in the Chemical f aboratory
...a
Edited by N O R M A N V. STEERE, 140 Melbourne Ave., S.E. Minneapolis, Minn. 5541 4
feature
CXXIX. Biological Safety Cabinets for Contamination Control C. L. Baldwln and P. H. Errico Dow Bio-Engineering Services 3 Choke Cherry Road Rockville, Maryland 20850
Figure 1.
Laboratory Infections The need for safety equipment and precautionary measures in the biomedical laborator" has been confirmed in a series of ratory-acquired infections. A recent summary of their reports (4) indicates nearly 3500 overt laboratory infections, 4.6% of which terminated in death. These authors, as well as Phillips (5), have observed that a~oroximatelv20% of the r e ~ o r t e dcases e m be rrlnted u, a known or reeognmd l~horntorvi n r ~ d m tthe , other 80% resulted from unknown muses T ~ rndentdlable P causes of infection were accidental ingestion of viable microbial agents by pipetting accidents, or inoculation of viable microhial agents by accidental needle punctures, scratches.. cuts.. and animal bites. The unidentifiable causes of the remaining laboratory infections are believed to be the result of unrecognized generation of aerosols and subsequent inhalation or ingestion of the infeetious airborne particulates; aerosol generation from common laboratory operations has been experimentally demonstrated (6, 7,8). The magnitude and seriousness of laboratary-acquired infections and analysis of the predominant causes and modes of transmission bas led to the development of some basic containment concepts. These containment concepts derived far the primary purpose of eliminating or minimizing exoosure of ~ersonnelto hazardous aeents may generally he placed in tuo categurm pr,jrrdural harrtrrr and p h ) ,wol harnvr, The procedural barr~ersarc g w d laboratry practices or aseptic techniques, general housekeeping, and personal hygiene. Also included as procedural barriers are sterilization and decontamination techniques, waste control procedures, material and personnel flow, and operating regulations. Separation of the infectious agent from the laboratory worker or the worker from the agent is also achieved by physical barriers such as walls, windows, doors and floors. Another physical barrier is the air control system whieh moves the air in de-
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sired directions (hy achieving differential atmospheric pressures) and removes airborne contaminants from air streams by filtration through high efficiency particulate air (HEPA) filters. Protective clothing or respiratory protection for the worker and filter bonnets for animal cages are also examples of physical harriers.
Biological Safety Cabinets
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Another verv, sienificant ohvsical barrier " , is the htolog~calsafety cabinet All safety rnhmets h a w three charsrterl~ticsin cummon: I I A solid enclosure whreh may be either absolute or partial and a window through whieh the operator can observe the work, 2) uentilation by forced flow whieh provides directional control over airborne particulates in the work area and dilution of the infectious aerosols that may be generated during the work, and 3) remoual of airborne particulates from the effluent air streams. Early in the development of biological safety cabinets the viable particulates were removed or inactivated by incineration of the exhaust air. Air filtration techniques evolving from the Atomic Energy Commission radioactivity control program have generally supplanted incineration and pyrolysis is now employed only for treating the most hazardous particulate effluents. Filtration removal of airborne particulates is ordinarily accomplished by HEPA filters (also called "ahsolute") which are capable of removing 99.97% of the particles 0.3 ilm or larger in diameter. The filter media is most frequently a sheet of glass, ceramic, or cellulose-asbestos fibers whieh is folded back and forth over corrugated metal dividers to present e maximum surface area and high air flow rates with minimal differential pressures. Velocities through these filters range upward from 5 linear feet per minute (Ifpm) but seldom exceed 10 lfpm. The same filtration systems used to remove viable airborne particulates are effective in the removal of toxic or hazardous nonviable particulates of corresponding size; the same filter systems are applied in the eontain-
