Operating Procedure for Steam Autoclaves

Physical hazards involved with steam autoclaves are heat, steam and pressure. The biological hazards involve potential exposure to viable human pathogens.

Due to the high heat and pressure created in autoclaves during operation, you must follow proper loading, use and unloading procedures to prevent burns and other accidents. Burns can result from physical contact with the structure of the autoclave, and steam burns can occur from contact with steam leaving the apparatus. Burns can also result from careless handling of vessels containing hot liquids. Explosive breakage of glass vessels during opening and unloading — as a result of temperature stresses — can lead to mechanical injury, cuts and burns.

Autoclave performance for sterilization purposes is dependent on proper use.

If you are a principal investigator (PI), it is your responsibility to ensure that:

• staff and all students working in your lab are trained before operating any autoclave unit
• everyone understands and follows procedural and instructional documents

Personnel who use an autoclave must be trained to understand the proper operation procedures as referenced in:  

• RMW Part 365 Management Plan vDRP(2)
• BSL-3 RWM Part 365 Management Plan

Video training is also available: “On-Site Biohazardous Waste Management: Autoclaving.”

Additionally, instruction sheets are posted in each autoclave room to assist with the operation of the equipment.

You must autoclave all potentially infectious materials before you wash, store or dispose of them.

You must label biohazardous materials as such, and you must sterilize them by the end of each work day, or you must secure them appropriately. Biohazardous materials should not be left in an autoclave overnight in anticipation of autoclaving the next day.

For the autoclave process to be effective in achieving sterilization, sufficient temperature, time and direct steam contact are essential. Air must be completely removed from the sterilizer chamber and from the materials to allow proper steam penetration.

Factors that affect air removal include:

• type and quantity of material to be autoclaved
• packaging load density and configuration
• container type, size, and shape

The potential safety risks for autoclave operators are:

• heat burns from hot materials and autoclave chamber walls and door
• steam burns from residual steam escaping the autoclave and from materials on completion of cycle
• hot fluid scalds from boiling liquids and spillage in autoclave
• hand and arm injuries when closing the door
• bodily injury if there is an explosion

You must wear personal protective clothing and equipment when loading and unloading the autoclave, including:

• heat-insulating gloves to protect hands and forearms
• face shields to protect face and neck
• lab coat to protect chest and legs
• closed-toed footwear to protect feet

Ensure that the material is able to be autoclaved. Items that should not be autoclaved include:

• oils and waxes
• some plastics
• flammable materials
• radioactive materials
• substances that may emit toxic fumes 
• samples containing solvents

Glassware should be heat-resistant borosilicate (Pyrex or Kimax), and you should inspect it for cracks prior to autoclaving.

Plastics should be heat resistant, for example:

• polycarbonate (PC)
• PTFE (“Teflon”)
• polypropylene (PP)

Prepare and package material suitably. You should wrap or bag loose dry materials in steam-penetrable paper or loosely cover them with aluminum foil. Wrapping too tightly will impede steam penetration, decreasing the efficiency of the process.

Containers of liquid should be a maximum volume of 2/3 filled.

Cover all containers with a loosened lid or steam-penetrable bung to prevent pressure buildup and to prevent bottles from shattering during pressurization.

Use plain, unmarked containers for items that are not hazardous.

You should tag items or baskets with autoclave temperature tape.

Place items in containers to secure and contain spills. Place the following items inside a secondary pan (must be autoclavable plastic or a stainless-steel container) in the autoclave:

• containers of liquid
• bags of agar plates
• other materials that may boil over or leak

The pan must be large enough to contain a total spill of the contents. Open, shallow, metal pans are more effective in conducting heat and allowing air removal than tall plastic tubs. Adding some water to the secondary pan will help to heat items more evenly.

• Ensure the drain strainer in the bottom of the autoclave’s sterilizer chamber is clean before loading the autoclave.
• Use a cart to transfer items to be autoclaved. To avoid back injuries, push the cart up to the autoclave door and gently slide the load into the autoclave.
• Never place autoclave bags or glassware in direct contact with the bottom of the autoclave.
• Place the secondary pan containing the items to be sterilized on the shelf or rack of the autoclave.
  • You must use secondary containment pan under the bag to catch any leaks that may occur during autoclaving.
• Do not overload the autoclave. It is important to leave sufficient room for thorough steam circulation. 
• Do not mix loads of liquids with solids.
• Make sure that you have selected the correct cycle before starting the autoclave (see below).

The parameters for the sterilization cycle will depend upon the amount and type of material. Usually 121 degrees Celsius at 15 pounds-force per square inch (lbf/in² or psi) for a minimum of 30 minutes is recommended. However, you can increase the temperature and cycle time depending on the size and type of load.

