Biosafe Environs For Biohazardous Operations

biosafe facilities, equipment and practices
biosafe environs for biohazardous operations
adapted with kind permission from the CDC/NIH 4th edition of Biosafety in Microbiological and Biomedical Laboratories
preamble Every year about 90 million babies are born in the world. If these infants are protected from infection and malnutrition in the first five years of their lives, they can hope to live to 70 and beyond. Vaccinating the infant early enough does the job of protecting the child from fatal infections such as diarrhoea, cholera, TB, mumps, measles, rubella and other viruses. Given these numbers, it is apparent that vaccines are not just health aids but can also be money-spinners. Indeed, the revenue from vaccine sales across the world today amounts to $8 billion every year. And vaccines are not for children alone. Hepatitis, malaria, dengue, typhoid, flu, yellow fever and HIV affect all ages and effective vaccines against these can earn much revenue.
current good manufacturing practices: sterilisation & aseptic processing
A point to note in this connection is that keeping the water and environment clean aids in reducing (almost eliminating) many infective agents that cause life-threatening diseases. The developed world has taken these steps while over 100 nations of the South across the globe have not. The wealthier nations have also cut their population to static or even negative growth rates while the South grows at anywhere between 2 to 4 per cent every year. A result of this is that revenues from vaccines are not large in the developed world. Like any investor in any other industry, for the average investor in pharmaceuticals cGMP translates to can you Guarantee My Profits? Since the threat to health and life in developed countries is not from infection but from obesity, hypertension, diabetes, cancer and other systemic diseases, major multinational pharma companies do not find it worth investing money in vaccine research, whereas they are desperate to invent blockbuster drugs against systemic diseases and ways to reverse the process of ageing. This augurs well for India, which has already invested in research and development of vaccines, and a tradition of vaccine production that dates back to 100 years, to take the lead and become the vaccine supplier to the globe in fighting infections diseases worldwide. India is already among the largest producers of vaccine for human use in the world, perhaps the largest. And a wide variety of vaccines are made here. Several factors have converged to make this possible. Adequate infrastructure and expertise have been put in place in different institutions across India. The emergence of a new and dynamic biotech industry with capability both in vaccinology and a keen sense of the market has helped boost this realisation. Indeed, the vaccine portfolio of lndia is as impressive as it is successful. At last count, there were 21 vaccines being manufactured or under clinical trials. These are DPT, BCG, MMR, HBV, OPV, FMDV, Rabies vaccine, Leprosy vaccine - all of which are manufactured, and vaccines against Anthrax, HPV, HIV, diarrhoea, Typhoid, IEV, malaria, cholera, rotavirus, HIB, meningitis, improved version of TB vaccine, dengue, Hepatitis C virus and DNA vaccine are in various stages of clinical trails. Many of these have been developed in house in India, and as collaborations with academia and R&D centres in the country. Given the constraints and directions of multinational major pharma, it is clear that small players from developing nations can occupy a niche and gain profits through low cost-high volume sales of dozens of vaccines across the South.
However, mere making of vaccines the classical way is not enough. DNA vaccinology needs to be explored more rigorously. And focusing on antibodies is not enough. Methods must be found and formulated to activate T-cells in order to improve the effectiveness of vaccines. Also, attention needs to be focussed on methods to beat or eliminate the cold chain, which requires the storage and transport of vaccines under refrigeration. While DNA vaccines might not need the cold chain, even classical vaccines need to be packaged in novel and cost-effective ways. There is much R&D to be done yet. India has taken initial steps that have proved successful and rewarding. Building on these, the R&D community and the burgeoning biotech industry can work together to make India the vaccine suppliers of the world. issues of concern Despite the 100 years of our vaccine research, deveopment and production traditions, there is still, in many organisations, at both laboratory as well as shop floor, an alarming sense of complacency, and apalling disregard for fundamentals of biosafety, due either to incomplete or incorrect information, or to lack of comprehension about the dangers involved in working with pathogenic organisms. The reasons for this vary: O Working with strains that induce the required immuno response, but do not cause disease O The organism is attenuated and lacks the virulence to cause disease O The concentration needed to induce disease is several orders of magnitude higher than that which may be accidentally ingested O The organism does not have any effect on adults O The organism does not cause disease in humans These notions, widespread as they are, are disturbing, and something that should cause deep concern. For example, Bordetella pertussis, a human respiratory pathogen of worldwide distribution, is the causative agent of whooping cough. The disease is typically a childhood illness; however, the agent has increasingly been associated with adult illness. Several outbreaks in health-care workers have been reported in the literature. Adolescents and adults with atypical or undiagnosed disease can serve as reservoirs of infection and transmit the organism to infants
and children. Eight cases of infection with B.pertussis in adults have been documented at a large research institution. The individuals involved did not work directly with the organism, but had access to common laboratory spaces where the organism was manipulated. One case of secondary transmission to a family member was documented. A similar incident occurred at a large Midwestern university resulting in two documented cases of laboratory-acquired infection and one documented case of secondary transmission. Other laboratory-acquired infections with B. pertuss is have been reported, as well as adult-to-adult transmission in the workplace. Laboratory-acquired infections resulting from the manipulation of clinical specimens or isolates have not been reported. The attack rate of this airborne infection is influenced by intimacy and frequency of exposure of susceptible individuals. Genetic mutations, transgenic mutations and trans-species mutations are all eminently plausible, and should not be discounted. It is well recognised that virus in attenuated vaccine for birds can, after five successive passages, can regain full virulence. Opportunistic pathogens and compromised hosts are ubiquitous, and no precaution is too much in our endeavours to provide biosafe working environs. Though guidelines for biological production have been reproduced elsewhere, the biosafety aspects of biohazardous operations are covered here, drawing heavily from the guidelines and recommendations of Center for Disease Control (CDC), USA; National Institute of Health (NIH), USA and a host of other International Agencies concerned with Biosafety in Medical and Biological Laboratories. As a consequence, much of the material here is directed towards biosafe laboratories; but the same principles also apply for manufacturing. The reader is also cautioned about the differences in perception of risks across countries for the same organism. For example, Foot and Mouth Disease (FMD) is classified at Risk Level 2 in India; but at Risk Level 5 in Canada and elsewhere. Tuberculosis is routinely treated in India at Level 2, while in this presentation where material has been borrowed from USA and Canada, the risk is indicated at Level 3. What we commonly refer to as P3 Facilities are described instead as P4 here.
biosafety Microbiology laboratories and biological production centres are special, often unique, work environments that may pose identifiable infectious disease risks to persons in or near them. Bacteria, viruses, fungi or other infectious agents are studied because they may cause disease, they can help us understand the natural world, and for many other reasons including the possibility of industrial applications. Since many of the agents can be pathogenic to humans, animals or other forms of life, their use poses risks which vary with each agent and the way it is used. Biotech laboratories, therefore, are special, often unique, work environments that may pose identifiable infectious disease risks to persons in or near them. Infections have been contracted in the laboratory and production areas throughout the history of microbiology and immunology. As a result, safety norms, standards and practices have been designed and developed over the years to reduce to an acceptable level the risks inherent in the use of dangerous materials. Stringent standards are set for hazardous agents and less stringent ones for agents which cause only minor problems. Safety standards are therefore compromises designed to allow needed work to proceed without exposing those involved or others to more than minimal risk. Besides the attitudes and actions of those who work in these hazardous environs determine their own safety, and that of their colleagues and of the community. Facility, equipment and design can contribute to safety only if they are used properly by people who are genuinely concerned and knowledgeable about safety issues. definitions Biohazard An agent of biological origin that has the capacity to produce deleterious effects on humans, i.e. microorganisms, toxins and allergens derived from those organisms; and allergens and toxins derived from higher plants and animals. Biosafe facilities Controlled Environments, known by the generic name of Bio Safety or Protection facilities are graded at Levels such as BSL 1/2/3/4 (where batch sizes exceed 10 litres of culture cells) or P1/2/3/4/5, depending on the virulence, toxicity or pathogenicity of the harmful agent.
Biosafety The application of combinations of laboratory practice and procedure, laboratory facilities, and safety equipment when working with potentially infectious microorganisms. Chain of Infection
Figure 1: Chain of infection
risk assessment The assessment of risks associated with laboratory activities involving the use of infectious microorganisms is ultimately a subjective process. The risks associated with the agent, as well as with the activity to be conducted, must be considered in the assessment. The described risk assessment process is also applicable to laboratory operations other than those involving the use of primary agents of human disease. Microbiological studies of animal host-specific pathogens, soil, water, food, feeds, and other natural or manufactured materials, by comparison, pose substantially lower risks for the laboratory worker. Microbiologists and other scientists working with such materials may, nevertheless, find the practices, containment equipment, and facility recommendations described in this publication of value in developing operational standards to meet their own assessed needs. classification of biological agents according to risk General Judgements of the inherent risks of a pathogen are made on the basis of such factors as the severity of the disease it causes, the routes of infection, its virulence and infectivity. This judgement should take into account the existence of effective therapies (e.g. antibiotic resistance), immunization, the presence (or absence) of vectors, quantity of agent and whether the agent is indigenous to our country as well as possible effects on other species, including plants and animals. Emerging
pathogens and novel agents, because of their unknown characteristics, may require specialized practices and procedures for handling. With these factors as the prime consideration, biological agents are classified according to risk groups which are analogous to the levels of containment described below. These classifications presume ordinary circumstances in the research laboratory, or growth in small volumes for diagnostic and experimental purposes. The classifications of biological agents primarily reflect the judgements made on their inherent risks. Agents not listed should be classified on the basis of similarity to those listed. criteria for classification of biological agents by risk group Risk Group 1 (low individual and community risk) A biological agent that is unlikely to cause disease in healthy workers or animals. Risk Group 2 (moderate individual risk, limited community risk) A pathogen that can cause human or animal disease but, under normal circumstances, is unlikely to be a serious hazard to laboratory workers, the community, livestock, or the environment. Laboratory exposures rarely cause infection leading to serious disease; effective treatment and preventive measures are available and the risk of spread is limited. Risk Group 3 (high individual risk, low community risk) A pathogen that usually causes serious human or animal disease, or which can result in serious economic consequences but does not ordinarily spread by casual contact from one individual to another, or that can be treated by antimicrobial or antiparasitic agents. Risk Group 4 (high individual risk, high community risk) A pathogen that usually produces very serious human or animal disease, often untreatable, and may be readily transmitted from one individual to another, or from animal to human or vice-versa directly or indirectly, or by casual contact. recombinant DNA and genetic manipulations Genetic methods such as natural selection, cross breeding, conjugation and transformation have been used for many years to change biological species and organisms. These methods have recently been supplemented
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by newer and much more efficient ones, of which the best known are the techniques of recombinant DNA. This technology allows scientists to transfer genes between unrelated organisms and species, and has spawned the recent surge in biotechnology. The initial fear of possible risks arising from organisms altered by this technology led Canada, the United States and Great Britain, among other countries, to develop stringent biosafety guidelines. Experience rapidly showed that the initial fears were not justified. By 1980, many of the containment requirements of 1975-1977 had been removed. Guidance in how to assess potential risks in recombinant DNA research can only be very general; each case needs individual assessment. It is not realistic to try to define in advance all of the possible genetically engineered organisms which might be created or used in the laboratory. The vast majority of this research involves only the remotest possibility of creating a hazard because the source of the DNA being transferred, the vector and the host are all innocuous. However, some genetic manipulation does raise significant possibility of risk. In general, if none of the components of the genetic manipulation presents any known hazard, and none can be reasonably foreseen in their combination, then no biohazard restrictions are needed. If one of the components of the reaction is hazardous, then, in general, discussion of the containment level required should start at the level appropriate to the known hazard. Its containment level might be increased or decreased according to such considerations as: the particular gene being transferred; the expression of the gene in the recombinant organism; the biological containment offered by the host vector system; the envisaged interactions between the gene being transferred and the host vector system; and other such factors. In any research with genes coding for hazardous products, host vector systems of limited ability to survive outside the laboratory (i.e. offering biological containment) should be used; their use will reduce the level of containment required. transgenic plants and animals There is considerable potential for production of bioproducts in transgenic plants or animals. The potential release of transgenics into the environment and transmission of novel genes to other plants and animals need to be considered when designing both the production system and facilities to contain the transgenics. In each case, the risk level needs to be determined.
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This section will focus on transgenic plants and animals that are used for production of bioproducts and the containment required for these activities. In the case of transgenic animals, the first consideration is that they be handled according to the Guidelines set forth by various regulating agencies. An important consideration is the ability of the animal to transmit genes by interbreeding with the same species or any related species. Under these conditions, it is important that the transgenics are well-contained to prevent the spread of genetic modifications. It is recommended that, if at all possible, transgenics be created using methodology which restricts the potential for transmission of the genes from one host to another. Transgenic plants may transmit novel characteristics to other plants, thereby modifying the gene pool of existing species. Since this transmission is mediated by pollen, transgenic plants should be made sterile or contained in a growth chamber or greenhouse designed to prevent pollen release either by air or insects. If plants are allowed to mature, care must be taken to contain the seeds in the green house or growth chamber. If live microorganisms are used as vehicles for transfection, the containment level for the plants or animals inoculated with these viable recombinant microorganisms must be at least as high as that required for work with that specific microorganism. Transgenics (eg. produced by micro-injection, by use of replication defective vectors, or other sequences that are not horizontally transmitted) can be normally handled at Containment Level 1. The following recommendations should be considered prior to the initiation of transgenic studies: a Complete copies of the genome or replication competent genome should not be used. b The constructs should not contain genes capable of causing neoplastic transformation of animals. c The probability of recombination with extraneous microorganisms should be minimal or non- existent. The biological hazards associated with the use of mammalian or other cells in culture fall into 3 categories: a. Primary cultures of mammalian or other cells may harbour infectious agents or integrated DNA originally present in the animal or person from which the cultures were derived. Whenever possible,
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the donor should be tested for any suspect pathogens prior to the preparation of the culture, and the culture should be considered to be contaminated until proven to be free of suspect agents. Such primary cultures should be handled in a manner appropriate to the risk class of the suspected contaminant, and precautions should be taken against parenteral or other means of exposure of laboratory personnel. b. Cell lines known to contain infectious agents or integrated DNA should be handled according to the risk class of the agent. c. Cell lines that are deemed to be free of infectious agents would, rarely, pose a biological hazard. If there is unintentional parenteral inoculation, normal immune response should provide protection, prevent progressive growth and cause rejection of accidentally transplanted cells. use of mammalian cells in culture The biological hazards of mammalian cells arise from the possibility that they might contain or transmit infectious agents. It is prudent to consider all cell lines to be potentially infectious. Cells known or suspected to contain such agents, or primary cultures from animals and humans known or reasonably suspected to be infected, should be in the risk group for the suspected agent. Primate cell lines derived from lymphoid or tumor tissue, all cell lines exposed to or transformed by a primate oncogenic virus, all samples of human tissues and fluids, all primate tissue, all cell lines new to the laboratory (until proven to be free of adventitious agents), all virus-containing primate cell lines, and all mycoplasma-containing cell lines should be handled at containment Level 2. risk levels associated with the use of laboratory animals The use of experimental animals and insects poses special problems. Animals can harbour infectious organisms which are acquired naturally. These infections can give rise to a chronic carrier state, or the agent might persist in a latent non-infective form which can be reactivated periodically or as a result of certain stimuli. If the possibility that such an agent may be excreted by an animal during the course of an experiment cannot be excluded, all those animals should be kept at a containment level appropriate to the risk. Animals may also be deliberately inoculated with microorganisms in each of the four risk groups or with viable materials (i.e. transformed cells) suspected of containing these organisms. Under these circumstances, the animal should be kept at the containment level appropriate to the risk of the organism, recognizing that, in some cases, in vivo work may increase that risk.
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In all situations, it is the responsibility of the scientist and the host institution in consultation with the Government and the Animal Care authorities, to determine the risk levels inherent in the proposed activity. principles of biosafety The term containment is used in describing safe methods for managing infectious agents in the laboratory environment where they are being handled or maintained. The purpose of containment is to reduce or eliminate exposure of laboratory workers, other persons, and the outside environment to potentially hazardous agents. Primary containment, the protection of personnel and the immediate laboratory environment from exposure to infectious agents, is provided by both good microbiological technique and the use of appropriate safety equipment. The use of vaccines may provide an increased level of personal protection. Secondary containment, the protection of the environment external to the laboratory from exposure to infectious materials, is provided by a combination of facility design and operational practices. Therefore, the three elements of containment include laboratory practice and technique, safety equipment, and facility design laboratory practice and technique The most important element of containment is strict adherence to standard microbiological practices and techniques. Persons working with infectious agents or potentially infected materials must be aware of potential hazards, and must be trained and proficient in the practices and techniques required for handling such material safely. The director or person in charge of the laboratory is responsible for providing or arranging for appropriate training of personnel. Each laboratory should develop or adopt a biosafety or operations manual which identifies the hazards that will or may be encountered, and which specifies practices and procedures designed to minimize or eliminate risks. Personnel should be advised of special hazards and should be required to read and to follow the required practices and procedures. A scientist trained and knowledgeable in appropriate laboratory techniques, safety procedures, and hazards associated with handling infectious agents must direct laboratory activities.
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When standard laboratory practices are not sufficient to control the hazard associated with a particular agent or laboratory procedure, additional measures may be needed. The laboratory director is responsible for selecting additional safety practices, which must be in keeping with the hazard associated with the agent or procedure. Laboratory personnel, safety practices, and techniques must be supplemented by appropriate facility design and engineering features, safety equipment, and management practices. safety equipment ( primary barriers) Safety equipment includes biological safety cabinets (BSCs), enclosed containers, and other engineering controls designed to remove or minimize exposures to hazardous biological materials. The biological safety cabinet (BSC) is the principal device used to provide containment of infectious splashes or aerosols generated by many microbiological procedures. Safety equipment also may include items for personal protection such as gloves, coats, gowns, shoe covers, boots, respirators, face shields, safety glasses, or goggles. Personal protective equipment is often used in combination with biological safety cabinets and other devices which contain the agents or materials being worked with. facility design (secondary barrier) The design of the facility is important in providing a barrier to protect persons working inside and outside of the laboratory within the facility, and to protect persons in the community from infectious agents which may be accidentally released from the laboratory. Laboratory management is responsible for providing facilities commensurate with the laboratorys function and the recommended biosafety level for the agents being manipulated. The recommended secondary barrier is determined by the risk of transmission of specific agents. Secondary barriers in these laboratories will include separation of the laboratory work area from public access, availability of a decontamination facility (e.g., autoclave), and hand washing facilities. Design features could include airconditioning, controlled access zones, airlocks at laboratory entrances, or separate buildings or modules for isolation of the laboratory.
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biosafety levels Four biosafety levels (BSLs) are described which consist of combinations of laboratory practices and techniques, safety equipment, and laboratory facilities. Each combination is specifically appropriate for the operations performed, the documented or suspected routes of transmission of the infectious agents, and for the laboratory function or activity. The recommended biosafety level(s) for the organisms in Section VII (Agent Summary Statements) represent those conditions under which the agent can ordinarily be safely handled. The laboratory director is specifically and primarily responsible for assessing risks and for appropriately applying the recommended biosafety levels. Generally, work with known agents should be conducted at the biosafety level recommended in Section VII. When specific information is available to suggest that virulence, pathogenicity, antibiotic resistance patterns, vaccine and treatment availability, or other factors are significantly altered, more (or less) stringent practices may be specified. biosafety level 1 Biosafety Level 1 practices, safety equipment, and facilities are appropriate for undergraduate and secondary educational training and teaching laboratories, and for other facilities in which work is done with defined and characterized strains of viable microorganisms not known to cause disease in healthy adult humans. Bacillus subtilis, Naegleria gruberi, and infectious canine hepatitis virus are representative of those microorganisms meeting these criteria. Many agents not ordinarily associated with disease processes in humans are, however, opportunistic pathogens and may cause infection in the young, the aged, and immunodeficient or immunosuppressed individuals. Vaccine strains which have undergone multiple in vivo passages should not be considered avirulent simply because they are vaccine strains. Biosafety Level 1 represents a basic level of containment that relies on standard microbiological practices with no special primary or secondary barriers recommended, other than a sink for handwashing. biosafety level 2 Biosafety Level 2 practices, equipment, and facilities are applicable to clinical, diagnostic, teaching and other facilities in which work is done with the broad spectrum of indigenous moderate-risk agents present in the community and associated with human disease of varying severity.
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With good microbiological techniques, these agents can be used safely in activities conducted on the open bench, provided the potential for producing splashes or aerosols is low. Biosafety Level 2 is appropriate when work is done with any human-derived blood, body fluids, or tissues where the presence of an infectious agent may be unknown. Primary hazards to personnel working with these agents relate to accidental percutaneous or mucous membrane exposures, or ingestion of infectious materials. Extreme precaution with contaminated needles or sharp instruments must be emphasized. Even though organisms routinely manipulated at BSL2 are not known to be transmissible by the aerosol route, procedures with aerosol or high splash potential that may increase the risk of such personnel exposure must be conducted in primary containment equipment, or devices such as a BSC or safety centrifuge cups. Other primary barriers should be used as appropriate, such as splash shields, face protection, gowns, and gloves. Secondary barriers such as hand washing and waste decontamination facilities must be available to reduce potential environmental contamination. Biosafety Level 2 is the recommended level for work with bloodborne pathogens such as hepatitis B virus and HIV. The containment elements described in Biosafety Level 2 are consistent with the Occupational Exposure to Bloodborne Pathogens Standard from the Occupational Safety and Health Administration (OSHA), that requires the use of specific precautions with all clinical specimens of blood or other potentially infectious material (Universal Precautions). Additionally, other recommendations specific for clinical laboratories may be obtained from the National Committee for Clinical Laboratory Standards. Biosafety Level 2 recommendations and OSHA requirements focus on the prevention of percutaneous and mucous membrane exposures to clinical material. biosafety level 3 Biosafety Level 3 practices, safety equipment, and facilities are applicable to clinical, diagnostic, teaching, research, or production facilities in which work is done with indigenous or exotic agents with a potential for respiratory transmission, and which may cause serious and potentially lethal infection. Mycobacterium tuberculosis, St. Louis encephalitis virus, and Coxiella burnetii are representative of microorganisms assigned to this level. Primary hazards to personnel working with these agents relate to autoinoculation, ingestion, and exposure to infectious aerosols.
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At Biosafety Level 3, more emphasis is placed on primary and secondary barriers to protect personnel in contiguous areas, the community, and the environment from exposure to potentially infectious aerosols. For example, all laboratory manipulations should be performed in a BSC or other enclosed equipment, such as a gas-tight aerosol generation chamber. Secondary barriers for this level include controlled access to the laboratory and a specialized ventilation system that minimizes the release of infectious aerosols from the laboratory. biosafety level 4 Biosafety Level 4 practices, safety equipment, and facilities are applicable for work with dangerous and exotic agents which pose a high individual risk of life-threatening disease, which may be transmitted via the aerosol route, and for which there is no available vaccine or therapy. Additionally, agents with a close or identical antigenic relationship to Biosafety Level 4 agents should also be handled at this level. When sufficient data are obtained, work with these agents may continue at this level or at a lower level. Viruses such as Marburg or Congo-Crimean hemorrhagic fever are manipulated at Biosafety Level 4. The primary hazards to personnel working with Biosafety Level 4 agents are respiratory exposure to infectious aerosols, mucous membrane exposure to infectious droplets, and autoinoculation. All manipulations of potentially infectious diagnostic materials, isolates, and naturally or experimentally infected animals pose a high risk of exposure and infection to laboratory personnel, the community, and the environment. The laboratory worker's complete isolation of aerosolized infectious materials is accomplished primarily by working in a Class III BSC or a full-body, air-supplied positive-pressure personnel suit. The Biosafety Level 4 facility itself is generally a separate building or completely isolated zone with complex, specialized ventilation and waste management systems to prevent release of viable agents to the environment.
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Figure 2: A simple isolation facility
Figure 3: A typical BSL level 3 facility
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Figure 4: Steam barrier used in biological production
Figure 5: A typical facility with pressure profile for biotech
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Figure 6: A typical cytotoxic manufacturing facility
Figure 7: HVAC in a cytotoxic manufacturing facility
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Table 1: Facility design checklist

Location - General Containment Level 1 2 3 4
Separated from public areas by door ...................... Laboratory doors labeled with biohazard signs ..... Access limited to authorized personnel ................. In separate sealed room(s) with restricted access away from public thoroughfares ............................. In separate building or sealed room with independent air supply and exhaust restricted access ...................................................... Containment labs located away from outside building envelope walls .......................................... Containment labs located adjacent to or nearby mechanical rooms to minimize lengths of containment ducts ................................. Office areas can be located within lab if next to access or egress door. .............................................. Office areas must be outside laboratory containment zone .................................................... Facility must be kept locked when not in use (consistent with local fire and safety regulations) . Location - Containment Perimeter A. Walls Reinforced structural masonry ............................... Reinforced non-load-bearing masonry ................... Steel frame reinforced non-load-bearing masonry Reinforced concrete ................................................
M N N N
M M R N
M M M M
M M M M
N N
N R
R R
M M
N Y N N
N Y N N
R N M M
M N M M
N N N N
N N N N
R R R R
M M M M
Key: M = Mandatory
R = Recommended
Y = Yes N = No/Not applicable
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Table 1: Facility design checklist (contd)

Location - Containment Perimeter Containment Level 1 2 3 4
B. Ceilings Steel frame gypsum partition or impervious ceiling acoustic tile ................................................. Reinforced steel frame and gypsum ceiling, filler primer and paint finish ........................................... C. Coatings and sealants Seamless, gas- and chemical-resistant wall and ceiling coatings ....................................................... Chemical- and gas-resistant (disinfectant), nonhardening sealants .................................................. Containment seals for mechanical/electrical service openings ...................................................... D. Doors Doors lockable ........................................................ Doors self-closing ................................................... Doors to provide restricted access via keycard system or equivalent ............................................... Ventilated airlock required for the separation of higher and lower containment areas with interlocking pneumatic or compressible sealed doors .. Doors and frames of solid finish construction ....... Door openings should be of sizes to allow passage of all anticipated equipment ................................... Doors to have fire ratings as required and be located as per fire safety standards ........................ Entrance doors interlocked with manual overrides All exits marked and illuminated .......................... Egress to fire exits set out so that travel through any high-hazard areas is minimized or to conform to applicable codes ..................................................
Key: M = Mandatory R = Recommended
Y N
Y N
N M
N M
N N N N N N
R R R R R N
M M M M M R
M M M M M M
N N M M N M
N R M M N M
N M M M R M
M M M M M M
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Location - Containment Perimeter Containment Level 1 2 3 4
E. Windows Windows, if openable, protected by fly screens ..... Windows of safety glass, of proven performance, solid stops sealed in place ...................................... F. Floors Slip-resistant flooring ............................................. Seamless, gas- and chemical-resistant (e.g. epoxy) coating with integral cove base .............................. Seamless, rolled or resilient tile flooring (eg.vinyl) Air Handling System
Y N M R M
Y N M R M
N M M M N
N M M M N
A. Room Air Supply Air supply independent from adjoining laboratory zones ........................................................................ Air supply HEPA-filtered or provided with bubble tight dampers .......................................................... Equipped with pressure maintaining gauges at entry (e.g. magnehelics) ......................................... Directional inward, non-recirculated airflow ........ Interlocked with exhaust ventilation to prevent positive pressurization ............................................ Equipped with audible alarms to detect depressurization (i.e. failure of the exhaust system) Air supply ductwork sealed airtight and independent from other laboratory zones and accessible from outside the containment zone ....... Equipped with bubble tight damper to permit sealing for decontamination procedures ................
N N N N N
N N R N N
R R M M R
M M M M M
N N
N N
R M
M M
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Air Handling System Containment Level 1 2 3 4
B. Room Exhaust Ventilation Room equipped with pressure monitoring devices at entry .................................................................... Sealed airtight independent-exhaust ductwork and fan system accessible from outside the containment zone which meets performance and verification testing requirement All exhaust ventilation HEPA-filtered and connected to audible alarm to detect failure of exhaust system ........................................................ Interlocked with air supply to prevent positive pressurization of the laboratory .............................. Equipped with bubble tight damper to permit sealing for decontamination ................................... Exhaust from laboratory at a minimum of 10 room volumes/ hour ......................................................... Air vertically discharged to the outside, clear of buildings or supply air intakes at 12 mps .............. Recirculated HEPA-filtered room air permitted ... Minimization of dead spaces where contaminated air can accumulate .................................................. Ventilation sufficient to remove vapours of flammable liquids and dangerous chemicals before they reach hazardous concentrations ..................... Exposed ductwork to stand clear of walls to allow access for maintenance, leak testing and access to equipment filters and lighting ................................
N N N N R R R
N R R R R M R
R M M M R N M
M M M M R N M
Key: M = Mandatory
R = Recommended
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Air Handling System Containment Level 1 2 3 4
C. Biological Safety Cabinets Class I ...................................................................... Class II .................................................................... Class III ................................................................... Class I and II cabinets permitted with use of positive-pressure suits ............................................. Cabinet air can be recirculated in laboratory if HEPA-filtered .......................................................... D. Fume Hoods Recommended when necessary .............................. HEPA and charcoal filters (if required) ................. Air flow alarms ....................................................... Decontamination & Waste Disposal A. Decontamination Laboratory floors, walls and ceilings to be treated with disinfectant-resistant, cleanable coatings ...... All laboratory furnishings and surface materials disinfectant-resistant and cleanable. Bench tops with coved splash backs, seamless, or with unions sealed with non-shrinking sealant ..... Surfaces of plastic laminate .................................... Laboratory perimeter sealed to allow gaseous decontamination of whole room ............................. R N R R N R R R R R M R N N N N N R R N N M M M N N M N M M M N
R R N
R R N
M M M
M M M
Key: M = Mandatory
R = Recommended
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Decontamination & Waste Disposal Containment Level 1 2 3 4
B. Sterilization Interlocking double-door pass-through autoclave in laboratory. ........................................................... Autoclave in laboratory .......................................... Autoclave in building ............................................. Exposed steam pipes covered with insulating material ................................................................... Incinerator in building ........................................... C. Waste Disposal Liquids Drainage traps filled with disinfectant specified by laboratory operator .................................................. All liquid effluents must be sterilized in mechanically and biologically monitored tanks located adjacent to the containment area prior to disposal .................................................................... Solids Provide space for support stands for biomedical waste bags ............................................................... Provide refrigerated space for lockable, closed storage for biomedical waste which will be disposed of off site ........................... Provide disinfectant dunk tanks for all nonautoclavable materials and equipment exiting the containment area as a part of the cabinet line. In the case of a suited lab, this function may be provided by a dunk tank or via the chemical shower at the containment perimeter .....................
N N R M N
N N M M N
R M M M N
M M M M R
Key: M = Mandatory
R = Recommended
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Personal Hygiene & Safety Containment Level 1 2 3 4
Facilities Handwashing facilities in laboratory ..................... Dedicated handwashing facilities with foot, knee or automatic controls in laboratory (not applicable for positive-pressure-suit mode) ............................. Chemical deluge shower at the laboratory perimeter (for positive-pressure-suit mode) .............................. Eye/face wash facilities equipped with in-use audio/visual alarm (not applicable for positive pressure suit mode) ................................................. Body shower in containment area .......................... Clothing change area adjacent to containment area (0.5 m2 per person) .......................................... Provide storage space for laboratory clothing in lab or adjacent change area (minimum 300 linear mm. for each peg) ................................. Provide space adjacent to exit door for laundry hamper (minimum 0.900m2) for used laboratory clothing to be autoclaved prior to laundering ........ A. Plumbing And Drainage All drains connected to sterilization system .......... All drains connected directly to sanitary sewer ..... Autoclave chamber condensate directed to sewer through a closed system ............................... All piping penetrations sealed with non-shrinking sealant at laboratory perimeter ............................... All supply water services fitted with backflow preventers ................................................................
N N
R N
M N
M M
R N N
R N N
R R M
M M M
R N N N N N
R N N N N N
M N R R M M
M M N M M M
Key: M = Mandatory
R = Recommended
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Piping to be exposed and stand clear of walls within high containment area to allow access f or maintenance ........................................................ N Main water supply control to be located outside laboratory perimeter ............................................... N All exposed hot and cold water pipes are to be covered with insulating material and protected from movement ....................................................... M All vent lines equipped with HEPA filters or equivalent ................................................................ N B. Compressed Gases Air supply lines HEPA filtered or equivalent as backflow protection ................................................ N All gas lines to have backflow preventers ............. N All vacuum lines HEPA-filtered or equivalent *. .. N No vacuum lines should lead from facility (needs to be met by pumps inside laboratory) ................... N All supply line penetrations sealed with non-shrinking sealant at laboratory perimeter ...... N Compressed gas cylinder storage outside laboratory ................................................................ N * Vacuum lines must not leave Containment Levels 3 or 4 C. Electrical All supply conduit and wiring to be sealed at the containment barrier with non-shrinking sealant ... N Fluorescent light ballasts and starters to be located outside containment area ........................................ N
N N
M R
M M
M N
M R
M M
R N R N N N
M M M M M R
M M M M M M
N N
R R
M R
Key: M = Mandatory
R = Recommended
30

Circuit breakers located outside biocontainment area .......................................................................... Building security systems integrated with laboratory safety and monitoring systems ............. 70 ft.-candles of light at work surface level (metric) minimum maintained at work surface ..... All circuit-breaker switches, panels and controls to be appropriately labeled ..................................... Electrical system is to be equipped with standby generator for emergency support of essential equipment, which includes biological safety cabinets .................................................................... Laboratory to be equipped with fire alarm system Lab to be equipped with a communication system between containment area and outside support area Closed-circuit TV monitoring of entire work area Emergency & Monitoring Provisions A. Air Handling Directional inward, non-recirculated airflow, monitored at entrance to lab ................................... Biological safety cabinets equipped with pressure monitoring gauges for all HEPA filters .................. Provision of access for decontamination of HEPA filters ............................................................ Entry to laboratory via ventilated air-lock with interlocking doors ................................................... Entry to laboratory via sealed air-lock ...................
N R R M
N R R M
R R R M
M M R M
N M N N
R M N N
R M R N
M M M R
N N M N N
R R M N N
M M M R N
M M M N M
31

Emergency & Monitoring Provisions Containment Level 1 2 3 4
Rooms in isolation area to be maintained at pressure negative to corridor with greatest negative pressure in most hazardous room ............ B. Fire Prevention And Containment Equipped with fire alarms ...................................... Equipped with appropriate fire extinguishers ....... Doors to have appropriate fire ratings ................... All fire exits marked and illuminated .................... Biocontainment area designated as burn out. Firemen to enter only to save life, not to extinguish fire, which should be controlled from outside, to prevent spread ....................................... Equipped with suitable storage cabinets or explosion-proof refrigerators which are clearly labeled for flammable liquids ................................. Storage for flammable liquids to be located outside biocontainment area ................................... Sprinkler systems (where required by law) to be preactioned type; other fire suppression systems may be considered for level 4 ................................. C. Personal Emergency Equipment Equipped with bottled back-up breathing air sufficient to provide 30 minutes per person. (Level 4 suit lab only) ............................................. Equipped with positive-pressure hood respirators with compressed breathing air cylinders located in support area ........................................................ Eye/face wash facilities in laboratory (not applicable for positive-pressure-suit mode, Level 4) ...................................................................
N M M M M
N M M M M
M M M M M
M M M M M
M R
M R
M R
M R
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Emergency & Monitoring Provisions Containment Level 1 2 3 4
Body shower in support area .................................. Equipped with communication system between containment zone and support area ....................... Emergency lighting ................................................ D. Backup Service Equipped with standby generator for support of essential equipment, including biological safety cabinets ......................................................... Performance Verification & Testing Construction of laboratory perimeter to be leakproof and able to withstand loading characteristics imposed by negative air pressure required in laboratory operation: ........................... a) Integrity of seals demonstrated by visual inspection ................................................................ b) Integrity of room tightness demonstrated by physical testing (pressure decay 0.05 wg loss/min) at 2"wg .................................................................... All air supply and exhaust ductwork tested in situ to be leak-tight by pressure decay:
Level 3 not > 0.2% duct volume per min at 2"wg (500Pa); Level 4 not > 0.1% duct volume per min at 2"wg (500Pa)
R N M
R N M
R R M
M M M
N N N N
N N N N
R M M R
M M M M
All air supply and exhaust ductwork verified t o have back-draft protection ................................... All HEPA filters tested to meet required specification after installation ................................ All HEPA-filter housings tested to be leak tight: not > 0.2% of volume per min at 10"wg. (2500Pa)
33

Performance Verification & Testing Containment Level 1 2 3 4
Testing of biological safety cabinets meets required specifications after installation ................ Testing of autoclaves to meet specified standards after installation by the use of biological indicators Testing of fume hoods according to CSA Standard Z316.5-94 ....................................... Drainage and liquid-waste-disposal systems including sampling ports tested to ensure efficacy by use of biological indicators ................................ Verification of integrity of sewage lines ................ Verification of alarm systems for air systems failure (exhaust, supply, room pressure, breathing air) .......................................................... Verification of alarm systems for electrical failure Verification of fire alarm systems .......................... Verification of communication systems ................. Testing of directional airflow demonstrated by field tests with visual smoke .................................. Verification of integrity positive pressure suits ..... Testing of breathing air as per CSA Standard Z180.1-M85 .................................... Testing of regular and emergency air system ........
N M M
M M M
M M M
M M M
N N
N N
N N
M M
N N M N N N N N
N M M N R N N N
M M M R M N N N
M M M M M M M M
Key: M = Mandatory
R = Recommended
Table 2: Summary of biosafety (levels 1-3) for infectious agents Practices None required Open bench top sink Safety Equipment Facilities
BSL
Agents
Not known to cause disease in healthy adults Class II BSC for all manipulations of agents that cause splashes or aerosols of infectious materials; PPEs: Protective laboratory coats; gloves; face protection as needed BSL-2 safety equipment plus: Respiratory protection as needed Open bench top sink Autoclave
Standard Microbiological Practices
Associated with human disease, hazard, autoinoculation ingestion, mucous membrane exposure Decontamination of all waste
BSL 1 practices plus: Limited/ Controlled access Biohazard warning signs Sharps precautions Biosafety manual
Indigenous or exotic agents with potential for aerosol transmission; disease may have serious or lethal consequences.
BSL-2 practice plus: Decontamination of lab clothing before laundering Medical surveillance policies, such as baseline serum monitoring
BSL-2 facilities plus: Physical separation from access corridors; self-closing, double- door access; exhaust air must not be recirculated; negative air pressure maintained within laboratory so that airflow is always inwards
selection, use, testing and maintenance of biological safety cabinets
adapted from the CDC/NIH 2nd edition of Selection and use of Biological Safety Cabinets
rom the earliest laboratory-acquired typhoid infections to the hazards posed by todays antibiotic-resistant bacteria and rapidly-mutating viruses, threats to worker safety have stimulated the development and refinement of cabinets in which infectious microorganisms could be handled safely. Work with tissue cultures, the need to maintain sterility of cell lines, and efforts to minimize cross-contamination contributed to concerns regarding product integrity. The use of proper procedures and equipment cannot be overemphasized in providing primary personnel and environmental protection. For example, high-speed blenders designed to reduce aerosol generation, needle-locking syringes, microburners, and safety centrifuge cups or sealed rotors are among the engineering devices that protect the laboratorian. However, the most essential piece of containment
36
equipment is the biological safety cabinet in which manipulations of microorganisms are performed. This section presents information on the selection, function and use of biological safety cabinets (BSCs), which are the primary means of containment developed for working safely with infectious microorganisms. Brief descriptions of the facility and engineering concepts for the conduct of microbiological research are also provided. BSCs are only one part of an overall biosafety program which requires consistent use of good microbiological practices. BSCs are designed to provide personnel, environmental and product protection when appropriate practices and procedures are followed. Three kinds of biological safety cabinets, designated as Class I, II and III have been developed to meet varying research and clinical needs. This section is not meant to be definitive or all encompassing. Rather, an overview is provided to clarify the expectations, functions and performance of these critical primary barriers. This has been written for the laboratorian, engineer or manager who desires a better understanding of each type of cabinet and the rationale for selecting the most appropriate BSC to meet specific operational needs. class I BSC The Class I BSC provides personnel and environmental protection, but no product protection. It is similar in air movement to a chemical fume hood, but has a HEPA filter in the exhaust system to protect the environment. In the Class I BSC, unfiltered room air is drawn across the work surface. Personnel protection is provided by this inward airflow as long as a minimum velocity of 75 linear feet per minute (lfpm) is maintained through the front opening. With the product protection provided by the Class II BSCs, general usage of the Class I BSC has declined. However, in many cases Class I BSCs are used specifically to enclose equipment (e.g., centrifuges, harvesting equipment or small fermenters), or procedures (e.g. cage dumping, aerating cultures or homogenizing tissues) with a potential to generate aerosols. The Class I BSC is hard-ducted to the building exhaust system, and the building exhaust fan provides the negative pressure necessary to draw room air into the cabinet. Cabinet air is drawn through a HEPA filter as
37
Figure 1: Class I BSC
it enters the exhaust plenum. A second HEPA filter may be installed at the terminal end of the exhaust. Some Class I BSCs are equipped with an integral exhaust blower; the cabinet blower must be interlocked with the building exhaust fan. In the event that the building exhaust fan fails, the cabinet exhaust blower must turn off so that the exhaust ducts are not pressurized. Filters should be installed on the intake side of the fan. Also note that use of two filters increases the static pressure on the fan. If the ducts are pressurized and the HEPA filter develops a leak, contaminated air could be discharged into other parts of the building or the environment. A steel panel with arm holes to allow access to the work surface can be added to the Class I cabinet. The restricted opening results in increased inward air velocity, thereby increasing worker protection. For added safety, arm-length gloves can be attached to the paned. Makeup air is then drawn through an auxiliary air supply opening (which may contain a filter) and/or around a loose-fitting front panel. class II BSC The Class II (Types A, Bl, B2, and B3) biological safety cabinets provide personnel, environmental and product protection. Airflow is drawn around the operator into the front grille of the cabinet, which provides personnel protection. In addition, the downward laminar flow of HEPAfiltered air provides product protection by minimizing the chance of cross-contamination along the work surface of the cabinet. Because cabinet air has passed through the exhaust HEPA filter, it is contaminantfree (environmental protection), and may be recirculated back into the laboratory (Type A BSC) or ducted out of the building (Type B BSC).
38
HEPA filters are effective at trapping particulates and infectious agents, but not at capturing volatile chemicals or gases. Only BSCs that are ducted to the outside should be used when working with volatile toxic chemicals. All Class II cabinets are designed for work involving microorganisms assigned to biosafety levels 1, 2 and 3. Class II cabinets provide the microbe-free work environment necessary for cell culture propagation, and also may be used for the formulation of nonvolatile antineoplastic or chemotherapeutic drugs. class II, type A BSC An internal blower draws sufficient room air through the front grille to maintain a minimum calculated or measured average inflow velocity of at least 75 lfpm at the face opening of the cabinet. The supply air flows through a HEPA filter and provides particulate-free air to the work surface. Laminar airflow reduces turbulence in the work zone and minimizes the potential for cross-contamination. The downward moving air splits as it approaches the work surface; the blower draws part of the air to the front grille and the remainder to the rear grille. Although there are variations among different cabinets,
Figure 2: Class II type A BSC
this split generally occurs about half-way between the front and rear grilles, and two to six inches above the work surface.
39
The air is then discharged through the rear plenum into the space between the supply and exhaust filters located at the top of the cabinet. Due to the relative size of these two filters, approximately 30% of the air passes through the exhaust HEPA filter and 70% recirculates through the supply HEPA filter back into the work zone. Most Class II, Type A cabinets have dampers to modulate this 30/70 division of airflow. An unducted Class II Type A BSC is not to be used for work involving volatile or toxic chemicals. The buildup of chemical vapors in the cabinet (by recirculated air) and in the laboratory (from exhaust air) could create health and safety hazards. It is possible to duct the exhaust from a Type A cabinet out of the building. However, it must be done in a manner that does not alter the balance of the cabinet exhaust system, thereby disturbing the internal cabinet air flow. The typical method of ducting a Type A cabinet is to use a thimble, or canopy hood, which maintains a small opening (usually 1 inch) around the cabinet exhaust filter housing.
Figure 3: Thimble unit over exhaust for class II type A BSC
The volume of the exhaust must be sufficient to maintain the flow of room air into the space between the thimble unit and the filter housing (contact manufacturers for any additional specifications). The thimble must be removable or be designed to allow for operational testing of the cabinet. The performance of a cabinet with this exhaust configuration is unaffected by fluctuations in the building exhaust system. Hard-ducting (i.e., direct connection) of Class II Type A cabinets to the building exhaust system is not recommended. The building exhaust system must be precisely matched to the airflow from the cabinet in both volume and static pressure. However, fluctuations in air volume and pressure that are common to all building exhaust systems make it
40
difficult, if not impossible, to match the airflow requirements of the cabinet. class II, type B1 BSC Some biomedical research requires the use of small quantities of certain hazardous chemicals, such as carcinogens. The powdered form of these carcinogens should be weighed or manipulated in a chemical fume hood or a static-air glove box. Carcinogens used in cell culture or microbial systems require both biological and chemical containment.
Figure 4: Class II type B1 BSC - floor-standing version
The Class II, Type B cabinet originated with the National Cancer Institute (NCI)-designed Type 2 (later called Type B) biological safety cabinet, which was designed for manipulations of minute quantities of these hazardous chemicals with in vitro biological systems. The National Sanitation Foundation (NSF) Standard 49 definition of Type Bl cabinets includes this classic NCI design Type B, as well as cabinets without supply HEPA filters located immediately below the work surface, and/or those with exhaust/recirculation downflow splits other than 70/30%. The cabinet supply blowers draw room air (plus a portion of the cabinets recirculated air) through the front grille and then through the supply HEPA filters located immediately below the work surface. This particulate-free air flows upward through a plenum at each side of the
41
cabinet and then downward to the work area through a back-pressure plate. In some cabinets there is an additional supply HEPA filter to remove particulates that may be generated by the blower/motor system.
Figure 5: Class II type B1 BSC - table-top version
Room air is drawn through the face opening of the cabinet at a minimum inflow velocity of 100 lfpm. As with the Type A cabinet, there is a split in the down-flowing air stream just above the work surface. In the Type B cabinet, approximately 70 percent of the downflow air exits through the rear grille, passes through the exhaust HEPA filter, and is discharged from the building. The remaining 30 percent of the downflow air is drawn through the front grille. Since the air which flows to the rear grille is discharged into the exhaust system, activities that may generate hazardous chemical vapors or particulates should be conducted towards the rear of the cabinet. Type Bl cabinets must be hard-ducted, preferably to their own dedicated exhaust system, or to a properly-designed laboratory building exhaust. As indicated earlier, blowers on laboratory exhaust systems should be located at the terminal end of the duct work. A failure in the building exhaust system may not be apparent to the user, as the supply blowers in the cabinet will continue to operate. A pressure-independent monitor should be installed to sound an alarm and shut off the BSC supply fan, should failure in exhaust airflow occur. Since this feature is not supplied by all cabinet manufacturers, it is prudent to install a sensor in the exhaust system as necessary. To maintain critical operations, laboratories using
42
Type B BSCs should connect the exhaust blower to the emergency power supply. class II, type B2 BSC
Figure 6: Class II type B2 BSC
This BSC is a total-exhaust cabinet; no air is recirculated within it. This cabinet provides simultaneous primary biological and chemical containment. The supply blower draws in room air or outside air at the top of the cabinet, passes it through a HEPA filter and down into the work area of the cabinet. The building or cabinet exhaust system draws air through both the rear and front grilles, capturing the supply air plus the additional amount of room air needed to produce a minimum calculated or measured inflow face velocity of 100 lfpm. All air entering this cabinet is exhausted, and passes through a HEPA filter (and perhaps some other air-cleaning device such as a carbon filter) prior to discharge to the outside. Exhausting as much as 1200 cubic feet per minute of conditioned room air makes this cabinet expensive to operate.
43
Should the building or cabinet exhaust fail, the cabinet will be pressurized, resulting in a flow of air from the work area back into the laboratory. Cabinets built since the early 1980s usually have an interlock system installed by the manufacturer to prevent the supply blower from operating whenever the exhaust flow is insufficient. Presence of such an interlock system should be verified; systems can be retrofitted if necessary. Exhaust air movement should be monitored by a pressure-independent device. class II, type B3 BSC This biological safety cabine is a ducted Type A cabinet having a minimum inward airflow of 100 lfpm. All positive pressure contaminated plenums within the cabinet are surrounded by a negative air pressure plenum. Thus, leakage in a contaminated plenum will be into the cabinet and not into the environment. Special applications - Class II BSCs can be modified to accommodate special tasks. For example, the front sash can be modified by the manufacturer to accommodate the eye pieces of a microscope, or the work surface can be designed to accept a carboy, a centrifuge, or other equipment that requires containment. A rigid plate with arm holes can be added if needed. Good cabinet design, microbiological aerosol tracer testing of the modification, and appropriate certification are required to ensure that the basic systems operate properly after modification. Maximum containment potential is achieved only through strict adherence to proper practices and procedures.
Figure 7: Class II type B3 BSC - table-top version
44
Table 1: Comparative specifications for biological safety cabinets

Airflow Pattern Applications Nonvolatile Toxic Volatile Toxic Chemicals and Chemicals and Radionuclides Radionuclides Yes Yes Yes Yes Yes Yes No Yes (minute amounts)2 Yes (small amounts) Yes (minute amounts)2 Yes (small amounts) Yes1
BSC Class
Face Velocity (fpm)
75
IIA
75
IIB1
100
IIB2
100
IIB3
100
III
NA
In at front; exhausted through HEPA to the outside or into the room through HEPA 70% recirculated to the cabinet work area; balance 30% exhausted through HEPA back into the room or outside through a thimble unit Exhaust cabinet air must pass through a dedicated duct to the outside through a HEPA filter No recirculation; total exhaust to the outside through hard-duct and a HEPA filter Same as II, A, but plenums are under negative pressure to room; exhaust air is thimble-ducted to the outside through a HEPA filter Supply air inlets and hard-duct exhausted to outside through two HEPA filters in series
(1)
(2)
Installation may require a special duct to the outside, an in-line charcoal filter, and a spark proof (explosion proof) motor and other electrical components in the cabinet. Discharge of a Class I cabinet in to a room should not occur if volatile chemicals are used. In no circumstances should the chemical concentration approach the lower explosion limits of the compound.
45
class III BSC The Class III biological safety cabinet was designed for work with biosafety level 4 microbiological agents, and provides maximum
Figure 8: Class III BSC
protection to the environment and the worker. It is a gas-tight enclosure with a non-opening view window. Access for passage of materials into the cabinet is through a dunk tank (that is accessible through the cabinet floor) or double-door pass-through box (such as an autoclave) that can be decontaminated between uses. Reversing that process allows for safe removal of materials from the Class III biosafety cabinet. Both supply and exhaust air are HEPA filtered. Exhaust air must pass through two HEPA filters, or a HEPA filter and an air incinerator, before discharge to the outdoors. Airflow is maintained by a dedicated independent exhaust system exterior to the cabinet, which keeps the cabinet under negative pressure (usually about 0.5 inches of water pressure). Long, heavy-duty rubber gloves are attached in a gas-tight manner to ports in the cabinet and allow for manipulation of the materials isolated inside. Although these gloves restrict movement, they prevent the users direct contact with the hazardous materials. The trade-off is clearly on the side of maximizing personal safety. Depending on the design of the cabinet, the supply HEPA filter provides particulate-free, albeit somewhat turbulent, airflow within the work environment. Several Class III cabinets can be joined together in a line to provide a larger work area. Such cabinet lines are custom-built; the equipment installed within the cabinet line (e.g., refrigerators, small elevators, shelves to hold small animal cage racks, microscopes, centrifuges, incubators, etc.) is generally custom-built as well. Furthermore, Class III cabinets are usually only installed in maximum containment
46
Table 2: Safety performance requirements and specifications for biological safety cabinets
Leak rate Exhaust requirements through HEPA filter
Cabinet Type
Use classification NCIa DNAb Virus Carcinogen 0 100% Not applicable
CDCc
Performance requirements Face Recycle Exhaust velocity air air fpm (%) (%)
Class I
P1-P3 1-3
75
Class II Type A 30 70
P1-P3 1-3
75
70
30
Class II Type B1 0 100
Low- No moderate Low- No moderate Low- Yes High Gas tight. Leak rate < 1 x 10-5 lps at 500 Pa (2 wc) Pressure tight. No air/soap bubble at 500 Pa (2wc) Pressure tight as for Type B1 Pressure tight as for Type B1 Gas tight. Leak rate < 1 x 10-9 lps at 750 Pa (3 wc)
P1-P3 1-3
100
LowHigh
Yes
P1-P3 1-3
100
LowHigh
No
P1-P3 1-3
100
Direct exhaust from work area, vented outdoor Exhaust airflow from common plenum, vented into room or outdoor Exhaust directly from rear of area, vented outdoor Exhaust directly from work area, vented outdoor Exhaust from common plenum, vented outdoor Direct exhaust from work area, vented outdoor
Class III
High
Yes
P4
Key to superscripts etc.
a b c $
National Cancer Institute (US Public Health Service) For work with recombinant DNA molecules Centre for Disease Control (US Public Health Service) Not applicable
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laboratories that have controlled access and require special ventilation or other support systems (such as steam for autoclaves). The reader should consult more definitive literature on these systems. risk assessment The potential for untoward events must be evaluated to reduce or eliminate worker exposure to or release of infectious organisms. Through the process of risk assessment, the work procedures are evaluated for potential exposure to the microorganism. The hierarchy of controls to prevent or minimize exposure to hazardous materials includes engineering controls, administrative and procedural controls, and work practices which may involve use of additional personal protective equipment. Having a properly operating BSC available is an effective engineering control; requiring its use is an administrative control. Some suggested work practices and procedures associated with working safely in a BSC are detailed here. preparing for work within a class II BSC Preparing a written checklist of materials necessary for a particular activity and placing necessary materials in the BSC before beginning work serves to minimize the number of arm-movement disruptions across the fragile air barrier of the cabinet. The rapid movement of a workers arms in a sweeping motion into and out of the cabinet will disrupt the air curtain and may compromise the partial barrier containment provided by the BSC. Moving arms in and out slowly, perpendicular to the face opening of the cabinet, will reduce this risk. Other personnel activities in the room (e.g., rapid movement, open/closing room doors, etc.) may also disrupt the cabinet air barrier. Laboratory coats should be worn buttoned over street clothing; latex gloves are worn to provide hand protection. A solid front, back-closing lab gown provides better protection of personal clothing than a traditional lab coat. Gloves should be pulled over the knitted wrists of the gown, rather than worn inside. Elasticized sleeves can also be worn to protect the investigators wrists. Before beginning work, the investigator should adjust the stool height so that his/her face is above the front opening. Manipulation of materials should be delayed for approximately one minute after placing the hands/ arms inside the cabinet. This allows the cabinet to stabilize and to air sweep the hands and arms to remove surface microbial contaminants.
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Table 3: Selection of a safety cabinet through risk assessment Biological Protection Provided Product Environment BSC Class
Risk Assessed Personnel
BSL 1/ P1 BSL 2/ P2
Yes Yes
No Yes
Yes Yes
I II A, B1, B2, B3
BSL 3/ P3
Yes
Yes
Yes
II B1, B2 III
Table 9.4: Comparison of biosafety cabinet characteristics

BSC Class Face Velocity Airflow Pattern Applications
Nonvolatile Toxic Chemicals and Radionuclides I 75 In at front; exhausted through HEPA to the outside or into the room through HEPA 70% recirculated to the cabinet work area through HEPA; 30% balance can be exhausted through HEPA back into the room or to the outside through a thimble unit Exhaust cabinet air must pass through a dedicated duct to the outside through a HEPA filter No recirculation; total exhaust to the outside through hard-duct and a HEPA filter Same as II, A, but plenums are under negative pressure to room; exhaust air is thimble-ducted to the outside through a HEPA filter Supply air inlets and hard-duct exhausted to outside through two HEPA filters in series YES Volatile T Chemica Radionuc
YES (
II, A
75
YES
NO
II, B1
100
YES
YES (m amounts
II, B2
100
YES
YES (s amoun
II, B3
100
YES
YES (m amounts
III
N/A
YES
YES (s amoun
Notes (1) Installation may require a special duct to the outside, an in-line charcoal filter, and a spark proof (explosion proof) motor and other electrical components in the cabinet. Discharge of a Class I cabinet in to a room should not occur if volatile chemicals are used. (2) In no circumstances should the chemical concentration approach the lower explosion limits of the compound
49
When the users arms rest flatly across the front grille, room air may flow directly into the work area, rather than being drawn through the front grille. Raising the arms slightly will alleviate this problem. The front grille must not be blocked with research notes, discarded plastic wrappers, pipetting devices, etc. All operations should be performed at least four 4 inches from the front grille on the work surface. Materials or equipment placed inside the cabinet may cause disruption to the airflow, resulting in turbulence, possible cross-contamination, and/ or breach of containment. Extra supplies (e.g., additional gloves, culture plates or flasks, culture media) should be stored outside the cabinet. Only the materials and equipment required for the immediate work should be placed in the BSC. BSCs are designed to be operated 24 hours per day, and some investigators find that continuous operation helps to control the laboratorys level of dust and other airborne particulates. Although energy conservation may suggest BSC operation only when needed, especially if the cabinet is not used routinely, room air balance is an overriding consideration. In some instances, room exhaust is balanced to include air discharged through ducted BSCSs. Cabinet blowers should be operated at least three to five minutes before beginning work to allow the cabinet to purge. This purge will remove any particulates in the cabinet. The work surface, the interior walls (not including the supply filter diffuser), and the interior surface of the window should be wiped with 70% ethanol (EtOH), a 1:100 dilution of household bleach (i.e., 0.05% sodium hypochlorite), or other disinfectant as determined by the investigator to meet the requirements of the particular activity. When bleach is used, a second wiping with sterile water is needed to remove the residual chlorine, which may eventually corrode stainless steel surfaces. Wiping with non-sterile water may recontaminate cabinet surfaces, a critical issue when sterility is essential (e.g., maintenance of cell cultures). Similarly, the surfaces of all materials and containers placed into the cabinet should be wiped with 70% ETOH to reduce the introduction of contaminants to the cabinet environment. This simple step will reduce introduction of mold spores and thereby minimize contamination of cultures. Further reduction of microbial load on materials to be placed or used in BSCs may be achieved by periodic decontamination of incubators and refrigerators.
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material placement inside the BSC Plastic-backed absorbent toweling can be placed on the work surface (but not on the front or rear grille openings). This toweling facilitates routine cleanup and reduces splatter and aerosol formation during an overt spill. It then can be folded and placed in an autoclavable biohazard bag when work is completed.
Figure 9: Material placement inside the BSC
All materials should be placed as far back in the cabinet as practical, toward the rear edge of the work surface and away from the front grille of the cabinet. Similarly, aerosol-generating equipment (e.g., vortex mixers, tabletop centrifuges) should be placed toward the rear of the cabinet to take advantage of the air split . Active work should flow from the clean to contaminated area across the work surface. Bulky items such as biohazard bags, discard pipette trays and suction collection flasks should be placed to one side of the interior of the cabinet. Certain common practices interfere with the operation of the BSC. The autoclavable biohazard collection bag should not be taped to the outside of the cabinet. Upright pipette collection containers should not be used in BSCs nor placed on the floor outside the cabinet. The frequent inward/ outward movement needed to place objects in these containers is disruptive to the integrity of the cabinet air barrier and can compromise both personnel and product protection. Only horizontal pipette discard trays containing an appropriate chemical disinfectant should be used within the cabinet. Furthermore, potentially contaminated materials should not be brought out of the cabinet until they have been surface decontaminated. Alternatively, contaminated materials can be placed into a closable container for transfer to an incubator, autoclave or for other decontamination treatment.
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operations within a class II BSC Many common procedures conducted in BSCs may create splatter or aerosols. Good microbiological techniques should always be used when working in a biological safety cabinet. For example, techniques to reduce splatter and aerosol generation will minimize the potential for personnel exposure to infectious materials manipulated within the cabinet. Class II cabinets are designed so that horizontally nebulized spores will be captured by the downward flowing cabinet air within fourteen inches of travel. Therefore, as a general rule of thumb, keeping clean materials at least one foot away from aerosol-generating activities will minimize the potential for cross-contamination. The general work flow should be from clean to contaminated (dirty). Materials and supplies should be placed in such a way as to limit the movement of dirty items over clean ones. Several measures can be taken to reduce the chance for crosscontamination when working in a BSC. Opened tubes or bottles should not be held in a vertical position. Investigators working with Petri dishes and tissue culture plates should hold the lid above the open sterile surface to minimize direct impaction of downward air. Bottle or tube caps should not be placed on the toweling. Items should be recapped or covered as soon as possible. Open flames are not required in the near microbe-free environment of a biological safety cabinet. On an open bench, flaming the neck of a culture vessel will create an upward air current which prevents microorganisms from falling into the tube or flask. An open flame in a BSC, however, creates turbulence which disrupts the pattern of air supplied to the work surface. When deemed absolutelv necessary, touch-plate microburners equipped with a pilot light to provide a flame on demand mav be used. Internal cabinet air disturbance and heat buildup will be minimized. The burner must be turned off when work is completed. Small electric furnaces are available for decontaminating bacteriological loops and needles and are preferable to an open flame inside the BSC. Disposable sterile loops can also be used. Aspirator bottles or suction flasks should be connected to an overflow collection flask containing appropriate disinfectant, and to an in-line HEPA or equivalent filter.
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Figure 10: Working with aspirator bottles and suction flasks
This combination will provide protection to the central building vacuum system or vacuum pump, as well as to the personnel who service this equipment. Inactivation of aspirated materials can be accomplished by placing sufficient chemical decontamination solution into the flask to kill the microorganisms as they are collected. Once inactivation occurs, liquid materials can be disposed of appropriately as noninfectious waste. Investigators must determine the appropriate method of decontaminating materials that will be removed from the BSC at the conclusion of the work. When chemical means are appropriate, suitable liquid disinfectant should be placed into the discard pan before work begins. Items should be introduced into the pan with minimum splatter, and allowed appropriate contact time as per manufacturers instructions. Alternatively, liquids can be autoclaved prior to disposal. Contaminated items should be placed into a biohazard bag or discard tray inside the BSC. Water should be added to the bag or tray prior to autoclaving. When a steam autoclave is to be used, contaminated materials should be placed into a biohazard bag or discard pan containing enough water to ensure steam generation during the autoclave cycle. The bag should be taped shut or the discard pan should be covered in the BSC prior to removal to the autoclave. The bag should be transported and autoclaved in a leakproof tray or pan. surface decontamination All containers and equipment should be surface decontaminated and removed from the cabinet when work is completed. At the end of the work day, the final surface decontamination of the cabinet should include a wipe-down of the work surface, the cabinets sides and back, and the interior of the glass. If necessary, the cabinet should also be monitored for radioactivity and decontaminated when necessary. Investigators
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should remove their gloves and gowns and wash their hands as the final step in safe microbiological practices. Small spills within the BSC can be handled immediately by removing the contaminated absorbent paper toweling and placing it into the biohazard bag. Any splatter onto items within the cabinet, as well as the cabinet interior, should be immediately wiped with a towel dampened with decontaminating solution. Gloves should be changed after the work surface is decontaminated and before placing clean absorbent toweling in the cabinet. Hands should be washed whenever gloves are changed or removed. Spills large enough to result in liquids flowing through the front or rear grilles require more extensive decontamination. All items within the cabinet should be surface decontaminated and removed. After ensuring that the drain valve is closed, decontaminating solution can be poured onto the work surface and through the grille(s) into the drain pan. Twenty to thirty minutes is generally considered an appropriate contact time for decontamination, but this varies with the disinfectant and the microbiological agent. Manufacturers directions should be followed. The spilled fluid and disinfectant solution on the work surface should be absorbed with paper towels and discarded into a biohazard bag. The drain pan should be emptied into a collection vessel containing disinfectant. A flexible tube should be attached to the drain valve and be of sufficient length to allow the open end to be submerged in the disinfectant within the collection vessel. This procedure serves to minimize aerosol generation. The drain pan should be flushed with water and the drain tube removed. Should the spilled liquid contain radioactive material, a similar procedure can be followed. Radiation safety personnel should be contacted for specific instructions. gas decontamination BSCs that have been used for work involving infectious materials must be decontaminated before HEPA filters are changed or internal repair work is done. Before a BSC is relocated, a risk assessment which considers the agents manipulated within the BSC must be done to determine the need for decontamination. The most common decontamination method uses formaldehyde gas, although more recently hydrogen peroxide vapor has been used successfully. This
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environmentally benign vapor is useful in decontaminating HEPA filters, isolation chambers and centrifuge enclosures. facility and engineering requirements: secondary barriers Whereas biological safety cabinets are considered to be the primary safety barrier for manipulation of infectious materials, the laboratory room itself is considered to be the secondary safety barrier. Inward directional air flow is established exhausting a greater volume of air than is supplied to a given laboratory and by drawing makeup air from the adjacent space. This is optional at biosafety level 2 but must be maintained at BSL-3. The air balance for the entire facility should be established and maintained to ensure that air flow is from areas of least- to greater contamination. building exhaust At BSL-3 and BSL-4, exhaust laboratory air must be directly exhausted since it is considered potentially contaminated. This concept is referred to as a dedicated single-pass exhaust system. The exhausted room air can be HEPA-filtered when a high level of aerosol containment is needed, which is always true at BSL-4 and is optional at BSL-3. When the building exhaust system is used to vent a ducted BSC, the system must have a sufficient capacity to maintain the exhaust flow if changes in the static pressure within the system should occur. Otherwise, each cabinet must have a dedicated exhaust system. The room exhaust system should be sized to handle both the room and all containment devices vented through the system. Adequate supply air must be provided to ensure appropriate function of the exhaust system. The facility engineer should be consulted before locating a new cabinet requiring connection to the building exhaust system. Right angle bends, long horizontal runs, and transitional connectors within the systems will add to the demand on the exhaust fan. The building exhaust air should be discharged away from supply air intakes, to prevent entrainment of exhausted laboratory air back into the building air supply system. utility services Utility services needed within a BSC must be planned carefully. Protection of vacuum systems has already been addressed. Electrical outlets inside the cabinet must be protected by ground fault circuit interrupters and should be supplied by an independent circuit. When propane gas is
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provided, a clearly marked emergency gas shut-off valve outside the cabinet must be installed for fire safety. All nonelectrical utility services should have exposed, accessible shut-off valves. ultraviolet lamps Ultraviolet (UV) lamps are not required in BSCs. If installed, UV lamps must be cleaned weekly to remove any dust and dirt that may block the germicidal effectiveness of the ultraviolet light. The lamps should be checked periodically with a meter to ensure that the appropriate intensity of UV light is being emitted. UV lamps must be turned off when the room is occupied to protect eyes and skin from UV exposure, which can burn the cornea and cause skin cancer. BSC placement Biological safety cabinets were developed as work stations to provide personnel, product and environmental protection during the manipulation of infectious microorganisms. Certain considerations must be met to ensure maximum effectiveness of these primary barriers. Whenever possible, a 12-inch clearance should be provided behind and on each side of the cabinet to allow easy access for maintenance, and to ensure that the air return to the laboratory is not hindered. A 12- to 14- inch clearance above the cabinet may be required to provide for accurate air velocity measurement across the exhaust filter surface with a thermoanemometer and for exhaust filter changes. When the BSC is hard-ducted or connected by a thimble unit to the ventilation system, adequate space must be provided so that the configuration of the duct work will not interfere with air flow. The thimble unit must provide access to the exhaust filter for testing of the HEPA filter. The ideal location for the biological safety cabinet is remote from the entry (e.g., the rear of the laboratory away from traffic), since people walking parallel to the face of a BSC can disrupt the air curtain. The air curtain created at the front of the cabinet is quite fragile, amounting to a nominal inward and downward velocity of 1 mph. Open windows, air supply registers, or laboratory equipment that creates air movement (e.g., centrifuges, vacuum pumps) should not be located near the BSC. Similarly, chemical fume hoods must not be located close to BSCs. HEPA filters HEPA filters, whether part of a building exhaust system or part of a cabinet, will require replacement when they become so loaded that
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sufficient air flow can no longer be maintained. Filters must be decontaminated before removal. To contain the formaldehyde gas typically used for microbiological decontamination, exhaust systems containing HEPA filters require airtight dampers to be installed on both the inlet and discharge side of the filter housing. This ensures containment of the gas inside the filter housing during decontamination.
Figure 11: Bag-in/bag-out of contaminated HEPAs
Access panel ports in the filter housing also allow for performance testing of the HEPA filter. A bag-in/bag-out (BIBO) filter assembly can be used in situations where HEPA filtration is necessary for operations involving biohazardous materials and hazardous or toxic chemicals. This protects the technician handling the filter as well as the environment. The BIBO system is used when it is not possible to decontaminate the HEPA filters with formaldehyde gas, or when hazardous toxic chemicals have been used in the BSC. Note, however, that this requirement must be identified at the time of purchase and installation; a BIBO assembly cannot be added to a cabinet after-the-fact. certification of biological safety cabinets Development of containment standards The evolution of containment equipment for varied research and diagnostic applications created the need for consistency in construction, certification and performance. A Federal standard was developed to establish classes of air cleanliness and methods for monitoring clean work stations and clean rooms where HEPA filters are used to control airborne particulates. The first standard to be developed specifically for BSCs served as a Federal procurement specification for the NIH Class II, Type 1 (now called Type A) biological safety cabinets, which had a fixed or hinged
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front window or a vertical sliding sash, vertical downward laminar airflow and HEPA-filtered air supply and exhaust. This guideline specified design criteria and defined prototype tests for microbiological aerosol challenge, velocity profiles, and leak testing of the HEPA filters. A similar procurement specification was generated when the Class II Type 2 (now called Type B1) cabinet was developed to control both airborne particulates and some gaseous contaminants. The National Sanitation Foundation (NSF International) Standard No. 49 for Class II (Laminar Flow) Biohazard Cabinetry was first published in 1976, providing the first independent standard for design, manufacture and testing. This standard replaced the NIH specifications which had been used by other institutions and organizations when purchasing BSCs. NSF Standard 49 incorporates specifications regarding design, materials and construction. This Standard for biological safety cabinets establishes performance criteria for biological safety cabinets and provides the minimum requirements that are accepted in the United States. Cabinets which meet the standard and were certified by the NSF bear an NSF 49 Seal. Standard No. 49 pertains to all models of Class II cabinets (Type A, Bl, B2, and B3) and lists a series of specifications regarding: design/construction, performance, installation recommendations, recommended microbiological decontamination procedure, and references and specifications pertinent to Class II Biohazard Cabinetry.
While the NSF standard does not cover field testing of BSCs, it is common for many of its test methods and parameters to be applied in the field, and these are included in Annex F of the document. Most recently revised in 1992, this Standard is reviewed every five years by a steering committee to ensure that it remains consistent with developing technologies. The operational integrity of a new BSC must be validated by certification before it is put into service or after a cabinet has been repaired or relocated. Relocating a BSC may break the HEPA filter seals or otherwise damage the filters or the cabinet. Each BSC should be tested and certified at least annually to ensure continued proper operation.
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Table 5: Biological safety cabinets: references for applicable containment tests
Note: Parenthetical references are to the NSF 49; letters and numerals indicate specific sections and subsections.
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On-site testing following the recommendations for field testing (NSF Standard 49) must be performed by experienced, qualified personnel. Some basic information is included here to assist in understanding the frequency and kinds of tests to be performed. Selecting competent individuals to perform testing and certification is important, and it is suggested that the institutional biosafety officer be consulted in identifying companies qualified to conduct the necessary field performance tests. It is strongly recommended that accredited field certifiers be used to test and certify BSCs whenever possible. If in-house personnel are performing the certifications, then these individuals should become accredited. The importance of proper certification cannot be emphasized enough, since persons who manipulate infectious microorganisms are at increased risk of acquiring an occupational illness when their BSCs are functioning improperly. BSCs perform consistently well when proper annual certification procedures are followed; cabinet or filter failures tend to occur infrequently. performance testing BSCs in the field BSCs are the primary containment device that protect the worker, product and environment from exposure to microbiological agents, and their performance as specified by Standard No. 49 needs to be verified at the time of installation and annually thereafter. The purpose and acceptance level of the performance tests are to ensure the balance of inflow and exhaust air, the distribution of air onto the work surface, and the integrity of the cabinet. Other tests check electrical and physical features of the BSC. A. Downflow velocity and volume test This test is performed to measure the velocity of air moving through the cabinet workspace, and is to be performed on all Class I and II biosafety cabinets. B. Inflow velocity test This test is performed to determine the calculated or directly measured velocity through the work access opening, to verify the nominal set point average inflow velocity and to calculate the exhaust airflow volume rate.
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Table 6: Biological safety cabinets performance testing

Biosafety Cabinet Tests Performed For Class I Class Class II III Acceptance Value
Primary Containment Cabinet Integrity HEPA Filter Leak Downflow Velocity Profile Inflow (Face) Velocity N/A Req A Req A Req Req Req A Req Req Req Hold 500 Pa (2 wc) within 10% for 30 min 0.01% +/- 0.025 mps (+/- 5 fpm) of set point value Type A: 0.38 mps (75 fpm) Type B: 0.5 mps (100 fpm) Variance for both: +/- 0.005 mps (+/-5 fpm) Type A: 76 m3/m (45 CFM/ft) of table width Type B: 110 m3/m (65 CFM/ft) of table width
Exhaust Flow Volume
Req
Req
Req
Negative Pressure/ Ventilation Rate Airflow Smoke Patterns Alarms and Interlocks Electrical Safety Electrical Leakage, etc. Ground Fault Interupter Other Lighting Intensity UV Intensity Noise Level Vibration Temperature Rise Notes
B Req C, D E, D D E C, E E E E
A Req C, D E, D D E C, E E E E
Req Req C, D E, D < 500 ma. Ground circuit resistance < 0.15 D E 860 - 1600 lux (80 - 150 ft candles)
C, E E 67 dBA E 0.0002 rms @ 10 - 250 Hz E 8.5 oC (15 oF)
Required during certification. Required for proper certification if the cabinet is new, has been moved or panels have been removed for maintenance. B If used with gloves. C If present. D Encouraged for electrical safety. E Optional, at the discretion of the user. F Used to determine air distribution within cabinet for clean to dirty procedures. N/A Not applicable Cabinet integrity should be checked with soap bubble and/or halogen leak testing methods
Req A
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C. Airflow smoke patterns test This test is performed to determine if the airflow along the entire perimeter of the work access opening is inward, if airflow within the work area is downward with no dead spots or refluxing, if ambient air passes onto or over the work surface, and if there is refluxing to the outside at the window wiper gasket and side seals. The smoke test is an indicator of airflow direction, not velocity. D. HEPA filter leak test This test is performed to determine the integrity of supply and exhaust HEPA filters, filter housing, and filter mounting frames while the cabinet is operated at the nominal set point velocities. An aerosol in the form of generated particulates of dioctylphthalate (DOP), 10 mg / litre, or an accepted alternative is required for leak-testing HEPA filters and their seals. Although DOP has been identified as a potential carcinogen, competent service personnel are trained to use this chemical in a safe manner. The aerosol is generated on the intake side of the filter, and particles passing through the filter or around the seal are measured with a photometer on the discharge side. This test is suitable for ascertaining the integrity of all HEPA filters. E. Cabinet leak test The pressure holding test is performed to determine if exterior surfaces of all plenums, welds, gaskets, and plenum penetrations or seals are free of leaks. It need only be performed just prior to initial installation when the BSC is in a free-standing position (all four sides are easily accessible) in the room in which it will be used, after a cabinet has been relocated to a new location, and again after removal of access panels to plenums for repairs or a filter change. This test may also be performed on fully installed cabinets. Test is performed with soap water and looking for soap bubbles or or dichlorodifluoromethane gas and halogen leak detector. F. Electrical leakage and ground circuit resistance and polarity test These safety tests are performed to determine if a potential shock hazard exists by measuring the electrical leakage, polarity ground fault interrupter function, and ground circuit resistance to the cabinet connection. They may be performed by an electrical technician other than the field certification personnel at the same time the other field
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certification tests are conducted. The polarity of electrical outlets are checked using a polarity tester. The ground fault circuit interrupter should trip when approximately 5 milliamperes (ma) is applied. G. Lighting intensity test This test is performed to measure the light intensity on the work surface of the cabinet as an aid in minimizing cabinet operators fatigue. Acceptance range: 860 - 1600 lux (80 - 150 ft candles) at the work surface. H. Vibration test This test is performed to determine the amount of vibration in an operating cabinet as a guide to satisfactory mechanical performance, as an aid in minimizing cabinet operators fatigue, and to prevent damage to delicate tissue culture specimens. I. Noise level test This test is performed to measure the noise levels produced by the cabinets, as a guide to satisfactory mechanical performance and an aid in minimizing cabinet operators fatigue. Acceptance value is 67 dBA with ambient below 57 dbA. J. UV lamp test A few BSCs have UV lamps. When used, they must be tested periodically to ensure that their energy output is sufficient to kill microorganisms. After having been turned off and allowed to cool, the surface on the bulb should be cleaned with 70% ethanol prior to performing this test. Five minutes after the lamp has been turned on, the sensor of the UV meter is placed in the center of the work surface. The radiation output should not be less than 40 microwatts per square centimeter at 254 nanometers.
Table 7: Microbial challenge tests for BSC
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Finally, accurate test results can only be assured when the testing equipment is properly maintained and calibrated. lt is appropriate to request the calibration information for the test equipment being used by the certifier. microbial challenge tests A. Purpose These tests determine if aerosols will be contained within the cabinet, outside contaminants will not enter the cabinet work area, and aerosol contamination of other equipment in the cabinet will be minimised. The cabinet shall be operated at the airflow velocities indicated in the specific test methods. The cabinet shall be turned on at least 30 mins before the start of any test, and operated continuously throughout all test methods. Cabinets meeting these tests shall then meet airflow characteristics as specified. B. Materials 1. Spores of Bacillus subtilis var. niger (B. subtilis), American Type Culture Collection (ATCC), USA, No. 9372 or National Collection Type Culture (NCTC), UK, No. 10073. 2. Sterile diluent prepared as follows: a. Final diluent phosphate buffer solution (PBS) (step 1) Dissolve 34 gms KH2PO4 in 500 ml distilled H2O. Adjust pH to 7.2 +/- 0.5 with 1 N NaOH at 25oC (77oF). Dilute to 1 L with distilled H2O Final diluent PBS (step 2) Distilled H2O - 1 L Stock PBS (step 1) - 1.25 ml Final pH - 7.2 +/- 0.5 Autoclave at 121oC (250 oF) for 15 minutes Optional - Magnesium sulphate (50 gms MgSO4.7H2O per litre of distilled H2O) - 5.0 ml OR b. Distilled H2O - 1 L. Adjust pH to 7.0 +/- 0.1 at 25 oC (77 o F). Autoclave at 121 oC (250 oF) for 15 minutes Note: Formula b is suitable for diluent when spore suspension is prepared for immediate use. When storage of diluent suspension at 4 oC (39.2 oF) is required, Formula a should be used.
