1. Wear Proper Personal Protective Equipment (PPE)

The high pressure and temperature operating conditions encountered during autoclaving pose potential heat burn or explosion risks. Appropriate PPE includes a laboratory coat, safety glasses, heat resistant gloves, and closed-toe shoes; these must be worn when loading and unloading materials from the autoclave. When dealing with liquid samples, wear an apron and face shield as additional PPE.

2. Preparation of Materials to be Loaded

1. Identify Autoclave Compatible Material and Autoclave Incompatible Material
   Although autoclaving represents a commonly used and economic method for decontamination, not all materials are suitable for autoclaving due to the high pressure and temperature working conditions. In general, all materials that will evaporate or melt under the high-pressurized steam are not compatible with autoclaving. This includes materials like organic solvents, poorly heat resistant plastic such as polystyrene, polyethylene, and metals excluding stainless-steel. Never autoclave flammable, corrosive, toxic, reactive, or radioactive chemicals. Materials compatible for autoclaving include Pyrex or Type I borosilicate glass, polypropylene, polycarbonate, gloves, stainless steel, pipette tips, paper (put inside autoclave bags), and media solutions.
2. Packaging Material
   After identifying autoclave-compatible materials, suitable packaging is also important since it ensures safety and efficient autoclaving. Here are the basic principles when packaging materials:
   1. Always use a secondary container made of polypropylene or stainless steel.
   2. For liquid samples, fill the vial around ½ full and loosen the caps or use vented closures. Never fill the containers over 2/3 full with the cap fully tightened.
   3. Inspect the glass vessel to make sure there are no cracks. If cracks are identified, dispose of glassware in a proper receptacle. Never autoclave a known broken glass vessel.
   4. Always use autoclave-compatible bags to package waste. The bags should have indication tapes on it and should be opened at the ends before loading to allow steam inside the bag.
   5. Never overload the bags or container to allow sufficient steam flow for complete sterilization. Leave space between items.
   6. Don't mix incompatible materials.
   7. Never place sharp items in the waste bag.

3. Load Material

1. Check the autoclave's interior to make sure there is no potential hazard left from the previous user.
2. Clean the drain before loading materials to allow good circulation.
3. Make sure the bags do not touch the interior walls of the autoclave to avoid melting.
4. Liquids and dry materials require different cycles and need to be autoclaved separately.
5. Close the door firmly.

4. Operation of Autoclave

1. Only trained personnel should be allowed to operate autoclaves. Refer to the equipment manual if needed, since operation protocols vary among different autoclaves.
2. After making sure the autoclave door is firmly closed, set the autoclaving temperature to 121 °C and maintain a pressure of at least 15 psi.
3. To set the autoclave time and cycle, the following factors need to be considered:
   1. Equipment recommendations from the manufacturer's manual.
   2. The type of loaded materials inside: dry or liquid.
   3. The amounts of material(s) loaded.
   4. Shape and size of container used.
   5. Heat conductivity of container and materials.
   6. Purpose of decontamination.

5. Unloading of Autoclaved Material(s)

1. Wear proper PPE as described above.
2. Make sure the temperature and pressure of the autoclave have returned to a safe range.
3. Stand behind the door and carefully open the door to release any leftover steam inside the autoclave into the room.
4. Allow the materials inside the autoclave to stand for 10 min to release any steam or hot air trapped inside the material.
5. Do not shake any liquid material while removing it from the autoclave. If necessary, label the material as hot to avoid heat burns to an individual who may be potentially unaware of the fact that the material has recently been autoclaved.
6. Wait for the material to cool down to room temperature before transporting within the laboratory or outside the laboratory. In both instances, be sure to utilize appropriate containment for transporting.
7. Record the details of the materials and autoclaving conditions in a user's log.