Biosafe Environs for Biohazardous Operations

biosafe facilities, equipment and practices

biosafe environs for biohazardous operations

preamble

Every year about 90 million babies are born in the world. If these infantsare protected from infection and malnutrition in the first five years oftheir lives, they can hope to live to 70 and beyond. Vaccinating theinfant early enough does the job of protecting the child from fatalinfections such as diarrhoea, cholera, TB, mumps, measles, rubella andother viruses. Given these numbers, it is apparent that vaccines are notjust health aids but can also be money-spinners. Indeed, the revenuefrom vaccine sales across the world today amounts to $8 billion everyyear. And vaccines are not for children alone. Hepatitis, malaria, dengue,typhoid, flu, yellow fever and HIV affect all ages and effective vaccinesagainst these can earn much revenue.

adapted with kind permission from the CDC/NIH 4th edition ofBiosafety in Microbiological and Biomedical Laboratories

4 current good manufacturing practices: sterilisation & aseptic processing

A point to note in this connection is that keeping the water andenvironment clean aids in reducing (almost eliminating) many infectiveagents that cause life-threatening diseases. The developed world hastaken these steps while over 100 nations of the South across the globehave not. The wealthier nations have also cut their population to staticor even negative growth rates while the South grows at anywherebetween 2 to 4 per cent every year. A result of this is that revenues fromvaccines are not large in the developed world.

Like any investor in any other industry, for the average investor inpharmaceuticals cGMP translates to can you Guarantee My Profits?Since the threat to health and life in developed countries is not frominfection but from obesity, hypertension, diabetes, cancer and othersystemic diseases, major multinational pharma companies do not find itworth investing money in vaccine research, whereas they are desperateto invent blockbuster drugs against systemic diseases and ways to reversethe process of ageing.

This augurs well for India, which has already invested in research anddevelopment of vaccines, and a tradition of vaccine production that datesback to 100 years, to take the lead and become the vaccine supplier tothe globe in fighting infections diseases worldwide.

India is already among the largest producers of vaccine for human usein the world, perhaps the largest. And a wide variety of vaccines aremade here. Several factors have converged to make this possible.Adequate infrastructure and expertise have been put in place in differentinstitutions across India. The emergence of a new and dynamic biotechindustry with capability both in vaccinology and a keen sense of themarket 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 clinicaltrials. These are DPT, BCG, MMR, HBV, OPV, FMDV, Rabies vaccine,Leprosy vaccine - all of which are manufactured, and vaccines againstAnthrax, HPV, HIV, diarrhoea, Typhoid, IEV, malaria, cholera, rotavirus,HIB, meningitis, improved version of TB vaccine, dengue, Hepatitis Cvirus and DNA vaccine are in various stages of clinical trails. Many ofthese have been developed in house in India, and as collaborations withacademia and R&D centres in the country. Given the constraints anddirections of multinational major pharma, it is clear that small playersfrom developing nations can occupy a niche and gain profits throughlow cost-high volume sales of dozens of vaccines across the South.

5biosafe facilities, equipment and practices

However, mere making of vaccines the classical way is not enough. DNAvaccinology needs to be explored more rigorously. And focusing onantibodies is not enough. Methods must be found and formulated toactivate T-cells in order to improve the effectiveness of vaccines. Also,attention needs to be focussed on methods to beat or eliminate the coldchain, which requires the storage and transport of vaccines underrefrigeration. While DNA vaccines might not need the cold chain, evenclassical 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 biotechindustry can work together to make India the vaccine suppliers of theworld.

issues of concern

Despite the 100 years of our vaccine research, deveopment and productiontraditions, there is still, in many organisations, at both laboratory aswell as shop floor, an alarming sense of complacency, and apallingdisregard for fundamentals of biosafety, due either to incomplete orincorrect information, or to lack of comprehension about the dangersinvolved in working with pathogenic organisms. The reasons for thisvary:

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 causedisease

O The concentration needed to induce disease is several orders ofmagnitude higher than that which may be accidentally ingested

O The organism does not have any effect on adultsO The organism does not cause disease in humans

These notions, widespread as they are, are disturbing, and somethingthat should cause deep concern. For example, Bordetella pertussis, ahuman respiratory pathogen of worldwide distribution, is the causativeagent 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 theliterature. Adolescents and adults with atypical or undiagnosed diseasecan serve as reservoirs of infection and transmit the organism to infants


and children. Eight cases of infection with B.pertussis in adults havebeen documented at a large research institution. The individuals involveddid not work directly with the organism, but had access to commonlaboratory spaces where the organism was manipulated. One case ofsecondary transmission to a family member was documented. A similarincident occurred at a large Midwestern university resulting in twodocumented cases of laboratory-acquired infection and one documentedcase of secondary transmission. Other laboratory-acquired infections withB. pertuss is have been reported, as well as adult-to-adult transmissionin the workplace. Laboratory-acquired infections resulting from themanipulation of clinical specimens or isolates have not been reported.The attack rate of this airborne infection is influenced by intimacy andfrequency of exposure of susceptible individuals.

Genetic mutations, transgenic mutations and trans-species mutationsare all eminently plausible, and should not be discounted. It is wellrecognised that virus in attenuated vaccine for birds can, after fivesuccessive passages, can regain full virulence. Opportunistic pathogensand compromised hosts are ubiquitous, and no precaution is too muchin our endeavours to provide biosafe working environs.

Though guidelines for biological production have been reproducedelsewhere, the biosafety aspects of biohazardous operations are coveredhere, drawing heavily from the guidelines and recommendations ofCenter for Disease Control (CDC), USA; National Institute of Health(NIH), USA and a host of other International Agencies concerned withBiosafety in Medical and Biological Laboratories. As a consequence,much of the material here is directed towards biosafe laboratories; butthe same principles also apply for manufacturing.

The reader is also cautioned about the differences in perception of risksacross countries for the same organism. For example, Foot and MouthDisease (FMD) is classified at Risk Level 2 in India; but at Risk Level 5in Canada and elsewhere. Tuberculosis is routinely treated in India atLevel 2, while in this presentation where material has been borrowedfrom USA and Canada, the risk is indicated at Level 3. What wecommonly 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 infectiousdisease risks to persons in or near them.

Bacteria, viruses, fungi or other infectious agents are studied becausethey may cause disease, they can help us understand the natural world,and for many other reasons including the possibility of industrialapplications. Since many of the agents can be pathogenic to humans,animals or other forms of life, their use poses risks which vary witheach agent and the way it is used. Biotech laboratories, therefore, arespecial, often unique, work environments that may pose identifiableinfectious disease risks to persons in or near them.

