Fundamental Autoclave Techniques

Version 3, September 2010

Veronika Tatarinoff GSBmE University of NSW Brandy Nelson University of Kentucky

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

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

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.

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.

Micro-organisms
Include bacteria, viruses, fungi & spores Found on every external surface including

respiratory tract and 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.

Sterilisation methods
There are a number of 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 (liquids only)

Autoclave use basics

Fundamental autoclave information

Personal Protective Equipment (PPE)

Packaging Loading / Unloading

Operating cycles
User logs Hazards

Improper Autoclave Practices

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.

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 20-60 minutes are generally used. Condensation creates negative pressure and draws in additional steam. Moist heat kills microorganisms via coagulation of proteins.

What can be autoclaved?

Surgical Instruments

Glassware
Plastic tubes and pipette tips Solutions and water

Animal food and bedding

Waste

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 in this way or they will melt. Items that may be damaged by steam, should be sterilised by another method. Chemicals (e.g. bleach) emit toxic fumes when autoclaved Incompatible chemicals e.g. acids , bases , organics solvents It is essential to ensure that your material/s can be autoclaved.

PPE for Autoclave Users

Eye Protection

Lab Coat, Buttoned

Closed-toed Shoes Heat-resistant Gloves

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 Autoclave paper bags/roll Metal bins or containers Pyrex or Shott glassware Plasticware Do not overfill containers Do not tighten lids Label Identify contents, owner & date

Loading the Autoclave

Load material to allow

efficient steam penetration Autoclave clean items and waste separately Do not allow material being autoclaved to touch the sides or top of the chamber Ensure load probe cord is not in trolley path to prevent damage

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 SWPI005 & I006 and will be demonstrated to you.

GSBME Autoclaves

Getinge HS55 (automated)

Atherton Benchtop model (small manual)

Getinge HS 55 Automated
This Autoclave is a large stand alone model. It is controlled by a microprocessor and has both set programs and manual parameter selection. If your material requires specific parameters please see autoclave custodian.

P02 - POROUS CYCLE

USE: DRAPES / WRAPPED PACKAGES / BOTTLES Items that are required to be dry at the completion of the cycle and/or requiring greater steam penetration.

PARAMETER SETTINGS STERILISE 121C 16 minutes DRYING TIME 5 minutes DRYING AIR PULSE 10 minutes

P09 - HARD GOODS CYCLE

USE: INSTRUMENTS/SOLID ITEMS Items of equipment that do not require wrapping, drying or prolonged cooling.

PARAMETER SETTINGS STERILISE 121C 16 minutes DRYING TIME 5 minutes

P04 FLASH (HARD GOODS) CYCLE

USE: INSTRUMENTS/SOLID ITEMS Items of equipment for surgical procedures that do not require wrapping, drying or prolonged cooling .

PARAMETER SETTINGS STERILISE 126C 12 minutes DRYING TIME 3 minutes

Liquids
Liquids take longer to heat up and cool down than

most solid materials. A control (similar fluid) at the maximum volume of sample load is required for autoclaving liquids. The load probe is placed in the control bottle to ensure that the liquids reach the correct temperature for the correct time to ensure sterilisation. Lids must be loose to prevent explosion. Liquids must be in heat treated laboratory glassware with room for fluid expansion. A liquid cycle must be used to prevent boiling over.

LIQUID CYCLE P05 Fo Liquids, open

USE: SALINE, WATER Non heat sensitive liquids that require a probe in a control volume to ensure specified parameters are reached. A slow pressure release is used to avoid boiling over and allow cool down PARAMETER SETTINGS STERILISE 121C 16 minutes DRYING RAMP TIME 0.05/m

Ensure that lid is loose on the bottle/s during sterilisation

P10 - WASTE CYCLE

USE: OGTR/PC2 TISSUE CULTURE WASTE BINS Items in the bins are contaminated with biological materials and do not require wrapping, drying or prolonged cooling. PARAMETER SETTINGS STERILISE 121C 60 minutes DRYING TIME 5 minutes Following completion & verification of sterilisation, waste must be double bagged, labeled and placed into yellow bins in 4C cold room pending disposal by UNSW contractor.

Bench top Autoclave

The GSBME small benchtop model is a 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.

Primarily this autoclave is a back-up if

required.

Autoclave User Log

User Log must be completed with every use
User Logs are maintained for at least five years
Date Time Authorised User Cycle Type
e.g. fluid, hard goods

Items /comments eg drapes,

bottles, waste, validation

Unloading the Autoclave

Put on Personal Protective Equipment
Verify cycle conditions were met Stand clear of door when opening

Allow contents to cool before removal

Carefully remove items

Be especially careful with fluids and plastic bins

How can you tell if something has been sterilised?

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

* Chemical

* Biological * Physical

Indicators
Chemical indicators, can be Biological indicators include

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

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

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.

these separate indicators, autoclaves have temperature and pressure gauges visible from the outside. A printout maybe available to confirm conditions were met.

Example of a Chemical indicator

Autoclave tape - colour change(bottom) indicates a temp of 80C has been reached.

Examples of Physical indicators

Untreated physical indicators Class 5 & 6

Colour change in treated (bottom) indicator, sterilisation confirmed

Examples of Biological indicators

Unprocessed

Processed - Failed

Processed - Passed

Autoclave Cycle Verification

Each Autoclave Load (automatic documentation) Have the sterlisation conditions been met? Document all autoclave runs in the User Log Review the printout report & any other indicator that you may have used. If conditions were met, remove the load. Printout report can be filed in lab book. For validation cycles indicator and print our are filed in the equipment validation log. If conditions were not met, do not remove the load, review and repeat cycle. If conditions are not met again, contact Autoclave custodian.

Packaging OGTR/PC 2 Waste for Autoclaving

Tie waste bag with string, leave bag in metal biohazard

bin Fix autoclave tape to bag Leave bin lid off to one side 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.
Indicator in the centre of the load

OGTR/PC2 Biowaste Bins

Over full bin

Hazards associated with using autoclaves

Burns from hot autoclave, sterilised items, steam. Sharp/hot/heavy items falling trolley onto operator. Cuts from broken glass Possible noxious/toxic fumes released from melted

plastics Biohazard contamination (OGTR/PC2 waste) Chemical incompatibility In an emergency call x56666 security.

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

Autoclave Explosion

Results of Improper Autoclave Use

Improper Autoclave Practices

Additional Information
It is essential that you read and understand the following GSBME documents prior to your FAT quiz. 1. SOP S006 - Guideline to sterilisation at GSBME 2. SWPI005- Operation of getinge autoclave SWPI006- Operation of small autoclave 3. Risk Assessment - 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.

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 by the trainer.

Dont forget your PPE!