Introduction
Despite a greater awareness of biosafety and biocontainment practices, infection
of laboratory workers with the same agents used by them in the lab continues to
result in sickness and mortality of these same workers. 1 Incidences of the
transfer of biohazardous material into the general population may also be
occurring.
The SARS scare of 2002 in Toronto ON, coupled with the increased
concern of bioterrorism and an increased awareness in the general public
regarding acceptable levels of risk, have created a climate of concern with
respect to the use, transportation, storage and disposal of potentially
biohazardous substances.
Laboratory Biosafety Guidelines have been published by both Health
Canada (HC) and the Canadian Food Inspection Agency (CFIA) to address many
of these concerns. The most recent editions of these guidelines (HC 3 rd edition 2,
and CIA 2nd edition 3) are essentially mirror images with the requirements for
Containment labs being the same.
In Canada, work with Biohazardous materials which may infect humans is
governed by Health Canada and would follow the guidelines and the
requirements of the “Peach Book” which is the nickname given to HC Laboratory
Biosafety Guidelines. Work with Biohazardous material which may affect wildlife
or agriculture is governed by the CFIA guidelines and the requirements of the
“Red Book” which is the nickname given to the CFIA Containment Standards for
Veterinary Facilities which governs work in laboratories.
The guidelines and requirements listed in this safety manual are the
minimum expected to work with pathogens at Trent University. Researchers are
expected to follow this manual as well as the appropriate governmental
standards in all the work that they do. Working with pathogens at Trent
University requires the submission of an application to work with pathogens to
the Biosafety Committee. The application must be approved by this committee
prior to work commencing. Any changes to the work procedures described in
that application must have the approval of the Biosafety Committee or be as a
result of increased safety and containment procedures. Certifications and
inspections of labs are carried out by the Biosafety Officer on behalf of the
committee (see pg. 25)
What is a Biohazard?
-Any organism or its toxin that is known or suspected to cause disease in animals
or humans.
-Biohazardous or infectious materials fall under class D, division 3 of WHMIS
1
Collins, C.H., and Kennedy, D.A. Laboratory-acquired infections. IN: Laboratory-acquired infections:
history, incidence, causes and preventions. Oxford, UK: Butterworth-Heinemann, 1999; 1-37.
2
Laboratory biosafety guidelines 3rd edition. Ottawa: Health Canada: 2004.
3
Containment standards for veterinary facilities. Ottawa: Canadian Food Inspection Agency; 1996.
1
-Includes microorganisms such as viruses, bacteria, fungi and parasites and their
toxins
-Also includes blood and body fluids, as well as tissues from humans and
animals
-Transformed cell line and certain types of nucleic acids also are considered as
biohazardous.
What is Biological Safety?
The combination of measures employed when handling biohazardous materials
to avoid infecting oneself, others or the environment. This includes:
-Engineering Controls (e.g., HVAC, BSC’s)
-Administrative Controls (e.g., Biosafety Committee)
- Practices and Procedures (e.g., Biosafety Manual, Laboratory Safety plan)
-Personal Protective Equipment (e.g., Gloves, Lab coats,
The primary operating theory behind Biosafety is that ultimately the safeguarding
of a potentially hazardous infectious agent is based on a) the infectiousness and
toxicity of the pathogen and b) the containment strategy use to control for
unplanned exposures.
Risk Groups
The classification of organisms into risk groups reflects an attempt to categorize
the relative hazards of infective organisms. The factors used to determine which
risk group an organism falls into are: the pathogenecity, the infectious dose, the
mode of transmission, host range, availability of preventative measures and the
availability of effective treatment. The definitions below have been paraphrased
for both human and animal/plant pathogens. See the appropriate guidelines for
the specific definitions.
Risk Group 1. (low individual and community risk)
Any biological agent that is unlikely to cause disease in healthy workers or
animals
Risk Group 2. (moderate individual risk, low community risk)
Any pathogen that can cause human or animal disease but under normal
circumstances is unlikely to be a serious hazard to laboratory workers, the
community, livestock or the environment. Laboratory exposures rarely cause
infection leading to serious disease; effective treatment and preventative
measures are available, and the risk of spread is limited
2
Risk Group 3. (high individual risk, low community risk)
Any pathogen that usually causes serious human or animal disease or can result
in serious economic consequences but does not ordinarily spread by casual
contact from one individual to another, or that causes diseases treatable by
antimicrobial or anti-parasitic agents
Risk Group 4 (high individual risk, high community risk)
Any pathogen that usually produces very serious human or animal/plant disease,
often untreatable, and may be readily transmitted from one individual to another,
or from animal to human or vice-versa, directly or indirectly, or by casual contact.
A list of human pathogens categorized according to Risk group can be obtained
by contacting the Office of Laboratory Security, Health Canada or by contacting
the Canadian Food Inspection Agency.
Unconventional Pathogens
Some progressive neurological diseases (spongiform encephalopathies) are
caused by agents referred to as unconventional agents, slow viruses or priors
(proteinaceous infectious particles). Examples of such diseases are:
Creutzfeldt-Jakob disease in humans, Mad Cow disease (BSE) in cattle and
Scrapie in sheep and goats.
These agents are resistant to destruction by chemical (10% formalin,
gluteraldehyde, 70% ethanol, iodine) and physical (UV light, ionizing radiation,
boiling) procedures that normally inactivate viruses.
While there have been no documented cases of laboratory acquired infections,
the following precautions should be observed when handling neurological tissue
from infected or potentially infected humans or animals.
-Handle tissue as Risk Group 2 or higher
- Handle formalin-fixed tissues and paraffin-embedded blocks as if still infectious
-Follow up-to-date disinfection protocols
Tissue Culture
All mammalian lines are to be considered infectious due to the possibility that
they may contain or transmit infectious agents.
Cells or primary cultures from animals and humans known or reasonably
suspected to be infected should be in the risk group for the suspected agent.
All primate cell lines, all cell lines exposed to or transformed by primate
oncogenic virus and all mycoplasma containing cell lines should be handled at
Level 2 containment.
Any cell line containing mycoplasma should be likewise treated as Level 2
All other tissue culture work should be considered as Level 1.
3
All waste generated from tissue culture must be autoclaved or disinfected by
other means prior to disposal.
Animal Work
By definition all work involving animals is considered biohazardous.
Animals can harbour infectious organisms (naturally or through their use in
research) which can be transmitted to humans
The Trent University Animal Care Committee ensures that all work involving
vertebrates and cephlapods meets all the standards and regulations of the
Canadian Council on Animal Care and the Animals for Research Act.
All work involving vertebrates and cephlapods use must receive prior approval
from the Trent University Animal Care Committee (see
www.trentu.ca/animalcare).
Containment Levels
The term containment levels (CL or AP) are selected to provide the end-user
with a description of the minimum containment required for handling the
organism safely in a laboratory setting. The characteristics of each containment
level refer to the systems and operational procedures in place and may include,
engineering controls, HVAC, work procedures etc… These levels are more fully
defined in either the HC Laboratory Biosafety Guidelins (peach book), or the
CFIA Containment Standards for Veterinary Facilities (red book). It is important
to understand that the Risk Groups do not specify the containment level required
to work with an organism. The containment level of the lab required for a specific
pathogen is based on a Risk Assessment which is a critical step in the selection
of the appropriate containment required. While the risk group can supply some
of this information, the risk assessment may also include things like: potential for
aerosol generation, quantity, concentration, stability in the environment, type of
work (in vitro, in vivo, and in situ) as well as the use of recombinant organisms.
Containment Level 1 (CL1)
CL1 applies to the basic laboratory that handles agents requiring containment
level 1. A CL1 lab has no special requirements beyond those suitable for a well
designed and functional lab. Biological Safety Cabinets (BSC) are not required
and work may be performed on the open bench.
Containment Level 2 (CL2)
CL2 applies to a laboratory in which agents requiring containment level 2 are
handled. The primary exposure hazards associated with organisms requiring
CL2 are through the ingestion, inoculation and mucous membrane route.
Agents handled in a CL2 are generally not transmitted by airborne routes, but
care must be taken to avoid the generation of aerosols which can become
ingestion risks. Primary containment devices such as BSCs and centrifuges with
4
sealed rotors should be used as well as the appropriate Personal Protective
Equipment (PPE). As well, environmental contamination must be minimized by
the use of hand-washing and decontamination facilities such as autoclaves.
Level 2 labs include clinical and diagnostic facilities, as well as research and
teaching laboratories with level 2 agents. They require a class I or class II
biological safety cabinet if any potential for aerosol or splash exists. An
emergency plan must be developed for handling spills. All personnel who enter
the facility must be informed of the hazards present. The biohazard sign with
appropriate information must be posted.
Containment Level 3 (CL3)
CL3 applies to the laboratory that handles agents requiring containment level 3.
These agents may be transmitted by the airborne route, often have a low
infectious dose to produce effects and can cause serious or life-threatening
disease. CL3 emphasizes additional primary and secondary barriers to minimize
the release of infectious organisms into the immediate laboratory and the
environment. Additional features to prevent transmission of Cl3 organisms are
appropriate respiratory protection, HEPA filtration of exhausted laboratory air and
strictly controlled laboratory access.
Level 3 labs are specially designed and constructed and must undergo annual
performance testing and verification. All work must be performed in type II or type
III biosafety cabinets. All centrifugation must be performed in closed trunnion
cup centrifuges. Staff must receive specific training for the agents employed,
above the basic hazard information. PPE is very specific, including solid front
clothing and dedicated footwear, for use only within the facility. Written protocols
must be provided and posted. A medical surveillance program must be in effect.
A reporting system for accidents must be in place.
Containment Level 4 (CL4)
CL4 labs have the maximum containment available and are suitable for facilities
manipulating agents requiring containment level 4. These agents have the
potential for aerosol transmission, often have a low infectious dose and produce
very serious and often fatal disease; there is generally no treatment or vaccine
available. The level of containment represents an isolated unit, functionally and,
when necessary, structurally independent of other areas. CL4 emphasizes
maximum containment of the infectious agent by complete sealing of the facility
perimeter with confirmation by pressure decay testing; isolation of the researcher
from the pathogen by his or her containment in a positive pressure suit or
containment of the pathogen in a Class III BSC line; and decontamination of air
and other effluents produced in the facility.
There is only one CL4 lab in Canada in Winnipeg, Manitoba.
5
Risk Assessment
Risk assessment is a critical step in the selection of the appropriate containment
level for the microbiological work to be carried out. A detailed local risk
assessment should be conducted to determine whether work requires
containment level 1, 2, 3 or 4 facilities and operational practices. Individuals with
varying expertise and responsibilities should be involved in the assessment and
may include the facilities manager, the biosafety committee, a microbiologist, and
the biosafety officer.
Available information can be used as a starting point to assist in the identification
of risk factors, including the recommended Risk Group of the organism.
Additional information about specific organisms may also be obtained from the
MSDS for the pathogen. A list of human pathogens can be obtained by calling
the Office of Laboratory Security directly at (613) 957-1779 or by accessing their
Web site at http://www.phac-aspc.gc.ca/ols-bsl/pathogen/organism_e.html.
These MSDS‘s are produced by Health Canada and are written from the
perspective of protecting human health only. Depending on the nature and
purpose of the research, additional information and precautions may be
necessary when working with animal and zoonotic pathogens.
In addition to the Risk Group classifications, which are based on the risk factors
inherent to the organism, the following factors associated with the laboratory
operation should also be examined:
- potential for aerosol generation
-quantity
-concentration
-agent stability in the environment (inherent biological decay rate)
-type of work proposed (e.g., in vitro, in vivo, aerosol challenge studies)
-use of recombinant organisms (e.g., gene coding for virulence factors or toxins;
host range alteration; oncogenicity; replication capacity; capability to revert to
wild type)
The containment level required for work with a particular agent is based on the
manipulations generally associated with laboratory scale research and clinical
procedures. If a particular procedure, such as preliminary identification, poses a
lower hazard than manipulation of a live culture, then a lower containment level
may be appropriate. On the other hand an increase in containment may be
required if the local risk assessment indicates that the procedures pose a higher
risk than routine laboratory scale and diagnostic manipulations.
6
Primary Containment
Primary containment is the first line of defence against unplanned exposures. It
usually involves a protective envelope that effectively encapsulates the infection
agent or animal, or conversely the envelope may encapsulate the worker. The
envelope ensures the protection of personnel and the immediate laboratory
environment from exposure to the infectious agent. Primary containment is
usually made up of biological safety cabinets, glove boxes and animal caging
equipment. It also includes simple measures like stoppered bottles.
Secondary Containment
Secondary Containment protects the environment external to the laboratory from
exposure and this includes the design of the facility and the operational practices
of mechanical system and of the lab.
Personal Protection
The effective use of vaccines is often an overlooked form of protection
against exposures. If vaccines exist for the pathogen being used, get immunized
against it.
Although the preferred method of protection is by reducing exposure at the
source, to protect against the failure of the primary containment, PPE can
become an important line of defense.
In considering the use of PPE two criteria should be used: 1. degree of
protection offered and 2. the ease of use. Once the PPE has been identified it is
the responsibility of both the supervisor and the user to ensure that PPE is used
and used properly.
Each type of containment level (1, 2, 3, and 4) requires specific protective
laboratory clothing.
Personal Protective Equipment

