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Autoclaving Instructions

INDEX

 

Purpose and Principles

Autoclaving is used for:

  • sterilisation of media and equipment required for growing microorganisms.
  • sterilisation of cultures and waste materials prior to disposal.

During autoclaving the contents (liquid or solid) become exposed to saturated steam at the required temperature for the appropriate length of time.

Air Removal

For efficient sterilisation exclusion of air is a primary goal. If air is present steam may become superheated with a relative humidity less that 100% and sterilisation efficiency is decreased.

Air removal is achieved by:

  1. Downward displacement of steam, or
  2. Evacuation by pump prior to sterilisation cycle

Downward Displacement Steriliser

As steam enters the chamber, it fills the upper areas as it is less dense than air. This compresses the air at the bottom, forcing it out through the strainer and drain pipe past the temperature sensing device to waste. Only when air evacuation is complete should discharge stop. (This can be done manually or automatically).

High Vacuum Autoclaves

High Vacuum Autoclaves are not suitable for the sterilisation of liquids and are primarily used for non aqueous materials or porous loads where air is likely to be trapped in cavities or gaps. The vacuum line in pre-vacuum sterilisers should be fitted with appropriate air filters to prevent the release of infectious aerosols into surrounding areas.

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Sterilisation Times

The sterilisation time must be the sum of the steam penetration time (time required for the entire load to reach the set temperature) and the holding time (minimum time required for complete sterilisation at the set temperature). The time-temperature profile of infectious waste being autoclaved varies with the load and type of container used in the process.

THE TEMPERATURE ATTAINED BY THE CONTENTS AND THE HOLDING TIME ARE CRITICAL PARAMETERS IN STERILISATION.

Cycle times based on the temperature of the chamber have no relevance to the efficacy of the process.

Cycle times can only be determined for any given load type by the use of thermocouples (see below) placed at centre of the load and inside large volumes of liquids.

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Monitoring Sterilisation

In most states autoclaves are classified as boilers or pressure vessels and require registration and inspection under state law (Victorian WorkCover Authority). An accredited inspector must inspect large autoclaves every 3 years.

Temperature controllers, recording charts and timers must be calibrated every six months (eg. use NATA calibrated thermometers to calibrate thermocouples).

Pressure gauges need not be calibrated but must read true with respect to the required pressure at the nominated calibrated temperature.

Autoclave Tape

Autoclave tape only shows that the tape has been exposed to heat (80C). No information on time, steam penetration or temperature/pressure can be inferred.

Biological Indicators

Biological indicators are recommended as an adjunct to the daily monitoring of autoclave cycle parameters.

Biological indicators can be used to confirm thermocouple data when checking heating profiles and validating autoclave operational parameters. They should not be used in isolation as a measure of sterilisation efficacy.

Manufacturers of biological indicators using Bacillus stearothermophilus claim that spores will survive 5 minutes at 121C but not 15 minutes at 121C.

Check each lot by confirming survival for 5 minutes at 121C.

Biological indicators have several limitations:

  • Results are retrospective (several days). Autoclaved materials may be required earlier.
  • They cannot be used for checking the centre of large liquid loads.
  • They are less accurate in determining holding times than temperature measurement.
  • They cannot be used for sterilisation cycles below 121C.

Chemical Indicators

There are many types of chemical indicators in use and one must check the performance of a particular type and use it in conjunction with temperature and time measurements only.

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Safe Operating Procedures

A risk assessment for autoclave use is available.

Training MUST be given to any autoclave user.

Loading of downward displacement autoclaves:

  1. Place material in small shallow containers
  2. Pack small bottles and tubes in open mesh baskets
  3. Loosen screw caps
  4. Open autoclave bags
  5. Do not use large containers which may trap air to sterilise small articles
  6. Sterilise large empty containers by placing them on their side
  7. Materials and objects should be loosely packed to allow free circulation of steam and easy removal of air

Before opening any autoclave that has completed a cycle check the following points:

  1. The chamber pressure has returned to 0 (the autoclave pressure can be read from the pressure gauge). Do not attempt to open an autoclave with pressure in the chamber. Call someone from the maintenance staff or the Departmental Manager if the pressure will not come down to 0.
  2. In the case of liquids, do not attempt to remove them from the autoclave until the temperature has dropped to 80C. This reduces the problem of liquids boiling over due to residual pressure in the vessels at higher temperatures. Even if the chamber temperature is less than 100C, the liquid in larger volumes can be superheated and boil over when moved.

