CSULB Department of Biological Sciences Common Use Equipment Training Autoclaves

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

Steris Century Gravity Sterilizers 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

“Gravity” Sterilizer Operation Steam jacket is charged with steam Steam is injected into the chamber and air is forced from the chamber by gravity Once air is completely evacuated, an automatic valve closes Steam pressure builds increasing the chamber temperature Pressurized steam penetrates objects and kills all microorganisms Extra information: -Boiling water (100°C) will kill most vegative bacteria, viruses, fungi in approximately 10 minutes -Longer time (0.5-20 hours) is necessary to kill bacterial endospores and some viruses 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 121oC – will kill microorganisms in ~ 15 min

General Operating Guidelines Prior to entering the Autoclave Room 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 _______________________________________________________________________________________________________ Once in the Autoclave Room Make an entry on the user log (next to each autoclave) detailing your usage Verify the chamber is empty & clean prior to putting your items into the chamber Never place items to be autoclaved directly on the chamber surface Always verify that the door closes & seals properly at the start of your cycle Pick up your items in a timely manner. Use caution when handling hot materials Before removing ALLOW HOT ITEMS TO COOL ~10 minutes for liquids & ~5 minutes for other materials

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)

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 needs to 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!!!

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

Considerations When Autoclaving, continued 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

MLSC Common Use Autoclave Cycles:, Unwrapped - materials that are not wrapped or packaged 15 min at 1210C + 5 minute drying step Instant pressure release Metal equipment, tubing, mortar/pestle sets Wrapped - materials that are contained within packaging 15 min at 1210C + 30 minute drying step Pipette tips, microfuge tubes, etc. Glassware - For items made of glass

MLSC Common Use Autoclave Cycles: Liquids – (Liquid 15 or Liquid 20) 15 or 20 minutes at 1210C + slow pressure exhaust to prevent super-heated liquids from boiling over Caps on vessels containing liquids MUST be vented or loosely capped to allow steam to enter and expand Cycles  Liq 15 < 500ml Liq 20 < 1L Waste – For items prior to disposal 45 minute at 1210C + instant pressure release Bags should be loosely closed to allow steam to enter Waste bags must be completely contained in secondary containment!!!

Caution Autoclaves are HOT Steam will burn Autoclaved items are HOT Always wear personal protective equipment (PPE) When loading & 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. Please advise students to the student health center or to take appropriate actions for the severity of the burn.

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)

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.edu Diane Graham – Microbiology Tech Microbiology Building Common Use Autoclaves 562-985-4857, Diane.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.edu

Trainings MLSC Training Location  MLSC 100 Training Present by Michelle Giffin 1st Tuesday of each month @ 10:00am 3rd Tuesday of each month @ 4:00pm no appointment needed – you must be on time MICRO Training Location  Micro 104 Training Presented by Diane Graham Dates: Tuesdays @ 8:30am by appointment only Individual trainings may be requested via your research advisor in the eventuality of time conflict due to class schedules.