Autoclave Sterilization and Decontamination

Autoclave Sterilization and Decontamination, v1.0 Kathryn E. Caruso · 0009-0003-2436-1791 Foreman Lab · Center for Biofilm Engineering, Montana State University Updated March 2026

Autoclave Sterilization and Decontamination

Standard Operating Procedure

Autoclave cycle summary:

Autoclave liquids at a rate of 20min/L

1 Safety

PPE:

• Lab coat
• Heat-resistant gloves (when unloading autoclave)
• Safety glasses
• Closed-toe shoes

Hazards:

• Steam burns — never open the autoclave immediately after cycle ends
• Superheated liquids — media and liquid waste can boil over violently if disturbed before cooling; do not agitate bottles immediately after cycle completion
• Pressure buildup — tightly sealed containers can burst; always loosen caps before autoclaving liquids
• Bag rupture — overfilled or tightly sealed autoclave bags can burst; leave bags loosely closed
• Hot items immediately after cycle completion
• Odor — autoclaved biological waste produces strong odors; ensure adequate ventilation

General principles:

• Always apply autoclave tape to every item or load — verify color change after the cycle
• Never autoclave tightly sealed containers
• Use the correct cycle type for the load (see sections below)
• Allow pressure to reach 0 psi before opening the autoclave door
• Allow loads to cool inside the autoclave before handling

2 Equipment Sterilization

Items to autoclave:

• Tube caps/closures
• Milli-Q water (for use in filter-sterilized reagents, e.g., urea stock — use liquid cycle)
• Reusable tools that contact cultures
• Media bottles (empty, before filling)
• Any non-glass, non-pre-sterilized item contacting sterile media or cultures

Materials:

• Autoclave
• Aluminum foil or autoclave bags
• Autoclave tape
• Items to be autoclaved

Procedure:

1. Wrap or bag each item loosely in aluminum foil or autoclave bags — ensure steam can penetrate
2. Apply autoclave tape to each package
3. Autoclave at 121°C, 15 psi, 15–20 min (dry cycle)
4. Allow to dry and cool completely inside the autoclave
5. Store sealed/wrapped until use
6. Label autoclave tape with date and initials

3 Liquid Media Sterilization

Key parameters:

Autoclave liquids at a rate of 20 min/L.

Materials:

Reagents:

• Prepared liquid media (pre-mixed to desired recipe, pH-adjusted if needed)
• Heat-sensitive additives if applicable (see below)

Equipment:

• Autoclave (liquid cycle capable)
• Autoclave-safe glass bottles (borosilicate, e.g., Pyrex/Duran) with loose caps
• Autoclave tape
• Autoclave tray or secondary containment pan
• Stir plate (for post-autoclave mixing, if adding supplements)

Media preparation before autoclaving:

1. Dissolve all heat-stable components in the appropriate volume of deionized water
2. Adjust pH if required by the recipe — note that autoclaving may shift pH slightly (~0.1–0.2 units)
3. Transfer media to autoclave-safe glass bottles
4. Fill bottles to no more than 2/3 capacity to allow for expansion and boiling
5. Loosen caps — leave caps resting on top or turn only 1/4 turn closed to allow steam venting

Autoclave procedure:

1. Apply autoclave tape to each bottle
2. Place bottles upright on an autoclave tray — do not stack
3. Select liquid cycle (slow exhaust) — this prevents boil-over during depressurization
4. Set cycle parameters based on volume (see table above):
   • ≤ 1 L: 121°C, 15 psi, 20 min
   • 1 – 2 L: 121°C, 15 psi, 40 min
5. Start cycle and record start time
6. When cycle is complete, wait for pressure to reach 0 psi before opening door
7. Crack door slightly and allow bottles to cool for 10–15 min inside the autoclave before handling
8. Carefully transfer bottles to bench using heat-resistant gloves
9. Tighten caps once media has cooled to ~60°C or below
10. Verify autoclave tape has changed color (dark stripes)
11. Label each bottle with media type, date, and initials

Heat-sensitive additives:

Some media components degrade at autoclave temperatures and must be filter-sterilized (0.2 µm) and added aseptically after the media has cooled to ≤ 55°C.

Common heat-sensitive additives:

Procedure for adding supplements:

1. Prepare stock solutions of heat-sensitive components and filter-sterilize through a 0.2 µm syringe filter
2. Allow autoclaved media to cool to ≤ 55°C (warm to touch but not hot)
3. Add supplements aseptically near a flame or in a laminar flow hood
4. Swirl gently to mix — do not vortex

Expected results:

• Media should be clear (or appropriately colored for the recipe) with no precipitate or browning
• Autoclave tape should show dark indicator stripes
• Media should remain sterile when stored properly at room temperature (weeks) or 4°C (months)

Troubleshooting:

4 Waste Decontamination

BSL-1

Waste segregation:

Separate waste into the following categories before autoclaving:

Solid waste (gravity cycle):

• Used agar plates
• Contaminated consumables (pipette tips, microcentrifuge tubes, Falcon tubes, culture tubes)
• Paper towels or wipes from culture work
• Used swabs, applicators, inoculating loops (disposable)

Liquid waste (liquid cycle):

• Spent liquid culture media
• Liquid culture waste from flasks, tubes, or well plates
• Rinse water from washing culture vessels

Materials:

• Autoclave bags (orange or clear biohazard-rated, polypropylene)
• Autoclave-safe secondary containment tray (metal or polypropylene)
• Autoclave tape
• Liquid waste containers (if decontaminating liquid cultures — use autoclave-safe bottles or flasks with loosened caps)
• Permanent marker or labels

Procedure — Solid waste:

1. Place solid waste into an autoclave bag — fill no more than 3/4 full
2. Do not compact or crush waste tightly — steam must penetrate the load
3. Leave the bag open (fold top over loosely but do not seal or tie shut)
4. Place the open bag upright in a secondary containment tray
5. Apply autoclave tape across the bag opening
6. Run on gravity cycle: 121°C, 15 psi, 60 min
7. When cycle is complete, wait for pressure to reach 0 psi
8. Allow load to cool for 10–15 min before removing
9. Verify autoclave tape has changed color
10. Seal the bag (tie or tape shut) and dispose in regular trash

Procedure — Liquid waste:

1. Collect liquid waste in autoclave-safe glass bottles or flasks
2. Fill to no more than 2/3 capacity
3. Loosen caps — do not seal tightly
4. Place bottles upright on an autoclave tray
5. Apply autoclave tape
6. Run on liquid cycle (slow exhaust) at 121°C, 15 psi:
   • ≤ 1 L: 20 min
   • 1 – 2 L: 40 min
7. When cycle is complete, wait for pressure to reach 0 psi
8. Allow bottles to cool for 15–20 min inside the autoclave — do not agitate
9. Verify autoclave tape has changed color
10. Once cooled to room temperature, decontaminated liquid can be poured down the drain with running water (check local regulations)
11. Rinse bottles and return to service or recycle

Autoclave log:

Record each decontamination run in the lab’s autoclave log (paper or digital):

Expected results:

• Autoclave tape shows dark indicator stripes
• All biological material is considered decontaminated and safe for regular waste disposal
• Liquid waste is safe to pour down the drain (if allowed by local regulations)

Troubleshooting: