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Developing Your Lab’s Soil Sterilization Protocol

soil sterilization for research and restoration

Soil sterilization can be an ordeal. Its density and the heartiness of endospores and other biological material present in soil make it a truly vexing challenge. Many labs run their soil through multiple  the autoclave times, just to be sure. But that’s less than ideal, consuming time and resources better spent elsewhere.

We’ve spoken with lab staff from major North American research universities and an industry-leading soil testing lab about the reliable soil sterilization protocol development procedures that they use. Considering approaches like these is a great place to start in developing the soil sterilization protocols that may work best in your lab.

You’ll note we said “developing the soil sterilization protocols.” Most labs will need several different approaches for the different ways they are handling soil.

But, regardless of sample size, sample origin, or its future use, the fundamental things apply: always use a tray! Any leak from a ruptured bag or bottle is a mess in your autoclave, but the last thing you want is mud in the bottom of your chamber—or worse, all of that grit circulating through the plumbing. It’s much easier to rinse out a bin or tray than to try and dismantle and clean your autoclave. 

Validating Your Steam Autoclave Soil Sterilization Process

Regardless of soil sterilization method, every lab will need to develop its own soil sterilization protocols for the various substrates they are using and load types they prepare. You will need to validate each of these 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 sterilization validation, most labs prefer spore ampoules.

The following proven process will help you establish the validity of any steam autoclave-based soil sterilization process:

  1. Prepare several identical soil samples (this may be trays of loose soil, trays of small flasks filled with soil, red bags, etc.)
  2. Embed biological indicators in the load, being sure to have one at the center of mass (i.e., the middle of a bagged waste load, the centermost flask, etc), and several along the perimeter and length of the load at various depths
  3. Process the tray for 30min at 121ºC or 20min at 135ºC
  4. Remove your indicators, noting the location of each, and incubate
  5. If the samples show no activity (i.e., doesn’t change color/turbidity after 48 hrs of incubation), your procedure is validated
  6. In all likelihood, some samples will show some activity; note the locations of these “cold spots” or problem areas and adjust your loading and soil preparation process to avoid them.

If the initial sterilization procedure did not inactivate any of your indicators, repeat with an identical load (e.g., same style tray, same soil type, same number of flasks or brand of bags, same soil depth, same number and placement of indicator ampoules), but process it for twice as long as the first. Incubate those indicators. 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.)

Examples of Procedures for Soil Sterilization

Here are several sample soil sterilization protocols labs that regularly process soil use on a daily basis.

Sterilizing Open Trays of Soil

This protocol was developed at a major North American research university for processing large trays of soil:

  1. Load the soil into open polyethylene autoclave bags
  2. Arrange the bags in autoclave trays so that the soil is a uniform 2 to 3 inches deep
  3. Roll down the bag tops so that the surface of the soil is exposed
  4. Load the trays into the autoclave
  5. Process the soil using the “waste” cycle (120ºC for 50 minutes with both pre- and post-cycle vacuum stages)

Sterilizing Smaller Soil Samples

Smaller samples undergo a similar process (this based on the protocol used at a prominent soil testing lab):

  1. Place a 1 to 2 inch deep soil sample in a ~200 mL glass bottle or flask
  2. Place sample containers in a tray
  3. Load tray into the autoclave
  4. Run autoclave for 30 minutes at 121º or 20 minutes at 135º 
  5. Cool samples and cap with sterilized lid

Sterilizing Red Bag Waste

Red bag waste gets an additional step to be sure it’s safe to dispose of, but follows a similar procedure:

  1. Bag your disposal-ready soil (if it is not already in a red bag)
  2. Place a biological indicator in the bag of waste
  3. Do not seal the bag! Instead, folder over the top (this prevents grit from blowing out into the chamber during any vacuum or freesteaming stages of the sterilization cycle, but still allows for steam penetration into the dense soil load)
  4. Place bagged waste in an impermeable container (almost anything that will contain spills is fine; one lab used an old stockpot)
  5. Load into the autoclave
  6. Process the soil using the “waste” cycle (120ºC for 50 minutes with both pre- and post-cycle vacuum stages)
  7. Test the biological indicator to confirm sterilization
  8. If the indicator is inactivated, cool, seal, and dispose of the bagged waste; if not, place a fresh indicator and run a longer waste cycle

Reliable Tools, Reliable Protocols, Reliable Results

Reliable tools are the key to a consistent process. Every Priorclave is optimized for long-term reliability and low-energy/low-water/low-maintenance operation. Each unit is backed by our free lifetime technical support—including advice on validating custom cycles and protocols for soil sterilization. 

Are you frustrated by an unreliable autoclave or do you need help finding the right sterilization systems for your lab? Let us know, we’d love to help.