Laboratory Coats and Gowns
The lab coat has two uses: to protect street clothing from biological or chemicals
spills, and to offer some additional body protection
Level 2 lab: lab coat, gown or smock or uniform. It is recommended that it be of
100% cotton due to flame retardancy and resistance to a number of chemicals.
Level 3: solid front or wrap around gowns. Some institutions recommend a 2
piece scrub suit as mandatory under the solid front gown.
7

Head coverings
Generally not required in most biological areas, unless a complete change of
clothing is required for access, and where product protection is required.

Shoes and shoe coverings
Any area where there is a significant risk of dropping heavy objects should
require the use of industrial safety shoes. For general biological use,
comfortable shoes such as tennis shoes or nurses shoes are recommended.
Sandals are not allowed in laboratories using biohazards due to the potential
exposure to infectious agents. A change from street shoes is strongly
recommended for those working in level 3 facilities and for those working with
infected animals in animal rooms. Alternately, shoe coverings may be used in
level 2 and level 3 labs when a complete change of shoes and a dedicated pair
of shoes are not required.

Gloves
Gloves are the most widely used form of PPE. They are used for a wide variety
of hazards including protection from heat, cold, solvents, caustics, toxins,
infectious microorganisms, radioisotopes, cuts and animal bites and acids.
Unfortunately, there is no ideal glove that will protect against all hazards. Gloves
are made from a variety of materials including, rubber (latex), neoprene,
neoprene-latex, nitrile, polyurethane, PVC etc... . Selection should depend on
the hazard encountered. If biological work will include the use of chemical
solvents such as toluene, benzene or carbon tetrachloride; rubber, neoprene,
neoprene-latex, and PVC will be graded. Choose the glove appropriate to the
task. If you are unsure, contact the vendor as to the correct choice of gloves for
the application you intend.
In microbiological labs, surgical gloves or latex, rubber or vinyl are generally the
preferred choice. (Latex gloves are slowly being removed from service due to
the increase in allergic responses to latex). They offer a high level of dexterity
and a higher level of sensitivity; unfortunately they offer very little if any protection
against needle sticks, sharps and animal bites. Remember that gloves are the
weakest component of the PPE. Gloves should overwrap the cuff and lower
sleeve of the laboratory clothing. When handling infected tissues during
necropsies, often stainless steel mesh gloves are worn to protect against
accidental cuts.

Respiratory Protection
Two types of respiratory protection exist. Those which supply clean air or those
which remove the hazardous particulates. A respirator such as a full face or half
face cartridge respirator, require quantitative fit testing according to OSHA 29 (of
8
the US). These type of respirators would be worn in atmospheres that pose and
infectious or toxic hazard such as an animal room where infectious agents could
be excreted in urine.
Singe use paper dust masks are not classified as true respirators. They do not
offer adequate respiratory protection in infected animal rooms or other areas
where infectious aerosols may be present, because one cannot be ensured of an
adequate fit. They are acceptable for use during necropsies and other surgical
procedures in order to maintain a sterile surgical field. The second type of
respirator which supplies clean air includes hoods, helmets and full suits or self
contained breathing apparatus. These are more bulky and ease of movement is
often restricted.

Eye or Face Protection
Eye or facial protection is important because biological work may often use
concentrated alkalis, and acids, concentrated disinfectants, including phenolics
and quaternary ammonium compounds which can cause severe eye damage
and blindness if splashed into the eye. Infection can also occur through the
conjunctiva if certain pathogenic microorganisms are splattered into the eye. Full
Face respirators or half face respirators plus splash goggles are often
recommended when respirable aerosols or droplets may be produced.
Safety glasses are intended to provide impact protections, but should not be
used to protect against splashes. Fro these hazards, safety goggles or face
shields should be used. Ordinary glasses offer better splash protection than
nothing at all, however, they do not replace the need for approved safety
eyewear. The use of Contact Lenses in laboratories is discouraged at Trent as in
the event of an accident where the lens needs to be removed (for flushing) the
time required the lens prior to flushing may result in additional injury. In addition
some chemical vapours may be absorbed through the lens and trapped behind it.
Biological Safety Cabinets
Biological Safety Cabinets (BSCs) are the single most important safety device in
the microbiology laboratory and second in overall importance only to safe work
practices.
There are three classes of BSCs. Class I cabinets have un-recirculated air flow
away from the operator that is discharged through a HEPA filter. Class I cabinets
provide good operator protections but do not protect the material within the
cabinet (the product) from contamination. Class II cabinets have inward air flow
for personnel protection, downward HEPA-filtered air for product protection and
HEPA filtered exhaust air for environmental protection. They are divided into two
types (A and B) based on construction, air flow velocities and patterns, and
exhaust systems. Class III cabinets are totally enclosed and gas-tight with HEPA
9
filtered supply and exhaust air. Work is performed with attached long-sleeved
gloves. Class III cabinets protect the worker and the product.
NOTE: Horizontal Clean Benches which direct air towards the operator are not
biological safety cabinets and must not be used for handling infectious, toxic or
sensitizing agents. Laminar flow hoods are not such a cabinet, they protect a
process not a person.
All three classes of cabinets serve to minimize contact between the operator and
the infectious agent by the use of directional airflows. All BSCs contain HEPA
filters to ensure the exhaust from the cabinets is free of infectious material.
BSCs should be located away from doors, drafts, convection currents, diffusers
and high traffic areas.
Selection of the proper class of BSC requires careful evaluation of the work
involved. Class II (i.e., Type A and Type B) cabinets are designed for work
involving microorganisms in CL 2, 3 and 4 facilities. Cabinet air from Type A
cabinets may be recirculated back into the laboratory in level 2 and 3 facilities.
Ducting a Type A cabinet out of the building is possible, providing the method of
ducting uses “thimble” connections (i.e., small opening around the cabinet
exhaust filter housing) and the balance of the cabinet exhaust system is not
disturbed. The thimble must be removable or be designed to allow for proper
certification of the cabinet (i.e., bubble tight damper to seal off the cabinet for
decontamination, access port to allow scan testing of the HEPA filter).
Class II, Type B cabinets can be used when manipulating small quantities of
chemicals as an adjunct to work with microorganisms in CL2, 3 and 4 labs. Type
B1 cabinets recirculate 30% of the air within the cabinet and are suitable for work
with minute amounts of chemicals and radionuclide. These cabinets must be
“hard ducted” (i.e., direct connection), preferably to their own dedicated exhaust
system. The exhaust canopy must allow for proper BSC certification. Type B2
cabinets are total-exhaust cabinets with no air recirculation within them and are
suitable for work with small amounts of volatile chemicals and radionuclides.
These cabinets are also hard-ducted.
Class III cabinets are designed for work with CL 4 level pathogens.
Only cabinets which meet the National Sanitation Foundation (NSF) Standard
No. 49 Class II Biohazard Cabinetry (1992) and bear an NSF 49 seal are to be
purchased and installed in university labs.
BSCs must be tested and verified before they are used upon installation and
after repairs or relocation. Testing must be performed by qualified individuals
and it is recommended that only NSF accredited field certifiers be used.
10
General Laboratory Biosafety Guidelines


















All Personnel must understand the hazards and be trained.
A laboratory safety manual must be prepared.
The laboratory must be kept neat, orderly and clean.
Appropriate protective equipment must be worn. It must not be worn in
non-laboratory areas. This includes gloves, which must be worn for all
procedures involving direct skin contact; safety eyewear, which must be
worn to protect against splashes; and lab clothing.
Eating and drinking, inserting or removal of contact lenses, application of
cosmetics are not permitted.
Oral pipetting is strictly prohibited
Long hair must be tied back.
Hands must be washed after gloves are removed and before leaving the
lab.
Work surfaces must be cleaned and decontaminated daily
Aerosol production is to be minimized
All contaminated or infectious materials are to be decontaminated.
Access to laboratories should be restricted.
Hazard warning signs must be posted
The use of needles, syringes and other sharp objects should be limited
and disposed of into the appropriate “Sharps” Container and not into the
regular garbage.
Laboratory workers should be protected by immunization where
appropriate
A BSO and Biosafety Committee should be established.
All work with Biohazardous agents must be approved by the Biosafety
Committee
All rooms designated as Containment labs will be done so by the
Biosafety Committee
OPERATIONAL PRACTICES
GENERAL REQUIREMENTS
The following general practices are required when working in any containment
laboratory or animal facility:


entry must be restricted to laboratory staff, animal handlers, maintenance
staff and other persons on official business
only persons meeting specific entry requirements (e.g. immunization,
serum screening) may enter containment laboratories unless the facility
11













has been appropriately decontaminated
a health and medical surveillance program must be provided as
recommended by Health Canada
personnel must receive training on the potential hazards associated with
the work involved and the necessary precautions to prevent exposures to
zoonotic agents and release of non-indigenous agents; personnel must
show evidence that they understood the training provided; training must
be documented and signed by both the employee and supervisor
a documented procedural manual must be written and followed
all persons (including visitors, maintenance staff, etc.) entering the
containment area must be trained and know and follow the operational
protocols for the project in process; trainees must be accompanied by a
trained staff member
persons entering a containment facility must be well prepared and bring all
materials they will need with them; if something has been forgotten, 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 via proper protocols)
employees working in the containment area must have general knowledge
of the physical operation and design of the facility (e.g. air pressure
gradients between zones, directional air flow patterns, alarm signals for air
pressure failure, containment perimeter)
traffic flow patterns from clean to dirty areas must be established and
adhered to (i.e. move from least to most contaminated areas)
smoke testing (i.e. with a smoke pencil) should be done periodically by lab
staff to verify correct airflow
entry/exit protocols for persons, animals, equipment, samples, waste, etc.
must be written, posted and followed; general protocols must be
supplemented with protocols specific for each project in progress
emergency procedures for entry/exit, spill clean-up, air handling/biosafety
cabinet failure, fire, animal escape and other emergencies must be
written, posted and followed
in the event of life-threatening emergencies, personal health and safety
are a priority; exit protocols must be established whereby routine
procedures are bypassed; a reporting area must be identified where
further steps must be taken (e.g. disinfecting footwear, changing,
showering) prior to leaving
all spills, accidents, overt or potential exposures to infectious materials,
and losses of containment (e.g. lab positive pressurization) must be
reported immediately to the laboratory supervisor; written records of such
incidents must be maintained
an effective rodent and insect control program must be maintained
12
LABORATORIES
The following describes the minimum operational practices required for
containment level 2:


















laboratory personnel must be trained in and follow the safe use of
laboratory equipment, biological safety cabinets, procedures to minimize
the production of aerosols, decontamination and emergency response
open wounds, cuts, scratches and grazes should be covered with
waterproof dressings
eating, chewing gum, drinking, smoking, storing food, and applying
cosmetics are prohibited
personal items such as purses and outdoor clothing should be kept
separate from work areas
the work area containing hazardous materials should be kept free from
materials not pertinent to the work and that cannot be easily
decontaminated (e.g. journals, books, correspondence); paperwork and
report writing should be kept separate from such work areas
laboratory reference material should be kept in the laboratory zone
hands should be washed frequently (after handling infectious materials,
after removing gloves, and before leaving the laboratory)
open-toed and high-heeled shoes must not be worn in the laboratory
long hair should be tied back so that it cannot come into contact with
hands, specimens, containers, or equipment
gloves (e.g. intact vinyl or latex) must be worn when handling infectious
materials; metal mesh gloves can be worn underneath the latex or vinyl
glove to provide protection from sharps and needles
laboratory coats, gowns or coveralls must be worn when working in the
laboratory; this clothing must not be worn in non-laboratory areas (e.g.
offices, staff rooms, canteens, libraries)
protective lab clothing should not be stored in the same locker as street
clothing
contaminated clothing must be decontaminated prior to laundering (unless
laundering facilities are within the laboratory zone and have been proven
to be effective in decontamination)
eye and face protection must be worn when it is necessary to guard
against splashing hazardous materials, flying particles, and harmful light
or other rays
laboratory doors must be kept closed as required by the facility design
biological safety cabinets must be used for procedures with potential for
producing infectious aerosols (e.g. with zoonotic agents) and with high
concentrations or large volumes of zoonotic materials
contaminated work surfaces must be decontaminated
all contaminated materials must be decontaminated before disposal or
cleaning for reuse
13


contaminated equipment leaving the laboratory for servicing or disposal
must be appropriately decontaminated
efficacy monitoring of autoclaves using biological indicators must be done
at least weekly, depending on the frequency of use of the autoclave, and
records of the results kept on file; cycle log records (i.e. time, temperature
and pressure) must also be kept on file
In addition to the general operational practices listed for level 2, the following
describes the minimum operational practices required at containment level 3:













a protocol specific to the operation of the lab must be developed and read
by personnel; employees must certify in writing that they have understood
the material in the protocol
the laboratory zone must be kept locked
infectious agents should be stored inside the laboratory zone; agents
stored outside the zone must be kept locked, in leak proof containers
personnel must have demonstrated proficiency in microbiological practices
and techniques (e.g. experience in handling infectious organisms or cell
cultures)
personal items such as purses and outdoor clothing must not be brought
into the laboratory zone
a containment check must be performed prior to entering the laboratory
zone (i.e. verify negative lab pressurization as designed)
water seals must be maintained in drainage traps (i.e. through regular
sink/shower usage and/or by filling traps in areas that are not being used)
laboratory samples and supplies may be carried into the laboratory zone
or passed through a ventilated pass-box; where the barrier autoclave is
used to pass materials into the laboratory, the autoclave must have been
cycled prior to opening the outer "clean side" door
personnel entering the laboratory zone must remove street clothing and
jewellery, and change into dedicated laboratory clothing and shoes
where full body protective clothing is not worn a shower is required on exit
from the laboratory; where a known or suspected aerosol exposure has
occurred (e.g. dropping infectious materials) a shower is required on exit
from the laboratory zone
a shower (including washing hair, beards) is required on exit from a
laboratory zone handling non-indigenous animal pathogens; eye glasses
must be disinfected at the containment barrier
a second layer of protective clothing (i.e. solid-front gowns with tight-fitting
wrists, gloves) should be worn over laboratory clothing when directly
handling infectious materials (e.g. dedicated for use at the biological
safety cabinet)
contaminated clothing must be decontaminated prior to laundering (unless
laundering facilities are within the laboratory zone and have been proven
to be effective in decontamination of the microorganisms likely to be
encountered)
14




all activities with infectious materials are conducted in a biological safety
cabinet; where this is not possible, other physical 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
centrifugation of infectious materials must carried out in sealed safety
cups or rotors that are loaded and unloaded in a biological safety cabinet
all contaminated waste materials leaving the laboratory zone must be
decontaminated through a double-door autoclave at the barrier before
disposal; both doors of the autoclave must not be opened simultaneously
heat sensitive materials that cannot be autoclaved out of the laboratory
zone must be decontaminated at the containment barrier (e.g. fumigated
with formaldehyde or vaporized hydrogen peroxide, disinfected using
liquid chemicals, or other technology proven to be effective)
ANIMAL FACILITIES
Work with animals poses a variety of special hazards including exposure to
zoometric agents (naturally occurring or experimentally infected), animal bites
and scratches, kicks and crushing injuries, physical hazards (e.g. noise,
temperature) and chemical hazards (e.g. cleaning agents, disinfectants). Allergic
conditions can result from contact with animal fur or hair, bedding, and animal
wastes. At least one-fifth of people who work with laboratory rodents, guinea pigs
and rabbits develop allergies. Protection from allergens must be provided
through engineering controls, ventilation, use of isolators and cages with filter
tops and appropriate use of respiratory protection.
Animal handlers must have knowledge of the species' general characteristics
such as behaviour, instincts and physical attributes. Consideration should also be
given to their natural ecto- and endoparasites and the zoonotic diseases to which
they are susceptible including their route of excretion and dissemination.
The following describes the minimum operational practices required in small
animal facilities (SA facilities):





coveralls and footwear must be worn when working in AP containment
level 2 SA facilities
personnel entering AP containment level 3 and 4 SA facilities must
change into dedicated facility clothing and shoes
gloves must be worn when handling infected animals
hands must be washed after handling animals, after removing gloves and
before leaving the facility
HEPA-filtered respirators are required for handling animals in AP
containment level 2 and 3 SA facilities where infectious aerosols of
zoonotic agents may be generated and cannot be contained within a
primary containment device
15
















positive pressure ventilated suits are required for handling animals
infected with HC containment level 4 zoonotic agents
a shower (including washing hair, beards) is required on exit from AP
containment level 3 SA facilities handling non-indigenous agents and
when handling other agents where aerosols cannot be contained in
primary containment devices; eye glasses must be disinfected at the
containment barrier
a shower is required on exit from AP containment level 4 SA facilities; a
chemical shower is required when suits are worn
where a clothing change is not performed on exit from each animal room,
disinfectant footbaths are required (the disinfectant must be effective
against the microorganisms of concern and changed regularly in
accordance with the active life of the disinfectant)
contaminated clothing must be decontaminated prior to laundering (unless
laundering facilities are within the laboratory zone and have been proven
to be effective against the microorganisms likely to be encountered)
each animal room must be labeled with unique hazards and entry
requirements (e.g. respiratory protection)
animal room doors must be kept closed as required by facility design
water seals must be maintained in drainage traps (i.e. through regular
sink/shower/floor drain usage and/or by filling traps in areas that are not
being used)
cages housing infected animals must be appropriately labeled
containment caging systems should be used in AP containment level 3 SA
facilities to contain aerosols (e.g. laminar flow cabinets, solid wall and
bottom cages covered with filter bonnets)
containment caging systems must be used in AP containment level 4 SA
facilities
careful handling procedures must be employed so as to minimize the
creation of aerosols and dissemination of dust from cages, refuse and
animals
proper methods of restraint must be used to minimize scratches, bites and
accidental self-inoculations
necropsy of small animals infected with a zoonotic agent and intranasal
inoculation of animals in AP containment level 3 and 4 SA facilities should
be conducted in a biological safety cabinet; animals must be securely
transported to the biological safety cabinet
supplies and materials are brought into the AP containment level 3 and 4
SA facilities by carrying them in or by way of a ventilated pass-through;
barrier autoclaves, fumigation chambers or airlocks may also be used
providing that they have been decontaminated before opening the outer
"clean side" door
animal bedding must be removed in a manner that minimizes the
generation of aerosols and dust; for AP containment level 3 and 4 SA
facilities handling zoonotic agents, cages must be decontaminated prior to
removing bedding
16


all contaminated materials must be decontaminated before disposal or
cleaning for reuse
autoclaving is the preferred method of decontaminating cages prior to
washing; temperature of the final rinse water in mechanical cage washers
should be at least 82oC
animal carcasses and tissues must be incinerated or processed through new
technology proven to be effective (e.g. tissue autoclave); carcasses must be
transported from the animal room for disposal in leak proof containers that are
appropriately labeled.
POST-MORTEM ROOMS
Hazards in the post-mortem (PM) room are not limited to splashes and aerosols
of infectious materials. Accidents can be caused by cutting instruments, sharp
ends of cracked bones, slippery floors, electrical equipment, chemical fixatives
and disinfectants.
General precautions:





only authorized staff are allowed to use the necropsy facilities
staff must be trained in the use of all equipment and tools (e.g. electric
hoist/monorail, tools, PM table, incinerator)
staff must be trained in proper disinfection and cleaning procedures
the area must be kept neat and tidy; equipment, paper, reports, etc.
should be stored securely and not be accumulated in the PM room to
facilitate cleaning and decontamination; floors should be clear of
obstructions
specific protocols for each project must be developed and followed; these
include entry/exit protocols (for people, animals, equipment and samples),
protective clothing and equipment, disinfection and cleaning protocols, use
of the incinerator and autoclaves, and emergency procedures
Preparation for necropsy:

protective clothing appropriate to the AP containment level and potential
hazards must be worn in the PM room; this should include the removal of
street clothing and donning of protective clothing and footwear; HEPAfiltered respirators are required when the potential for infectious aerosols
exist; waterproof aprons, gloves and eye/face protection (face shield,
goggles) should also be worn; a safety helmet is required when operating
an electrical hoist/monorail
17

specific protocols must be developed for the movement of animals and
carcasses into the PM room (e.g. hoist for large animals, cart for small
livestock, secure containers for poultry and laboratory animals)
Necropsy Procedures:



necropsy safety procedures specific to the species involved must be
followed (i.e. use of cutting instruments to avoid injury)
the animals (especially birds and small lab animals) should be wetted with
water and/or disinfectant prior to necropsy
skilful technique is required to prevent excessive spread of contamination
and the formation of aerosols originating from fluids and tissues (this is
particularly important for work with zoonotic agents); every effort should be
made to confine the spread of contamination; this is especially true when
there is a likelihood of material being dropped from an elevated position
Cleaning and disposal procedures:






upon completion of the post mortem, all necropsy tools and instruments
must be decontaminated by autoclaving or disinfection (the disinfectant
must be effective against the microorganisms of concern); as some
disinfectants are inactivated in the presence of organic materials, gross
contamination should be removed prior to disinfection
disposable sharps, needles, blades, glass slides, etc. must be discarded
into an appropriate sharps container for decontamination
the necropsy table, floor and other contaminated work areas must be
cleaned and disinfected at the end of an experiment using an appropriate
procedure; preliminary washing using a general purpose
disinfectant/detergent should be done; special care must be exercised
when using a hose to wash the area (i.e. prevent the spread of
contamination and formation of aerosols); decontamination of the PM
room can then be achieved by spraying or fumigating with a disinfectant
effective against the microorganisms of concern
specimens (fresh, frozen or fixed) for further study should be placed in
leak proof containers, appropriately labeled; the outside of the container
must be cleaned and disinfected at end of necropsy or upon exit from the
PM room; samples may only be opened in a laboratory zone of the same
AP containment level
all animal waste must be incinerated or processed through new
technology proven to be effective (e.g. tissue autoclave); the incinerator or
tissue autoclave should be located adjacent to the PM room
where large specimens must be divided into smaller pieces and
transported to the incinerator, pieces should be placed carefully into leak
proof containers to avoid splashes and aerosols; the outsides of
containers must be cleaned down and disinfected prior to transport
18

out of the PM room; the containers must be labeled with the contents and
the name and phone number of a contact person
Exit procedures:


the requirement for showering out of the PM room is dependent on the
microorganism of concern; a full shower out of the facility (including
washing hair, beards and glasses) is mandatory when working with
zoonotic level 3 and 4 agents, and non-indigenous agents
contaminated protective clothing must be decontaminated prior to disposal
or re-use; contaminated laundry is autoclaved prior to processing (unless
using a pass-through laundry machines proven to be effective against the
microorganism of concern)
Specific Techniques
Good Microbiological Technique (GMT)
Just like Chemists have Good Laboratory Practices, (GLP). Professionals who
work with microorganisms have Good Microbiological Techniques (GMT).
These are standard operating procedures developed from years of experience
which usually not only provide the best and most consistent results but also
typically meet the highest health and safety standard as well.
The following page will cover some of the most frequently used manipulations
and procedures used in a microbiology lab. As with all things planning your
sequence of events is perhaps the most important step one can make.
1. Working in BSCs
Before using the cabinet
1. Turn off the UV lamp; turn on the fluorescent lamp
2. Disinfect work surfaces with appropriate disinfectant (see chapter on
disinfection)
3. Place essential items inside cabinet
4. Allow the blower to run for 5 – 10 minutes before work.
After Completion of work
1. Leave blower on at least 5 minutes to purge cabinet
2. Remove and decontaminate equipment and materials with the appropriate
disinfectant
3. Disinfect the cabinet surfaces
4. Turn off blower and fluorescent lamp, turn on UV lamp.
19
Maintenance





Twice daily: Work surfaces wiped down
Weekly: UV lamp should be wiped clean (dust decrease the intensity of
the lamp)
Monthly: All vertical surfaces wiped down
Annually: UV lamp intensity verified (intensity decrease with time)
Annually: Decontamination with formaldehyde gas and certification
2. Safe Use of Centrifuges
 Check centrifuge tubes for stress lines prior to use
 Avoid overfilling
 Ensure Caps or stoppers are properly in place
 Use sealed buckets or rotors which can be loaded and unloaded in BSCs.
 Ensure all buckets are properly balanced
 Ensure centrifuge achieves run conditions before leaving
 Ensure centrifuge completely stopped before opening lid
 Ensure equipment interlocks working properly
 Check immediately for spills or leaks prior to removing samples
 Clean all spills promptly and completely
3. Needles and Syringes
 Avoid the use of needles and syringes whenever possible
 Perform all operations within a BSC
 Fill syringes carefully, avoid frothing or introduction of bubbles
 Shield needles with disinfectant soaked cotton when withdrawing from
stoppers
 Do not bench Shear or recap needles. Dispose of in Biohazard Sharps
Containers (Yellow labeled containers which may or may not contain
disinfectant)
 If needles must be recapped, use one handed scoop method or holder
4. Hand washing





One of the best defenses to prevent exposure
In CL 1 lab, a non antiseptic soap can be used
In CL 2 and 3 labs, antiseptic hand washing solutions are required. Most
of the common antiseptic solutions contain chlorhexine gluconate or
trichlosan. An alternative is to use alcoholic hand rubs. They contain an
emollient to counteract the drying action of alcohol.
Liquid dispensers should be used rather than bars.
When to wash?
Before starting any manipulations
Before leaving the lab
When hands are obviously soiled
20
Before and after completing any task in a BSC
Every time gloves are removed.
Before contact with one’s face and hands
At the end of the day
How to Hand wash Properly
1.
2.
3.
4.
Turn on faucets and wet hands with tepid water
Dispense soap into a cupped hand
Spread soap or compound around both hands and between fingers.
Wash (lather hands for at least 10 sec. Vigorously rub both sided of
hands starting a few inches above the wrist, extending downwards
between the fingers and around and under the fingernails. Beware of the
creases in your hands
5. Rinse thoroughly under the tepid running water, Rinsing should start
above the wrist area and proceed to the tips of the fingers. Note if faucets
are not knee or foot-operated do not turn of water yet.
6. Dry hands thorough with paper towels. If hand operated, turn faucets off
once hands dry, using the paper towel to protect hands.
Decontamination, Disinfection and Sterilization
Definitions

Decontamination: Destruction or removal of microorganisms to a lower
level, such that there is no danger of infection to unprotected individuals.

Sterilization: Use of physical or chemical means to bring about the total
destruction of al viable microorganisms.

Disinfection: Use of physical or chemical agents to destroy pathogens
and potential pathogens on inanimate objects.
It is a basic biosafety principle that all contaminated materials be decontaminated
prior to disposal. Decontamination includes both sterilization (the complete
destruction of all microorganisms, including bacterial spores) and disinfection
(the destruction and removal of specific types of microorganisms). It is the
responsibility of all laboratory workers to ensure the effective use of products for
decontamination of materials, equipment and samples from containment zones;
of surfaces and rooms; and of spills of infectious materials.
There are basically two methods for accomplishing decontamination, sterilization
and disinfection; Physical methods and Chemical methods.
21
Physical Methods

Heat:
Autoclaves: most practical and recommended
Incineration: for disposal of sharps and tissues

Irradiation:
U.V. light: wavelength of 253 nm is germicidal
Gamma: disrupts DNA and RNA

Filtration:
HEPA: Biological safety cabinets, ventilation 0.2 micron
physically removes particulates
Chemical Methods

Generally used for disinfection rather than sterilization

Choice depends on many factors (number and nature of organism, type of
item to be disinfected, purpose of treatment, other chemicals, contact time
required for disinfection, toxicity, cost)

Most common are chlorine compounds and alcohols
Disinfection: What to use for my organism?

Vegetative bacteria
(Ecoli, ect.)
1% domestic bleach
75% Ethanol
Quaternary ammonia
6% formulated Hydrogen
peroxide

Mycobacteria and fungi
1 % domestic bleach
Phenolic compounds
75% Ethanol
6% formulated Hydrogen
peroxide

Spore forming bacteria
(Bacillus)
(Time extended for several
hours to ensure sporicidal)
10% domestic bleach
Gluteraldehyde
Formaldehyde
6% formulated Hydrogen
peroxide
 Enveloped viruses
(HIV, Herpes)
1 % domestic bleach
75% Ethanol
22
Quatenary ammonia
6% formulated Hydrogen
peroxide

Non enveloped viruses
(Hepatitis, Adenovirus)
10% domestic bleach
6% formulated Hydrogen peroxide
Gluteraldehyde
Formaldehyde
Disinfection for Prions
Autoclave at 132-136 oC for 60 minutes, or Autoclave at 121
o C for 4.5 hours, or;
Soak in 1N sodium hydroxide for 1 hour, then autoclave at
121 o C for 1.5 hours, or Soak in 3% SDS for 1 hour, then
autoclave at 121 o C for 1 hour
Treat work surfaces with 10 % sodium hypochlorite (industrial
strength bleach) for at least 30 minutes.
2N sodium hydroxide may also be used to treat surfaces.
If skin becomes contaminated, treat for 5-10 minutes with 1N
sodium hydroxide followed by extensive washing with water.
23
Chemical Decontamination Methods Halogen Releasing Chemical
Germicides
Chemical
Type
Compound
Chlorine
Compounds
Effective
Advantages
Concentratio
n; Contact
Times
Disadvantages
Examples of Uses
Toxic, corrosive to skin
and metals Unstable at
optimum pH of 6
Inactivated by organic
matter
General disinfectant
Emergency spill clean up
temperatures
As for liquid bleach but
more stable
Deteriorates under light
and heat; shelf life of
dilutions: I week
As for liquid bleach, but
shelf life is longer
As for liquid
bleach
Stable at pH 6. More
stable than hypochlorites.
No strong smell.
Toxic, corrosive
inactivated by organic
matter
As for liquid bleach
As for liquid
bleach
More stable, less affected Deteriorates under
As for liquid bleach
by organic matter than
humidity, light and heat.
hypochlorites
Leaves powdery
Longer activity than
residue.
hypochlorites
Demand release
of chlorine in situ
Longer activity than
Aqueous solutions
chlorine compounds.
decompose under light
Less corrosive, toxic than
other chlorine compounds
Effective at pH 6-10
Broad spectrum
Not consistently
Instrument disinfection.
Gas sterilization of germfree animal chambers
Germicidal over a wide
pH range Less toxic and
less irritating than
aqueous or alcoholic
iodine solutions
sporocidal
antiseptics
Readily neutralized by
organic matter May
allow growth of
Pseudomonas species
and other bacteria
Surface decontamination of
skin prior to procedures
Sodium Hypochlorite 0.1-1% free
solution (domestic
chlorine
bleach)
10-60 minutes
Broad spectrum
Inexpensive
Widely available
Bactericidal at low
Calcium hypochlorite As for liquid
(granules, powder,
bleach
tablets)
Sodium
Dichloisocyanu-rate
Waste liquids
Surface decontamination
Instrument disinfection
As for liquid bleach
(NaDCC)
(tablets, powder)
Chloramine~T
(Sodium
tosylchloramide)
(powder)
—
Chlorine Dioxide
Iodine
Preparations
Iodophors
0.003-1% free
iodine
10-30 minutes
Germicidal soap and
Household sodium hypochlorite (Javex) is recommended for most
applications. A 10% dilution for most disinfection purposes, with full
strength used for spills.
24
Heat Decontamination Methods
Type
Principle/Conditions
Advantages
Dry Heat
Thermal inactivation:
destroys by oxidation
Non-corrosive Simple
design and principle
Hot Air Oven
160-180°C for 2-4 hours
Red-heat
Flame
Oxidation to ashes (fuming)
Penetrates water-insoluble
materials (e.g.,grease and
oil)
Less corrosive to metals
and sharp instruments than
steam ~
Rapid
Incineration
Oxidation to ashes
Reduces volume of waste
(burning)
by up to 95%
1 -60 minutes: temperatures
may exceed 1000°C
Moist Heat
Irreversible coagulation of
microbial proteins
Pasteurization Heating to below boiling
point (normally 60-709C) for
up to 30 minutes
Tyndallization Heating to 80-i009C for 30
mm on 3 consecutive days
with incubation periods in
between
Boiling
Autoclaving
Uses
Materials that are
damaged by, or are
impenetrable to, moist
heat
Slow diffusion, penetration.
Anhydrous materials,
Loading, packing critical to
such as oils, greases and
performance.
powders
Not suitable for reusable plastics Laboratory
glassware,instruments
~
Initial contact with flame can
Inoculating loops, needles
produce a viable aerosol
Possibility of accidental fire
Improper use may lead to
For decontamination of
emission of pathogens in smoke
Requires transport of infectious
waste. Excess plastic (>20%)
content reduces combustibility
More rapid and more
effective than dry heat
Can be used on heat
Not reliably sporicidal
sensitive liquids and
medical devices Low cost
Resistant spores germinate Time consuming Not reliably
and are killed on the second sporicidal
and third day
Maximum temperature
Minimal equipment required
obtainable is I OOLIC at sea
level. Minimum 10 minutes
Steam under pressure 121
Minimal time required
o C at 15 psi for 15-90
Most dependable sterilant
for lab use
mins.
Disadvantages
Less effective than moist heat;
requires longer times and/or
higher temperatures
Cumbersome: not practical for
everyday use
Not reliably sporicidal
Loading and packing critical to
performance
Shielding dirt must first be
removed
Maintenance and quality control
essential
Damages heat sensitive items
waste items prior to
disposal in landfill
Milk and dairy products
Some heat-sensitive
medical equipment
Heat sensitive materials
such as bacteriological
media, solutions of
chemicals, biological
materials
Small instruments and
equipment
Preparation of sterile
glassware, media and
instruments.
Decontamination of
reusable material
Decontamination of
infectious waste
25
Chemical Decontamination Methods
Non Halogen Chemical Germicides
Type
Effective
concentration;
contact times
Advantages
Disadvantages
Alcohols
70-85% ethanol
60-95%
isopropanol
3-30 minutes
Low toxicity
Low residue
Non corrosive
Rapid evaporation, reduces Skin disinfectant
contact time Flammable,
Surface decontamination
skin desiccant Non
Bench top, cabinet wipe down
sporicidal, ineffective with
unconventional agents
0.04-5%
1 0-30 minutes
Leaves an active residue
Biodegradable
0.05 -1.5%
10-30 minutes
Has combined detergent
and germicidal activity
Stable
Working dilutions have low
toxicity
Rapid action
no residue
Low toxicity
Environmentally safe
30% is sporicidal
Broad spectrum sporicidal
at low temperatures Can
tolerate organic load Rapid
action
Broad spectrum
Does not corrode metal
Can tolerate organic load
-
Phenolic
compounds
-
Quaternary
Ammonium
compounds
----
Hydrogen
peroxide
3-30%
1 -60 minutes
New “formulated”
6% has additives
Peracetic acid
0.001-0.3%
10-60
minutes
gas phase 2-4%
5-120
minutes
0.5-2.5% aqueous
2-30 minutes (up to
12 hours to kill all
spores)
Aldehydes
Gluteraldehyde
Aldehydes
Formalin
(37%
formaldehyde)
3-27% formalin in
70-90% water
10-30 minutes
Broad spectrum
Inexpensive
Does not corrode metal
Can tolerate organic load
Aldehydes
Formaldehyde
1-3 hours
As for formalin Effective
penetration
gas
(paraformaldehy
de)
Ethylene Oxide 50-200 mg/L
Gas
1-12 hours
Broad spectrum
No heat or moisture
Released
Penetrates packaging
materials
Examples of uses
-
~
Pungent odour, corrosive ,
toxic
Non sporicidal, limited
activity against viruses
Not sporicidal, limited
against viruses,
myeobaeteria
Not readily biodegradable
Disinfection of floors and other
surfaces
Antiseptic soaps
~
Surface decontamination
Equipment wipe down antiseptic
Limited sporicidal activity
Corrosive to some metals,
irritant
Concentration dependant
Surface decontamination
Instruments and equipment
Pungent odour Corrosive to
some metals Shelf life of
dilution is less than 1 week
irritant to skin and eyes
Expensive Requires good
ventilation pH, temperature
dependant Pungent odour
Toxic
Less than 2 week shelf life
Pungent odour
Irritant
Potential carcinogen
May require 24 hours to
kill spores
As for formalin
Toxic
Flammable
Poor penetration of
covered surfaces
Flammable, reactive
Toxic; potential carcinogen
and mutagen. Some
sterilized items may require
24 hours for out gassing
Instruments and equipment Gas
phase sterilization of chambers for
germ free animals
Cold sterilant and fixative Surface
decontamination Instruments,
equipment, glassware
Cold sterilant and fixative
Surface decontamination
Instruments and equipment
~
On site decontamination of
biological safety cabinets, HEPA
filters
Enclosed area
Heat or moisture sensitive
supplies, equipment and
instruments
Requires specialized
equipment and training
26
Autoclaves
Infectious laboratory wastes (Petri dishes, pipettes, culture tubes, glassware etc...) can be
effectively decontaminated in either a gravity displacement or prevacuum autoclave. The
effectiveness of decontamination by steam autoclaving depends upon various loading
factors that influence the temperature to which the material is subjected and the contact
time. Particular attention must be given to packaging, including the size of containers
and their distribution in the autoclave. Containers of waste must allow steam penetration
and must be arranged in the autoclave in a manner that permits free circulation of steam.
Tight-fitting containers do not permit steam penetration. Piling containers above one
another and overloading can result in decontamination failure.
Effective operating parameters for autoclaves should be established by developing
standard loads and their processing times through the use of thermocouples and
biological indicators placed at the centre of the load (i.e., the most difficult areas of the
load to decontaminate). Biological indicators are also used for routine monitoring (e.g.,
weekly based on the frequency of use) of the sterilization process. A biological indicator
is a standardized population of bacterial spores intended to demonstrate favourable
sterilization conditions in the load. Attention must be paid to appropriate selection of the
indicator, as the design and construction vary depending on its intended use (i.e., liquid
versus dry load, self-contained system, enzyme-based rapid method). Chemical
indicators are meant to be used in conjunction with biological indicators and physical
monitors (i.e., pressure and temperature readings). They provide instant results for dayto-day monitoring that the load has been processed; however, they must not be used as
the sole indicator of sterility.
Spill Response
Spill response will vary depending on the nature of the spill, the size, the location and the
material. Spills should be cleaned up immediately to ensure proper decontamination. If
you must leave the scene, the door must be posted informing other of the spill and the
hazards. All spills must be reported to Security and the BSO.
Spills within Biological Safety Cabinets
 Leave the ventilation on
 Flood the spill with 10 % bleach and cover with absorbent
 Leave for 20 minutes
 Pick up absorbent material
 All items within the cabinet should also be disinfected (wiped down or
autoclaved)
 All waste should be autoclaved
 Ventilation should run 10-15 mins
Spill outside of BSCs, in a lab (large spills)
27






Evacuate the area for 30 minus to allow aerosols to settle
Assemble cleaning supplies and PPE
Using Concentrated disinfectant such as 5% hypochlorite (undiluted bleach) pour
disinfectant from outside, towards inside.
Cover with absorbent and allow disinfectant to act for 20 min. If spill is of blood
or other heavy organic material, wipe up most of the material, then allow
disinfectant to act for 20 min)
All adjacent areas should also be disinfected or wiped down
All waste should be autoclaved
Spills occurring in Centrifuges






Leave lid closed and allow aerosols to settle for at least one (1) hour
Thoroughly wipe down inside of centrifuge, including the lid with paper towels
soaked in disinfectant
Disinfect the entire rotor, especially the bucket where spill occurred within the
centrifuge
Remove rotor from centrifuge and repeat disinfection.
Rinse both rotor and inside of centrifuge with water if bleach was used
All waste should be autoclaved.
Spills occurring during transport


Clean-up must be initiated immediately (as hallways are not negatively pressured)
Follow directives for spills outside of biological safety cabinet.
Biological Waste
Biological Waste includes the following
 Sharps and biomedical waste
 Tissue Culture Waste
 Microbiological waste, including media
Sharps should be disposed of in the proper containers and once full, will be picked up for
disposal.
Tissue culture and microbiological waste is decontaminated prior to disposal.
Material can be either autoclaved or chemically decontaminated
All biological waste is to be decontaminated.
28
Procedures for Working with Biohazardous Material at Trent
The tricouncil granting agencies require that Biosafety Guidelines (Health Canada and
the Canadian Food Inspection Agency) for work with biohazardous material are met. To
accomplish this, the University has set up a Biosafety Committee to oversee the Biosafety
Program at the University.
The terms of reference are attached (Appendix 1.) and membership is made up of a
broad range of stakeholders including qualified faculty, staff, physical resources and
members of the Office of Research and Graduate Studies.
The Biosafety committee is responsible for ensuring that all work with biohazardous
materials meets or exceeds the guidelines set out by Health Canada or the Canadian Food
Inspection Agency. Personnel proposing work with biohazardous substances are required
to get approval from the Biosafety Committee.
The Biosafety committee will ensure that: rooms for proposed work meet the appropriate
containment standards, that the work procedures for the specific pathogen are appropriate
and meet the guidelines, and that a training program for working with Biohazardous
organisms is available.
How Do I Get Approval to Work with Biohazardous Substances?
Primary investigators are responsible for completing the Biosafety Protocol
application attached (Appendix 2). Much of the information requested will require
the applicant to read the relevant sections of either the Red or Peach Book.
Applicants are encouraged to submit this protocol to the committee with as much lead
time as possible as certification of rooms, import permits etc… require considerable
time to prepare.
The Biosafety committee will review all applications for work with biohazards in as
timely fashion as possible. The Biosafety committee may request changes, and
alterations to work procedures if it determines they are necessary to meet the
guidelines.
Research applications which require Biosafety Committee approval prior to
submission should ensure that there is appropriate time available to the committee to
do its work.
Laboratory Biosafety Program
Each lab involved in work with biohazardous substances must have a documented
Biosafety program in place. For assistance with this contact the BSO and read the
“Peach Book” or “Red Book”. Typically Biosafety programs address cradle to grave
issues of working with the pathogen and specific safety requirements beyond the
scope of this document.
29
Biosafety officer
Currently the role of Biosafety Officer is being fulfilled by the Science Facilities
Manager. The BSO is responsible to ensure that the guideline and regulations
involving working with biohazardous substances are being met and that safe work
conditions and practices are being adhered to. The BSO has the authority to
temporarily suspend work, if it is deemed unsafe. This suspension, upon review by
the Biosafety committee may be revoked or permanently installed if necessary
without prejudice.
Procedure for the importation of pathogens from outside of Canada.
The importation of pathogens to Canada has become considerably more complicated
over the last couple of years. The increase in border security (on both sides of the
border) as well as the new terrorism legislation in the U.S. has resulted in a situation
where considerable lead time is required for the application and approval process. In
addition, it has become apparent, that even when Canadian approval is granted, the
U.S. export laws have made the process additionally complicated. Contact the BSO
well in advance (as much as two to three months) for assistance with this. The
university will not authorize the importation of a pathogen, however without a Project
Approval in advance.
30
Appendix 1.
Biosafety Committee Terms of Reference
Membership
A.V.P. Research *Chair*
Research Officer
H and S officer
1 other faculty science
Director Physical Resources
Assoc. Dean of Science
Faculty with microbiology or cell biology
background
1 rep MNR who has responsibility for
Biosafety within MNR operations at Trent.
Biosafety officer (BSO)
Director Health Services
(Faculty shall be appointed by the V.P. academic for 2 year terms and should have a thorough background
in microbiology or cell biology. If possible, faculty appointments should be staggered so that a measure of
stability is achieved)
Mandate
1. To set policy for the University on actions or policies related to Biosafety
2. Oversee the university’s biosafety program( the acquisition, use, storage, transfer and disposal of
biohazardous or potentially biohazardous material), recommend changes and provide direction as required.
3. To review, assess and approve all work at the university which may represent a Biosafety hazard for
personnel or animals in and around the university. Certification of such research programs will follow
policies as laid out by the Tri-Council funding organizations.
4. The committee will approve applications for research involving the use of genetically modified
organisms.
5. To develop and recommend policies and procedures to meet or exceed the requirements for each
containment level as stated in the most current version of the HC Laboratory Biosafety Guidelines or the
most current version of the CFIA Containment standards or veterinary facilities.
6. In collaboration with the Biosafety officer, conduct inspections of CLII and above labs annually with a
written report including deficiencies and recommendations with respect to the operation of the labs.
7. To recommend to the university the appropriate changes as required to the Occupational Health and
Safety Program of the university.
8. The committee will review its Terms of reference annually and make changes as necessary.
Meetings
The biosafety committee should meet a minimum of 4 times a year.
Quorum
A quorum shall consist of the majority plus one but must include the biosafety officer.
31
Appendix 2
Office of the Associate Dean Science
Office of the Associate Vice President of
Research
Biosafety Project Approval and Materials Registration
This protocol must be completed by each Principal Investigator holding a grant
administered by Trent University or supervising a research project where the use of
biohazardous infectious materials are described and used. This form must also be
completed if animal work is proposed involving the use of biohazardous agents or
animals carrying zoonotic agents infectious to humans or wildlife. Completed forms are
to be sent to the Office of Research for distribution to the Biosafety Committee. For
questions regarding the completion of this form please contact the Biosafety Officer at
ext 7061. Any changes to this form or to the projects described within must be
completed and forwarded on to the Biosafety Committee for reassessment.
Information on Biosafety at Trent can be accessed on the Web at
www.trentu.ca/sciencedean or www.trentu.ca/research
Please attach MSDS from Public Health Agency Canada (if available) or any other
information regarding the pathogenecity of the organism. Other sources of
information on Pathogens include U.S. CDC, UN Biosafety Program
Project # (for internal use only) _______________
Principal Investigator:
Department:
Office Number:
Phone Number:
Email Address:
Project(s) Title(s):
protocol.
Give all the Project(s) title(s) which will be covered by the
32
Lay Summary(s): Give a separate lay summary for each project included in this
Protocol. List each pathogen in each project.
Location where experimental work to be carried out:
Building
Room(s)
**For work being performed at affiliated institutions or away from Symons Campus
please indicate full address. If the affiliated institution has a safety officer, their signature
of approval will be required prior to review by the Biosafety Committee review**
Funding Agency/Agencies:
Names of all personnel working under the Principal Investigator in the location listed
above.
1.
3.
5.
2.
4.
6.
Note: All personnel (students and staff) who will be working with Biohazardous
material rated as risk group 2 or above are required to take the Biosafety course.
Contact the Biosafety Officer or Science Facilities Manager (ext. 7061) for course
information.
33
Methods: (Describe in detail the methods you will be using in the proposed project.
Attach additional pages if necessary).
1.0 Microorganisms
Does your work involve the use of microorganisms?
If not proceed to 2.
Yes
No
Is this for active work;
Yes
No
Or is this for a storage permit
Yes
No
(Note: All substances considered as biohazardous risk group 2 or greater must be
permitted and an inventory maintained, even if no experimental work is on-going).
34
Please complete the table below:
Name of
Is the
Microorganism
microorganism
(Family/genus/sp. known to be a
e.g., Pox
human
viridae/Vaccinia) pathogen?
Yes/No
(if yes give
name of
disease)
Is the
microrganism
known to be
an animal
pathogen?
Is the
microorganism
known to be a
zoonotic
agent?
Yes/No
(if yes give
name of
disease)
Yes/No
(if yes explain
the risk)
Risk
Group
(1,2,or
3)
Maximum
quantity
to be
cultured
or in
possession
at any one
time?
Yes/No
For each substance listed above what is the source of the microorganism?
1
2
3
4
5
6
2.0 Cell Culture
Does your work involve the use of cell cultures?
If no proceed to 3.
Please complete the table below:
Cell Type
Name of Cell
Line
Yes
Is this cell type used
in work? Yes/No
No
Established or
Primary*
(print one)
Human
Rodent
Other
* derived from fresh tissue
For each cell line list the supplier of primary cell culture tissue?
35
For each cell types give the HC or CFIA risk group (1 2 3).
3.0 Use of Human Source Materials
Does your work involve the use of human source materials?
If no proceed to 4.0
Indicate if the following will be used in the lab.
 Human blood (whole) or other bodily fluids?
If Yes please Specify:
Yes
No
Yes
No

Human blood (fraction) or other bodily fluids?
If Yes please specify:
Yes
No

Human Organs (unpreserved) ?
If Yes please specify:
Yes
No

Human tissues (unpreserved) ?
If yes please specify:
Yes
No
Yes
No
Yes
No
Yes
No
Yes
No
Yes
No
Yes
No
4.0 Genetically Modified Organisms and Cell lines
Will genetic modifications be made to the organism or cell line?
If no please proceed to 5.0
Will genetic sequences from the following be involved?
 Genes from any CDC class 1 pathogens
If yes pleas specify:

Other human or animal pathogen and/or their toxins
If yes please specify:
Will intact genetic sequences be used from:
 SV 40 Large T antigen
If Yes please specify:

Known oncogenes
If Yes please specify:
Will a live vector(s) (viral or bacterial) be used for gene
transduction
36
If Yes name vector:
Will virus be replication defective
Will virus be infectious to humans or animals
Will this be expected to increase the Containment level required
Yes
Yes
Yes
No
No
No
5.0 Animal Experiments
Will any of the agents listed be used in live animals
Yes
No
Name of animal species to be used
ACC protocol number (All experiments with vertebrate animals requires approval f the
Animal Care Committee).
If using murine cell lines, was the cell line tested for murine or mammalian pathogens.
Yes
No
Yes
No
Yes
No
Yes
No
Yes
No
6.0 Biological Toxins
Will toxins of biological origin be used
If no please proceed to 7.0
If yes, please name the toxin:
What is the LD 50 of the toxin:
7.0 Import Requirements
Will the agent(s) be imported (or were they imported originally
prior to your acquisition)?
If no proceed to 8.0
Has an Import permit been obtained from HC for human
pathogens?
Has an Import permit been obtained from CFIA and has a
letter from HC indicating that an HC permit is not required,
been obtained
Has a copy of the permit been sent to the BSO
Yes
No
Note: Importation requirements for human, animal and plant pathogens vary
considerably contact the BSO for assistance in obtaining these permits. Persons
receiving hazardous substances will require Transport Dangerous Goods training.
Contact the Science Facilities Manager for details.
All imports require the approval and signature of the BSO prior to acquisition.
37
8.0 Radioactive Material.
Will Radioactive material be used.
If yes please give the Radiation Permit #.
If no proceed to 9.0.
Yes
No
Note: Work with Radioactive substances at Trent University requires a Radiation Work
Permit and that all personnel handling Radioactive Material have taken the Radiation
Safety Course.
Contact the RSO or the Science Facilities Manager at ext. 7061.
9.0 Training Requirements for Personnel named on Form
All personnel named on the above form who will be using any of the above named agents
are required to attend the following training course given by the Health and Safety cocoordinator and the BSO as a minimum. The use of animals or radioactive substances
will require additional training. Contact the Environmental Health and Safety Officer or
the Science Facilities Manager for additional information.


Biosafety
WHMIS
As the principal investigator, I have ensured that all of the personnel named on the form
who will be using any of the biohazardous agents in Sections 1-8 have been trained.
Signature:
Date:
9. Containment levels
For the work described above please and based on your Hazard Risk Assesment circle the
highest containment level required
1
2
3
Has the facility (Building and room number) been certified by the Biosafety Committee
for this level of containment?
Yes
No
38
10. Methodology
Attach the specific methodology you will use for your work. Include descriptions of
techniques such as any method which may produce aerosols (i.e., vortexing, centrifuging,
mixing of solutions). Include the specifics of your decontamination of instruments,
equipment, waste products and sample destruction.
11. Principal Investigator Commitment
I
agree to conduct my research in accordance with all of the
regulations and guidelines (Federal, Provincial and Institutional) which this project may
fall under. I agree that all of the information above is accurate to the best of my
knowledge and I agree not to change the project, with the exception of improving safety,
without notifying and getting the approval of the Biosafety Committee first.
Signature of Principal Researcher:
Approvals
Date:
Trent Bio-safety Committee
Chair, Bio-Safety Committee
Signature:____________________________
Date:________________
Dr. James Parker, AVP Research
Bio-Safety Officer
Signature:_____________________________
Date:________________
Chris Williams
Health & Safety Officer
Signature:_____________________________
Date:________________
Bill Gibson
External Safety Officer
Signature:
Date:________________
39
12. Suggested Readings
1. The Laboratory Biosafety Guidelines, 3rd ed. Health Canada, Minister of Public
Works and Government Services, Ottawa, Ontario, Canada, 2004.
2. Containment Standards of Veterinary Facilities, 2nd ed. Ottawa: Agriculture and
Agri-Food Canada, Minister of Supply and Services Canada, No. 1921/E, 1996.
Canada.
3. Laboratory Biosafety Manual. Geneva: World Health Organization, 1993.
4. Richmond, J.Y. and McKinnery, R.W. Biosafety in microbiological and
biomedical laboratories. Washington, DC: U.S. Government Printing Office 1999.
5. Biosafety, University of Ottawa. Environmental and Health Services. 2003
Web sites of interest.
Office of Laboratory Security
www.phac-aspc.gc.ca/ols-bsl/
Canadian Food Inspection Agency
www.inspection.gc.ca/english/toce.shtml
Health Canada Biohazard MSDS
www.phac-aspc.gc.ca/msds-ftss/index.html
National Center Disease Control
www.cdc.gov/ncidod
World Health Organization
www.who.int/csr/labepidemiology/projects/biosafetymain/en/
40