Unloading:

  1. Wear laboratory coat and eye/face protection
  2. Heat resistant gloves must always be worn (elbow length preferable) when items are unpacked from the autoclave. This will prevent your arms from being burnt.
  3. Open the door slowly to let steam escape
  4. When opening autoclaves, particularly the low level type, do not allow your face to get close to the door, as residual steam can rush out and burn unprotected skin and eyes.
  5. Do not attempt to stretch your body into the autoclave with exposed arms to reach contents.
  6. Always wear protective shoes, as vessels containing liquids may break during unpacking and pour hot liquids onto your legs and feet.

In the event of receiving a burn, the immediate first-aid treatment is to hold the burnt area under running COLD tap water for 15 minutes.

Routine "housekeeping":

  • Use indicators regularly
  • Determine operating cycles with thermocouples
  • Keep chamber and drain clean

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Temperature/Pressure Units and Conversion Factors

Temperatures Pressures Conversion Factors
C F kPa lb/in2 (p.s.i.)
100 212 69 10 1C= 1.8C
116 240 101 15 1F= 0.56C
121 250 138 20 1kPa= 0.145 lb/in2 (p.s.i.)
132 270 185 27 1lb/in2 (p.s.i)= 6.9 kPa

 

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Operating Procedures for Departmental Ground floor Autoclaves

Note: Autoclaves are available to authorised users only

Extracted form Media Preparation Unit SOPs

Operating Instructions For Autoclave A (Rubbish):

(Extract from MPU1009, SOP#1020 authorised Alida Scholtes 28-4-2000, Reviewed 5/6/2001)

  1. Autoclave A is directly connected to a steam supply.
  2. Autoclave tins x 12 is the maximum number per load. Lids must be removed before placing in autoclave. Lids are autoclaved separately.
  3. Full autoclave bags may only be one layer high. If they are only 1/2 full then they can go on both shelves as there is enough room for steam to penetrate. Each autoclave bag must have a "water bomb" in it. ("Water bomb"=200 ml of water in an old petri dish bag which melts/disintegrates at low heat allowing water to be dispersed throughout autoclave bag before autoclave bag collapses). The water bomb allows steam to be generated within the autoclave bag after it collapses. Ensure that autoclave bag is fully inside the chamber before adding water bomb. If there are any leaks, they are confined within the autoclave and thus sterilised.
  4. Document all information concerning the autoclave cycle on the Autoclave Record Form MPU-FM102
  5. After loading, close door and do as follows:
    1. Set temperature controller to required temperature exactly (121C usually).
    2. Set timer to time required for sterilisation. Use push buttons.
    3. Autoclave bags: 121C for 80 mins (and water bomb).
      Autoclave bins: 121C for 60 mins.
    4. Turn steam control to STER (sterilise).
    5. Timer is activated, when chamber is filled with steam and pre-set temperature is reached (sterilising point).
    6. Rubbish will be sterilised for pre-set time. When time has lapsed, the timer will cut out. Autoclave will not automatically start to go down.
    7. When timer is complete, turn steam control to either:
      FAST EXH (fast exhaust) for autoclave bags or
      SLOW EXH (slow exhaust) for autoclave tins.
    8. When pressure gauge reads zero and temperature is below 80C, autoclave door can be opened.
    9. Wear laboratory coat and long safety gloves when removing collapsed autoclave bags and hot autoclave bins.
    10. Dispose of all non-recyclable rubbish directly into green industrial bins.
      Recyclables must go to wash-up.
  6. Record relevant information on the Autoclave Record Form MPU-FM102.

Operating Instructions For Autoclave B (Usually media)

(Extract from MPU1009, SOP#1020 authorised. Alida Scholtes 28-4-2000, Reviewed 5/3/2001)

