Micro Practical 3 PRINCIPLE AND WORKING OF AUTOCLAVE Dr. Shahzad Ali Assistant Professor Department of Wildlife and Ecology UVAS, Ravi Campus, Pattoki

Autoclave  An autoclave is a pressure chamber used to sterilize equipment and supplies by subjecting them to high pressure (15 psi) saturated steam at 121 °C for around 15–20 (or more) minutes depending on the size of the load and the contents

Uses of Autoclave  Surgical Instruments  Plastic Sharps containers  Glassware  Plastic tubes and pipette tips  Solutions and water  Animal food and bedding  Biohazardous waste

Principles of Autoclave Operation Steam penetrates objects in the autoclave Condensation creates negative pressure and draws in additional steam Moist heat kills microorganisms via coagulation of proteins Two types of autoclaves Gravity Displacement Vacuum/Gravity Assisted

Types of Autoclaves Other Manufacturers: NOTE: Even though autoclaves on UNC campus operate similarly in the context of mechanics, training is required in the use of each autoclave since not all autoclaves are programmed the same. SterisCastle Getinge

PROCEDURE/Working OF AUTOCLAVE

Preparation and Loading of Materials  Prepared the material for autoclaving with indicator paper/tape  Fill he bottom of autoclave with water, up to support  Place the material to be sterilized in the autoclave  Always put bags of biological waste into pans to catch spills.  Position biohazard bags on their sides, with the bag neck taped loosely.  Leave space between items to allow steam circulation.  Household dishpans melt in the autoclave. Use autoclavable polypropylene or stainless steel pans  Close the lid and make sure that the rubber washer is in the groove  Screw down the clamps firmly  Open the air outlet valve

Preparation and Loading of Materials  Turn on the heating. Do not leave autoclave un-attended  Close the outlet valve when a constant jet of steam issue from it  Once the expected temperature is reached, reduce the heat to maintain temperature  Do not touch the drainage tape or the outlets or safety valves while heating under pressure  When the required time is up, turn off the heating completely  When temperature falls below 100 C, open the outlet valve slowly. Do not leave outlet valve opened for too long  Never unscrew the lid clamps and open the lid until the hissing sound has stop  Leave the sterilized material to cool before removing it from autoclave  Check whether he autoclave tape (used as indicator) has turn black

 Indicator Tape (Heat Sensitive Tape): Indicator tape in an “X” pattern. Not autoclaved (no markings on tape) Autoclaved (markings on tape)

PPE for Autoclave Users  Eye Protection  Lab Coat, Buttoned  Closed-toed Shoes  Heat-resistant Gloves

Precautions for Time Selection  Take into account the size of the articles to be autoclaved. A 2-liter flask containing 1 liter of liquid takes longer to sterilize than four 500 mL flasks each containing 250 mL of liquid.  Material with a high insulating capacity (animal bedding, high sided polypropylene containers) increases the time needed for the load to reach sterilizing temperatures.  Autoclave bags containing biological waste should be autoclaved for 1 hour to assure decontamination.  In case of culture media mostly autoclaved at high pressure (15 psi) saturated steam at 121 °C (or higher) for around 15–20 minutes

MaterialTemperatureTime Laundry121°C (250° F)30 Minutes Trash (Biohazard bags containing infectious waste.) 121°C (250° F)1 Hour Glassware121°C (250° F)1 Hour Liquids121°C (250° F) Minutes Animals121°C (250° F)8 Hours Autoclave Waste Decontamination Procedures

Precautions for Removing the Load Put on Personal Protective Equipment Allow the autoclave to completely finish cycle Pressure gauge must read zero Verify cycle conditions were met Open door slightly to allow steam to escape While slowly opening the autoclave door, make sure to keep head, face, and hands away from the opening. Verify that heat sensitive tape has changed color or word “autoclaved” has appeared. Allow contents to cool before removal When removing biohazard bags, always pick up from the top, taped area of the bag. Never handle biohazard bags by grabbing from the sides or bottom.