Chapter 3: The Laboratory Biosafety Guidelines: 3rd Edition 2004 – Handling infectious substances

Chapter 3 Handling Infectious Substances

Individuals who work in a laboratory that handles infectious substances are at risk of exposure to the substances they handle. Laboratory-acquired infections (LAIs) are not uncommon -- over 5,000 cases and 190 deaths had been reported up to 1999(1) , although these figures are believed to be a significant underestimate because of underreporting(2,3). Additionally, only about 20% of infections can be attributed to any known, single exposure event(4).

There are a number of ways in which infectious substances can enter the body and cause infection, including ingestion, inhalation, or contact with mucous membranes, including conjunctivae (transfer of microorganisms to the eyes by contaminated hands), or with non-intact skin.

The types of events that can lead to an infection include the following: exposure to infectious aerosols; spills and splashes; accidental needlestick injuries; cuts from sharp objects and broken glass; bites and scratches from animals or ectoparasites; oral pipetting (which is prohibited); centrifuge accidents; secondary spread of infectious materials to nonlaboratory areas. Exposure to aerosols may be the greatest biohazard facing laboratory workers(5). Aerosols can present a risk in terms of inhalation, ingestion, mucous membrane contact, etc. Operational practices and techniques must be used to minimize the creation of aerosols associated with common laboratory procedures.

As assessed in Chapter 2, the following are operational practices for activities involving laboratory scale use of human pathogens at the four containment levels.

3.1 Operational Practices for Laboratories

3.1.1 General Practices

The following general practices are required for all laboratories handling infectious substances.

  1. A documented procedural (safety) manual must be available for all staff, and its requirements followed; it must be reviewed and updated regularly.
  2. Personnel must receive training on the potential hazards associated with the work involved and the necessary precautions to prevent exposure to infectious agents and release of contained material; personnel must show evidence that they understood the training provided; training must be documented and signed by both the employee and supervisor; retraining programs should also be implemented.
  3. Eating, drinking, smoking, storing of either food, personal belongings, or utensils, applying cosmetics, and inserting or removing contact lenses are not permitted in any laboratory; the wearing of contact lenses is permitted only when other forms of corrective eyewear are not suitable; wearing jewelry is not recommended in the laboratory.
  4. Oral pipetting of any substance is prohibited in any laboratory.
  5. Long hair is to be tied back or restrained so that it cannot come into contact with hands, specimens, containers or equipment.
  6. Access to laboratory and support areas is limited to authorized personnel.
  7. Doors to laboratories must not be left open (this does not apply to an open area within a laboratory).
  8. Open wounds, cuts, scratches and grazes should be covered with waterproof dressings.
  9. Laboratories are to be kept clean and tidy. Storage of materials that are not pertinent to the work and cannot be easily decontaminated (e.g., journals, books, correspondence) should be minimized; paperwork and report writing should be kept separate from such biohazardous materials work areas.
  10. Protective laboratory clothing, properly fastened, must be worn by all personnel, including visitors, trainees and others entering or working in the laboratory; suitable footwear with closed toes and heels must be worn in all laboratory areas.
  11. Where there is a known or potential risk of exposure to splashes or flying objects, whether during routine operations or under unusual circumstances (e.g., accidents), eye and face protection must be used. Careful consideration should be given to the identification of procedures requiring eye and face protection, and selection should be appropriate to the hazard.
  12. Gloves (e.g., latex, vinyl, co-polymer) must be worn for all procedures that might involve direct skin contact with biohazardous material or infected animals; gloves are to be removed when leaving the laboratory and decontaminated with other laboratory wastes before disposal; metal mesh gloves can be worn underneath the glove.
  13. Protective laboratory clothing must not be worn in nonlaboratory areas; laboratory clothing must not be stored in contact with street clothing.
  14. If a known or suspected exposure occurs, contaminated clothing must be decontaminated before laundering (unless laundering facilities are within the containment laboratory and have been proven to be effective in decontamination).
  15. The use of needles, syringes and other sharp objects should be strictly limited; needles and syringes should be used only for parenteral injection and aspiration of fluids from laboratory animals and diaphragm bottles; caution should be used when handling needles and syringes to avoid auto-inoculation and the generation of aerosols during use and disposal; where appropriate, procedures should be performed in a BSC; needles should not be bent, sheared, recapped or removed from the syringe; they should be promptly placed in a puncture-resistant sharps container (in accordance with Canadian Standards Association [CSA] standard Z316.6-95(R2000))(6) before disposal.
  16. Hands must be washed after gloves have been removed, before leaving the laboratory and at any time after handling materials known or suspected to be contaminated.
  17. Work surfaces must be cleaned and decontaminated with a suitable disinfectant at the end of the day and after any spill of potentially biohazardous material; work surfaces that have become permeable (i.e., cracked, chipped, loose) to biohazardous material must be replaced or repaired.
  18. Contaminated materials and equipment leaving the laboratory for servicing or disposal must be appropriately decontaminated and labelled or tagged-out as such.
  19. Efficacy monitoring of autoclaves used for decontamination with biological indicators must be done regularly (i.e., consider weekly, depending on the frequency of use of the autoclave), and the records of these results and cycle logs (i.e., time, temperature and pressure) must also be kept on file.
  20. All contaminated materials, solid or liquid, must be decontaminated before disposal or reuse; the material must be contained in such a way as to prevent the release of the contaminated contents during removal; centralized autoclaving facilities are to follow the applicable containment level 2 requirements.
  21. Disinfectants effective against the agents in use must be available at all times within the areas where the biohazardous material is handled or stored.
  22. Leak-proof containers are to be used for the transport of infectious materials within facilities (e.g., between laboratories in the same facility).
  23. Spills, accidents or exposures to infectious materials and losses of containment must be reported immediately to the laboratory supervisor; written records of such incidents must be maintained, and the results of incident investigations should be used for continuing education.
  24. An effective rodent and insect control program must be maintained.

