Research Compliance and Tech Transfer

Chapter 13: Decontamination and Sterilization

Decontamination is a process or treatment that renders a device, instrument, or work surface safe to handle. A decontamination procedure can range from sterilization by autoclave or hydrogen peroxide vapor (i.e., VHP) to simple cleaning with soap and water. Sterilization, disinfection, and antisepsis are all forms of decontamination. Sterilization is the use of a physical or chemical procedure to destroy all microbial life, including highly resistant spores.

Disinfection eliminates virtually all pathogenic, non-spore-forming microorganisms but not necessarily all microbial forms on inanimate objects (work surfaces, equipment, etc.). Effectiveness is influenced by the kinds and numbers of organisms, the amount of organic matter, the object to be disinfected, and chemical exposure time, temperature, and concentration.

Antisepsis is the application of a liquid antimicrobial chemical to skin or living tissue to inhibit or destroy microorganisms. It includes using germicidal solutions to swab an injection site on a person or animal, as well as for handwashing. Although some chemicals may be utilized as either a disinfectant or an antiseptic, adequacy for one application does not guarantee adequacy for another. Manufacturers’ recommendations for appropriate use of germicides should always be followed.

General Procedures

Decontamination of cultures and objects contaminated by biological agents is routinely performed in laboratories and is a vital component of microbiological safety practice. It not only serves to protect laboratory personnel (as well as any bystanders) from infection, but also prevents the release of infectious organisms to the outside environment. Decontamination of media, work surfaces, and equipment is also necessary to prevent contamination of experimentally cultured organisms.

Infectious waste materials such as liquid and solid will be handled, treated, and disposed of according to hazardous waste policies and procedures. Liquid wastes such as bacterial or viral culture media from BSL-2 labs will be treated with appropriate disinfectants prior to sink disposal. Solid waste from BSL-2 laboratories will be segregated and placed in biohazard containers lined with biohazardous waste bags and disposed of as biological waste.

• Autoclaving is the preferred method for treating biological waste. 
• A disinfectant must be appropriate for the organism in use. 
• All liquid biological cultures must be inactivated with appropriate disinfectant for the appropriate contact time.
• All solid biological waste must be disposed of in the biohazard waste containers. 

Methods of Decontamination

The three main categories of physical and chemical decontamination are heat, liquid disinfection, and vapors/gases.

• Heat
  • Wet heat is the most dependable method of sterilization. Autoclaving (saturated steam under pressure of approximately 15 psi with a chamber temperature of at least 121° C/250° F for a prescribed time) is the best method of rapidly achieving destruction of all forms of microbial
  • Sterilization requires that materials come in direct contact with the steam and heat. Indicators of proper autoclave operation (e.g., autoclave tape or autoclave-sensitive labels) must be used with each load to visually confirm successful processing. 
  • Use of autoclave tape alone is not an adequate monitor of autoclave performance. As mentioned previously, the Autoclave Quality Assurance Program requires the use of a well-documented, monthly sporulation test to confirm sterilization. 
• Liquid disinfection
  • A liquid disinfectant (e.g., 1:10 solution of household bleach yielding a final hypochlorite concentration of 0.5%) is used to wipe or soak potentially contaminated materials to kill all pathogenic agents present. Each specific disinfectant requires its own specific contact time. 
• Gas and vapor
  • Potentially contaminated articles are exposed to a sterilizing gas (e.g., ethylene oxide, or ETO) or vapors from a chemical (e.g., formaldehyde). Because of the hazardous nature of the gases and vapors used, this requires specially designed equipment and facilities. 

Autoclaving

Autoclaving uses saturated steam under pressure to achieve a high temperature in the autoclave. Autoclaving can be used to destroy vegetative bacteria, bacterial spores, and viruses. When decontaminating biohazardous waste, it is recommended that the temperature in the waste reach a minimum of 121°C. The total processing time required to meet these conditions depends on several loading factors (see below); however, it is recommended that a minimum autoclave cycle of one hour be used when decontaminating waste.

