A laboratory working with biohazardous materials will inevitably require certain laboratory surfaces and equipment to be disinfected or decontaminated, to mitigate the possibility of transmission of pathogens to laboratory workers, the public, and the environment. The terms cleaning, disinfection, decontamination, and sterilization are often misunderstood and incorrectly used interchangeably. This chapter will clarify and define these terms, discuss considerations for the disinfectants often utilized in research laboratories, and identify suitable methods of decontamination and disinfection for these surfaces and items.
The appropriateness of a decontamination procedure depends on your goal. Do you wish to disinfect or sterilize? Will you be using the disinfectant on hard surfaces, in a biosafety cabinet, on instruments, or on waste? When choosing a disinfectant one should consider the organism, the item to be disinfected, the cost and ease of use of the disinfectant, and what level of disinfection will accomplish your goal.
Antimicrobial pesticides (ex. disinfectants) are classified as pesticides and are regulated by both the United States Environmental Protection Agency (EPA) and the United States Food and Drug Administration (FDA). A laboratory is responsible for selecting an appropriate EPA-registered product and for using it according to the manufacturer’s specific instructions for that product. The Office of Biological Safety is available to assist laboratories in selecting an appropriate product based on a risk assessment. A list of EPA-registered disinfectants is available at https://www.epa.gov/pesticide-registration/selected-epa-registered-disinfectants.
The FDA defines three types of liquid chemical germicides for processing medical devices (also applicable to laboratory equipment utilized with biohazardous materials in the case of UK research laboratories), and these germicides are regulated as auxiliary devices per the FDA 1977 Policy Manual.
Low-Level Disinfection – A lethal process utilizing an agent that kills vegetative forms of bacteria, some fungi, and enveloped viruses.
Intermediate-Level Disinfection – A lethal process utilizing an agent that kills viruses, mycobacteria, fungi, and vegetative bacteria, but no bacterial spores.
High-Level Disinfection – A lethal process utilizing a sterilant under less than sterilizing conditions (ex. 10-30 minutes contact time instead of 6-10 hours needed for sterilization). The process kills all forms of microbial life except for large numbers of bacterial spores.
Disinfectant solutions should be made and stored according to manufacturer directions for maximum stated effectiveness. It is crucial to remember that a clean surface is more effectively decontaminated than a soiled surface, the required contact time for a given disinfectant product may vary, and disinfectants are not necessarily detergents. However, some disinfectants do include a detergent or surfactant to aid in the removal of gross contamination from a surface during the disinfection or decontamination process.
There are several types of liquid chemical disinfectants and sterilants, including but not limited to household bleach (sodium hypochlorite), peroxides, quaternary ammonium compounds, aldehydes, phenolic compounds, alcohols, and iodine/iodophors. The general characteristics of these categories are outlined here.
*Note: Sterilization with a liquid chemical sterilant is not considered by the FDA to produce as sterile a result as the processes of steam/vapor/gas sterilization. Sterilization is still achieved by liquid chemical sterilants but is not as readily maintained immediately after processing due to environmental factors and application.
Chlorine, a fast-acting oxidant, is a widely available and broad-spectrum chemical disinfectant. It is normally sold as household bleach, an aqueous solution of sodium hypochlorite (NaOCl), which can be diluted with water to provide various concentrations of available chlorine. Chlorine is highly alkaline and can be corrosive to metal, which must be considered when it is used on metal clad benches and laboratory equipment (ex. Incubators, Biological Safety Cabinets, etc.). The disinfectant activity of chlorine is considerably reduced by organic matter (ex. protein).
Storage of stock or working solutions of bleach in open containers, particularly at high temperatures, may release chlorine gas thus weakening their disinfectant potential. Undiluted household bleach stored at room temperature in the original container has a shelf-life of approximately one year. Working solutions of bleach should be prepared on a daily basis. Household bleach (typically 5.25% NaOCl, please check the label) should be diluted 1:10 to obtain final concentration of 0.5% NaOCl. Industrial solutions of bleach have a higher sodium hypochlorite concentration and must be diluted accordingly to obtain the correct concentration.
Chlorine gas is highly toxic. Bleach must therefore be stored and used in well-ventilated areas only. Undiluted bleach must not be mixed with acids or other incompatible chemicals, such as ammonia containing compounds, to prevent the rapid release of chlorine gas.
