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National Research Council (US) Committee on Hazardous Biological Substances in the Laboratory. Biosafety In The Laboratory: Prudent Practices for the Handling and Disposal of Infectious Materials. Washington (DC): National Academies Press (US); 1989.

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Biosafety In The Laboratory: Prudent Practices for the Handling and Disposal of Infectious Materials.

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3Safe Handling of Infectious Agents

A— GUIDELINES FOR HANDLING PATHOGENIC MICROORGANISMS

In 1984, the Centers for Disease Control (CDC) and the National Institutes of Health (NIH) jointly published a set of guidelines for the safe handling of pathogenic microorganisms[105 ]. These guidelines, developed over a period of several years in consultation with experts in the field, remain the best judgments available; they are reproduced here in their entirety, as Appendix A. The reader should consult these guidelines in deciding on the appropriate level of precaution to use in the handling of a particular organism.

Guidelines for handling agents identified after the CDC/NIH publication are published as Agent Summary Statements in Morbidity and Mortality Weekly Report (MMWR), issued by the CDC. The Agent Summary Statement for human immunodeficiency virus (HIV)[36 ]is reprinted here as Appendix B, and additional MMWR articles on HIV ("Recommendations for Prevention of HIV Transmission in Health-Care Settings"[34 ][38 ]) are reprinted here as Appendix C.

Throughout this and the following chapters, frequent reference is made to Biosafety Levels 1 through 4. These levels are described in the CDC/NIH publication (Appendix A). Table A.1 of this appendix summarizes the practices, techniques, and safety equipment prescribed for each level.

B. ORGANISMS POSING SPECIAL RISKS

The risk of acquiring an infection in the laboratory is influenced by many variables. Among these factors are the health and immune status of the laboratory worker, the suitability of the laboratory for work with highly pathogenic agents, the characteristics and the concentrations of the microbe being handled, and the specific manipulations involved in its handling.

Studies of infections acquired by personnel working in microbiological laboratories have been carried out by several investigators over the past half-century[42 ][84 ][101 ][105 ][120 ][121 ]and have identified a number of potential human pathogens that are clearly more frequent causes of laboratory-acquired illnesses than are others (see Chapter 2, above). Organisms falling in this category are to be found among viruses, bacteria, rickettsiae, and fungi. Awareness of those species with a high potential for invading normal humans should lead to the use of appropriate precautions to minimize the risk of infection.

Among the agents that have been identified in recent years as posing the greatest risk of infection to laboratory and ancillary personnel of diagnostic laboratories are the virus of hepatitis B, Mycobacterium tuberculosis, and Shigella spp. [60 ][70 ][121 ]. A partial list of other agents known to pose greater than average risk to laboratory workers includes Brucella spp., Salmonella spp., leptospires, Coxiella burnetii, Rickettsia spp., and Coccidioides immitis. The recently identified virus of AIDS (HIV), on the other hand, poses a low risk of occupational infection to laboratory workers, except to those working with concentrated virus suspensions[37 ][143 ]. The supplement to the CDC/NIH guidelines recommends, therefore, that HIVs be handled according to the standards and special practices of Biosafety Level 2 or 3, depending on the concentration or quantity of virus or the type of laboratory procedure used (see Appendix B).

No agent that is a component of the normal or abnormal microbial flora of man should be regarded as lacking totally in pathogenic potential, and all microorganisms should be handled with appropriate techniques. With the increase in research in virology in the past half-century, laboratory infections with viruses have increased relative to those caused by bacteria and mycoplasmas.

An important defense against infection with some viral agents is immunity induced by vaccination. Whenever a vaccine is available (see Table 5.2), its use should considered for those at risk of exposure prior to their handling of the virus in question. Under certain circumstances, when work with highly virulent agents is contemplated, it may be necessary to consider the administration of an experimental vaccine. Because of the potential risk of injury to the fetus from apparent or inapparent viral infection, special precautions, including temporary reassignment, may be considered for female personnel who are pregnant or are contemplating pregnancy. (See Chapter 5, Section D.)

All personnel working with infectious agents should have documented evidence of immunization with the vaccines required by most jurisdictions for admission to elementary school, e.g., diphtheria, tetanus, pertussis, poliomyelitis, measles, mumps, and rubella. In addition, vaccines for preventing infections with other agents to which they may be exposed, if available, should be offered, and in certain circumstances consideration should be given to making such immunization mandatory.

Acceptance of immunization against, or demonstration of proven immunity to, hepatitis B virus should be a precondition for the employment of all workers who will be handling human blood or body fluids. If the medical program of the hiring organization includes a serum bank, a sample should be obtained at the time of employment and stored in the frozen state, to provide a baseline for subsequent immunologic assays as required (see Chapter 5, Section D).

C. HAZARDS FROM VERTEBRATE ANIMALS AND INSECTS IN THE LABORATORY

Personnel who work with experimental vertebrate animals in the laboratory, or who receive and handle specimens from vertebrate animals, should be cognizant of the potential for exposure to zoonotic pathogens and to allergenic animal danders, urine, and saliva.

A list of zoonotic pathogens and potential animal sources of infection for humans is included in Appendix D; the information for this table was derived from references 6, 17, 21, 26, 53, and 69. While it is recognized that many of the agents listed are not significant hazards under ordinary laboratory circumstances, laboratory staffs should recognize the dangers of zoonotic pathogens and should realize, for example, that protozoan cysts and larval stages of certain helminths in fecal material can be infectious[26 ]. Application of the seven basic rules of biosafety cited in Section F of this chapter will greatly reduce the risks of infection while handling vertebrate animals or specimens obtained from them (see also Section G of this chapter).

Strong consideration should be given to immunizing employees with appropriate vaccines against zoonotic agents, if available (see Table 5.2).

Numerous agricultural, veterinary, and human disease research laboratories are involved in the production and maintenance of insects. Insects are also produced for regulatory and control activities (e.g., screwworm control, which involves the release of insects into the environment). The human health hazards of insect production have been recognized recently. In addition to the hazards associated with insect bites, allergic reactions and respiratory diseases may result from contact with, or aerosol exposure to, various insect developmental stages, insect waste products (e.g., body hairs and feces), ingredients used in insect diets, or mold spores and bacteria that contaminate larval diets. Repeated exposure over a period of months or years may produce respiratory ailments or other manifestations of allergic reactions in susceptible individuals.

During the preplacement medical evaluation at the time of hiring or job assignment, a history of allergies to vertebrate animals or insects that the prospective employee is likely to encounter should be elicited. After hiring, the periodic monitoring medical examinations should include an evaluation for the development of allergies (see Chapter 5, Section D). The prevalence of allergies among personnel who work with or are exposed to vertebrate laboratory animals has been estimated to be 11 to 30 percent[14 ]. Some individuals may become very sensitive to low concentrations of allergens[150 ][151 ]. More than 300 cases of allergic reactions that probably resulted from the inhalation of insect-derived materials have been reported[11 ]. More than 40 species (among eight orders) of insects were associated with work-related allergic symptoms among U.S. Department of Agriculture employees working with insects[10 ]. Insect allergy questionnaires and surveys indicate that respiratory symptoms (e.g., sneezing, coughing, and chest tightness) and eye and skin irritation or skin rash are the major symptoms in those with complaints of insect allergy[21 ][146 ]. Inhalation of airborne material was reported as the mechanism most frequently responsible for allergic symptoms in persons working in insect-rearing facilities[21 ][146 ].

