4.1 Containment Levels: Facility Design and Work Practices
4.1.1 Level 1
4.1.2 Level 2
4.1.3 Level 3
4.1.4 Level 4
4.2 Biological Safety Cabinets
4.2.1 HEPA Filters
4.2.2 Classes of Biological Safety Cabinets
188.8.131.52 Class I
184.108.40.206 Class II
220.127.116.11 Class III
4.2.3 Placement of Biological Safety Cabinets
4.2.4 Working Safely in Biological Safety Cabinets
4.2.5 Cabinet Start Up and Shut Down Procedures
4.2.6 BSC Failure
4.2.7 Maintenance/Certification of Biological Safety Cabinets
6. Sterilization and Disinfection in the Laboratory
6.1 Microbial Resistance to Physical and Chemical Agents
6.2 Physical Sterilants and Disinfectants
6.2.1 Heat Sterilization and Decontamination
6.2.2 Other Physical Agents of Sterilization and Disinfection
18.104.22.168 UV Light (Germicidal Lamps)
22.214.171.124 Miscellaneous Physical Methods
6.3 Chemical Sterilants and Disinfectants
7. Biohazards Associated with Animal Handling
7.2 Laboratory Acquired Allergies to Animals
7.3 Theory and Practical Training Requirements for Animal Users
7.4 The Occupational Health Program
7.4.1 Steps Involved in Medical Monitoring
7.4.2 Features of the Occupational Health Program
7.4.3 How to Enroll in the Occupational Health Program
8. Safe Handling of Laboratory Equipment
8.0.2 Selection and Use of Equipment
8.3 Mixing Apparatus
8.4 Freezing Apparatus
8.5 Vacuum/Aspirating Equipment
8.6 Needles and Syringes
8.7.1 Selection of a Mechanical Pipetting Aid
8.7.2 Safe Use of Pipettes
8.9 Miscellaneous Equipment
11. Transport of Containment Levels 1 and 2 Material
11.1 Transport Within or Between Labs
11.2 Transport Between Buildings
11.3 National and International Transportation Regulations
11.3.1 Shipping of Biohazardous Material
11.3.2 Importation of Biohazardous Material
126.96.36.199 Human and Terrestrial Animal Pathogens
188.8.131.52 Other Pathogens
The McGill Laboratory Biosafety Manual has been prepared for the benefit of those who handle or work in proximity to potentially infectious biological agents. This manual attempts to address the concerns most frequently expressed to Environmental Health & Safety personnel by those who work in areas where biohazardous materials are used.
As this manual does not address the chemical and physical hazards commonly encountered in the lab, it is to be regarded as an addendum to the Laboratory Safety Manual. The hazards presented by radiation are of physical rather than biological origin and thus are not covered in the Biosafety Manual; information on working safely with radiation can be obtained from the McGill Radiation Safety Policy and Manual or by consulting the Radiation Safety Officer at Environmental Health & Safety.
A biohazard can be defined as any organism, or material produced by such an organism, that is known or suspected to cause human or animal disease. Biohazardous/infectious material falls under Class D, Division 3 of the Workplace Hazardous Materials Information System (WHMIS), and includes:
- microorganisms such as viruses, fungi, parasites, and bacteria and their toxic metabolites
- mammalian blood and body fluids
- unfixed and fixed tissues and specimens from humans and non-human primates
- cell lines and other tissue cultures
- certain types of nucleic acids, such as DNA derived from pathogenic organisms, human oncogenes or transformed cell lines
- genetically altered organisms, including plants
- zoonotic agents
Exposure to biohazardous agents may occur via puncture wounds or as a result of absorption through the respiratory tract, digestive system, skin and mucous membranes: such exposures may result while handling microorganisms, animals, cell cultures and tissues or diagnostic specimens. Investigators who are uncertain as to whether a material is biohazardous or not should consult the Biosafety Officer at Environmental Health & Safety.
Environmental Health & Safety must approve any activities carried out involving biohazardous agents. The certification requirement is not restricted to research activities, but also includes biological agents used for testing, diagnostic or teaching purposes. Approval is required for all containment levels, including Level 1. Investigators must complete the Application to Use Biohazardous Materials form and submit it for approval to Environmental Health & Safety prior to:
- starting new projects
- changing a protocol (i.e., use of a new biohazardous material)
- expiry of a previously approved application
It is the responsibility of the investigator to send a copy of the first page of the approved license to the Research Grants Office and to the granting agency.
For further information, review the McGill Biohazards Policy and section D of the McGill University Administrative Handbook, or contact Environmental Health & Safety.
Basic requirements for a laboratory using infectious materials are:
- Ensure that all laboratory personnel, including service and custodial staff and visitors, understand the chemical and biological dangers associated with the lab. Affix biohazard signs to Laboratory Information Cards on doors outside laboratories where biohazardous material is handled or stored. Post the spill response protocol in a visible location in the laboratory.
- Restrict laboratory access and keep doors locked when the laboratory is unattended.
- Keep the facility clean and free of clutter. Ensure that emergency safety devices (e.g., fire extinguishers, eyewashes, etc.) are easily accessible and in working order.
- Ensure that all personnel, students and visitors adhere to University policies for eye and face protection and for protective clothing (Refer to Sections 11, 11.1 and 11.2 of the Lab Safety Manual). Remove lab coats or gowns and gloves before leaving the laboratory; never wear lab clothing in eating facilities.
- Avoid eating, drinking, smoking, storage of food and food utensils, application of cosmetics or lip balm and insertion or removal of contact lenses in the laboratory.
- Restrain long hair. Avoid wearing loose clothing or jewelry, shorts and open-toed shoes or sandals.
- Observe "Universal Precautions" when collecting, processing, storing, shipping or transporting human blood and body fluids; i.e., handle such specimens as if infected with a bloodborne pathogen such as hepatitis B or C or human immunodeficiency virus (HIV).
- Carry out procedures so as to minimize risks of splashes, spills and generation of aerosols.
- Refrain from pipetting by mouth.
- Use hypodermic needles only when absolutely necessary. Do not bend, break, shear or recap used needles.
- Wash hands after handling infectious material (even when gloves have been worn) and before leaving the laboratory.
- Decontaminate all contaminated materials before disposal or reuse.
- Decontaminate laboratory surfaces following any spill of biohazardous materials and at the end of each workday.
- Report all spills and accidents/incidents.
Criteria for classification of infectious agents are outlined in the document Laboratory Biosafety Guidelines, published by the Public Health Agency of Canada. Essentially, microbiological pathogens are classified according to their impact upon the individuals who manipulate them, upon their colleagues, and upon the surrounding community. Agents that pose little or no risk are assigned to Risk Group 1, while those with the greatest hazardous potential are in Risk Group 4. Risk assessment is based upon several factors, including:
- severity of induced disease
- route(s) of infection
- virulence and infectivity of the microorganism
- antibiotic resistance patterns
- availability of effective medical treatment (e.g., antibiotic therapy) or vaccine
- presence of vectors (e.g., arthropods)
- whether the pathogen is indigenous to Canada
- possible effects on other animals and plants
Before setting up experiments involving new biohazards, consideration should also be given to conditions under which the infectious agent is used. For example, manipulation of large volumes and high concentrations of an infectious microorganism in culture media presents a greater risk than smearing the same pathogen on a slide. Work involving release of microbial aerosols, passage in animals and infection of arthropod vectors also increase the hazard. In these cases, pathogens should be handled as if they were in the next highest risk group: i.e., if the experimental procedure is likely to generate large amounts of aerosolized Risk Group 2 agent, the physical and operational requirements applicable to Risk Group 3 agents should be observed.
An outline of the characteristics of agents in each Risk Group is presented in Table 1.
TABLE 1 -
Risks and characteristics associated with pathogens from Risk Groups 1 to 4, and recommended containment level and class of biological safety cabinet.
|Risk group||Risk assessment||Characte ristics||Examples||Contain ment level||Bio safety cabinet|
|1||Low individual; low community.||Unlikely to cause disease in animals or humans||Lactobacillus spp., Bacillus subtilis, Naegleria gruberi, Micrococcus spp., E. coli K12||1||Not required|
|2||Moderate individual; low community.||Rarely cause serious human or animal disease; effective prevention and treatment available; limited risk of spreading.||Hepatitis B virus, Toxoplasma spp, HIV (non-cultured), Ascaris, Salmonella typhimurium||2||Class I or Class II|
|3||High individual; Low community||May cause serious disease in humans or animals; effective prevention and treatment available; unlikely to be spread by casual contact.||Lassa fever virus, Hantavirus, Yersinia pestis, Histoplasma capsulatum, Bacillus anthracis, cultured isolates of HIV*||3||Class I or Class II|
|4||High individual; high community.||Likely to cause very serious disease in humans or animals; readily transmitted from one indvidual to another, or between animals and humans; preventative vaccines or effective treatment not available.||Marburg virus, Ebola virus, Crimean-Congo hemorrhagic fever virus, Herpesvirus simiae||4||Class I or II plus positive pressure suits or Class III|
Some progressive neurological diseases are caused by unconventional "slow viruses". Among the slow viruses, prions (proteinaceous infectious particles) have been associated with transmissible degenerative diseases of the central nervous system in humans (Creutzfeldt-Jacob, kuru) and animals (transmissible encephalopathy of mink and scrapie in sheep and goats). These unconventional viruses are resistant to destruction by chemical (10% formalin, glutaraldeyhye, 70% ethanol, iodine) and physical (UV light, ionizing radiation, boiling) procedures. While there have been no documented cases of laboratory-acquired infections, the following precautions should be observed when handling neurological material from infected or potentially infected humans and animals:
- Handle as Risk Group 2 or higher, depending on the nature of work and amount of agent being manipulated.
