1in the Chemical laboratory I 1
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Edited bv NORMAN V. STEERE. 140 Melbourne Ave., S.E. Minneapolis, Minn. 5541 4
CXVII. Safety in the Biological Laboratory* J. R. S o n g e r a n d J. F. Sullivan, National Animal Disease Center,
OBJECTIVES The three essential objectives of biological laboratory safety are: (A) t o protect the laboratory worker from infection, ( B ) t o insure the integrity of the experimental studies or clinical tests being carried on, and (C1 t o protect the surrounding cammunity from infectious agents being studied. A. The first and most important aspect of a biological laboratory safety programthe protection of the laboratory workerswill be covered in detail. Primary emphasis will be given t o the use of proper laboratory techniques and safety equipment. B. The importance of protecting the integrity of experimental studies or clinical procedures being carried on in a biological labaratnrv cannot be overemohasized. animals not only wastes many man hours, but all too frequently results in the publication of misleading information, the release of contaminated vaccines, or other acts with dire consequences. When working with disease agents noninfectious for man, the temptation is t o be less cautious than with those of potential human hazard. A good safety program encourages techniques and precautions fitting each situation C. Every effort should be made to prevent the escape of infectious agents from the laboratory. Systems far treating sewage, either with chemicals or heat, should be employed. Exhaust air from contsminated buildings should be filtered or heat sterilized. Infected animals should not leave the premises. Proper procedures for personnel entry and exit should be established t o minimize the possibility of agent escape on personnel or their clothing. Controlled methods for receiving and shipping equipment and materials to and from the laboratory should be established. A good safety program promotes employee morale and is an excellent means of establishing and maintaining desired public relations.
*Presented a t the Sixteenth Biological Safety Conference
MANAGEMENT OF BIOLOGICAL LABORATORY SAFETY PROGRAM Phillips a n d Jemski (1) list several suggestions for proper management of bialogical laboratory safety programs. Their suggestions are directed toward the focal point of laboratory safety-the laboratory worker. They include the following: 1. Establish written safety regulations which are read and understood by all. 2. Keep safety needs in mind when screening and selecting new employees. 3. Train each new employee until certain t h a t he understands the rules and whv. ~~, 4. Inasmuch as possible, design safety into techniques and procedures as they are developed. 5. Establish responsibility for safety. Each supervisor should be responsible for the safety of his people, but each employee should have a personal responsibilitysafety should be a part of every job. 6. Establish a reporting system for accidents, lass-time injuries, and labaratoryacquired infections and insist an prompt reporting. 5. Investigate each accident and illness t o determine what can be done t o prevent reoccurrence. 8. Encourage workers a t all levels t o suggest means of eliminating laboratory hazards. Safety, in its proper perspective, cannot be separated from laboratory techniques or procedures. In a real sense, it is an attitude or frame of mind. No technique, procedure, or piece of equipment should be accepted until it is safe. If it is right, it is safe.
BIOLOGICAL LABORATORY SAFETY REGULATIONS Wedum has prepared a n excellent list of laboratory regulations applicable for work with infectious agents (2). They are as follows: 1. There will be no direct mouth pipetting of infectious or toxic fluids. 2. Pipettes will be plugged with cotton. 3. No infectious material will be blown out of pipettes.
