Marywood University Standard Operating Procedure Autoclave Effective Use of Autoclaves Overview An autoclave is a commonly used piece of equipment in microbiology laboratories. Autoclaves (steam sterilizers) are metal pressure vessels that are used for steam sterilization of media, instruments and labware and for decontamination of biological waste. Autoclaves function by pressurizing steam in an airtight chamber, which significantly increases the steam temperature. The superheated steam kills all microorganisms and degrades most macromolecules rapidly. Autoclaves are generally easy to use but they pose many hazards to the operator including physical hazards (e.g. heat, steam and pressure) and biological hazards (ineffective treatment of waste). Autoclave waste packaging, treatment, and handling are also subject to regulation which carries substantial penalties if waste is handled incorrectly. This Standard Operating Procedure provides a review of the proper use of autoclaves to ensure that these devices are used safely, effectively, and in compliance with applicable regulations. Factors in autoclave function The energetics of steam makes it far more efficient for sterilization and decontamination that dry heat at the same temperature. Effective steam sterilization depends on the interaction of temperature, pressure and time, but additional conditions inside the autoclave chamber such as material, containers, container placement and total volume of the materials also influence sterilization success. Each of these factors must be controlled within a narrow range of values or conditions: Pressure/temperature relationship: Pressurization to 15 psi typically “superheats” steam to about 121oC (250 oF), which is adequate to kill all microorganisms and to decontaminate or sterilize in reasonable time. Time: Other factors being equal, autoclave loads up to about 2.0 ft³ in volume require 30-60 to sterilize at 15 psi and 121oC. Larger loads and tightly packed materials may require much more time. Contact: To sterilize or decontaminate uniformly, superheated steam must contact all areas of the load. Volume: “Dense” material such as media in bottles to be treated in the autoclave should occupy no more than half of the autoclave chamber volume, so that steam can circulate completely around and into the load. Less dense materials such as bagged waste can occupy somewhat more space but should never contact that autoclave chamber wall. Primary Containers Autoclave waste bags - Autoclave waste bags are used for bulk dry disposable materials such as paper towels, contaminated gloves, plastic centrifuge tubes, plastic Pasteur transfer pipettes and plastic petri dishes (with or without agar-based culture media). Autoclave bags used for biological waste at MU are made if translucent, colorless, steam resistant plastic film with no markings. Autoclave bags should be loosely taped or tied during decontamination to allow steam to penetrate into the bag. Air pockets in tightly sealed bags may cause localized sterilization failure. Bottles – Glass bottles or glass tubes with screw or push tops are used for autoclaving liquids such as water, some buffers and culture media. Always loosen the screw or push top Page 1 2/18/2015 Marywood University Standard Operating Procedure Autoclave so that it barely engages the thread or neck of the bottle or tube. If using plastic bottles and or caps, always verify that the plastic is heat-resistant before using them in an autoclave. Miscellaneous – Steam and heat-resistant pouches are commonly used to sterilize surgical instruments. Metal cans with snug-fitting metal lids may be used to sterilize glass serological pipettes (although this is less common nowadays because disposable pipettes are widely used). Hard-walled plastic sharps containers should not be autoclaved. These should be disposed of through the sharps waste stream. Report full sharps containers to the Lab Manager so they may be transported to the medical/pathological waste accumulation site. Medical waste bags are red or orange with a preprinted universal biohazard symbol. They contain medical or pathological waste such as cell lines, tissues and body fluids or consumables contaminated with body fluids. Red bag waste should not be autoclaved. Report full red bag waste to the Lab manager so they may be transported to the medical waste/ pathological waste accumulation site. Secondary Containers Autoclave bag contents may spill out of liquefy, block internal ports and seriously damage the autoclave during the autoclave cycle, so it is critically important to place autoclave bags in secondary containers during decontamination cycles. Likewise, liquid media bottles must always be autoclaved leakproof tubs. Polypropylene plastic or