- Handle formalin-fixed tissues and paraffin-embedded blocks as if still infectious.
- Keep up-to-date on disinfection protocols
The term "biotechnology" describes a variety of techniques for manipulation of cells; biotechnology has long been used for purposes such as selective breeding of animals and food production (bread, yogurt, beer).
More recently, in vitro incorporation of segments of genetic material from one cell into another ("recombinant DNA technology") has resulted in altered organisms that can manufacture products such as vaccines, hormones, interferons and enzymes. Genetically engineered organisms are used for treatment of waste and spills, and plants can be made resistant to cold, disease, pests and drought.
However, biotechnology carries with it the potential for harm. A genetically altered organism may be directly pathogenic or toxic or, if released into the environment, crowd out beneficial organisms, transfer undesirable genetic traits to wild species or mutate into a pathogenic form.
The risks associated with recombinant DNA technology are to be assessed by the investigator when submitting the Application to Use Biohazardous Materials form to McGill Environmental Health & Safety. Such assessments should be based upon the:
- source of the DNA to be transferred
When assessing the risk of, and containment level required by, a genetic engineering protocol, the following approach is recommended: if the components of a genetic manipulation are not hazardous, then the altered organism is unlikely to present a risk, and no restrictions are needed. However, if one of the components is potentially hazardous, a risk level appropriate for the known hazard is assigned and modified as required. Subsequent modifications depend on factors such as:
- expression of the transferred gene in the recombinant organism
- ability of the vector to survive outside the laboratory
- expected interactions between transferred gene, host and other factors
Cell cultures derived from humans or animals known to be infected with a pathogen, as well as cultures known or suspected to contain infectious microorganisms (e.g., herpesvirus or EBV-transformed cultures) should be assigned to the risk group appropriate for the suspected or known pathogen and handled using the relevant containment level and work practices. Risk groups and containment levels for specific pathogens can be obtained from the federal Laboratory Biosafety Guidelines.
In addition, cell cultures may carry unsuspected oncogenic, allergenic or infectious particles. It is impractical, if not impossible, to screen such cultures for all potentially harmful microorganisms: even well characterized lines with a history of safe use can become contaminated by adventitious, possibly infectious, microorganisms. For this reason, it is prudent to treat all eukaryotic cultures as moderate risk agents (i.e., Risk Group 2) and to use containment level 2 facilities and work practices whenever working with them.
The term "containment" is used in describing measures used to provide a barrier between the infectious organism(s) being handled and the worker (and, ultimately, the community at large). Containment is achieved through the use of appropriate safety equipment, facility design and lab procedures and practices.
Careful consideration must be given to both facility design and work practices to ensure protection of laboratory personnel, their colleagues and the community as a whole. Four containment levels are outlined in the Health Canada guidelines: of the four containment levels, the highest safety standards (Level 4) are reserved for the most hazardous pathogens (Risk Group 4), and the least stringent (Level 1) for those which have minimal impact on health (Risk Group 1).
Level 1 containment is used when working with agents (Risk Group 1) that pose no risk to healthy adults:
- The laboratory may be near a public area but doors should be kept closed.
- Work may be carried out on an open bench top.
- Lab surfaces (walls, ceilings, furniture and floors) should be cleanable.
- Open windows should have insect screens.
- Eyewash stations and handwashing facilities should be available.
- Street clothes and lab coats should not be kept together.
- Disinfection should be carried out as required, using effective concentrations and contact times; solutions should be replaced regularly.
Level 2 containment is appropriate for work with Risk Group 2 agents. The following precautions, in addition to those for containment Level 1, are recommended:
- The facility should be away from public areas and should have self-closing doors.
- A biohazard sign with relevant information should be posted at the entrance.
- Service and custodial staff should be informed of the hazards; the latter should be expected to clean floors only.
- Items should be autoclaved or chemically decontaminated before removal from the facility.
- Use Class I or II biological safety cabinets for procedures that generate infectious particles.
- Procedures should be carried out such that aerosol generation is minimized.
- An emergency spill response plan should be in place and posted in a visible location.
- Vacuum lines should be equipped with HEPA filters.
- Lab coats may be front-closing, but should not be worn outside the lab.
- Wear gloves to prevent skin contamination.
Level 3 containment is recommended for work with Risk Group 3 agents. Measures should include the recommendations outlined for levels 1 and 2, plus the following:
- The lab should be away from general work areas, with controlled access.
- There should be a change and shower area within the containment facility perimeter.
- The area should be kept at negative pressure relative to surrounding areas.
- Supply and exhaust air should be HEPA-filtered or provided by dedicated systems.
- A hands-free handwashing sink should be located near the exit.
- Lab windows should be unbreakable and sealed shut.
- Lab personnel should be trained in handling, disposal, and emergency procedures. Written protocols for these procedures should be developed and posted in a visible location.
- Personnel should wear solid-front lab clothing, which should be autoclaved before laundering or disposal.
- A medical surveillance program is recommended.
Level 4 is used for work with Risk Group 4 agents. Additional recommendations include:
- physical isolation of the laboratory, with an airlock for access
- entry restricted to authorized personnel and recorded in a log book: no one should work alone
- use of class III biological safety cabinets and/or positive-pressure protective suits
- additional safety measures for ventilation, waste treatment, and gas and water services
Biological safety cabinets reduce the risk of airborne infection by reducing the escape of aerosolized infectious agents into the laboratory environment. In addition to protecting workers, some biological safety cabinets protect the work inside the cabinet from airborne contamination (product protection). Biological safety cabinets minimize contact between the operator and pathogens through the use of directional airflow, HEPA filtration of supply and/or exhaust air, and, in some cases, a physical barrier such as a plastic or glass shield.
HEPA (High Efficiency Particulate Air) filters are an essential component of the biological safety cabinet, and have particle removal efficiencies of 99.97% or better for 0.3 micron diameter particles. This size particle is used as the basis for filter definition because it is considered the most difficult to remove. Thus, a filter that can trap 0.3-micron diameter particles can easily eliminate other sizes.
HEPA filters consist of continuous sheets of glass fiber paper pleated over rigid corrugated separators and mounted in a wooden or metal frame. The filter medium is delicate and should never be touched. As well, the gaskets used to seal the filter frame to the cabinet must not be disturbed; thus the biological safety cabinet should not be moved without subsequently being tested and certified.
While HEPA filters remove particulates from an airstream, they are not effective at collecting chemical gases or vapours. Thus, it is inadvisable to use recirculating Class II cabinets with agents which have significant amounts of hazardous volatile or radioactive components. Although Class III and 100% exhaust Class II cabinets can be used in experiments which involve use of chemicals of moderate toxicity, it should be remembered that these cabinets are not explosion-proof. Use of flammable or explosive products is to be avoided unless the cabinet has been specifically designed for their use.
Horizontal and vertical clean benches are not biological safety cabinets: HEPA- filtered air is directed over the work surface and then discharged directly into the room. Thus, these units provide product protection, but do not protect the operator from exposure to the materials being handled; they must not be used for work with potentially infectious or toxic materials.
There are three basic types of biological safety cabinet, each providing different levels of containment:
- protects operator and environment
- for work with low and moderate risk agents (Risk Groups 2 and 3) where product protection is not critical
General principle of operation: An inward flow of room air through the work opening, away from the operator, prevents the escape of airborne pathogens into the laboratory. Negative cabinet pressure is created by a blower that exhausts the air, either into the room or to the outside, through a HEPA filter. It is this HEPA filtration of exhaust air that provides environmental protection. A disadvantage of this type of cabinet is that the product is exposed to contaminants that are pulled in from the room environment. In addition, internal air turbulence may result in cross-contamination within the cabinet.
- protects operator, product and environment from particulate contamination
- for work with low to moderate risk agents (Risk Groups 2 and 3)
General principle of operation: Escape of pathogens into the worker's environment is prevented by an inward flow of room air which enters the front opening without crossing the work area and by HEPA filtration of exhaust air (this provides environmental protection), while downward flow of HEPA-filtered air through the work area removes work zone contaminants and protects the product. The amounts of room air drawn into the intake grille and the amount of air exhausted through the exhaust filter are equal. This balance is critical: positive pressure will allow the outflow of pathogens, while negative pressure will result in inflow of room contaminants.
The different types of Class II cabinets (e.g., Type A, Type B or 100% exhaust) vary in:
- airflow velocities
- amount of cabinet air recirculated (from 0 to 70%)
- amount of cabinet air exhausted (from 30 to 100%)
- destination of exhaust air (back to lab or outside)
- exhaust ducting (building system versus dedicated ducts)
It should be kept in mind that toxic or radiolabelled chemicals must not be handled in cabinets that recirculate air within the cabinet or exhaust into the laboratory.
- totally enclosed, gas tight, with glove ports for manipulation of pathogens
- provides the greatest level of operator and product protection
- for work with high risk pathogens (Risk Group 4)
General principle of operation: These cabinets form a physical barrier between the operator and microbiological agent. Internal negative pressure confines any leaks to the inside of the cabinet. Supply and exhaust air is HEPA-filtered; a dedicated exhaust fan, separate from that of the facility ventilation system, discharges directly to the outdoors. There is no recirculation of air within the cabinet.
A Class III cabinet system must be designed to allow for safe introduction, handling and removal of all materials throughout the procedure. Equipment such as the incubator, refrigerator, centrifuge, autoclave and chemical dunk tank are connected to the cabinet system.
Since an uninterrupted curtain of inward flowing air at the front is critical to cabinet performance, the biological safety cabinet should be situated in an area where there will be no interference with this air barrier. Interfering room air currents may be caused by:
- pedestrian traffic
- room ventilation such as overhead supply diffusers, fans, fume hoods, heating and air conditioning registers
- drafts from open windows
- operation of doors
The ideal location would be a "dead end" corner of the lab, away from doorways, throughways, windows, room air supply diffusers, fume hoods and heating equipment.
Biological safety cabinets must be combined with good work practices for optimum safety and contamination control. Recommended practices when using a biological safety cabinet include the following:
- Movement of arms into and out of the cabinet can disrupt airflow, adversely affecting cabinet performance. Whenever possible, place all materials needed for a procedure inside the cabinet before starting. Avoid bringing non-essential equipment and supplies into the cabinet.
- Place supplies, equipment and absorbent towels so that air intake or exhaust grilles are not obstructed.
- Keep opening and closing of lab doors and other personnel activity to a minimum.
- Open flames contribute to the heat load, generate convection currents that disrupt airflow patterns and may damage the HEPA filter. Gas can escape from loose connections or damaged tubing and may be ignited by sparks or heat from cabinet motors and switches. The use of an open flame in the presence of flammable alcohol-based disinfectants further increases the risk of fire or explosion. Pre-sterilized loops, needles, etc. or a micro-incinerator (e.g. Bacti-Cinerator) should be used instead of a flame. Installation of new natural gas lines into biological safety cabinets will only be considered under exceptional circumstances and upon providing written justification to Environmental Health & Safety demonstrating that there are no other viable methods available and the use of an open flame cannot be avoided.
- Attach a HEPA filter cartridge between the vacuum trap and the source valve.
- Work at least 4-6 inches inside the cabinet window.
- Carry out work on an absorbent pad to contain small spills. Clean up spills as soon as they occur; remove and disinfect the grille if contaminated.
- Designate separate areas within the cabinet for contaminated and clean materials.
Before using the cabinet:
- Turn off the UV lamp; turn on the fluorescent lights.
- Disinfect the work surface (refer to Section 6).
- Place essential items inside the cabinet.
- Allow the blower to run for at least five minutes before starting work.
After completion of work:
- Leave blower on for at least five minutes to purge the cabinet.
- Remove and decontaminate equipment and materials, and disinfect cabinet surfaces.
- Turn off the blower and fluorescent lamp, and turn on the UV light.
- Cap cultures, surface decontaminate and return them to the incubator.
- Surface decontaminate all materials inside the BSC before removing them.
- Remove waste to biohazard bag and autoclave, or place in biohazard waste box for incineration.
- Close the sash and turn off the blower motor switch.
- Turn on the UV lamp if it still works.
- If the failure is cause by a power outage, restart and decontaminate the BSC as described in Section 4.2.5 when the power returns.
- If the failure is due to BSC malfunction, decontaminate cabinet surfaces and contact your service provider for repair. Ensure that the representative decontaminates the cabinet before carrying out any repairs.
- Affix a warning sign (eg. "OUT OF ORDER. DO NOT USE") to the cabinet.
Biological safety cabinets must be tested and certified annually. Cabinet performance must also be evaluated:
- upon initial installation in the laboratory
- when moved from one building or laboratory to another
- when moved from one area to another within the same room
- whenever maintenance is carried out on internal parts, and whenever filters are changed
Annual certification is provided at no charge, and can be arranged by contacting Environmental Health & Safety (local 4563). Costs of certification of new installations, relocated cabinets, and units that have been repaired are the responsibility of the user.
All individuals who work in a lab where pathogens are used must know how to handle these agents safely and what to do in case of a spill. An emergency spill response protocol specific for the microorganisms in use should be prepared and posted in a visible location within the laboratory.
An accident prevention plan should be the first priority. General safety precautions include:
- Limit access to rooms where microbiological agents are used.
- Wear appropriate protective clothing.
- Use the appropriate biological safety cabinet.
- Use plastics rather than breakable glassware to reduce likelihood of puncture wounds, cuts and generation of aerosols in the event of an accident.
- Transport materials on carts that have lipped shelves, using secondary containers (i.e. tubs) to catch spills.
- Disinfect waste.
Response procedures should be established before a spill occurs. Assessment of the hazards presented by the pathogen(s) in use should be based upon:
- virulence and infectivity of the agent
- viability - e.g., does the organism become inactive when dried?
- route of entry - e.g., can the organism enter the body via aerosols or splash to the eye?
- quantity and location of possible spill
- immune status of the individuals at risk
The necessary clean-up materials should be available on site. In preparing a spill response kit, ascertain that it contains the appropriate clean-up materials, protective clothing and equipment. The kit should be stored in a visible and accessible location immediately outside the facility and should include:
- disposable protective clothing (e.g., long-sleeved coat or gown, mask, gloves)
- absorbent paper
- autoclavable container and bags
- disinfectant appropriate for the pathogen(s) handled: be sure to replace the disinfectant before it expires
- autoclavable squeegee or forceps and dustpan
The appropriate spill response depends on the nature of the spilled organism and on the size of the spill. Sections 5.3.1 and 5.3.2 outline suitable approaches to handling minor and major spills.
Small spills can be cleaned up immediately by lab personnel, provided that the organism does not pose a health risk (i.e., if the spill consists of low to moderate risk agents). Cover with a disinfectant-soaked towel (using a spray bottle for distributing the disinfectant generates aerosols and is to be avoided). Autoclave or discard contaminated material in a biomedical waste container.
For spills of large volumes of moderate risk agents or small volumes of high risk agents, proceed as follows:
- Treat serious injuries before attempting to contain the spill.
- Evacuate the area immediately if exposure to the aerosolized microorganism presents a potential health hazard; close the facility door(s) and allow aerosols to settle for 30 minutes. Remove contaminated clothing and place it in an autoclave bag or other sealed container; disinfect and wash exposed skin. Report the spill by dialing local 3000 downtown or local 7777 at Macdonald campus.
- Don the appropriate protective clothing and cover the spill with absorbent material such as paper towels to reduce splashing. Pour disinfectant around the perimeter of the spill rather than directly onto it to minimize creation of aerosols. Work the disinfectant toward the centre and let it sit for at least 20 minutes.
- If the spilled material has leaked through the grilles of a biological safety cabinet, leave the cabinet running and pour in enough disinfectant (avoid alcohol due to explosion hazard) to dilute the spill tenfold. Drain the catch tray after the time interval appropriate for the disinfectant.
- Wipe down any adjacent walls, cabinets, furniture and equipment that may have been splashed.
- Use forceps/squeegee and dustpan to pick up and transfer the contaminated material into an autoclavable bag or biomedical waste container.
- Decontaminate the waste and cleaning utensils.
There is an important distinction between sterilization and disinfection. Whereas sterilization results in destruction of all forms of microbial life, disinfection results in destruction of specific pathogenic microorganisms. A more detailed description of disinfection levels can be found in the Glossary of this manual (Section 13).
Microorganisms vary in their resistance to destruction by physical or chemical means. A disinfectant that destroys bacteria may be ineffective against viruses or fungi. There are differences in susceptibility between gram-negative and gram-positive bacteria, and sometimes even between strains of the same species. Bacterial spores are more resistant than vegetative forms, and non-enveloped, non-lipid-containing viruses respond differently than do viruses which have a lipid coating.
Information on the susceptibility of a particular microorganism to disinfectants and physical inactivation procedures can be found in the material safety data sheet (MSDS) for that agent. MSDSs provide additional details such as health hazards associated with the microorganism, mode of transmission, containment requirements and spill response procedures. Environmental Health & Safety has available, and can provide to individuals, MSDSs on a number of infectious microorganisms.
Generally, sterilization is best achieved by physical methods such as steam or dry heat, which are less time-consuming and more reliable than chemical germicides. A summary of physical agents that employ heat for control of microorganisms can be found in Table 2. Of these physical procedures, steam autoclaving is the most practical option for the majority of laboratories for both sterilization and decontamination purposes.
Details on the use of an autoclave are given in Section 8.8.
TABLE 2 -
Outline of the properties of heat decontamination methods. For everyday laboratory purposes, autoclaving is the preferred method, unless the item cannot withstand the heat and/or moisture of autoclaving.