Peroxides are non-chlorine based strong oxidizers with many desirable characteristics for a liquid chemical disinfectant. They are capable of a wide range of bactericidal, fungicidal, and virucidal activity, are capable of protein and lipid denaturation, and degrade into relatively harmless byproducts; hydrogen peroxide (H2O2) degrades into oxygen & water. Hydrogen peroxide possesses these properties but also ranges from an intermediate level of disinfection up to sterilization* depending on the concentration and contact time employed.
At lower concentrations, hydrogen peroxide is not fully sporicidal and an organism's ability to produce the peroxidases (ex. catalase) can reduce its effectiveness. However, when used at the concentrations found in most peroxide-based disinfectants (typically ≥3%), peroxidases have limited impact on effectiveness. A stronger peroxide disinfectant is Peracetic acid (C2H4O3) which is a mixture of hydrogen peroxide and acetic acid. Acetic acid acts as a strong acid catalyst enabling peracetic acid to have robust bactericidal, sporicidal, fungicidal, and virucidal properties while also maintaining activity in the presence of peroxidases. Peracetic acid offers a high level of disinfection up to sterilization* depending on concentration and contact time, and it decomposes into acetic acid and oxygen.
Both hydrogen peroxide and peracetic acid can be corrosive to metal surfaces, though stainless-steel surfaces are minimally affected. Additionally, hydrogen peroxide and peracetic acid-based disinfectants do not require inactivation after application.
Many types of quaternary ammonium compounds are used as mixtures and often in combination with other germicides, such as alcohols, or detergents. They have good activity against some vegetative bacteria and lipid-containing viruses. The germicidal activity of certain types of quaternary ammonium compounds is considerably reduced by organic matter, water hardness, and anionic detergents. Care is therefore needed in selecting agents for pre-cleaning when quaternary ammonium compounds are to be used for disinfection. Potentially harmful bacteria are able grow in quaternary ammonium compound solutions, requiring careful attention to their storage and usage. Quaternary ammonium compounds, when properly diluted, have a low odor and are generally non-irritating.
Aldehyde based compounds (ex. glutaraldehyde, formaldehyde, paraformaldehyde) are capable of a wide range of antimicrobial activity levels, from low-level disinfection to sterilization* with the appropriate combination of concentration and contact time. These compounds are frequently found in laboratories as fixative agents for tissues and microscopy preparations. However, they are NOT recommended for the disinfection or decontamination of research laboratory surfaces or equipment due to their known chemical hazards. For example, formaldehyde is classified as a known human carcinogen with a low permissible exposure limit and there are currently no FDA cleared liquid chemical disinfectants that contain it as an active ingredient. These compounds should be reserved only for specific experimental procedure purposes.
Phenolic compounds, a broad group of agents, were among the earliest germicides. However, more recent safety concerns restrict their use. They are active against vegetative bacteria and lipid-containing viruses and, when properly formulated, also show activity against mycobacteria. They are not active against spores and their activity against non-lipid-containing viruses is variable. Many phenolic products are used for the decontamination of environmental surfaces, and some (ex. triclosan and chloroxylenol) are among the more commonly used antiseptics. Some phenolic compounds are sensitive to and may be inactivated by water hardness and therefore must be diluted with distilled or deionized water. They may be absorbed by latex gloves and can also penetrate the skin. Phenolic compounds can be irritating to the skin and eyes and may have an associated odor.
Ethanol (ethyl alcohol, C2H6O) and 2-propanol (isopropyl alcohol, (C3H8O) have similar disinfectant properties. They are active against vegetative bacteria, fungi, and lipid-containing viruses but not against spores. Their action on non-lipid-containing viruses is variable. For highest effectiveness they should be used at concentrations of approximately 70% (v/v) in water. Higher or lower concentrations are not as germicidal as 70% (v/v). A major advantage of aqueous solutions of alcohols is that they do not leave any residue on treated items. Since alcohols tend to evaporate rapidly, they are generally most effectively used in circumstances where items can be submerged, allowing the appropriate contact time to be achieved. A 70% (v/v) aqueous solution of ethanol can be used to soak small pieces of surgical instruments, with a contact time of ten minutes or more required.
