Sterilising soil is a tremendous challenge. Different soil compositions have different densities, microbial loads, nutrient concentrations, and chemical profiles. But they’re all heavy, hard to penetrate, hard to validate, and prone to recontamination.
But sterilising soil samples is an important first step if we want to study the degradation of pesticides and xenobiotics, the sorption and mobility of nutrients, landscape re‐colonisation, microbiological mechanisms, microbial processes vs. abiotic reactions, or explore the intricacies of regenerative/restoration agriculture and permaculture and other advanced agricultural practices.
Three Soil Sterilisation Options
You have a variety of options for soil sterilisation. Broadly, these can be broken into three categories:
- chemical treatment (e.g., propylene oxide, sodium azide, etc.)
- gamma radiation
- heat (usually steam heat; in a lab setting, dry heat sterilisation of soil is often far too slow to be practical)
In general, chemical treatments alter soil pH and chemistry fairly drastically and still may not fully sterilise the soil samples. Sodium azide, for example, tends to inhibit bacteria and drastically reduce fungal populations, but doesn’t achieve consistent sterilisation and makes the soil significantly more alkaline. Propylene oxide is more effective at sterilisation but likewise increases pH by a unit or more. Studies have also found propylene oxide potential unsuitable for agricultural research: Germination and growth of wheat and alfalfa appear to be retarded in propylene oxide treated soil.
Similarly, gamma radiation may not be an appropriate method for all experiments, as it can influence soil chemical properties (in particular soil nitrate and ammonium levels). Also, some bacteria and fungi are known to be radio-resistant, and regularly survive gamma sterilisation-and then dominate the soil sample since their competitors have been removed.
Heat has long been used for sterilisation and soil is no exception. In many agricultural settings, soil may be sterilised by the simple expedient of laying it in a thin layer exposed to full sun for an extended period. That’s not practical for most research, although dry-heat sterilisation ovens accomplish essentially the same thing, albeit with smaller sample sizes. Neither dry-heat approach is appropriate to the lab.
As for steam heat, there are two broad categories of soil steam sterilisation procedures: Greenhouse “steam sterilisers” (which apply steam to soil samples under ambient pressure) and laboratory autoclaves (which penetrate soil samples with steam in a sealed high-pressure chamber).
With respect to microorganisms and macromolecules, autoclaving is much more destructive than other lab strategies (i.e. chemical and radiation), while altering soil pH very little. That said, it does tend to disrupt soil organic matter and will often significantly change the physical texture of the soil.
Steam Heat-based Soil Sterilisation in the Lab
Greenhouse steam sterilisers pump steam through soil at normal atmospheric pressure; this is cost effective and usable on larger scales, low-labour, and effective at sterilising soil (if properly validated and monitored). It is, however, also extremely slow when compared to other methods. That tends to make it unsuitable for most research lab situations, although possibly desirable when piloting new plant propagation or soil reclamation approaches in later-stage research.
For many lab applications, autoclave-based steam sterilisation proves to be the most cost-effective, consistent, and quick strategy. It’s also the simplest to validate (which is important since resting spores can be so resilient in soil).
Reliable Soil Steam Sterilisation Procedures
Regardless of soil sterilisation method, every lab will need to develop its own soil sterilisation protocol (if not several, for the various substrates they are using). Each of these will need to be validated carefully, using a quality biological indicator (e.g., Self-Contained Biological Indicators (SCBI), Mini Self-Contained Biological Indicators (MSCBI), or self-contained spore ampoules). For soil sterilisation validation, most labs seem to prefer spore ampoules.
Here’s a good starting point to establish your steam autoclave-based soil sterilisation process:
- Prepare several identical metal trays of soil, layering 2-4 inches of soil evenly in the tray
- Embed spore ampoules in the soil, being sure to have one at the centre of mass, and several along the length of the tray at various depths
- Process the tray for 30 min at 121ºC or 20min at 135ºC
- Incubate your spore samples
- If the samples show no activity (i.e., doesn’t change colour/turbidity after 48 hrs of incubation), your procedure is validated
- If the initial sterilisation procedure did not inactivate the spore ampoule, repeat with an identical tray of soil (same style tray, same soil type, same soil depth, same number and placement of spore ampoules), but process it for twice as long as the first. Incubate those ampoules. If spore activity is still evident, repeat again, processing for three times the original cycle time. (NOTE: Many labs find a single 30 minute cycle sufficient, although 90 minute cycles aren’t unusual.)
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