When using an autoclave, the following guidelines should be taken into consideration:

• Biohazardous materials should not be placed in autoclaves overnight in anticipation of autoclaving the next day.
• Autoclaves must not be operated by untrained personnel. 
• Precautions must be taken to prevent accidental removal of material from an autoclave before it has been sterilized.
• Dry hypochlorite, or any other strong oxidizing material, must not be autoclaved with organic materials such as paper, cloth, or oil.  

Temperature: an autoclave uses steam under a pressure of approximately 15 psi to achieve a chamber temperature of at least 121°C. Although the autoclave chamber may reach 121°C, this does not necessarily mean that the interior of the load will reach this temperature.

Time: a minimum autoclave cycle time of 20 minutes at a chamber temperature of 121°C (time does not begin as soon as the autoclave cycle is initiated) is commonly recommended for sterilization of clean items. However, the total processing time required to achieve decontamination depends on several loading factors, including the load container (heat transfer properties); the amount of water added to the load; and the weight of the load. For increased loads, an increased cycle time will be required to ensure effective decontamination.

Contact: steam saturation is essential for maximum heat transfer. Steam must contact all areas of the load. Autoclave bags and other containers should be left partially open (or otherwise permit entry of steam) to ensure adequate contact. Studies have shown that adding water to the interior of the bag improves the time- temperature profile of the autoclave cycle, thereby increasing the autoclave’s sterilization efficiency.

Dry Heat

Requiring higher temperature and longer contact time, dry heat is less effective than moist heat (autoclaving). Nevertheless, dry heat is preferable to moist heat for decontamination of anhydrous materials and closed containers because the moisture component of the steam used in an autoclave will not effectively penetrate anhydrous materials and closed containers.

A temperature of 160^o-180^o C/320 ^o-356 ^o F for three to four hours is recommended for decontamination of waste using a dry heat oven.

Chemical Disinfection

Disinfection is the decontamination of work surfaces, equipment, BSCs, and other inanimate objects using antimicrobial agents. Several chemical agents are used as disinfectants.

Laboratory workers should remember that there are hazards associated with all chemical disinfectants.

• Inhalation and skin contact should be minimized, and eye contact avoided; PPE is essential. 
• Appropriate gloves and safety eyewear should always be worn when handling these chemicals. 

Disinfectant Selection

Disinfectant selection is based on several factors :

• What is the target organism that you wish to inactivate?
• What are the physical characteristics of the surface which will be disinfected? (porous surfaces may absorb disinfectants; some disinfectants may corrode metal surfaces).
• How long will the contact time be between the disinfectant and the target organism? (high concentrations of biological organisms may require longer contact times).

Note that the disinfection of prions and prion-like proteins must follow specific guidelines.

It is important to note that ‘bleach’, a very common and effective disinfectant, is not stable at dilute concentrations. Working solutions of 10% bleach (10% Bleach solution, prepared using household bleach, results in a final hypochlorite concentration of 0.5% to 0.9%) must be made fresh prior to use and is effective in most situations. Note that undiluted bleach must not go down the drain.

Alcohol based disinfectants will also evaporate over time and should be made up at appropriate intervals.

The following list of disinfectants, their efficiencies, contact times and recommended dilutions are general guidelines—please follow specific manufacturer’s recommendations if available.

Quaternary Ammonium Compounds are commonly used in floor cleaning solutions. Quaternary ammonium compounds are effective in inactivating most vegetative bacteria, fungi, and lipid-containing viruses. Quaternary ammonium compounds are NOT effective when used to disinfect Mycobacterium tuberculosis (TB), bacterial spores, and many viruses such as HBV.

Ethanol is commonly used on equipment whose surfaces are susceptible for corrosion if other disinfectants are applied. Ethyl alcohol is effective in inactivating most vegetative bacteria, fungi, and lipid-containing viruses. Ethanol is NOT effective when used to disinfect HBV, Mycobacterium tuberculosis (TB) and bacterial spores.

