Fundamental Autoclave Techniques

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Fundamental Autoclave Techniques

• June, 2008
• Veronika Tatarinoff
• GSBME University of NSW
• Brandy Nelson
• University of Kentucky

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The following information is directed at
aseptic/sterile surgical, tissue culture (PC1
PC2)and QC1 techniques at the GSBME.
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GSBME Autoclave Training Program

• This presentation forms part of the GSBME
  Autoclave training program.
• 1. Fundamental Autoclave Techniques (FAT)
• 2. Review of supporting literature (SOP,
  SWP, RA)
• 3. FAT quiz
• 4. Demonstration of GSBME Autoclave use
• 5. Competency assessment
• 6. Authorisation sign off in WITA

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Learning outcomes

• At the end of this session participants will be
  able to
• Describe the mechanisms of autoclave
  sterilisation
• Identify autoclave/s at GSBME
• Identify materials that can be autoclaved
• Package and label items for sterilization
• Correctly load/unload the autoclave
• Identify and select appropriate sterilisation
  cycle/s
• Verify autoclave sterilisation process
• Maintain log of sterilisation
• Dispose of biowaste appropriately
• Identify and use the Personal Protective
  Equipment (PPE) and devices in a safe manner.
• Identify hazards and risks to self and others,
  improper autoclave techniques and identify
  appropriate responses to injury.

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What does aseptic mean?preventing
putrefaction rotting, decomposition by
securing the absence of bacteria Oxford
Dictionary

• Disinfection.
• The process of destroying or inhibiting the
  growth of micro-organisms on a surface.

• Sterilisation.
• The destruction of all living micro-organisms,
  including viruses and spores. This is an absolute
  state.

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Micro-organisms

• Include bacteria, viruses, fungi spores
• Found on every external surface including
  respiratory tract, alimentary canal
• A micro-organism infection occurs when the
  micro-organism grows in an area where it isnt
  normally found or in numbers greater than
  normal.
• Surgical and cell culture procedures provide an
  ideal opportunity for micro-organisms to cause
  problems.

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Sterilisation methods

• There are a number or methods which can kill or
  remove micro-organisms including
• Heat (dry or steam autoclave)
• Gas (eg Ethylene Oxide)
• UV or gamma irradiation
• H202 plasma
• Liquid sterilisation (eg glutaraldehyde)
• Filter sterilisation (fluids only)

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Autoclave use basics

• Fundamental autoclave information
• Personal Protective Equipment (PPE)
• Packaging
• Loading / Unloading
• Operating cycles
• User logs
• Hazards
• Improper Autoclave Practices

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Critical Sterilisation parameters

• Autoclaving uses high temperature, high pressure
  and steam to kill organisms.
• The critical autoclaving parameters are
• ? temperature
• ? steam under pressure
• ? time
• Items have to be hot enough for long enough in
  the presence of steam to disrupt the proteins of
  the micro-organisms.

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Autoclave (steam) sterilisation

• Water is boiled under pressure and the steam is
  enclosed within the chamber.
• Objects placed in the chamber and the steam
  circulates around and penetrates them.
• Temperatures of 121-134C at set pressure for
  20-50 minutes are generally used.
• Condensation creates negative pressureand draws
  in additional steam.
• Moist heat kills microorganismsvia coagulation
  of proteins.

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What can be autoclaved?

• Surgical Instruments
• Glassware
• Plastic tubes and pipette tips
• Solutions and water
• Animal food and bedding
• Waste

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What cant be autoclaved?

• The effectiveness of autoclaving depends on
  steam, the steam must be able to penetrate the
  material being autoclaved. Goods that are packed
  in impervious packaging cannot be autoclaved.
• Because damp heat is used, heat labile products
  (such as some plastics) cannot be sterilised this
  way or they will melt. Some paper or other
  products that may be damaged by the steam, should
  also be sterilised another way.
• Chemicals (e.g. bleach)
• It is essential to ensure that your material/s
  can be autoclaved.

