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Environment, Health, & Safety Division

Appendix F

Decontamination and Antimicrobials
Text Box:


F.1    Introduction and Scope

This appendix primarily provides information and guidance on decontamination principles, decontamination terms, and the variety of chemical and physical agents used to decontaminate. In a few cases, requirements are stated using the words should or must. See Section 5.7 of this manual for requirements and additional information regarding decontamination, waste, and decommissioning. Information used to develop this appendix was taken from a wide variety of Web pages and documents. Primary sources are listed in the reference section at the end of this appendix.

F.2    Decontamination Principles and Terms

Decontamination is a process that uses an antimicrobial to reduce or inactivate biological contaminants or components to an acceptable level so as to reduce or eliminate the possibility of transmitting pathogens to undesired hosts. An antimicrobial is the chemical or physical agent that is used in a decontamination process to prevent microbial growth. Prevention of microbial growth and pathogen transmission is needed to control contamination of the work and prevent disease in hosts such as laboratory workers, the general public, and other organisms in the environment. The decontamination process, level, antimicrobial, frequency, and specific method are based on the work activity, agents that need inactivation, and decontamination objective or requirements.

Sterilization, disinfection, sanitization, and antisepsis are decontamination processes that result in different levels of decontamination or decontamination of different types of objects. These processes are discussed in Section F.2.1 below. A variety of terms are also used to describe the antimicrobials that are used in sterilization, disinfection, sanitization, and antisepsis. These antimicrobial terms are discussed in Section F.2.2 below.

F.2.1    Decontamination Processes and Levels

F.2.1.1 Sterilization

Sterilization is the process of completely destroying all living microorganisms and viruses on an object. Any item, device, or solution is considered to be sterile when it is completely free of all living microorganisms and viruses. Sterility is an absolute term (an item is either sterile or it is not), but sterilization procedures must be defined to achieve sterility. A sterilization procedure is a treatment process to which an item is subjected after which the probability of a microorganism or virus (including a high number of bacterial endospores) surviving on the item is less than 1 in 1 million. This level of killing efficacy is referred to as the sterility assurance level.

Sterilization can be accomplished by heat (e.g., autoclave or incineration), ethylene oxide gas, hydrogen peroxide gas, plasma, ozone, and radiation. Solid biohazardous waste is typically sterilized prior to disposal.

F.2.1.2 Disinfection

Disinfection is generally a less lethal process than sterilization. Disinfection is the process of generally eliminating nearly all recognized pathogenic microorganisms but not necessarily all microbial forms (e.g., bacterial spores) on inanimate objects (e.g., work surfaces, equipment). Disinfection does not ensure “overkill'' and therefore lacks the margin of safety achieved by sterilization procedures. Longer disinfection times or higher concentrations of disinfectant may be needed if the effectiveness of a disinfection procedure is reduced significantly by a number of factors such as:

  1. More resistant microorganisms (especially bacterial spores)
  2. Higher microbial concentrations
  3. Presence of more organic matter (e.g., soil, feces, or blood)
  4. Rougher surfaces or more porous equipment or material
  5. Lower temperatures

Disinfection may involve chemical or physical agents, but the term disinfection more commonly implies the use of chemical germicides or disinfectants on inanimate objects. See Section F.2.2 below for additional explanation of germicides and disinfectants.

Disinfection is a process that reduces the level of microbial contamination, but there is a broad range of activity that extends from sterility at one extreme to a minimal reduction in the number of microbial contaminants at the other. By definition, chemical disinfection and in particular, high-level disinfection differs from chemical sterilization by its lack of sporicidal power. This is an oversimplification of the actual situation because a few chemical germicides used as disinfectants do, in fact, kill large numbers of spores even though high concentrations and several hours of exposure may be required. Nonsporicidal disinfectants may differ in their capacity to accomplish disinfection or decontamination. Some germicides rapidly kill only the ordinary vegetative forms of bacteria such as staphylococci and streptococci, some forms of fungi, and lipid-containing viruses, whereas others are effective against such relatively resistant organisms as Mycobacterium tuberculosis var. bovis, nonlipid viruses, and most forms of fungi.

Levels of chemical disinfection and activity levels for chemical disinfectants (or germicides) on inanimate surfaces may be used to assist in categorizing and selecting disinfection methods and disinfectants. Levels of chemical disinfection are categorized in Table F-1, and activity levels of selected disinfectants are shown in Table F-2.

Table F-1
Levels of Chemical Disinfection

Level

Level Definition and Description

High

High-level disinfection kills vegetative microorganisms and inactivates viruses, but not necessarily high numbers of bacterial spores. Such disinfectants are capable of sterilization when the contact time is relatively long (e.g., 6 to 10 hours). As high-level disinfectants, they are used for relatively short periods of time (e.g., 10 to 30 minutes). These chemical germicides are potent sporicides and, in the United States, are classified by the Food and Drug Administration (FDA) as sterilant/disinfectants. They are formulated for use on medical devices, but not on environmental surfaces such as laboratory benches or floors.

Intermediate

Intermediate-level disinfection kills vegetative microorganisms, including Mycobacterium tuberculosis, all fungi, and inactivates most viruses. Chemical germicides used in this procedure often correspond to Environmental Protection Agency (EPA)-approved "hospital disinfectants" that are also "tuberculocidal." They are used commonly in laboratories for disinfection of laboratory benches and as part of detergent germicides used for housekeeping purposes.

Low

Low-level disinfection kills most vegetative bacteria except M. tuberculosis, some fungi, and inactivates some viruses. The EPA approves chemical germicides used in this procedure in the U.S. as "hospital disinfectants" or "sanitizers."

Source: adapted from Biosafety in Microbiological and Biomedical Laboratories (BMBL), fifth edition, Appendix B.