Infections have been contracted in the laboratory and production areasthroughout the history of microbiology and immunology. As a result,safety norms, standards and practices have been designed and developedover the years to reduce to an acceptable level the risks inherent in theuse of dangerous materials. Stringent standards are set for hazardousagents and less stringent ones for agents which cause only minorproblems. Safety standards are therefore compromises designed to allowneeded work to proceed without exposing those involved or others tomore than minimal risk.

Besides the attitudes and actions of those who work in these hazardousenvirons determine their own safety, and that of their colleagues and ofthe community. Facility, equipment and design can contribute to safetyonly if they are used properly by people who are genuinely concernedand knowledgeable about safety issues.

definitions

Biohazard

An agent of biological origin that has the capacity to produce deleteriouseffects on humans, i.e. microorganisms, toxins and allergens derivedfrom those organisms; and allergens and toxins derived from higherplants and animals.

Biosafe facilities

Controlled Environments, known by the generic name of Bio Safety orProtection facilities are graded at Levels such as BSL 1/2/3/4 (wherebatch sizes exceed 10 litres of culture cells) or P1/2/3/4/5, dependingon 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 potentiallyinfectious microorganisms.

Chain of Infection

risk assessment

The assessment of risks associated with laboratory activities involvingthe use of infectious microorganisms is ultimately a subjective process.The risks associated with the agent, as well as with the activity to beconducted, must be considered in the assessment. The described riskassessment process is also applicable to laboratory operations other thanthose 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. Microbiologistsand other scientists working with such materials may, nevertheless, findthe practices, containment equipment, and facility recommendationsdescribed in this publication of value in developing operational standardsto 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 ofsuch factors as the severity of the disease it causes, the routes of infection,its virulence and infectivity. This judgement should take into accountthe existence of effective therapies (e.g. antibiotic resistance),immunization, the presence (or absence) of vectors, quantity of agentand whether the agent is indigenous to our country as well as possibleeffects on other species, including plants and animals. Emerging

Figure 1: Chain of infection


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 areclassified according to risk groups which are analogous to the levels ofcontainment described below. These classifications presume ordinarycircumstances in the research laboratory, or growth in small volumesfor diagnostic and experimental purposes.

The classifications of biological agents primarily reflect the judgementsmade on their inherent risks. Agents not listed should be classified onthe 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 oranimals.

Risk Group 2 (moderate individual risk, limited community risk)A pathogen that can cause human or animal disease but, under normalcircumstances, is unlikely to be a serious hazard to laboratory workers,the community, livestock, or the environment. Laboratory exposuresrarely cause infection leading to serious disease; effective treatment andpreventive 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, orwhich can result in serious economic consequences but does notordinarily spread by casual contact from one individual to another, orthat 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 individualto 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, conjugationand transformation have been used for many years to change biologicalspecies and organisms. These methods have recently been supplemented


by newer and much more efficient ones, of which the best known are thetechniques of recombinant DNA. This technology allows scientists totransfer genes between unrelated organisms and species, and has spawnedthe recent surge in biotechnology.

The initial fear of possible risks arising from organisms altered by thistechnology led Canada, the United States and Great Britain, among othercountries, to develop stringent biosafety guidelines. Experience rapidlyshowed that the initial fears were not justified. By 1980, many of thecontainment requirements of 1975-1977 had been removed.

Guidance in how to assess potential risks in recombinant DNA researchcan only be very general; each case needs individual assessment. It isnot realistic to try to define in advance all of the possible geneticallyengineered organisms which might be created or used in the laboratory.The vast majority of this research involves only the remotest possibilityof creating a hazard because the source of the DNA being transferred,the vector and the host are all innocuous. However, some geneticmanipulation does raise significant possibility of risk. In general, if noneof the components of the genetic manipulation presents any knownhazard, and none can be reasonably foreseen in their combination, thenno biohazard restrictions are needed. If one of the components of thereaction is hazardous, then, in general, discussion of the containmentlevel required should start at the level appropriate to the known hazard.Its containment level might be increased or decreased according to suchconsiderations as: the particular gene being transferred; the expressionof the gene in the recombinant organism; the biological containmentoffered by the host vector system; the envisaged interactions betweenthe gene being transferred and the host vector system; and other suchfactors. In any research with genes coding for hazardous products, hostvector systems of limited ability to survive outside the laboratory (i.e.offering biological containment) should be used; their use will reducethe level of containment required.

transgenic plants and animals

There is considerable potential for production of bioproducts in transgenicplants or animals. The potential release of transgenics into theenvironment and transmission of novel genes to other plants and animalsneed to be considered when designing both the production system andfacilities to contain the transgenics. In each case, the risk level needs tobe determined.


This section will focus on transgenic plants and animals that are usedfor production of bioproducts and the containment required for theseactivities.

In the case of transgenic animals, the first consideration is that they behandled according to the Guidelines set forth by various regulatingagencies. An important consideration is the ability of the animal totransmit genes by interbreeding with the same species or any relatedspecies. Under these conditions, it is important that the transgenics arewell-contained to prevent the spread of genetic modifications. It isrecommended that, if at all possible, transgenics be created usingmethodology which restricts the potential for transmission of the genesfrom one host to another.

Transgenic plants may transmit novel characteristics to other plants,thereby modifying the gene pool of existing species. Since thistransmission is mediated by pollen, transgenic plants should be madesterile or contained in a growth chamber or greenhouse designed toprevent pollen release either by air or insects. If plants are allowed tomature, care must be taken to contain the seeds in the green house orgrowth chamber.

If live microorganisms are used as vehicles for transfection, thecontainment level for the plants or animals inoculated with these viablerecombinant microorganisms must be at least as high as that requiredfor work with that specific microorganism. Transgenics (eg. producedby micro-injection, by use of replication defective vectors, or othersequences that are not horizontally transmitted) can be normally handledat Containment Level 1. The following recommendations should beconsidered prior to the initiation of transgenic studies:

a Complete copies of the genome or replication competent genomeshould not be used.

b The constructs should not contain genes capable of causingneoplastic transformation of animals.

c The probability of recombination with extraneous microorganismsshould be minimal or non- existent.