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PPE for Autoclave Users

• Eye Protection
• Lab Coat, Buttoned
• Closed-toed Shoes
• Heat-resistant Gloves

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Packaging for Autoclaving

• Be sure the material can be autoclaved
• No volatile chemicals, acids, bases, sharps
• Utilize containers and autoclave bags appropriate
  for autoclaving
• Clear or yellow biohazard bags
• Sterilope paper bags
• Metal bins or containers
• Pyrex or Shott glassware
• Plasticware
• Do not overfill containers
• Do not tighten lids
• Label Identify contents, owner date

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Loading the Autoclave

• Load material to allow efficient steam
  penetration
• Autoclave clean items and waste separately
• Do not allow material to be autoclaved to touch
  the sides or top of the chamber

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GSBME AUTOCLAVE USE

• Consult with the Animal House manager if animal
  surgery is in progress before using the
  autoclave.
• Surgery has priority.
• ALWAYS CHECK Sterilisation parameter SETTINGS
  BEFORE USE.
• Different materials or quantities may require
  different sterilisation temperatures and times.
  There is a guide on the wall opposite the
  autoclave and in SOP S006.
• Specific instruction on operation of each GSBME
  autoclave is given in SWPs and will be
  demonstrated to you.

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GSBME Autoclaves
Atherton Centenary Series (automated)
Atherton Benchtop model (manual)
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POROUS CYCLE

• USE DRAPES / LINEN WRAPPED PACKAGES
• Items that are required to be dry at the
  completion of the cycle. A drying cycle at the
  end uses a vacuum to dry the load .
• PARAMETER SETTINGS
• STERILISE 121?C
• 20 minutes
• DRYING 20 minutes drapes OR
• 10 minutes single paper wrapped packages.
• DATA LOG 5 minutes

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HARD GOODS CYCLE

• USE INSTRUMENTS (FLASH), BOTTLES
• Items of equipment that do not require wrapping,
  drying or prolonged cooling. Exhaust may also be
  called vapour removal.
• PARAMETER SETTINGS
• STERILISE 126?C
• 12 minutes
• EXHAUST 5 minutes
• DATA LOG 5 minutes

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FLUIDS CYCLE

• USE SALINE, WATER, MEDIA
• Items that require prolonged cooling to ensure
  that the rapid temperature change on removal does
  not
• PARAMETER SETTINGS
• STERILISE 121?C 20 minutes
• COOL DOWN 20 minutes
• DATA LOG 5 minutes
• Ensure that the lid is not tightly closed on the
  bottle/s during sterilisation

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WASTE HARD GOODS CYCLE

• USE PC1 PC2 TISSUE CULTURE WASTE
• Items that are contaminated with biological
  materials and do not require wrapping, drying or
  prolonged cooling.
• PARAMETER SETTINGS
• STERILISE 121?C
• 60 minutes
• EXHAUST 5 minutes
• DATA LOG 5 minutes
• Following completion verification of
  sterilisation, waste must be double bagged and
  removed from the Animal Facility. Waste bags are
  labelled and placed into yellow bins in 4C cold
  room pending disposal by UNSW contractor.

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Atherton Centenary Series

• Specific instructions will be given in swp and
  demonstrated.
• Warm up run
• The first autoclave run of any day is likely to
  come up with an aborted message .
• This is because the jacket and chamber of the
  autoclave have not warmed up.
• You must do a warm up run at the beginning of any
  autoclaving day.
• PARAMETER SETTINGS
• Hard goods cycle
• TEMPERATURE 126?C
• TIME 5 minutes
• EXHAUST 2 minutes
• DATA LOG 5 minutes

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Bench top Autoclave

• The GSBME small benchtop model is totally manual,
  gravity displacement style of autoclave with no
  drying or cool down cycle.
• Modification of parameter settings is made by
  adjusting gauges directly on the autoclave.
• Specific instructions will be given in swp and
  demonstrated.

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Autoclave User Log

• User Log must be completed with every use
• User Logs are maintained for at least two years

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Unloading the Autoclave

• Put on Personal Protective Equipment
• Allow the autoclave to completely finish cycle
• Pressure gauge must read zero
• Verify cycle conditions were met
• Open door slightly to allow steam to escape
• Allow contents to cool before removal
• Carefully remove items
• Be especially careful with fluids and plastic bins

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How can you tell if something has been sterilised?