Table F-2
Activity Levels of Selected Liquid Germicides a

Procedure/Product

Aqueous Concentration

Disinfection Activity Level

Sterilization

glutaraldehyde

variable

N/A

hydrogen peroxide

6–30%

N/A

formaldehyde

6–8% b

N/A

chlorine dioxide

variable

N/A

peracetic acid

variable

N/A

Disinfection

glutaraldehyde

variable

high to intermediate

ortho-phthalaldehyde

0.5%

high

hydrogen peroxide

3 to 6%

high to intermediate

formaldehyde

1 to 8%

high to low

chlorine dioxide

variable

high

peracetic acid

variable

high

chlorine compounds c

500 to 5,000 mg/L available chlorine (or 1 to 10% household beach in water)

intermediate

alcohols(ethyl,isopropyl) d

70%

intermediate

phenolic compounds

0.5 to 3%

intermediate to low

iodophor compounds e

30 to 50 mg/L free iodine up to
10,000 mg/L available iodine
0.1 to 0.2%

intermediate to low

quaternary ammonium
compounds

 

low

Source: adapted from BMBL, fifth edition, Appendix B.

Footnotes:

  1. This list of chemical germicides centers on generic formulations. A large number of commercial products based on these generic components can be considered for use. Users should ensure that commercial formulations are registered with the EPA or by the FDA.
  2. Because of the ongoing controversy of the role of formaldehyde as a potential occupational carcinogen, the use of formaldehyde is limited to certain specific circumstances under carefully controlled conditions, e.g., for the disinfection of certain hemodialysis equipment. There are no FDA- cleared liquid chemical sterilant/disinfectants that contain formaldehyde.
  3. Generic disinfectants containing chlorine are available in liquid or solid form (e.g., sodium or calcium hypochlorite). Although the indicated concentrations are rapid acting and broad spectrum (tuberculocidal, bactericidal, fungicidal, and virucidal), no proprietary hypochlorite formulations are formally registered with EPA or cleared by FDA. Common household bleach is an excellent and inexpensive source of sodium hypochlorite. Concentrations between 500 and 1,000 mg/L (or ppm) chlorine are appropriate for the vast majority of uses requiring an intermediate level of germicidal activity. Higher concentrations are extremely corrosive as well as irritating to personnel, and their use should be limited to situations where there is an excessive amount of organic material or unusually high concentrations of microorganisms (e.g., spills of cultured material in the laboratory).
  4. The effectiveness of alcohols as intermediate-level germicides is limited because they evaporate rapidly, resulting in short contact times, and also lack the ability to penetrate residual organic material. They are rapidly tuberculocidal, bactericidal, and fungicidal, but may vary in spectrum of virucidal activity (see text). Items to be disinfected with alcohols should be carefully precleaned and then completely submerged for an appropriate exposure time (e.g., 10 minutes).
  5. Only those iodophors registered with EPA as hard-surface disinfectants should be used, closely following the manufacturer's instructions regarding proper dilution and product stability. Antiseptic iodophors are not suitable for disinfecting devices, environmental surfaces, or medical instruments.

An undertanding of the resistance of organisms to chemical germicides should also be considered when selecting the disinfection methods and disinfectants. Table F-3 shows the resistance of selected organisms to decontamination, from most to least resistant.

Table F-3
Descending Order of Organism Resistance to Germicidal Chemicals

BACTERIAL SPORES
Bacillus subtilis, Clostridium sporogenes

MYCOBACTERIA
Mycobacterium tuberculosis var. bovis, nontuberculous mycobacteria

NONLIPID OR SMALL VIRUSES
Poliovirus, Coxsackievirus, Rhinovirus

FUNGI
Trichophyton spp., Cryptococcus spp., Candida spp.

VEGETATIVE BACTERIA
Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella choleraesuis, Enterococci

LIPID OR MEDIUM-SIZE VIRUSES
Herpes simplex virus, cytomegalovirus, respiratory syncytial virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), Hantavirus, Ebola virus

Source: adapted from BMBL, fifth edition, Appendix B

Note: There are exceptions to this list. Pseudomonas spp. are sensitive to high-level disinfectants, but if they grow in water and form biofilms on surfaces, the protected cells can approach the resistance of bacterial spores to the same disinfectant. The same is true for resistance to glutaraldehyde by some nontuberculous mycobacteria, some fungal ascospores of Microascus cinereus and Cheatomium globosum, and the pink-pigmented Methylobacteria. Prions are also resistant to most liquid chemical germicides and are discussed in the last part of this section.

F.2.1.3 Sanitization

Sanitization is the process of generally reducing microorganisms by the use of general cleaning agents. Sanitization is less effective than disinfection at reducing the number of microorganisms. General cleaning of laundry or laboratory, restroom, room, and equipment surfaces with soap and water or another cleaning agent are examples of sanitization. A particular cleaning method might use a chemical germicide or disinfectant, but the cleaning process is considered sanitization if the process only generally reduces the number of microorganisms. See Section F.2.2 below for additional explanation of germicides and disinfectants.

In the food industry, the term sanitization has a more specific meaning. According to the California Retail Food Code (CRFC), sanitization means the application of cumulative heat or chemicals on cleaned food-contact surfaces that, when evaluated for efficacy, is sufficient to yield a reduction of five logs, which is equal to a 99.999% reduction, of representative disease microorganisms of public health importance.

F.2.1.4 Antisepsis

Antisepsis is the application of a liquid antimicrobial chemical to human or animal living tissue. The purpose of antisepsis is to prevent sepsis by destroying potentially infectious organisms or by inhibiting their growth and multiplication. Sepsis is the presence of infectious organisms in the blood or other tissue of the body. No sporicidal activity is implied. Examples of antisepsis include application of a germicide to the injection site on a research animal, and handwashing with germicidal solution. With handwashing, the objective includes preventing the spread of infectious or contaminating agents for safety and quality control.

F.2.2    Antimicrobial Categories

Chemical or physical agents or substances that can decontaminate under ideal conditions have specific terms with specific meanings. The broadest term for such agents is the term antimicrobial. Antimicrobial is a chemical or physical agent that can prevent microbial growth either by some static action or by the direct killing of microbes. Categories of antimicrobials include:

  • Sterilant. An antimicrobial chemical or physical agent that is capable of killing all microbes including their spores to the sterility assurance level.
  • Germicide. An antimicrobial substance or physical agent that kills microbes. Germicides are a broader category of antimicrobials than disinfectants, since some germicides are active against endospores and viruses. Germicides, which are also known for the specific microorganisms they kill, end with the suffix –cidal (e.g., bacteriocide, sporicide, fungicide, virucide).
  • Disinfectant. A chemical germicide or physical agent that is applied to inanimate objects to kill microbes, but is not capable of killing endospores, some viruses, or mycobacterium. Disinfectants are typically chemical germicides.
  • Antiseptic. A disinfecting chemical agent applied to living tissue and used to prevent sepsis. Antiseptics are a subset of disinfecting chemical agents. A few agents are suitable as both disinfectants and antiseptics, although most disinfectants are too harsh for use on delicate skin.

