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 isn’t 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)

Fundamental autoclave information

Personal Protective Equipment (PPE)

Packaging

Loading / Unloading

Operating cycles

User logs

Hazards

Improper Autoclave Practices

Autoclave use basics

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.

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 121-134°C 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.

Autoclave (steam) sterilisation

What can be autoclaved?

Surgical Instruments

Glassware

Plastic tubes and pipette tips

Solutions and water

Animal food and bedding

Waste

What can’t 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 4°C 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

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

How can you tell if something

has been sterilised?

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 121°C 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.

Example of a Chemical indicator

Autoclave tape - colour change(bottom) indicates a

temp of 80°C 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.

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

Packaging OGTR/PC 2 Waste for

Autoclaving

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.

Don’t forget your PPE!