• INDICATORS can be used to ensure an autoclave
  reached the correct temperature and pressure for
  the correct amount of time.
• They fall into 3 broad categories Chemical
  Biological Physical

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Indicators

• Chemical indicators, can be found on medical
  packaging and autoclave tape, and these change
  colour once the correct temperature conditions
  have been met. This indicates that the object
  inside the package, or under the tape, has been
  exposed to heat.
• Physical indicators often consist of an alloy
  designed to melt only after being subjected to
  121C for 15 minutes. If the metal has melted,
  the change will be visible.

• Biological indicators include Attest devices.
  These contain spores of a heat resistant
  bacterium. If the autoclave did not reach the
  right temperature, the spores will germinate, and
  their metabolism will change the colour of a
  pH-sensitive chemical.
• Printout gauges. As well as these separate
  indicators, autoclaves have temperature and
  pressure gauges visible from the outside. A
  printout maybe available to confirm conditions
  were met.

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Example of a Chemical indicator
Autoclave tape - colour change in treated
(bottom) indicator, right temperature has been
reached.
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Examples of Physical indicators
Colour change in treated (bottom) indicator,
sterilisation confirmed
Untreated physical indicators Class 5 6
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Autoclave Cycle Verification

• Each Autoclave Load (automatic documentation)
• Review the printout report any other indicator
  that you may have used.
• If conditions were met, remove the load, sign,
  date, User Log. Printout report can be filed in
  lab book.
• If conditions were not met, do not remove the
  load, repeat autoclave cycle.
• If conditions are not met again, Contact Lab or
  Animal Facility manager

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Autoclave Verification Program

• Monthly Verification for autoclaves
• Physical Indicator Test (Class 5 or 6)
• Checks all conditions of autoclave cycle, time,
  temperature, and pressure
• Program will ensure that all potentially
  hazardous biological waste is properly
  decontaminated prior to deposition in landfill.
• Program requires testing of the ability of
  autoclaves to kill microorganisms

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Packaging Waste for Autoclaving

• Untie string used in PC waste transfer
• Carefully add water to autoclave bags
• Add approximately 500ml of water per waste bag
• Aids in reaching appropriate temperature quickly,
  creates steam in the autoclave bag
• Insert Physical indicator on a string or rod into
  center of each load.
• Check that all containers including bags are
  vented
• Leave bin lid off for autoclaving
• It is essential that each biowaste load has been
  verified as sterile prior to transfer to cold
  room pending removal by UNSW waste contractor.

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PC1 PC2 Biowaste Bins
Over full bin
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Hazards associated with using autoclaves

• Burns from hot autoclave, sterilised items,
  steam.
• Stackable trolley falling off mobile stand
  dropping sharp/hot/heavy items onto operator.
• Cuts from broken glass
• Possible noxious/toxic fumes released from melted
  plastics
• Biohazard contamination (PC2 waste)
• Chemical incompatibility
• In an emergency call x56666 security.

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Importance of Proper Autoclave Use

• Tremendous pressure from steam in chamber
  provides explosive potential
• High temperatures and presence of extremely hot
  water creates potential for burns and scalding
• Inadequate decontamination allows for the
  potential of biological hazards and personnel and
  environmental contamination

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Autoclave Explosion
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Results of Improper Autoclave Use
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Improper Autoclave Practices
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Additional Information

• It is essential that you read and understand the
  following GSBME documents prior to your FAT quiz.
• SOP S006 Guideline to sterilisation at GSBME
• SWP Basic Autoclave operation at GSBME
• Risk Assessment RA Basic use of Autoclaves
• It is essential that you have completed PC2
  training, quiz and authorisation prior to entry
  to PC labs at GSBME.
• It is essential that you have completed UNSW
  Animal Care and Ethics training and have ACE
  approval to conduct any animal work.

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Where do I go from here?

• This fundamental autoclave techniques is
  followed by a quiz. Download from OHS page on
  GSBME School Website
• http//www.gsbme.unsw.edu.au/ohs/autoclave
• submit your completed quiz to either the Lab or
  Animal Research Facility Manager who will
  arrange
• Demonstration of autoclave/s use
• You will then complete a competency assessment
• Authorisation for autoclave use occurs when
  Signed off as competent in WITA by either
  manager.

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Dont forget!