Autoclave Operation

I. Purpose/Overview
The purpose of this document is to provide standard operating procedures for the use of
autoclaves. Autoclaving is a process used to destroy microorganisms and decontaminate
biohazardous waste and microbiological equipment at Biosafety Level 1,2,3, and 4.
II. Risk Management
A. Potential Risks (high pressure, high heat)
1. Heat burns - from hot materials and autoclave chamber walls and door
2. Steam burns - from residual steam coming out from autoclave and
materials on completion of cycle
3. Hot fluid scalds from boiling liquids and spillage in autoclave and
during transport of superheated materials
4. Hand and arm injuries when closing the door
5. Body injury if there is an explosion
B. Health and Safety
1. Name of person responsible for autoclave is posted near autoclave
(Nancy, Room D350, 5-3838)
2. SOP posted on outside of autoclave
3. Users are trained prior to using autoclave
4. Personal protective clothing and equipment must be worn when
loading/unloading autoclave
C. Personal Protective Equipment
1. Heat insulating gloves that provide complete coverage of hands and
2. Lab coat
3. Eye protection
4. Closed-toe footwear
III. Operator Instructions
A. Training
1. All personnel who use autoclave must have successfully completed a
training session
B. Material Preparation
1. Ensure that material is safe for autoclaving
a. Samples containing solvents or substances that emit toxic fumes
should not be autoclaved
1) Sodium hypochlorite (bleach) - may release hazardous
chlorine gas when autoclaved. Bleach solutions must be
neutralized with sodium thiosulfate before being placed in
autoclave (1ml of 5% sodium thiosulfate per ml of 5%
hypochlorite ion)
2) Agar treated with formaldehyde in aqueous solutions
will produce formaldehyde gas
b. Glassware
1) Inspect for cracks prior to autoclaving
2) Must be heat resistant borosilicate

ethyl benzene ACL: Acetal 121 yes formaldehyde d. Radioactive Materials 2. vinyl chloride = human carcinogen 2) Polystyrene . styrene and ethyl benzene 3) Acetal . All containers must be covered by a loosened lid. Prepare and package material suitably a. .c.releases formaldehyde 4) Nalgene Max Use Temp (oC) Autoclavable Possible emissions PC: polycarbon ate 135 yes PP: polypropyl ene 135 yes HDPE: high density polyethylen e 120 with caution at 1200 for 20 min LDPE: low density PE 80 no PS: polystyrene 90 no styrene. Wrapping too tightly will impede steam penetration. Loose dry items must be wrapped or bagged in steampenetrating paper or loosely covered with aluminum not autoclave. Loosen all lids to prevent pressure build-up. Containers of liquid must not exceed two-thirds full. with lids loosened c. Plastic 1) PVC .do not autoclave. b.

Pan must be large enough to contain a total spill of the contents d. or other materials that may boil over or leak. heat-insulating gloves and closed-toe shoes b. Do not overload. Make sure that the door of the autoclave is fully closed and latched c. into a secondary container 1) Liquid items should have about an inch of tap water in pan c. Check inside autoclave chamber for any items left by the previous user that could pose a hazard c. Ensure that the door gaskets have not deteriorated. Bags should be loosely tied so that steam can penetrate C. and push that button twice Button # Cycle Temp Sterilization Time . bags of agar plates. Place material in autoclave. Place containers of liquid.never placed directly on the autoclave chamber floor b. Items in secondary containers to secure and contain spills a. Do not mix incompatible materials. it does not indicate that it was heated at the proper pressure for the appropriate length of time 3. Prior to loading a. Wear lab coat. Loading a. Operating the Autoclave 1. but are still intact and pliable 2. items should be placed in a stainless steel pan or other autoclavable container for stability and ease of handling b. Tag all items with lead-free autoclave tape 1) Note: Tape only indicates that the proper temperature for the cycle has been reached. Choose appropriate cycle for the material.d. 1) Liquids a) should be within a heat resistant steel tray containing an inch of water b) Bottles should not be filled more than ⅔ and loose capped c) Keep 1-2 inches of space between bottles 2) Dry a) Individual glassware pieces should be within a heat resistant tray on a shelf or rack . Ensure that the drain strainer is clean before loading the autoclave d. leave sufficient room for steam circulation.

and closed-toe shoes 2. Post a sign alerting others not to use the autoclave. Do not attempt to open the door while autoclave is operating f. This will release steam and allow pressure within liquids and containers to normalize. 2. shut autoclave door (do not lock) F. . Autoclave Use Log (Room ) 1. contact Nancy E. 4. Autoclave Failure 1. Contact the department’s safety representative. 15 dry 2 Liquid 250oC 20 min 3 Liquid 250oC 30 min 4 Gravity (Dry/wrapped) 250oC 15 min. Wearing heat-insulated gloves. Equipment Malfunction 1. Discontinue use immediately if an autoclave is not working properly. Maintenance and Repair 1. G. No person shall operate the autoclave unless the autoclave is in good repair. These records are used for maintenance/service and reporting incidents/accidents and/or faults. Do not agitate containers of super-heated liquids or remove caps before unloading. Allow the autoclaved load to stand for 10 minutes in the chamber to allow the steam to clear and trapped air to escape from hot liquids 5.see Nancy in room D350 or call her at 5-3838 d. 2. eye protection. 3. Unloading Autoclave 1. 10 dry 1) Customizable cycle . Only qualified professionals are permitted to make repairs. 2. lab coat. Ensure that cycle has completed and both temperature and pressure of chamber have returned to a safe range 3. Report possible malfunctions to : Nancy IV. remove items from the autoclave and place them in an area which clearly indicates that items are “hot” 7. stand back from the door as a precaution and carefully open door no more than one-inch. Logbook is kept adjacent to autoclave. Contingency Plans A. Entries must be placed in the log book each time the autoclave is used. Contact Nancy to report the problem. If problems are perceived. Start cycle e. Nancy H. Wear heat-insulating gloves. Wearing PPE.1 Gravity (Dry/wrapped) 250oC 20 min. 6.

stanford.html . or burns over large areas): seek medical attention immediately (Call 9-911) 2. Report any accidents by using a SU-17 from at : http://www. After 5PM. Room B15 650-725-5308 b. H126 in the Stanford University Hospital. Non-life threatening burns a. removing clothing from the burn area and keeping the injured area cool for at least 5 minutes 3. notify your PI C.2. Major burns (burns to the face. Minor burns should be treated by using first aid procedures. Emergency Contact 1. including immersing the burn in cool water immediately. Regardless of severity. Post an alert sign to warn others. Incident Response 8AM-5PM: Stanford Occupational Health Center 480 Oak Road. B. third degree burns. next to cafeteria 650-723-2670 2. before 8AM Stanford Emergency Room.