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					Autoclaves Safe and Effective Use
Environmental Health and Safety

University of Central Florida

Topics
Sterilization Principles  Safety Instruction  Proper Use of Cycles  Special Situations  Autoclave sterilization-techniques  Safety concerns  Research concerns, and  UCF Biohazard Waste Policy
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Principles of Steam Sterilization
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Sterilization: the complete destruction of all forms of microbial life, including bacterial spores
Steam under pressure reaches high temperatures

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Principles of Steam Sterilization (cont.)
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The “killing power” of steam is due to its latent heat of vaporization  80 calories: 1 L water to boiling  540 calories: 1 L boiling water to steam

Latent Heat of Vaporization
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For example:

Action of Steam
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Steam contacts a cooler surface, condenses, causing a huge decrease in volume and setting up a negative pressure that draws more steam Condensation occurs as long as there is a temperature differential Action of steam ensures: Surface heating, penetration, and protein coagulation

Thermal Death Time (TDT)
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TDT is the time required to kill a known population of microorganisms in a specific suspension at a particular temperature

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Increasing temperature decreases TDT
Lowering the temperature increases TDT

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Thermal Death Time ( cont.)
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Acidic or basic pHs decrease TDT Fats and oils slow penetration and increase TDT

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Standard Temperature and Pressure

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250 °F (121 °C) and 15 p.s.i.

Safety Instructions
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PPE - heat proof  Gloves  Face Shield  Apron

Safety Instructions (cont.)
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Proper procedures  Close door tightly  Check chamber pressure before opening  Crack door before opening  Wear loose fitting thermal gloves  Let hot liquids cool

Hazard Warnings
Explosion hazard: Autoclaves are not designed to process flammable liquids

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Burn Hazard When Sterilizing Liquids
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Before loading containers of liquids into the autoclave, the caps must be loosened to avoid having the bottles shatter during pressurization. Use vented closures on bottles. Use Type 1 borosilicate glass bottles. Avoid sudden full opening of door at the end of the cycle. Crack one inch, wait 10 minutes.

Burn Hazard When Sterilizing Liquids (cont.)
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Use LIQUID cycle only. Do not jolt hot bottles. Do not move bottles if bubbling or boiling. Allow bottles to cool to touch before attempting to move them from the sterilizer shelf.

Hazard Warnings
Slip hazard: Wipe up spills on slippery floors.  Burn hazard: Remember sterilizer racks and shelves will be HOT after cycle is run. Wear protective gloves, apron, and face shield if needed.
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Hazard Warnings (cont.)
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When removing items from the autoclave, always wear a rubber apron in addition to rubber sleeve protectors, heat resistant gloves and a face shield. Remove the load and let the glassware cool for 15 minutes before touching it with ungloved hands.

Hazard Warnings
Allow sterilizer and everything else to cool before cleaning or performing maintenance.  Burn and shock hazard: Repairs and adjustments should be performed by authorized personnel only.  Never clean sterilizer with abrasives, wire brush, or steel wool.
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Vocabulary
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Gravity  jacket is steam reservoir for chamber Isothermal (“same temperature”)  jacket and chamber charge with steam at the same time and discharge at the same time Pre-vacuum  air is removed from the chamber before steam is allowed in.

Sterilization Techniques
Fabrics and Hard Goods  use gravity cycle (or pre-vacuum if available)  Liquid  use liquids cycle only
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Fabrics and Hard Goods (Gravity Cycle)
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Wrap in “blue surgical wrap”(available from Boundary or Health Center Stores) Limit size and density Arrange load for maximum exposure

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When Mixing Loads
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hard goods on lower shelves fabrics on higher shelves Sterilize liquids separately!

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Liquids (Liquid Cycle Only)
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Use vented closures - no screw caps or rubber stoppers Borosilicate glass (Pyrex) only Liquids cycle only Containers should be no more than two-thirds full When done, crack door 1/2 inch, wait 10 minutes Do not jolt hot bottles Move out cool bottles only

Operating Instructions
Checklist before operating  Automatic Operation  Gravity  Liquids  Laboratory/Isothermal
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Checklist Prior to Operation of Autoclave
Check settings  Choose Gravity or Isothermal  Check date/time  Check paper tape roll  Check printer cartridge
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How Long to Autoclave (1)
Dry goods @ 121 °C
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Time (min) 15 30

Glassware, empty, inverted Instruments, wrapped

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Utensils, wrapped

30

How Long to Autoclave (2)
Biohazardous waste bags, @ 121 °C, loosely tied Time (min) 100 + 90 60

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2 or more bags 1 bag, full 1 bag, half full

Testing available for individual situations

How Long to Autoclave (3)
Liquids, in bottles with vented caps Size (ml) Time (minutes) 75 25 250 30 500 40 1000 45 1500 50 2000 55

Special Situations
Media boils out of bottles  differs among media  Very dense or closed dry objects  beaker full of microfuge tubes with aluminum foil covering  SPF rodent cages
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Summary
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We have gone over sterilization, safety, use of cycles, and special cases Users should feel more confident about their safety as well as their ability to assess their loads and to choose the proper cycle

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Where to get more information
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EH&S, Biological Safety
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José Vázquez - 823-6301


				
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posted:8/28/2009
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