4.
STANDARD OPERATING
PROCEDURES
RYERSON UNIVERSITY
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Biological Safety Training – Certificate Holder and User Training
BIOLOGICAL SAFETY
4. Standard Operating Procedures
Infection Control
Containment of Aerosols
Working in CL 1 areas
Working in CL 2 areas
Biological Safety
Cabinets
Human Blood and Body
Fluids
Needle Stick Injury
Prevention
Signs and Labels
Personal Protective
Equipment
Purchasing/Transfer
Inventory Control
Decontamination/Waste
Disposal
Large Scale Work
RYERSON UNIVERSITY
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Biological Safety Training – Certificate Holder and User Training
4. Standard Operating Procedures
Infection Control
Good microbiological practice:
is a basic code of practice to handle biological agents
Involves specific sterile techniques of product
protection, contamination control
- Danger
- Radiation
applied to all Rayonnement
types of work
involving
microorganisms
irrespective of containment level to reduce the risk of
exposure and infection
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Biological Safety Training – Certificate Holder and User Training
4. Standard Operating Procedures
Infection Control
Good microbiological practices prevent contamination of:
laboratory workers
the environment
experimental samples
Good work practices can significantly reduce the risk of:
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aerosol production
contamination of experimental equipment surfaces
contains the biological agent
reduces the risk of infection
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Biological Safety Training – Certificate Holder and User Training
4. Standard Operating Procedures
Infection Control – Hand Washing
When to wash?
Before starting any manipulations
Before leaving the lab
When hands are obviously soiled
Before and after completing any task in a
biosafety cabinet
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- Danger
- Radiation
Every time
gloves are
removed
Before contact with one’s face or mouth
At the end of the day
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Biological Safety Training – Certificate Holder and User Training
4. Standard Operating Procedures
Infection Control – Hand Washing
Frequent hand washing has proven to be the
single most effective means of avoiding infection if
done properly and frequently
liquid dispensers should be used rather than bars
RYERSON UNIVERSITY
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Biological Safety Training – Certificate Holder and User Training
4. Standard Operating Procedures
Infection Control – Hand Washing
Wet hands with warm water
Dispense soap into a cupped hand
Spread around hands and between fingers
Wash hands for at least 10 sec.
Rinse thoroughly under warm water.
Dry hands thoroughly with paper towels.
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Containment of Aerosols
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4. Standard Operating Procedures
Containment of Aerosols
Aerosols are gaseous suspensions of fine solid or liquid
particles ranging in sizes from 0.01 to 100 µm and can
remain suspended in air for extended periods of time.
Pathogens such as viruses and bacteria are so small
that they can travel within one aerosol droplet and be
dispersed by building ventilation.
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Aerosols can settle on many surfaces where personnel
may unwittingly be exposed to a potentially infectious
material through the risk of direct contact.
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4. Standard Operating Procedures
Containment of Aerosols
When appropriate, other primary barriers such as splash
shields, face protection or gowns should also be used.
When conducting procedures that create considerable
aerosols or when using agents classified at Containment
Level 2, work should be conducted in a Biological Safety
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Cabinet
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4. Standard Operating Procedures
Containment of Aerosols
The generation and dispersal of aerosols must be
minimized and controlled.
Numerous procedures and devices which can result in the
generation of aerosols:
pouring liquids,
using centrifuges, shakers, blenders,
Rayonnement -vessels,
Danger - Radiation
opening pressurized
inserting a hot loop into a culture,
pipetting, etc.
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4. Standard Operating Procedures
Containment of Aerosols
Sterilization of inoculation loops in an open flame
may create aerosols which may contain viable
microorganisms.
Use a shielded electric incinerator.
Shorter handles minimize vibrations.
Disposable plastic loops are good alternatives.
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Biological Safety Training – Certificate Holder and User Training
4. Standard Operating Procedures
Containment of Aerosols
Mouth pipetting is prohibited.
All biohazardous materials should be pipetted in BSC’s.
Never force fluids out, use ‘to deliver’ pipettes.
To avoid splashes, allow discharge to run down
dispense the receiving container wall.
Never mix material
by suction
expulsion.
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- Dangerand
- Radiation
Reusable pipettes should be placed horizontally in a
disinfectant filled pan. Autoclave before reuse.