3.1.2 Containment Level 2

In addition to the general practices required for all laboratories handling infectious substances, the following describe the minimum operational practices required for containment level 2.

  1. Good microbiological laboratory practices intended to avoid the release of infectious agents are to be employed.
  2. BSCs must be used for procedures that may produce infectious aerosols and that involve high concentrations or large volumes of biohazardous material. Laboratory supervisors, in consultation with the Biological Safety Officer/Institutional Biosafety Committee, should perform a risk assessment to determine which procedures and what concentrations and volumes necessitate the use of a BSC.
  3. Appropriate signage indicating the nature of the hazard being used (e.g., biohazard sign, containment level) must be posted outside each laboratory; if infectious agents used in the laboratory require special provisions for entry, the relevant information must be included on the sign; the contact information of the laboratory supervisor or other responsible person(s) must also be listed.
  4. Entry must be restricted to laboratory staff, animal handlers, maintenance staff and others on official business.
  5. All people working in the containment area must be trained in and follow the operational protocols for the project in process. Trainees must be accompanied by a trained staff member. Visitors, maintenance staff, janitorial staff and others, as deemed appropriate, must also be provided with training and/or supervision commensurate with their anticipated activities in the containment area.
  6. Emergency procedures for spill clean-up, BSC failure, fire, animal escape and other emergencies must be written, easily accessible and followed. A record must be made of other people entering the facility during an emergency.

3.1.3 Containment Level 3

In addition to the operational practices for all laboratories handling infectious substances and those minimum requirements for containment level 2, the following describe the minimum operational practices required at containment level 3.

