Effective Use of s Overview An autoclave is a commonly used piece of equipment in microbiology laboratories. s (steam sterilizers) are metal pressure vessels that are used for steam sterilization of media, instruments and labware and for decontamination of biological waste. s 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. s 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). waste packaging, treatment, and handling are also subject to regulation which carries substantial penalties if waste is handled incorrectly. This 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: Page 1 Pressure/temperature relationship: Pressurization to 15 psi typically superheats steam to about 121 o C (250 o F), 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 121 o C. 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 waste bags - 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). bags used for biological waste at MU are made if translucent, colorless, steam resistant plastic film with no markings. 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
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 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 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. 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. s 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
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 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. Page 3 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 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 4 3. Liquids (flask symbol). B. Accessory program 4. Dry only (asterisk symbol). All programs are preset to a temperature of 121 o C (250 o F). 9. It is possible at this time to change preset parameters: To increase or decrease sterilization temperature (TEMP), sterilization time (STE TIME) or dry time (DRY TIME), simply press the desired key. To raise or lower temperature or time, press the UP and DN keys until the desired temperature or time is reached. 10. Press the START key to put the autoclave in operation. WATER INLET is displayed until the correct volume of water is automatically introduced. Entry of water into the chamber may be accompanies by a noise for approximately 30 seconds. This is normal noise generate by regular operation of the pump. The autoclave starts performing the sequence of operations. The actual measured values of pressure and temperature are displayed continuously (and printed with optional printer). The indicator lights HEAT, STE, EXH AND DRY are turned on and off as each stage is started and completed. Note after loading and starting the autoclave, processing time starts after the autoclave reaches normal operating conditions of 121 C and 15 psi pressure. For example, 60 minutes of sterilizing averages 2 hours for full cycle which includes an average 30 minute heat time, and 30 minute cool down time. When up to temperature the autoclave will read 121 C, the timer begin count down from 60 minutes and the psi will average 15. 11. During any program that has a drying stage scheduled, the dry stage begins after the steam exhaust stage. To obtain better drying results from the E models, during the dry stage open the door partially (as described in the autoclave manual page 38) at an angle of approx. 20-25 degrees. The heaters will be on the lower power level and the water vapor and humidity will be evacuated from the chamber efficiently. Without moving the closing device from its position, unscrew the closing device as far as it will go. Allow the door to open to the maximum that the closing device will allow (see picture on page 38 of autoclave manual). 12. At the end of the cycle a buzzer rings for approximately 5 seconds, the START light switches OFF. The air outlet valve is opened to prevent formation of a vacuum. In the event of a program failure, the exhaust valve is opened to release pressure from the chamber and a continuous buzz will sound for 5 seconds followed b an interrupted buzz of 7 seconds. 13. Open the autoclave door. At a minimum lab coat, goggles and long cuffed heat-resistant autoclave gloves should be worn. Always open the hinged autoclave door just 2-3 inches and allow the steam to escape from within the autoclave before opening the door all the way. Stand well back from the autoclave door. Always open the door as slowly as possible. 14. Before removing sterilized items, wait 5 minutes for loads containing only dry glassware, and 10 minutes for autoclaved liquid loads. For non-liquid loads, let the glassware cool for 15 minutes before touching it with ungloved hands.