F.2.3    Antimicrobial Selection and Registered Disinfectants

When using a chemical or physical antimicrobial to ensure decontamination is accomplished for biosafety purposes (i.e., protection of workers, public, agriculture, or environment):

  • There should be information indicating that the selected antimicrobial will be effective when used in a certain manner for the biological materials or agents and equipment or surfaces that need to be decontaminated; and
  • The antimicrobial should be used in accordance with its antimicrobial activity capabilities and conditions of use.

Antimicrobial information in this appendix, information provided by manufacturers (e.g., labels or technical specifications), and other information may be used for selecting and using the appropriate antimicrobial. Selecting a commercially available chemical antimicrobial product registered with the EPA or cleared by the FDA and using the product within its manufacturer-specified limits also ensure effective decontamination. The following lists of antimicrobials registered with EPA and FDA are available online:

The Occupational Safety and Health Administration (OSHA) Bloodborne Pathogens (BBPs) Standard requires that work surfaces that are contaminated with BBP material (as defined in Section 3.3.4 of this manual) must be cleaned with an “appropriate disinfectant.” Appropriate disinfectants include:

  • Household bleach (i.e., approximately 5.25% sodium hypochlorite) diluted to concentrations ranging from 1% (1:100) to 10% (1:10) in water.
  • EPA-registered products as sterilants (List A)
  • EPA-registered products as tuberculocides (List B)
  • EPA-registered products effective against HIV/HBV (List D), or
  • FDA-cleared sterilants and high-level disinfectants

Any of the above products are considered effective when used according to the manufacturer's instructions, provided the surfaces have not become contaminated with agents, or volumes or concentrations of agents for which higher level disinfection is recommended. Also note that the EPA lists contain the primary registrants' products only. The same formulation is frequently repackaged and renamed and distributed by other companies. These renamed products will not appear on the list, but their EPA Registration Number must appear on the label. Products cleared solely by the FDA will not have an EPA Number.

F.3    Chemical Antimicrobials

This section summarizes basic types and characteristics of antimicrobials that are chemical agents. Section F.4 below summarizes antimicrobials that are physical agents.

All chemical antimicrobials harm microorganisms in some manner, but different chemical antimicrobials have different mechanisms of action. Mechanisms of harm include protein denaturation, membrane disruption, nucleic acid damage, and inhibition of metabolism. Chemical antimicrobials that are summarized in this section include surfactants, halogen-containing compounds, alcohols, phenol and phenol derivatives, oxidizing agents, and alkylating agents.

F.3.1    Surfactants (Soaps and Detergents)

A surfactant is a surface active agent that is usually an organic compound that possesses both hydrophilic (water-loving) and lipophilic (fat-liking) properties that make the compound soluble in water and lipids. Surfactants therefore increase the solubility of lipids in water solutions and increase the ability of water solutions to wet (i.e., move across or penetrate) lipid surfaces. Soaps and detergents are examples of surfactants.

F.3.1.1 Soaps

Soap is sodium or potassium salts of fatty acids. Soaps are therefore alkaline (pH greater than 7). Soaps either harm bacteria that are sensitive to high pH, or remove pathogens from surfaces by cleaning the surface.

F.3.1.2 Detergents and Quaternary Ammonium Compounds

Detergent is a synthetic surfactant. A detergent may be cationic (positively charged) or anionic (negatively charged). Cationic detergents are better at inactivating bacteria than anionic detergents.

One commonly used type of cationic detergent disinfectant is a quaternary ammonium compound. Quaternary ammonium compound or quat is a cationic detergent compound derived from ammonia by replacing the hydrogen atoms with organic radicals, and the compound is especially important as surface-active agents or disinfectants, or in drugs. Quats have strong surface activity and can be used for general cleaning and low-level disinfection. Additional properties of quaternary ammonium compounds include the following:

  • Active against Gram-positive bacteria and lipid-containing viruses. They are less active against Gram-negative bacteria and are not active against nonlipid-containing viruses and bacterial spores.
  • Less effective or inactivated by organic materials, soaps or anionic detergents, or salts of metals found in water. Quats are often mixed with another agent to overcome some of these problems.
  • Built-in cleaning properties and relatively nontoxic (e.g., can be used for general cleaning and food equipment).
  • Has no odor but acts as a deodorizer.
  • Effective at temperatures up to 212°F.
  • More effective in alkaline than in acid solutions.
  • Typically nonirritating to the skin when used in proper dilution, but prolonged skin or eye contact should be avoided.
  • Stable in storage.

F.3.2    Halogens (Chlorine and Iodine)

Halogens are a group of elements on the periodic table. Chlorine and iodine are two halogens that are routinely used as antimicrobials.

F.3.2.1 Chlorine and Sodium Hypochlorite

Chlorine-containing solutions are commonly used disinfectants, and sodium hypochlorite in the form of household bleach is the most common solution used for chlorine disinfection. These solutions have broad-spectrum antimicrobial activity, but their decay rates and corrosive nature limit their use. The following bullets provide additional information:

  • Concentrations and Effectiveness: Chlorine-containing solutions have broad spectrum activity, but the concentration of the chlorine-active ingredient in the solution at time of use affects germicidal activity. Low concentrations of available chlorine (2 to 500 ppm) are active against vegetative bacteria, fungi, and most viruses. Effectiveness increases with concentration of available chlorine. Rapid sporicidal action can be obtained at about 2,500 ppm.
  • Active Ingredient Decay: The chlorine-active ingredient typically decays or is consumed. Decay or decomposition typically occurs over time and is accelerated by unfavorable storage conditions. Chlorine is also consumed by excess organic materials. Use of sufficient concentrations and quantity of chlorine, along with precleaning items to be disinfected, ensures sufficient chlorine is available for disinfection.
  • Corrosiveness: Chlorine-containing solutions are strong oxidizers and are very corrosive to personnel and some surfaces. Personnel handling these solutions must wear required hand, eye, and body protection (see Section 5.4 of this manual). Surfaces such as stainless steel may be corroded and should be wiped or rinsed with water following disinfection.

Text Box:  One of the most common and effective disinfectants used in the laboratory is sodium hypochlorite (NaOCl) in water or “bleach.” Household bleach is a water-based solution of sodium hypochlorite with a typical concentration of 5.25% by weight (or 52,500 ppm) of the active sodium hypochlorite ingredient. Commercial supplies are also available in the 12 to 15% dilution range, but household bleach is typically sufficient for laboratory use. Many brands and formulations of bleach are registered with the EPA as a disinfectant that is effective against bloodborne and other common human pathogens (see Section F.2.3 above). Clorox® is the best-known brand of bleach in the U.S.

Common applications and mixtures of household bleach are listed below.

  • Work Surfaces and Equipment: Hard work surfaces and equipment may be disinfected with 1% solution of fresh household bleach (or 500 ppm sodium hypochlorite). A 1% household bleach solution can be made by mixing 1 part household bleach with 99 parts water, or 1/8 to 1/4 cup household bleach with water in a gallon container, or 10 ml of household bleach with water in a 1 L container. Contact time for bleach is generally considered to be the time it takes the product to air dry.
  • Spills and Liquid Waste: Biohazardous spills and liquid waste may be decontaminated by adding household bleach to water or the liquid to be decontaminated until a 10% concentration of household bleach is achieved (or 5,000 ppm sodium hypochlorite). A 10% household bleach solution can be made by mixing one part household bleach with 9 parts water, or 1.5 cups household bleach with water in a gallon container, or 100 ml of household bleach with water in a 1 L container. The bleach should remain in contact with the spill or waste material for approximately 20 minutes to ensure adequate germicidal action. See Appendix G of this manual for additional information on spill cleanup.

Sodium hypochlorite solutions are not very stable, and the antimicrobial activity of the chlorine typically decays over time. This decay is accelerated by unfavorable storage conditions and must be compensated by mixing fresh solutions. Favorable storage conditions include: temperature below 70°F, plastic container (not metal or glass), opaque container (to minimize exposure to light), and closed container (to minimize exposure to air). It is common to measure 50% decay within one month under favorable storage conditions. Since bleach antimicrobial activity decays over time, bleach solutions must be sufficiently fresh so that the solution to be used for decontamination has sufficient antimicrobial activity. Fresh solutions of diluted household bleach made up daily are recommended for disinfection of work surfaces.

F.3.2.2 Iodine and Iodophors

Iodine is another halogen that is routinely used as an antimicrobial (at 70 to 150 ppm total iodine), and iodine has properties similar to chlorine. Iodophor is a preparation containing iodine complexed with a solubilizing agent, such as a surfactant or povidone (a type of water soluble polyvinyl polymer). The resulting iodophor is a water-soluble material that increases penetration (as a surfactant) and slows the release of free iodine over long periods (as a disinfectant) when in solution. Iodophors are prepared by mixing iodine with the solubilizing agent. Wescodyne® is a common laboratory disinfectant iodophor.

Additional properties of iodophors include:

  • Rapid germicidal action. Effective against vegetative bacteria, Gram-positive bacteria, Gram-negative bacteria, fungi, viruses, and tubercle bacilli. Poor activity against bacterial spores.
  • Most effective in acid solutions.
  • Should not be used in hot water, since iodine is vaporized at 120 to 125°F. For optimal germicidal activity, dilute with warm acidic water. Resulting solutions are less stable but have a higher germicidal activity.
  • Effectiveness reduced by organic matter (but not as much as hypochlorites).
  • Stable in storage if kept cool and tightly covered.
  • Relatively harmless and nontoxic to humans.
  • The solution has germicidal activity if the color is brown or yellow.
  • Solutions of sodium thiosulfate can be used to inactivate iodophors and remove iodophor stains.

Iodophors may also be used as antiseptics. Betadine and isodine are examples of antiseptic iodophors. Iodine may also be used in an alcohol solution (i.e., or tincture) as an antiseptic.

 F.3.3   Alcohols

Ethyl or isopropyl (rubbing) alcohol concentrations of 70 to 90% in water are good general-use disinfectants with some limitations. Alcohol-water mixtures are more penetrating than pure alcohols, and therefore provide better disinfection. Alcohol concentrations above 90% are less effective than 70 to 90% concentrations.

Alcohols have some positive and negative characteristics, including:

  • Alcohols are effective against a broad spectrum of bacterial species and many viruses, but they are less active against nonlipid viruses and ineffective against bacterial spores.
  • Alcohols evaporate quickly and leave no residue. These characteristics often make alcohols convenient and efficient, but provide limited penetration and disinfection time.

 F.3.4   Phenol and Phenol Derivatives (Phenolics)

Phenol and phenol derivatives (or phenolics) come in various concentrations ranging mostly from 5 to 10% phenol-based compounds. These disinfectants are especially useful for disinfecting materials contaminated with organic materials and contaminated surfaces. Lysol® is an example of a phenol-based disinfectant.

Additional properties of phenol and phenol derivatives include the following:

  • Effective at killing Gram-negative and Gram-positive bacteria including Mycobacterium tuberculosis, fungi, and lipid-containing viruses. Not active against spores or most nonlipid viruses.
  • Low solubility in water unless combined with detergent.
  • Stable in storage.
  • Less adversely affected by organic matter than other common disinfectants.
  • Effective over a relatively large pH range.
  • Prolonged contact deteriorates rubber.
  • Can cause skin and eye irritation.
  • Not for use on food contact surfaces.
  • Some phenolics are mild enough for use as antiseptics whereas others are too harsh or otherwise dangerous to be employed on living tissue.

F.3.5    Oxidizing Agents (Hydrogen Peroxide)

Hydrogen peroxide is an oxidizing agent and may be used as a liquid or vapor antimicrobial. Hydrogen peroxide vapor may be used for decontamination of equipment such as biosafety cabinets or high-containment (Biosafety Level 3) rooms that may be sealed during the decontamination process.

F.3.6    Alkylating Agents (Formaldehyde, Glutaraldehyde, Ethylene Oxide)

Formaldehyde, glutaraldehyde, and ethylene oxides are alkylating agents. These agents add carbon-containing functional groups to biological molecules.

F.3.6.1 Formaldehyde

Formaldehyde may be used as a liquid or gaseous antimicrobial. When used as a liquid, formaldehyde may be mixed with water as formalin or mixed with alcohol. Formaldehyde is also a human carcinogen, creates respiratory problems, and has a very low occupational exposure ceiling and short-term exposure limits that are approximately equal to the odor threshold.
Additional information on formaldehyde antimicrobials are listed below:

  • Formalin is 37% solution of formaldehyde in water. Dilution of formalin to 5% results in an effective disinfectant. A concentration of 8% formaldehyde exhibits good activity against vegetative bacteria, spores, and viruses.
  • Formaldehyde and alcohol solutions (8% formaldehyde in 70% alcohol) are considered very good disinfectants because of their effectiveness against vegetative bacteria, fungi, spores, and viruses. This is the disinfectant of choice for many applications.
  • Formaldehyde gas may be generated by heat-accelerated depolymerization of flake paraformaldehyde. The resulting gas may be used to decontaminate equipment such as biosafety cabinets that may be sealed prior to decontamination.

F.3.6.2 Glutaraldehyde

Gluteraldehyde may be used for cold sterilization of equipment (e.g., medical) that cannot be steam sterilized, but sterilization often requires many hours of exposure. Two percent solutions exhibit good activity against vegetative bacteria, spores, and viruses. Its use, however, must be limited and controlled due to its toxic properties and ability to damage the eyes.

Glutaraldehyde is slightly acidic in aqueous solution and typically used at ambient temperature. When these solutions are adjusted by sodium bicarbonate (or other buffers) to a pH of 7.5 to 8.5, glutaraldehyde is considered to be activated and the antimicrobial activity enhanced. Activated glutaraldehyde has limited stability after activation.

F.3.6.3 Ethylene Oxide

Ethylene oxide is a gaseous chemical antimicrobial used to sterilize laboratory, medical, and pharmaceutical products and equipment that would be damaged by high-temperature steam sterilization (e.g., prepackaged plastic petri dishes). This gas is especially useful because it penetrates very well into small crevices.

F.4    Physical Antimicrobials

This section summarizes basic types and characteristics of antimicrobials that are physical agents. Physical antimicrobials summarized in this section include dry heat, wet heat, ultraviolet radiation, ionizing radiation, visible light, and filtration.

F.4.1    Heat

Dry heat (e.g., oven) and moist heat (e.g., autoclave) may be used to sterilize materials and equipment. The following principles and comparisons generally apply to sterilization with dry and moist heat:

  • Moist heat is more effective than dry heat at a given temperature or length of exposure.
  • Moist heat is more penetrating than dry heat.
  • Temperature and length of exposure are inversely related, and penetration is critical.
  • Temperature and length of exposure needed to achieve sterilization are inversely related (i.e., lower temperatures require longer exposure times).
  • Time to achieve sterilization does not start until heat has penetrated into the item and the required temperature in the item has been achieved.

F.4.1.1 Dry Heat (Baking and Incineration)

Dry heat sterilization may include baking or incineration.

  • Baking in an oven to achieve sterilization typically requires 171°C for at least 1 hour, 160°C for at least 2 hours, or 121°C for at least 16 hours.
  • Incineration may also be used to achieve dry heat sterilization. Examples include off-site incineration of biohazardous or pathological waste by an LBNL subcontractor or heating an inoculating loop in an infrared heat chamber at 815°C (1,500°F).

Specific times and temperatures must be determined for each type of material being sterilized. Generous safety factors are usually added to allow for variables that can influence the efficiency of dry heat sterilization, such as:

  • The moisture of the sterilization environment as well as the moisture history of organisms prior to heat exposure.
  • The heat transfer properties and the spatial configuration or arrangement of articles in the load.

F.4.1.2 Wet Heat (Boiling and Autoclaving)

Use of wet heat may include boiling an item in water or processing the item in an autoclave. Boiling water is a common means of applying moist heat, but boiling does not kill endospores and all viruses. Boiling water is 100°C (212°F) at standard atmospheric pressure. Higher wet-heat temperatures and sterilization efficacy may be achieved with a pressurized autoclave.

Autoclaves are commonly used to sterilize laboratory equipment or materials such as glassware, media, reagents, or waste. See Section F.5 below for general information and guidelines on autoclave principles, operation, and maintenance.

F.4.2    Ultraviolet (UV) Radiation

UV radiation or UV light is electromagnetic radiation with a wavelength shorter than that of visible light but longer than X-rays. They are in the range of 10 nanometers (nm) to 400 nm, and energies from 3 electron volts (eV) to 124 eV. UV radiation is so named because the spectrum consists of electromagnetic waves with frequencies higher than those that humans identify as the color violet.

F.4.2.1 UV Light Health Effects and Categories

UV radiation may affect or damage the skin and eyes depending on the wavelength, intensity, and duration of exposure. Other organs are typically not affected because UV light does not penetrate deeply into tissue. Acute effects to the skin and eyes are generally not permanent but can be quite painful.

The UV spectrum is divided into three wavelength bands primarily based on their biological effects:

  • UVA (315 to 400 nm) is long-wave UV or “back light” and is used in dentistry and tanning. UVA rays can penetrate the middle layer of skin (dermis) and cause darkening and toughening of the skin. Overexposure to UVA has also been associated with suppression of the immune system and cataract formation.
  • UVB (280 to 315 nm) is medium-wave UV and is used for fade testing and photocuring of plastics. UVB rays reach the outer layer of skin (epidermis) and cause skin burns, erythma (reddening of the skin), and darkening of the skin. Prolonged exposures increase the risk of skin cancer.
  • UVC (100 to 280 nm) is short-wave UV and is used as a germicidal (e.g., inside biosafety cabinets). UVC poses the most risk to skin. Although UVC from the sun is absorbed by the atmosphere, manmade sources of UVC need to restrict their intensity and control exposure.