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Continue on to:
Working in Containment
Level 1 & 2 Areas
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4. Standard Operating Procedures
Working in CL 1 areas
The following requirements are basic for any laboratory
using biological agents. These requirements follow
Health Canada’s Laboratory Biosafety Guidelines 3rd ed
2004:
Good microbiological laboratory practices intended to avoid the
release of infectious agents are to be used.
A documentedRayonnement
procedural (safety)
must be available for all
- Dangermanual
- Radiation
staff and its requirements followed. The manual must be reviewed
and updated regularly
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4. Standard Operating Procedures
Working in CL 1 areas
PROHIBITED:
• eating, drinking, smoking
• storing food or utensils
• applying cosmetics
• inserting or removing contact lenses
• oral pipetting
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• loose long hair - must be tied back or restrained.
• doors to laboratories must not be left open (does not
apply to an open area within a laboratory)
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4. Standard Operating Procedures
Working in CL 1 areas
WORK AREA
• laboratory must be kept neat, and clean
• storage of materials not pertinent to the work and cannot be easily
decontaminated must be minimized
• paperwork and report writing should be kept separate from
biohazardous work areas.
• access to laboratory and support areas is limited to authorized
personnel
• extreme caution Rayonnement
must be used- Danger
when handling
needles and syringes to
- Radiation
avoid autoinoculation and the generation of aerosols during use and
disposal. Needles must not be bent or sheared.
• disposable needles and syringes must not be replaced in their cap but
placed into a puncture-resistant container
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4. Standard Operating Procedures
Working in CL 1 areas
WORK AREA
• technical procedures must be performed in a manner that minimizes
the creation of aerosols
• work surfaces must be cleaned and decontaminated with the
appropriate disinfectant at the end of the day and after any spill of
potentially hazardous material.
• loose or cracked work surfaces must be repaired or replaced.
• contaminated materials and equipment leaving the laboratory for
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Danger - Radiation
servicing or disposal
must be-appropriately
decontaminated and
labelled or tagged as contaminated
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4. Standard Operating Procedures
Working in CL 1 areas
PERSONAL PROTECTIVE EQUIPMENT
• laboratory coats must be worn and fastened by all personnel
working in the laboratory.
• suitable footwear with closed toes and heels and preferably with
non-slip soles must be worn in laboratory
• protective laboratory clothing must not be worn in non-laboratory
areas;
• lab coats must not be stored in contact with street clothing
• suspected contaminated clothing must be decontaminated before
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laundering
• eye protection must be worn when necessary to protect the eyes
• hands must be washed after gloves are removed, before leaving the
laboratory, and after handling materials known or suspected to be
contaminated, even when gloves have been worn
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4. Standard Operating Procedures
Working in CL 1 areas
TRAINING:
Lab personnel must receive training on the potential hazards and the
necessary precautions to prevent exposure to infectious agents and
release of contained material
personnel must show evidence that they understood the training
provided
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- Danger
- Radiation
training must be
documented
and signed
by both the employee and
supervisor
retraining programs should also be implemented.
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4. Standard Operating Procedures
Working in CL 1 areas
DISINFECTION & WASTE DISPOSAL
Disinfectants effective against the agents in use must be available at
all times within the areas where the biohazardous material is handled
or stored
effective concentrations and contact times must be used
all contaminate materials must be decontaminated before disposal or
reuse
efficacy monitoring of autoclaves used for decontamination with
biological indicators
must be
done regularly
Rayonnement
- Danger
- Radiation(i.e. consider weekly,
depending on the frequency of use of the autoclave)
records of these results and cycle logs (temperature, pressure) must
also be kept on file
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Biological Safety Training – Certificate Holder and User Training
4. Standard Operating Procedures
Working in CL 1 areas
ACCIDENTS
All spills, accidents (needlesticks, punctures, cuts, etc.) and overt or
potential exposures must be reported in writing to the laboratory
supervisor as soon as circumstances permit.
must file two reports:
Ryerson University’s Internal Accident Investigation Report filed
with the Centre for Environmental Health and Safety Management
(CEHSM) within 24 hours of incident
Workers’ Compensation Form (WSIB Form 7) form must be sent
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to Human Resources
with 24 hours of incident.
Appropriate medical evaluation, surveillance, and treatment must be
sought and provided as required. Actions taken to prevent future
occurrences should be documented.
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Biological Safety Training – Certificate Holder and User Training
4. Standard Operating Procedures
Working in CL 2 areas
In addition to the safety precautions outlined for Containment Level 1
laboratory additional operational practices are outline below:
biological safety cabinets must be used for procedures that may
produce infectious aerosols and that involve high concentrations or
large volumes of biohazardous material.
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appropriate door sign must be posted outside each laboratory
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Continue on to:
Biological Safety Cabinets
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4. Standard Operating Procedures
Biological Safety Cabinets
Biological Safety Cabinets (BSC) provide an effective means
of physical containment for biological agents, especially
when aerosols are generated.
The main role is to provide protection to personnel, and the
environment and product.
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4. Standard Operating Procedures
Biological Safety Cabinets
Protection is achieved through the control of air
movement within and prior to leaving the cabinet, and
through the use of HEPA (high-efficiency particulate
air) filtration
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- Danger - Radiation
HEPA filters
are designed
to remove particles with
a minimum size of 0.3 microns with an efficiency of
99.97%.
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4. Standard Operating Procedures
Laminar Flow Cabinets
Laminar flow cabinets (LFC) are similar in appearance, but
are not Biological Safety Cabinets:
•only protect the product
• intake room air which is passed through a pre-filter and a HEPA
filter to remove contaminants, dust and other particles
•purified air then enters the work surface in a laminar flow (nonturbulent) which is directed out of the cabinet or down into intakes
• these cabinets provide product protection only and must not be
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used when working with any form of biohazard
•sometimes referred to as Clean Benches.
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4. Standard Operating Procedures
Biological Safety Cabinets
Cabinet Certification
BSCs must be certified annually or after cabinet has
been moved
laminar flow hoods should be certified every second
year
 undertaken to ensure the HEPA filter has not be
damaged, leaking or plugged.
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- Danger - Radiation prior to any
 cabinet should
be decontaminated
certification activity.
HEPA filters & cabinets must be decontaminated prior to
disposal
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4. Standard Operating Procedures
Biological Safety Cabinets
The effectiveness of a BSC is dependent upon:
the integrity of the cabinet – (the risk of exposure
increases if the integrity is jeopardized)
location of cabinet in a room
proper microbiological technique and work practices
that do not disturb established airflow velocity and cause
reverse currents
that can -re-introduce
contaminants into
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the work area
continued maintenance and certification.
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4. Standard Operating Procedures
Biological Safety Cabinets
Before using the cabinet:
Ensure BSC is certified
Disinfect work surfaces with
disinfectant
Place essential items inside
cabinet
Allow the blower to run for 5-10
min before work
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4. Standard Operating Procedures
Biological Safety Cabinets
During use of a Biological Safety Cabinet:
ensure material and aerosol-generating
equipment is placed near the back of the hood
do not block any vents
use techniques that reduce splatter and aerosols
general work flow should be from clean to
contaminated areas
minimize movement so as not to impede air flow
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4. Standard Operating Procedures
Biological Safety Cabinets
After completion of work:
Leave blower on at least 5
minutes to purge cabinet
Remove and decontaminate
equipment and materials
Disinfect cabinet surfaces
Turn off blower and fluorescent
lamp, turn on UV lamp
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4. Standard Operating Procedures
Biological Safety Cabinets
Flames in Biological Safety Cabinets
The use of flame to sterilize equipment within a BSC is controversial. An
open flame in a BSC creates several major problems:
Flame creates turbulence, which disrupts the pattern of air supplied to
the work surface, therefore reducing maximum efficiency.
Heat build-up inside the BSC that may damage the HEPA filters
Creates a fire hazard.
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- Danger
Radiation
It is recommended
that flame
should-not
be used in BSC's.
Alternatives to flames include using disposable sterile inoculating
loops and needles, or pre autoclaved equipment in sterile packaging.
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4. Standard Operating Procedures
Biological Safety Cabinets
Flames in Biological Safety Cabinets
If a flame is required, a touch-plate micro-burner equipped with a
pilot light to provide a flame on demand may be used.
This device will minimize internal cabinet air disturbances and
heat build-up.
During use, the heat source should be placed to the rear of the
workspace where resulting air turbulence will have minimal effect.
- Danger
An emergencyRayonnement
shut off valve
should- Radiation
be placed just outside the
BSC gas supply line and during the use of any burner, all
combustible materials and solvents must be removed.
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4. Standard Operating Procedures
Biological Safety Cabinets
Ultraviolet Lamps:
Intended to destroy microorganisms in air or on exposed surfaces
Have limited penetrating power and only effective when properly
cleaned, maintained
Dust is attracted to the lamps which reduces the transmission of
the germicidal effect
Have a limited life span – even if the blue-violet glows, the lamps
are not effective if the terminal ends are blackened even slightly
Rayonnement
- Danger - hazards
Radiationsince UV light can be
UV lamps themselves
are potential
harmful to the eyes and skin and should therefore be turned off
when work is being conducted in the cabinet.