For liquid loads, let liquids stand for a full hour before touching with ungloved hands. Be sure others in the area know a heat hazard is present. A minimum time interval of 10 minutes needs to be observed between the end of a cycle and the start of a new cycle, to prevent overheating of the autoclave. 15. Record all autoclave runs in autoclave notebook located on top of autoclaves. Record date, time in, time out, items sterilized, autoclave letter, initials of operator and comments. Save autoclave printout. If applicable, record the performance of gasket (daily) and tray (weekly) cleaning. 16. Leave the autoclave door open. Summer and Holiday Procedure Drain autoclaves before summer and breaks to avoid mold growth in water reservoir. Performance of monthly autoclave validation All autoclaves used for waste decontamination must be tested monthly for effectiveness. indicator tape does not prove decontamination effectiveness. Tape only indicates that the outside of the container came to temperature, it does not reflect time or conditions inside the load. Monthly testing is performed by using a biological indicator (BI). 1. Record the autoclave number and date on the BI label. 2. Attach the BI to a stick or string and put in the center of the load. 3. Process the load according to the Biological Waste SOP. 4. Remove the BI and confirm the chemical indicator printed on the label has turned brown. Caution: After processing, the BI is hot and under pressure. Always allow to cool for ten minutes before crushing. Failure to so could cause the glass ampule inside the BI vial to burst which may result in injury. For this reason, safety goggles should be worn when handling and crushing a processed BI. 5. Activate the processed BI within 8 hours after processing by gently crushing the inner glass media tube using a vial crusher. A positive control must also be used. As the control, an unprocessed BI (from the same lot) should be crushed using a vial crusher and incubated each day the autoclaves are tested. 6. Incubate at 55-60ºC (in heat block) for 24 hours checking for spore growth (visual color change from purple to yellow) at regular intervals (i.e. 3, 5, and 8 hours) Growth of surviving spores has been documented in as little as 2 ½ hours. 7. Record all results in autoclave validation log. No color change in the purple medium after full incubation indicates proper sterilization - Negative (no growth). Color change from purple to yellow indicates a Positive (growth) result. This should be reported to the Lab Manager immediately. The autoclave will be taken out of service until the problem is found and resolved. Page 5
Maintenance Instructions All daily, weekly, periodic and yearly maintenance as described in the Operation and Maintenance Manual is recorded in the autoclave notebook. Air jet cleaning, external surface cleaning, replacing water weekly or after 20 cycles, cleaning the water outlet strainer and cleaning the autoclaves with chamber brite is performed bimonthly and recorded in the autoclave notebook. Air Jet Cleaning see page 43 of Operation and maintenance manual (Located in the water reservoir) A dirty air jet is the number one cause of failed spore tests. Procedure: 1. Remove the water reservoir cover. 2. Clean the hole of the jet by manipulating the air trap wire back and forth 10 times. Cleaning water outlet strainer see pages 47 and 47 of Operation and Maintenance Manual WARNING: The strainer s cover is HOT. Do not touch the strainer s cap, mounted on the exhaust line, during and shortly after operation. Touching the hot strainer s cap may cause server injuries. Procedure: 1. Open the strainer cap. 2. Remove the strainer element. 3. Rinse the strainer with water, using a brush if necessary. 4. Reinstall the strainer element. 5. Close the strainer cap. Cleaning with Chamber Brite see pages 49 and 50 of Operation and Maintenance Manual NEVER use bleach, steel wool, a steel brush or anything abrasive to scrub or clean the Chamber. Cleaning Procedure: 1. IMPORTANT All steps in the procedure must be completed without interruption. 2. When autoclave is cold, remove instrument and trays from the autoclave 3. Open the door and spread the contents of a packet of Clean Brite in a straight even line alone the bottom of the chamber from back to front. Model 3870E and 3454 EP in CNHS 107 and 300 require two packets of Chamber Brite 4. Select and start program No. 1 (Scissors without dry). When the cycle is finished it will automatically exhaust. 5. At the end of the exhaust cycle drain the water from the reservoir. 6. Fill the water reservoir with distilled water. Page 6
7. Repeat the sterilization cycle without Chamber Brite powder, to remove any excessive dirt in the pipes. Select and start program No. 1 (Scissors without dry). When the cycle is finished it will automatically exhaust. 8. At the end of the exhaust cycle drain the water from the reservoir. 9. Turn the autoclave off and allow chamber to cool. 10. Remover the tray holder; rinse and wipe the interior of the chamber with a damp cloth. 11. Fill the reservoir with distilled water only. 12. The instrument is read to use. Page 7