Principle / condition
Thermal inactivation: destroys by oxidation
|Non-corrosive simple design and principle||Less effective than moist heat; requires longer times and/or higher temperatures||Materials that are damaged by, or are impenetrable to, moist heat|
|Hot Air Oven:
160-180oC for 2-4 hours
|penetrates water-insoluble materials (e.g., grease and oil)
less corrosive to metals and sharp instruments than steam
|slow diffusion, penetration; loading, packing critical to performance; not suitable for reusable plastics||anhydrous materials, such as oils, greases and powders; laboratory glassware, instruments; closed containers|
oxidation to ashes (burning)
|rapid||initial contact with flame can produce a viable aerosol; possibility of accidental fire||inoculating loops, needles|
oxidation to ashes (burning) 1-60 mins: temperatures may exceed 1000oC
|reduces volume of waste by up to 95%||improper use may lead to emission of pathogens in smoke; requires transport of infectious waste; excess plastic (>20%) content reduces combustibility||for decontamination of waste items prior to disposal in landfill|
Principle / condition
Irreversible coagulation of (microbial) proteins
|More rapid and more effective than dry heat|
heating to below boiling point (generally 77oC) for up to 30 mins
|can be used on heat sensitive liquids and medical devices; low cost||not reliably sporicidal||milk and dairy products; some heat-sensitive medical equipment|
|Tyndallization (Fractional Sterilization):
heating to 80-100oC for 30 mins on 3 successive days, with incubation periods in between
|resistant spores germinate and are killed on the second and third days||time consuming; not reliably sporicidal||heat sensitive materials such as bacteriologic media, solutions of chemicals, biological materials|
maximum temperature obtainable is approx. 100oC
|minimal equipment required||cumbersome; not practical for everyday lab use; not reliably sporicidal||small instruments and equipment|
steam under pressure 121oC/15 psi for 15-90 mins (gravity displacement autoclave); 132oC/27 psi for 4-20 mins (pre-vacuum autoclave)
|minimal time required; most dependable sterilant for lab use||loading and packing critical to performance; shielding dirt must first be removed; maintenance and quality control essential; damages heat-sensitive items||preparation of sterile glassware, media and instruments; decontamination of reusable supplies and equipment; decontamination of infectious waste|
The light (approximately 260 nm wavelength) emitted by UV lamps is germicidal, and can reduce the number of pathogenic microorganisms on exposed surfaces and in air. However, UV light has poor penetrating power; accumulations of dust, dirt, grease or clumps of microorganisms may shield microorganisms from the direct exposure required for destruction. UV light can cause burns to skin and eyes, and factors such as lamp age and poor maintenance can reduce performance. For safe and reliable use of germicidal lamps:
- Clean the bulb at least every 2 weeks; turn off power and wipe with an alcohol-moistened cloth.
- Blue light output is not an indication of the lamp's effectiveness; measure radiation output at least twice yearly with a UV meter or replace the bulb when emission declines to 70% of its rated output.
- Post warning signs to discourage personnel from entering areas where there is risk of exposure to UV light.
- Wear UV protective goggles, caps, gowns and gloves in rooms with UV installations.
The procedures listed below are included for the reader's interest:
- Infrared radiation: used for heat treatment of small metal and glass items.
- Microwaves: used for treatment of liquids, nonmetallic objects, and biohazardous waste.
- Gamma irradiation: disrupts DNA and RNA in living organisms, and is used by hospital and laboratory suppliers for materials that do not tolerate heat and pressure (i.e., autoclaving) or chemical treatments.
- Membrane filtration: physically removes particulates (e.g., microorganisms) from heat-sensitive pharmaceutical and biological fluids. The size of the particles removed is determined by the pore size of the filter membrane.
Instruments or materials that cannot withstand sterilization in a steam autoclave or dry-air oven can be sterilized with a gas such as ethylene oxide or a broad spectrum liquid chemical germicide. Chemical decontamination of surfaces may also be necessary for very large or fixed items. Since liquid chemical germicides generally require high concentrations and several hours of exposure time for sterilization purposes, they are usually used for disinfection rather than for sterilization purposes. The majority of chemical disinfectants have toxic properties: follow the manufacturer's directions for use and wear the appropriate personal protective equipment (e.g., gloves, eye protection, apron), especially when handling stock solutions.
Choice of a chemical germicide for use on contaminated equipment, supplies, laboratory surfaces or biohazardous waste depends upon a number of factors, including:
- number and nature of microbes to be destroyed (e.g., spores vs vegetative cells, bacteria vs viruses)
- type and configuration of item to be disinfected (fissures, crevices and enclosures may shield organisms)
- purpose of treatment (e.g., disinfection vs sterilization)
- interaction with other active chemicals
- whether the item is covered with soil which might inactivate the disinfectant
- contact time required for disinfection
- toxicity to individuals, culture systems, environment, residual toxicity on items
- pH, temperature, hardness of available dilution water
Direct contact between germicide and microorganism is essential for disinfection. Microorganisms can be shielded within air bubbles or under dirt, grease, oil, rust or clumps of microorganisms. Agar or proteinaceous nutrients and other cellular material can, either directly (through inactivation of the germicide) or indirectly (via physical shielding of microorganisms) reduce the efficacy of some liquid germicides.
No one chemical germicide is effective for all disinfection or sterilization purposes. A summary of chemical germicides, their use, effective concentrations, advantages and disadvantages are outlined in Tables 3, 4A and 4B.
TABLE 3 - halogen-releasing chemical germicides
Summary of concentrations used, contact times, advantages and disadvantages and uses of some of the halogen-releasing chemical germicides. The wide ranges of effective concentrations and contact times cited are due to a number of factors, including the interdependence of time and concentration, the variability in resistance of different microorganisms, the amount of organic material present and the desired effect (e.g., low-level vs. high-level disinfection).
|Sodium hypochlorite solution1 (liquid bleach)|
|Effective concentrations, contact times||100-10,000 ppm (.01-1%) free chlorine
10-60 min (3,000 ppm for broad spectrum)
|Advantages||Broad spectrum; inexpensive; widely available; bactericidal at low temperature|
|Disdvantages||Toxic, corrosive to skin and metals; efficacy decreases as pH increases; inactivated by organic matter; deteriorates under light and heat: shelf life of dilutions is less than 1 week|
|Some uses||General disinfectant; waste liquids; surface decontamination; emergency spill clean up; instrument disinfection|
|Calcium hypochlorite2 granules, powder, tablets|
|Effective concentrations, contact times||As for liquid bleach|
|Advantages||As for liquid bleach, but more stable|
|Disdvantages||As for liquid bleach above, except shelf life is longer|
|Some uses||As for liquid bleach|
|NaDCC3 (Sodium dichloroisocyanurate) powder, granules, tablets|
|Effective concentrations, contact times||as for liquid bleach|
|Advantages||More stable than hypochlorites|
|Disdvantages||Toxic; corrosive; inactivated by organic matter|
|Some uses||as for liquid bleach|
|Chloramine-T4 (Sodium tosylchloramide) powder or tablets|
|Effective concentrations, contact times||as for liquid bleach|
|Advantages||More stable, less affected by organic matter than hypochlorites; longer activity than hypochlorites|
|Disdvantages||Deteriorates under humidity, light and heat|
|Some uses||As for liquid bleach|
|Effective concentrations, contact times||Demand-release of chlorine dioxide in situ|
|Advantages||Longer activity than other chlorine compounds; less corrosive, less toxic than other chlorine compounds; effective at pH 6-10|
|Disdvantages||Aqueous solutions decompose under light|
|Some uses||Instrument disinfection; gas sterilization of germ-free animal chambers|
|Effective concentrations, contact times||30-1,000 ppm (.003-.1%) free iodine
|Advantages||Broad spectrum; germicidal over a wide pH range; generally nonstaining, less toxic and less irritating than aqueous or alcoholic iodine solutions|
|Disdvantages||Not consistently sporicidal; efficacy reduced by organic matter; some iodophor solutions support growth of Pseudomonas 7|
|Some uses||Germicidal soaps and antiseptics; surface decontamination; work surface wipedown; instrument disinfection|
1 a 1/10 dilution of 5.25% bleach provides 5,250 ppm available chlorine
2 "high tested" provides 70-72% available chlorine; chlorinated lime or bleaching powder provides approximately 35% available chlorine
3 approximately 60% available chlorine
4 approximately 25% available chlorine
5 To avoid shipping of this extremely reactive product, reagents ("base" and "activator") from commercially available kits are mixed with water to generate chlorine dioxide immediately prior to use
6 10% povidone-iodine provides 1% available iodine
7 Iodophor stock solutions can be less effective germicide than dilutions. For example, full-strength (10%) povidone-iodine provides approximately 10 times less free available iodine than a 1/100 dilution. Iodophors must be used at the manufacturer's recommended concentrations.