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4. Standard Operating Procedures
Biological Safety Cabinets
Maintenance of a Cabinet:
Twice daily - Work surfaces wiped down
Weekly - UV lamp should be wiped clean
Monthly - All vertical surfaces wiped down
Annually - certification
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Human Blood and Body Fluids
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4. Standard Operating Procedures
Human Blood and Body Fluids
blood borne pathogens, which are microorganisms that are
present in blood and bodily fluids and are capable of causing
disease in exposed individuals
exposure to human blood and bodily fluids (e.g., semen,
cerebrospinal, amniotic,), tissue cultures, or organ cultures
increases the risk
pathogens of greatest concern are hepatitis B virus, hepatitis C
virus and the Human Immunodefiency Virus (HIV)
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4. Standard Operating Procedures
Human Blood and Body Fluids
RISK OF EXPOSURE DEPENDENT ON:
pathogen involved
type of body fluid
route of exposure
duration of exposure
volume of blood involved in exposure
concentration of virus at time of exposure
PPE worn
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4. Standard Operating Procedures
Human Blood and Body Fluids
UNIVERSAL PRECAUTIONS:
The minimum standard of practice to prevent exposure to blood
borne pathogens and includes:
•
•
•
•
education
personal protective equipment
hand washing
safe work practices
Universal precautions assumes the material to be infectious and
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required to use control measures to prevent skin and mucous
membrane exposure.
These precautions must always be used when handling blood or
body fluids.
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4. Standard Operating Procedures
Universal Precautions
1. Gloves should be worn when handling potentially contaminated
surfaces.
2. Avoid touching items that are NOT contaminated when gloves are
being worn.
3. PPE should be worn during procedures that are likely to generate
droplets of blood or bodily fluids.
4. Contaminated lab coats and gloves should be removed immediately
after procedureRayonnement
is completed.- Danger - Radiation
5. Hand washing is the most important preventative tool - hands should
be washed immediately after gloves are removed and before leaving a
work area.
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Needle Stick Injury Prevention
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4. Standard Operating Procedures
Needle Stick Injury Prevention
Injuries from sharps (needles, syringes, etc.)
may result from:
• lack of training on proper work practices
• crowded work conditions
• incorrect recapping of non retracting
needles
• poor disposal
practices
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[risk of injury exists not only to the user, but also
support staff such as caretaking or hazardous waste
disposal personnel]
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4. Standard Operating Procedures
Needle Stick Injury Prevention
Should a needle stick injury occur it is important
to report it immediately and seek a medical
assessment.
Such injuries must not be ignored - potential
Rayonnement - Danger
- Radiationcould develop
laboratory associated
infections
without appropriate treatment.
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4. Standard Operating Procedures
Needle Stick Injury Prevention
To avoid needle stick injury use:
•
•
•
•
•
needle-less techniques where possible
retractable sharps
fill syringes carefully
do not bend, or recap needles
Rayonnementsharps
- Dangercontainers
- Radiation
approved designated
should be
used for disposal that will not allow penetration or
direct access to sharps
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Continue on to:
Signs and Labels
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4. Standard Operating Procedures
Signs & Labels
Biohazardous or infectious materials fall under;
Class D, division 3 of WHMIS
(Poisonous and Infectious
Material - Biohazardous Infectious
Material)
Class 6.2 of Transport Canada
Transport of Dangerous Goods
Act (Infectious Substances)
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4. Standard Operating Procedures
Signs & Labels
ENTRANCES
Biohazard signs must be posted on doors to rooms
where biohazardous materials are used (Level 2 or
greater)
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4. Standard Operating Procedures
Signs & Labels
Biohazard labels must also be placed on containers, equipment and storage units
(e.g., fridges, freezers) used for biological agents.
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4. Standard Operating Procedures
Signs & Labels
INSIDE LAB
(posted
prominently)
Internal Ryerson Biosafety Certificate
Current list of authorized users
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Personal Protective Equipment
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4. Standard Operating Procedures
Personal Protective Equipment
•
•
•
•
Lab coat (buttoned)
Disposable gloves
Safety glasses
Footwear
Responsibility of both the user and the supervisor to
ensure that PPE is appropriately worn
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4. Standard Operating Procedures
Personal Protective Equipment
Personal protective equipment (PPE) is only effective if
correctly selected, fitted, used and cared for, and the
individual is trained
Criteria for consideration
• routes of exposure that need to be blocked
• degree of protection offered
• specific to each level of containment
Ensure PPE is removed before leaving the lab.