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3. Suspension of B. subtilis var. niger spores prepared as follows: Method A (using previously harvested B. subtilis spores) a. Aseptically inoculate by streak plating technique several tryptic soy agar petri plates (100 x 15 mm) b. Incubate for 48 +/- 2 hrs at 37 +/- 0.5 oC (99 +/- 1 oF) c. Remove characteristic (pigmented dark orange) colonies and transfer to ten 220 ml sterile screw-capped bottles containing approximately 50 ml of tryptic soy agar. d. Incubate for 48 +/- 2 hrs at 37 +/- 0.5 oC (99 +/- 1 oF) e. Add 10 ml of PBS to each slant, and gently wash the bacteria from the agar surface. f. Transfer the bacterial suspensions to yield approximately 100 ml in a sterile 150 ml screw-cap bottle. If cell debris interferes with nebuliser dissemination, the suspension may be clarified by washing three times in PBS by centrifugation at 1500 rpm for 10 min. Re-suspend in PBS to the original volume. g. Heat shock culture at 65 +/- 0.5 oC (149 +/- 1 oF) for 15 min. h. Determine spore concentration by standard dilution- plate methods using PBS and tryptic soy agar. Spores prepared as above should yield an average count of 2 to 4 x 10 9 per ml. i. Incubate for 48 +/- 2 hrs at 37 +/- 0.5 oC (99 +/- 1 oF) j. Dilute the spore suspension with PBS to obtain final spore concentration of 5 to 8 x 10 8 per ml if spores are to be used immediately. k. Store the stock spore suspension (2 to 4 x 10 9 per ml) at 4 oC (39.2 oF), or divide into aliquots to store in screw-capped vials at 70 oC (- 94 oF). l. Make frequent checks of spore viability by surface plating; and spore predominance by an acceptable spore staining technique. Method B a. Inoculate 250 ml portions of sterile tryptose broth with aliquots of previously harvested B. subtilis spores; or rehydrated freeze-dried cultures as per ATCC or NCTC instructions. b. Incubate on a reciprocating shaker for 48 +/- 2 hrs at 37 +/- 0.5 oC (99 +/- 1 oF) c. Heat shock culture at 65 +/- 0.5 oC (149 +/- 1 oF) for 15 min.
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d. Transfer the suspensions to screw-cap test tubes and wash at least three times in sterile distilled H2O by centrifugation at 1500 rpm for 10 min. Use PBS in last washing if storage is required. e. Determine spore concentration by standard dilution- plate methods using PBS and tryptic soy agar. Spores prepared as above should average 1.5 x 10 9 per ml. f. Incubate for 48 +/- 2 hrs at 37 +/- 0.5 oC (99 +/- 1 oF) g. If spore suspension is to be used promptly, dilute the spore suspension with PBS to obtain final suspension concentration of 5 to 8 x 10 8 per ml h. To store the stock spore culture, divide into aliquots and store at 4 oC (39.2 oF) in screw-capped vials, or store in freezer at - 70 oC (- 94 oF). Before use, check viability of spore suspension as in Method A, item l. 4. Petri plates (100 x 15 mm and 150 x 22 mm) containing nutrient agar, tripticase soy agar, or other suitable growth medium with no inhibitors or other additives. 5. Six AGI-30 (or equivalent) samplers (flow rate calibrated at 12.5 lpm) containing 20 ml of sterile diluent. 6. Two slit-type air samplers operating at a rated flow of 1 +/0.000472 m3/sec (0.05 cfm) 7. Refluxing 6-jet modified MRE-type short-form collision nebuliser or equivalent that can be demonstrated to produce a bacterial aerosol of equivalent characteristics. 8. One 63 mm (2.5) outside diameter stainless steel, steel, or aluminium cylinder with closed ends shall be used to disrupt the airflow. The length to be determined by size of cabinet interior. One end butts against the back end of the cabinet, and the other end protrudes at least 15 cm (6) into the room through the work access opening of the cabinet. C. Personnel Protection Test: system challenged with 1 to 8 x 10 8 per ml B. subtilis spores in five min. 1. Method a. A nebuliser containing up to 55 ml of spore suspension (5 to 8 x 10 8 per ml) is centered between the side walls of the cabinet. The horizontal spray axis is placed 35 cms (14) above the work surface, the opening of the nebuliser is 10 cms (4) behind the front window. The spray axis is parallel to the work surface and directed toward the front window.
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b. The cylinder is placed at the cabinet centre. The axis of the cylinder is 6.9 cms (2.75) above the work surface. Around the cylinder, four AGI-30s are positioned with the sampling inlets 6.3 cms (2.5) outside the cabinet front. Two AGIs are placed so that their inlet axes are 15 cm (6) apart, and in a horizontal plane, tangent to the top of the cylinder. Two AGIs are positioned so their inlet axes are 5 cm (2) apart, and lie in a horizontal plane 2.5 cms (1) below the cylinder. As a positive control, an agar plate is placed under the centre of the cylinder, and supported 1 cm (0.4) above or below the front intake grille, to minimise the obstruction of airflow to the grille. c. Two slit-type air samplers are placed so the horizontal plane of the air inlets is at the work surface elevation, the vertical axes of the inlets are 15 cms (6) in front of the cabinet, and 20 cms (8) from each interior side wall. Two AGI-30 samplers are placed so the horizontal plane of the air inlets is 35 cms (14) above the work surface, the vertical axes are 5 cms (2) outside the front edge of the cabinet, and 15 cms (6) on each side of the cabinet centreline. d. The cabinet shall be set at the nominal set point. e. Duration of the test is 30 min. The test sequence is as follows: Time (mins) Activity 30 Start slit samplers 25 Start nebuliser 24 Start impingers 19 Stop impingers 18.5 Stop nebuliser 0 Stop slit samplers Three replicate tests shall be performed. f. Filter the sampling fluid from all of the AGI-30 samplers through a 47 mm diameter 0.22 membrane filter, remove the filter aseptically and place on appropriate media. Plates containing the filters and plates from the slit-type air samplers shall be incubated at 37 oC (98.6 oF). Examine at 24 - 28 hrs and, if negative, reincubate and read at 44 - 48 hrs. For research and field applications, the sampling fluid from each AGI-30 sampler may be filtered separately to provide information on specific areas within the cabinet.
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g. Repeat the above steps setting the cabinet airflow velocities at - 5 x 10- 4 +/- .01 mps (- 10 +/- 2 fpm) inflow and + 5 x 10- 4 +/- .01 mps (+ 10 +/- 2 fpm) supply from nominal set point. Airflow velocity adjustments shall be made by the adjustment of voltage, blower speed and dampers (if applicable). If the 5 x 10- 4 +/- .01 mps (10 +/- 2 fpm) adjustments cannot be reached based on cabinet design, the maximum deviation from the nominal set point shall be used. h. Repeat the above steps setting the airflow velocities at - 5 x 10- 4 +/ .01 mps (- 10 +/- 2 fpm) from the nominal set point for both supply and inflow. 2. Acceptance: The number of B. subtilis colony forming units (CFU) recovered from the six AGI-30 samplers shall not exceed 10 CFU per test. Total slittype air sampler plate counts shall not exceed 5 B. subtilis CFU for a 30-minute sampling period. Three replicate tests shall be performed. The control plate shall be positive. A plate is positive when it contains greater than 300 CFU of B. subtilis. D. Product Protection Test: system challenged by 1 to 8 x 10 6 B. subtilis spores per ml in 5 min. 1. Method a. Cover the work surface with open agar plates (100 x 15 mm) with the cylinder at the mid-point. b. Position the horizontal spray axis of the nebuliser containing 55 ml of 5 to 8 x 10 6 B. subtilis spores per ml at the level of the top edge of the work opening, and center between the two sides of the cabinet, with the opening of the nebuliser 10 cms (4) outside the window. The spray axis is parallel to the work surface and directed toward the open front of the cabinet. c. A 6.3 cms (2.5) outside diameter cylinder, with closed ends, is placed in the centre of the cabinet. The cylinder is positioned in the cabinet so one end butts against the back wall of the cabinet, the other end extends at least 15 cms (6) into the room through the front opening of the cabinet, and the axis of the cylinder is 6.9 cms (2.75) above the work surface. d. As a positive control, an agar plate is placed under the centre of the cylinder, and supported 1cm (0.4) above or below the front intake grille. e. The cabinet shall be set at the nominal set point.
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f. Operate nebuliser for 5 mins. 5 mins after nebulisation is terminated, place lids on agar plates. g. Incubate plates at 37 oC (98.6 oF) and examine them at 24 - 28 hrs. If negative, reincubate and read at 44 - 48 hrs. h. Repeat the above steps at airflow velocities - 5 x 10- 4 +/- .01 mps (- 10 +/- 2 fpm) supply and + 5 x 10- 4 +/- .01 mps (+ 10 +/- 2 fpm) inflow from nominal set point. Airflow velocity adjustments shall be made by the adjustment of voltage, blower speed and dampers (if applicable). If the 5 x 10- 4 +/- .01 mps (- 10 +/- 2 fpm) adjustments cannot be reached based on cabinet design, the maximum adjustment from the nominal set point shall be used. 2. Acceptance The number of B. subtilis colony forming units (CFU) on agar settling plates shall not exceed 5 CFU for each test. Three replicates shall be performed. The control plates shall be positive. A plate is positive when it contains greater than 300 CFU of B. subtilis. E. Cross-Contamination Test: system challenged by 1 to 8 x 10 4 B. subtilis spores per ml for 5 min. 1. Method a. Position the horizontal spray axis of the nebuliser containing 55 ml of 5 to 8 x 10 4 B. subtilis spores per ml 76 - 127 mm (3 - 5 ) above the work surface, with the back of the nebuliser located against the mid-point of the left interior side wall. The spray axis is parallel to the work surface and is directed toward the opposite side wall. b. The cabinet shall be set at the nominal set point. c. Place open agar plates (100 x 15 mm) on the work surface in the following manner: two rows of control plates with the centre-line under the outlet of the nebuliser; one row of plates with their centres on a line drawn front to back 36 cms (14) from the side wall being tested. Nest at least one more row of plates beyond the 36 cms (14) row; two rows when there is room. d. Start the nebuliser. After 5 mins stop the nebuliser, but continue to operate the cabinet for 15 mins. e. Let the cabinet motor run while placing the covers on the open agar plates. Incubate the plates at 37 oC (98.6 oF) and examine them at 24 - 28 hrs. If negative, reincubate and read at 44 - 48 hrs.
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f. Perform the same procedure (a - e), but place nebuliser against the mid-point of the right interior wall. 2. Acceptance Some agar plates, from the challenge side wall to 36 cms (14) from the side wall, will recover B. subtilis CFU and shall be used as controls. The total number of CFU recovered on agar plates with centres greater than 36 cms (14) shall not exceed 2 CFU per test. Three replicates each shall be performed from the left and right sides of the cabinet. effective use of biological safety cabinets Exposure to airborne microorganisms can result in infection of laboratory workers or contamination of research materials. Biomedical engineering and technology have provided safeguards, but these safeguards do not prevent mistakes or human errors. Danger to personnel and to the success of scientific investigation from carelessly or improperly used equipment cannot be overly emphasized. The Laminar Flow Biological Safety Cabinet, designed to prevent escape of pathogens into the workers' environment and to bar contaminants from the research work zone, is a key element to safe, successful experimentation with biological materials. Escape of pathogens into the workers' area is prevented by an air barrier at the front opening and the cleaning action of the exhaust air filter. Inward flow of room air into the front air intake grill creates the air barrier. The amount of air drawn into the air intake grill and the amount of air exhausted through the exhaust filter are equal. The exhaust filter removes airborne biological contaminants which may be released in the cabinet. It does not remove chemical or radiological contaminants. Contamination of the work area inside the cabinet is prevented by the cleaning action of the supply filters. Air flows through the cabinet work area in a downward direction at a uniform velocity. The air continues to be recirculated by the fan through the air flow plenum. Airborne biological contaminants are removed by the filters as the air is returned to the cabinet work area. Certification and advance planning are of prime importance to safe operation. Only qualified personnel using approved test methods and equipment should provide performance certification at initial installation, after maintenance, and on an annual basis thereafter. Certification is also necessary after the cabinet has been moved and after filters have been replaced. Many cabinets have gauges to indicate pressure differential
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across the supply filters. If the filters must be replaced, the cabinet MUST be decontaminated first. This is the responsibility of the researcher to do or have done by a qualified contractor. Procedures must follow those outlined in the National Sanitation Foundation Standard Number 49. After decontamination, only qualified Site Support personnel should replace filters. Fan speed must also be readjusted by qualified maintenance technicians. It is the responsibility of individual researchers and/or departments to insure this process is accomplished at least annually. In a survey performed by a cabinet manufacturer, 65 of 100 cabinets failed to pass filtration system leak tests. The operators of these cabinets were unaware of the malfunction. Maximum safety and full use of the cabinet can be best achieved by adequate advanced planning. Ideally, advanced planning should follow a procedural check list to anticipate equipment, apparatus, media, order of events and the many other details necessary for the completion of the assignment. When planning is completed, start-up procedures may be initiated. There are three start-up steps: 1 Turn on the lights 2 Check the air intake and exhaust grill to make sure they are unobstructed 3 Turn on the fan Allow the fan to operate a minimum of five minutes before manipulations are begun in the cabinet. In addition, the following points should be considered: 1 Some cabinets are equipped with ultraviolet light. These must be turned off during the day while laboratory personnel are occupying the room. 2 Hands and arms should be washed well with germicidal soap before and after work in the cabinet. 3 Technicians are encouraged to wear long-sleeve gowns with knit cuffs and rubber gloves. This minimizes the shedding of skin flora into the work area and protects the hands and arms from contamination by viable agents. 4 Interior surfaces of the work area should be disinfected by wiping them thoroughly with 70% alcohol. 5 The cabinets should not be overloaded. Everything needed for the complete procedure should be placed in the cabinet before starting
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6 7
10
11
12
13
14
15
so that nothing passes in or out through the air barrier until the procedure is completed. Do not place anything over the front intake or rear exhaust grill in units having a solid work surface. As a general rule, keep equipment at least four inches inside the cabinet window and perform transfer of viable materials as deeply into the cabinet as possible. After all materials have been placed in the cabinet, wait 2-3 minutes before beginning work. This will allow sufficient time for the cabinet air to purge airborne contamination from the work area. Hold the activity in the room to a minimum. Unnecessary activity may create disruptive air currents. The ideal location for a cabinet is in a quiet end of the laboratory, removed fromdoorways, air conditioning and heating vents. Opening and closing laboratory doors can cause disruptive drafts that allow microorganisms to penetrate the air barrier. Schedule uninterrupted work periods. The movement of objects including hands and arms causes turbulent air currents which disrupt the air barrier and allow escape and entrance of airborne contaminants. Air turbulence caused by rotating laboratory equipment, such as a small clinical centrifuge, disrupt airflow within the cabinet and at the work opening. This is sufficient for contaminated air to escape to the laboratory environment. If a centrifuge must be used in the cabinet, do not perform other research activities in the cabinet while the centrifuge is operating. Normal laboratory contamination control procedures and aseptic techniques are still necessary while working in the biological safety cabinet. Equipment in direct contact with the biological agent should not be removed from the cabinet until enclosed or until the surface is decontaminated. Trays of discarded pipettes and glassware must be covered before removal from the cabinets. If an accident occurs which spills or splatters the biological agent in the work area, all surfaces in the cabinet must be surface decontaminated before being removed. Do not use a Bunsen Burner in a biological safety cabinet. The flame causes turbulence in the air stream and the heat generated may damage the HEPA filter. If a procedure requires the use of a
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flame, a burner with a pilot light should be used. It should be placed to the rear of the workspace where resulting air turbulence will have a minimal effect. 16. Do not mouth pipette. Following completion of the work, the following steps must be performed: 1 Allow the cabinet to run 2-3 minutes with no activity. This will allow sufficient time for cabinet airflow to purge airborne contaminants from the work area; 2 Decontamination of the interior surfaces should be repeated after removal of all materials, cultures, apparatus, etc. A careful check of the work area should be made for spilled or splashed nutrients. They may support fungus growth and result in spore liberation that contaminates the protected work environment; and 3 Shut down by turning off the fan and lights. Use UV lights according to manufacturer's recommendations. 4. Do not use the cabinet to store excess laboratory equipment. bsc:faqs* * Adapted from Labconco Biological Safety Cabinet Training Program, Version 1.0, 3/91. "I've got to use a Bunsen Burner in my biohazard cabinet..." Using a Bunsen Burner in a biohazard cabinet compromises the performance of the unit and may be dangerous. During operation, the flame of a burner is very disruptive to the air flow patterns of the cabinet, and may actually increase the dispersion of aerosols in the work area. In addition, if the flame of the burner is too large, the excessive heat may melt in adhesive holding the HEPA filter together or literally burn holes in the filter media. (Yes, it does happen on a regular basis.) Finally, a Bunsen Burner in a biological safety cabinet is just plain dangerous. An unattended burner may blow out. If in a Type A or A/B3 cabinet, the recirculating gas may reach explosive concentrations (that has also happened on several occasions). Labconco recommends using alternative methods such as electric incinerators, or disposable inoculating hoops, for instance. The practice of flaming bottle mouths is unnecessary, as the work area of a Biohazard Cabinet should be a sterile environment, if used properly. "I can use a biological safety cabinet just as if it were a fume hood..."
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No. The biohazard cabinet and the chemical fume hood are two distinctly different pieces of equipment and MUST be used differently. The fume hood is designed to remove noxious or toxic fumes and aerosols away from the operator. It should be constructed of materials that are inert to a wide variety of chemical agents. The biohazard cabinet's primary purpose is to protect the operator, environment, and often the product from biohazardous contaminants. The biohazard cabinet and its HEPA filters are constructed of materials that are inert to the chemicals used in connection with biological research, but may be damaged by some of the more corrosive chemicals commonly used in fume hoods. Don't try to use a Biohazard Cabinet as a Fume Hood! "If I work in a biohazard cabinet, I don't have to be as careful with my technique..." Wrong. The biohazard cabinet will provide personal and product protection only if used properly. Aseptic technique must be practiced at all times while working in a biohazard cabinet. "I use the cabinet's UV light, so I don't need to decontaminate the work area..." Wrong. The UV light is only good as an adjunct, to minimize contamination of the work area when the cabinet is not in use. Ultraviolet light has virtually no penetrating power, and as such, will not kill microbes protected by dust, dirt, or organic material. The best method to prevent contamination in the cabinet is regular decontamination of the work area surfaces, before and after the cabinet is used. "Can I put a centrifuge in the biohazard cabinet?" Large objects placed in the biohazard cabinet will impede the airflow in the work area, reducing the efficiency of the cabinet. Electrical appliances like centrifuges, blenders, etc., will often disrupt the airflow around them due to their cooling fans. It is better to use a primary barrier on the appliance (such as a sealed safety cup in the centrifuge) rather than a biohazard cabinet to provide containment. "There's nothing wrong with using the biohazard cabinet to store material when not in use." Yes there is. Storing chemicals and materials in the biohazard cabinet make it more difficult to use when the need arises. If chemicals leak while stored in the cabinet, the work area of the cabinet could be damaged. Don't use the biohazard cabinet as a storage area.
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"All biohazard cabinets should operate continuously, 24 hours-a-day." Some applications of the biohazard cabinet require that the unit operate continuously. When used to prepare cytotoxic drugs, for example, the unit should operate continuously, to prevent toxic residue form migrating out of the cabinet ductwork and into the laboratory. If the cabinet is not used in such an application, there is no need to leave it operating continuously. This will only reduce the life of the cabinet blower and HEPA filters. "If I leave my Type A cabinet running continuously, it will clean all the air in the room to Class 100 conditions." Not necessarily. Assuming you had an air-tight room, with no ventilation system, an air-tight door seal, and no activity in it, then a recirculating Type A cabinet might clean the room to Class 100 levels. This would also unfortunately shorten the operating life of the motor and HEPA filters (and heat up the room considerably). Regardless, as soon as the operator opens the room door to enter, particulate-laden air will contaminate the room, raising it far above Class 100 conditions. "Is there any alternative to exhausting so much of costly tempered room air?" Yes. To ensure that the BSC is maintained at negative pressure with respect to the room, you must draw room air at least sufficient to sustain the face velocity at the specified level. This will work out to approximately 20 to 35 cfm per linear foot of the workspace width. If your cultures do not require any special temperature or RH control, then you may draw untempered air from any adjacent room or corridor through prefilters and make up your exhaust air volume. For example, if you are using a Type II B3 BSC, it is designed for 30% exhaust, which works out to throwing out 360 CFM. If you are able to sustain the face velocity with 160 cfm drawn from the room, you may make up the balance 200 cfm required from an adjacent room. Do not draw air from outside the building. If the room in which you work is to be maintained at negative pressure with respect to the outside environment, do not use the BSC exhaust arrangement as the only means of achieving that condition. The room should have its own separate exhaust system. Exhaust air from BSCs and Biosafe facilities should not be connected to the buildings general exhaust system. A separate dedicated exhaust should be used.
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Biosafe exhaust design should factor in such possibilities as power outage and fan failure. "Are the biological test methods different for different types of Class II BSCs?" Unfortunately, no. All four types of Class II BSCs are qualified using the same microbiological test method described earlier. The User is advised to devise more aggressive challenge methods, if deemed necessary, appropriate to the application. handling and disposal of waste A. Biohazard waste The following information regarding biohazard waste is being provided to eliminate any misunderstandings about the requirements for proper disposal of biohazard wastes. Biohazard Wastes are discarded materials "that are biological agents or conditions (as an infectious organism or unsecure laboratory condition) that constitutes a hazard to man or his environment." This definition includes "any and all substances which contain materials to which organisms may cause injury or disease to man or his environment, but which are not regulated as controlled industrial waste". B. Infectious wastes include the following categories: 1 2 3 4 5 6 7 cultures and stocks of infectious agents and associated biologicals; human blood and blood products, pathological wastes, contaminated sharps, contaminated animal carcasses, body parts, and bedding, wastes from surgery, necropsy and other medical procedures, laboratory wastes, 8 isolation wastes, unless determined to be non-infectious by the infection control committee, 9 any other material and contaminated equipment which, in the determination of the facility infection control staff, presents a significant danger of infection because it is contaminated with, or may reasonably be expected to be contaminated with, etiologic agents.
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C. Chemical wastes Chemical Wastes subject to the requirements of biohazard waste regulations include wastes from the following categories: 1 pharmaceutical wastes 2 laboratory reagents contaminated with infectious body fluids 3 all the disposable materials which have come into contact with cytotoxic/antineoplastic agents during the preparation, handling, and administration of such agents 4 other chemicals that may be contaminated by infectious agents, as designated by experts at the point of generation of the waste. D. Treated biohazard wastes Treated Biohazard Wastes are all biohazard wastes that have been treated by one of the following methods and rendered harmless and biologically inert: incineration in an approved incinerator, steam sterilization at sufficient time and temperature to destroy infectious agents in waste ("autoclaved"), chemical disinfection where contact time, concentration, and quantity of the chemical disinfectant are sufficient to destroy infectious agents in the waste, and any other method approved and generally recognized as effective.
E. Sharps Sharps are used in animal or human patient care or treatment or in medical research, or industrial laboratories, including: hypodermic needles, syringes, (with or without the attached needle) pasteur pipettes scalpel blades suture needles blood vials needles with attached tubing and culture dishes (regardless of presence of infectious agents). other types of broken or unbroken glassware that were in contact with infectious agents, such as used slides and cover slips.
The following guidelines should be followed for biohazard waste disposal:
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1. If any infectious waste is also a chemical waste, call for assistance with disposal AFTER disinfection. All waste of this type must be non-human, non-infectious, and non-viable. 2. Biohazard wastes that are also radioactive shall be treated according to requirements for both biohazard and radioactive waste. 3. Untreated biohazard waste shall NEVER be disposed of in the municipal solid waste stream. All laboratories shall evaluate their waste stream to ensure that all biohazard wastes, including sharps and syringes, are treated in a manner as described earlier before disposal in the municipal waste stream. 4. Prior to any treatment all biohazard wastes, including those to be incinerated, shall be enclosed in a puncture-proof, red BIOHAZARD BAG that is marked with the universal biological hazard symbol. 5. All sharps intended for disposal, whether contaminated or not, shall be enclosed in a sharps container. Recapping needles is dangerous and shall be avoided. Treat syringes as you would a controlled substance. It is recommended that all unwanted syringes be destroyed after disinfection but before disposal in the solid waste stream. Destroying an infectious sharp or syringe before disinfection could spread contamination. Special consideration should also be given to the disposal of contaminated pipettes. 6. After disinfection but before disposal in the municipal waste stream, all treated biohazard wastes shall be enclosed in an unmarked outer bag that is NOT red. Any biohazard waste that has been treated as described above, packaged such that it is clearly evident that the waste had been effectively treated AND contains no chemical or radioactive waste is NOT subject to regulation as biohazard waste and may be collected, transported, and disposed of as MUNICIPAL WASTE. F. Guidelines for disposal 1. If any infectious waste is also a chemical waste, call for assistance with disposal after disinfection. Antineoplastic/cytotoxic agents require special disposal. 2. Biomedical wastes that are also radioactive should be treated according to requirements for both biomedical and radioactive waste.
3. Prior to any treatment, all biomedical wastes, including those to be incinerated, should be enclosed in a puncture-resistant, red biohazard bag that is color-coded or labeled with the biological hazard symbol. 4. All sharps intended for disposal, whether contaminated or not, must be enclosed in a specially designed sharps container. Never clip or recap needles before putting them in the sharps container. The sharps container should be puncture-resistant, leak proof on the sides and bottom, and color-coded or labeled with the biohazard symbol. When selecting sharps containers, look for special safety features such as lids that lock tight for safe disposal, a container that can be sterilized by steam, gas, or chemicals, and a clear top that would allow inspection. If sharps containers are not specifically constructed to be autoclaved, the resulting mass of melted plastic is extremely hazardous due to the needles that often protrude. 5. Untreated biomedical waste is not to be disposed of in the municipal waste stream. All biomedical waste, including sharps and syringes, must be treated by incineration, steam sterilization, or chemical disinfection before disposal in the municipal waste stream. 6. After disinfection, but before disposal in the municipal waste stream, all treated biomedical wastes should be enclosed in an unmarked outer bag that is not red or labeled with the biohazard symbol. Any biomedical waste that has been treated as described above and packaged such that it is clearly evident that the waste has been effectively treated, is not subject to regulation as biomedical waste and may be collected, transported, and disposed of as municipal waste.

Biosafe Environs For Biohazardous Operations

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