CSULB Department of Biological Sciences Common Use Equipment Training Autoclaves
Outline/Learning Objectives Understand how gravity autoclaves work Learn how autoclaves are SAFELY operated What materials can & can not be autoclaved The different autoclave cycle types How to keep autoclaves in operational condition Contact info for people that can help with issues Lab is not a test. When in doubt: Ask questions, look it up, refer back to this document
Autoclaves Uses pressurized steam to & elevated temperatures to kill all microorganisms Steam pressures creates a potential for explosion High temperatures create a potential for burns & scalding Improper use can lead to injury costly repairs inconvenient equipment down time 2 units in MLSC 4 units in Micro MLSC Autoclaves: Steris Century Gravity Sterilizers
Gravity Sterilizer Operation 1b) 2) 3) 1) Jacket is charged with steam from the steam generator 2) Steam is injected into the chamber & air is forced from the chamber by gravity 3) Once air is evacuated, an automatic valve closes 4) Steam & pressure build, in the chamber 5) Pressurized steam kills all microorganisms 1a) Note: * Steam heat kills in less time than dry heat - by degrading nucleic acids & proteins via hydrogen bond disruption * Pressure + 121 o C for ~ 15min = kills microorganisms (Endospores & some viruses my take longer)
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!!!! (more on this later) In the Autoclave Room 1) Make an entry on the user log (next to each autoclave) detailing your usage 2) 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 Your item must be in heat stable secondary containment 3) Always verify that the door closes & seals properly at the start of your cycle 4) Pick up your items in a timely manner * The count down will help you estimate when you should return to pick up your items 5) Never leave your items in the autoclave overnight!! *This disrupts the shutdown procedure performed at midnight. Required for flushing!!!
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 a heat stable secondary containment * Must be able to catch ALL broken & spilled material * Needs to be a minimum of 2x larger than the volume you wish to autoclave * Must be able to withstand elevated temperatures (heat stable) NOTE: Not all heat stable containment is marked Never assume your containment is heat stable When in doubt, ASK a knowledgeable person Melted Plastic someone used non-heat stable plastic as secondary containment
Considerations When Autoclaving Remember - Steam Kills Standard Trash - no small particles or dirt Leave unsealed so steam can circulate Trash bags with small particles need to be sealed Small particles blow around & end up in the air removal hole Dry trash w/particles place some water into the bag Do not overfill trash bags 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 Never pour hot liquids down any sink
Considerations When Autoclaving, continued Do not overfill bags, containers or liquids Always loosen the lid of a sealable container If the lid is tight it will crack glass It takes longer to sterilize water as volumes increase Specific heat (SH) of water is 4.184 Joules!!! 4.184 J required to raise 1 g water 1 degree 0 C For example the SH of glass is.84 Joules How to autoclave different volumes of liquid Slide 12 Never fill a vessel greater than 1/2 of its capacity Keeps from liquid boiling over
MLSC Common Use Autoclave Cycles Do not mix your cycles at CSULB Unwrapped - materials that are not wrapped or packaged Metal equipment, tubing, mortar/pestle sets 15 min at 121 0 C + 5 minute drying step Quick pressure release Wrapped - materials that are contained within packaging Pipette tips, microfuge tubes, etc. 15 min at 121 0 C + 30 minute drying step Quick pressure release Glassware - For items made of glass 15 min at 125 0 C + 30 minute drying step Quick pressure release
MLSC Common Use Autoclave Cycles, 2 Liquids (Liquid 15 or Liquid 20) 15 or 20 minutes at 121 0 C + slow pressure exhaust to prevent super-heated liquids from boiling over Caps on vessels containing liquids MUST be loosened Cycles Liq 15 < 500ml Liq 20 < 1L Waste For items prior to disposal 45 minute at 121 0 C + quick pressure release Bags should be loosely closed to allow steam to enter Waste bags must be completely contained in secondary containment!!! See next slide.
Waste Containment Proper Waste Containment Improper Waste Containment
Autoclaves are HOT Caution Always wear personal protective equipment (PPE) and loading & unloading Steam will burn When opening the chamber keep head and arms away Autoclaved items are HOT Allow hot items to cool for ~5-10 min prior to touching If you are burned seek medical treatment as soon as possible & inform the science safety office
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 / melts report them immediately Michelle Giffin MLSC Biology Tech MLSC Building Common Autoclaves 562-985-4038, michelle.giffin@csulb.edu Diane Graham Microbiology Tech Microbiology Building Common Autoclaves 562-985-4857, Diane.Graham@csulb.edu When no other is available contact CNSM Safety Office John de la Cuesta or Chris Frost 562-985-5623, John.delaCuesta@csulb.edu, c.frost@csulb.edu
Training Part 2 MLSC Training Location MLSC 100 Training by Michelle Giffin 1 st Tuesday of each month @ 10:00am 3 rd Tuesday of each month @ 4:00pm No appointment needed you must be on time Individual trainings may be requested via your research advisor in the eventuality of time conflict due to class schedules. MICRO Training Location Micro 104 Training by Diane Graham Dates: Tuesdays @ 8:30am By appointment only