• Recommended contact time: 10 minutes
• Recommended Working Dilution: 70-85%
• Recommended for: Stainless steel surfaces. CAUTION: Do not use 70% ethanol to clean a Class II, type A recirculating biosafety cabinet. The vapors from ethanol are flammable and the lower explosive limit (LEL) for ethanol is easily attained

Phenolics are commonly used to decontaminate surfaces such as lab bench tops. Phenolics are effective in inactivating vegetative bacteria, fungi, TB, lipid containing viruses and have some effect on HBV. However, phenolics will not inactivate bacterial spores.

• Recommended contact time: 10 minutes
• Recommended Working Dilution: 1.0-5.0%
• Recommended for: an alternative to bleach as a broad-spectrum disinfectant for bench tops, floors, and metal surfaces. Phenolics will not corrode metal surfaces as readily as bleach

Iodine-containing compounds or iodophors are commonly used to decontaminate metal surfaces or equipment. Iodophors are effective in inactivating vegetative bacteria, fungi, TB and lipid containing viruses and have some effect on HBV. However, iodophors will not inactivate bacterial spores.

• Recommended contact time: 10 minutes
• Recommended Working Dilution: 25-1600 ppm, 0.47%
• Recommended for: biosafety cabinets, dental equipment, bench tops, floors and lab equipment in general

Chlorine compounds such as bleach (sodium hypochlorite) are commonly used in the lab because of the relative ease in accessibility and low cost. Chlorine (hypochlorite) compounds are effective in inactivating vegetative bacteria, fungi, lipid and non-lipid viruses, Coxiella burnetii and TB. Chlorine compounds have some effect in inactivating bacterial spores.

• Recommended contact time: 30 minutes
• Recommended Working Dilution: 500 ppm (1:10 dilution of household bleach, 5% hypochlorite ion)
• Recommended for: floors, spills (inactivating liquid specimens), bench tops and contaminated clothing. Do not use bleach on electronic equipment, optical equipment or unpainted stainless steel. Undiluted bleach and other disinfectants must not go down the drain
• Can be destructive to materials such as clothing. At industrial strength

Paraformaldehyde, glutaraldehyde, and formaldehyde have often been used to decontaminate large pieces of laboratory equipment, such as biosafety cabinets (but only by professionals!). Paraformaldehyde/formaldehyde and glutaraldehyde will inactivate vegetative bacteria, fungi, lipid and non-lipid viruses, HBV, TB, Coxiella burnetii, and bacterial spores. However, paraformaldehyde and formaldehyde are registered carcinogens in the State of California and are very toxic to use without the accessibility of a vented fume hood and/or personal protective equipment. Do not use paraformaldehyde, formaldehyde, glutaraldehyde in the lab to decontaminate equipment. The approved biosafety cabinet contractor will use paraformaldehyde to decontaminate your biosafety cabinet prior to changing the HEPA filters. Be sure to avoid using the biosafety cabinet while this operation is in effect!

Characterisitcs of Select Disinfectant Classes 

This table provides general microbial spectrums for disinfectant chemical classes. Antimicrobial activity and characteristics vary with formulation and concentration. 

Always read and follow directions on the product label.

Microbial Spectrum Legend: + Effective; +/- Variable or Limited Effectiveness; - Not Effective; A-requires High Concentration 

Data compiled from: Maillard JY. 2013. Factors Affecting the Activities of Microbiocides. IN: Fraise AP et al. (eds). Russell, Hugo & Ayliffe’s Principles and Practice of Disinfection, Preservation and Sterilization, 5th ed. 2013; McDonnell G. 2020. Microorganisms and resistance. IN: Block’s Disinfection, Sterilization, and Preservation, 6th edition;

Quinn PJ et al. Disinfection and biosecurity in the prevention and control of disease in veterinary medicine. IN: Block’s Disinfection, Sterilization, and Preservation.

© CFSPH 2008-2023 For more information read Disinfection 101 on the CFSPH disinfection website.