BiosafetyChapter One

The purpose of this manual is to be a resource for information, guidelines, policies, and procedures that will enable and encourage those working in the laboratory environment to work safely and reduce or eliminate the potential for exposure to biological, chemical and radioactive materials hazard.

‘Laboratory biosafety’ is the term used to describe the containment principles, technologies and practices that are implemented to prevent unintentional exposure to pathogens and toxins, or their accidental release.

Practices, safety equipment, and facility design and construction are appropriate for undergraduate and secondary educational training and teaching laboratories, and for other laboratories in which work is done with defined and characterized strains of viable microorganisms not known to consistently cause disease in healthy adult humans. Bacillus subtilis, Naegleria gruberi, infectious canine hepatitis virus, and exempt organisms.

Practices, equipment, and facility design and construction are applicable to clinical, diagnostic, teaching, and other laboratories in which work is done with the broad spectrum of indigenous moderate-risk agents that are present in the community and associated with human disease of varying severity. With good microbiological techniques, these agents can be used safely in activities conducted on the open bench, provided the potential for producing splashes or aerosols is low. Hepatitis B virus, HIV, the Salmonella, and Toxoplasma are representative of microorganisms to this containment level. BSL-2 is appropriate when work is done with any human-derived blood, body fluids, tissues, or primary human cell lines where the presence of an infectious agent may be unknown.

Practices, safety equipment, and facility design and construction are applicable to clinical, diagnostic, teaching, research, or production facilities in which work is done with indigenous or exotic agents with a potential for respiratory transmission, and which may cause serious and potentially lethal infection. Mycobacterium tuberculosis, St. Louis encephalitis virus, and Coxiella burnetii are representative of the microorganisms assigned to this level. Primary hazards to personnel working with these agents relate to autoinoculation, ingestion, and exposure to infectious aerosols. At BSL-3, more emphasis is placed on:

Practices, safety equipment, and facility design and construction are applicable for work with dangerous and exotic agents that pose a high individual risk of life-threatening disease, which may be transmitted via the aerosol route and for which there is no available vaccine or therapy. Agents with a close or identical antigenic relationship to BSL-4 agents also should be handled at this level. When sufficient data are obtained, work with these agents may continue at this level or at a lower level. Viruses such as Marburg or Congo-Crimean hemorrhagic fever are manipulated at BSL-4.

Classification of infective microorganisms by risk group:

The backbone of the practice of biosafety is risk-assessment. While there are many tools available to assist in the assessment of risk for a given procedure or experiment, the most important component is professional judgment. Risk assessments should be performed by the individuals most familiar with the specific characteristics of the organisms being considered for use, the equipment and procedures to be employed, animal models that may be used, and the containment equipment and facilities available. The laboratory director or principal investigator is responsible for ensuring that adequate and timely risk assessments are performed, and for working closely with the institution's safety committee and biosafety personnel to ensure that appropriate equipment and facilities are available to support the work being considered. Once performed, risk assessments should be reviewed routinely and revised when necessary, taking into consideration the acquisition of new data having a bearing on the degree of risk and other relevant new information from the scientific literature. One of the most helpful tools available for performing a microbiological risk assessment is the listing of risk groups for microbiological agents. However, simple reference to the risk grouping for a particular agent is insufficient in the conduct of a risk assessment. Other factors that should be considered, as appropriate, include:

On the basis of the information ascertained during the risk assessment, a biosafety level can be assigned to the planned work, appropriate personal protective equipment selected, and standard operating procedures (SOPs) incorporating other safety interventions developed to ensure the safest possible conduct of the work.

The risk assessment procedure described above works well when there is adequate information available. However, there are situations when the information is insufficient to perform an appropriate risk assessment, for example, with clinical specimens or epidemiological samples collected in the field. In these cases, it is prudent to take a cautious approach to specimen manipulation.

The affected individual should remove protective clothing, wash the hands and any affected area(s), apply an appropriate skin disinfectant, and seek medical attention as necessary. The cause of the wound and the organisms involved should be reported, and appropriate and complete medical records kept.

Protective clothing should be removed and medical attention sought. Identification of the material ingested and circumstances of the incident should be reported, and appropriate and complete medical records kept.

All persons should immediately vacate the affected area and any exposed persons should be referred for medical advice. The laboratory supervisor and the biosafety officer should be informed at once. No one should enter the room for an appropriate amount of time (e.g. 1 h), to allow aerosols to be carried away and heavier particles to settle. If the laboratory does not have a central air exhaust system, entrance should be delayed (e.g. for 24 h).