Most insect-related health problems develop after repeated exposure, and severity often increases with continued exposure. Sensitivity and susceptibility vary greatly among individuals. The allergic symptoms of conjunctivitis, rhinitis, sinusitis, asthma, or pruritus and dermatitis can develop in from less than one year to many years after initial exposure. Whether or not people with allergies are more likely to develop additional allergies to animal products is controversial. Precluding allergic individuals from employment does not eliminate the problem, since nonallergic individuals also can become sensitized.

Reducing contamination levels and reducing exposure are the best preventive measures. This may be accomplished by engineering controls such as filtration and directional control of airflow, or by the use of filter-top cages and directional airflow racks to prevent the allergens from reaching the worker. The selection, design, and utilization of such equipment are the most important steps in controlling respiratory hazards. Respirators should be used only for temporary or intermittent work, such as during maintenance work on the ventilation equipment, and should not be relied upon as a permanent solution[151 ]. It may be appropriate for vertebrate animal caretakers, insect production workers, laboratory personnel, and others who work with animals or who enter the animal holding areas to wear gloves, eye protection, and a mask covering the nose and mouth. It is good practice to change from street clothing to laboratory garb. All persons who enter the animal holding area should adhere to the protocols and the regulations that apply to activities in the vivarium.

D. PRIMARY AND CONTINUOUS CELL CULTURES

Cell cultures, in general, present few biohazards in the laboratory, as evidenced by their extremely wide usage and the rare cases of transmitted infections to laboratory personnel. Primary cell cultures initiated with tissues from infected humans or animals are recognized hazards. Thus macaques, and possibly other Old World monkeys, may have latent Herpesvirus simiae (B-virus) infections and present a hazard to personnel handling these animals and their tissues. At least 24 documented cases of infections of laboratory workers handling primary cell culture tissues (e.g., primary rhesus monkey kidney cells) have occurred in the past 30 years[46 ]. A particularly noteworthy instance of the laboratory infection of a number of workers by an adventitious agent from monkeys occurred in 1967 in Marburg and Frankfurt, Germany, and in Yugoslavia. Laboratory workers handling tissues and cell cultures from African green monkeys developed an acute febrile illness. Seven deaths occurred among 31 documented cases due to a previously unknown virus, subsequently named Marburg virus. It has not occurred in laboratory workers since those incidents[115 ]. Tissues from mice infected with lymphocytic choriomeningitis (LCM) virus or from chickens carrying Newcastle Disease virus (NDV) also present potential hazards, but such laboratory infections have not been reported. Clearly, primary cell cultures prepared from humans infected with hazardous agents (e.g., HIV) present danger of infection, and such tissues must be handled with the precautions required of the known or suspected infectious agent (see Appendix A).

Continuous cell cultures present no real documented risk in the laboratory unless they are carelessly contaminated with an infectious agent. All continuous cell lines should be regularly monitored for contamination with infectious agents, and it should be emphasized that all nutrient media or other reagents that may contain ingredients of biologic origin must be treated as though they contain potentially infectious agents.

E. HANDLING OF NECROPSY AND SURGICAL SPECIMENS

1. Introduction

Necropsy and surgical pathology expose health care workers to various infectious agents that may be in human tissues or associated body fluids. Proper handling can minimize the risk of infection. Because the consequences of infection are grave, agents of principal concern are hepatitis B virus (HBV), human immunodeficiency virus (HIV), Creutzfeldt-Jakob agent (CJA), and M. tuberculosis, although a number of other infectious agents, including viruses, rickettsiae, bacteria, fungi, and parasites, pose potential risks. The principal means of acquiring infections when performing anatomic examinations are through breaks in the skin caused by needle punctures, cuts, or severe dermatitis, by contamination of mucous membranes, and by inhalation. The risk of infection is decreased by preventing breaks in the body surfaces, preventing the formation of droplets that might contaminate surface breaks or mucous membranes, inserting barriers such as rubber gloves, goggles, and masks between the infectious hazard and the potential site of entry, and preventing the generation of aerosols.

Fresh tissue may be infected with agents such as HBV or HIV even if there is no history of such infection. Those who perform autopsies or handle fresh tissue or blood on a regular basis should have immunity to HBV.

To date, in the United States there are few recommendations for biosafety in necropsy and surgical pathology[9 ][79 ][90 ], although such have been proposed in Great Britain[47 ]. Recommendations have been developed for Creutzfeldt-Jakob disease (CJD)[109 ], and a number of publications have helped to define the risks associated with this agent[9 ][24 ][55 ][56 ]. The National Committee for Clinical Laboratory Standards (NCCLS) has published proposed guidelines, entitled Protection of Laboratory Workers from Infectious Disease Transmitted by Blood and Tissue, that include necropsy and tissue handling recommendations[90 ]. Independently, a committee of the College of American Pathologists is developing recommendations for necropsy and surgical pathology.

In a given institution, there should be a clear definition of the responsibility for biosafety in the handling of a body from the time of death until it is transferred to the mortician or incinerated, and for surgically removed tissue. For an autopsy, the prosector (generally a pathologist) is responsible for biosafety. It is beyond the scope of this publication to discuss autopsy biosafety in detail, but some major points are considered below.

2. Necropsy

a. Routine Necropsies

Because of the high incidence of asymptomatic carriers of HBV and HIV in hospital or forensic autopsies, all cases should be considered potentially infectious and the necropsy performed carefully. Care should be taken to minimize chances of needle sticks, cuts, or abrasions. Risk of contamination of mucous membranes should be decreased by wearing safety goggles and a surgical mask, or a face shield.

b. Necropsies on Bodies Known to Be Infected

Bodies for necropsy should be appropriately labeled if they are known to be infected with such agents as HBV, CJA, HIV, or M. tuberculosis . The medical record should also indicate the diagnosis. Before beginning a dissection, it may be helpful to discuss the case with the clinician to clarify the extent of examination required. Autopsy assistants should be informed of the nature of the clinical diagnosis so that special disinfectants, such as sodium hypochlorite solution (household bleach diluted 1:100 in tap water), can be prepared prior to beginning the dissection.

In addition to the prosector and autopsy assistant, it is helpful to have a "circulating" assistant who remains "uncontaminated," thus preventing contamination of telephones, cameras, drawer pulls, cultures, papers, and other items by those doing the dissection, and confining the contamination to the necropsy table area.

Protective clothing should include the following:

  • a scrub suit covered with a long-sleeved gown or a long-sleeved coverall suit plus an impervious apron;
  • impervious shoe covers;
  • head covering;
  • goggles or eyeglasses to prevent conjunctival contamination;
  • face mask to decrease risk of droplet contamination of mucous membranes, or inhalation of aerosols; and
  • double gloves (preferably including one pair of heavy-duty gloves).

In performing the autopsy, it may be helpful to cover rib ends with towels to decrease risk of cuts. Dissection in the body should be limited to one prosector at a time. Use of scissors when possible will decrease the risks of cuts. Production of droplets and aerosols should be minimized. Use of a Stryker saw to open the skull or to cut bone is controversial because of the potential for generation of droplets and aerosols. Some authorities advocate using a hand saw, whereas others recommend using the Stryker saw with a HEPA-filtered vacuum attachment or covering the equipment with a wet towel. The saw and aerosol control apparatus should be adequately disinfected after use. In cases of CJD, there should be special care not to cut the brain. A new technique for the removal of the brain from cases of AIDS at autopsy has been developed in which the sawing is done inside a plastic bag[78 ][79 ]. The British Committee on Dangerous Pathogens has suggested performing limited postmortem examinations with discrete tissue sampling for most AIDS cases[1 ].

Any spills of blood or body fluid should be cleaned immediately with a solution of household bleach diluted 1:100 in tap water.

Specimens for culture or other clinical laboratory examinations should be handled in the same fashion as in patient care areas, with care being taken not to contaminate the outside of the container.

Disposable syringes and needles and knives should be placed in a leak- and puncture-resistant container for subsequent disposal.

If persons are cut or punctured while dissecting or handling tissues or body fluids, the wound should be encouraged to bleed, flushed with abundant water, and treated with an antiseptic such as povidone-iodine. The accident should be reported to the appropriate persons such as the safety officer, employee health director, or laboratory supervisor, depending on the institutional requirements.

At completion of the autopsy, the body should be packed with absorbent material to prevent seepage of liquids and should be washed with a 1:100 dilution of household bleach or other appropriate disinfecting agent. Tags on the body should note the infectious hazard. The body should then be placed in a plastic bag, which is also labeled with the appropriate hazard warning (e.g., "Blood and Body Fluid Precautions"). In addition to labels on the body, the mortician should be notified specifically of the infection hazard. As discussed below in Section F of this chapter, however, the use of special hazard warning labels should not lead to the misconception that other bodies are not potentially infectious.

When finished, prosectors and autopsy assistants should remove protective clothing in the autopsy room and place it in appropriate containers for incineration or transport to the isolation laundry, and should then shower. Soiled disposable items should be placed in biohazard bags for incineration. Soiled linens should be double-bagged in durable, labeled isolation bags and handled in the same manner as hospital isolation linen.

Tissues that are to be saved should be placed in formalin (1 part tissue to 10 parts formalin) and should be cut thin enough (<2 cm thickness) to ensure penetration. Fixation in 10 percent formalin will inactivate most infectious agents; mycobacteria and CJA are exceptions (see below).

Instruments should be autoclaved or soaked in a 1:100 dilution of household bleach, or other appropriate disinfectant, for 30 minutes to 1 hour. Only stainless steel can be placed in hypochlorite solution. The table and the floor around the table should be cleaned with a 1:100 dilution of household bleach, or with a germicide approved (by FDA) for use as a "hospital disinfectant" that is also tuberculocidal. If a mop is used, it should be autoclaved.

Creutzfeldt-Jakob agent is particularly resistant to killing, requiring autoclaving at 121°C for at least 30 minutes; it can survive in 10 percent formalin for many months[9 ][56 ]. Paraffin blocks may therefore contain infectious CJA. CJA is usually inactivated by household bleach at 0.5 to 5 percent concentrations, with the higher concentration being more effective but also more corrosive[24 ]. The agent is most susceptible to 1N NaOH. Contaminated material should be autoclaved as above, inactivated with one of the chemicals cited above, or incinerated. It has recently been noted that formalin-fixed brain tissue can be autoclaved to inactivate CJA and then processed for histologic sections[80 ].

HIV and HBV are readily inactivated by a variety of agents, including formalin, hypochlorite, and iodine-based disinfectants. Special care should be exercised when performing autopsies on patients who died of infections with these agents.

Mycobacterium tuberculosis is moderately resistant to 10 percent formalin, requiring prolonged exposure for complete killing[110 ]; formalin-fixed tissue from recent cases may therefore be infective. The usual route of infection is the inhalation of aerosols generated during necropsy, or the trimming of tissue for histologic processing. Occasionally, the organism is introduced into a cut ("prosector's wart").

3. Surgical Pathology

The hazards of surgical pathology are similar to those of autopsy. Many tissues have been fixed in formalin when received and are thus not infectious, with the exceptions noted above. Such tissues are best disposed of by incineration, more for aesthetic reasons than those related to biohazard.

Cryostats used for frozen sections present a particular problem[123 ]. The operator should wear gloves, gown, and mask when cutting the section, whether or not the patient is known to have a disease transmitted by blood or tissue. In addition, the cryostat should be disinfected periodically (at least weekly). If it is known that the patient has an infection that represents a hazard such as AIDS or tuberculosis, frozen sections should be prepared only when absolutely necessary. The cryostat should be disinfected with an appropriate disinfectant as soon as possible after the sections have been cut, to remove contaminated tissue fragments and to decontaminate surfaces.

All human anatomical waste and cadavers should be disposed of by burial or incineration. The incinerator must be appropriately designed for handling anatomical laboratory waste. Cadavers containing radioactive isotopes or antineoplastic drugs require special handling during autopsy and for disposal (see Chapter 4).

F. GOOD LABORATORY PRACTICES

1. Introduction

A number of reports and studies[8 ][15 ][40 ][67 ][68 ][77 ][84 ][96 ][101 ] attest to the potential for occupationally acquired infection by laboratory personnel working directly with microbial agents. The significant element to be derived from these reports is that the exact source or cause of the infection could be documented in fewer than 20 percent of the cases. This finding provides strong evidence that exposures and consequent infection occur not as the result of overt accidents but during the performance of routine procedures.

2. Routes of Exposure

The nature of infective contaminants dispersed during the performance of any laboratory procedure is a direct function of the amount of energy applied during the procedure. Low-energy procedures (e.g., removal of screw caps and pouring of liquid medium) principally yield droplets that are dispersed onto body and work surfaces. Exposure of personnel in these instances occurs usually through breaks in the skin surface caused by cuts, scratches, and other cutaneous lesions, or by ingestion of infectious material transferred to the mouth by hands or objects. On the other hand, procedures involving application of large amounts of energy, such as homogenization and centrifugation, have the potential for generating respirable aerosols. It should be recognized that a large number of procedures may result in the generation of a mixture of droplets and aerosols with the result that exposure by more than one route is possible.

While it has been typical to focus on respirable aerosols as the primary source of infection for laboratory personnel, it is essential that other routes of exposure be considered: contact, oral, ocular, and inoculation.

a. Contact Route

The control of potential exposure by the contact route requires that procedures be conducted in a manner that avoids contamination of body or work surfaces. This is accomplished through the use of gloves and other personal protective clothing, protection of work surfaces with appropriate absorbent disposable covering, use of care in the performance of procedures, and cleaning and disinfecting work surfaces. Procedures that can result in the generation of droplets include decanting of liquids, pipetting, removal of screw caps, vortex mixing of unsealed containers, streaking inocula on agar surfaces, and inoculation of animals.

It should be recognized also that dispersal of contaminants to other surfaces can occur by their transfer on the gloves of the laboratory worker, by the placement of contaminated equipment or laboratory ware, and by the improper packaging of contaminated waste.

b. Oral Route

A number of procedures carried out in the laboratory and animal facility offer the potential for either direct or indirect exposure by the oral route. The procedure that offers the greatest potential for exposure by ingestion is mouth pipetting. Clearly, such exposures are completely avoidable through the use of mechanical pipetting devices. Indirect oral exposures can be avoided through the use of the personal hygienic practice of regular hand washing, and by not placing any objects, including fingers, into the mouth. The wearing of a surgical mask or face shield will serve to protect the worker against the splashing of infectious material into the mouth.

c. Ocular Route

The wearing of a face shield, safety glasses, or goggles will protect the worker against splashing infectious material into the eyes.

d. Inoculation Route

The single procedure that presents the greatest risk of exposure through inoculation is the use of a needle and syringe. These are used principally for the transfer of materials from diaphragm-stoppered containers and for the inoculation of animals. Their use in the transfer of materials from diaphragm-stoppered containers can, in addition, result in the dispersal of infectious material onto surfaces and into the air. Depending upon the route of inoculation of animals, the use of a needle and syringe may also result in the contamination of their body surfaces. Because of the imminent hazard of self-inoculation, the use of the needle and syringe should be limited to those procedures where there is no alternative, and then the procedure should be conducted with the greatest of care. Inoculation can also result from animal bites and scratches.

e. Respiratory Route

Several procedures have the potential for generating respirable aerosols. Included are sonication, homogenization, centrifugation, vigorous discharge of fluids from pipettes, heating inoculating loops, opening lyophilized preparations, and changing of the litter in animal cages (see Chapter 3, Section I).

