Chapter 26

BIOSAFETY

____________________

 

Appendix F. Decontamination and Antimicrobials

F.1 Introduction and Scope

Text Box:  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

Text Box:   
Sterilization processes: autoclaving or disposal as medical/biohazardous waste. Source: Berkeley Lab EHS.
Sterilization processes: autoclaving or disposal as medical/biohazardous waste. Source: Berkeley Lab EHS.

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 one in one 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

 

Text Box:        
Surface disinfectants and disinfection processes. Lower-level disinfection of a laboratory bench top with 70% ethanol. Intermediate-level disinfection of a biosafety cabinet with fresh 1% household bleach and animal cage shelves with chlorine dioxide solution. Source: Berkeley Lab EHS.
Surface disinfectants and disinfection processes. Lower-level disinfection of a laboratory bench top with 70% ethanol. Intermediate-level disinfection of a biosafety cabinet with fresh 1% household bleach and animal cage shelves with chlorine dioxide solution. Source: Berkeley Lab EHS.

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., six 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 sterilants/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 and 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 Germicidesa

 

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 compoundsc

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 compoundse

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:

a   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.

b   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 sterilants/disinfectants that contain formaldehyde.

c   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. Chlorine concentrations between 500 and 1,000 mg/L (or ppm) 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).

d   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. Items to be disinfected with alcohols should be carefully precleaned and then completely submerged for an appropriate exposure time (e.g., 10 minutes).

e     Only iodophors registered with the 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 understanding 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

 

Descending Order of Resistance to Germicides

 

Agent Type

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

Text Box:  
Restroom sanitization with a disinfectant Source: Berkeley Lab EHS.
Restroom sanitization with a disinfectant Source: Berkeley Lab EHS.

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.

 

Text Box:     
Research sanitization processes: Laboratory coat, glassware, and animal cage washing. Fermenter cleaning using a clean-in-place process unit. Source: Berkeley Lab EHS and Public Affairs.
Research sanitization processes: Laboratory coat, glassware, and animal cage washing. Fermenter cleaning using a clean-in-place process unit. Source: Berkeley Lab EHS and Public Affairs.


F.2.1.4 Antisepsis

Text Box:  
Antiseptic for animal surgery. Source: Berkeley Lab EHS.
Antiseptic for animal surgery. Source: Berkeley Lab EHS.

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:

F.2.3  Antimicrobial Selection and Registered Disinfectants

Text Box:  

 
Antimicrobial capabilities and conditions of use. Source: Berkeley Lab EHS.
Antimicrobial capabilities and conditions of use. Source: Berkeley Lab EHS.

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

 

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 contaminated with BBP material (as defined in Work Process B.3.f, Bloodborne Pathogens and Human Materials, of this program) must be cleaned with an “appropriate disinfectant.” Appropriate disinfectants include:

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. Quaternary ammonium compounds are also:

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:

 

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.

 

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 using fresh supplies and mixing fresh solutions. Favorable storage conditions include: temperatures between 50 and 70°F, plastic container (not metal or glass), opaque container (to minimize exposure to light), and closed container (to minimize exposure to air). According to Clorox, manufactured bottles of bleach (a) can be stored for about six months under favorable conditions (after this time, bleach will begin to degrade at a rate of 20% each year until it has totally degraded to salt and water) and (b) should be disposed of after three months if full-strength bleach is required. 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 should be mixed daily for proper disinfection of work surfaces.


How to mix, use, and store bleach so that it is effective

 

Wipe hard work surfaces and equipment with 1% solution of fresh household bleach and allow to air dry

 

Add household bleach to liquid biohazardous spills or liquid waste until a 10% concentration of bleach is achieved for 20 minutes.

Household bleach is a water-based solution of sodium hypochlorite (NaOCl) with a typical concentration of 5.25% by weight of the NaOCl active ingredient. Manage bleach’s decay in antimicrobial activity by:

  • Mixing fresh, daily solutions of diluted bleach
  • Storing bleach under favorable conditions, dating bottles, rotating stock, and disposing of older bleach (e.g., greater than 6 to 12 months) 
Source: Berkeley Lab EHS

 

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). 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:

 

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

Text Box:  
70% Ethanol. Source: Berkeley Lab EHS.
70% Ethanol. Source: Berkeley Lab EHS.

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:

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:

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:

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:

F.4.1.1 Dry Heat (Baking and Incineration)

Dry heat sterilization may include baking or incineration:

 

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:

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. UV wavelengths are in the range of 10 nanometers (nm) to 400 nm, and their energy ranges from 3 electron volts (eV) to 124 eV. UV radiation is so named because the spectrum consists of electromagnetic waves with frequencies that are higher than visible violet light.

F.4.2.1  UV Light Health Effects and Categories

Text Box:  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:

 

Chart023

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). Blue UV light inside a biosafety cabinet. Source: Berkeley Lab EHS.

 

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 

Text Box:  
BSC UV light. Source: Berkeley Lab EHS.
BSC UV light. Source: Berkeley Lab EHS.

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:

 

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.

 

 

EM-spectrum

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 Work Process D.6.d.ii, Hoods and Biosafety Cabinets, and Appendix E of this program 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

Text Box:  

 
Tabletop and
floor-standing autoclaves.
Source: Berkeley Lab EHS.
Tabletop and floor-standing autoclaves. Source: Berkeley Lab EHS.

This section provides general information and guidelines on autoclave principles, operation, and maintenance typically needed to sterilize materials or equipment and ensure operator safety. An 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:

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

Text Box:  
Autoclave warning and procedure posting.
Source: Berkeley Lab EHS.
Autoclave warning and procedure posting. Source: Berkeley Lab EHS.

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:

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:

F.5.2.4 Autoclave Preparation and Loading

F.5.2.5 Autoclave Cycle and Time Selection

Text Box:  
Autoclave cycle selections. 
Source: Berkeley Lab EHS.
Autoclave cycle selections. Source: Berkeley Lab EHS.

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:

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

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:

F.5.2.7 Autoclave Material Staging

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

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 (i.e., 911). 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 five 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:

Text Box:    
Autoclave monitoring. Mechanical monitoring of cycles, temperature, pressure, and time. Chemical monitoring with autoclave tape to indicate load temperature. Biological monitoring with bacteria spores in a vial to indicate load sterilization. Source: Berkeley Lab EHS.
Autoclave monitoring. Mechanical monitoring of cycles, temperature, pressure, and time. Chemical monitoring with autoclave tape to indicate load temperature. Biological monitoring with bacteria spores in a vial to indicate load sterilization. Source: Berkeley Lab EHS.

 

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

 

 

_____________________

<< Chapter 25 || Table of Contents || Chapter 27 >>