Broken containers contaminated with infectious substances and spilled infectious substances should be covered with a cloth or paper towels. Disinfectant should then be poured over these and left for the appropriate amount of time. The cloth or paper towels and the broken material can then be cleared away; glass fragments should be handled with forceps. The contaminated area should then be swabbed with disinfectant. If dustpans are used to clear away the broken material, they should be autoclaved or placed in an effective disinfectant. Cloths, paper towels and swabs used for cleaning up should be placed in a contaminated-waste container. Gloves should be worn for all these procedures.

If a breakage occurs or is suspected while the machine is running, the motor should be switched off and the machine left closed (e.g. for 30 minutes) to allow settling. If a breakage is discovered after the machine has stopped, the lid should be replaced immediately and left closed (e.g. for 30 minutes). In both instances, the biosafety officer should be informed.

All sealed centrifuge buckets should be loaded and unloaded in a biological safety cabinet. If breakage is suspected within the safety cup, the safety cap should be loosened the bucket autoclaved. Alternatively, the safety cup may be chemically disinfected.

For the purposes of this manual, the guidance and recommendations given as minimum requirements pertaining to laboratories of all biosafety levels are directed at microorganisms in Risk Groups 1–4. Although some of the precautions may appear to be unnecessary for some organisms in Risk Group 1, they are desirable for training purposes to promote good (i.e. safe) microbiological techniques GMT. Diagnostic and health-care laboratories (public health, clinical or hospital-based) must all be designed for Biosafety Level 2 or above. As no laboratory has complete control over the specimens it receives, laboratory workers may be exposed to organisms in higher risk groups than anticipated. This possibility must be recognized in the 8development of safety plans and policies. In some countries, accreditation of clinical laboratories is required. Globally, standard precautions should always be adopted and practiced.

This code is a listing of the most essential laboratory practices and procedures that are basic to GMT. In many laboratories and national laboratory programs, this code may be used to develop written practices and procedures for safe laboratory operations. Each laboratory should adopt a safety or operations manual that identifies known and potential hazards, and specifies practices and procedures to eliminate or minimize such hazards. GMT is fundamental to laboratory safety. Specialized laboratory equipment is a supplement to but can never replace appropriate procedures. The most important concepts are listed below:

In designing a laboratory and assigning certain types of work to it, special attention should be paid to conditions that are known to pose safety problems. These include:

Together with good procedures and practices, the use of safety equipment will help to reduce risks when dealing with biosafety hazards. This section deals with 11basic principles related to equipment suitable for laboratories of all biosafety levels.

Historical evidence indicates that the microorganisms handled at this level are unlikely to cause human disease or animal disease of veterinary importance. Ideally, however, all laboratory workers should undergo a pre-employment health check at which their medical history is recorded. Prompt reporting of illnesses or laboratory accidents is desirable and all staff members should be made aware of the importance of maintaining GMT.12

Human error and poor technique can compromise the best of safeguards to protect the laboratory worker. Thus, a safety conscious staff, well-informed about the recognition and control of laboratory hazards, is key to the prevention of laboratory acquired infections, incidents and accidents. For this reason, continuous in-service training in safety measures is essential. An effective safety program begins with the laboratory managers, who should ensure that safe laboratory practices and procedures are integrated into the basic training of employees. Training in safety measures should be an integral part of new employees' introduction to the laboratory. Employees should be introduced to the code of practice and to local guidelines, including the safety or operations manual. Measures to assure that employees have read and understood the guidelines, such as signature pages, should be adopted. Laboratory supervisors play the key role in training their immediate staff in good laboratory techniques. The biosafety officer can assist in training and with the development of training aids and documentation.

Waste is anything that is to be discarded. In laboratories, decontamination of wastes and their ultimate disposal are closely interrelated. In terms of daily use, few if any contaminated materials will require actual removal from the laboratory or destruction. Most glassware, instruments and laboratory or clothing will be reused or recycled.

Steam autoclaving is the preferred method for all decontamination processes. Materials for decontamination and disposal should be placed in containers, e.g. autoclavable plastic bags, that are color-coded according to whether the contents are to be autoclaved and/or incinerated. Alternative methods may be envisaged only if they remove and/or kill microorganisms.

Identification and separation system for infectious materials and their containers should be adopted. National and international regulations must be followed. Categories should include:

After use, hypodermic needles should not be recapped, clipped or removed from disposable syringes. The complete assembly should be placed in a sharps disposal container.