Electromagnetic spectrum. Source: CCOHS, OSH Answers, Physical Agents, Ultraviolet Radiation
(February 2010).


http://www.fda.gov/ucm/groups/fdagov-public/documents/image/ucm116427.jpg

UV light that penetrates skin. Source: FDA, Radiation-emitting Products, Ultraviolet Radiation
(February 2010).


The eyes are particularly sensitive to UV radiation. Even a short exposure of a few seconds can result in painful but temporary inflammatory conditions known as photokeratitis and conjunctivitis. Examples of eye disorders resulting from UV exposure include "flash burn," "ground-glass eye ball," "welder's flash," and "snow blindness.” The symptoms are pain, discomfort similar to the feeling of sand in the eye, and an aversion to bright light.

The eyes are most sensitive to UV radiation from 210 nm to 320 nm (UVC and UVB). Maximum absorption by the cornea occurs around 280 nm. UVA absorption by the lens may be a factor in producing a cataract (a clouding of the lens in the eye).

All wavelengths less than 320 nm (UVB and UVC) are actinic, which means they are capable of causing chemical reactions. Wavelengths below 180 nm are of little practical biological significance since the atmosphere readily absorbs them.

F.4.2.2 Biosafety Cabinet UV Light

Long-term exposure to UV light may be used for disinfecting surfaces and air; however, UV light is not recommended or necessary for use inside biosafety cabinets (BSCs). This is because UV light is limited by many factors (see bulleted list below) as a disinfectant and harmful to human tissue. Other means of disinfection (e.g., chemical) are recommended for use inside BSCs.

UV light’s ability to disinfect inside BSCs is limited by a number of factors including:

  • Penetration: UV light lacks penetrating power. Microorganisms beneath dust particles or beneath the work surface are not affected by the UV radiation.
  • Relative Humidity: Humidity decreases the effectiveness of UV light. Antimicrobial effects of UV light drops off precipitously above 70% relative humidity.
  • Temperature and Air Movement: Optimum temperature for UV light output is 77 to 80°F. Temperatures below this optimum temperature result in reduced output of the antimicrobial wavelength. Moving air tends to cool the lamp below its optimum operating temperature and results in reduced output.
  • Lamp Cleanliness: Dust and dirt can block the antimicrobial effectiveness of UV lights. UV lamps need to be cleaned weekly with an alcohol and water mixture.
  • Lamp Age: The intensity of UV light emitted from UV lamps decreases with age, and bulb ratings (hours of use) may vary by manufacturer. UV lamps need to be checked periodically (approximately every six months) to ensure the intensity and wavelength of UV light needed for antimicrobial activity is being emitted.

See Appendix E, Section E.5, of this manual for additional information on using UV light inside BSCs. If UV light is used as an antimicrobial but is not a required biosafety control, then maintenance and testing of the UV lights is not required for biosafety purposes. For example, germicides are used as the primary means of BSC disinfection, so maintenance and testing of the UV light inside the BSC is not required for biosafety purposes.

F.4.3    Ionizing Radiation   

Ionizing radiation is radiation of sufficiently high energy to cause ionization in the medium through which it passes. This radiation may be of a stream of high-energy particles (e.g. electrons, protons, alpha particles) or short-wavelength electromagnetic radiation (e.g., ultraviolet, X-rays, gamma rays). This type of radiation can cause extensive damage to the molecular structure of a substance either as a result of the direct transfer of energy to its atoms or molecules, or as a result of the secondary electrons released by ionization. The effect of ionizing radiation in biological tissue can be very serious, usually as a consequence of the ejection of an electron from a water molecule and the oxidizing or reducing effects of highly reactive species. Biological effects on living cells may include DNA damage and mutations.

 


Ionizing and nonionizing radiation. Source: Wikipedia, “Nonionizing Radiation” (February 2010).


Different types of ionizing radiation display different degrees of penetration and may be used to sterilize equipment (e.g., medical instruments) or biological materials (e.g., inside human cadaver bones). Use of ionizing radiation as an antimicrobial requires established and specialized methods known to sterilize specific items.

F.4.4    Visible Light

Strong visible light can decrease bacterial viability. Drying laundry on a clothesline is an example of disinfection by using detergents and strong visible light.

F.4.5    Filtration (HEPA Filters)

Filtration is used as an antimicrobial treatment for air and liquids.

  • High-efficiency particulate air (HEPA) filters are used to filter air flowing into aseptic areas (e.g., the work area inside a BSC) and out of potentially contaminated areas (e.g., exhaust from a BSC). See Section 5.6.4.2(a) and Appendix E of this manual for additional HEPA filter and BSC information.
  • Filtration is commonly used when materials are heat labile, but sterilization is not necessarily achieved unless the filter has very small filter pores. Smaller filter pores will also slow filtration speed.

F.5    Autoclave Sterilization and Safety

This section provides general information and guidelines on autoclave principles, operation, and maintenance typically needed to sterilize materials or equipment and ensure operator safety. Autoclave is a piece of equipment with a chamber that is used to sterilize items by applying wet heat (i.e., high-pressure steam) at temperatures above the normal boiling point of water and pressures above normal atmospheric pressure.

Autoclaves are used to sterilize laboratory equipment or materials such as glassware, media, reagents, or waste. Autoclaves are commonly used because they are a dependable means of achieving the necessary level of killing efficacy (or sterility assurance level) for most biological materials. In addition, autoclaves do not generate other chemical antimicrobial waste or sources of contamination. See Section F.2.1.1 for general information on sterilization and killing efficacy.

Autoclaves must be operated and monitored properly to achieve sterility and safety. Operator safety is a concern because autoclaves may pose physical hazards (e.g., heat, steam, pressure) and biological hazards.

F.5.1 Autoclaves and Sterilization

Autoclaves achieve higher sterilization efficacy in part because they generate wet-heat temperatures (e.g., 121°C or 250°F) higher than those achieved under standard atmospheric pressure (i.e., 100°C or 212°F). Exposure of material in an autoclave to 121°C (250°F) for 15 or more minutes is typically sufficient for sterilization, but the material’s temperature must be 121°C before the time to achieve sterilization is started. Large items, large volumes, and items that are poorly penetrated by the autoclave’s steam may take much longer than 15 minutes to sterilize. If penetration of moisture into the item is blocked, sterilization may not be achieved.