Ethanol should never be used to disinfect hands since ethanol can dry the skin, reducing its effectives as a barrier. Alcohol-based hand-rubs, alcohol mixed with emollients, are recommended for the disinfection of lightly soiled hands in situations where proper handwashing is inconvenient or not possible. However, it must be remembered that ethanol is ineffective against spores, Hepatitis B Virus (HBV), Mycobacterium tuberculosis (TB), and may not kill all types of non-lipid-containing viruses.
Alcohols are volatile and flammable and must not be used near open flames. Do not use 70% ethanol to clean a Class II, Type A recirculating Biological Safety Cabinet (BSC). The vapors from ethanol are flammable and the lower explosive limit (LEL) for ethanol is easily attained with the amount of ethanol required to clean a BSC. Working solutions should be stored in proper containers to avoid evaporation and prevent vapor build-up in an area. Additionally, alcohols may harden rubber and dissolve certain types of glue. Bottles with alcohol-containing solutions must be clearly labeled and must not be autoclaved.
The action of these disinfectants is similar to that of chlorine, although they may be slightly less inhibited by organic matter. Iodine can stain fabrics and environmental surfaces and is generally unsuitable for use as a disinfectant. On the other hand, iodophors and tinctures of iodine are good antiseptics. Povidone-iodine is a reliable and safe surgical scrub and preoperative skin antiseptic. Antiseptics based on iodine are generally unsuitable for use on medical/dental devices. Iodine should not be used on aluminum or copper and can be toxic. Organic iodine-based products must be stored at 4–10°C to avoid the growth of potentially harmful bacteria in them. No liquid chemical disinfectant with iodophors as the main active ingredient has received clearance by the FDA as of 2023.
Utilizing the information earlier in this section, informed decisions can be made regarding the most effective selection and application of a liquid chemical disinfectant or sterilant in the laboratory. A decontamination procedure needs to be developed by each laboratory that corresponds to the specific work with biohazardous materials and is determined during a risk assessment. For example, a laboratory conducting work with spore forming bacterial pathogens or prions will have very different decontamination procedure considerations than a laboratory only conducting work with uninfected human-derived cell lines. This decontamination procedure for laboratory surfaces and equipment will need to be utilized after work with biohazardous materials is completed. Your UK Institutional Biosafety Committee (IBC) protocol registration will specify the disinfectant(s) to be utilized in your laboratory.
An effective decontamination procedure is required for surfaces and equipment that have been utilized with biohazardous materials and is to be employed before the removal/surplus of any such contaminated equipment from the laboratory and a laboratory closeout. Hard, non-porous surfaces can effectively be decontaminated with surface applied liquid chemical disinfectants. Porous objects and surfaces are not recommended to be used with biohazardous materials due to the drastically increased difficulty in their decontamination.
In most circumstances, lab surfaces and equipment cannot readily be steam sterilized in an autoclave and will require the use of liquid chemical disinfectants. In UK research laboratories, 10% (final concentration) household bleach is one of the most utilized disinfectants. After application of 10% bleach, allow a 20-minute contact time, and follow with a final wipe down of the equipment or surface with 70% ethanol or water that is needed to remove residual disinfectant.
The effectiveness of household bleach is considerably reduced by the presence of organic matter (ex. protein, tissues, bacterial cultures, etc.). Heavily soiled items or surfaces will require cleaning prior to achieving decontamination. Household bleach can also be utilized in the cleaning step of the decontamination procedure, but more than one application of household bleach may be required to sufficiently remove enough organic matter to achieve effective decontamination of the surface or item. If a surface has minimal organic matter contamination, a single application of household bleach as denoted above is generally sufficient (excluding prion contamination) to achieve decontamination unless otherwise determined in the risk assessment of your work with the biological agent. Please contact the Office of Biological Safety if you require guidance on the decontamination of a surface or item.
For the decontamination of spills involving biohazardous materials, see the Biological Spill SOP HERE.
Prions are abnormal transmissible pathogenic agents that can induce the formation of certain abnormally folded proteins, causing pathology primarily in the brain (ex. transmissible spongiform encephalopathy). Prions are notably resistant to conventional decontamination methods (ex. heat and chemical germicides), making decontamination procedures especially challenging. For guidance on effective decontamination of prion contaminated surfaces or items, please contact the Office of Biological Safety.
