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9. Sterilize discarded pipets and syringes in the pan where they were first placed after use. 10. Before centrifuging, inspect tubes for cracks. Inspect the inside of the trunnion cup for rough walls caused by erosion or adhering matter. Carefully remove all bits of glavs from the rubber cushion. A germicidal solution added betweenthe tube and the trunnion cup not only disinfects the surfaces of both of these, but also provides an excellent cushion against shocks that otherwise might break the tube. 11. Use centrifuge trunnion cups with screw caps or equivalent. 12. Avoid decanting centrifuge tubes; if you must do so, afterwards wipe off the outer rim with a disinfectant. Avoid filling the tube to the paint that the rim ever becomes wet with culture. 13. Wrap a lyophilized culture vial with disinfectanGwetted cotton before breaking. Wear gloves. 14. Never leave a discarded tray of infected material unattended. 15. Sterilize all contaminated discarded material. 16. Periodically, clean out deep freese and dry-ice chests in which cultures are stored to remove broken ampoules or tubes. Use rubber gloves and respiratory protection during this cleaning. 17. Handle diagnostic sernm specimens carrying a risk of infections hepatitis with rubber gloves. 18. Develop the habit of keeping your hands away from your mouth, nose, eyes, and face. This may prevent self-inoculation. 19. Avoid smoking, eating, and drinking in the laboratory. 20. Make special precautionary arrangements for respiratory, oral, intranasal, and intratracheal inoculation of infectious material. 21. Give preference to operating room gowns that fasten a t the back. 22. E v d u a t r the extent to which the hands may became contaminated. With some agents and operations, forceps or rubber gloves are advisable. 23. Wear only clean laboratory clothing in the dining room, library, etc. 24. Shake broth c u l t u ~ sin a manner that avoids wetting the plug or cap.
Loboratory Safety Equipment
Both experimental evidence and practical experience have shown that good techniques alone will not prevent infection of laboratory people. Engineering can provide a physical separation of the workers' environment from that of the microorgsnism. The most important type of safety equipment is the ventilated, protective cabinet ( B ) . Figure 1 shows one example of a single bacteriological work cabinet that is adequate for many laboratory manipulations. This type of cabinet can be used with attached armlength gloves, or it can be used with the gloves removed and the air sweeping away from the operator. Figure 2 shows a similar cabinet but without an entrance air-lock and with the glove panel removed.
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Table 6. Correlation of Estimation of Risk with Recommendations for Use of Protective Cabinets* Cabinet systemc Single cabinetsd aerosol Aerosol Other Disease or agent studies studies techniques ... Brucellosis Coccidioidamycasis ++f ... Russian s-s encephalitis ... Tuberculosis ... Monkey B virus f+f Glanders Melioidosis f Rift Valley fever ++f Arhoviruses, general ... f+f Encephalitides, various ... f+f Psittacosis f f+f Rocky Mt. spotted fever Q fever Typhus f Tularemia f ++f Tularemiah Teuezuelan encephalitisb Anthrax Botulismb Histoplasmosis ... Loptospirosis i Plague ... j , Poliomyelitis ... Rabies ... Smsllpoxd Typhoid ... 0 Adeno, entero viruses f i Diphtheriab ... 0 Fungi, various ... f 0 Influenza. ... i Meningococcus ... 0 Pne,tmococcus ... 0 Streptococcus ... 0 Tetanusb ... f 0 Vacciniab ... 0 Yellow f e v d ... 0 Salmnnellosis .. . i Shigellosis ... Infectious hepatitis .. . ... 3= Newcastle virus . .. 0 = mandatary; = strongly advised; = optional, but in absence of s. cabinet a. few infections wlll occur; zt = depending upon technique and supervision. 0 = not required. For perems receiving live vaccine or toxoid. Figwe 3, or equivalent. Figures 1, 2, or equivalent.
+++ +++ +++ +++ ++
+++ +++
+++++ ++ ... +'++ ++
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++ +++ + +'i'+ + +++ + +++
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+
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+
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Figure 1 Closed Mictobiologicol Sofety Cabinet with Air Lock.
The basic minimum requirements for surh a cshinet are: sufficient inward an' flow (50 to 100 linear feet per minute) or onarnt,ion a t a neeative Dresswe. filtration -r ~-~ of contaminated exhaust air, and means of sterilizing both the exhaust filter and the interior of the cabinet. Figure 3 illustrates s. mare complicated cabinet system for laboborstory operations of higher risk. These cabinets are modular units that are gas-tight and can be internally eqnipped with incubators, refrigerators, deep freezes, and centrifuges. D e
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pending upon t,heir complexity, they can include all equipment needed to carry out microbiological research. These ~ystems, however, are obvio~lslyneeded only for work s t a very high hazard level. (Continued on page A 180) Volume 42, Number 2, February
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I ' , , I Figure 3.
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Gas-Tight Modular Cabinet Syrtemr.
To msist in selecting the proper type of proteotive cabinets Wedum and Phillips (26) have made estimates of the relative risks of various types of laboratory research and have formulated recommends tions for the types of cahitrets to use with the agents of a. number of diseases. The scale of laharatory risks starts a t the highest with a laboratory that wishes to perform any type of experiment with any infectious agent ming animals as large as chimpanzees. Use of dry powders af infectious a p n t s presents the next highest degree of risk followed by experiments with highly infectious a e n d s . Lower i l l risk i laborstory work with highly infectiom organisms in a Hl~id or liquid culture state. General rrediaation of the relative order of infectious risks call be osefd in selecting the proper t.ype of cabinet system. Table 6 lists a number of disease agents and provides recomme~idations for appropriate protective cabinets. I n general, experiments with miemhisl aerosuls and those using the more serious or more infectious disease organisms are those that should be done in gas-tight cabinet systems. These recommendations are also based on the use of vacoines or toxoids with some diseases.
Figure 4.
Anirnol Rock for Ventilated Cager
According to the environments1 stability and infectionsness of the at hog ens and (Continued on page A 123)
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the method of snimsl challenge, va.riaus degrees of isolrttion of experimentally infected animals are needed. In a, complete containment system animals may be housed within gas-tight cabinets. The next level of containment would be provided by closed ventilated cages such as are shown in Figure 4. A still lower level is provided by the use of open cages screened by ultraviolet radiatiuu ($9). Other types of containment equipment for speeifio procedures are available and are recommended for use, expecially if gastight cabinet systems are not available for centrifuging, blending, shaking, or lyophilising dangerous pathogens.
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Figure 5. Safety Centrifuge Cup.
Revolving centrifuge tubes may produce m~crobial aerosols w~thout breaking the tube, if the rim of the tube b wet with the cultnre dming preparation (85). Although strict attentloll to technique can avoid this and the m e of plastics will reduce tube breakage, these hazards are better avoided by the use of safety centrifnge cups as shown in Figure 5. These, of course,
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Centrifuge Cobinel with Pull-Out
should be loaded and nnloaded in a ventilated safety cabinet. Figure 6 shows a ventilated, ultraviolet irradiated chamber for centrifuges used in some Swedish laboratories. Small centrifuges ran often be placed in ordinary cabinets for use, but the Sharples supercentrifuge should be enclosed in a. specially designed cabinet when used with highly infectious microorganisms. Figure 7 shows a ventilated cabinet designed to sccommodate a refrigerated eentrifoge. The shaking of cultures during incuhsi tion can present substantial infectious risks if flasks or tubes break or if the stoppers fall off. If shakers are not enclosed in ventilated cabinets, aerosol tight shaking containers with viewing (Catinued a page Al84)
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Figure 7. Ventiloted Cabinet for Refrigerated Centrifuge.
windows should be constructed for each shaker (30). A lyophilizing apparatus is likewise hazardous when used with pathogens. I t should either be operated in a closed cabinet or constructed so as to he sterilized without dismantling. I n either ease a bacterial filter should belocsted in the exhaust air line before the VhCUUm pump (31)
Building Design Modern construction criteria applied in huildine laboratories and animal rooms
new or renovated facilities have been discussed along with typical recommended floor plans (B).t Some engineering features commonly wed for microbiological environmental control in infections laboratories and animal rooms include (a) ventilated cabinets and cages to oontain microbes a t their point of use; (b) differential, increasingly negative air pressures as one moves from clean areas to those of greater infectious risk; (c) appropriately effective filtration of air from rooms, cabinets, and ventilated cages; (d) change rooms and showers for personnel; ( e ) ultraviolet air lacks and door bsrrien to separate areas of unequal risk; (f) treatment of cantaminated liquid effluents; (g) room arrangement or layout to achieve traffic control dong a clean-contaminated axis; and ( h ) an effective intercommuniestion system. For those faced with initiating a design plan, the problem is one of determining whioh of the above items are to be used and to what extent. Moreover, it is usually necessary to make the determinations in the e d y stages of planning a new or renovated laboratory facility. The prohlem is difficult. The dangers are that the facility design will provide mare protection than needed, less protection than needed, will fail to protect the surrounding cammunity, or will be too inflexible in the future. consideration of some basic t Copies of a bibliography on design ot mierc biological fsoilities oan be obtained from Mr Phiilipg on request.
(Continued on page A1%)
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policy decisions before the design is begun mU provide a basis for selecting specific construction details. A comprehensive list of such policy questions has been published (86). Control of sir in the laboratory is especially important because the most common source of occupationally-acquired infection is the inhalation of accidentally or experimentally-produoed microbial aaerosol. Control should begin a t the laboratory bench level where the aerosol is formed, which is why there should he emphasis on primary containment devices such as ventilated cabinets. To the extent that the microbiological hazards are controlled a t their source, other building features such as differential air pressures in rooms, protective respiratory equipment, ultrzviolet irradiation, and personnel showers become less important in protecting laboratory employees. There is the necessity for a reasonable balance in hboborrttorv w u i ~ m e n t and
air from a room in which there are no ventilated microbiological safety cabinets or snimal isolation oages. If the room air is a t a hazard level requiring its filtration before discharge, it is inconsistent to have unprotected human occupants in the room. Major decisions in selecting engineering safety features should he based on the fact that safety begins a t the laboratory work surface because that is where most infections originate. Education in Microbiological Safety
I n spite of the considerations, a p preaches, and equipment discussed thus far, human factors in accident prevention occupy a dominant position and the educational process is essential in safety. As we are speaking primarily about safety in the campus situation, education takes on a double significance. The responsibility for the safety of the individual working in the infectious disease laboratory rests with the teaching institution that provided his initial training in laboratory procedures. Endowing the student with heuristic desires and technical knowledge is not enough. He must be taught how to use the instruments and apparatus of the laboratory. He must, in the learning process, he made to understand the importance of the manipulations and impressed with the notion that a good scientist is also a safe scientist. Too often school authorities have avoided the need for microbiological safety education and salved infectious hazards problems simply by forbidding the use of pathogenic agents in the school's laboratories. I t is true that not d l microbiologists handle or need to handle infectious argrtnisms in their work. But it is academic "buck-passing" to tell the student taking a course in infectious disesses that if he is required to handle pathogens later in his career his employer or someone else will give him the proper instructions. If (Continued on page AZb8)
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microbialagists and others who work with infectious microorganisms are to he given every opportunity to protect themselves from acquiring occupational diseases, should not safety education in the hazards associated with handling highly virulent microbes he included in the college curriculum? If safe behavior is a concept of life and important eontribations to attitudinal outlook are formed early is it realistic to wait until after the completion of professional training to institute education in safety?
Conclusions ond Future Prospects As has heen uoted the w e of animals and infectious agents often leads to occupational disease among laboratory people. From the point of view of each safety administmtor, it is important that an evaluation he made of sctual, potentid, or future microhiologiesl hazsrds before deciding if and how much of a prevention effort is required. I t has been principally during the last two decades that attention has been given to the prohlem of correcting or reducing laboratary-acquired illnesses. Former traditions of personal sacrifice are gradually becoming outdated by economic, moral, and legalpressures. Also, in the last few years, it has become eminently clear that laboratory determinations will be xewrate only if controlled to the oxteut that concurrent culture crosscontamination or animal crass-infection can be prevented. This has prompted research hrlpfol in developing techniques and methods which reduce human illfectious risks in the laboratory. The mast important single conclusion from this research is that preventing the release of accidental microbial aerosols s t the laboratory working smface through enreid techniques sud through the use of containment devices is the best way of sehieving microbiological environment,al control. The specific tools for controlling microbiological hazards me: The required management slipports and administrative techniques of reporting, analyzing, selecting, regulating, and training. The use of correct teohniques. The use of safety equipment. Properly designed laboratories. Vaccination of personnel.
On a campus, it is essential that the school discharge its responsibility for the safety education of strtdents who are exposed to infectious disease hazards. Increased national expenditures for education and increased emphasis on microbiological research portends sn inareasinelv greater demand far microstudents, researchers, and scientists. Education in laboratory safety methodology requires, as a background, a n adequate body of facts about laboratory hazards, their prevention, and most particolsrly, their causes. I n a time when there is a. clearly recognized shortage of educators and teachers it is appropriate that scientific methodology be applied in efforts to control and reduce laboratary (Continued on page AI30)
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infections among teachers, researchers, and students. Moreover, future demands on the educational system signal a need for research information an this subject. Of significance is the trend toward team research in which persons trained in fields other than microbiology use infectious cultures 8s tools in the solving of life science problems. Should the effort to isolate and identify virus strains as the etiologicd agents of certain cancers be successful to any degree, the need to protect research workers handling such strains would come under consideration. Perhaps it should be considered now. Likewke, in the space satellite research program it has been recognized that uncontrolled transfer of microbes between planets is undesirable. I n the medical field, a. more immediate hospital problem is that of the spread of staphylococci and other infections among hospital personnel snd patients. The principles of environmental control applicable in laboratory microbiological safety are helpful in solving these problems. Before microbiological safety control can be successfully integrated into needed areas in colleges and universities, it will be necessary, through safety education, to impart knowledge about these hazards to the university staff and through them to the students. The task promises to be formidable but nonetheless it should be made a part of future planning. Because of present emphasis on the teaching of science, many new and enlarged laboratories and teaching facilities are being constructed. Expanded courses in specialized areas of microbiology are being planned and larger teaching staffs are being sought. Now is the time to incorporate microbiological laboratory hazard control programs into needed areas. Literature Cited
Citations (1-$5) will be found on pages A47-A48 of the January, 1966, issue, accompnnying Part One of this article. (26) WEDUM, A. G., AND PHILLIPS,G. B., J. Am. Soc. Heating, Refrig., and Air-conditioning Engineers, Feb., (1964). (27) WEDUM,A. G., "Laboratory Safety in Research With Infectious Aerosols," Public Health Rept., 76, 619 (1964). (28) GREMILLION, G. G., "The Use of Baeteria-Tight Cabinets in the Infectious Disease Laboratom," Proc. of the Second Symposium on Gnotobiotie Technology, Univ. of Notre Dame Press, Notre Dame, Indiana, pp. lil-182. (%S) PHILLIPS, G. B., REITMAN, M., MULLICAN, C. L., A N D GARDNER, G. D., Pme. Animal Care Panel, 7 , 235 (1957). (30) DECKER,H. M., GEILE,F. A,, HARSTAD, J. B., AND GROSS,N. H., J. Bacterial., 63, 377 (1952). (51) REITMAN,IM., MOSS, M. L.. HARSTAD,J. B., ALG,R. L., AND GROSS, N. H., J . Baetel-iol., 68, 541 (1954). (3%) WEDUM,A. G., HANEL.E., JR., PHILLIPS, G. B., AND MILLER, 0. T., .4m.J . Pub. Health, 46, 1102 (1956).
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