ment of non-gaseous chemical carcinogens. Biological safety cabinets fall into three categories: Class I, Class I1 or laminar flow biological safety eahinets, and Class 111. The Class I cabinets (Fig. 1) were probably the first type developed and are direct descendants of the more familiar chemical fume hoods. These are considered partial containment cabinets because of the opening in the face through which the operator extends his arms to perform the work. Inflow of air through the work opening may range from 70 lfpm to 150 lfpm. A minimum of 70 lfpm is required to keep work space aerosols from escaping to the laboratory environment; the higher the inward veloeity, the greater the personnel proteetion. Although the operator is protected from the work-generated aerosols, the work zone is penetrated by airborne contaminants from the laboratory; therefore, the biological experiment or "product" is frequently exposed to both viable and nonviable particulate contamination. Another disadvantaee of the Class I cabinet is the nossihility ot cross ronlorninafion within the rnclorure itielf. Because of air turbulence rrented by expansmn of the air stream as it passes into the cabinet through the narrow work opening, one item in the cabinet may be contaminated with particulates originating from another item. The exterior environment or communitv is oratected from the contaminants by the filtration of rabi. net exhaurt air before its release. A concept of contamination control which utilizes the undirectional flow of HEPA-filtered air was advanced in 1962 by Whitfield (9).The undireetianal flow is achieved by laminar air flow which is defined as "air flow in whieh the entire body of air within a confined area essentially moves with uniform velocity along parallel flow lines" (10). The concept was suceessfully applied first to whole rooms and then to workbench stations for critical assembly operations in the National Aeronautin and Space Administration space vehicle proerams. The horizontal flow clean bench and ;he vertical flaw clean bench are still used extensively in the pharmaceutical, elec(Continued on page A546)
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Volume 52, Number 12, December 1975 1 A545
Safety
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tronie, food, and other industries. Obviously these clean benches do an exceptional joh of protecting the product from contamination-a capability not found in the usual biological safety cahinet--hut present a very great hazard to the operator who mav he workine" with infectious aeents. " Safety equipment engineers, recognizing the great potential of the laminar flow eoneept of contamination control for work with pathogenic agents, designed cabinets which recirculated 80-90% of the cahinet air and had an inflow of air a t the work opening to minimize operator exposure: These uniquely-designed cabinets were originally called laminar flow biological safety cabinets hut are now referred to as Class I1 cabinets. One of the early sueeessful attempts to establish both operator and product protection through precise control of air flow utilized a ten percent free area perforated work surface combined with a 20 oercent free area oerforated slot alone the frcmt edge of the work .,pening and a mrsed Ilp at the leading w g r r t the slot to turn the rnroming air pam.lel to rhe interior downflow to create an efficient air barrier at the opening. The establishment of true laminar flow was enhanced by orienting the window parallel to the rear wall of the work space. The efficiency of the desien was oroven bv tests usine a microbial indicator system 1111, and demonstrated not only excepttonal product pn,tertim hut operator protection as well,
Variations with a slanted window and solid work surface have slightly reduced the overall efficiency because of increased air turbulence, but the containment capabilities remain s t least equivalent to the Class I cabinet and retain most of the product protection characteristics. There are two types of Class 11 eabinet. The speeifieations which standardize the design and ~erformanceparameters of the Class 11, Type I safety cabinet (Fig. 2) have been formalized in a National Institutes of ~.~~~~ -~ Health Specitication NIH-03.112~( 1 2 ) and are currently heing mudilied fur adoption hy the National Saniuttmn Foundation for promulgation by that group among commercial cabinet fabricators. Briefly, the eriteria for the Class 11, Type I cabinets are: 1)a minimum average downflow velocity in the work space of 75 Lfpm, 2) a minimum inflow velocity at the work opening of 75 Ifpm, 3) discharge of approximately 30% of the circulating air through a HEPA filter (compensated by equal supply through the ~~
front opening), and 4) a permanently fixed 8" high front opening. The restricted front opening is a disadvantage in that it is a restraint to equipment placement and operator manipulations in the work space. Another undesirable feature of the design is the oositive nressure of the contaminated air plenum and ducts lying hetween the fan and the supply filter. (Refer to Fig. 2). This pressure permits contamination of the laboratory environment through any leaks in these chamber walls. A modification of the Class I1 cabinet, designated Type I1 (13) (Fig. 3), has been designed to overcome the disadvantages of the Type I. The principal characteristics of this modification are: 1) an average downflow of 50 Lfpm (*lo), 2) a minimum inflow of 100 lfpm (+lo) with an 8" high work opening, 3) exhaust of approximately 70% of the circulating air volume, and 4) a sliding front sash for enhanced access. The sash must he a t the 8" level far maximum
Figure 3.