TABLE 4A - non-halogen chemical germicides
Summary of recommended concentrations, contact times,advantages and disadvantages of non-halogen chemical germicides. The wide ranges of effective concentrations and contact times cited reflect the interdependence of time and concentration as well as factors such as resistance of the particular class or strain of target microorganism(s) and desired effect.
|Effective concentrations, contact times||70-80% ethanol
|Advantages||Low toxicity; rapid action; low residue; non-corrosive|
|Disadvantages||Rapid evaporation limits contact time; flammable, eye irritant; may damage rubber, plastic, shellac; ineffective against bacterial spores|
|Some uses||Skin disinfectant (antiseptic); surface decontamination; benchtop, cabinet wipedown|
|Effective concentrations, contact times||400-50,000 ppm (.04-5%)
|Advantages||Tolerant of organic load; "hard" dilution water leaves an active residue (may be desirable on some surfaces); biodegradable|
|Disadvantages||Pungent odour; corrosive; some forms toxic; not sporicidal; limited activity against viruses; leaves a residual film (undesirable in culture systems); may support growth of bacteria 1|
|Some uses||Instruments and equipment disinfection; disinfection of floors and other surfaces; antiseptic soaps and lotions|
|QUATERNARY AMMONIUM COMPOUNDS|
|Effective concentrations, contact times||500-15,000 PPM (.05-1.5%)
|Advantages||Combined detergent and germicidal activity; stable; working dilutions have low toxicity|
|Disadvantages||not sporicidal; limited activity against viruses, mycobacteria; most formulations not readily biodegradable; may support growth of bacteria 2|
|Some uses||Surface decontamination; equipment wipedown; antiseptic formulations available; floors and walls|
|Effective concentrations, contact times||3-30% aqueous solution
10-60 min 6% for 30 min may kill spores
|Advantages||Rapid action; no residue; low toxicity; environmentally safe|
|Disadvantages||Limited sporicidal activity; corrosive to some metals; potentially explosive at high concentrations; stock solutions irritating to skin and eyes|
|Some uses||Surface decontamination; instruments and equipment|
|PERACETIC ACID (PAA)|
|Effective concentrations, contact times||.001-.3% aqueous solution
gas phase: 2-4%
|Advantages||Broad spectrum; sporicidal at low temperature; can tolerate organic load; rapid action; nontoxic decomposition products; leaves no residue|
|Disadvantages||Pungent odour; corrosive to some metals; shelf life of dilutions is less than 1 week; stock solutions irritating to skin and eyes; stock must be protected from heat & light; gas phase: respiratory irritant; fire hazard above 55oC|
|Some uses||instruments and equipment; gas phase sterilization of chambers for germ-free animals|
TABLE 4B - non-halogen chemical germicides
Summary of recommended concentrations, contact times, advantages and disadvantages of non-halogen chemical germicides. The wide ranges of effective concentrations and contact times cited reflect the interdependence of time and concentration as well as factors such as resistance of the particular class or strain of target microorganism(s) and desired effect.
|Effective concentrations, contact times||0.5-2.5% alkalinized aqueous solution
2-30 mins; up to 12 hours to kill all spores
|Advantages||Broad spectrum; does not corrode metal; can tolerate organic load|
|Disadvantages||Expensive; pH, temperature dependent; pungent odour; toxic: skin, eye, respiratory tract irritant; activated solutions have less than 2-week shelf life|
|Some uses||Cold sterilant and fixative; surface decontamination; instruments, equipment, glassware|
|Formalin (37% aqueous formaldehyde)|
|Effective concentrations, contact times||3-27% formalin (1-10% formaldehyde) in 70-90% alcohol
|Advantages||Broad spectrum; inexpensive; does not corrode metal; can tolerate organic load|
|Disadvantages||Pungent odour; skin, eye and respiratory tract irritant; potential carcinogen (animal studies); may require 24 hrs or more to kill all spores|
|Some uses||Cold sterilant and fixative; surface decontamination; instruments and equipment|
|Effective concentrations, contact times||1-3 hours|
|Advantages||As for formalin; effective penetration|
|Disadvantages||As for formalin; flammable; poor penetration of covered surfaces|
|Some uses||On site decontamination of biological safety cabinet HEPA filters; enclosed areas|
|ETHYLENE OXIDE GAS|
|Effective concentrations, contact times||50-1200 mg/L
|Advantages||Broad spectrum; no heat or moisture evolved; penetrates packaging materials|
|Disadvantages||Flammable, reactive; toxic: potential carcinogen and mutagen; some sterilized items may need more than 24 hours for outgassing|
|Some uses||Heat or moisture sensitive supplies, instruments and equipment|
Individuals whose work involves exposure to or handling of animals and animal tissues, body fluids and cell cultures should be aware of the possibility of acquiring, and take measures to avoid contracting, a zoonosis. Zoonoses are diseases that can be transmitted from animals to humans and may be acquired through:
- animal bites and scratches
- contact with animal tissues and cultures, body fluids and excreta
- exposure to aerosols produced as a result of activities such as cleaning of cages
Over 150 diseases have been classified as zoonoses, some of which are listed in Table 5 below. A more complete listing can be found in the "Guide to the Care and Use of Experimental Animals", published by the Canadian Council on Animal Care.
TABLE 5 -
Examples of laboratory-acquired zoonoses, causative microorganisms, and animals most commonly associated with transmission to humans.
|Disease||Agent||Means of spread||Host animal|
|Anthrax||Bacillus anthracis||Contact, inhalation, ingestion||Farm animals|
|Brucellosis||Brucella spp.||Contact, ingestion||Swine, dogs, cattle, sheep, goats|
|Q Fever||Coxiella burnetii||Contact, inhalation, ingestion||Cattle, sheep, goats|
|Tuberculosis||Mycobacterium spp.||Contact, inhalation, ingestion||Primates|
|Salmonellosis||Salmonella spp.||Contact, inhalation, ingestion||Farm animals, rodents, reptiles, amphibia|
|Tetanus||Clostridium tetani||Bite and soil-contaminated puncture wounds||Horses, other equinae (also carried by other mammals, and present in soil)|
|Rabies||Rabies virus||Bites, saliva contact||Dogs, bats, other feral animals|
|Monkey B Virus||Herpesvirus simiae||Bite wounds, contact||Old World monkeys|
|Lymphocytic choriomeningitis (LCM)||Lymphocytic choriomeningitis virus||Contact, inhalation||Mice, guinea pigs, hamsters, monkeys|
|Toxoplasmosis||Toxoplasma gondii||Ingestion of oocytes, inhalation||Cats|
|Ringworm||Dermatophytes||Contact||Dogs, cats, guinea pigs, cattle|
|Histoplasmosis||Histoplasma capsulatum||Inhalation of fungi||Dogs, other domestic and wild species|
1 especially bis-phenols, which have reportedly been contaminated with gram-negative bacteria such as Pseudomonas spp. and Serratiamarcescens
2 especially benzalkonium chloride, reported to be contaminated with gram-negative bacteria
Exposure to laboratory animals can result in allergic responses in susceptible individuals. Allergies can develop following inhalation of airborne animal allergens or after eye or skin contact with hair, dander, urine, saliva, and serum or body tissues of laboratory animals. Estimates of the prevalence of animal allergy among laboratory workers range from ten to thirty percent. Symptoms of allergy can be mild (itchy eyes, runny nose, sneezing, red raised itchy patches on skin) to severe (wheezing, chest tightness, shortness of breath). Consult a physician if you experience allergy symptoms when working with laboratory animals.
Measures to reduce exposure to laboratory animal allergens include:
- engineering controls (e.g., ventilation)
- filtered cage systems
- respiratory protection (face mask)
- protective clothing such as gloves, gowns and shoe covers, which are reserved for use inside the animal facility
- regular handwashing and showering after handling laboratory animals, their serum or other body tissues
- regular cleaning and decontamination of animal facilities
The University Animal Care Committee (UACC), in conjunction with the Office of the V-P-Research and International Relations, provides online theory training in animal use for research and teaching. This training is mandatory for all individuals who intend to work with animals at McGill and its affiliated hospitals. In addition, everyone who plans to work with live wild or laboratory animals is required to attend and pass a practical Animal Methodology Workshop specific to the species which he or she will handle. The practical training is provided at the Comparative Medicine and Animal Resources Centre, as well as several McGill-affiliated institutions. Certifications for both courses are valid for a period of 5 years. Detailed course information is available on the UACC website at: http://www.animalcare.mcgill.ca
While prevention is the most desirable means of minimizing the risk of transmission, it is not always completely effective. Thus, verifying the health status of those involved in animal studies or animal care is a necessary safety check. In recognition of this, McGill University has developed an occupational health program that includes medical monitoring for students and staff who, as part of their duties, are potentially exposed to zoonotic agents.
|Pre-placement assessment; medical history questionnaire; and, if clinically indicated, medical examination||everyone in direct* or indirect** contact with animals|
|Tetanus immunization (if not up to date); booster every 10 years||Everyone in direct contact with animals|
|Selective pre-placement rabies immunization; repeated immunizations as required||Everyone in direct contact with non-domestic mammals and carnivores|
|Pre-placement PPD skin testing (2-step)||Everyone in direct contact with non-human primates|
|Hepatitis A vaccination; booster at 1 year||Everyone in direct contact with non-human primates|
|Q-fever immunization||Everyone in direct or indirect contact with sheep|
*Direct contact: handling live animals, unpreserved tissues or body fluids, animal cages, cage accessories, animal waste or carcasses
**Indirect contact: working in areas where animals are used or housed
The program is free of charge for McGill employees and students who are involved in animal studies or animal care. Participation in the program is mandatory for persons in contact with non-human primates, and is optional for those in contact with all other species.
Medical records remain confidential, will be maintained only by the administering physician, and will be shared only with the patient. The physician will inform the University only in instances where active zoonoses are diagnosed: such cases will be handled no differently than any other illness that compromises the safety of the individual or that of others.
All procedures are specific to the species of animal involved and the nature of contact, are designed to be relevant only to the diagnosis of zoonoses, and are not used for any other purpose.
A detailed description of McGill's Occupational Health Program, as well as registration instructions and links, is available here.
Persons working in affiliated hospitals or Research Institutes should contact their local Occupational Health Offices, which have their own programs.
In addition to avoiding obvious sources of infection such as splashes, cuts, accidental inoculation or ingestion, laboratory workers should also be aware that some pathogens, when airborne, may cause infection if inhaled.
Aerosolized microorganisms are generated during most routine laboratory procedures involving manipulation of liquid suspensions: another potential source of aerosolized pathogens is from animal cages where bedding is contaminated with feces and urine (Refer to Section 7 for a discussion of some of the biohazards associated with animal handling). Not all pathogens can infect via the aerosol route, but for those that do, the risk of infection depends on the type and concentration of agent and on the health status of the exposed individual.
The degree of penetration and retention of airborne pathogens in the respiratory tract is determined primarily by size: particles which are 10 m m in diameter or smaller are most efficiently inhaled, deposited and retained in the upper respiratory tract or in lung alveoli. Larger particles (140 m m or greater diameter) are also of concern because they can settle and contaminate work surfaces, equipment and personnel.
Whenever lab equipment is purchased, preference should be given to equipment that
- limits contact between the operator and the infectious agent
- is corrosion-resistant, easy to decontaminate and impermeable to liquids
- has no sharp edges or burrs
Every effort should be made to prevent equipment from becoming contaminated. To reduce the likelihood of equipment malfunction that could result in leakage, spill or unnecessary generation of aerosolized pathogens:
- Review the manufacturer's documentation. Keep for future reference.
- Use and service equipment according to the manufacturer's instructions.
- Ensure that anyone who uses a specific instrument or piece of equipment is properly trained in setup, use and cleaning of the item.
- Decontaminate equipment before it is sent out for repairs or discarded.
The following sections outline some of the precautions and procedures to be observed with some commonly used laboratory equipment.
Improperly used or maintained centrifuges can present significant hazards to users. Failed mechanical parts can result in release of flying objects, hazardous chemicals and biohazardous aerosols. The high-speed spins generated by centrifuges can create large amounts of aerosol if a spill, leak or tube breakage occurs.
To avoid contaminating your centrifuge:
- Check glass and plastic centrifuge tubes for stresslines, hairline cracks and chipped rims before use. Use unbreakable tubes whenever possible.
- Avoid filling tubes to the rim.
- Use caps or stoppers on centrifuge tubes. Avoid using lightweight materials such as aluminum foil as caps.
- Use sealed centrifuge buckets (safety cups) or rotors which can be loaded and unloaded in a biological safety cabinet. Decontaminate the outside of the cups or buckets before and after centrifugation. Inspect o-rings regularly and replace if cracked or dry.
- Ensure that the centrifuge is properly balanced.
- Do not open the lid during or immediately after operation, attempt to stop a spinning rotor by hand or with an object or interfere with the interlock safety device.
- Decant supernatants carefully and avoid vigorous shaking when resuspending packed cells.
- Clean spills promptly.
When using high-speed or ultra centrifuges, additional practices should include:
- Connect the vacuum pump exhaust to a disinfectant trap.
- Record each run in a log book: keep a record of speed and run time for each rotor.
- Install a HEPA filter between the centrifuge and the vacuum pump.
- Never exceed the specified speed limitations of the rotor.
Aerosols may be produced during operation of a freeze drier and when material is being removed from the chamber. When lyophilizing biohazardous materials:
- Load samples in a biological safety cabinet.
- Check glass vacuum containers for nicks and scratches.
- Use only glassware that was designed for high vacuum use.
- Use a disinfectant-containing trap for the vacuum pump exhaust.
- After completion of the run, decontaminate all accessible surfaces.
Homogenizers, shakers and sonicators can release significant amounts of aerosols during their operation and should be operated in a biological safety cabinet if possible.
When using any mixing equipment, remember to:
- Check condition of gaskets, caps and bottles before using.
- Allow aerosols to settle for at least one minute after use before opening containers, opening in a biological safety cabinet if possible.
- Cover tops of blenders with a disinfectant-soaked towel during operation.
- Immerse sonicator tip into solution to a depth sufficient to avoid creation of aerosols.
- Disinfect all exposed surfaces after use.
Spills inside freezing equipment may place laboratory and maintenance personnel at risk; for safe use of such equipment:
- Periodically check freezers, liquid nitrogen tanks and dry ice chests for broken ampoules, tubes etc.
- To minimize breakage and leaks, place primary containers such as test tubes inside secondary containers prior to storage in freezing units.
- For electrical safety, remember to shut down units before proceeding with decontamination.
Glass vacuum vessels may rupture and shower laboratory personnel with glass fragments and flask contents. To reduce these risks:
- Use metal flasks and vacuum traps whenever possible.
- Tape glass containers with duct or adhesive tape to contain glass shards in case of rupture or, use a secondary metal container that is at least as tall as the vacuum flask.
To prevent exposure of lab personnel or maintenance employees who may be required to repair the central vacuum system, vacuum line connections that draw biohazardous aerosols or fluids should be fitted with:
- a HEPA filter in the line leading into the vacuum line: cartridge-type in-line filters provide an effective barrier to escape of aerosols into vacuum systems, and are commercially available for this purpose (discard used filters as biomedical waste)
- an overflow flask in case of accidental aspiration of liquids out of the collection vessel. This flask should:
- be of sufficient capacity
- be placed between the collection flask and the air filter
- contain the appropriate disinfectant
- contain an antifoam agent whenever air bubbling generates excessive foam
Hypodermic needles and syringes present hazards of spill, autoinoculation and aerosol generation, and should be used only when absolutely necessary, such as for parenteral injection or withdrawal of body fluids. When working with syringes and needles, the following precautions are recommended:
- Perform all operations with infectious material in a biological safety cabinet.
- Fill syringes carefully; avoid frothing or introduction of air bubbles.
- Shield needles with disinfectant-soaked cotton pledgets when withdrawing from stoppers.
- Use luer-lock needles and syringes or units in which needles are integral to syringes. Better still, use one of the newer "safe" alternatives to needles and syringes.
- Do not bend, shear by hand, or recap needles.
- Place used needles and syringes in puncture-resistant containers and decontaminate before disposal.
- When withdrawing liquids from septum-capped or diaphragm bottles, consider using an opener made especially for this type of bottle; this allows for use of a pipette rather than a syringe/needle assembly.
- Use cannulas or blunt-end needles for introduction or removal of fluids through small apertures in equipment.
Improper handling of pipettes can lead to contamination of the user and/or to generation of hazardous aerosols. Mechanical pipetting aids should be used for all pipetting procedures: never pipette by mouth.
Selection of a pipetting device should be based upon:
- intended use
- ease of handling
- delivery accuracy
- user preference
- quality of seal formed with pipettes to be used; liquid should not leak from the pipette tip
- whether the pipetting aid can be sterilized
If infectious aerosols are likely to be generated, perform pipetting operations in a biological safety cabinet. Handling pipettes as described below will reduce splashing and aerosolization:
- Plug pipettes with cotton.
- Check pipettes before using; cracked or chipped suction ends may damage the seals of the pipetting aid.
- Keep pipettes upright while in use and between steps of a procedure to prevent contamination of the mechanical aid.
- Gently expel contents close to the surface of a liquid or allow to flow down the side of the container.
- Avoid mixing fluids by alternate suction and blowing, or by bubbling air from the pipette.
- Avoid forceful ejection of the contents; use TD (short for "to deliver", also referred to as "mark-to-mark") rather than TC ("to contain") pipettes, as the last drop of fluid does not have to be expelled with TD pipettes.
- Use easier-to-handle shorter pipettes when working inside a biological safety cabinet.
- Submerge used non-disposable pipettes horizontally in disinfectant solution; dropping them in vertically may force out any liquid remaining in the pipette.
BACK TO 6.2.1
Autoclaves are ideal for decontaminating biohazardous waste and for sterilizing surgical dressings, glassware and microbiological media and liquids. They must be loaded carefully to allow for steam penetration, since steam must contact pathogens in order to destroy them. Longer times are needed for larger loads, large volumes of liquid and denser materials. Proper loading and packing procedures include the following precautions:
- Wrap packages to allow for steam penetration; aluminum foil does not allow steam penetration, and should not be used for wrapping.
- Do not overload the chamber.
- Avoid overpacking of autoclave bags.
- Do not seal bags or close bottles and other containers tightly.
- Do not stack containers.
The changes that are seen on autoclave indicator tapes following an autoclave cycle do not guarantee that the contents of containers are sterile: they indicate only that the tape on the outside of the packages has been exposed to a certain amount of heat or steam. The time required for effective sterilization depends on the size of the load, volumes of liquid and density of materials to be autoclaved. Regular use (at least monthly) of a heat-resistant biological indicator such as Bacillus stearothermophilus should be used to ensure that the cycle in use really achieves sterilization. The indicator is placed in the area least likely to reach sterilizing conditions, such as in the middle of the largest or densest package. A subsequent colour change indicates that the load has been exposed to the required conditions for a sufficient length of time.
Safe work practices when using an autoclave include the following:
- Read the operating manual and post proper work procedures near the autoclave.
- Never autoclave hazardous chemicals.
- Open the door slightly to allow escape of steam before unloading.
- Wear insulated gloves or mitts when unloading.
- Microscopes: disinfect the stage, eyepieces, knobs and any other contaminated parts. Select a disinfectant that will be effective on the pathogens and non-corrosive to the microscope.
- Microtomes: disinfect knives and anti-roll plates after use.
- Water baths:
— Clean regularly; add disinfectant, such as a phenolic detergent, to the water. Avoid using sodium azide to prevent growth of microorganisms (sodium azide forms explosive compounds with some metals).
— Raise the temperature to 90oC or higher for 30 minutes once a week for decontamination purposes.
— To prevent electrical shocks, unplug the unit before filling or emptying and have the continuity-to-ground checked on a regular basis.
- Tissue grinders: use in a biological safety cabinet; wrap glass grinders in a wad of absorbent paper and wear gloves. Polytetrafluoroethylene (PTFE, "Teflon") grinders are safer, as they will not break.
- Microbiological transfer loops: to eliminate the spattering and aerosolization associated with flaming of loops, char the material before fully inserting the loop into the flame: i.e., before flaming, hold the loop close to (but not into) the flame. Alternatively, use disposable loops or a microincinerator.
All accidents, dangerous incidents, workplace exposures to infectious material, or suspected occupational diseases should be reported using the blue form available from building porters, area personnel officers, or from Environmental Health & Safety. Forms should be completed and submitted to Environmental Health & Safety within 24 hours of the accident: these accident reports aid in determining the cause of the accident and in developing measures for preventing recurrence. Any near-accidents or incidents which could result in an accident should also be reported, as these reports are useful in evaluating hazards for prevention of future accidents.
To protect individuals and the community from unnecessary exposure to biohazardous agents, biomedical waste must not be disposed of with regular waste. Disposal of biomedical waste is governed by the Regulation Respecting Biomedical Waste (Quebec), and encompasses the following categories:
- human anatomical waste (body parts or organs)
- animal anatomical waste (carcasses, body parts, organs)
- non-anatomical waste, which includes:
- sharps which have contacted animal or human blood, biological fluids or tissues
- tissue or microbial cultures, and material contaminated by such cultures
- live vaccines
- containers or materials saturated with blood products
Biomedical waste should be disposed of frequently to reduce accumulation of these materials in work areas. Disposal service for biomedical waste is provided to users in McGill buildings by the Waste Management Program, local 5066. The service is provided at no charge and includes provision of waste containers and regular pick ups. Waste boxes are filled by those who generate the waste and must be packed and labeled as follows:
- Line boxes with two biohazard plastic bags.
- Affix a biohazard warning sign and user identification to the outside.
- Ensure that liquids are in leakproof unbreakable containers.
- Place sharps in a plastic puncture-proof container prior to disposal in the biomedical waste box. Refer to Section 184.108.40.206 of the Laboratory Safety Manual for a definition of Sharps.
- Store the box at 4oC or lower in a locked refrigerator.
- Use separate boxes for each category of waste, e.g., human anatomical should not be mixed with animal anatomical or non-anatomical waste.
Whenever biohazardous materials are moved, whether it be within the lab, between labs or buildings or by public carrier, precautions must be taken to control the risks associated with a spill or leak. Arrangements should be made to:
- Limit the number of moves,
- Reduce the possibility of breakage, and
- Contain the material in the event of a leak or spill.
When transporting within or between laboratories:
- Place specimens in leakproof and breakage-resistant receptacles. Close with screwcaps rather than snap caps whenever possible.
- Use unbreakable leakproof secondary containers; for light loads that are to be carried, ensure that the secondary containers have solid handgrips. Small tubes can be sealed inside zipper-lock freezer bags, which are inexpensive, leakproof and will not break if dropped.
- For heavier items, use a cart with guard rails or raised edges. Load so that the contents will not dislodge if the cart should bump into a wall or door.
When moving biohazardous substances from one building to another:
- Ensure that the substance is in a closed and sealed primary receptacle such as a test tube, vial or flask.
- Place cushioning absorbent material around the primary container.
- Use a secondary leakproof container that can withstand dropping or crushing while in transit.
- If the material must be kept refrigerated or frozen during transport, place the coolant (e.g., dry ice, crushed ice) inside an insulated tertiary vessel. To prevent rupture of the package, ensure that dry ice is able to release carbon dioxide gas.
Within Canada, transport of biohazardous material is regulated by federal and provincial laws and acts. Use of regular mail for shipment of material that is known to be infectious is prohibited by Canada Post.
Agencies and associations such as the World Health Organization, the United Nations Committee of Experts on the Transport of Dangerous Goods, the Universal Postal Union (UPU), the International Civil Aviation Organization (ICAO) and the International Air transport Association (IATA) have developed standards for the safe international shipment of infectious substances. It is the responsibility of the sender and the recipient to ensure that all regulations are enforced and that permit requirements have been met. Contact an EHS Biosafety Officer (local 1657 or 4818) for additional information.
Biohazardous materials are classified as Class 6, Division 6.2 Infectious Substances under Transport of Dangerous Goods Regulations. The regulations stipulate that all individuals involved in the transport of hazardous materials must be trained, tested and certified.
All biological material must be packaged so that there will be no leakage during transport. Packaging requirements may differ according to destination, carrier, mode of transport and whether the material is fully or partially controlled: contact your carrier and an EHS Biosafety Officer (local 1657 or 4818) for assistance. The norms generally stipulate that biohazardous substances must be packaged as described below:
- Place the specimen inside an appropriately labeled leakproof primary (inner) container; close with screw caps or seal with stoppers and tape or other suitable material.
- Wrap the container in enough absorptive material (e.g. paper towels, tissue, cotton wool) to absorb all fluid in the event of a leak.
- Several samples or cultures can be sent together provided each is inside a primary container, packed to prevent contact with each other, and surrounded by sufficient absorbent in case of breakage.
- Place the wrapped container inside a secondary watertight receptacle, using enough absorbent material to cushion the primary container.
- Place the secondary container inside a third (outer) package for protection from physical damage and water while in transit.
- If the shipment must be kept cold or frozen, notification to that effect should appear on the accompanying documents and on the outer package. Containers shipped with dry ice must be able to release carbon dioxide gas that could otherwise build up and cause the package to rupture.
Infectious substances should not be sent until arrangements have been made with the sender (the "consignor"), the carrier and the recipient (the "consignee"). To ensure that the material is transported safely and as quickly as possible, the sender should:
- Observe national and international transport regulations.
- Communicate with carrier and consignee to coordinate transport and receipt.
- Obtain and complete shipping documents and declaration forms.
- Arrange dispatch by direct route whenever possible.
- Send all transportation documentation to the receiving lab.
Importation of animal and human pathogens is overseen by the Canadian Food Inspection Agency (CFIA) Office of Biohazard Containment Safety (OBCS) and the Public Health Agency of Canada (PHAC) Pathogen Regulation Directorate (PRD).
Import Permits for human and most terrestrial animal pathogens, as well as Containment Level 2 Compliance Letters for laboratories handling these pathogens, are issued by PHAC. Please contact the EHS Biosafety Officer to obtain the Application for Permit to Import Human and/or Terrestrial Animal Pathogens and Application for a Containment Level 2 Compliance Letter forms.
To facilitate passage through customs, complete and fax the Declaration for Import of Biological Products form to Procurement Services at 514-398-1885.
Import Permits for the following are issued through CFIA:
- Pathogens causing foreign animal and emerging animal diseaes (i.e. pathogens not established or indigenous to Canada);
- Animals, animal tissues, sera and blood infected with animal pathogens;
- Aquatic animal pathogens;
- Plant pathogens
Please contact the EHS Biosafety Officer to obtain the appropriate forms.
The procedure for importing biohazardous substances is summarized below:
- Obtain the import permit and compliance letter.
- Provide copies of the import documents to the sender, who must make certain that it accompanies the shipment into Canada.
- Ensure that the sender packs and labels the infectious materials according to regulations.
- Arrange to have someone available on the delivery day to accept and examine the package.
- Have the necessary supplies and equipment on hand for decontamination and disposal in case of leakage during transport.
- Acknowledge receipt to the sender.
Note that both sender and receiver are required to keep copies of shipping documents for at least 2 years.
Biological Safety: Principals and practices, 3rd Edition. Fleming DO and Hunt DL, eds. American Society for Microbiology, ASM Press, Washington DC, 2000.