3. Prevention of Exposure

The time-honored approach for the safe handling of infectious agents involves the use of a combination of strategies. This is accomplished by

  • controlling the hazardous material at the source to prevent release into the workplace,
  • minimizing accidental release of the material, and
  • protecting the worker against contact with the material.

However, the safe conduct of work with infectious material is primarily dependent upon the application of good laboratory practices by the laboratory worker (see below).

4. The Seven Basic Rules of Biosafety

The most common means of exposure can be essentially eliminated as occupational hazards by following the seven basic rules of biosafety:

  • Do not mouth pipette.
  • Manipulate infectious fluids carefully to avoid spills and the production of aerosols and droplets.
  • Restrict the use of needles and syringes to those procedures for which there are no alternatives; use needles, syringes, and other ''sharps" carefully to avoid self-inoculation; and dispose of "sharps" in leak- and puncture-resistant containers.
  • Use protective laboratory coats and gloves.
  • Wash hands following all laboratory activities, following the removal of gloves, and immediately following contact with infectious materials.
  • Decontaminate work surfaces before and after use, and immediately after spills.
  • Do not eat, drink, store food, or smoke in the laboratory.

These simple and effective work practices can be implemented readily by laboratory management at minimal cost and with no loss of employee efficiency or productivity. Even in the absence of more sophisticated means for providing safety in the laboratory, these practices can achieve a major reduction in the risk of accidental infection.

Laboratory activities that pose the risk of infection via airborne aerosols or droplets demand the use of special safeguards. For many "airborne pathogens," the human infectious dose may be as low as one viable microorganism, as demonstrated for tuberculosis[107 ][108 ]. It is recommended that biological safety cabinets or other primary containment devices be used for all manipulations of materials, including clinical specimens, known to contain or suspected of containing microorganisms capable of infecting by the respiratory route. In laboratories where such materials are handled, the ventilation system should provide directional airflow from "clean" to "contaminated" areas, and the air should not be recirculated.

The recommended procedures listed above, targeted at minimizing overt occupational exposures, constitute the basic essentials of good laboratory practice. Furthermore, these procedures are also effective in reducing or eliminating overt exposure to the variety of indigenous bacterial, viral, fungal, and parasitic agents present in the community and commonly found in clinical material submitted to the laboratory for examination. The ultimate responsibility for assessing the risk of occupational infections and for implementing appropriate practices, as well as for providing adequate facilities and containment equipment, rests with the laboratory director.

5. Summary

Virtually all laboratory procedures have the potential to disperse infectious material into the workplace. Laboratory workers should be aware of these potential hazards and exercise a high degree of care during all manipulations of infectious materials. As evidenced by the data accumulated in the review of laboratory-acquired infections by Pike[101 ], exposure of laboratory workers is not often associated with overt accidents. More than 80 percent of laboratory-associated infections could not be ascribed to any specific event. It is critical, therefore, that laboratory workers recognize that good microbiological practices are required to prevent exposure to infectious agents. These practices are described in more detail in subsequent sections of this chapter.

G. TRANSPORTATION AND SHIPMENT OF SPECIMENS

1. Introduction

Although it is obvious that biological specimens should be properly packaged, labeled, shipped, and received, concerned national and international organizations have found it necessary to develop recommendations and guidelines because of the fear of accidents and spills involving such materials[71 ][86 ][147 ][148 ]. Federal regulations govern the packaging and shipping of hazardous materials. The importation and subsequent transfer between laboratories of etiologic agents and vectors of plant, animal, and human diseases (including zoonotic agents) are controlled through permit systems.

2. Packaging. Shipping, and Handling of Biological Specimens

The shipment of diagnostic specimens, biological products, and etiologic agents concerns everyone involved in the process. Infectious materials that are properly packaged and handled may pose considerably lower risks of accidental exposure for nonlaboratory personnel who come in contact with the shipment in transit. Proper packaging also may ensure considerate and prompt handling of valuable specimens.

The shipping of unmarked and unidentified etiologic agents is prohibited. Requirements for the proper method of containment in the packaging and the use of the hazardous warning label are stipulated in the U.S. Public Health Service Interstate Shipment of Etiologic Agents Regulation[129 ]. Comparable requirements of the International Civil Aviation Organization (ICAO) apply to the international shipment of diagnostic specimens and infectious agents.

The containment packaging and hazard warning labeling specified in the U.S. Public Health Service Regulation[129 ] for the shipment of etiologic agents is illustrated below in Figure 3.1. The package should consist of

FIGURE 3.1. Containment packaging and hazard warning labeling specified by the U.

FIGURE 3.1

Containment packaging and hazard warning labeling specified by the U.S. Public Health Service. Reprinted from U.S. Code of Federal Regulations, Title 42, U.S. Public Health Service, Part 72.

  • a securely closed, watertight primary container (test tube, vial, or ampoule);
  • a durable, watertight secondary container; and
  • a tertiary or outer shipping container.

The space between the primary and secondary container must be filled with absorbent material sufficient to absorb the contents of the primary container should there be leakage during transit. The outside of the primary container should be examined and cleaned to remove blood, feces, or other contaminants before it is packaged for shipment.

The exteriors of packages containing cultures of, or suspensions of, etiologic agents should have affixed to them the "Etiologic Agent—Biomedical Materials" hazard warning label illustrated in Figure 3.1. The packaging and the labeling requirements of the regulation cited also apply to the local transport of etiologic agents and diagnostic specimens by courier or by other delivery services. Similar requirements and restrictions applicable to the shipment of etiologic agents, diagnostic specimens, and biological products by all modes of transportation (i.e., air, motor, rail, and water) are imposed by the Department of Transportation[131 ] and the U.S. Postal Service (Postal Service Manual), as well as by airline carriers and pilots' associations.

The importation of etiologic agents of human diseases, as well as their subsequent transfer within the United States, is regulated by the U.S. Public Health Service (USPHS)[128 ]. The U.S. Department of Agriculture (USDA) similarly regulates the importation and transfer of etioloic agents of plant and animal diseases[125 ]. In addition, the USDA Animal and Plant Health Inspection Service (APHIS)/Veterinary Services (VS) Memorandum 593.1 establishes procedures for the "Importation of Cell Cultures Including Hybridomas." Examples of the appropriate USPHS and USDA application forms and permits are included in Appendix E.

A summary of the requirements of the federal agencies involved in the shipment of biological specimens has been published recently by the American Type Culture Collection (ATCC) (Rockville, MD 20852-1776). This document also describes the procedures used for packaging and shipping the different types of cultures of microorganisms and cells maintained by the ATCC[3 ].

Procedures for receiving and unpacking etiologic agents or other potentially infectious materials should be established by laboratories receiving these items. Often, such materials are received initially by shipping, clerical, or other nonlaboratory personnel. These employees should be given specific instructions to notify laboratory staff promptly of the arrival of such materials, and to deliver packages unopened directly to a designated area or person. Shipments of etiologic agents or diagnostic specimens should never be opened in offices or in shipping and other nonlaboratory areas.

In the laboratory, the designated specimen-receiving area should meet the facilities recommendation for Biosafety Level 2 [86 ][105 ]. Microbiological practices, including the wearing of laboratory coats, gloves, or other protective clothing, should be followed as applicable. A Class I or Class II biological safety cabinet provides the most suitable work station for opening packages and for the initial handling of incoming specimens[86](see Chapter 3, Section J). Specimens that show any evidence of damage or leakage should be opened in a biological safety cabinet only by trained personnel wearing appropriate protective clothing.

Laboratories should have emergency contingency plans[147 ][148 ] for handling damaged shipments. Such plans are best prepared by the laboratory supervisor in conjunction with the laboratory staff and the safety officer. Emergency plans should be posted in a conspicuous place in the laboratory for immediate reference. Emergency plans should provide written procedures for dealing with

  • breakage or spillage of infectious materials,
  • exposure of personnel to infectious materials by accidental injection, cuts, or other injuries,
  • accidental ingestion or contact of mucous membranes with potentially hazardous material, and
  • aerosols.

Such emergency plans should include the following:

  • decontamination procedures,
  • emergency services (whom to contact), and
  • emergency equipment and its location.

H. LABELING OF SPECIMENS WITHIN THE LABORATORY

Some form of labeling is necessary to maintain the identity of specimens in the laboratory and to ensure that the analytical results obtained are properly recorded and reported. In addition, it is the practice in many cases (and may be required as a condition for accreditation) that special hazard warning labels be affixed to specimens that are known to be hazardous (e.g., specimens obtained from patients known to be infected with hepatitis B virus (HBV) or human immunodeficiency virus (HIV), or from patients in high-risk groups for these infections, or when previous tests of the specimen have shown it to contain an etiologic agent).

The need for such special labeling is concerned more with ethical or regulatory issues (e.g., workers' right-to-know) than with laboratory safety. Unfortunately, the use of special hazard warning labels can inadvertently lead to the dangerous misconception that other clinical specimens, not so labeled, can be handled with less caution. Two levels of laboratory practice may thus evolve: one for handling hazard-labeled specimens and another for unlabeled samples. This must be scrupulously avoided: all clinical material must be considered to be infectious, and must be handled with exactly the same precautions as are used for processing specimens with hazard warning labels.

It is generally recognized that any clinical specimen may contain infectious agents (such as HBV or HIV) regardless of its source, the working clinical diagnosis, or the testing requested. For example, published reports indicate that from 1.0 to 1.5 percent of the adults in the United States have serological markers indicative of current or previous HBV infection[66 ]. Thus, even though the percentage of specimens containing an infectious agent may be higher among samples collected from hepatitis patients, the total number will probably be greater among routine specimens, which make up the vast majority of the materials received in most laboratories. The potential for worker exposure may, therefore, be actually greater from the more numerous routine specimens, which would not be identified with a hazard warning label. From the above considerations, it is clear that the "Universal Precautions" described by the CDC[34 ][38 ] must be followed when handling all clinical specimens, whether labeled or unlabeled.

I. PREVENTION OF AEROSOL AND DROPLET GENERATION

1. Introduction

Exposure to microorganisms dispersed or spread in the form of infectious aerosols or droplets is an important source of laboratory-acquired infection. Infectious aerosols may be composed of dry or liquid particles typically less than 5 microns in diameter, which can be produced during the course of many common laboratory processes. Such aerosols do not settle quickly and can be dispersed widely through a ventilation system or otherwise carried long distances by air streams. If inhaled, the particles in an aerosol are carried to the alveoli of the lungs. In contrast, droplets (particles typically larger than 5 microns in diameter) remain airborne only for a short period of time and are nonrespirable. Because of their mass, droplets tend to settle quickly on inanimate surfaces, or may be deposited on skin or mucous membranes of the upper respiratory tract. Accordingly, droplets pose risks of infection associated with direct or indirect contamination of the mucous membranes of the eyes, nose, or mouth as well as of skin, clothing, and laboratory equipment.

2. Control of Aerosols and Droplets

Almost any handling of liquids or of dry powders is likely to generate aerosols and droplets; certain operations such as pipetting, mixing, shaking, grinding, filtering, sonicating, flaming, and centrifuging have a high potential for aerosol production. Of these, pipetting may be the most important. Various reports indicate that pipettes are associated with many laboratory-acquired infections[98 ][99 ][100 ][102 ][104 ][120 ]. Hazards relating to pipetting include the production of aerosols, aspiration of fluid into the mouth, and contamination of the mouthpiece by the operator's finger. The last two of these dangers can be avoided if mouth pipetting is strictly prohibited, as required by good laboratory practice. A wide variety of mechanical pipetting devices are available[63 ], and mouth pipetting under any circumstances is absolutely unacceptable.

To minimize aerosol production, pipettes should be drained gently with the tip against the inner wall of the receiving tube or vessel. No infectious material should be expelled forcibly from the pipette, and air should never be bubbled through a suspension of infectious agents in an open container. When handling organisms for which Biosafety Level 3 precautions are indicated (e.g., etiologic agents of tuberculosis, systemic mycoses, or Q fever), it is recommended that pipetting procedures be carried out in a biological safety cabinet. The equipment used in the other operations mentioned above should be selected for features designed to contain infectious liquids or aerosols. For example, blenders should have leakproof bearings and a tight-fitting gasketed lid. Blender bowls, tubes, and other devices likely to contain aerosols should be opened, filled, and emptied in a biological safety cabinet.

Centrifuges with sealed buckets, safety trunnion cups, or sealed heads are effective in preventing escape of liquids and aerosols (Figure 3.2). If fluid should escape from a cup or rotor during high-speed operation, the potential for extensive contamination and multiple infections is great. For many specimens, however, such as urine, the standard clinical centrifuge is satisfactory. There have been comparatively few centrifuge accidents reported as the cause of laboratory-acquired disease, but some of these caused multiple infections because the accident created a large volume of infectious aerosol[141 ].

FIGURE 3.2. If a fluid containing an infectious agent were to escape from a centrifuge rotor or cup during high-speed operation, the potential for extensive contamination and multiple infections would be great.

FIGURE 3.2

If a fluid containing an infectious agent were to escape from a centrifuge rotor or cup during high-speed operation, the potential for extensive contamination and multiple infections would be great. The use of sealed buckets, safety trunnion caps, or (more...)

Instruments should be checked regularly to ensure that leakage does not occur during operational procedures. For ultracentrifuges, a HEPA filter should be installed between the chamber and the vacuum pump. If circumstances require such precautions, centrifuges and other laboratory instruments that can be enclosed and operated in specially designed safety cabinets are available. Only those instruments and cabinets intended for such a combined system should be used together, otherwise the expected containment may not be achieved. For example, the airstreams created by an ordinary benchtop centrifuge operating in the work space of an ordinary Class II biological safety cabinet can easily overwhelm the protective air curtain.

Sputum and other clinical specimens submitted for culture may contain unsuspected microorganisms, such as mycobacteria, which are highly infectious by the airborne route. Every effort should be made, therefore, to minimize the risk of their aerosolization. If generation of an aerosol is likely to occur during the processing of these specimens, the use of a biological safety cabinet is recommended strongly for these procedures.

Improper technique in the flaming of inoculating loops can result in the spread of infectious agents. Spatter and release of droplets or aerosols can be prevented by such methods as heating the shaft until the sample has been heat-dried before flaming the loop itself (Figure 3.3). Spatter can also be controlled effectively by using a side-arm burner or electric microincinerator. Flaming itself can be avoided by using sterile, disposable plastic loops.

FIGURE 3.3. Proper technique in the flaming of inoculating loops is an important way to prevent the spread of infectious agents.

FIGURE 3.3

Proper technique in the flaming of inoculating loops is an important way to prevent the spread of infectious agents. Courtesy, National Institutes of Health.

Early models of certain laboratory instruments, such as cell sorters and other automated devices, were not designed for containment and may be a source of inadvertent contamination in the workplace. Later models generally have overcome the problem, but users are advised to test all equipment carefully in order to identify any biological hazards associated with its operation.

Regardless of the type of equipment used or the task performed, the objective is to prevent aerosol release and to avoid exposure of personnel. These ends can be accomplished by the laboratory practices described above and by the use of appropriate equipment, especially biological safety cabinets. Leaks or escape of aerosols can be detected by using an indicator such as fluorescein. It may be added to a sham specimen or to water, and processed with the system or procedure being tested. Its presence can then be determined on surfaces, on material collected from key locations, or in specimens from air samplers, by using an ultraviolet lamp for excitation. Other suitable methods may be devised.

J. CONTAINMENT EQUIPMENT

1. Introduction

The risk of exposure of laboratory personnel can be minimized by the use of carefully selected safety equipment. A primary objective of containment is to control aerosols, but in a broader sense safety equipment should serve effectively to isolate the worker from the toxic or infectious material being processed. In many situations, however, the need is just the reverse: i.e., to protect the product or the work from contamination originating with the worker or the environment. Finally, there is often the need to protect both the worker and the product, as in handling cell cultures and some clinical specimens, or in surgical procedures. The following examples are representative of the types of equipment designed to avoid the most common laboratory hazards, and these types of equipment are, for that reason, among the most important.

2. Biological Safety Cabinets

Most laboratory procedures generate aerosols that may spread infectious material in the work area and pose a risk of infection to the worker. Biological safety cabinets are used extensively to prevent the escape of aerosols or droplets and to protect materials from airborne contamination (Figure 3.4). There are three major types of this very useful safety device, referred to as Class I, Class II, and Class III. These instruments are distinct from horizontal or vertical laminar flow "clean benches," which should never be used for handling infectious, toxic, or sensitizing material. The Class I biological safety cabinet is an enclosure with an inward airflow through the front opening. It may be configured with a full-width open front or with an installed front closure panel to which arm-length rubber gloves may be attached. The exhaust air from the biological safety cabinet is passed through a HEPA filter so that the equipment provides protection for the worker and environment. The product in the cabinet, however, is subject to contamination by organisms that may be present in the air supply.

FIGURE 3.4. Biological safety cabinets, combined with protective gloves and laboratory coats, provide effective isolation of the worker from the toxic or infectious material being handled.

FIGURE 3.4

Biological safety cabinets, combined with protective gloves and laboratory coats, provide effective isolation of the worker from the toxic or infectious material being handled. Courtesy, John H. Richardson.

Class II biological safety cabinets provide protection to the worker, the environment, and the product. The airflow velocity at the face of the work opening is at least 75 linear feet per minute (lfpm), and both the supply and the exhaust air are HEPA-filtered. Class I and Class II cabinets are partial containment devices, which, if used in conjunction with good laboratory practices, can dramatically reduce the risk of exposure of operators to infectious aerosols and droplets.

Figure 3.5[73 ] shows the airflow patterns and operating velocities for the five types of Class I and Class II biological safety cabinets produced in the United States. All of these biological safety cabinets provide a comparable level of protection for the user against exposure to infectious aerosols and droplets, in that the velocity of the protective inward airflow through the work opening is essentially the same. The air quality within the Class I cabinets reflects that of the laboratory room from which it is drawn, since there is no filtration of the supply air. The Class II types provide a very high quality, low-particulate or particulate-free atmosphere within the work chamber. Class IIA cabinets are generally suitable for procedures involving clinical specimens, and thus are the most commonly used biological safety cabinet.

FIGURE 3.5. Airflow characteristics of Class I (negative pressure) and Class II (vertical laminar flow) biological safety cabinets.

FIGURE 3.5

Airflow characteristics of Class I (negative pressure) and Class II (vertical laminar flow) biological safety cabinets. Adapted from National Sanitation Foundation Standard 49 (revised May 1983) by G.P. Kubica, Centers for Disease Control.

It is emphasized that biological safety cabinets are not chemical fume hoods. Some of the air (30 to 70 percent) drawn in through the work opening of Class IIA, IIB1, and IIB3 cabinets is recirculated within the cabinet. Accordingly, users should be aware of the possible buildup of hazardous concentrations within the cabinet if toxic, flammable, or explosive materials are used. In addition, users of Class IIA cabinets should know that nonparticulate toxic, flammable, or explosive materials are not removed by HEPA filters, and are thus discharged back into the laboratory room.

Class IIB3 units are functionally the same as those of Class IIA except that the exhaust air from the former is ducted to the outside directly or via a nonrecirculating exhaust system rather than back into the laboratory room. Class IIB1 and IIB2 cabinets exhaust 70 percent and 100 percent, respectively, of the intake air and provide containment of infectious aerosols. Those contemplating the purchase of Class IIB1 or IIB2 cabinets should be aware of their high air demand (700 to 1200 ft3/min), increased energy requirements, and higher purchase and operating costs.

The Class III cabinet is a totally enclosed, gastight work space equipped with protective gloves. It is ventilated with HEPA-filtered air and operated with a negative air pressure of at least 0.5 inches of water in the cabinet work space. The exhaust air is passed through two HEPA filters, installed in series, before being discharged to the outside of the building, usually through a dedicated exhaust system. Class III cabinets provide the highest level of worker, product, and environmental protection and are appropriate for work with exotic high-risk biological agents, including those in the Biosafety Level 4 category.

The operational efficiency of each biological safety cabinet should be specifically tested and the system certified before the instrument is placed in operation after installation, and subsequently on an annual basis. Recertification is also required if the unit is relocated or if maintenance that may affect performance is done. Maintenance work on biological safety cabinets should be performed by trained service personnel only (see Chapter 5, Section B). In addition, cabinet users should understand the operation of the equipment, its limitations, and the proper procedures to be followed. Laboratory directors are responsible for providing such training.

3. Pipetting Devices

Pipettes are among the most commonly used pieces of equipment in the biomedical laboratory, and their misuse has been related to a significant number of laboratory-acquired infections[100 ]. Regrettably, many laboratory workers were taught to pipette by mouth, even after the associated hazards were recognized. These individuals should be required to give up the old practice and learn to use the pipetting aids that are now available for any application[63 ](Figure 3.6). The importance of these aids cannot be overemphasized, and any device requiring mouth suction should be considered unsafe and inappropriate for use in the biological laboratory. Mouth pipetting of any material under any circumstances should be explicitly prohibited.

FIGURE 3.6. The wide variety of available pipetting aids make mouth pipetting an unnecessary and obsolete practice.

FIGURE 3.6

The wide variety of available pipetting aids make mouth pipetting an unnecessary and obsolete practice. Courtesy, John H. Richardson and National Institutes of Health. (Figure continued on next page.)

4. Sonicators, Homogenizers, and Mixers

Operation of these or similar instruments may create hazardous aerosols and lead to exposure of personnel unless extreme caution is exercised. If indicated by the characteristics of the material being processed or the agents involved, the instrument should be operated in a biological safety cabinet. Blenders should be designed to prevent leakage from the rotor bearing or at the cover. Caps and gaskets should be in good condition and the system checked to ensure that leakage does not occur during operation. When the risk of exposure to infectious aerosols is present, blender bowls, tubes, and other containers should be opened in a biological safety cabinet.

5. Clothing, Masks, and Face Shields

Laboratory coats, gowns, safety glasses, face shields, masks, and gloves offer some personal protection and are often used in combination with other safety devices such as biological safety cabinets. Special laboratory clothing protects street wear from contamination. It should not be worn outside of the laboratory. Each of these items has a particular use in protecting the worker and should be used when circumstances require. Gloves are especially important when handling any potentially infectious material such as blood or other biological specimens. Safety glasses, face shields, and masks may protect mucous membranes of the eye, nose, and mouth from splash or droplet hazards during operations performed outside of a biological safety cabinet.

K. BIOSAFETY IN LARGE-SCALE PRODUCTION

1. Introduction

Microbial cultures of greater than 10 liters in volume (defined as large-scale[136 ]) present biosafety concerns very similar to those described for small-scale (<10 liters) experiments in the laboratory. All the recommendations described in the CDC/NIH guidelines (Appendix A) for laboratory-scale research should, therefore, be followed for large-scale production, with the addition of the recommendations described below. These recommendations are equally applicable to cultures as small as 20 liters and as large as 10,000 liters.

This section addresses only issues of biological safety relative to infectious agents and does not deal with other major areas of potential risk in large-scale production (e.g., end-products, by-products, media components, and nonviable biological agents). These subjects exceed the scope of this book, but other sources[118 ], and references contained therein, provide pertinent information. Similarly, this section does not address the biological safety issues that pertain to the large-scale growth of mammalian cell cultures. This topic is discussed briefly in Section D of this chapter.

The physical containment typically implemented for large-scale production serves to protect the worker. The self-contained design used for large-scale production should essentially eliminate the generation of aerosols, one of the most common causes of laboratory infections[100 ]. Standard operating procedures (SOPs)—including validation of equipment's function, biological disposal, and specific methods of operation—should be written and closely followed for all large-scale productions. Implementation of these protocols should facilitate the maintenance of a safer environment in which to work and produce the highest quality of resultant product.

Special circumstances inherent in large-scale production may actually reduce risks to levels lower than those encountered in laboratory research. Potential biological hazards associated with the specific organisms used for large-scale production will typically have been defined and assessed in the research and developmental stages before large-scale production is initiated. Because fewer biological unknowns or variables are present, the biological hazards should be reduced. This situation is especially true when comparing large-scale culture of well-characterized microorganisms with laboratory-scale culture of clinical isolates or of soil isolates, where a tremendous diversity of unknown organisms is present. When the proper precautions are implemented and maintained, large quantities of potentially hazardous microorganisms may be grown safely[65 ][135 ]. Many of the organisms or cell lines of interest for production purposes (e.g., Bacillus and yeast) have had an extended industrial history of safety on the basis of which risks can be accurately and confidently evaluated.

Image img00010.jpg

Where large-scale production is initiated in the process of commercialization of a desired product, the constraints enforced to ensure product integrity typically result in increased safety to personnel as well. As numerous laboratories have developed large-scale production facilities for the commercialization of biological products, the need for carefully designed and implemented procedures has become increasingly important for the safety of laboratory workers, the community, and the environment. The following procedures are intended to allow handling of suspected or known hazardous organisms at large-scale levels with proper concern for health and safety. The recommendations are not intended to apply rigidly to all situations. As experience is gained in scale-up operations, more or less stringent guidelines for individual operations may be indicated by the safety department, biological safety officer, or biosafety committee. The procedures reflect the best judgment of acceptable techniques and applicable legal requirements[4 ][13 ][18 ][51 ][82 ][105 ][124 ][129 ][130 ][132 ][133 ][134 ][136 ]. The recommendations rely heavily on the NIH ''Guidelines for Research Involving Recombinant DNA Molecules"[136 ], which have proved dependable as a foundation for laboratory safety programs whether or not recombinant DNA is involved. These recommendations are broadly applicable because they are based on the potential pathogenicity or infectivity for the host. These procedures incorporate safety concepts and guidelines published in documents of the National Institutes of Health[133 ][134 ][136 ], the Centers for Disease Control, in conjunction with the National Institutes of Health[105], the U.S. Department of Agriculture[124 ], the American Industrial Hygiene Association[4 ], and the Medical Research Council of Canada[82 ].

2. Organization and Responsibilities

Institutions planning to scale up the production of biotechnology-related products should have, at least, a safety department, a biological safety officer, and a biosafety committee, with the responsibilities described in Chapter 5. Scientists, technicians, equipment workers, and maintenance and custodial personnel with access to the large-scale production area should all be considered candidates for medical surveillance, depending upon the organism being grown and the product produced.

3. Containment

Containment levels for organisms such as fungi, bacteria, and viruses are grouped into classes according to their perceived or potential hazard to humans. Selection of an appropriate biosafety level for work with a particular agent depends upon such factors as the virulence, pathogenicity, biological stability, mode of transmission, endemic nature, and communicability of the agent; the function of the laboratory; the procedures and manipulations of the agent; the availability of effective vaccines or therapeutic measures; and the quantity and concentration of the agent[105 ]. The NIH "Guidelines for Research Involving Recombinant DNA Molecules"[136 ] considers cultures greater than 10 liters in volume to be large-scale and, for specific organisms, to require higher levels of physical containment than cultures less than 10 liters in volume. Increased containment for large-scale culture is typically reflected in the requirement for more extensive physical design, and not for operating at a higher biosafety level.

The risks associated with the use of microbial cultures are controllable through two containment approaches. For some work, an appropriate approach is biological containment: the use of species or strains that pose a reduced risk to workers or to the environment. The more widely applicable approach, however, is physical containment: a combination of laboratory practices, design, and equipment. Physical containment, through the implementation of good laboratory practices and proper monitoring, certification, and maintenance of the facility, leads to a significantly safer and more productive work environment.

A National Institute for Occupational Safety and Health (NIOSH) survey of six biotechnology companies[51 ]showed that companies with many years of experience in fermentation technology tended to emphasize more sophisticated, more effective, and safer practices for handling infectious agents than newly established companies.

All facilities and equipment used to provide containment should be tested before initiation of a program and periodically thereafter by trained personnel. Such monitoring should include checks of room air balance, biological safety cabinets, supply and exhaust filters, sterilizers, and centrifuges. Laboratory personnel should ensure that biological safety cabinets are appropriately certified prior to use. A systematic, scheduled program of preventive maintenance should be implemented for agitator seals, control valves, pressure relief valves, and equipment and facility safeguards[51 ]. Sterilization of fermentors, feed lines, feed tanks, inoculating devices, exhaust ports, sampling ports, and extraction devices should be validated routinely.

Maintaining, testing, or cleaning of facilities or equipment should not be allowed until all surfaces needing servicing have been decontaminated. Spills should be disinfected by laboratory personnel before housekeepers are allowed to give assistance.

Typically, large-scale production facilities are specifically engineered for maximal physical containment to ensure both personnel safety and product integrity. The specific minimum requirements for physical containment are described in the NIH "Guidelines for Research Involving Recombinant DNA Molecules"[136 ]. The large-scale containment classifications, BSL1-LS, BSL2-LS, and BSL3-LS (Biosafety Levels 1, 2, and 3, large-scale, respectively) are required for organisms for which the corresponding containment levels BSL1, BSL2, and BSL3 (Biosafety Levels 1, 2, and 3, respectively) are required for small-scale (<10 liters) research. No provisions are made for large-scale growth of microorganisms that require the Biosafety Level 4 containment at the laboratory scale. (Consult Appendix A for the small-scale containment levels for specific microorganisms.) The appropriate implementation of safeguards and of protective engineering controls at the time of the design of the facility or laboratory can reduce human exposure and avoid expensive retrofitting. Specific details of engineering and design features that reduce the potential biological risks associated with large-scale microbial production can be found in the NIH guidelines[136 ].

4. Inactivation

Vessels used for large-scale production are typically steam sterilized in place, in the absence or presence of medium, prior to fermentation. Temperature-sensitive tapes, crayons, or, preferably, thermocouples, may be used to monitor sterilization procedures. The growth medium is either sterilized in the system or sterile medium is introduced via a closed system designed to maintain physical containment. Connections should be sterilizable in place. Metal containers, designed to preclude escape or entry of viable organisms during inoculation, should be used. These inoculation vessels should be sterilizable after the transfer process, and prior to their removal from the production vessel, to minimize release.

Following growth, cultures in the production vessel should be inactivated either chemically or thermally prior to initiating the product recovery processes. Inactivation is used in this context to refer to the reduction in the total number of viable target microorganisms in a large-scale culture to a number comparable to or less than that obtained in a small-scale laboratory culture (<10 liters). This reduction enables a culture that is grown in a large-scale, closed system to be handled according to the containment level applicable for the corresponding laboratory-scale culture. Inactivation, in this context, is therefore an interim action that is to be followed by decontamination prior to the disposal of microbial cultures. As an alternative to inactivating cultures in the production vessel, cultures can be inactivated by cell rupture or by further steps in subsequent stages of the process, if these are designed into the same closed system. When the viable biological agent itself is the desired end-product (e.g., some vaccines), the culture would not be inactivated but would be maintained within a closed system to avoid human exposure. The specific method of inactivation (see Chapter 4) depends on the organism or cell culture employed and upon the product to be isolated. Inactivation efficiencies should be validated as described in the SOPs. Physical containment during processing steps following culture inactivation (i.e., steps involved in the recovery and purification of the defined product) is not required but is recommended when possible and feasible. All liquid and solid biological waste should be decontaminated chemically or thermally prior to disposal. In-line thermocouples are especially effective in validating the thermal inactivation of large quantities of liquid cultures prior to disposal. If other primary equipment, such as centrifuges, is used in-line for harvesting cells prior to inactivation, this equipment should also be decontaminated appropriately.

Water supplies or storage tanks provide ideal entry points for biological contaminants. Treatment of water with ultraviolet light, ozone, or passage through specifically selected filters significantly reduces the potential for its contamination. The quality and type of water processing should be specified in the SOPs.

5. Disposal

All biological waste generated through routine procedures or as the result of accidents should be decontaminated prior to its disposal, and should be segregated from nonbiological waste and from radioactive waste. Solid trash and small volumes of waste should be disposed of by using the procedures described for laboratory research. Contents of the production vessel should be physically contained (e.g., with dikes or decontamination tanks) to confine spills and leaks and to allow for their rapid and efficient decontamination. All liquid collected by these procedures should be properly decontaminated by using validated procedures to prevent the release of viable organisms or cells into the environment. The contents and all associated materials and equipment should be inactivated either chemically or physically prior to disposal. If decontamination tanks are used, the method of inactivation should be validated for each organism employed. Once inactivated, even large quantities of liquid culture can be disposed of by discharge to the sewer lines, provided that such action is permitted by the relevant state and local agencies. Air discharged from fermentors should be filtered through a HEPA filter, incinerated, or otherwise treated prior to release.

Personnel involved in the cleanup of accidentally spilled waste should proceed as described in Chapter 5 for spills with laboratory-scale cultures. Particular precautions should be taken to handle and decontaminate these large quantities of culture, as well as the absorption materials used for cleanup.

6. Exposure

The self-contained design of the large-scale production system, the inoculation method, and the in-place inactivation of the vessel contents following production essentially eliminate the release of aerosols, thereby reducing human exposure. Nonetheless, air and surfaces should be monitored to validate the integrity of the systems during the fermentation or processing procedures. Surfaces can be monitored for microbial release by any of five basic methods: rinse, swab-rinse, agar contact, direct surface agar plating, or vacuum probe surface method[4 ]. Other procedures are modifications of these basic methods. In a survey of six genetic engineering companies, agar contact was found to be the method most frequently used[51 ]. Organism detecting and counting (RODAC) plates are commercially available with a variety of media and are used routinely. The method works well and rapidly as a qualitative procedure.

Numerous methods are available to sample airborne microorganisms[4 ]. The more frequently used methods include settling plates, air impingers, and filters. Settling plates provide qualitative information and consist simply of an open petri dish containing appropriate culture medium onto which particles settle due to gravity. Air impingers are intrinsically more quantitative because they pull air at a fixed rate and volume onto the surface of the medium. Large and defined quantities of airborne microorganisms can be concentrated and analyzed. The most common method for sampling air is filtration. A fixed volume of air can be passed through a filter of a selected pore size. Particles and organisms can be flushed from the filter, and the filtrate analyzed on an appropriate medium, or the filter itself can be overlaid directly with an appropriate medium. The use of filters is limited, because of dehydrating effects, to the detection of spores and of resistant vegetative cells. Selection of an appropriate sampling method depends upon the nature of the particles of interest, their expected concentration, and the need for quantification. The reader can consult Table XVIII in reference 6 for a more detailed description of these and alternative sampling methods.

Surface and air sampling methods should be selected to detect biological contaminants, as well as the production organism itself. Adventitious agents expected in large-scale production are similar to those found in laboratory research (e.g., viruses, bacteria, and fungi). Potential candidates depend upon the specific organism under study and the medium employed.

7. Conclusion

Large-scale microbial culture operations can be done safely, despite the risks associated with some microorganisms. Personnel can function safely and efficiently at any scale by using the proper facilities and equipment.

L. BIOSAFETY IN PHYSICIANS' OFFICE LABORATORIES AND OTHER SMALL VOLUME CLINICAL LABORATORIES

Each small laboratory, including those in physicians' offices, should have a safety program even if the laboratory has only one employee (see Chapter 5). For those employees without training in the biological sciences, special effort should be made to provide simple but effective instruction. The program should be tailored to the laboratory function, with proper emphasis on providing training in the seven basic rules of good microbiological practices as outlined in Section F of this chapter. Indeed, all of the general principles outlined in this chapter for the safe handling of infectious agents apply to small-volume laboratories as well as large-volume ones.

Many of these small-volume laboratories may be handling human blood or blood components that could be infectious. If this is the case, the standard biological practices as well as the special practices recommended by the CDC/NIH guidelines for Biosafety Level 2 should be used (see Appendix A), and the Universal Precautions described in Appendix C should be followed. Prudence should also be exercised to minimize exposure to toxic chemicals and radionuclides.

Special precautions should be taken to ensure that waste is managed in a safe, responsible manner. Potentially infectious waste should preferably be decontaminated on site. Liquid waste, which may be contaminated with an infectious agent, can be steam autoclaved or decontaminated by using a chemical disinfectant that is effective for the intended use. Decontaminated liquids can than be poured carefully down a drain connected to the sanitary sewer. Solid waste, including contaminated "sharps" (e.g., hypodermic needles and broken glassware), should be packaged in sealed, leak- and puncture-resistant containers for transport and disposal. Such properly contained waste presents minimal risk to waste handlers and can be safely managed as municipal solid waste. If this practice is prohibited by local regulations, then the prevailing practices should be followed. Human excreta should be disposed of through the sanitary sewer. The practices just described are appropriate for the small laboratory that is involved in testing patients' specimens. The safe disposal methods discussed in Chapter 4 should be followed in the small biomedical laboratory where infectious agents are isolated and grown in cultures.

Further information on safety in the office laboratory may be found in references 12 and 54.

Copyright © 1989 by the National Academy of Sciences.
Bookshelf ID: NBK218635
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