Autoclave manufacturers generally provide several pre-set cycle options that vary in the pre-set sterilization temperature, sterilization time and dry time.

• Gravity cycle: air removal from the autoclave chamber is achieved by gravity air purge; this is appropriate for loads where air removal from porous materials or penetration of steam into wrapped/packaged items is not required
• Vacuum cycle: air from the autoclave chamber is removed by pulsing between pressure and vacuum; this is suitable for wrapped or difficult-to-penetrate items
• Liquids cycle: gravity air purge removes air from the chamber as in the gravity cycle; deep vacuum is not used since liquids to be autoclaved would be expelled from their vessels

After a run is complete, check the pressure gauge to ensure that the pressure in the chamber is “0.” If pressure is not released, do not open the door. Inform your PI / supervisor about the malfunction.

Before opening the door, wear eye protection and heat-resistant gloves or mitts. Be sure to wear closed-toed shoes (hot condensate may drip from the door). Rubber aprons in addition to rubber sleeve protectors are advisable.

Use caution when removing liquids, molten agar, etc. Liquids, especially large volumes, may continue boiling for some time after autoclaving. To avoid being splashed with scalding liquid:

• do not agitate containers of super-heated liquid
• do not remove caps before unloading 

Slide a cart to the opening of the autoclave and pull the autoclave secondary container onto the cart for transport. Place the cart in a low-traffic area while additional cooling occurs.

Before touching items with ungloved hands, allow for the following cooling times:

• glassware: 15 minutes
• liquid loads: one full hour 

Please note: If a faulty condition exists (e.g., sterilizer did not finish the cycle, or water leaks out when the door is unlocked), inform the person responsible for contacting the autoclave service technician.

When you are autoclaving biohazardous waste materials inside heat-resistant autoclavable bags, be sure that you:

• use heat resistant autoclavable bags labeled “biohazard” (usually red or orange) for waste materials that contain or may be contaminated with potentially infectious agents
• store biohazard waste bags in rigid leak-proof secondary containment pans
• store waste inside the autoclave room — not in hallways.
• do not double bag waste or tightly seal bags; this will impede steam penetration
• avoid compressing bags; this may create aerosols
• do not put sharp objects such as broken glassware into an autoclave bag
• use a secondary containment pan under the bag to catch any leaks that may occur during autoclaving

It is advisable to add some water to bags of solid wastes (the water will vaporize into steam that will drive out residual air once sterilization temperature has been reached inside the bag).

Label the autoclavable bag with a “biohazard” symbol on it with commercially available autoclave temperature tape that changes color, e.g., visible black stripes or the word “autoclaved” appear once sterilizing temperature has been reached. Autoclave temperature tape only indicates that the desired temperature was reached — it does not indicate any information about time and pressure. Apply this tape across the “biohazard” symbol on the bag before autoclaving. Do not throw away any biohazard bags without covering all biohazard symbols.  

Autoclave 50 to 60 minutes, at temperature and pressure of 121 degrees Celsius (250 degrees Fahrenheit) and 15 psi. When cycle is finished, inspect autoclave temperature tape and visually check autoclaved bags. Bags should be left to cool for several minutes before removing from autoclave.

Once cool, remove the Steam Chemical Integrator from the autoclave bag and interpret the results to ensure that the autoclave worked correctly. If this test indicates a successful sterilization, then:

• securely close biohazard bag with tape or tie wrap
• attach a label to autoclaved waste bag that states: “Disinfected”
• place bag inside another opaque bag (black, green, etc.) to be placed in regular trash

Please note:

• parameter monitoring (pressure, time and temperature) is important to ensure efficacy of an autoclave
• do not pour melted agar down the drain as it will congeal and clog the plumbing      
• biohazardous waste should not be left for “someone else” to autoclave

Document the treatment of each load of biohazardous waste on the Autoclave Waste Treatment Log (available at each equipment location), which lists:

• date of treatment
• amount of waste treated
• method/conditions of treatment
• printed name/initials including contact info of the person autoclaving the waste
• results of Steam Chemical Integrator tests

Keep printout strips with the log sheets as documentation of the autoclave operation.  

No person shall operate an autoclave unless it has been inspected by a qualified inspector and a certificate of inspection has been issued. A current inspection certificate is posted near the autoclave.

Users are not to make repairs. Autoclaves shall be maintained and repaired by qualified persons.

If the autoclave does not operate exactly as expected, Facilities Planning and Management will place a notice on the autoclave indicating that it is not to be used until the problem is diagnosed and corrected.