  1. Autoclave B is directly connected to a steam supply.
  2. Thermolog indicator must be placed in the chamber in the most inaccessible part of the load preferably in the middle of the load. Autoclave tape must also be placed on the articles/load.
  3. Document all information on the Autoclave Record Form MPU102.
    Namely: Date. Batch no:
    Description of load Autoclave: time and temperature, pressure
    Time: in and out Autoclave tape result
    Thermolog result Operator
  4. After loading, close door and do as follows:
    1. Set buzzer to FAST EXH (on).
    2. Set temperature controller red needle to required temperature (usually 121C) exactly.
    3. Set timer to time required for sterilisation. Use outside markings.
    4. Turn steam control to STER (sterilise). Listen to hear the steam come in.
    5. When chamber is filled with steam and pre-set temperature is reached, i.e. sterilising point, the timer will be activated.
    6. Autoclave will sterilise for pre-set time. When time has lapsed, the timer will cut out and a buzzer will sound.
    7. When buzzer sounds, turn steam control to SLOW EXH (slow exhaust) so autoclave slowly exhausts the steam. All media must be slow exhausted otherwise bottles will explode or liquid boil over.
    8. Wait until pressure gauge reads zero and temperature is below 80C before opening door.
  5. The buzzer on Autoclave "B" has a number of variables.
    1. To leave autoclave going after work hours, the buzzer has to be set to the off position.
      Set buzzer to SLOW EXH (off). When the timer cuts out, instead of the buzzer sounding, the autoclave will very slowly exhaust and cool down.
      Autoclave may go into vacuum and will have to put back up for a minute to allow steam into the chamber to return the pressure to zero and allow the door to be opened.
    2. To enable the autoclave to be fast exhausted:-
      Set buzzer to FAST EXH (on) and when timer cuts out and buzzer sounds, set steam control to FAST EXH (fast exhaust). The autoclave will evacuate the steam rapidly. Media must never be exhausted this way. Solids may be exhausted in this fashion i.e. steriliser leads, pipette tips, contaminated petri dishes, etc.
  6. Some autoclaved materials need drying when removed from autoclave i.e. paper autoclave bags, sterile glassware, boxes of tips etc. Place in drying cabinet located in autoclave room.
  7. Wear laboratory coat and safety gloves when removing load from autoclave. Allow a couple of minutes for any liquids to stop bubbling (boiling) before removing from autoclave.
  8. Remove thermolog indicator and record relevant information on the Autoclave Record Form MPU FM102

Operating Instructions For Autoclave C (Rubbish/Media)

(Extract from MPU1009, SOP#1020 Authorised Alida Scholtes 28-4-2000, Reviewed 5/6/2001)

  1. Autoclave C is directly connected to a steam supply.
  2. Autoclave tins x 5 is the maximum number per load. Lids must be removed before placing in autoclave. Lids are autoclaved separately.
  3. Autoclave bags may only be one layer high. Each autoclave bag must have a "water bomb" in it. ("Water bomb" = 200 ml of water in an old petri dish bag which melts/disintegrates at low heat allowing water to be dispersed throughout autoclave bag before autoclave bag collapses). The water bomb allows steam to be generated within the autoclave bag after it collapses. Ensure that autoclave bag is fully inside the chamber before adding water bomb. If there are any leaks, they are confined within the autoclave and thus sterilised.
  4. Document all information concerning the autoclave cycle on the Autoclave Record Form MPU-FM102
  5. After loading, close door and do as follows:
    1. Set temperature controller to required temperature exactly (121C usually).
    2. Set timer to time required for sterilisation.
    3. Autoclave bags: 121C for 80 mins (and water bomb).
      Autoclave bins: 121C for 60 mins.
      Media: As required by instructions.
    4. Set buzzer to ON. (When set to OFF, autoclave will automatically go down)
    5. Turn steam control to STER (sterilise).
    6. Timer is activated, when chamber is filled with steam and pre-set temperature is reached (sterilising point).
    7. Rubbish will be sterilised for pre-set time. When time has lapsed, the timer will cut out and buzzer will sound.
    8. When buzzer sounds, turn steam control to either:
      FAST EXH (fast exhaust) for autoclave bags or
      SLOW EXH (slow exhaust) for autoclave tins.
    9. When pressure gauge reads zero and temperature is below 80C, autoclave door can be opened.
    10. Wear laboratory coat and long safety gloves when removing collapsed autoclave bags and hot autoclave bins.
    11. Dispose of all non-recyclable rubbish directly into green industrial bins.
      Recyclables must go to wash-up.
  6. Record relevant information on the Autoclave Record Form MPU-FM102.

 

Protocol For Recording Autoclave Runs:

(Extract from MPU1009, SOP#1020 Authorised Alida Scholtes 28-4-2000, Reviewed 5/6/2001)

  • Each run of the autoclave must be recorded. This is a requirement for Safety Map, NATA Accreditation, ISO9000 Certification and Good Common Sense.
  • A clipboard is provided to record a number of parameters of each run. These parameters are: Date, Items in autoclave, sterilisation time and temperature, time of commencement of run and completion, person operating autoclave or doing this run. It also has columns for recording if the autoclave tape changed colour, that the thermolog (if used) was correct for this run and a thermocouple chart recording observation (Not available presently)
  • Please record all relevant information on the clipboard. When the sheet is full, start another one.
  • Thermolog's are available in the autoclave area. They can be found near where the spare tins and autoclave bags are kept. They are to be used with every run that is not a waste run. Ie: for media, containers etc. Results are to be recorded on the clipboard and then the Thermolog may be discarded.

 

Document: SP022 (Master)
Created By: J. P. Gorry, 22/02/02
Authorised By: J. P. Gorry, 22/02/02
Last Modified By: 

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