  1. There must be a program for the management of biological safety issues in place with appropriate authority to oversee safety and containment practices (see Chapter 2, Section 2.5).
  2. Everyone entering the containment laboratory must have completed a training course in procedures specific to the containment laboratory and must show evidence of having understood the training; training must be documented and signed by the employee and supervisor.
  3. Employees working in the containment area must have knowledge of the physical operation and design of the facility (e.g., air pressure gradients between zones, directional airflow patterns, alarm signals for air pressure failure, containment perimeter).
  4. A protocol specific to the operation of the laboratory must be developed and read by personnel; employees must certify in writing that they have understood the material in the protocol. This should include entry and exit protocols for people, animals, equipment, samples and waste. General protocols must be supplemented with protocols specific to each project in progress.
  5. Personnel must have demonstrated proficiency in microbiological practices and techniques.
  6. Smoke testing (i.e., using a smoke pencil held at the door between the anteroom and the containment facility, and other doors as required) should be done periodically by laboratory staff to verify correct airflow; a containment check must be performed before entering the containment laboratory (e.g., verify correct reading on the pressure monitoring device).
  7. People entering a containment facility must be well prepared and bring all materials they will need with them; if something has been forgotten, established traffic patterns must still be adhered to (i.e., do not go back to get it; either phone for someone to bring it or exit using proper protocols).
  8. Routine laboratory cleaning must be done by personnel using the containment facility or by specific personnel dedicated and trained for this task.
  9. The containment laboratory must be kept locked.
  10. Infectious agents should be stored inside the containment laboratory; agents stored outside of the zone must be kept locked, in leakproof containers; emergency response procedures are to take into account the existence of such infectious agents outside of the containment level 3 laboratory.
  11. Personal items such as purses and outdoor clothing must not be brought into the containment laboratory.
  12. Drainage traps must be filled with liquid (i.e., through regular sink usage, automatic primers or by filling traps in areas that are not frequently used).
  13. Laboratory samples and supplies may be carried into the containment laboratory or passed in through a pass-box; if the barrier autoclave is used to pass materials into the laboratory, the autoclave must have been cycled before the outer "clean side" door is opened.
  14. Personnel entering the containment laboratory must remove street clothing and jewelry, and change into dedicated laboratory clothing and shoes; dedicated laboratory clothing and shoes must be removed before leaving the containment laboratory in a manner that minimizes any contamination of the skin with the potentially contaminated dedicated laboratory clothing. The use of full coverage protective clothing (i.e., completely covering all street clothing) is an acceptable alternative. When a known or suspected exposure may have occurred, all clothing, including street clothing, requires appropriate decontamination. Laboratories manipulating organisms, such as HIV, that are not infectious via inhalation, are not required to remove street clothing.
  15. An additional layer of protective clothing (i.e., solid-front gowns with tight-fitting wrists, gloves, respiratory protection(7) ) may be worn over laboratory clothing when infectious materials are directly handled and should be removed after completion of work (e.g., dedicated for use at the BSC).
  16. Centrifugation of infectious materials must be carried out in closed containers placed in sealed safety cups or rotors that are unloaded in a BSC.
  17. Animals or arthropods that have been experimentally infected must remain in the laboratory or appropriate animal containment facility.
  18. When a known or suspected aerosol exposure may have occurred, protocols based on a local risk assessment must be in place to determine whether showering is required on exit from the laboratory.
  19. All activities with infectious materials are conducted in a BSC; if this is not possible, other primary containment devices in combination with personal protective clothing and equipment must be used; no work with open vessels containing infectious materials is conducted on the open bench.
  20. Heat-sensitive materials that cannot be autoclaved out of the containment laboratory must be decontaminated at the containment barrier (e.g., fumigated with formaldehyde, vaporized hydrogen peroxide or a suitable alternative; disinfected using liquid chemicals; or subjected to other technology proven to be effective).
  21. Emergency procedures for failure of air handling systems and other containment emergencies must be written, easily accessible and followed.
  22. In the event of life-threatening emergencies, personal health and safety are a priority; exit protocols must be established whereby routine procedures might be bypassed; a reporting area must be identified where further steps must be taken (e.g., disinfecting footwear, changing, showering).

3.1.4 Containment Level 4

In addition to the operational practices for all laboratories handling infectious substances and those minimum requirements for containment level 2 and 3, the following describe the minimum operational practices required at containment level 4.