No precleaning should be attempted of any contaminated materials to be autoclaved and reused. Any necessary cleaning or repair must be done only after autoclaving or disinfection.

Apart from sharps, which are dealt with above, all contaminated materials should be autoclaved in leak proof containers, e.g. autoclavable, color-coded plastic bags, before disposal.

The containment laboratory, Biosafety Level 3 is designed and provided for work with Risk Group 3 microorganisms and with large volumes or high concentrations of Risk Group 2 microorganisms that pose an increased risk of aerosol spread. Biosafety Level 3 containment requires the strengthening of the operational and safety program over and above those for basic laboratories, Biosafety Levels 1 and 2.

Laboratories in this category should be registered or listed with the national or other appropriate health authorities.

The code of practice for basic laboratories, Biosafety Levels 1 and 2 applies except where modified as follows:15

The laboratory design and facilities for basic laboratories—Biosafety Levels 1 and 2 apply except where modified as follows:

The principles for the selection of laboratory equipment, including biological safety cabinets are the same as for the basic laboratory—Biosafety Level 2. However, at Biosafety Level 3, manipulation of all potentially infectious material must be conducted within a biological safety cabinet or other primary containment device. Consideration should b e given to equipment such as centrifuges, which will need additional containment accessories, for example, safety buckets or containment rotors. Some centrifuges and other equipment, such as cell-sorting instruments for use with infected cells, may need additional local exhaust ventilation with HEPA filtration for efficient containment.

The maximum containment laboratory, Biosafety Level 4 is designed for work with Risk Group 4 microorganisms. Before such a laboratory is constructed and put into operation, intensive consultations should be held with institutions that have had experience of operating a similar facility. Operational maximum containment laboratories, Biosafety Level 4 should be under the control of national or other appropriate health authorities. The following information is intended only as introductory material.17

The code of practice for Biosafety Level 3 applies except where modified as follows:

The features of a containment laboratory, Biosafety Level 3 also apply to a maximum containment laboratory, Biosafety Level 4 with the addition of the following:

Personal protective equipment and clothing may act as a barrier to minimize the risk of exposure to aerosols, splashes and accidental inoculation. The clothing and equipment selected is dependent on the nature of the work performed. Protective clothing should be worn when working in the laboratory. Before leaving the laboratory, protective clothing should be removed, and hands should be washed.

Laboratory coats should preferably be fully-buttoned. However, long-sleeved, back-opening gowns or coveralls give better protection than laboratory coats and are preferred in microbiology laboratories and when working at the biological safety cabinet. Aprons may be worn over laboratory coats or gowns where necessary to give further protection against spillage of chemicals or biological materials such as blood or culture fluids. Laundering services should be provided at/near the facility. Laboratory coats, gowns, coveralls, or aprons should not be worn outside the laboratory areas (Table 1.2).

The choice of equipment to protect the eyes and face from splashes and impacting objects will depend on the activity performed. Prescription or plain eye glasses can be manufactured with special frames that allow lenses to be placed in frame from the front, using shatterproof material either curved or fitted with side shields (safety glasses). Safety spectacles do not provide for adequate splash protection even when side shields are worn with them. Goggles for splash and impact protection should be worn over normal prescription eye glasses and contact lenses (which do not provide protection against biological or chemical hazards). Faces shields (visors) are made of shatterproof plastic fit over the face and are held in place by head straps or caps. Goggles, safety spectacles, or face shields should not be worn outside the laboratory areas.

Respiratory protection may be used when carrying out high hazard procedures (e.g. cleaning up a spill of infectious material). The choice of respirator will depend on the type of hazard(s). Respirators are available with interchangeable filters for protection against gases, vapors, particulates and microorganisms.

Those who use animals for experimental and diagnostic purposes have a moral obligation to take every care to avoid causing them unnecessary pain or suffering. The animals must be provided with comfortable, hygienic housing and adequate wholesome food and water. At the end of the experiment they must be dealt with in a humane manner.

With respect to agents to be used in the animal laboratory, factors for consideration include:

With respect to animals to be used in the animal laboratory, factors for consideration include:

As with laboratories, the requirements for design features, equipment and precautions increase in stringency according to the animal Biosafety Level.

This is suitable for the maintenance of most stock animals after quarantine (except nonhuman primates, regarding which national authorities should be consulted), and for animals that are deliberately inoculated with agents in Risk Group 1. GMT is required. The animal facility director must establish policies, procedures and protocols for all operations, and for access to the vivarium. An appropriate medical surveillance program for the staff must be instituted. A safety or operations manual must be prepared and adopted.