The biological hazards associated with the use of mammalian or othercells in culture fall into 3 categories:

a. Primary cultures of mammalian or other cells may harbourinfectious agents or integrated DNA originally present in the animalor person from which the cultures were derived. Whenever possible,


the donor should be tested for any suspect pathogens prior to thepreparation of the culture, and the culture should be considered to becontaminated until proven to be free of suspect agents. Such primarycultures should be handled in a manner appropriate to the risk class ofthe suspected contaminant, and precautions should be taken againstparenteral or other means of exposure of laboratory personnel.b. Cell lines known to contain infectious agents or integrated DNAshould 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 parenteralinoculation, normal immune response should provide protection,prevent progressive growth and cause rejection of accidentallytransplanted cells.

use of mammalian cells in culture

The biological hazards of mammalian cells arise from the possibilitythat they might contain or transmit infectious agents. It is prudent toconsider all cell lines to be potentially infectious. Cells known orsuspected to contain such agents, or primary cultures from animals andhumans known or reasonably suspected to be infected, should be in therisk group for the suspected agent. Primate cell lines derived fromlymphoid or tumor tissue, all cell lines exposed to or transformed by aprimate oncogenic virus, all samples of human tissues and fluids, allprimate tissue, all cell lines new to the laboratory (until proven to befree of adventitious agents), all virus-containing primate cell lines, andall mycoplasma-containing cell lines should be handled at containmentLevel 2.

risk levels associated with the use of laboratory animalsThe 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 agentmight persist in a latent non-infective form which can be reactivatedperiodically or as a result of certain stimuli. If the possibility that suchan agent may be excreted by an animal during the course of an experimentcannot be excluded, all those animals should be kept at a containmentlevel appropriate to the risk. Animals may also be deliberately inoculatedwith microorganisms in each of the four risk groups or with viablematerials (i.e. transformed cells) suspected of containing these organisms.Under these circumstances, the animal should be kept at the containmentlevel appropriate to the risk of the organism, recognizing that, in somecases, in vivo work may increase that risk.


In all situations, it is the responsibility of the scientist and the hostinstitution in consultation with the Government and the Animal Careauthorities, to determine the risk levels inherent in the proposed activity.

principles of biosafety

The term containment is used in describing safe methods for managinginfectious agents in the laboratory environment where they are beinghandled or maintained. The purpose of containment is to reduce oreliminate exposure of laboratory workers, other persons, and the outsideenvironment to potentially hazardous agents.

Primary containment, the protection of personnel and the immediatelaboratory environment from exposure to infectious agents, is providedby both good microbiological technique and the use of appropriate safetyequipment. The use of vaccines may provide an increased level of personalprotection.

Secondary containment, the protection of the environment external tothe laboratory from exposure to infectious materials, is provided by acombination of facility design and operational practices.

Therefore, the three elements of containment include laboratory practiceand technique, safety equipment, and facility design

laboratory practice and techniqueThe most important element of containment is strict adherence tostandard microbiological practices and techniques. Persons working withinfectious agents or potentially infected materials must be aware ofpotential hazards, and must be trained and proficient in the practicesand techniques required for handling such material safely. The directoror person in charge of the laboratory is responsible for providing orarranging for appropriate training of personnel.

Each laboratory should develop or adopt a biosafety or operations manualwhich identifies the hazards that will or may be encountered, and whichspecifies practices and procedures designed to minimize or eliminaterisks. Personnel should be advised of special hazards and should berequired to read and to follow the required practices and procedures. Ascientist trained and knowledgeable in appropriate laboratory techniques,safety procedures, and hazards associated with handling infectious agentsmust direct laboratory activities.


When standard laboratory practices are not sufficient to control the hazardassociated with a particular agent or laboratory procedure, additionalmeasures may be needed. The laboratory director is responsible forselecting additional safety practices, which must be in keeping with thehazard associated with the agent or procedure.

Laboratory personnel, safety practices, and techniques must besupplemented by appropriate facility design and engineering features,safety equipment, and management practices.

safety equipment ( primary barriers)Safety equipment includes biological safety cabinets (BSCs), enclosedcontainers, and other engineering controls designed to remove orminimize exposures to hazardous biological materials. The biologicalsafety cabinet (BSC) is the principal device used to provide containmentof infectious splashes or aerosols generated by many microbiologicalprocedures.

Safety equipment also may include items for personal protection suchas gloves, coats, gowns, shoe covers, boots, respirators, face shields,safety glasses, or goggles. Personal protective equipment is often usedin combination with biological safety cabinets and other devices whichcontain the agents or materials being worked with.

facility design (secondary barrier)The design of the facility is important in providing a barrier to protectpersons working inside and outside of the laboratory within the facility,and to protect persons in the community from infectious agents whichmay be accidentally released from the laboratory. Laboratory managementis responsible for providing facilities commensurate with the laboratorysfunction and the recommended biosafety level for the agents beingmanipulated.

The recommended secondary barrier is determined by the risk oftransmission of specific agents.

Secondary barriers in these laboratories will include separation of thelaboratory work area from public access, availability of a decontaminationfacility (e.g., autoclave), and hand washing facilities. Design featurescould include airconditioning, controlled access zones, airlocks atlaboratory entrances, or separate buildings or modules for isolation ofthe laboratory.


biosafety levels

Four biosafety levels (BSLs) are described which consist of combinationsof laboratory practices and techniques, safety equipment, and laboratoryfacilities. Each combination is specifically appropriate for the operationsperformed, the documented or suspected routes of transmission of theinfectious 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 whichthe agent can ordinarily be safely handled. The laboratory director isspecifically and primarily responsible for assessing risks and forappropriately applying the recommended biosafety levels. Generally,work with known agents should be conducted at the biosafety levelrecommended in Section VII. When specific information is available tosuggest that virulence, pathogenicity, antibiotic resistance patterns,vaccine and treatment availability, or other factors are significantlyaltered, more (or less) stringent practices may be specified.

biosafety level 1

Biosafety Level 1 practices, safety equipment, and facilities areappropriate for undergraduate and secondary educational training andteaching laboratories, and for other facilities in which work is donewith defined and characterized strains of viable microorganisms notknown to cause disease in healthy adult humans. Bacillus subtilis,Naegleria gruberi, and infectious canine hepatitis virus are representativeof those microorganisms meeting these criteria. Many agents notordinarily 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 strainswhich have undergone multiple in vivo passages should not be consideredavirulent simply because they are vaccine strains.

Biosafety Level 1 represents a basic level of containment that relies onstandard microbiological practices with no special primary or secondarybarriers recommended, other than a sink for handwashing.

biosafety level 2

Biosafety Level 2 practices, equipment, and facilities are applicable toclinical, diagnostic, teaching and other facilities in which work is donewith the broad spectrum of indigenous moderate-risk agents present inthe community and associated with human disease of varying severity.


With good microbiological techniques, these agents can be used safelyin activities conducted on the open bench, provided the potential forproducing splashes or aerosols is low. Biosafety Level 2 is appropriatewhen work is done with any human-derived blood, body fluids, or tissueswhere the presence of an infectious agent may be unknown. Primaryhazards to personnel working with these agents relate to accidentalpercutaneous or mucous membrane exposures, or ingestion of infectiousmaterials. Extreme precaution with contaminated needles or sharpinstruments must be emphasized. Even though organisms routinelymanipulated at BSL2 are not known to be transmissible by the aerosolroute, procedures with aerosol or high splash potential that may increasethe risk of such personnel exposure must be conducted in primarycontainment equipment, or devices such as a BSC or safety centrifugecups. Other primary barriers should be used as appropriate, such assplash shields, face protection, gowns, and gloves.

Secondary barriers such as hand washing and waste decontaminationfacilities must be available to reduce potential environmentalcontamination.

Biosafety Level 2 is the recommended level for work with bloodbornepathogens such as hepatitis B virus and HIV. The containment elementsdescribed in Biosafety Level 2 are consistent with the OccupationalExposure to Bloodborne Pathogens Standard from the OccupationalSafety and Health Administration (OSHA), that requires the use ofspecific precautions with all clinical specimens of blood or otherpotentially infectious material (Universal Precautions). Additionally,other recommendations specific for clinical laboratories may be obtainedfrom the National Committee for Clinical Laboratory Standards.Biosafety Level 2 recommendations and OSHA requirements focus onthe prevention of percutaneous and mucous membrane exposures toclinical material.

biosafety level 3

Biosafety Level 3 practices, safety equipment, and facilities are applicableto clinical, diagnostic, teaching, research, or production facilities inwhich work is done with indigenous or exotic agents with a potentialfor respiratory transmission, and which may cause serious and potentiallylethal infection. Mycobacterium tuberculosis, St. Louis encephalitis virus,and Coxiella burnetii are representative of microorganisms assigned tothis level. Primary hazards to personnel working with these agents relateto autoinoculation, ingestion, and exposure to infectious aerosols.


At Biosafety Level 3, more emphasis is placed on primary and secondarybarriers to protect personnel in contiguous areas, the community, andthe environment from exposure to potentially infectious aerosols. Forexample, all laboratory manipulations should be performed in a BSC orother enclosed equipment, such as a gas-tight aerosol generation chamber.Secondary barriers for this level include controlled access to thelaboratory and a specialized ventilation system that minimizes the releaseof infectious aerosols from the laboratory.

biosafety level 4

Biosafety Level 4 practices, safety equipment, and facilities are applicablefor work with dangerous and exotic agents which pose a high individualrisk of life-threatening disease, which may be transmitted via the aerosolroute, and for which there is no available vaccine or therapy. Additionally,agents with a close or identical antigenic relationship to Biosafety Level4 agents should also be handled at this level. When sufficient data areobtained, work with these agents may continue at this level or at a lowerlevel. Viruses such as Marburg or Congo-Crimean hemorrhagic feverare manipulated at Biosafety Level 4.

The primary hazards to personnel working with Biosafety Level 4 agentsare respiratory exposure to infectious aerosols, mucous membraneexposure to infectious droplets, and autoinoculation. All manipulationsof potentially infectious diagnostic materials, isolates, and naturally orexperimentally infected animals pose a high risk of exposure andinfection to laboratory personnel, the community, and the environment.

The laboratory worker's complete isolation of aerosolized infectiousmaterials is accomplished primarily by working in a Class III BSC or afull-body, air-supplied positive-pressure personnel suit. The Biosafety Level4 facility itself is generally a separate building or completely isolatedzone with complex, specialized ventilation and waste managementsystems to prevent release of viable agents to the environment.


Figure 2: A simple isolation facility

Figure 3: A typical BSL level 3 facility


Figure 4: Steam barrier used in biological production

Figure 5: A typical facility with pressure profile for biotech


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Separated from public areas by door ...................... M M M MLaboratory doors labeled with biohazard signs ..... N M M MAccess limited to authorized personnel ................. N R M MIn separate sealed room(s) with restricted accessaway from public thoroughfares ............................. N N M MIn separate building or sealed room withindependent air supply and exhaust -restricted access ...................................................... N N R MContainment labs located away from outsidebuilding envelope walls .......................................... N R R MContainment labs located adjacent to or nearbymechanical rooms to minimizelengths of containment ducts ................................. N N R MOffice areas can be located within lab if next toaccess or egress door. .............................................. Y Y N NOffice areas must be outside laboratorycontainment zone .................................................... N N M MFacility must be kept locked when not in use(consistent with local fire and safety regulations) . N N M M

Location - Containment Perimeter

A. WallsReinforced structural masonry ............................... N N R MReinforced non-load-bearing masonry ................... N N R MSteel frame reinforced non-load-bearing masonry N N R MReinforced concrete ................................................ N N R M

Key: M = Mandatory R = Recommended Y = Yes N = No/Not applicable

Table 1: Facility design checklistLocation - General Containment Level

1 2 3 4


B. CeilingsSteel frame gypsum partition or imperviousceiling acoustic tile ................................................. Y Y N NReinforced steel frame and gypsum ceiling, fillerprimer and paint finish ........................................... N N M MC. Coatings and sealantsSeamless, gas- and chemical-resistant wall andceiling coatings ....................................................... N R M MChemical- and gas-resistant (disinfectant), non-hardening sealants .................................................. N R M MContainment seals for mechanical/electricalservice openings ...................................................... N R M MD. DoorsDoors lockable ........................................................ N R M MDoors self-closing ................................................... N R M MDoors to provide restricted access via keycardsystem or equivalent ............................................... N N R MVentilated airlock required for the separation ofhigher and lower containment areas with inter-locking pneumatic or compressible sealed doors .. N N N MDoors and frames of solid finish construction ....... N R M MDoor openings should be of sizes to allow passageof all anticipated equipment ................................... M M M MDoors to have fire ratings as required and belocated as per fire safety standards ........................ M M M MEntrance doors interlocked with manual overrides N N R MAll exits marked and illuminated .......................... M M M MEgress to fire exits set out so that travel throughany high-hazard areas is minimized or to conformto applicable codes .................................................. M M M MKey: M = Mandatory R = Recommended Y = Yes N = No/Not applicable

Table 1: Facility design checklist (contd)Location - Containment Perimeter Containment Level

1 2 3 4


E. WindowsWindows, if openable, protected by fly screens ..... Y Y N NWindows of safety glass, of proven performance,solid stops sealed in place ...................................... N N M MF. FloorsSlip-resistant flooring ............................................. M M M MSeamless, gas- and chemical-resistant (e.g. epoxy)coating with integral cove base .............................. R R M MSeamless, rolled or resilient tile flooring (eg.vinyl) M M N NAir Handling System

A. Room Air SupplyAir supply independent from adjoining laboratoryzones ........................................................................ N N R M

Air supply HEPA-filtered or provided with bubbletight dampers .......................................................... N N R MEquipped with pressure maintaining gauges atentry (e.g. magnehelics) ......................................... N N R MDirectional inward, non-recirculated airflow ........ N R M MInterlocked with exhaust ventilation to preventpositive pressurization ............................................ N N M MEquipped with audible alarms to detectdepressurization (i.e. failure of the exhaust system) N N R MAir supply ductwork sealed airtight andindependent from other laboratory zones andaccessible from outside the containment zone ....... N N R MEquipped with bubble tight damper to permitsealing for decontamination procedures ................ N N M MKey: M = Mandatory R = Recommended Y = Yes N = No/Not applicable

Table 1: Facility design checklist (contd)Location - Containment Perimeter Containment Level

1 2 3 4



B. Room Exhaust VentilationRoom equipped with pressure monitoring devicesat entry .................................................................... N N R MSealed airtight independent-exhaust ductwork andfan system accessible fromoutside the containment zone which meetsperformance and verification testing requirement N N R MAll exhaust ventilation HEPA-filtered andconnected to audible alarm to detect failure ofexhaust system ........................................................ N N R MInterlocked with air supply to prevent positivepressurization of the laboratory .............................. N R M MEquipped with bubble tight damper to permitsealing for decontamination ................................... N R M MExhaust from laboratory at a minimum of 10 roomvolumes/ hour ......................................................... N R M MAir vertically discharged to the outside, clear ofbuildings or supply air intakes at 12 mps .............. R R R RRecirculated HEPA-filtered room air permitted ... R M N NMinimization of dead spaces where contaminatedair can accumulate .................................................. R R M MVentilation sufficient to remove vapours offlammable liquids and dangerous chemicals beforethey reach hazardous concentrations ..................... M M M MExposed ductwork to stand clear of walls to allowaccess for maintenance, leak testing and access toequipment filters and lighting ................................ N N M M

Table 1: Facility design checklist (contd)Air Handling System Containment Level

1 2 3 4



C. Biological Safety CabinetsClass I ...................................................................... N R M NClass II .................................................................... N R M MClass III ................................................................... N N N MClass I and II cabinets permitted with use ofpositive-pressure suits ............................................. N N N MCabinet air can be recirculated in laboratory ifHEPA-filtered .......................................................... N M M N

D. Fume Hoods

Recommended when necessary .............................. R R R RHEPA and charcoal filters (if required) ................. N N R MAir flow alarms ....................................................... R R R R

Decontamination & Waste Disposal

A. Decontamination

Laboratory floors, walls and ceilings to be treatedwith disinfectant-resistant, cleanable coatings ...... R R M MAll laboratory furnishings and surface materialsdisinfectant-resistant and cleanable.Bench tops with coved splash backs, seamless, orwith unions sealed with non-shrinking sealant ..... R R M MSurfaces of plastic laminate .................................... R R M MLaboratory perimeter sealed to allow gaseousdecontamination of whole room ............................. N N M M

Table 1: Facility design checklist (contd)Air Handling System Containment Level

1 2 3 4



B. SterilizationInterlocking double-door pass-through autoclavein laboratory. ........................................................... N N R MAutoclave in laboratory .......................................... N N M MAutoclave in building ............................................. R M M MExposed steam pipes covered with insulatingmaterial ................................................................... M M M MIncinerator in building ........................................... N N N RC. Waste DisposalLiquidsDrainage traps filled with disinfectant specified bylaboratory operator .................................................. N R M MAll liquid effluents must be sterilized inmechanically and biologically monitored tankslocated adjacent to the containment area prior todisposal .................................................................... N N N MSolidsProvide space for support stands for biomedicalwaste bags ............................................................... M M M MProvide refrigerated space for lockable, closedstorage for biomedical wastewhich will be disposed of off site ........................... R R R MProvide disinfectant dunk tanks for all non-autoclavable materials and equipment exiting thecontainment area as a part of the cabinet line. Inthe case of a suited lab, this function may beprovided by a dunk tank or via the chemicalshower at the containment perimeter ..................... N N R M

Table 1: Facility design checklist (contd)Decontamination & Waste Disposal Containment Level

1 2 3 4



FacilitiesHandwashing facilities in laboratory ..................... M M M MDedicated handwashing facilities with foot, kneeor automatic controls in laboratory (not applicablefor positive-pressure-suit mode) ............................. N R M MChemical deluge shower at the laboratory perimeter(for positive-pressure-suit mode) .............................. N N N MEye/face wash facilities equipped with in-useaudio/visual alarm (not applicable for positivepressure suit mode) ................................................. R R R MBody shower in containment area .......................... N N R MClothing change area adjacent to containmentarea (0.5 m2 per person) .......................................... N N M MProvide storage space for laboratory clothing inlab or adjacent change area (minimum300 linear mm. for each peg) ................................. M M M MProvide space adjacent to exit door for laundryhamper (minimum 0.900m2) for used laboratoryclothing to be autoclaved prior to laundering ........ R R M MA. Plumbing And DrainageAll drains connected to sterilization system .......... N N N MAll drains connected directly to sanitary sewer ..... N N R NAutoclave chamber condensate directed tosewer through a closed system ............................... N N R MAll piping penetrations sealed with non-shrinkingsealant at laboratory perimeter ............................... N N M MAll supply water services fitted with backflowpreventers ................................................................ N N M M

Table 1: Facility design checklist (contd)Personal Hygiene & Safety Containment Level

1 2 3 4



Piping to be exposed and stand clear of wallswithin high containment area to allow access for maintenance........................................................ N N M MMain water supply control to be located outsidelaboratory perimeter ............................................... N N R MAll exposed hot and cold water pipes are to becovered with insulating material and protectedfrom movement ....................................................... M M M MAll vent lines equipped with HEPA filters orequivalent ................................................................ N N R MB. Compressed GasesAir supply lines HEPA filtered or equivalent asbackflow protection ................................................ N R M MAll gas lines to have backflow preventers ............. N N M MAll vacuum lines HEPA-filtered or equivalent *. .. N R M MNo vacuum lines should lead from facility (needsto be met by pumps inside laboratory) ................... N N M MAll supply line penetrations sealed withnon-shrinking sealant at laboratory perimeter ...... N N M MCompressed gas cylinder storage outsidelaboratory ................................................................ N N R M* Vacuum lines must not leave ContainmentLevels 3 or 4C. ElectricalAll supply conduit and wiring to be sealed at thecontainment barrier with non-shrinking sealant ... N N R MFluorescent light ballasts and starters to be locatedoutside containment area ........................................ N N R R


1 2 3 4



Circuit breakers located outside biocontainmentarea .......................................................................... N N R MBuilding security systems integrated withlaboratory safety and monitoring systems ............. R R R M70 ft.-candles of light at work surface level(metric) minimum maintained at work surface ..... R R R RAll circuit-breaker switches, panels and controlsto be appropriately labeled ..................................... M M M MElectrical system is to be equipped with standbygenerator for emergency support of essentialequipment, which includes biological safetycabinets .................................................................... N R R MLaboratory to be equipped with fire alarm system M M M MLab to be equipped with a communication systembetween containment area and outside support area N N R MClosed-circuit TV monitoring of entire work area N N N R

Emergency & Monitoring Provisions

A. Air HandlingDirectional inward, non-recirculated airflow,monitored at entrance to lab ................................... N R M MBiological safety cabinets equipped with pressuremonitoring gauges for all HEPA filters .................. N R M MProvision of access for decontamination ofHEPA filters ............................................................ M M M MEntry to laboratory via ventilated air-lock withinterlocking doors ................................................... N N R NEntry to laboratory via sealed air-lock ................... N N N M


1 2 3 4



Rooms in isolation area to be maintained atpressure negative to corridor with greatestnegative pressure in most hazardous room ............ N N M MB. Fire Prevention And ContainmentEquipped with fire alarms ...................................... M M M MEquipped with appropriate fire extinguishers ....... M M M MDoors to have appropriate fire ratings ................... M M M MAll fire exits marked and illuminated .................... M M M MBiocontainment area designated as burn out.Firemen to enter only to save life, not toextinguish fire, which should be controlled fromoutside, to prevent spread ....................................... N N N REquipped with suitable storage cabinets orexplosion-proof refrigerators which are clearlylabeled for flammable liquids ................................. M M M MStorage for flammable liquids to be locatedoutside biocontainment area ................................... R R R RSprinkler systems (where required by law) to bepreactioned type; other fire suppression systemsmay be considered for level 4 ................................. R R R RC. Personal Emergency EquipmentEquipped with bottled back-up breathing airsufficient to provide 30 minutes per person.(Level 4 suit lab only) ............................................. N N N MEquipped with positive-pressure hood respiratorswith compressed breathing air cylinders locatedin support area ........................................................ N N R MEye/face wash facilities in laboratory(not applicable for positive-pressure-suit mode,Level 4) ................................................................... M M M M

Table 1: Facility design checklist (contd)Emergency & Monitoring Provisions Containment Level

1 2 3 4



Body shower in support area .................................. R R R MEquipped with communication system betweencontainment zone and support area ....................... N N R MEmergency lighting ................................................ M M M MD. Backup ServiceEquipped with standby generator for support ofessential equipment, including biologicalsafety cabinets ......................................................... N N R M

Performance Verification & Testing

Construction of laboratory perimeter to beleakproof and able to withstand loadingcharacteristics imposed by negative air pressurerequired in laboratory operation: ...........................a) Integrity of seals demonstrated by visualinspection ................................................................ N N M Nb) Integrity of room tightness demonstrated byphysical testing (pressure decay 0.05 wg loss/min)at 2"wg .................................................................... N N N MAll air supply and exhaust ductwork tested in situto 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) N N R MAll air supply and exhaust ductwork verified to have back-draft protection ................................... N N M MAll HEPA filters tested to meet requiredspecification after installation ................................ N N M MAll HEPA-filter housings tested to be leak tight:not > 0.2% of volume per min at 10"wg. (2500Pa) N N R M

Table 1: Facility design checklist (contd)Emergency & Monitoring Provisions Containment Level

1 2 3 4



Testing of biological safety cabinets meetsrequired specifications after installation ................ N M M MTesting of autoclaves to meet specified standardsafter installation by the use of biological indicators M M M MTesting of fume hoods according toCSA Standard Z316.5-94 ....................................... M M M MDrainage and liquid-waste-disposal systemsincluding sampling ports tested to ensure efficacyby use of biological indicators ................................ N N N MVerification of integrity of sewage lines ................ N N N MVerification of alarm systems for air systemsfailure (exhaust, supply, room pressure,breathing air) .......................................................... N N M MVerification of alarm systems for electrical failure N M M MVerification of fire alarm systems .......................... M M M MVerification of communication systems ................. N N R MTesting of directional airflow demonstrated byfield tests with visual smoke .................................. N R M MVerification of integrity positive pressure suits ..... N N N MTesting of breathing air as perCSA Standard Z180.1-M85 .................................... N N N MTesting of regular and emergency air system ........ N N N M

Table 1: Facility design checklist (contd)Performance Verification & Testing Containment Level

1 2 3 4

Tabl

e 2:

Su

mm

ary

of b

iosa

fety

(lev

els 1-

3) fo

r inf

ectio

us ag

ents

BSL

Age

nts

Prac

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Safe

ty E

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icro

biol

ogic

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one

requ

ired

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Prac

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plu

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embr

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arn

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posu

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min

atio

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all

Bio

safe

ty m

anu

alm

ater

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;w

aste

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labo

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glo

ves;

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nee

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xotic

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equ

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line

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From the earliest laboratory-acquired typhoid infections to the hazardsposed by todays antibiotic-resistant bacteria and rapidly-mutatingviruses, threats to worker safety have stimulated the development andrefinement of cabinets in which infectious microorganisms could behandled safely. Work with tissue cultures, the need to maintain sterilityof cell lines, and efforts to minimize cross-contamination contributedto concerns regarding product integrity.

The use of proper procedures and equipment cannot be overemphasizedin providing primary personnel and environmental protection. Forexample, high-speed blenders designed to reduce aerosol generation,needle-locking syringes, microburners, and safety centrifuge cups orsealed rotors are among the engineering devices that protect thelaboratorian. However, the most essential piece of containment

selection, use, testing and maintenance ofbiological safety cabinets

adapted from the CDC/NIH 2nd edition ofSelection and use of Biological Safety Cabinets


equipment is the biological safety cabinet in which manipulations ofmicroorganisms are performed.