stainless steel tubs with 6-12 inch sides are commonly used to contain material in the autoclave. Do not use polyethylene or any other type of plastic tube except polypropylene because many other plastics will melt or distort permanently in the autoclave. Repeated autoclaving will eventually crack polypropylene – watch for this and replace these tubs at the first sign of deterioration. Stainless steel containers with leakproof seams are durable and good conductors of heat, and are a good alternative to polypropylene tubs. Each autoclave has its unique characteristics. Review and understand the owner’s manual located near each autoclave in the Autoclave Binder. Contact the manufacturer to request on-site training as needed. Ensure the owner’s manual is readily available in case questions arise during operation. Autoclave maintenance is an important aspect of a properly functioning autoclave. Follow the manufacturer’s recommendations for preventative maintenance and ensure all contractors are approved by the manufacturer. Maintenance should include periodic efficiency tests (e.g. Bacillus stearothermophilus spore testing) to ensure the autoclave is functioning properly. Autoclaves should be professionally serviced at least annually. Do not autoclave items containing corrosives (e.g. acids, bases, and phenol), solvents or volatiles (e.g. ethanol, methanol, chloroform) or radioactive materials. Applicability/Training/Recordkeeping This procedure applies to all University Faculty, Staff, and Students and other university employees who are involved with the preparation of microbiological media, sterilization of equipment or processing of biological waste. Page 2 2/18/2015 Marywood University Standard Operating Procedure Autoclave All individuals who use autoclaves must be trained. Training occurs before initial use, and reviewed as needed and when procedures are updated. The Laboratory Manager maintains training records. Records of maintenance, logs, calibration results and biological indicator load test should be kept for at least three years. General Procedure for Autoclave Operation 1. Before using the autoclave, check inside the autoclave for any items left by the previous user that could pose a hazard. 2. Clean the door gasket with a soft cloth. The gasket should be clean and smooth. 3. Make sure the autoclave has water in the reservoir. The add water light will be red when water is low. To fill water reservoir, remove the water reservoir cover. Pour distilled water in to the reservoir through the opening on the top of the autoclave until it reaches the base of the safety valve holder, approximately 3 liters. Replace water reservoir cover. Use only distilled (not pure) or mineral free water. Water sources may be: de-ionization column, reverse osmosis device, water distiller or rain water. Do not use tap water. 4. Load the autoclave in keeping with the proper established loading techniques (see pages 33-35 of Autoclave Manual). Ensure that all items put into the autoclave are properly labeled and have autoclave tape. Use secondary trays designed for the autoclave to hold the items to be autoclaved. Secondary trays and autoclave trays and tray holders should be cleaned weekly with detergent or a non-abrasive stainless steel cleaner and water using a cloth or sponge. Rinse immediately with water to avoid staining the metal. 5. Liquids should only be sterilized in heat proof glass or plastic. The bottle/beaker should only be filled 2/3 and lid should be on loosely to allow for expansion. Never place tightly sealed containers such as screw-capped bottles or tubes in an autoclave – they will very likely explode during the autoclave cycle. Bottles with narrow necks can also explode if filled with too much liquid. Check plastic materials to ensure they are compatible with the autoclave. Individual glassware pieces should never be placed directly on the autoclave bottom or floor. 6. Clear autoclave bags should not be packed tightly. Do not pack bags or other materials tightly into the autoclave chamber – leave some space between bags or bottles in the autoclave. 7. Once the autoclave is loaded, close the autoclave door making sure the “Close Door” indicator is illuminated. In order to ensure the door is fully sealed, tighten the door bolt until “hand tight”. Do not overtighten the bolt as this may result in damage to the gasket. Should the autoclave fail to reach sterilizing temperature/pressure, always check first that the door is fully sealed. 8. Select one of the sterilization programs offered with or without the accessory: A. Three sterilization programs: 1. Unwrapped instruments (scissors symbol) 2. Wrapped instruments and porous loads (dress symbol). Page 3 2/18/2015 Marywood University Standard Operating Procedure