Autoclave conditions critical to ensuring reliable sterilization methods are proper temperature and time and the complete replacement of autoclave chamber air with steam (i.e., no entrapment of air). Some autoclaves utilize a steam-activated exhaust valve that remains open during the replacement of air by live steam until the steam triggers the valve to close. Others utilize a precycle vacuum to remove air prior to steam introduction.

Standard autoclave conditions for the types of materials that need sterilization should be established. Autoclave treatment conditions to achieve sterility will vary in relation to the volume of material treated, volume of the autoclave, the contamination level, the moisture content, and other factors. Treatment conditions for typical materials are listed below:

  • Laundry: 121°C (250°F) for a minimum of 30 minutes.
  • Trash: 121°C (250°F) for at least 45 minutes per bag. Size of the autoclave and size of the bags greatly affect sterilization time. Large bags in a small autoclave may require 90 minutes or more.
  • Glassware: 121°C (250°F) for a minimum of 25 minutes.
  • Liquids: 121°C (250°F) for 25 minutes for each gallon.
  • Animals and bedding: Steam autoclaving is not recommended (sterilization time required would be at least 8 hours). Incineration in an approved facility is the recommended treatment of these wastes.

F.5.2 Autoclave Operation and Safety

This section provides general autoclave operation information and guidelines that should be used when applicable to the operation and as needed to ensure operator safety and sterilization. In addition, specific requirements and operational procedures noted in the autoclave owner’s manual should be followed since each autoclave may have unique characteristics. The owner’s manual should be readily available to answer autoclave operational questions.

F.5.2.1 Autoclave Instruction

The supervisor and work lead must ensure that the autoclave operator understands the autoclave hazards, controls needed to protect themselves, and any procedures necessary to accomplish sterilization for biosafety purposes.

F.5.2.2 Autoclave Operation Modes

Autoclaves typically use different combinations and patterns of high heat, vacuum, and pressure to sterilize the load. These combinations and patterns are used in autoclave run cycles or runs and are based on the type of material to be sterilized. General types of runs include liquids for any type of water-based solutions, dry goods with vacuum, and dry goods without vacuum. Autoclaves often have an additional drying cycle in which hot air is drawn through the chamber to dry materials after sterilization. Controls for different autoclaves vary, so the manufacturer’s instructions regarding loading, load sizes, cycle types, and settings should be carefully followed. Additional information typical of these different run cycles is listed below:

  • Liquids Run. This run is longer than the other two runs, but uses lower temperatures to minimize evaporation of the liquids being sterilized.
  • Dry Goods with Vacuum Run. This run moves steam and heat into the deepest parts of large bags or bundles of materials and provides the best conditions for killing resistant organisms. During this type of run, the chamber alternates between cycles of high pressure, steam, and vacuum. It is important that steam and pressure be able reach the entire load, so bag closures should be carefully loosened once they are in the autoclave.
  • Dry Goods without Vacuum Run. This run pressurizes the chamber with steam for the duration of the cycle and then returns to normal. This process is used primarily for items that have been cleaned but need to be sterilized. Materials should be packed so that the heat and pressure can readily reach the whole load.

F.5.2.3 Autoclave Container Selection

Bags, pans, and other containers are used in the autoclave to provide primary and secondary containment for the materials and items that need to be autoclaved. Additional considerations and practices regarding these containers include:

  • Polypropylene Autoclave Bags. Autoclave or biohazard bags that may be used to contain solid materials are tear-resistant but can be punctured or burst in the autoclave. These bags should therefore be placed in a rigid container during autoclaving. Bags are available in a variety of sizes, and some are printed with an indicator that changes color when processed.
  • Polypropylene Containers and Pans. Polypropylene is a plastic capable of withstanding autoclaving, but it is resistant to heat transfer. Materials contained in a polypropylene pan will therefore take longer to autoclave than the same materials in a stainless steel pan. The time required to sterilize material in a polypropylene container may be reduced by removing the container’s lid, turning the container on its side, or selecting a container with the lowest sides and widest diameter that will fit in the autoclave.
  • Stainless Steel Containers and Pans. Stainless steel is a good conductor of heat and is less likely to increase sterilizing time, but it is more expensive than polypropylene.

F.5.2.4 Autoclave Preparation and Loading

  • Wear long pants, closed-toe shoes, body protection such as a lab coat, gloves, and safety glasses or goggles.
  • Before loading the autoclave, check inside the autoclave for any items left behind by the previous user that could pose a hazard (e.g., sharps), and then clean the drain strainer.
  • Load the autoclave properly according to manufacturer’s recommendations. Typical loading practices are listed below.
  • Do not autoclave items containing materials such as corrosives, solvents, volatiles, or radioactive materials that may contaminate the autoclave, create an inhalation hazard, or explode.
  • Use autoclave bags and autoclavable polypropylene or stainless steel pans. Other plastics may melt.
  • Load liquids as follows:
    • Fill liquid containers only half full.
    • Loosen caps or use vented closures so that heated and expanding liquids and vapors do not cause explosion of bottles or tubes.
    • Use only borosilicate glass (e.g., PyrexTM or KimaxTM) that can withstand the high autoclave temperature.
    • Use a pan with a solid bottom and walls to contain the liquid and catch spills.
  • Load autoclave bags as follows:
    • Put bags into pans to catch spills.
    • Gather bags loosely at the top and secure the top with a large rubber band or autoclave tape. This will create an opening through which steam can penetrate. Bags are impermeable to steam and therefore should not be twisted and taped shut.
  • Load dry goods such as glassware as follows:
    • Check plastic materials to ensure they are compatible with the autoclave.
    • Put individual glassware pieces within a heat-resistant plastic tray on a shelf or rack and not on the autoclave bottom or floor.
    • Add 1/4 to 1/2 inch of water to the tray so the bottles will heat evenly.
  • Leave space between items in the load to allow steam circulation.

F.5.2.5 Autoclave Cycle and Time Selection

Ensure the door to the autoclave is fully closed and latched, and the correct cycle and time has been selected before starting the cycle. Cycle selection should be based on the type of items and packs to be autoclaved:

  • Use liquid cycle with slow exhaust when autoclaving liquids to prevent contents from boiling over.
  • Use fast exhaust cycle for glassware.
  • Use fast exhaust and dry cycle for wrapped items.

Time selection should be based on the items’ sizes, volumes, insulating capacity, and other characteristics as follows:

  • Take into account the size of the items to be autoclaved. Larger items with more volume take longer to autoclave. For example, a 2-liter flask containing 1 liter of liquid takes longer to sterilize than four 500 ml flasks that each contain 250 ml of liquid.
  • Materials with a high insulating capacity such as animal bedding or high-sided polypropylene containers increase the time needed for the load to reach sterilizing temperatures.
  • Autoclave bags containing biological waste should be autoclaved for 50 minutes to ensure decontamination.

F.5.2.6 Removing Autoclave Loads

Practices that should be used to prevent the operator from being injured or burned while removing the load from the autoclave include:

  • Wear long pants, closed-toe shoes, body protection such as a lab coat, safety glasses or goggles, and heat-resistant gloves to open the autoclave door and remove nonliquid items from the autoclave.
  • When handling large volumes of liquid, wear waterproof boots (e.g., rubber), a rubber or plastic apron that extends past the top of the boots, and sleeve protectors in addition to the clothing and personal protective equipment listed above.
  • Check that the run cycle is finished and the chamber pressure is zero.
  • Open the door in the following manner to prevent burns caused by steam rushing out the door: Stand behind the door, slowly open the door a crack, and keep head and hands away from the opening.
  • Allow liquids to cool for 10 to 20 minutes before removing the load from the autoclave. Liquids removed too soon may boil up and out of the container and burn the operator. Then let the liquids cool for an extended period (e.g., one hour) before touching the load with ungloved hands. Be sure others in the area know a heat hazard is present.
  • Allow loads containing only dry glassware to cool for 5 minutes before removing the load from the autoclave. Then let the glassware cool for about 15 minutes before touching with ungloved hands.

F.5.2.7 Autoclave Material Staging

The following guidelines apply to staging materials for autoclaving and cleaning:

  • Materials or equipment that will be reused and are contaminated with biohazardous material or waste should be autoclaved before being washed and stored.
  • Laboratories and other areas where materials or equipment are staged for autoclaving or cleaning should have separate areas or containers for items designated as “Biohazardous—To Be Autoclaved” and “Not Biohazardous—To Be Cleaned.”
  • Biohazardous materials or equipment being staged for autoclaving should be sterilized or safely confined and identified at the close of each workday. Such items should not be placed in autoclaves overnight in anticipation of autoclaving the next day.

F.5.2.8 Burn Emergencies

If you are burned, seek medical treatment as soon as possible. Burns to the face, third-degree burns, or burns over large areas of the body should be treated as emergencies. The LBNL emergency phone number should be called. Minor burns should be treated by using first aid procedures. These procedures include immersing the burn immediately in cool water, removing clothing from the burn area, and keeping the injured area cool for at least 5 minutes and preferably longer. Any burns to the face or eye or any burns that blister should be seen by a physician. Regardless of the degree of severity, report the burn to your supervisor and Health Services as an occupational injury.

F.5.3    Autoclave Maintenance and Monitoring

Assurance is needed that the autoclave is operating properly and sterilizing the load. Assurance includes routine autoclave maintenance, monitoring autoclave conditions, and maintaining documentation.

Maintenance described in the autoclave owner’s manual should be performed to ensure the autoclave is operating properly. This maintenance typically includes periodic maintenance performed by a qualified technician and more frequent maintenance procedures performed by the operator.

Monitoring the sterilization process and efficacy typically includes the use of different monitoring methods including:

  • Mechanical Monitoring. Mechanical monitoring, a secondary method for ensuring sterilization, involves observing and recording physical aspects of the cycle such as temperature, pressure, or time. Thermometers, pressure gauges, clocks, and logs are commonly used to observe and record the run’s physical parameters. Some autoclaves have recording devices to assist in recording run cycle conditions.
  • Chemical Monitoring. Chemical monitoring uses chemical indicators that change color or physical form when an autoclave bag or pack is exposed to certain autoclave temperatures. Examples include autoclave tape and special markings on autoclave bags that are used as external indicators on the outside of the load. These indicators are typically considered process indicators since they only show that the item has been processed through the autoclave at a certain temperature, but they do not show that:
    • Sterilization has been achieved or that a complete sterilization cycle has occurred.
    • Temperature was achieved in the innermost parts of the load unless they are carefully placed in the load. An easy way to check interior temperature is to wrap an item such as a plastic test tube or pipette tip with autoclave tape, attach string to the item, and put the item deep into the load. Then, tape the other end of the string to the outside of the bag so that the indicator can be pulled out of the bag. Recover the indicator after the run and confirm that it has also changed color. Warning: do not open a bag of material that may present a hazard to the operator (e.g., Risk Group 2 material) to bury an indicator inside.
  • Biological Monitoring. Biological monitoring (or spore testing) uses live, resistant bacterial spores on strips or in self-contained vials as biological indicators that sterilization has been achieved as demonstrated by the death of the bacterial spores. Use of appropriate biological indicators at locations throughout the autoclave is considered the best and most direct indicator of sterilization. The biological indicator most widely used for wet heat sterilization is Bacillus stearothermophilus spores. Biological indicators must be used to test the efficiency of the autoclave when the autoclave is used as the final treatment of the item prior to disposal as medical waste/biohazardous waste, or when the item will be reused and is contaminated with RG2 biological materials. In these cases, tests should be performed periodically, and test records should be maintained for three years.

The autoclave and process should be evaluated and corrected if monitoring indicates that the autoclave run conditions were not correct, temperatures were not sufficient as shown by chemical indicators, or spores on biological indicators were not killed. Discontinue use of the autoclave if it is not working properly and post a “do not use” sign. Mechanical failures need to be attended by a qualified autoclave technician. When the problem is corrected, the load should be re-autoclaved to ensure sterility.

F.6 References