  1. Protocols must be established for emergencies, including damage to positive pressure suits, loss of breathing air, and loss of chemical shower.
  2. Employees must immediately notify their supervisor of any unexplained febrile illness; supervisors must contact any employee with unexplained work absences.
  3. The employer must establish liaison with the local hospital/health care facility to ensure that in the event of an employee's accidental exposure to containment level 4 agents the hospital/health care facility is fully aware of the infectious agents involved and that the appropriate procedures are in place for the treatment of the employee (patient).
  4. A record of containment laboratory usage is to be maintained (i.e., log book of all entry and exits) with date and time.
  5. Cultures and stocks of infectious agents must be stored in a secure area inside the containment laboratory and an inventory of pathogens maintained.
  6. A daily check of containment systems (e.g., directional airflow, disinfectant level in chemical shower, critical containment points for a class III BSC line) and life support systems (e.g., back-up breathing air) must be carried out before entering the laboratory.
  7. Personnel entering the laboratory must remove street clothing (including undergarments) and jewelry, and change into dedicated laboratory clothing and shoes.
  8. Positive pressure suits must be worn (for level 4 suit mode); the integrity of the suit must be routinely checked for leaks.
  9. A chemical shower of appropriate duration is required for personnel in suits who are leaving the containment laboratory; the disinfectant used must be effective against the agents of concern, be diluted as specified and prepared fresh as required; this is not applicable for class III BSC line level 4 containment facilities.
  10. A body shower is required on exit from the containment laboratory.
  11. Material can be removed from the containment laboratory only after appropriate decontamination or after specific approval from the Biological Safety Officer or other appropriate authority.
  12. A competent person must be available outside the containment level 4 laboratory when work is being conducted within the laboratory, to assist in case of emergency.
  13. Small laboratory animals, primates or insects infected with level 4 agents are to be housed in a partial containment system (e.g., cages placed in HEPA filtered containment enclosures).
  14. All laboratory procedures are to be conducted within a BSC in conjunction with a positive pressure suit or within a class III BSC line.
  15. Large animals require specialized care and handling not dealt with by these Guidelines. For details, please refer to the current edition of Containment Standards for Veterinary Facilities, by the Canadian Food Inspection Agency(8). This office can be contacted by calling the Biohazard Containment and Safety Division directly at (613) 221-7088 or accessing their Web site:


  1. Harding, A.L, and Brandt Byers, K. Epidemiology of laboratory-associated infections. In: Fleming, D.O., and Hunt, D.L. Biological safety: principles and practices. Washington, DC: ASM Press, 2000;35-54.
  2. Pike, R.M. Laboratory-associated infections: incidence, fatalities, causes and preventions. Annu. Rev. Microbiol. 1979; 33:41-66.
  3. Collins, C.H., and Kennedy, D.A. Preface. In: Laboratory-acquired infections: history, incidence, causes and preventions. Oxford, U.K: Butterworth-Heinemann, 1999; ix-xi.
  4. Pike, R.M. Laboratory-associated infections. Summary and analysis of 3921 cases. Health Lab Sci 1976;105-14, volume 13.
  5. Collins, C.H., and Kennedy, D.A. Exposure, sources and routes of infection. In: Laboratory-acquired infections: history, incidence, causes and preventions. Oxford, U.K: Butterworth-Heinemann, 1999; 38-53.
  6. Evaluation of single use medical sharps containers for biohazardous and cytotoxic waste. CSA standard Z316.6-95(R2000). Toronto: Canadian Standards Association, 2000.
  7. Selection, Use, and Care of Respirators. CSA Standard Z94.4-02. Toronto: Canadian Standards Association, 2002.
  8. Containment standards for veterinary facilities. Ottawa: Agriculture and Agri-Food Canada, Minister of Supply ans Services Canada, No. 1921/E, 1996.
Report a problem or mistake on this page
Please select all that apply:

Privacy statement

Thank you for your help!

You will not receive a reply. For enquiries, contact us.

Date modified: