preservation is possible by fixation of soil samples in ethanol or formalin at sampling or soon after. This will preserve the microinvertebrates, fungi and bacteria, but not protozoa.

Biological fixation with ethanol is not suitable for protozoa which do not have cell walls, because the alcohol dissolves membranes and cells are lysed. However, the ethanol fixation procedure is suitable for DNA extraction as the nucleic acids are preserved. This procedure has been tested with bacteria for DNA recovery (Harry et al., 2000) and is proba- bly suitable for other organisms with modifications.

Once soil has been collected from the environment, it is no longer part of the open ecosystem. It is a materially closed system, in that it is held inside a sealed container. The sample no longer receives nutrient input, or exchanges material with the outside of the container; only energy can be exchanged, in terms of temperature. Thus, organisms in the sample will respond to this new situation, where the only food and moisture available were those present at the time of sampling. Furthermore, since the respired, secreted and excreted materials cannot escape, they accumulate in the sample. Over time, the sample will become depleted of food and oxygen, and will accumulate CO₂and other by-products of respiration and growth. One can maintain the sample aerated and moist, or even add nutrients; but by-products of respiration and growth accumulate none the less. For this reason, over time, the active species in the collected soil will no longer be representative of the active species at the time of sampling. Some biological activity continues even in the cold, through cold-tolerant species, so that moist stored soil is not completely inactive.

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