Chapter 7: The Laboratory Biosafety Guidelines: 3rd Edition 2004 – Program specific guidelines

Chapter 7
Program Specific Guidelines

7.1 Laboratory Animals

7.1.1 General Requirements

Work with animals poses a variety of unique hazards, including exposure to infectious agents (naturally occurring or experimentally produced), animal bites and scratches, kicks and crushing injuries, allergies and physical hazards (noise, temperature). In addition to keeping infectious agents from spreading to laboratory workers there is a need to address, in the equipment and practices of animal facilities, the issues of cross-contamination between animals and of keeping adventitious agents from inadvertently infecting experimental animals (also referred to as "barrier" facilities). Animal facilities for work with small and large animals should be designed and operated in accordance with the Containment Standards for Veterinary FacilitiesFootnote 1 , published by the Canadian Food Inspection Agency, the Guide to the Care and Use of Experimental Animals , published by the Canadian Council on Animal CareFootnote 2  and other CCAC guidelines and policies (as revised from time to time). Institutions using animals for research, teaching and testing should consider obtaining a CCAC Certificate of GAP (Good Animal Practice®). There are other international recommendations which can provide further assistance with the assessment of hazards associated with the care and use of research animalsFootnote 3 Footnote 4 Footnote 5 .

Ideally, animal facilities should be a physically separated unit, but if they adjoin the laboratory the animal rooms should be separated from other activities in the laboratory to allow for isolation and decontamination as required. As general protocols cannot anticipate the specific requirements of each experiment, specific entry and exit protocols for scientific staff, animal handlers, animals, biological samples, equipment, feed and wastes should be developed for each project.

Animal rooms for small animals should be designed for ease of cleaning and disinfection, and have a minimum of built-in equipment. A small preparation area, storage area and handwashing sink are usually all that are required. As well, the design should facilitate the use of containment caging systems and support facilities for animal procedures, cage washing, waste disposal and food/bedding storage. Recent technological improvements have been incorporated into a wide variety of housing systems to provide control of micro-environmental factors such as temperature, air exchange and humidity. Descriptions of currently used caging and bedding disposal systems have been provided elsewhereFootnote 6 .

At least one-fifth of people who work with laboratory rodents, guinea pigs and rabbits develop allergiesFootnote 7 . Allergic conditions may result from contact with animal fur or hair, bedding and animal wastes. The allergy may manifest itself immediately or may be acquired over a succession of exposures to the allergen. Symptoms range from mild rashes to severe asthma. Unnecessary exposure to these allergens can be minimized through engineering controls, ventilation, use of isolators and containment caging systems, and appropriate use of respiratory and other personal protectionFootnote 3 Footnote 4 .

Containment facilities for large animals are unique, in part because of the large quantity of infectious microorganisms that may be present in the animal cubicle. Unlike a laboratory room, where the BSC provides primary containment, the large animal cubicle serves as both the primary and secondary barrier. Particular attention must be given to the use of protective clothing and equipment by staff entering an animal cubicle contaminated with large volumes of infected animal waste. Floor drains connected to an effluent sterilization system are employed at containment levels 3 and 4 to effectively remove and treat infected animal wastes. Special care must also be taken to avoid serious injuries (e.g., crushing) that could occur when handling large animals. Physical barriers, restraints and gating systems should be designed and used to prevent such injuries. The handler must have knowledge of the animal's general characteristics, such as mentality, instincts and physical attributes.

7.1.2 Non-human Primates

Working with non-human primates presents unique hazards related to naturally occurring pathogenic organisms and to the animals themselves. Their long canine teeth and powerful jaws can inflict serious and painful lacerations. The animals also have sharp fingernails and toenails that can scratch and abrade the skin of handlers. They are generally very messy, noisy and destructive animals, characteristics that must be considered when designing animal rooms used to house them.

Infectious hazards to people handling non-human primates include bacterial diseases ( Salmonella , Shigella , Campylobacter, tuberculosis), viral diseases (hepatitis A virus, simian immunodeficiency virus and especially Cercopithecine herpesvirus 1 (CHV-1), also known as herpes B virus), protozoan and metazoan parasites ( Entamoeba, Blastocystis , Trichomonas , Balantidium ) and other agents. For a more comprehensive list of infectious hazards see reference 5.

CHV-1 is an enzootic virus present in up to 70% of captive macaques, including rhesus and cynomolgus non-human primatesFootnote 8. Although the virus causes oral lesions in its natural simian host, asymptomatic shedding from the buccal mucosa and urogenital tract (though rare) and the presence of the virus in conjunctival fluid can occur without such clinical signs. Human infection has been documented in at least 50 instances, resulting in either severe disease or deathFootnote 9. Except for one case of person-to-person transmission, all have occurred in people exposed to non-human primates or non-human primate tissues. Transmission to humans is believed to occur primarily by exposure to contaminated non-human primate saliva through bites and scratches, although one fatal case following mucocutaneous exposure without injury has been reportedFootnote 8. Guidelines are available for working safely with macaques, for the prevention of CHV-1 infection and for the treatment of such infections in exposed people, and these should be consultedFootnote 5 Footnote 8 Footnote 10 Footnote 11 Footnote 12 . Risk of exposure to pathogenic agents can also be reduced through an adequate animal health surveillance program, with emphasis on identification and treatment of diseased animals. Further information on hazard identification and risk assessment can be found in reference 5. Animal handlers should be enrolled in a health and medical surveillance program (see Chapter 2.4 and reference 5).

Everyone who handles non-human primates must be trained in proper methods of restraint and in the use of protective clothing to help prevent bites, scratches and splash exposures. Such methods include the use of squeeze-back cages, where feasible, transfer boxes, chutes, tunnels and squeeze mechanisms for non-human primates housed in groups. Cages and other equipment should be free of sharp edges and corners that may cause scratches or wounds. When feasible, chemical restraint may be used before removing animals from cages, especially in the case of macaques and other larger non-human primates. Behavioural conditioning can also be effectively used in combination with restraint procedures. Handlers are to be protected with arm-length reinforced leather gloves and long-sleeved gowns/coveralls to prevent scratches. Protection against aerosol exposure and splashes of mucous membranes (e.g., with surgical mask, face shield, eye goggles) should be provided to handlers and everyone entering animal rooms where non-human primates are housed. Reusable, protective clothing that has been in contact with non-human primates should be decontaminated before being sent to laundry. Animal handlers must be instructed to cleanse immediately and thoroughly all bites, scratches and abraded skin and to report these exposures at once. Postexposure procedures should also be institutedFootnote 5 Footnote 12.

Facilities for housing non-human primates should conform to the recommendations for small animal containment facilities in the Containment Standards for Veterinary FacilitiesFootnote 1. Unless experimentally infected with or known to have an infectious organism requiring a higher containment level, non-human primates can be handled in containment level 2 animal facilities with the additional practices and personnel precautions described above for working safely with these animals. It is recommended that all macaque colonies be treated as naturally infected with CHV-1, even those that have been shown to be free of CHV-1 antibodyFootnote 5 Footnote 9. The Guide to the Care and Use of Experimental Animals also provides information on housing and handling requirements specific to non-human primatesFootnote 2. Additional information on non-human primate facilities, equipment and special practices can be found elsewhereFootnote 13. Generally, housing for non-human primates requires the following:

  1. Consideration to be given to the behavioural, emotional and social needs of laboratory primates when planning their housing.

  2. Contact information of experienced primate handlers and the person responsible for the facility, to be provided throughout the facility.

  3. Animal rooms to be provided with a vestibule or other arrangement to ensure that there are always two doors between the non-human primate cage and the building corridor; provision should be made to observe all cages before entering the room to ensure that animals are not loose.

  4. All lighting, electrical fixtures and exposed plumbing in non-human primate rooms to be protected against tampering by the animals.

  5. Because of the requirement for daily sanitation of animal rooms, floors to be constructed of slip-proof materials and workers to wear footgear that provides traction on wet, slippery floors. As well, walls and ceilings to be designed with a finish to withstand wash-down cleaning and disinfection procedures.

  6. Shower and changing facilities to be provided for workers having substantial animal contact to shower at the end of the work day.

  7. Animal rooms and cages to be kept locked at all times and to be accessible only to authorized people. Security locks and closing devices must take into consideration the persistent, creative, destructive and intellectual capacities of most non-human primates.

  8. The movement of equipment items (e.g., carts, scales, feed containers and scoops, gloves) between animal rooms to include proper disinfection practices on exit from each room as appropriate to the containment level of the work.

  9. Cages to have sufficient strength that they cannot be damaged by the non-human primates and to be maintained in proper working condition.

  10. Cages to be equipped with a squeeze mechanism to facilitate examination and immobilization. Transfer boxes and other special restraint apparatus can be used to hold primates safely while primary cages are being cleaned or to move primates from one room to another.

  11. For group caging, such factors as compatibility between animals and the population dynamics of the species to be considered in order to minimize fighting.

7.2 Recombinant DNA and Genetic Manipulation

Genetic methods, such as natural selection, cross breeding, conjugation and transformation, have been used for many years to change biological species and organisms. These methods have been supplemented by newer and much more efficient ones, of which the best known are the techniques of recombinant DNA. Some newer techniques include the production of transgenic plants and animals; the cloning of microbial toxin or other virulence genes in an expression vector or in a host background in which it may be expressed; and the production of full-length infectious viral clones, including the reconstruction of infectious virions from recombinant constructs (reverse genetic engineering).

The initial fear of possible risks arising from organisms altered by this technology led Canada, the United States and Great Britain, among other countries, to develop stringent biosafety guidelines. Experience rapidly showed that the initial fears were not justified and that most recombinant DNA research in itself does not pose any specific risks to biological safetyFootnote 14 .

Guidance in how to assess potential risks in recombinant DNA research is availableFootnote 15 Footnote 16  but can only be very general. Factors to consider when determining the containment level of a recombinant organism should include:

  1. the containment level of the recipient organism;

  2. the containment level of the donor organism;

  3. the replication competency of the recombinant organism;

  4. the property of the donor protein to become incorporated into the recombinant particle; and

  5. potential pathogenic factors associated with the donor protein.

Each case needs to have a risk assessment, as it is not realistic to try to define in advance all the possible genetically engineered organisms that might be created or used in the laboratory. Assistance with the risk assessment can be provided by the Office of Laboratory Security, telephone (613) 957-1779.

The vast majority of recombinant research involves only the remotest possibility of creating a hazard, because the source of the DNA being transferred, the vector and the host are all innocuous. However, some genetic manipulation does raise significant possibility of risk. In general, if none of the components of the genetic manipulation presents any known hazard and none can be reasonably foreseen to result from their combination, then no biohazard restrictions are needed. If one of the components of the reaction is hazardous, then, in general, discussion of the containment level required should start at the level appropriate to the known hazard. Its containment level might be increased or decreased according to such considerations as the particular gene being transferred; the expression of the gene in the recombinant organism; the biological containment offered by the host vector systems; the envisaged interactions between the gene being transferred and the host vector systems; and the viability of the host vector systems. In any research with genes coding for hazardous products, host vector systems with limited ability to survive outside the laboratory should be used; their use will reduce the level of containment required.

Examples of such considerations follow:

  1. A recombinant vesicular stomatitis pseudotype virus expressing a different viral glycoprotein would be at level 2 because the virus is replication-deficient.

  2. A recombinant vesicular stomatitis virus expressing a different viral glycoprotein would be at least at the level of vesicular stomatitis virus since the virus is replication-competent and could have an altered tropism.

  3. A recombinant vaccinia virus expressing a different viral glycoprotein would be at the containment level of wild type vaccinia virus since the protein does not get incorporated into the virus particle, and it is unlikely that this manipulation will change the biological properties of the recombinant virus.

7.3 Cell Lines

Cell lines (cell cultures) are commonly used in diagnostic and microbiology laboratories, and in industry for the production of pharmaceuticals. There have been cases of laboratory-acquired infections reported as a result of manipulation of primary cell culturesFootnote 17 Footnote 18 . Although cell lines do not inherently pose a risk to individuals manipulating them in the laboratoryFootnote 19 , because of their potential to contain pathogenic organisms - either naturally or through contamination by adventitious agents, transformation or recombination - an assessment must be made as to the level of hazard associated with a particular lineFootnote 20 . Cell lines can be contaminated with bacteria, fungi, mycoplasma, viruses and prions.

7.3.1 Risk Assessment

Non-recombinant cell lines

For every new cell line that is manipulated in a laboratory, a detailed risk assessment must be done in order to determine the appropriate level of precautions to be taken. A detailed risk assessment should include, but is not limited, to the following:

  • source of cell line: the closer phylogenetically to humans, the greater the potential risk (highest to lowest risk: human autologous, human heterologous, primate, other mammalian, avian, invertebrateFootnote 21  );
  • source tissue: provides an indication of possible contaminants and latent (oncogenic) viruses;
  • type of cell line - highest to lowest risk: primary cell cultures, continuous cell cultures, intensively characterized cell cultures;
  • quantity of cells per culture;
  • source population of the specimen from which the cell line was derived.

Recombinant cell lines (in addition to the above criteria)

  • properties of the host cell line (in the case of hybridomas, the properties of each of the contributing cells must be considered);
  • vector used for transformation (may increase containment level requirements);
  • transfer of viral sequences (may increase containment level requirements);
  • transfer of virulence factors (may increase containment level requirements);
  • activation of endogenous viruses (may increase containment level requirements);
  • recombinant gene product (may increase containment level requirements);
  • helper virus presence (may increase containment level requirements).

Once all the relevant information regarding the cell line has been obtained, including any hazards associated with the media to be used during manipulation of the cell culture, it can be assessed to ascertain the hazards posed by manipulating the particular cell line. The cell line is to be handled at the containment level appropriate to the level of risk determined by the assessment.

7.3.2 Contamination with Infectious Agents

Bacteria and fungi

Cell lines contaminated with bacteria and fungi are readily identified when grown in antibiotic-free media because they quickly overgrow the cellsFootnote 20.

Viral contamination

Unlike bacteria and fungi, viruses are not readily identified and so can pose a significant hazard to those manipulating primary cell lines. A documented laboratory-acquired hantavirus infection has been linked to the manipulation of rat tumour materialFootnote 22. Because of the varying risks associated with cell line material, the World Health Organization proposed a classification of cell lines based on each line's likelihood of carrying viruses pathogenic to humansFootnote 23.

  • Low likelihood : cell lines derived from avian and invertebrate tissues.
  • Medium likelihood : mammalian nonhematogenous cells, such as fibroblasts and epithelial cells.
  • High likelihood : blood and bone marrow cells derived from human or non-human primates; human pituitary cells, caprine and ovine cells, especially those of neural origin; and hybridoma cells when at least one fusion partner is of human or non-human primate origin.
  • Both viral and cellular oncogenes have been recognized, most notably the human T-cell leukemia virus (HTLV-I). HTLV-I is a human oncogenic virus that transforms normal cells into malignant cells(21).
  • Cell lines with known or potential viral contaminants are to be handled at the containment level appropriate for the contaminating agent of the highest risk.

One of the primary hazards of manipulating cell cultures is the expression of latent viruses. Endogenous viral sequences have been found in a variety of cell lines derived from mammalian species, including humansFootnote 20. Cell lines can be grown in an altered manner by applying various treatments (e.g., change in pH, serum level, temperature, medium supplements, co-cultivation). These treatments may cause altered expression of oncogenes, expression of latent viruses, interactions between recombinant genomic segments or altered expression of cell surface proteinsFootnote 21.

  • Manipulations that may alter the "normal" behaviour of cell lines to a more hazardous state are to be conducted at a containment level appropriate to the new hazardous state.

The biological hazards associated with primate cell lines must also be taken into consideration when determining the level of containment required. Primary cell lines derived from the genus Macaca may harbour herpesvirus simiae (Cercopithecine herpes virus, B-virus), and therefore tissues from Macaca must be manipulated as follows:

  • Containment level 2 is to be used when handling tissues or body fluids from macaques.
  • If material is suspected or known to contain herpesvirus simiae, containment level 3 is required.
  • In vitro primary diagnostic tests are to be done at containment level 3.
  • All propagation (culturing) of the virus is to be done at containment level 4.


The protein-only infectious particle, or prion, is accepted as the causative agent of transmissible spongiform encephalopathies, such as bovine spongiform encephalopathy (BSE)Footnote 24 Footnote 25.

  • Cell cultures derived from bovine sources known or suspected to be BSE positive, and in vitro primary diagnostic tests of cell cultures derived from bovine sources known or suspected to be BSE positive are to be handled using TSE specific guidelines. Information and the TSE guidelines can be found by contacting CFIA, Biohazard Containment and Safety Division directly at (613) 221-7074 or accessing their Web site:


Although mycoplasmas have commonly been identified as sources of cell culture contamination, mycoplasma-contaminated cultures have not yet been reported as a source of a laboratory-acquired infection. However, because of the presence of biologically active mycoplasma products and the stability of mycoplasma antigensFootnote 21 as well as the fact that a number of mycoplasmas are human pathogens, they are considered hazardous in cell cultures.

  • Cell lines with mycoplasma contaminants are to be handled at the containment level appropriate for the contaminating agent of the highest risk.


Freshly prepared primary cell lines may be at risk of parasite contamination if the cell line was obtained from a specimen known or suspected to be infected with a human parasite. Parasites have many life cycle stages, and not all stages are infective. This must be taken into consideration when determining the appropriate level of containment.

  • Cell lines in which the life-cycle stage of the infecting parasite is not known are to be manipulated at the containment level appropriate for the contaminating agent of the highest risk.

7.3.3 Self-self Experiments

Procedures or experiments with transformed human cells derived from the individual (human autologous) manipulating the cells is prohibited. Such experiments put the individual at risk, since any immune protection that is normally available to destroy foreign cells is now bypassedFootnote 20 Footnote 21 .



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