contemolated cahinet. Identifvine the via, ~~" hle agents and making n subsequent assessment of personnel risk is the second step. Ikfinition of rrak must include factors such as the pathogenicity of the agent, manipulative methods to he employed, the number of accidental laboratory infections reported for the agent, minimal human infective dose, horizontal transmission data in animals, and availability of prophylactic vaccines or anti-serums (14-16). After assessing the risk, the purchaser must decide what the control objectives are:-product protection, personnel protection, or a combination of hoth. Tahle 2 gives examples of safety cabinet use recommendations which might he applied in work with cancer viruses and chemical carcinogens. If a high risk agent is being employed, obviously only the absolute containment provided hy Class 111 systems is appropriate. If the risk level is lowto-moderate and the protection objectives are hoth product and personnel, the Class I1 cabinets are recommended. If product
efficiency hut acceptable containment is still achieved when it is a t the 15" level. The placement of the filters below the work surface assures that the positive pressure chambers beyond the fans do not contain viable particulates and therefore leakage from these plenums is inconsequential. Exhaust air is removed from the cahinet by an externally mounted fan which is a disadvantage in that the cabinet is not fully integrated or self-contained. A comparison of distinguishing characteristics of the Type I and Type I1 Class I1 cabinets is summarized in Tahle 1. In contrast to the partial containment of the Class I and Class I1 cabinets, the Class III cabinet or "glove box" (Fig. 4) is reserved for only the most hazardous etiologic agents; it offers absolute containment by comp:ete and lesk-tight enclosure of the work space. The cabinets are analogous to the AEC "dry boxes" used for working with radioactive isotopes. A cabinet does not qualify as a Class I11 safety cahinet unless the construction will allow no leaks a t any single point greater than 10-"illiliters per second when the interior is pressurized to three inches on a water manometer (w.g.1. The operator works with his hands and arms inside rubber eloves. .. . which are sttnehed to ports in the fare of the cabinet. Ventilation is achieved through HEPA-filtered supply and exhaust ports to provide approximately 20 changes of air per hour and s slightly negative pressure (less than 0.5" w.g.1 in the cabinet. Integrity of the harrier is frequently maintained by passing clean materials into the cahinet through double-door pass boxes and passing contaminated material out through douhledoor autoclaves. In addition to absolute protection of the operator, the system eliminates potential of product contamination from the outside; however, cross contamination from item to item is still possible inside the cahinet. The most negative aspect of the Class I11 system is the severe constraint imposed on operator freedom by the cumbersome gloves and the restrictive glove ports.
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Figure 4
protection is the only objective, as in sterile assembly of equipment or product sterility testing, then the horizontal or vertical flow clean bench would be appropriate. Not all of the partial containment safety cabinets can be used for work with proven earcinogens because the OSHA requirements (17) call for air inflow of 150 lfpm. Careful review of objectives, awareness of risks, and familiarity with cabinet capabilities will allow judicious selection of proper equipment. ( ~ o n i i n u e don page A548)
Selecting a Cabinet The prospective purchaser or user of a biological safety cahinet should make a systematic analysis of needs and objectives before selecting one for the laboratory. The first step is to define the proposed lahoratory program and methods for utilizing the
Table 1. Characteristics of Class I I Safetv Cabinets*
~ r o n t w o r kopening Average vertical air flow velocity Minimum inward flow velocitv v o i . of work zone air recirculited Exhaust blowerr Positive pressure plenums
TYPE 1
T Y P E I1
Fixed LFPM LFPM 70% Integral contaminated air
Vertically riidingrarh l a 1 50 L F P M 100 LFPM ma'' awning)
75 75
30% ....
Independent (b) Filtered air
(a) optimum operating level of 8 " o ~ e n i n g . (b) Cabinet requires connection to a sewrate exhaust system incorporated in the facility.
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'From A workshoo for certification of ~ i o l o a i c aSafetv l Cabinets. Rockville. ~ i o - E M .~ neering Service. Dow Chemical. U.S.A. Prepared under National Cancer Institute Contract 1-CP-33243. ~
(a1 Optimum operating l w e i of 8 " owning.
Volume 52, Number 12,December 1975 1 A547
Safety
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Cabinet Certification There are two elements essential to effective utilization of biological safety eahinets: personnel performance and eguipment performance. As is true with any mechanical equipment or tool, thorough understanding on the part of the operator regarding capabilities and limitations of the cabinets is necessary in order to realize maximum benefits. The laboratory technician must he trained in proper operating methods to obtain the contamination eontrol ohlertiver the mechan~cnldesign wnr intended to supply. Huwever, a tpehnirian must iirrt be provided equipment tnnr is performing to the design apecificationa, which means that the cahinet must he certified to be in proper working condition through a series of performance tests. The importance of certification was recently highlighted by a study performed for the National Institutes of Health by Dow Bio-Engineering Services. A group of Class I1 cahinets from nineteen manufacturers were tested early in 1972 for filter leaks and more than half the group were retested again six months later. The first testing showed 61% of the cahinets had leaking filter systems that had to he corrected. The second testing showed 40% with faulty filter systems. In 1973 the total group (approximately 230 to 240) was again
tested and 18% failed the filter system leak test; in 1974 the leak test failure level had again shown an increase to 27%. Much of the failure rate in the first certification tests can be attributed to faulty original installations. The disturbing fact is the failw e rate evident on subsequent testing. There are several explanations; the filter media itself was sometimes found to he punctured or hurned, perhaps by careless operators or maintenance personnel. Separation of the filter media from the frame and failure of the filter frame seal itself may result from continuous operational vibrations or stresses incurred while relocating the cabinet. The integrity of a filter is certified by the filter manufacturer. However, ahipping stresses and filter installation deficiencies by the cahinet fabricator make in-place testing essential a t the site of cahinet use. The on-site testing should be done by cahinet certification technicians who challenge the filters with small particulate aerosols of dioctylphthalate (DOP) or tobacco smoke, and determine penetration with a forwardscattering photometer or particle counter. I n situ filter integrity should he certified on all types of safety cahinets on an annual basis. Cabinet certification should also include some assurance that positive pressure chamhers, ducts, or plenums are free of leaks. Pressure in these spaces should he increased to 2 inches w.g. and all exterior seams or joints painted with a soap solution; buhhle formation locates leaks. Even though they are normally operated under slightly negative pressure, the leak test for Class I11 cahinets is much more stringent. The halance of air flow in hiolugiral safety cnhiners is very important, eiprrially for Clara I1 cabinets. If them is a positive pressure in the work area as a result of improper inflow-exhaust flow, the contaminants of the work space will spill out on the operator. Likewise, too great a negative pressure in the work area will result in room contaminants flowing into the work area. Excessive vertical air velocities or "hot spots" in discrete areas of the cahinet work space will cause turbulence conducive to eross-
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contamination. Air flow should he checked and balanced by certification technicians using correctly cslihrated air velometers. The owner or user of ventilated safety cahinets might ask, "With what frequency should my equipment he certified?" As a guideline, the National Cancer Institute makes certification mandatory on an annual basis and further recommends that the integrity of the systems he checked upon initial installation, a t filter replacement time, and upon relocation of the cabinet (18). A new safety standard for research with chemical carcinogens in the Department of Health, Education, and Welfare (19) will recommend a t least annual certification. As mentioned previously, personnel performance is as important as equipment performance. A certified cabinet still cannot prevent personnel errors which may cause cross-contamination or infection (20).It is important that personnel plan ahead and assemble all materials before beginning work. All the materials necessary for a process should he placed in the eahinet hefore work begins. In addition, the interior surfaces of the cahinet should he decontaminated before and after work and - personnel should wear long-sleeved gowns and gloves.