This section presents information on the selection, function and use ofbiological safety cabinets (BSCs), which are the primary means ofcontainment developed for working safely with infectiousmicroorganisms. Brief descriptions of the facility and engineeringconcepts for the conduct of microbiological research are also provided.BSCs are only one part of an overall biosafety program which requiresconsistent use of good microbiological practices.

BSCs are designed to provide personnel, environmental and productprotection when appropriate practices and procedures are followed. Threekinds of biological safety cabinets, designated as Class I, II and III havebeen 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 andperformance of these critical primary barriers. This has been written forthe laboratorian, engineer or manager who desires a better understandingof each type of cabinet and the rationale for selecting the most appropriateBSC to meet specific operational needs.

class I BSC

The Class I BSC provides personnel and environmental protection, butno product protection. It is similar in air movement to a chemical fumehood, but has a HEPA filter in the exhaust system to protect theenvironment.

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 aminimum velocity of 75 linear feet per minute (lfpm) is maintainedthrough the front opening. With the product protection provided by theClass 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), orprocedures (e.g. cage dumping, aerating cultures or homogenizingtissues) with a potential to generate aerosols.The Class I BSC is hard-ducted to the building exhaust system, and thebuilding exhaust fan provides the negative pressure necessary to drawroom air into the cabinet. Cabinet air is drawn through a HEPA filter as


it enters the exhaust plenum. A second HEPA filter may be installed atthe terminal end of the exhaust.

Some Class I BSCs are equipped with an integral exhaust blower; thecabinet blower must be interlocked with the building exhaust fan. In theevent that the building exhaust fan fails, the cabinet exhaust blowermust turn off so that the exhaust ducts are not pressurized. Filters shouldbe installed on the intake side of the fan. Also note that use of two filtersincreases the static pressure on the fan. If the ducts are pressurized andthe HEPA filter develops a leak, contaminated air could be dischargedinto other parts of the building or the environment.

A steel panel with arm holes to allow access to the work surface can beadded to the Class I cabinet. The restricted opening results in increasedinward air velocity, thereby increasing worker protection. For addedsafety, arm-length gloves can be attached to the paned. Makeup air isthen drawn through an auxiliary air supply opening (which may containa filter) and/or around a loose-fitting front panel.

class II BSC

The Class II (Types A, Bl, B2, and B3) biological safety cabinets providepersonnel, environmental and product protection. Airflow is drawnaround the operator into the front grille of the cabinet, which providespersonnel protection. In addition, the downward laminar flow of HEPA-filtered air provides product protection by minimizing the chance ofcross-contamination along the work surface of the cabinet. Becausecabinet air has passed through the exhaust HEPA filter, it is contaminant-free (environmental protection), and may be recirculated back into thelaboratory (Type A BSC) or ducted out of the building (Type B BSC).

Figure 1: Class I BSC


HEPA filters are effective at trapping particulates and infectious agents,but not at capturing volatile chemicals or gases. Only BSCs that areducted to the outside should be used when working with volatile toxicchemicals.

All Class II cabinets are designed for work involving microorganismsassigned to biosafety levels 1, 2 and 3. Class II cabinets provide themicrobe-free work environment necessary for cell culture propagation,and also may be used for the formulation of nonvolatile antineoplasticor chemotherapeutic drugs.

class II, type A BSC

An internal blower draws sufficient room air through the front grille tomaintain a minimum calculated or measured average inflow velocity ofat least 75 lfpm at the face opening of the cabinet. The supply air flowsthrough a HEPA filter and provides particulate-free air to the worksurface. Laminar airflow reduces turbulence in the work zone andminimizes 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 tothe rear grille. Although there are variations among different cabinets,

this split generally occurs about half-way between the front and reargrilles, and two to six inches above the work surface.

Figure 2: Class II type A BSC


The air is then discharged through the rear plenum into the space betweenthe supply and exhaust filters located at the top of the cabinet. Due tothe relative size of these two filters, approximately 30% of the air passesthrough the exhaust HEPA filter and 70% recirculates through the supplyHEPA filter back into the work zone. Most Class II, Type A cabinetshave dampers to modulate this 30/70 division of airflow.

An unducted Class II Type A BSC is not to be used for work involvingvolatile or toxic chemicals. The buildup of chemical vapors in the cabinet(by recirculated air) and in the laboratory (from exhaust air) could createhealth 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 ofthe cabinet exhaust system, thereby disturbing the internal cabinet airflow. 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) aroundthe 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 ofroom air into the space between the thimble unit and the filter housing(contact manufacturers for any additional specifications). The thimblemust be removable or be designed to allow for operational testing of thecabinet. The performance of a cabinet with this exhaust configuration isunaffected by fluctuations in the building exhaust system.

Hard-ducting (i.e., direct connection) of Class II Type A cabinets tothe building exhaust system is not recommended. The building exhaustsystem must be precisely matched to the airflow from the cabinet inboth volume and static pressure. However, fluctuations in air volumeand pressure that are common to all building exhaust systems make it


difficult, if not impossible, to match the airflow requirements of thecabinet.

class II, type B1 BSC

Some biomedical research requires the use of small quantities of certainhazardous chemicals, such as carcinogens. The powdered form of thesecarcinogens should be weighed or manipulated in a chemical fume hoodor a static-air glove box. Carcinogens used in cell culture or microbialsystems require both biological and chemical containment.

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 thesehazardous chemicals with in vitro biological systems.

The National Sanitation Foundation (NSF) Standard 49 definition ofType Bl cabinets includes this classic NCI design Type B, as well ascabinets without supply HEPA filters located immediately below the worksurface, and/or those with exhaust/recirculation downflow splits otherthan 70/30%.

The cabinet supply blowers draw room air (plus a portion of the cabinetsrecirculated air) through the front grille and then through the supplyHEPA filters located immediately below the work surface. Thisparticulate-free air flows upward through a plenum at each side of the

Figure 4: Class II type B1 BSC - floor-standing version


cabinet and then downward to the work area through a back-pressureplate. In some cabinets there is an additional supply HEPA filter to removeparticulates 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 minimuminflow velocity of 100 lfpm. As with the Type A cabinet, there is a splitin the down-flowing air stream just above the work surface. In the TypeB cabinet, approximately 70 percent of the downflow air exits throughthe rear grille, passes through the exhaust HEPA filter, and is dischargedfrom the building. The remaining 30 percent of the downflow air isdrawn through the front grille. Since the air which flows to the reargrille is discharged into the exhaust system, activities that may generatehazardous chemical vapors or particulates should be conducted towardsthe rear of the cabinet.

Type Bl cabinets must be hard-ducted, preferably to their own dedicatedexhaust system, or to a properly-designed laboratory building exhaust.As indicated earlier, blowers on laboratory exhaust systems should belocated at the terminal end of the duct work. A failure in the buildingexhaust system may not be apparent to the user, as the supply blowers inthe cabinet will continue to operate. A pressure-independent monitorshould be installed to sound an alarm and shut off the BSC supply fan,should failure in exhaust airflow occur. Since this feature is not suppliedby all cabinet manufacturers, it is prudent to install a sensor in the exhaustsystem as necessary. To maintain critical operations, laboratories using


Type B BSCs should connect the exhaust blower to the emergency powersupply.


This BSC is a total-exhaust cabinet; no air is recirculated within it. Thiscabinet provides simultaneous primary biological and chemicalcontainment. The supply blower draws in room air or outside air at thetop of the cabinet, passes it through a HEPA filter and down into thework area of the cabinet. The building or cabinet exhaust system drawsair through both the rear and front grilles, capturing the supply air plusthe additional amount of room air needed to produce a minimumcalculated or measured inflow face velocity of 100 lfpm. All air enteringthis cabinet is exhausted, and passes through a HEPA filter (and perhapssome other air-cleaning device such as a carbon filter) prior to dischargeto the outside. Exhausting as much as 1200 cubic feet per minute ofconditioned room air makes this cabinet expensive to operate.

Figure 6: Class II type B2 BSC


Should the building or cabinet exhaust fail, the cabinet will bepressurized, resulting in a flow of air from the work area back into thelaboratory. Cabinets built since the early 1980s usually have an interlocksystem installed by the manufacturer to prevent the supply blower fromoperating whenever the exhaust flow is insufficient. Presence of such aninterlock system should be verified; systems can be retrofitted if necessary.Exhaust air movement should be monitored by a pressure-independentdevice.


This biological safety cabine is a ducted Type A cabinet having aminimum inward airflow of 100 lfpm. All positive pressure contaminatedplenums within the cabinet are surrounded by a negative air pressureplenum. Thus, leakage in a contaminated plenum will be into the cabinetand not into the environment.

Special applications - Class II BSCs can be modified to accommodatespecial tasks. For example, the front sash can be modified by themanufacturer to accommodate the eye pieces of a microscope, or thework surface can be designed to accept a carboy, a centrifuge, or otherequipment that requires containment. A rigid plate with arm holes canbe added if needed. Good cabinet design, microbiological aerosol tracertesting of the modification, and appropriate certification are required toensure that the basic systems operate properly after modification.Maximum containment potential is achieved only through strictadherence to proper practices and procedures.

Figure 7: Class II type B3 BSC - table-top version


BSC

Face

Airfl

ow P

atte

rnAp

plic

atio

ns Cl

ass

Velo

city

Nonv

olat

ile To

xic

Vola

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m)

Chem

ical

s and

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Radi

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lides

Radi

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lides

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t fron

t; exh

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the

outs

ide

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to th

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omYe

sYe

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thro

ugh H

EPA

IIA75

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recir

cula

ted t

o the

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net w

ork a

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bala

nce 3

0% ex

haus

ted

Yes

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roug

h HEP

A ba

ck in

to th

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om o

r out

side

thro

ugh a

thim

ble un

itIIB

110

0Ex

haus

t cab

inet

air m

ust p

ass t

hrou

gh a

dedic

ated

duct

to th

e ou

tsid

eYe

sYe

s (m

inute

amou

nts)2

thro

ugh a

HEP

A filt

erIIB

210

0No

recir

cula

tion;

tota

l exh

aust

to th

e ou

tsid

e thr

ough

hard

-duc

t and

aYe

sYe

s (sm

all am

ounts

)HE

PA filt

erIIB

310

0Sa

me a

s II, A

, but

plen

ums a

re un

der n

egat

ive pr

essu

re to

room

;Ye

sYe

s (m

inute

amou

nts)2

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ir is

thim

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the

outs

ide

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HEP

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erIII

NASu

pply

air in

lets

and h

ard-

duct

exha

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d to

outs

ide t

hrou

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oYe

sYe

s (sm

all am

ounts

)HE

PA filt

ers i

n se

ries

(1)In

stal

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ay re

quire

a sp

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duct

to th

e out

side,

an in

-line c

harc

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nd a

spar

k pro

of (e

xplos

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oof) m

otor a

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her

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l com

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in th

e ca

bine

t. Disc

harg

e of a

Clas

s I ca

binet

in to

a ro

om sh

ould

not o

ccur

if vo

latile

chem

icals

are u

sed.

(2)In

no

circu

mst

ance

s sh

ould

the c

hem

ical c

once

ntra

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ppro

ach t

he lo

wer e

xplos

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its o

f the

com

poun

d.

Tabl

e 1:

Com

para

tive

spec

ifica

tions

for

bio

logi

cal s

afet

y ca

bine

ts


class III BSC

The Class III biological safety cabinet was designed for work withbiosafety level 4 microbiological agents, and provides maximum

Figure 8: Class III BSC

protection to the environment and the worker. It is a gas-tight enclosurewith a non-opening view window. Access for passage of materials intothe cabinet is through a dunk tank (that is accessible through the cabinetfloor) or double-door pass-through box (such as an autoclave) that canbe decontaminated between uses. Reversing that process allows for saferemoval of materials from the Class III biosafety cabinet. Both supplyand exhaust air are HEPA filtered. Exhaust air must pass through twoHEPA filters, or a HEPA filter and an air incinerator, before dischargeto the outdoors. Airflow is maintained by a dedicated independent exhaustsystem exterior to the cabinet, which keeps the cabinet under negativepressure (usually about 0.5 inches of water pressure).Long, heavy-duty rubber gloves are attached in a gas-tight manner toports in the cabinet and allow for manipulation of the materials isolatedinside. Although these gloves restrict movement, they prevent the usersdirect contact with the hazardous materials. The trade-off is clearly onthe side of maximizing personal safety. Depending on the design of thecabinet, the supply HEPA filter provides particulate-free, albeit somewhatturbulent, airflow within the work environment.

Several Class III cabinets can be joined together in a line to provide alarger work area. Such cabinet lines are custom-built; the equipmentinstalled 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, ClassIII cabinets are usually only installed in maximum containment

46 current good manufacturing practices: sterilisation & aseptic processingCa

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Biosafe Environs for Biohazardous Operations

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