Facilities will record the problem in autoclave log book.

When maintenance work or repairs are needed, the PI/LAB must provide a safe work environment for the service technician. Please remove all items from the sterilizer chamber, clean any spills or leaks inside the chamber, remove untreated biohazardous materials from the vicinity, etc.

If any injury occurs, seek first aid or, if necessary, seek medical assistance by dialing:

• security office at 716-829-2357
• University Police at 716-645-2222

If clothing is soaked in hot water/steam, remove clothing and place the injured area in cool water.

Place a notice on the autoclave indicating that it is not to be used until:

• the cause of the incident is determined
• procedures are enacted to prevent future incidents
• the autoclave is deemed safe for operation

Report all other non-emergency incidents to Facilities Planning and Management.

Spills may occur from a boil-over or breakage of containers. You are not allowed to operate the autoclave until the spill is cleaned.

If you are the operator, you are responsible for cleaning spills. The spilled material can be contained using materials from the spill kit to absorb or contain the spill.

Wait until the autoclave and materials have cooled to room temperature before starting the cleanup inside the autoclave.

Review the Safety Data Sheet(s) (SDS) of the spilled material(s), if appropriate, to determine which protective equipment, spill cleanup, and disposal protocols are necessary.

You must:

• clean the equipment and work area in order to collect and remove all spilled materials
• dispose of the waste following the protocol appropriate for the material

If materials have been intermingled, follow the cleanup and disposal protocol for the most hazardous component of the mixture.

Cracked clean glassware must be disposed of properly in the “broken glass” disposal container provided by Custodial Services / EH&S.

Record the spill and cleanup procedure in autoclave log book.

Inform Environment, Health and Safety so that the spill kit can be replenished.

1. Environment and Safety, University at Buffalo
2. Biosafety in Microbiological and Biomedical Laboratories, U.S. Department of Health and Human Services, Centers for Disease Control and Prevention/National Institutes of Health
3. Chemical Hygiene Plan, University at Buffalo
4. RMW Part 365 Management Plan vDRP (2) (002), Waste Management Plan, University at Buffalo, Office of Biosafety
5. Regulated Medical Waste and Other Infectious Wastes at the University at Buffalo/CUBRC BSL-3, University at Buffalo, Office of Biosafety

Proper personal protective equipment (PPE) must be worn when replacing the cleaning chemicals.

To achieve maximum results, the Auto Clave Glassware Washers (Lancer 1400LX) require two separate chemical items (Consumables) to function properly:

• Lancer Clean Detergent “LCD-P”
• and Lancer Acid Rinse “LCA-A”

These two chemicals have been primarily used at the Jacobs School. 

For the Lancer 1400LX: 2 (two) glassware washers per each autoclave room. (The 2.5-gallon container is required of LCD-P and LCA-A for each machine) – so this would be one box of each that contains 5 gallons total of each product as listed here:

The Lancer Clean Detergent is formulated and is specifically designed for our Lancer high-pressure washers to achieve maximum cleaning results. The solution is powerful enough to clean a variety of contaminants, including laboratory soils, "baked on" organic residues and petroleum applications. This detergent is formulated to safely clean all types of glassware, surgical instruments and other labware items. Use of the chemical is made easy with their pre-mixed solution for direct use from the bottle to the washer and their peristaltic pump enabling automatic dosing – no measuring needed. The cleaning chemical is produced with a double filtration system and excellent quality control, ensuring the integrity of these solutions is maintained.

The Lancer Acid Rinse is to be used as a rinsing agent following the detergent in the wash cycle. The solution is specifically designed for our Lancer high-pressure washers to achieve maximum rinsing results. The solution is powerful enough to rinse a variety of contaminants including laboratory soils, "baked on" organic residues and petroleum applications. Use of this chemical is made easy with their premixed solution for direct use from the bottle to the washer and their peristaltic pump enabling automatic dosing – no measuring needed. This cleaning chemical is produced with a double filtration system and excellent quality control, ensuring the integrity of the solutions is maintained.

If the 2.5-gal container is empty (zero material remaining) it is safe to bag and dispose of the container according to the normal recycling procedures for the building.

If the container has some liquid remaining, you must complete the following tasks:

• complete the Environmental, Health and Safety hazardous waste disposal request form
• scan the form and send it to: hazwaste@buffalo.edu
• send a courtesy email to Facilities Planning and Management (jsmbs-facilities@buffalo.edu) to notify us that a hazardous waste disposal pickup was requested

Environmental, Health and Safety will pick up the materials and complete proper disposal activities.