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. 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

4. “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 ( 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

5. 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

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 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!!!

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, 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

10. 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

11. 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!!!

12. 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.

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
Diane Graham – Microbiology Tech
Microbiology Building Common Use Autoclaves ,
When no other is available contact: John de la Cuesta and Chris Frost
CNSM Safety Office
, ,

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