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4. No mixture of infectious niaterial will be prepared by bubbling expiratory air through the liquid by a pipette. 5. Use an alcuhol-soaked pledget around the stupper and needle when re^ moving a syringe and needle from a rubber-stoppered bottle. 6. Use only nerdle-locking hypodermic syringes. 7. Expel excessive fluid and bubbles from a syringe vertically intu a cottun pledget soaked with disinfectant or into a small bottle of cutton. 8. Before and after injectiv~iof a n a n i ~ mal, swab the site of injectiun with a disinfectant. 9. Sterilize discarded pipettea and syringes in the pan in which they were first placed after use. 10. Before centrifuging. inspect tubes for cracks. Inspect the inside ui the trunnion cup for ruugh walls caused hy erasiun or adhering matter. Carefully remwe all bits of glass from the rubber cushiun. A germicidal solution added between the tube and the tl.unniun cup nut only disim fects the surface uf huth, but also pwvidea a cushion against shucks t h a t uthrrrisr might break the tube. 11. Use centrifuge trunniou cups with screw caps or equivalent. 12. Avoid decanting centrifuge tubes. U you must decant, afterwards wipe oft the outer rim with a disinfectant. Aruid filling the tube tu the point t h a t the rim becomes wet with culture. 13. Wrap a lyophilixd culture vial with disinfeetant-wetted cotton brfurr brtwking. 14. Never leave a discarded tray of infectious material unattended. 15. S t e r i l i ~ e all euntaiuimted discard material. 16. Periodically clean Deepfreeze and Dry Ice chests in which cultures are stored t o remove any broken ampoules or tubes. Use rubber gloves and respiratory prutec tion during this cleaning. 17. Use rubber gloves wheli handling dl agnostic serum specimens carl.yin:: a nsk of infectious hepatitis. 18. Develop the habit uf keeping your hands away from your mouth. nus" eyes. and face. This may prevent self inwulation. 19. Avoid smoking. eating and drinking in the laboratory. 20. Make special precautionary a r rangements for oral. intranasal. and intratracheal inoculation of infectiuus m a t e r i ~ als. 21. Give preference to use uf uperatin:: room gowns fastening a t the back. 22. Evaluate the extent t o which hands may become contaminated. With some agents and operatiuns. furceps or rubber gloves are advisable. 23. Wear only clean labwatury clothing in the dining room, lihiary. etc.. 24. Shake bruth cultures in a manner (Continued on puge A4821 Volume 5 1 . Number 10. October 1 9 / 4
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that avoids wetting the plug or cap
PREVENTIVE MEDICAL PROGRAM An integral part of any well-rounded laboratory safety program includes a continual check of the health and well-being of all workers. A comprehensive medical history and physical examination should he required a t the time of employment. The latter should include a full plate chest X-ray, as well as appropriate serological and skin tests. Reference serum samples, collected a t this time, are frequently helpful in establishing an early and accurate diagnosis of any subsequent laboratoryacquired illness. Immunization for agents under investigation should be provided when warranted and available. Personnel with predisposing physical conditions should not be emplayed in a hazardous area. Pregnant employees should not be allowed to work in a viruslaboratory.
PERSONNEL INDOCTRINATION AND TRAINING All employees should be oriented with respect to the existence of and need far general and specialized safety programs within a laboratory. Support personnel (secretaries, janitors, maintenance men, etc.) should he allowed to enter areas where biological agents are worked with
only when needed t o provide specific services. Such entry should always be authorized by the scientist in charge of work under way in the unit involved; individuals authorizing entry t o potentially contaminated areas of a laboratory should prescribe procedures t o be followed (clathing and footwear required, use of safety equipment, ete.) when entering, working within,or leaving such areas. Individuals assigned to units involved in work in microbiology, biochemistry or radiology should receive instructions based upon specific job assignments. Indactrination programs for new employees should identify patential hazards of all job assignments, as well as approved procedures far their prevention and control. Pertinent information concerning the biological characteristics of agents being studied should take into consideration the host range, pathogenicity for man and animals, mode of spread, and methods of inactivation or decontamination. Individuals assigned to chemistry units or groups employing radioactive materials should be oriented with respect to the patential hazard of the material and trained in the use of proper techniques for their handling and storage, as well as the proper use and maintenance of protective devices routinely employed in the work. Specialized instructions should he designed to assist in the development of reliable employees whose conduct affords a maximum of protection to themselves, their fellow employees, and the continuity of the programs they are engaged in.
LABORATORY DESIGN AND SAFETY Mast authorities agree that many policy decisions must he made prior to the development and selection of a specific plan for a biological laboratory. Once policy decisions have been made with respect to agents to be worked with, including their host range, pathogenicity, mode of transmission, techniques to be employed, proposed animal exposure parameters (species and route), concurrent studies of multiple agents within a single laboratary, etc., a more intelligent decision can he made on the level of security required to protect the scientist involved, as well a s the integrity of the research studies being carried on. The cost of modifying existing facilities or constructing new biological laboratories must be balanced against policy decision, funds, and manpower available for the proposed work. Modern, well-designed biological labaratories are usually subdivided into a number of separate zones or areas-each of which is characterized by a similar degree of hazard. Three such zones can usually he found in a biological laboratory complex. They include: 1. Clean areas-reception and conference rooms, offices for administrative personnel, libraries, lunch rooms, media and animal production facilities, shops, warehouses. 2. Moderate Hazard Areas-all areas where work with biological agents are earried out. Generally restricted to work with agents of law pathogenicity or studies where laboratary techniques or safety equipment employed can be relied upon for agent containment and control. The ever-present possibility of accidents ar equipment failure makes all such units "potentially contaminated." 3. High Hazard Areas-all areas where work with highly pathogenic or transmissihle agents is carried out or areas where the activities under way can logically be expected to contaminate the work area. Such areas include diseased animal holding facilities of the nonventilated type, large animal necropsy areas, animal crematory or contaminated waste incinerator areas, etc. Good laboratary design, supported by appropriate regulations governing employees' conduct, are designed to maintain the status of clean areas and provide far the safe movement of oersonnel., materials, and waste into or from moderate or high hazard areas. The status of clean areas, which require only conventional types of construction, can usually be maintained by procedures designed to prevent the introduction of viable agents from moderate or high hazard laboratory areas (see below). All work with biological agents should be expressly prohibited in clean areas. Moderate or high hazard laboratory areas should provide facilities and equipment needed for cantainment and control of all agents workeri with. Design features should include perronnel entries containing dressing rooms, washup or shower facilities, as well as space for equipment needed for decontamination of foot and rainwear. Pass-through autoclaves and (Continued onpogeA484) ~~~~
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double door air locks should be available far the introduction or removal of materials into or from the agent work area. The latter units should be designed so that they can serve as a vapor phase sterilization chamber (formaldehyde or ethylene oxide gas) far items which are damaged by heat or are too large for the pass-through sterilizer. Laboratary work areas require special ventilation, sewage and solid waste handling systems. Chatigny (3) in describing'basic services required in a biological laboratory points out the need to ventilate laboratory rooms with 100 percent fresh air a t 10 to 15 changes per hour (up to 20 in animal rooms). He stresses the need to control directional flaw of air from the potentially least contaminated to the mast contaminated areas within the laboratory and reviews conditions which control the need for and type of air supply and air exhaust filtration units selected. In a discussion of lahoratory waste disposal requirements, Chatigny points out the need to consider all liquid wastes as potentially contaminated and discusses methods for their thermal or chemical inactivation. The desirability of access to a solid hearth animal crematar is also mentioned.
DISEASE TRANSMISSION IN THE LABORATORY The means by which man becomes infected in the laboratory depends upon
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both the biological agents involved and the eiperimental procedures employed. First to be considered are those procedures which predispose the infection by the commonly accepted routes. Oral exposure while mouth pipetting, self-inoculation while injecting animals or eggs, and conjunctival exposure from an improperly attached hypodermic needle are examples. Techniques and procedures should he continually reviewed to minimize accidents of these types. Reports by Stein and Segrave (4) and Sulkin and Pike ( 5 ) revealed that many laboratory infections result from accidents or poor techniques. However, in most eases the mode is not known. In one report by Sulkin (6), only 16 percent of the laboratory infeetions could be traced t o a known cause. Exhaustive on-the-spat investigations of 90 laboratory acquired illnesses a t Fort Detrick could only reduce the unknown group to 65 percent (11. Many times it was known only that the individual had worked with the agent or tended infected animals. Another important factor pointed out by Sulkin is that laboratory infections do not always follow the pathway of transmission established for the naturally-occurring disease. There is a preponderance of evidence in the literature that indicates that if the traceable cause of laboratory infections are eliminated, the remaining can be considered due to aerogenie transmission. Wedum (2) paints out that common laboratory procedures using the inaculating needle, pipette, syringe, centrifuge,
lyaphilizer and hlendar create aerosols laden with infectious agents of particle size suitahle for infection by inhalation. Air samples collected within 2 feet of the work area contain varying numbers of organsims, depending an the work being performed. Air samples collected a t the time a Waring hlendor cover was removed after mixing a culture contained too many organisms to count. Eighty-six organisms were collected when a lyophile culture tube was opened. Decsnting centrifuged fluid into a flask produced 17 airborne organisms. Inserting a hot loop into a culture flask produced 9 airborne organisms (2). Laboratary accidents also liberate many organisms into the sir. Table 1, from Wedum (2) presents some typical lahoratory accidents and the airborne organisms they liberate. Table 2, from the same source. present some single source multiple infections from known laboratory accidents.
BIOLOGICAL LABORATORY SAFETY EQUIPMENT The primary objective of all biological laboratory safety equipment is to break the chain of events which may eventually result in unwanted infection of laboratory personnel or animals. This may be done by control of specific operation or technique being performed a t the bench-top level (automatic pipetting devices, safety centrifuge cups and hlendars, etc.), by containment systems which confine hazardous work to gas-tight cabinets or cages (16) (biological hoods, ventilated cages. etc.), or by use of personnel protection equipment (respirators, gas masks, ventilated hoods or suits) when containment is impractical. The use of autoclavable, heat-sealable, pliable plastic materials for isolating the laboratory worker from equipment harboring infectious materials offers substantial advantages a t minimal equipment cost. The fallowing equipment and techniques have been designed to facilitate greater lahoratary safety. A modification of the high speed hlendar is described by Reitman et al. (17). If properly used, this blendor will eliminate the hazard associated with the high speed blending of infectious material. A model that has been further modified is cammercially available and isshawn in (Fig. 1). Hazards associated with the centrifuee result from either tube breakace or loss of stopper$ r~sultcngin the production oi in. fectious aero.01~. \I'h~twell~1 nl 181 also found thst an infectious aerosol was produced when contaminated fluid was left in the threads during centrifugation. Safety cups which will eliminate this hazard are shown in (Fig. 2). Mouth pipetting should be discouraged even when handling noninfectious material. If the habit of mouth pipetting is practiced, the likelihood of inadvertently pipetting by mouth with infectious agents is greater. Any pipetting procedure conducted in a safety cabinet would, of course, require the use of a pipetting device. Several types of pipettors are shown in (Figs. 3-61, Many laboratory accidents have been associated with the use of the hypodermic syringe and needle. Some examples are: (1) separation of the needle from the syringe during injection, (21 aerosol forma-
Table 1. Bacteria recovered bv air samolina durtna common laboratorv accidents Colonies obtained Per
Accident
accldent
One 50-ml. tube breaking in centrifuge and culture splashing side of centrifupe: air sampled 7 inches (18 cm) above centrifuge One 50-ml. tube breaking in centrifuge but all 30 ml. of culture stavine in trunnion cuo ~ c c i d e n t h ybreaking one ampoule of lyophilized nutrient broth culture on floor; air-sampled a t nostril height, 18 inches (46 cm) each side of accident site, for 1hr Droo of culture falline 12 inches (30cm) onto steel surface: air sdmplad within 2 ,i?1cm, feel ofsite Petri p l a ~ p c u i ~ u rde w s p p e d m floor; air rampled 1 feet $1.2 m, above tloor. 70 feet 21 .( mj from a m d e n t
1,183 4 491
From Wedum (2) Table 2. Episodes of single-source multiple laboratory infections
Persons infected
Disease Brucellosis (8) Coccidioidomycosis (9) Coxsackie virus infeetion (10) Louping ill virus infectian (11) Murine tvmhus (12) . .
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Q fever (13) Tularemia (14) Venezuelan encephalitis (15)
Probable source of infection Centrifugation Culture transfer, solid media Soilled tube of infected 'mouse tissue an floor Intranasal inoculation of mice Intranasal inoculation of mice Centrifugation 20 petri plates dropped 9 lyophilized ampoules dropped
Maximum distance from source
Number infected
Basement to Sdfloor
94 13
2 building floors 5 feet (estimated)
2
(1.5.m) 2 feet (estimated) (61 cm) 6feet (estimated)
3
6
Note: Numbers in parentheses are references. Modified from Wedum (2).
tion when needle is withdrawn from the vial, (3) spray formation resulting from skin puncture during an interdermal exposure, and (4) hand contamination resulting from leakage of inoculum from inoculated embryonated eggs.
The use of an inoculating loop can be a hazardous bacteriological procedure. Anderson et al. (19) demonstrated that when s cold inoculating loop was inserted into a 250 ml. Erlenmyer flask of culture, 0.8 organisms were recovered from the air per operation. However, if the loop was hot, 8.7 organisms were recovered from the air per operation. Spattering of the iniectious organisms when the loop is flamed is also a potential source of laboratory infection. A micro-incinerator unit used for sterilizing contaminated culture loops is shown (Fig. 7). Lyophilizatian procedures often predispose to laboratory infection. When vacuum is applied, the contaminated air is withdrawn from the ampoules through the pump and into the room. By the use of bi(Continued on page A486) Volume 51. Number 70. October 1974
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ological air filters or air decontamination procedures, this hazard can be greatly reduced. Aerosols are also often created by opening lyaphilized ampoules. When the vacuum is released, the rushing air may cause s fine awosal to escape into the room. This hazard may also be greatly reduced by wrapping the ampoule in a disinfeetant-soaked pledge of cotton before breaking. Such protective measures as: (11 use of Lur-Loc syringes, (2) eye protection (Fig. 8). (31 respiratory protection (Fig. 91, and (4) use of alcohol-saturated pledge of cotton a t puncture of needle and inoculum
vial should be used when warranted. The safety cabinet is one of the most useful protective devices in the laboratory. The level of security needed should determine the design of the cabinet. The highest level of security is afforded by the
Figure 3.
Figure 4
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Frean-tight class III hood. This cabinet operates a t a negative pressure and all supply and exhaust air is filtered through bialogical filters. It is equipped with rubber gloves. Equipment and materials enter and exit the hood through an air lock, autoclave, or a dunk tank. The commercially-available microbiological safety cabinet, sometimes referred to as a class 11 cabinet, differs from the class III in that i t is not Freon4ight. It utilizes biological filters and gloves and operates a t a negative pressure (Fig. 101. (Continued onpogeA488)
Figure 5.
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minimize the possibility of exposure of personnel as well as other animals. Two types of high security animal cages have been designed. The ventilated type developed a t Fort Detriek (20) utilizes high efficienev suoolv and exhaust filters and a negative pressure, forced ventilation system. Kraft type cages (21) utilize a high efficiency diffusion type filter and relies on diffusion of air through a large surface area filter. Humane methods of euthanasia should be employed when disposing of laboratory animals. Necrapsy procedures should minimize the danger of crass infection. When possible, dead animals should be incinerated. Care must be taken when incinerating contaminated animal carcasses. Although preheating the incinerator assures sterility of smoke and vapor released into the atmosphere, charging preheated incinerator with highly contaminated materials may liberate large quantities of contaminated smoke or vapor into the charging room.
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References G. B. and J e w 3 i . J. V. Rinloeieal Seiefy in the Animal Laboratory. Loh An. Care 13: Ko. 1, 1961. 121 Wedum. A. G. Confro1 of Laboratory Airhorn= 1 " ~ feetian. Barferiol. RPI. 25: 1961. 131 Chatigny. M . A. Microbioloeical Laboratory Safety. Advsnees in Applied Microbiology. Vnl. 3. 1961. AcadcmicPress, New York. N.Y. 141 Stein. Leon and Seedove. M. Unpublished special report, Camp Detrick, Frederick. Md.. 1949. 151 Sulkm, S. E., and Pike. R. M. T h e Prevention of Laboratory Infection in Diagnostic Procedures hi Viral and Rnkottsiai Diseases. :lrd Ed, Am. PuhIieHealth Arm. Ine.. KerYnrk. NY.. 1961. 161 Sulkin, S. E. Laboratory Acquired Infections. Rortertoi. Rac 25: 1961. 171 Wedum. A. G. Policy. Responsibility and Practice in Laborstory Safety. I" Praceedinirr. Second Symposium on Gnotobiotic Teehnoloev. Notre Dame. I n d . U n i x o f NofreDamePres. 1959. 161 Huddleron. I. F. and Muneer. M. A Studv of an Epidemic o f B ~ l c e l l o s i n ' D ~ fa e ~ ~ ~ ~ rnrlie f i o L e u i s . Am. J . Puhllr Hrolfh30: 944-956. 19d0 191 Smith. C. E. T h e Hazard of Acquiring Mycoix Infections in the Laboratory. Address delivered hefore the Eoidemioloev and Laboraiarv Sections. American P u b l i e & d f h Arsneiati& Annual Meefine. Nov. Z 1950. St. Louis. Mu. M s m e n ~ prsphad. 1101 Shaw. E. W.. Melnick. J. L.. and Curnen. E. C. Inleciion of Laboratory Worker$ with Coxsackie Vi. ruses. Ann. intern. Med. 33: :12-111. 1950. 1111 Rirais. T . M, and Schwentker. F. F. Loupine ill in 69: W-Yi 19:U M a n J E m, Med -~ (121 Laffler. W. and Moore.. H. M d e of Tlsnrmlanilln of Typhus Fever: Study bared on infeetion of group of laboratory workers. Schiceir. Med. Wrrhr. 72: 755-761. 1912. (In) ~ " e b n c r . R. J. h p o r t on an outbreak of Q F W ~ at the National InJtitutes of Health. Am. J P u h ~ licHaolth31: 4:111440. 1947. 1141 Barbeito. M. S.. Alg. R. L.. and Wedum. A. G. Infectious Raeferisl A e r m l from Dropped Petri nish c u ~ t u r e ~~m . J. ~ e d ~. p i h n o f n: :xu-.:IPZ. 1961. Il51 Slepuahkin. A. N. E p i d o m i o l o e d Study of a Lahmatmy Infection with the Venezuelan Equine Enrephalomy~lifir virus. Vop. v i w r ,US,7R, d: :