Cabinet Decontamination Decontamination, by definition, is destruction or removal af the living organisms to some reduced Level, hut not necessarily to a zero level. The most efficient method of decontamination is exposure of the microorganism to an elevated temperaturethe higher the temperature, the more effective the decontamination. However, it is not practical to suhject all surfaces and filters of a safety cabinet to increased temperatures so it is customary to achieve the decontamination by inactivating the mieroh i d contaminants with chemicals which disrupt the physiology or vital metabolism of the cells. The chemical decontaminant is routinely applied a s a solution to accessible surfaces before and after work in the cahi-
Tabla 2. Laboratory Safety Cabinets*
TYPE S A F E T Y CABINET
ONCOGENIC V I R U S M i N . AVG. CLASSIFICAFACE V E L O C I T Y TlON la) ILFPM)
APPROX. EXHAUST AIR ICFMI 4' H O O D 6' HOOD
PRDTECTiON
1. Partial containment
a ) clarr I b ) Ciars I I . T y p e I
C)
Cia65 I I , T Y P ~ ll
2. Absolute Class i l l
L O Wand Moderate Risk L o w and Moderate Risk
L o w and Moderate Risk
75 75 (Work Opening Fixed) (Sliding SarhWork Opening at 8 inches)
200
300
User only.
200
300
User & product.
265
400
user & product.
(b)
lb)
User or!marily.
containment High Risk
(b)
(a) hatmnal Cancer I n r t t l ~ l eS a f e t y Stanaarar f o r Rcrearcn lnvolv ng Oncoqcn c V r s e r . D d E W Pub cation N o . l h l r l ) 75-790. Oclober 3. 1 9 7 n . l o ) O n e vo L r n e a r cnangeeacn 3 rn mter f o r general rent a t o n or r z e to neat load. ' ~ m r n~ e r a g nCr term f o r uwal Onco.oqy Research ~ a I~rner. c O f l l c e ot Rerearcn safety, Natoona* cancer InstofLre. Aor I . 1975.
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net and also when cleanine uo accidental spills. The ehemicnlr are s e l e r l e d fur their effect un speafic types of vryanisms and alsu for ahrenee of h a r d t o the uperator under conditions of use. Some of those commonly used are ethanol, iodophors, alkaline glutaraldehyde, chlorinated phenols, and quaternary ammonium compounds; each has advantages and disadvantages. There are occasions when it becomes necessary to decontaminate the inner inaecessihle surfaces and filters of the cabinet. This total decontamination is done when the filters are changed or other msintenanee work must be done on the interior of the cahinet (19).Total decontamination in which all of the interior spaces are decontaminated is also recommended when there are accidental spills of highly infective agent and when the cabinet is removed to another location. The only possible way to reach all of the inaccessihle crevices and spaces of the cabinet is to apply the chemical in its vapor or gaseous phase. Formaldehyde has become the chemical of choice for decontamination of cabinets and other spaces. I t exhibits good activity against a wide variety of viable agents, it is relatively fast-acting, has good penetration, and is easy to use. The most satisfactory method of dispersing formaldehyde gas for decontamination has been through depolymerization of parafarmaldehyde by the application of beat to the dry powder or flake (21). Complete depolymerization of 0.3 gram of paraformaldehyde per cubic foot of space to be treated will give an approximate gas eon-
centration of 0.8% which is adequate for most cabinet decontamination requirements if held a t room temperature and relative humidity of 60% to 75% for 1-2 hours (22). In conclusion, the reader is reminded that biological safety cabinets are effective physical barriers which, when used with good procedural harriers, can provide personnel protection, product protection or both. Cabinets can only provide maximum benefits, however, if laboratory personnel who are responsible for selection, certification, and use have an understanding of cahinet capability and limitations.
8. Anderson, R. E., et o l , ,'Potential Infeetiova Hazard8 of Common Bacteriolaeicsl Technioue? . . J . BOG.
rerid., u.4 7 ~ 8 (igsi). 1 "A New Appmaeh to Clean Rmm 9. Whitfield, W. .I., Design." Sandia Corp. AIbuquerquc, N.M., Technical Report No. SC-4673 (RR). 1962. LO. General Services Administration, Clean Raom and
of a Laminer Airnow Biological Cabinef" Appl. Microbial. 16,1086 (1968). 12. National Institutes of Health soecification. Class 11. TYWI safety cabinet, N I H - O ~ I1916. ~. 13. Nstionsl Csncer Institute Sweification. General PurP- Clean Air Riologbal Safety Cabinet. 1973. 14. Wedum, A. G.. Barkley, W. E., and Hellman, A.. '"Handling of Infectious Agents." JAYMA. 161.
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