1. CSULB Department of Biological Sciences Common Use Equipment Training Autoclaves

2. Outline/Learning Objectives Obtain an understanding of how “gravity” autoclaves work Gain practical knowledge of how MLSC common use autoclaves are SAFELY operated – Learn what materials can be autoclaved and what materials cannot – Learn about the different autoclave cycle types and their appropriate use Obtain contact information for individuals that can assist with problems or issues

3. Autoclaves Use pressurized steam to generate elevated temperatures necessary to kill all microorganisms – Steam pressures creates a potential for explosion – High temperatures create a potential for burns and scalding Improper use can lead to – injury – costly repairs – inconvenient equipment down time MLSC Autoclaves: Steris Century Gravity Sterilizers

4. “Gravity” Sterilizer Operation 1)Air is displaced from the chamber by gravity 2) Once air is completely evacuated, the automatic valve closes. 3) Steam pressure builds increasing the chamber temperature 4) Pressurized steam penetrates objects and kills all microorganisms Note: * Moist heat kills in significantly less time than dry heat Steam kills by degrading nucleic acids and proteins via hydrogen bond disruption * Under pressure & at 121 o C – will kill microorganisms in ~ 15 min

5. General Operating Guidelines ALWAYS use proper personal protective equipment (PPE) – At minimum, heat resistant gloves, lab coat, closed-toed shoes & eye protection ALWAYS verify that any materials you plan to autoclave can withstand the elevated temperatures, ESPECIALLY your secondary containment - Never place items to be autoclaved directly on the chamber surface 1)Make an entry on the user log (next to each autoclave) detailing your usage 2)Make sure the autoclave chamber is empty 3)Always verify that the door closes and seals properly at the start of your cycle and that settings match your desired parameters 4)Use caution when handling hot autoclaved materials Before removing hot items from the autoclave ALLOW HOT ITEMS TO COOL ~10 minutes for liquids & ~5 minutes for other materials

6. Autoclaving Materials Items/materials that CAN be autoclaved – Most aqueous liquids (buffers, media, etc.) – Glassware – Heat stable plastics (pipette tips, microfuge tubes, etc.) 7 major items/materials that CANNOT be autoclaved – Acids, bases, phenol, flammable liquids – Radioactive materials – Any non-heat stable materials (without proper containment) – BSL-2 or higher for final waste cycles (contact the CNSM Safety Office if you will be working with BSL-2 or higher materials)

7. Secondary Containment All items that are placed in the autoclave must be contained with in an autoclavable vessel Vessel must be able to catch all broken or spilled material. – Vessel should be a minimum of 2x larger than the volume you wish to autoclave – Vessel must be able to withstand elevated temperatures Not all vessels are marked as autoclavable!!!! Never assume your secondary containment is autoclavable – When in doubt, ASK a knowledgeable person!!!

8. Considerations When Autoclaving Remember Steam Kills – Trash bags should be left unsealed Sealed bags do not allow steam to adequate circulate – Leave plenty of room for steam circulation – Do not overload the chamber Make sure items are not touching sides or top of the chamber – Only surfaces that are contacted by steam will be sterilized Arrange items such that steam can penetrate & reach all surfaces

9. Considerations When Autoclaving Do not overfill bags, containers or liquids in vessels Never fill a vessel greater than 1/2 of its capacity Always loosen the lid of a sealable container – If the lid is tight it will explode Never seal the lid of a sealable container right after an autoclave – This creates a vapor lock It takes longer to sterilize large a volume of liquid than a small volume. – Take time for heat to penetrate the liquid Dense Air Steam

10. MLSC Common Use Autoclave Cycles: Wrapped, Unwrapped & Glassware Wrapped - materials that are contained within packaging – Gravity cycle with a 30 minute drying step – Pipette tips, microfuge tubes, etc. Unwrapped - materials that are not wrapped or packaged – Gravity cycle with a 5 minute drying step – Metal equipment, tubing, mortar/pestle sets Glassware - For items made of glass – Gravity cycle at 121°C with a 30 minute drying step

11. MLSC Common Use Autoclave Cycles: Liquids and Waste Liquids – (Liquid 15 or Liquid 20) – Following sterilization, chamber pressure is slowly exhausted to prevent super-heated liquids from boiling over – Caps on vessels containing liquids MUST be vented or loosely capped to allow steam to enter Waste – For used laboratory materials that must be autoclaved prior to disposal – A liquids cycle with a 45 minute sterilization step – Bags (containing Petri dishes, tubes, etc.) should be loosely closed to allow steam to enter

12. Caution Autoclaves are HOT even if they have not been used for that day yet – Always wear personal protective equipment (PPE) When loading and unloading Steam will burn – When opening the chamber keep head and arms away Autoclaved items are HOT – For all items allow to cool for the appropriate amount of time prior to touching with out hot gloves If you are burned you should seek medical treatment as soon as possible.

13. MLSC Common Use Autoclave Cycles: Flush Steam generator MUST be flushed to prevent buildup of mineral scale – Must be done before each day of use and recorded on the user log Critical for continued proper autoclave operation Follow the on-screen instructions 1) Check the generator Pressure Gauge (must be at 0 psi) 2) Turn the generator drain valve (yellow) so that it is parallel with the pipe (open) 3) Start the flush (on screen prompt) and wait 5 min 4) Turn yellow handle back perpendicular (Closed)

14. List of Important Contacts If you have any spills or leaks you must report them immediately Michelle Giffin – MLSC Biology Tech – MLSC Common Use Autoclaves – 562-985-4038 michelle.giffin@csulb.edumichelle.giffin@csulb.edu Diane Graham – Microbiology Tech – Microbiology Building Common Use Autoclaves – 562-985-4857, Diane.Graham@csulb.eduDiane.Graham@csulb.edu When no other is available contact: John de la Cuesta and Chris Frost CNSM Safety Office 562-985-5623, John.delaCuesta@csulb.edu, c.frost@csulb.eduJohn.delaCuesta@csulb.educ.frost@csulb.edu