Biosafety in microbiological and biomedical laboratories (BMBL), 4th edition. U.S. Department of Health and Human Services, Public Health Service, Centers for Disease Control and Prevention, and National Institutes of Health. U.S. Government Printing Office, Washington, 2009 (http://www.cdc.gov/biosafety/publications/bmbl5/index.htm)
Biosafety Reference Manual, second edition. Heinsohn PA, R Jacobs and BA Concoby, eds. American Industrial Hygiene Association. AIHA Publications, Fairfax VA, 1995.
Disinfection, Sterilization, and Preservation, 5th edition. Block SS, editor. Lippincott Williams & Wilkins, Philadelphia, 2001.
Human Pathogens Importation Regulations, 1994 (http://www.canlii.org/ca/regu/sor94-558/)
Laboratory Biosafety Guidelines, 4th edition. Public Health Agency of Canada, Office of Laboratory Secruity, Ottawa, Canada, 2004 (http://www.phac-aspc.gc.ca/publicat/lbg-ldmbl-04/index-eng.php).
Laboratory Biosafety Manual, 3rd edition. World Health Organization. Geneva, 2004 (http://www.who.int/csr/delibepidemics/ WHO_CDS_CSR_LYO_2004_11/en/print.html).
NIOSH Alert. Preventing Asthma in Animal Handlers. Publication No. 97-116, Jan 1998 (http://www.cdc.gov/niosh/animalrt.html).
Occupational Health and Safety in the Care and Use of Research Animals. Chapter 4: Allergens, Chapter 5: Zoonoses. National Research Council, National Academy Press, Washington DC, 1997.
Shematek G and W Wood. Biological Hazards. In: Laboratory Safety, CSLt guidelines, fouth edition. Canadian Society of Laboratory Technologists, Hamilton, Ontario, 1996.
Transport of Dangerous Goods, Transport Canada, Jan 2009(http://www.tc.gc.ca/eng/tdg/clear-menu-497.htm)
Aerosol: A suspension in air of liquid or solid microscopic particles.
Antiseptic: Acting against sepsis. An antiseptic agent is one that has been formulated for use on living tissue such as mucous membranes or skin to prevent or inhibit growth or action of organisms. Antiseptics should not be used to decontaminate inanimate objects.
Aseptic procedure: A procedure carried out in a manner that prevents contamination of material.
Autoclave: An apparatus which employs physical means (moist heat under pressure) to sterilize or decontaminate. Two types of autoclave are:
- Gravity displacement autoclave: this type of autoclave operates at 121oC. Steam enters at the top of the loaded inner chamber, displacing the air below through a discharge outlet.
- Vacuum autoclave: this type of autoclave can operate at 134oC, allowing for reduced sterilization cycle time. The air is pumped out of the loaded chamber before it is filled with steam.
Bacterial spore: See "Spore, bacterial".
Bactericide: An agent that kills vegetative bacteria but not mycobacteria or spores.
Bacteriostatic: Inhibiting growth of bacterial organisms without necessarily killing them or their spores.
Bacterium: A single-celled microorganism, ranging in size from .4 to 2.0 microns, which multiplies by subdivision.
Biocide: An agent that can kill all pathogenic and non-pathogenic living organisms, including spores.
Bloodborne pathogens: Infectious microorganisms that are carried in the blood of infected humans or animals and that can be transmitted through contact with infected blood, body fluids, tissues or organs. Bloodborne pathogens are implicated in diseases such as malaria, syphilis, brucellosis, tuberculosis, hepatitis B and acquired immunodeficiency syndrome (AIDS). Workplace transmission of a bloodborne pathogen can occur via:
- accidental inoculation with a contaminated "sharp"
- exposure through open cuts, skin abrasions, and mucous membranes of eyes and mouth
- indirect transmission (e.g., touching mouth, eyes, nose or open cuts with contaminated hands)
Broad spectrum: A wide range. A broad spectrum disinfectant is effective against a wide range of microorganisms, including bacterial spores, mycobacteria, non-lipid and lipid viruses, fungi and vegetative bacteria.
Decontamination: Removal of microorganisms to a lower level, such that there is no danger of infection to unprotected individuals. Sterilization and disinfection are decontamination procedures.
Disinfectant: An agent used to kill microorganisms on inanimate objects such as instruments or surfaces.
Disinfection: Use of physical or chemical agents to destroy pathogens and potential pathogens on inanimate objects. Disinfection does not necessarily result in sterilization.
- "High level disinfection" inactivates fungi, viruses and bacteria. High level chemical disinfectants may be ineffective against bacterial spores if they are present in large numbers. Extended exposure times may be required.
- "Intermediate level disinfection" destroys fungi, some viruses (lipid and most non-lipid medium-size and small viruses), mycobacteria and bacteria.
- "Low level disinfection" kills vegetative forms of bacteria, some fungi, and some medium-size and lipid-containing viruses. Low level disinfectants do not reliably kill bacterial spores, mycobacteria or small or non-lipid viruses.
Etiologic agent: A disease-causing organism or toxin.
Fungicide: An agent that destroys fungi.
Germicide: An agent which destroys microorganisms, especially pathogenic microorganisms ("germs"). Sterilants, disinfectants and antiseptics are germicides.
Gravity displacement autoclave: See Autoclave
Infectious: Able to cause disease in a susceptible host.
Infectious agent: A biological organism that can establish a process of infection.
Iodophor: Literally, an "iodine-carrying" compound. An iodophor is a combination of iodine and a solubilizing surface-active agent, or carrier.
Microorganism: A microscopic organism, such as a bacterium, protozoan, yeast, virus or alga.
Pathogenic organisms: Organisms capable of causing disease, either directly (by infecting) or indirectly (by producing a toxin that causes illness).
ppm: Abbreviation for parts per million, used to describe concentrations in liquids or gases, e.g., 10,000 ppm is approximately equivalent to 10 g/liter or a 1% W/V solution.
Pre-vacuum autoclave: See Autoclave
Prions: Virus-like proteinaceous infectious agents. Prions differ from viruses in that they are not known to contain either DNA or RNA.
Protozoa: Nucleated microorganisms, some of which are large enough to be detected with the naked eye. Sizes range from .01 to 200 microns.
psi: Abbreviation for pounds per square inch, a unit of pressure equal to the pressure exerted on an area of one square inch. 1 psi = 7.03 x 10-2 kilograms per square centimeter.
Recombinant DNA techniques: Procedures which transfer genetic material between organisms or species.
Sanitization: Reduction of microbiological load on objects and surfaces to a safe level.
Sharps: Sharp objects such as needles, scalpel blades, broken glass, pasteur pipettes or any other object that can penetrate skin.
Spore, bacterial: A bacterial spore is a resistant body formed as part of the life cycle of some bacteria. Bacterial spores are able to withstand severe environmental conditions (e.g., heat, drying, chemicals) for many years. When conditions are favourable, spores germinate into vegetative bacterial cells capable of replication.
Sporicide: An agent that destroys bacterial and fungal spores.
Sterilization: Use of physical or chemical means to bring about the total destruction of all viable microbes, including resistant bacterial spores.
Tincture of iodine: A germicidal solution of iodine in aqueous alcohol, used primarily as antiseptics on skin and tissue.
Universal Precautions: Precautions taken when handling, storing, transporting or shipping items or specimens containing or contaminated with human blood and body fluids: all such materials are treated as if infectious.
Vector: An agent, such as an insect, that can carry a disease-producing organism from one host to another.
Vegetative form: In bacteria, a stage of active growth, as opposed to a resting state or spore formation.
Viable: Able to grow and multiply.
Virucide: An agent that destroys or inactivates viruses.
Virus: A microorganism, ranging in size from .01 to .25 microns (10 - 250 nanometers), that can reproduce only within living cells.
Virulence: The disease-producing power of a microorganism.
Zoonosis: A disease that can be transmitted from animals to humans.
For Importation of Human Pathogens
Centre for Biosecurity
Public Health Agency of Canada
Ottawa, Ontario, K1A 0K9
Tel: (613) 957-1779; Fax: (613) 941-0596
Email: permit-permis [at] phac-aspc [dot] gc [dot] ca
For Importation of Animal or Plant pathogens
Canadian Food Inspection Agency (CFIA) Regional Office
2001 University, Room 746-S
Montreal, QC H3A 3N2
Tel: (514) 283-8888; Fax: (514) 283-3143
Email: Importzoopath [at] inspection [dot] gc [dot] ca
Canadian Food Inspection Agency
59 Camelot Drive
Nepean, ON K1A 0Y9
Tel: (613) 225-2342; Fax: (613) 228-6630
To order WHO publications
World Health Organization
Canadian Public Health Association
1335 Carling Avenue, Suite 210
Ottawa, Ontario, K1Z 8N8
Tel: (613) 725-3769
For information on transport of biohazardous materials
700, boul. René-Lévesque Est, 27e étage
Québec (Québec) G1R 5H1
Tel: (514) 873-2605
Transport Canada, Surface
685 Cathcart Street, Suite 701
Montréal, Québec, H3B 1M7
Tel: (514) 283-5722
Transport Canada, Air Carrier Operations, Dangerous Goods
700 Leigh Capreol Street
Dorval, Québec, H4Y 1G7
For information on mailing non-infectious bloods, diagnostic specimens and biological products
Canada Post Corporation
General Inquiries, Customer Service
"Application to Use Biohazardous Materials" form.
The PDF form can be downloaded from our site.