This is suitable for work with animals that are deliberately inoculated with micro-organisms in Risk Group 2. The following safety precautions apply:

This is suitable for work with animals that are deliberately inoculated with agents in Risk Group 3, or when otherwise indicated by a risk-assessment. All systems, practices and procedures need to be reviewed and recertified annually. The following safety precautions apply:

Work in this facility will normally be linked with that in the maximum containment laboratory—Biosafety Level 4, and national and local rules and regulations must be harmonized to apply to both. If work is to be done in a suit laboratory, additional practices and procedures must be used over and above those described here:

As with vertebrates, the animal facility biosafety level will be determined by the risk groups of the agents under investigation or when otherwise indicated by a risk-assessment. The following additional precautions are necessary with certain arthropods, particularly with flying insects:

Biological safety cabinets (BSCs) are designed to protect the operator, the laboratory environment and work materials from exposure to infectious aerosols and splashes that may be generated when manipulating materials containing infectious agents, such as primary cultures, stocks and diagnostic specimens. Aerosol particles are created by any activity that imparts energy into a liquid or semiliquid material, such as shaking, pouring, stirring or dropping liquid onto a surface or into another liquid. Other laboratory activities, such as streaking agar plates, inoculating cell culture flasks with a pipette, using a multi-channel pipette to dispense liquid suspensions of infectious agents into microculture plates, homogenizing and vortexing infectious materials, and centrifugation of infectious liquids, or working with animals, can generate infectious aerosols. Aerosol particles of less than 5 m in diameter and small droplets of 5–100 m in diameter are not visible to the naked eye. The laboratory worker is generally not aware that such particles are being generated and may be inhaled or may cross-contaminate work surface materials. BSCs, when properly used, have been shown to be highly effective in reducing laboratory-acquired infections and cross-contaminations of cultures due to aerosol exposures. BSCs also protect the environment.

28Note: Horizontal and vertical outflow cabinets (“clean-air work stations”) are not biological safety cabinets and should not be used as such.

The directional flow of air whisks aerosol particles that may be generated on the work surface away from the laboratory worker and into the exhaust duct. The front opening allows the operator's arms to reach the work surface inside the cabinet while he or she observes the work surface through a glass window. The window can also be fully raised to provide access to the work surface for cleaning or other purposes (Table 1.4).

As the use of cell and tissue cultures for the propagation of viruses and other purposes grew, it was no longer considered satisfactory for unsterilized room air to pass over the work surface. The Class II BSC is designed not only to provide personnel protection but also to protect work surface materials from contaminated room air. Class II BSCs, of which there are four types (A1, A2,

29B1 and B2), differ from Class I BSCs by allowing only air from a HEPA-filtered (sterile) supply to flow over the work surface. The Class II BSC can be used for working with infectious agents in Risk Groups 2 and 3. Class II BSCs can be used for working with infectious agents in Risk Group 4 when positive-pressure suits are used.

The Class II type A1 BSC is internal fan draws room air (Supply air) into the cabinet through the front opening and into the front intake grill. The inflow velocity of this air should be at least 0.38 m/s at the face of the front opening. The supply air then passes through a supply HEPA-filter before flowing downwards over the work surface. As the air flows downwards it “splits” about 6–18 cm from the work surface, one half of the downwards flowing air passing through the front exhaust grill, and the other half passing through the rear exhaust grill. Any aerosol particles generated at the work surface are immediately captured in this downward airflow and passed through the front or rear exhaust grills, thereby providing the highest level of product protection. The air is then discharged through the rear plenum into the space between the supply and exhaust filters located at the top of the cabinet. Owing to the relative size of these filters, about 70% of the air recirculates through the supply HEPA-filter back into the work zone; the remaining 30% passes through the exhaust filter into the room or to the outside. Air from the Class IIA1 BSC exhaust can be recirculated to the room or discharged to the outside of the building through a thimble connection to a dedicated duct or through the building exhaust system (Figure 1.2).

Class IIA2 vented to the outside, IIB1 and IIB2 BSCs (Figure 1.4) are variations of the type IIA1. Their characteristics, along with those of Class I and Class III BSCs, are indicated (Figure 1.3). Each variation allows the BSC to be used for specialized purpose.

This type provides the highest level of personnel protection and is used for Risk Group 4 agents. All penetrations are sealed “gas tight”. Supply air is HEPA-filtered and exhaust air passes through two HEPA-filters. Airflow is maintained by a dedicated exhaust system exterior to the cabinet, which keeps the cabinet interior under negative pressure (about 124.5 Pa). Access to the work surface is by means of heavy duty rubber gloves, which are attached to ports in the cabinet. The Class III BSC should have an attached pass-through box that can be sterilized and is equipped with a HEPA-filtered exhaust. The Class III cabinet may be connected to a double-door autoclave used to decontaminate all materials entering or exiting the cabinet. Several glove boxes can be joined together to extend the work surface. Class III BSCs are suitable for work in Biosafety Level 3 and 4 laboratories.

A “thimble” or “canopy hood” is designed for use with Class IIA1 and IIA2 vented to the outside BSCs. The thimble fits over the cabinet exhaust housing, sucking the cabinet exhaust air into the building exhaust ducts. A small opening, usually 2.5 cm in diameter, is maintained between the thimble and the cabinet exhaust housing. This small opening enables room air to be sucked into the building exhaust system as well.

A BSC should be selected primarily in accordance with the type of protection needed: product protection, personnel protection against Risk Group 1–4 microorganisms, personnel protection against exposure to radionuclides and volatile toxic chemicals, or a combination of these. Table 8 shows which BSCs are recommended for each type of protection. Volatile or toxic chemicals should not be used in BSCs that recirculate exhaust air to the room, i.e. Class I BSCs that are not ducted to building exhaust systems, or Class IIA1 or Class IIA2 cabinets. Class IIB1 BSCs are acceptable for work with minute amounts of volatile chemicals and radionuclides. A Class IIB2 BSC, also called a total exhaust cabinet, is necessary when significant amounts of radionuclides and volatile chemicals are expected to be used (Figure 1.5).

The velocity of air flowing through the front opening into a BSC is about 0.45 m/s.

At this velocity the integrity of the directional air inflow is fragile and can be easily disrupted by air currents generated by people walking close to the BSC, open windows, air supply registers, and opening and shutting doors. Ideally, BSCs should be situated in a location remote from traffic and potentially disturbing air currents.

The front intake grill of Class II BSCs must not be blocked with paper, equipment or other items. Materials to be placed inside the cabinet should be surface decontaminated with 70% alcohol. Work may be performed on disinfectant-soaked absorbent towels to capture splatters and splashes. All materials should be placed as far back in the cabinet, towards the rear edge of the work surface, as practical without blocking the rear grill. Aerosol-generating equipment (e.g. mixers, centrifuges, etc.) should be placed towards the rear of the cabinet. Bulky items, such as biohazard bags, discard pipette trays and suction collection flasks should be placed to one side of the interior of the cabinet. Active work should flow from clean to contaminated areas across the work surface.

Ultraviolet lights are not required in BSCs. If they are used, they must be cleaned weekly to remove any dust and dirt that may block the germicidal effectiveness of the light. Ultraviolet light intensity should be checked when the cabinet is recertified to ensure that light emission is appropriate. Ultraviolet lights must be turned off while the room is occupied, to protect eyes and skin from inadvertent exposure.

A copy of the laboratory's protocol for handling spills should be posted, read and understood by everyone who uses the laboratory. When a spill of biohazardous material occurs within a BSC, clean-up should begin immediately, while the cabinet continues to operate. An effective disinfectant should be used and applied in a manner that minimizes the generation of aerosols. All materials that come into contact with the spilled agent should be disinfected and/or autoclaved.

All items within BSCs, including equipment, should be surface-decontaminated and removed from the cabinet when work is completed, since residual culture media may provide an opportunity for microbial growth.

BSCs must be decontaminated before filter changes and before being moved. The most common decontamination method is by fumigation with formaldehyde gas. BSC decontamination should be performed by a qualified professional.

The negative-pressure flexible-film isolator is a self-contained primary containment device that provides maximum protection against hazardous biological materials. It may be mounted on a mobile stand. The workspace is totally enclosed in a transparent polyvinylchloride (PVC) envelope suspended from a steel framework. The isolator is maintained at an internal pressure lower than atmospheric pressure. Inlet air is passed through one HEPA-filter and outlet air is passed through two HEPA-filters, thus obviating the need to duct exhaust air outside the building. The isolator may be fitted with an incubator, microscope and other laboratory equipment, such as centrifuges, animal cages, heat blocks, etc. Material is introduced and removed from the isolator through supply and sample ports without compromising microbiological security. Manipulations are performed using gloved sleeves incorporating disposable gloves. A manometer is installed to monitor envelope pressure.

A pipetting aid must always be used for pipetting procedures. Mouth pipetting must be strictly forbidden. The importance of pipetting aids cannot be overemphasized. The most common hazards associated with pipetting procedures are the result of mouth suction. Oral aspiration and ingestion of hazardous materials have been responsible for many laboratory-associated infections.

Today the nation is facing a new challenge in safeguarding the public health from potential domestic or international terrorism involving the use of dangerous biological agents or toxins. Existing standards and practices may require adaptation to ensure protection from such hostile actions.

Human error, poor laboratory techniques and misuse of equipment cause the majority of laboratory injuries and work-related infections. This section provides a compendium of technical methods that are designed to avoid or minimize the most commonly reported problems of this nature.

Improper collection, transport and handling of specimens in the laboratory carry a risk of infection to the personnel involved.

Specimen containers may be of glass or preferably plastic. They should be robust and should not leak when the cap or stopper is correctly applied. No material should remain on the outside of the container. Containers should be correctly labeled to facilitate identification. Specimen request or specification forms should not be wrapped around the containers but placed in separate, preferably waterproof envelopes.

To avoid accidental leakage or spillage, secondary containers, such as boxes, should be used, fitted with racks so that the specimen containers remain upright. The secondary containers may be of metal or plastic, should be autoclavable or resistant to the action of chemical disinfectants, and the seal should preferably have a gasket. They should be regularly decontaminated.

Laboratories that receive large numbers of specimens should designate a particular room or area for this purpose.

Personnel who receive and unpack specimens should be aware of the potential health hazards involved, and should be trained to adopt standard precautions, 37particularly when dealing with broken or leaking containers. Primary specimen containers should be opened in a biological safety cabinet. Disinfectants should be available.

Care should be taken when ampoules of freeze-dried materials are opened, as the contents may be under reduced pressure and the sudden inrush of air may disperse some of the materials into the atmosphere. Ampoules should always be opened in a biological safety cabinet.

Ampoules containing infectious materials should never be immersed in liquid nitrogen because cracked or imperfectly sealed ampoules may break or explode on removal. If very low temperatures are required, ampoules should be stored only in the gaseous phase above the liquid nitrogen. Otherwise, infectious materials should be stored in mechanical deep-freeze cabinets or on dry ice. Laboratory workers should wear eye and hand protection when removing ampoules from cold storage. The outer surfaces of ampoules stored in these ways should be disinfected when the ampoules are removed from storage.

Fixing and staining of blood, sputum and fecal samples for microscopy do not necessarily kill all organisms or viruses on the smears. These items should be handled with forceps, stored appropriately, and decontaminated and/or autoclaved before disposal.

Hypochlorites and high-level disinfectants are recommended for decontamin­ation. Freshly prepared hypochlorite solutions should contain available chlorine at 1 g/L for general use and 5 g/L for blood spillages. Glutaraldehyde may be used for decontaminating surfaces.

Prions (also referred to as “slow viruses”) are associated with the transmissible spongiform encephalopathies (TSEs), notably Creutzfeldt-Jakob disease (CJD; including the new variant form), Gerstmann-Sträussler-Scheinker syndrome, fatal familial insomnia and kuru in humans; scrapie in sheep and goats; bovine spongiform encephalopathy (BSE) in cattle; and other transmissible encephalopathies of deer, elk and mink. Although CJD has been transmitted to humans, there appear to be no proven cases of laboratory-associated infections with any of these agents. Nevertheless, it is prudent to observe certain precautions in the handling of material from infected or potentially infected humans and animals.

Recommended additional steps prior to incineration include:

Transport of infectious and potentially infectious materials is subject to strict national and international regulations. These regulations describe the proper use of packaging materials, as well as other shipping requirements. Laboratory personnel must ship infectious substances according to applicable transport regulations. Compliance with the rules will:

Defined as substances which are known or are reasonably expected to contain pathogens. Pathogens are defined as microorganisms (including bacteria, viruses, rickettsiae, parasites, fungi) and other agents such as prions which can cause disease in humans or animals (Table 1.6 and Figure 1.7).

The package must include the following:

Transport of infectious substances requires a basic triple packaging system. It consists of three layers as follows:

Each completed package is normally required to be marked, labeled and accompanied with appropriate shipping which is available from the Department for Transport.

In the event of a spill of infectious or potentially infectious material, the following spill clean-up procedure should be used.

Recombinant DNA technology involves combining genetic material from different sources thereby creating genetically modified organisms (GMOs) that may have never existed in nature before. Initially, there was concern among molecular biologists that such organisms might have unpredictable and 50undesirable properties that could represent a biohazard if they escaped from the laboratory.

Biological expression systems consist of vectors and host cells. A number of criteria must be satisfied to make them effective and safe to use. An example of such a biological expression system is plasmid pUC18. Frequently used as a cloning vector in combination with Escherichia coli K12 cells, the pUC18 plasmid has been entirely sequenced. All genes required for expression in other bacteria have been deleted from its precursor plasmid pBR322. E. coli K12 is a non-pathogenic strain that cannot permanently colonize the gut of healthy humans or animals. Routine genetic engineering experiments can safely be performed in E. coli K12/pUC18 at Biosafety Level 1 provided the inserted foreign DNA expression products do not require higher biosafety levels.

Higher biosafety levels may be required when:

Viral vectors, e.g. adenovirus vectors, are used for the transfer of genes to other cells. Such vectors lack certain virus replication genes and are 51propagated in cell lines that complement the defect. Stocks of such vectors may be contaminated with replication-competent viruses, generated by rare spontaneous recombination events in the propagating cell lines, or may derive from insufficient purification. These vectors should be handled at the same biosafety level as the parent adenovirus from which they are derived.

Animals carrying foreign genetic material (transgenic animals) should be handled in containment levels appropriate to the characteristics of the products of the foreign genes. Animals with targeted deletions of specific genes (“knock-out”animals) do not generally present particular biological hazards.

Transgenic plants expressing genes that confer tolerance to herbicides or resistance to insects are currently a matter of considerable controversy in many parts of the world. The discussions focus on the food-safety of such plants, and on the long-term ecological consequences of their cultivation. Transgenic plants expressing genes of animal or human origin are used to develop medicinal and nutritional products. A risk-assessment should determine the appropriate biosafety level for the production of these plants.

Risk-assessments for work with GMOs should consider the characteristics of donor and recipient/host organisms. Examples of characteristics for consideration include the following:

Assessment is necessary in situations where the product of the inserted gene has known biologically or pharmacologically active properties that may give rise to harm, for example:52

The consideration of such cases should include an estimation of the level of expression required to achieve biological or pharmacological activity.

Many modifications do not involve genes whose products are inherently harmful, but adverse effects may arise as the result of alteration of existing non-pathogenic or pathogenic traits. Modification of normal genes may alter pathogenicity. In an attempt to identify these potential hazards, the following points may be considered (the list is not exhaustive).

The use of whole animals or plants for experimental purposes also requires careful consideration.

A basic knowledge of disinfection and sterilization is crucial for biosafety in the laboratory. Since heavily soiled items cannot promptly be disinfected or sterilized, it is equally important to understand the fundamentals of cleaning prior to disinfection (precleaning). In this regard, the following general principles apply to all known classes of microbial pathogens.

Many different terms are used for disinfection and sterilization. The following are among the more common in biosafety:

An agent that kills microorganisms or suppresses their growth and multiplication.

A substance that inhibits the growth and development of microorganisms without necessarily killing them. Antiseptics are usually applied to body surfaces.

A general term for any agent that kills organisms.

A chemical or a mixture of chemicals used to kill microorganisms.

Any process for removing and/or killing microorganisms. The same term is also used for removing or neutralizing hazardous chemicals and radioactive materials.

A chemical or mixture of chemicals used to kill microorganisms, but not necessarily spores. Disinfectants are usually applied to inanimate surfaces or objects.54

A physical or chemical means of killing microorganisms, but not necessarily spores.

A chemical or mixture of chemicals that kills microorganisms. The term is often used in place of “biocide”, “chemical germicide” or “antimicrobial”.

A chemical or mixture of chemicals used to kill microorganisms and spores.

A process that kills and/or removes all classes of microorganisms and spores.

Cleaning is the removal of dirt, organic matter and stains. Cleaning includes brushing, vacuuming, drydusting, washing or damp mopping with water containing a soap or detergent. Dirt, soil and organic matter can shield microorganisms and can interfere with the killing action of decontaminants (antiseptics, chemical germicides and disinfectants). Precleaning is essential to achieve proper disinfection or sterilization.

Decontamination of the laboratory space, its furniture and its equipment requires a combination of liquid and gaseous disinfectants. Surfaces can be decontaminated using a solution of sodium hypochlorite (NaOCl); a solution containing 1 g/L available chlorine may be suitable for general environmental sanitation, but stronger solutions (5 g/L) are recommended when dealing with high-risk situations. For environmental decontamination, formulated solutions containing 3% hydrogen peroxide (H₂O₂) make suitable substitutes for bleach solutions.

To decontaminate Class I and Class II cabinets, equipment that independently generates, circulates and neutralizes formaldehyde gas is available. Alternatively, the appropriate amount of paraformaldehyde (final concentration of 0.8% paraformaldehyde in air) should be placed in a frying pan on an electric hot plate. Another frying pan, containing 10% more ammonium bicarbonate than paraformaldehyde, on a second hot plate is also placed inside the cabinet. The hot plate leads are plugged in outside the cabinet, so that operation of the pans can be controlled from the outside by plugging and unplugging the hot plates as necessary. If the relative humidity is below 70%, an open container of hot water should also be placed inside the cabinet before the front closure is sealed in place with strong tape (e.g. duct tape).

Whenever possible, suitable gloves should be worn when handling biohazardous materials. However, this does not replace the need for regular and proper hand-washing by laboratory personnel. Hands must be washed after handling bio-hazardous materials and animals, and before leaving the laboratory.

Additional alcohol-based solutions (HICC approved hand rub) are recommended for use in setting where handwashing facilities are inadequate or inaccessible and hands are not visibly soiled. If these solutions are used as a substitute for handwashing, handwashing with soap and water should be performed as soon as possible after procedure.

It is not always possible to protect the skin against various contaminants in the workplace. Therefore, cleaning and taking care of the hands is an important part of developing a proactive, holistic stance against work related skin disorders.

Heat is the most common among the physical agents used for the decontamination of pathogens. “Dry” heat, which is totally non-corrosive, is used to process many items of laboratory ware which can withstand temperatures of 160° or higher for 2–4 hours. Burning or incineration (Figures 1.12 and 1.13) is also a form of dry heat. “Moist” heat is most effective when used in the form of autoclaving.

Saturated steam under pressure (autoclaving) is the most effective and reliable means of sterilizing laboratory materials. For most purposes, the following cycles will ensure sterilization of correctly loaded autoclaves:

Materials should be loosely packed in the chamber for easy steam penetration and air removal. Bags should allow the steam to reach their contents.

The following rules can minimize the hazards inherent in operating pressurized vessels:

Incineration is useful for disposing of animal carcasses as well as anatomical and other laboratory waste, with or without prior decontamination. Incineration of infectious materials is an alternative to autoclaving only if the incinerator is under laboratory control.

The disposal of laboratory and medical waste is subject to various regional, national and international regulations, and the latest versions of such relevant documents must be consulted before designing and implementing a program for handling, transportation and disposal of biohazardous waste. In general, ash from incinerators may be handled as normal domestic waste and removed by local authorities. Autoclaved waste may be disposed of by off-site incineration or in licensed land fill sites.

It is essential that each laboratory organization has a comprehensive safety policy, a safety manual, and supporting programs for their implementation. The responsibility for this normally rests with the director or head of the institute or laboratory, who may delegate certain duties to a biosafety officer or other appropriate personnel.

Wherever possible a biosafety officer should be appointed to ensure that biosafety policies and programs are followed consistently throughout the laboratory. The biosafety officer executes these duties on behalf of the head of the institute or laboratory. In small units, the biosafety officer may be a microbiologist or a member of the technical staff, who may perform these duties on a defined part-time basis.

The activities of the biosafety officer should include the following:

A biosafety committee should be constituted to develop institutional biosafety policies and codes of practice. The biosafety committee should also review research protocols for work involving infectious agents, animal use, recombinant DNA and genetically modified materials. Other functions of the committee may include risk assessments, formulation of new safety policies and arbitration in disputes over safety matters.

The biosafety committee should seek advice from different departmental and specialist safety officers (e.g. with expertise in radiation protection, industrial safety, fire prevention, etc.) and may at times require assistance from independent experts in various associated fields, local authorities and national regulatory bodies. 63Community members may also be helpful if there is a particularly contentious or sensitive protocol under discussion.

The risks of working with particular agents should be fully discussed with individual researchers. The local availability, licensing state and utility of possible vaccines and/ or therapeutic drugs (e.g. antibiotic treatments) in case of exposure should be evaluated before work with such agents is started. Some workers may have acquired immunity from prior vaccination or infection.

The telephone numbers and addresses of the following should be prominently displayed in the facility: