Guidelines for the Decontamination of Clinical/Biological Waste and Spill Management

  1. Decontamination - General Information
  2. Methods for the Decontamination of Clinical and Biological Waste
    2.1 Pressure steam sterilising (autoclaving)
    2.1.1 Times for sterilisation
    2.1.2 Monitoring of sterilisation cycles
    2.2 Chemical disinfection
    2.2.1 Choosing which chemical disinfectant to use
    2.2.2 Chemicals used for the decontamination of clinical and biological waste
  3. Decontamination Method for Prion Contaminated Material
  4. Spill Management
  5. References

1. Decontamination - General Information

Decontamination is carried out to prevent the spread of pathogens or other biological materials, which may cause disease in humans or animals.

All Clinical and Biological waste and waste containing Genetically Modified Organisms (GMOs) should be decontaminated prior to disposal. Clinical and biological waste usually includes the following sub-categories:

  • Laboratory and associated waste directly involved in specimen processing; this category includes all specimens used for laboratory testing; cultures or suspensions of micro-organisms in tissue culture; used Petri dishes; culture bottles; disposable equipment, used gloves etc. This category includes wastes contaminated with or potentially contaminated with Risk Group 1 and Risk Group 2 microorganisms. Wastes contaminated with Risk Group 3 or 4 microorganisms are beyond the scope of these guidelines. Contact the for advice on decontamination of these agents.
  • Human tissues including materials or solutions that contain free-flowing or expressible blood.
  • Animal tissue or carcasses that are contaminated or suspected to be contaminated by pathogenic organisms.
  • Prions

A variety of decontamination methods are available. These include use of steam sterilisers (autoclaves), liquid chemicals (germicides, disinfectants), gaseous chemicals and vapours, radiation methods and sometimes a combination of these methods.

The most effective decontamination method depends on several factors. Steam sterilisation is generally the preferred method, but it cannot be used for large spaces, surfaces, delicate instruments or large volumes of liquids. Chemical disinfection is often the most practical alternative.

Refer to the University’s Hazardous Waste Guidelines for information on the most appropriate method for decontamination of the different sub-categories of clinical waste and information on how to dispose of decontaminated waste.

2. Methods for the Decontamination of Clinical and Biological Waste

The two most common methods of decontamination are use of Pressure steam sterilisers (autoclaves) and use of liquid chemicals (germicides, disinfectants).

2.1 Pressure steam sterilising (autoclaving) - See AS/NZS2243.3 Section 10.6 for more detail
Autoclaves operate at high temperatures and pressures and are and are useful pieces of equipment for decontaminating microorganisms.

Persons using an autoclave must be trained so that they understand that correct loading of the autoclave is essential to ensure sterilisation or decontamination of the load. Operators must also be trained so that they understand the hazards associated with heat, steam and pressure. Operators must be provided with protective clothing, including heat-insulating gloves, for use when unloading the autoclave. Use of a face shield is recommended when unloading the autoclave.

It is important that the autoclave cycle is complete before attempting to open the autoclave chamber. Care needs to be taken when removing containers of liquid after completion of sterilisation, as sudden changes in pressure and temperature may occur and they may break or boil over when moved. Before removal of a load, the autoclave door should be partly opened and sufficient time allowed for the load to cool before handling.

It is also important to ensure the proper conditions for load sterilisation are produced in the chamber. To work effectively, steam must be able to contact the material being sterilised for an appropriate period of time and at an appropriate temperature. To ensure steam can contact the materials to be sterilised, all the air must first be removed from the load and the autoclave chamber. This is done either by downward displacement by the steam or by using a vacuum pump before the sterilisation stage (pre-vacuum method). Downward displacement is well suited to sterilisation of culture media and fluids. Porous loads and large empty containers are more readily sterilised by the pre-vacuum method since this prevents the entrapment of air in cavities as can occur with the downward displacement method. The pre-vacuum method is not suitable for vessels containing fluids. Autoclave bags should be carefully opened prior to commencement of the autoclave cycle, without disturbing the contents, to allow the penetration of steam and displacement of air.

Wastes being transported from a facility to the autoclave must be wholly contained within a primary sealed container (eg an autoclave bag) and the primary sealed container must be packed in a secondary sealed unbreakable container (eg unbreakable plastic container with a sealable lid or a garbage bin with a sealable lid). The secondary container must be able to be easily decontaminated. Transport routes should be planned to minimise possible exposure to the wastes by consideration of activity levels and population densities at various times of the day and places on the routes.

Appropriate chemical disinfectants must be provided in the vicinity of the autoclave to assist in the clean up of any spills outside the autoclave. Easy access to hand washing facilities, safety showers and eyewash facilities must also be provided.

2.1.1 Times for sterilisation
Sterilisation times need to be determined for each load. Time is required for all parts of the load to reach the desired temperature, in addition to the contact time at the temperature. Minimum recommended contact times after the temperature is reached are
a) 15 min at 121°and 103kPa; or
b) 3 min at 134°C and 203kPa

(Note that these conditions are not suitable for prions – see separate section on prion decontamination). In loads where large containers of liquids or solids are to be sterilised, additional time for heating should be allowed to ensure all parts of the load have been exposed to the required temperature for the required period of time.

2.1.2 Monitoring of sterilisation cycles
It is important that some sort of indicating or measuring system is used to ensure that the sterilisation cycles are effective.

Visual indicators, such as sensitive papers or tapes, should be used in each cycle. Such visual indicators are used to check that materials have been processed, but do not monitor the efficacy of the sterilisation procedure. Chemical indicators progressively change colour with the time exposed at specific temperatures, and their use is recommended as they give an immediate indication of the efficacy of treatment.

Biological indicators, such as spore strips, should be used at regular intervals (eg. monthly) to monitor the microbial killing power of the sterilisation process. They should be placed in several positions in a load, including those least likely to attain sterilisation conditions. Bacterial enzyme indicators may be used instead of biological indicators for the monitoring of sterilisation cycles. These indicators are designed so that the loss of enzyme activity parallels the loss of spore viability. Their advantage is that enzyme inactivation can be easily and rapidly determined, e.g. within minutes or hours, by the addition of a substrate and observation for absence of a coloured or fluorescent end point. In contrast, biological indicators require incubation for growth for periods of days.

2.2 Chemical disinfection
Chemical disinfectants have a range of properties, and no single one is effective in all situations. A number of factors should be considered in choosing a chemical for decontamination. Micro-organisms vary in their resistance to chemical agents. Viruses containing lipids and vegetative forms of many fungi and bacteria are usually susceptible to chemical attack, whereas spores of micro-organisms tend to be quite resistant. Prions are extremely resistant to chemical disinfection. Refer to section on prion decontamination for further detail.

2.2.1 Choosing which chemical disinfectant to use
To choose an appropriate disinfectant, account should be taken of the identity of the micro-organisms, their concentration and the degree of inactivation you wish to achieve. Physical factors such as whether a space or surface is to be decontaminated, the type of surface, and any interaction between the material and potential disinfectants will also be factors in the selection. Time available for disinfection and the time required for particular disinfectants to be effective need to be considered.

The effectiveness of a disinfectant depends not only on the properties of the micro-organisms against which it is used, but also upon factors in the environment in which it is to be used. Factors that may affect the action of chemical disinfectants include the following:

  • the concentration of the chemical in the disinfectant solution;
  • the temperature;
  • pH;
  • Relative humidity of the environment;
  • The presence of organic matter (which inactivates some chemicals);
  • Duration of contact.

Most disinfectant solutions need to be regularly prepared as fresh solutions to avoid growth of micro-organisms in the solution and to ensure optimum activity of the disinfectant chemical. For optimum contact with the chemical it is advisable to clean surfaces and equipment first to reduce organic matter which might interfere with the disinfection process.

Many disinfectants have toxic effects ranging from irritation of the skin and mucous membranes to carcinogenesis, and some have physical properties that make them dangerous to handle and use. These properties should be taken into consideration when selecting a disinfectant for a particular use. A Material Safety Data Sheet (MSDS) should be available for all disinfectants to be used and should be read and understood by all persons with access to the disinfectants.

2.2.2 Chemicals used for the decontamination of clinical and biological waste - (see AS/NZS 2243.3 Appendix F for more detail). Tables F1 and F2 should be consulted for assistance when selecting disinfectants. Table F1 provides recommended applications for chemical disinfectants in microbiological facilities. Table F2 provides a guide to the effectiveness of different classes of disinfectant against a range of microorganisms.

The four most common chemicals that can be used for the decontamination of clinical and biological waste are as follows:

Alcohols - 80% v/v ethyl alcohol or 60-70% v/v isopropyl alcohol solutions - are useful for cleaning surfaces and disinfecting skin. They are also active against vegetative bacteria and lipid containing viruses, but are inactive against spores. They are ineffective against Mycobacterium species and HIV dried on surfaces in the presence of sputum or serum. Alcohols are unsuitable for application to proteinaceous material as they tend to coagulate and precipitate surface proteins which may then result in protection of the microorganisms present. Alcohols are flammable, so they should not be used near flames or sparks. Because of their volatility, alcohol disinfectants should be used sparingly in biological safety cabinets and not with equipment that is likely to cause sparks. Alcohol disinfectants may be used from a dispensing bottle, but should never be sprayed as a mist. Both ethyl and isopropyl alcohol are toxic, with isopropyl being the more toxic of the two.

Chlorine – in the form of sodium hypochlorite or other chlorine releasing compounds – is active against vegetative forms of bacteria and viruses and is the preferred disinfectant for HIV and hepatitis viruses. It is less effective against spores. For effective disinfection, a pH range of 6-8 is optimum. The effective strength of chlorine solutions decreases on storage so working solutions should be freshly prepared daily. Chlorine combines rapidly with proteins, so, in the presence of organic materials, the concentration of chlorine needs to be increased. For example, an equal volume of 5000-10000 ppm (0.5%-1% sodium hypochlorite) available chlorine is required for the inactivation of HIV and hepatitis viruses in blood or serum or in areas that have been grossly soiled with blood or body fluids. Benches, floors, walls and other inanimate objects likely to be contaminated, but not visibly soiled, should be cleaned with 500ppm (0.05% sodium hypochlorite) available chlorine. The hypochlorite solution should be in contact for at least 10 minutes. Hypochlorite solutions are corrosive to stainless steel and other metal surfaces and tend to bleach and damage fabrics.

Quaternary ammonium compounds (QACs) – are effective against Gram-positive and lipid-containing viruses eg herpes and influenza, but are less active against Gram-negative bacteria and non-lipid-containing viruses and are inactive against Mycobacterium species and bacterial spores. QACs are cationic detergents with powerful surface-active properties. They are inactivated by proteins, soap and anionic detergents, and are not recommended as general disinfectants. They are non-flammable, relatively non-volatile, non-toxic to skin, inexpensive, non-corrosive and non-staining. This makes them useful as cleaning agents for floors and in the food and dairy industries.

Chlorhexidine - used as chlorhexidine gluconate in 70% alcohol - is a useful skin antiseptic that is active against HIV and Gram-positive organisms. Chlorhexidine is ineffective against non-lipid containing viruses and sporulating bacteria. It is not compatible with soap or anionic detergents.


In addition to the four chemicals described above, the following chemicals can also be used for decontaminating Clinical and Biological waste. These chemicals, however, have disadvantages over the four most commonly used chemicals.

Iodine - in a solution with water or alcohol - is a highly reactive chemical and this makes it effective against all types of micro-organisms including bacterial spores. Like chlorine, it is not effective in the presence of organic matter. Iodine is usually used diluted to 1% w/v free iodine at pH neutral to acid. Iodophors are formed when iodine is complexed with non-ionic surface active agents, and they are widely used as antiseptic agents. Disadvantages to the use of Iodine are that it stains the skin, is toxic and allergenic. Continual exposure to iodine compounds is to be avoided.

Formaldehyde - a 5% w/v solution is an effective decontaminant, but has an irritating odour. Formalin (37% w/v formaldehyde gas in water) when diluted to 8% v/v in 80% v/v alcohol is very effective against vegetative bacteria, spores and viruses. It is also active in the presence of organic material. Although it can be a potent germicide, it does not penetrate well. Since it is non-corrosive, formaldehyde is useful for disinfecting instruments. Formaldehyde gas is useful for decontaminating spaces such as rooms and biological safety cabinets. To be effective the relative humidity of the space needs to be between 70% and 90%. Use of formaldehyde gas should be restricted to those who have specific expertise and training in the use of the gas and must be in accordance with appropriate safety precautions. Disadvantages to the use of formaldehyde are that it is a toxic material and is a confirmed human carcinogen (IARC Group 1). It can also react with chlorine to form another carcinogen. The current exposure limits are 1 ppm or 1.2mg/m3 TWA (Time-weighted average for 8 hr day over a working lifetime) and 2 ppm or 2.5mg/m3 STEL (Short term exposure limit for 15 minutes exposure).

Glutaraldehyde - available as aqueous solutions such as 2% v/v with 0.3% m/v sodium bicarbonate to make it alkaline - can be effective against most vegetative bacteria, spores, fungi and many viruses. It is used for decontaminating equipment, for which contact times of 3 - 10 hours are needed to achieve sterility. As a vapour it can be used in biological safety cabinets. Like formaldehyde it is non corrosive and effective in the presence of organic matter. Disadvantages to the use of glutaraldehyde are that it has sensitising and irritating properties. The current exposure limit for Glutaraldehyde is 0.1 ppm or 0.41mg/ m3 TWA (Time-weighted average for 8 hr day over a working lifetime).

Phenolic compounds – are active against bacteria and lipid-containing viruses, but are inactive against spores and the non-lipid-containing viruses. A major advantage of the phenolics is that they are not deactivated by organic matter. The phenolics are inactivated by non-ionic detergents. They may cause toxic effects if ingested, however they do not have the pungent odours, highly corrosive and skin irritancy properties of the crude parent compounds, phenol and lysol.

3. Decontamination method for Prion Contaminated Material

Prions are infectious agents that produce slow, progressive and fatal diseases of the central nervous system. Prions are the causative agents for a number of degenerative brain diseases, including scrapie (a fatal disease of sheep and goats), mad cow disease, Creutzfeldt Jacob disease (CJD) and Gertsmann-Straeussler-Scheinker (GSS) disease.

Prions are resistant to most traditional methods of inactivation used for other microorganisms. Because of the difficulties in inactivating the infectivity, prions pose particular problems in the laboratory. Prions should only be handled in dedicated laboratories using dedicated equipment.

Current recommendations for the sterilisation of articles or specimens that could be contaminated by prions are 18 minutes at 134°C to 138°C in a pre-vacuum pressure steam steriliser or 1 hour at 132°C in a downward displacement pressure steam steriliser. The recommended chemical disinfectant for effective decontamination of prions is 20 000 ppm (2% sodium hypochlorite) available chlorine for 1 hour with sodium hypochorite as the chlorine releasing agent.

Current recommendations for decontaminating heat-resistant instruments that may be contaminated by prions involves a combination of pressure steam sterilising and chemical treatment. This can be either:
a) immerse in 1M sodium hydroxide and heat in a downward displacement autoclave at 121°C for 30 minutes, clean, rinse in water then subject to routine sterilisation (refer to pressure steam sterilising section); or
b) immerse in 1M sodium hydroxide or 20 000 ppm sodium hypochlorite for 1 hour, transfer instruments to water, heat in a downward displacement autoclave at 121°C for 1 hour, clean and subject to routine sterilisation (refer to pressure steam sterilising section).

Infectivity is strongly stabilised by drying or fixing, so contaminated material should be kept wet between the time of use and disinfection. Formalin-fixed and paraffin-embedded tissues, particularly of the brain, remain infectious for long periods, if not indefinitely. They should be assumed to remain infectious through the processes of embedding, sectioning, staining and mounting on slides. The most effective chemical treatment for decontaminating formalin-fixed tissue is 96% formic acid for 1 hour.

Refer to the University’s Hazardous Waste Disposal Guidelines for information on how to dispose of decontaminated prion waste.

4. Spill Management

Spill kits should be available in all laboratories where clinical/biological material is stored or handled. Spill kits should also be available where clinical/biological waste is stored awaiting decontamination and disposal. The contents of the spill kit should be appropriate for the type, nature and amount of material that could be spilled in an area. Spill kits should contain a means to collect and contain wastes, disinfectant or neutralising agent and personal protective equipment including gloves, laboratory coat and safety glasses. Materials used to clean up a spill (such as paper towel) should be decontaminated and disposed of according to the type of clinical/biological material cleaned up. Refer to AS2243.3:2010Safety in laboratories – Part 3: Microbiological safety and containment, Section 9, Microbiological Spills for more guidance on spill management. A complete copy of the Standard can be accessed via the University Library database under Standards Australia.

5. References

AS/NZS 3816 - 1998: Australian/New Zealand Standard Management of clinical and related wastes

Australian/New Zealand Standard 2243.3 2010 - Safety in Laboratories – Part 3: Microbiological safety and containment

BMBL 1999. Section V11-D. Agent Summary Statements – Prions. Biosafety in Microbiological and Biochemical Laboratories, 4th ed. Centres for Disease Control and Prevention, National Institute of Health, U.S. Department of Health and Human

CCH Laboratory Safety Manual

National Occupational Health and Safety Commission - National Exposure Standards Database.

NH&MRC, 1999 - National Guidelines for the Management of Clinical and related Wastes

WHO. 2000. WHO infection control guidelines for transmissible spongiform encephalopathies. Report of a WHO consultation, Geneva, Switzerland, 23-26 March 1999.

Guidelines for the Disposal of Hazardous Waste, University of Sydney