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Fundamental Autoclave Techniques

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Title: Fundamental Autoclave Techniques


1
Fundamental Autoclave Techniques
  • June, 2008
  • Veronika Tatarinoff
  • GSBME University of NSW
  • Brandy Nelson
  • University of Kentucky

2
The following information is directed at
aseptic/sterile surgical, tissue culture (PC1
PC2)and QC1 techniques at the GSBME.
3
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

4
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.

5
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.

6
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.

7
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)

8
Autoclave use basics
  • Fundamental autoclave information
  • Personal Protective Equipment (PPE)
  • Packaging
  • Loading / Unloading
  • Operating cycles
  • User logs
  • Hazards
  • Improper Autoclave Practices

9
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.

10
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.

11
What can be autoclaved?
  • Surgical Instruments
  • Glassware
  • Plastic tubes and pipette tips
  • Solutions and water
  • Animal food and bedding
  • Waste

12
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.

13
PPE for Autoclave Users
  • Eye Protection
  • Lab Coat, Buttoned
  • Closed-toed Shoes
  • Heat-resistant Gloves

14
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

15
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

16
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.

17
GSBME Autoclaves
Atherton Centenary Series (automated)
Atherton Benchtop model (manual)
18
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

19
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

20
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

21
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.

22
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

23
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.

24
Autoclave User Log
  • User Log must be completed with every use
  • User Logs are maintained for at least two years


25
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

26
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

27
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.

28
Example of a Chemical indicator
Autoclave tape - colour change in treated
(bottom) indicator, right temperature has been
reached.
29
Examples of Physical indicators
Colour change in treated (bottom) indicator,
sterilisation confirmed
Untreated physical indicators Class 5 6
30
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

31
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

32
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.

33
PC1 PC2 Biowaste Bins
Over full bin
34
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.

35
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

36
Autoclave Explosion
37
Results of Improper Autoclave Use
38
Improper Autoclave Practices
39
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.

40
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.

41
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