While use of UV light in Biological Safety Cabinets (BSCs) is not strictly prohibited, UV light may not be used as a primary means of decontamination.
National Institutes of Health (NIH) & Use of Ultraviolet (UV) Radiation in Laboratories “The NIH does not recommend or support the use of ultraviolet (UV) radiation in laboratories. Although UV is effective against most microbes, it requires an understanding of its abilities and limitations. The 253.7-nm wavelength emitted by the germicidal lamp has limited penetrating power and is primarily effective against unprotected microbes on exposed surfaces or in the air. It does not penetrate soil or dust. The intensity or destructive power decreases by the square of the distance from the lamp. Thus, exposure time is always related to the distance. The intensity of the lamp diminishes over time. This requires periodic monitoring with a UV meter. The intensity of the lamp is drastically affected by the accumulation of dust and dirt on it. The bulbs require frequent maintenance. In addition, there are safety hazards associated with the use of UV that require personal protective equipment or other safety devices to protect users. UV lights in biosafety cabinets require the cabinet be decontaminated prior to performing maintenance on the system. Past experience has proven that good techniques in conducting experiments are highly effective in preventing contamination. The use of UV radiation does not eliminate the necessity for using good practices and procedures." Source: https://www.ors.od.nih.gov/sr/dohs/safety/laboratory/BioSafety/Pages/decontamination.aspx
“The NIH does not recommend or support the use of ultraviolet (UV) radiation in laboratories. Although UV is effective against most microbes, it requires an understanding of its abilities and limitations. The 253.7-nm wavelength emitted by the germicidal lamp has limited penetrating power and is primarily effective against unprotected microbes on exposed surfaces or in the air. It does not penetrate soil or dust. The intensity or destructive power decreases by the square of the distance from the lamp. Thus, exposure time is always related to the distance. The intensity of the lamp diminishes over time. This requires periodic monitoring with a UV meter. The intensity of the lamp is drastically affected by the accumulation of dust and dirt on it. The bulbs require frequent maintenance. In addition, there are safety hazards associated with the use of UV that require personal protective equipment or other safety devices to protect users. UV lights in biosafety cabinets require the cabinet be decontaminated prior to performing maintenance on the system. Past experience has proven that good techniques in conducting experiments are highly effective in preventing contamination. The use of UV radiation does not eliminate the necessity for using good practices and procedures."
Source: https://www.ors.od.nih.gov/sr/dohs/safety/laboratory/BioSafety/Pages/decontamination.aspx
Typical laboratory equipment with the capacity to emit non-ionizing UV wavelengths includes: biological safety cabinets (BSCs), transilluminator boxes and UV crosslinkers. The BSCs usually contain a UV lamp used to help maintain a sterile environment. Transilluminator boxes are used to observe gels susceptible to electrophoresis and contain nucleic acids. UV crosslinkers are mainly used to crosslink DNA or RNA to membranes.
Exposure to UV light poses a serious threat to both the eye and skin. Diagnosis of exposure may vary but are commonly set into two categories, photokeratitis (eye injury) and erythema (sunburn). Photokeratitis is an inflammation of the cornea (outer protective coating of the eye) that is caused by exposure to ultraviolet radiation. Eye injury can occur due to very brief exposure or with just a flash of intense UV. Erythema is sunburn of the skin and can occur within a few seconds of exposure to a concentrated form of UV. Prolonged exposure to ultraviolet light also causes premature aging and cancer of the skin.
As a common rule, never allow your eyes or skin to be exposed to UV light in the laboratory. This “laboratory UV light” is heavily concentrated and can cause severe damage with very short exposure periods. Always wear personal protective equipment (PPE) such as gloves, face shields, and lab coats (long sleeves) when using UV light. Thick nitrile gloves are recommended, but latex gloves can be doubled for use. Biological Safety Cabinets (BSCs) are never to be occupied while the UV lamp is activated. Always lower sash and keep away from escaping rays. Mechanical safety devices should be standard on most new cabinets. If there is no safety shield or safety switch, these must be retro-installed in such a way as to prevent exposure and not interfere with the operation of the apparatus. Transilluminators are never to be used without the protective shield in place. A face shield, thick nitrile or double latex gloves along with a lab coat are the recommended PPE. Crosslinkers are not to be used if the door safety interlocking mechanism is not working properly.