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4. Standard Operating Procedures
Personal Protective Equipment
Lab Coats/Gowns
long-sleeved, knee length
buttoned or ideally with snaps
periodic cleaning required
lab coat should be worn in lab area only
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4. Standard Operating Procedures
Personal Protective Equipment
Gloves
nitrile & vinyl for work with biological agents
avoid latex gloves if possible
consider tensile characteristics, length of cuff
remove gloves when walking in hallways
double glove or do not touch uncontaminated
items in lab when wearing gloves
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4. Standard Operating Procedures
Personal Protective Equipment
Safety Glasses
safety glasses or goggles to protect the eyes
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4. Standard Operating Procedures
Personal Protective Equipment
Footwear
closed toed shoes should always be worn
sandals or open toed shoes are not allowed in a
biohazard lab
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Continue on to:
Purchasing/Transfer
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4. Standard Operating Procedures
Purchasing
ALL PURCHASES OF CL 2 BIO AGENTS MUST BE
APPROVED BY BSO PRIOR TO PURCHASE
Only Certificate Holders or designated authorized users
may purchase biological agents
Import permits required from United States for certain animal
or human pathogens
Records of all purchases must be maintained for inspection
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4. Standard Operating Procedures
Transfer
WITHIN UNIVERSITY
prior BSO approval to ensure appropriate containment
available
unauthorized lending or borrowing of biohazards not
permitted between labs
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4. Standard Operating Procedures
Transfer of Human Cells/Tissue
OUTSIDE UNIVERSITY
prior BSO approval for lending or borrowing of biohazards from other
institutions before arrival on campus
valid Research Ethics Board (REB) approval is required prior to the transfer of
material
receiving institution must have appropriate containment
materials must be properly packaged and documentation
inventory must be adjusted
if material is being received by Ryerson, then a copy of the other
institution’s Research Ethic Board approval is also required
Online applications for Research Ethic Board approval is available at:
http://www.ryerson.ca/about/vpresearch/ethics.html
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4. Standard Operating Procedures
Inventory Control
Certificate Holder required to use Inventory control
Form for all biological agents
Inventory records must be kept up to date and available
to the BSO for inspection
Types of biological organisms should be kept current
with the list noted in the Biosafety Certificate
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Changes require a revision to the Biosafety Certificate
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Continue on to:
Decontamination & Waste
Disposal
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4. Standard Operating Procedures
Decontamination
There is no universal decontamination method
for biological materials
A spectrum of chemical and physical methods
for decontamination exist
Environmental and other factors can influence
efficacy
There is a specific vocabulary for
decontamination
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4. Standard Operating Procedures
Decontamination Vocabulary
• Decontamination
• Disinfection or sterilization of contaminated surfaces
and/or articles to make them suitable for use
• Disinfectant
• An agent, usually chemical, that inactivates viruses or
kills vegetative microbes but not necessarily resistant
forms such as spores
• Sterilization
• Act or process (physical or chemical) that destroys or
eliminates all forms of life, especially microorganisms
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4. Standard Operating Procedures
Decontamination
laboratory bench tops, other surfaces, and all
equipment contaminated with biohazards must be
decontaminated prior to reuse, servicing, transfer or
disposal.
procedures must be in place to ensure the effectiveness
of the methods used to decontaminate items.
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4. Standard Operating Procedures
Chemical Decontamination
Choice depends on:
• Type of material to be disinfected
• Organic load
• Chemical characteristics
Most common are chlorine
compounds and alcohols (broad
range)
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4. Standard Operating Procedures
Chemical Decontamination
•Chemicals include:
2% -10% domestic bleach (hypochorite solution)
70% Ethanol
Quaternary ammonia
6% formulated Hydrogen peroxide
10% formalin
2% glutaraldeyhye
phenolic compounds
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4. Standard Operating Procedures
Physical Decontamination
Heat:
• Autoclaving (most practical and recommended)
• Incineration (for disposal of sharps and tissues)
Irradiation:
• UV light (wavelength of 253 nm is germicidal)
• Gamma (disrupts DNA and RNA)
Filtration
• HEPA (biological safety cabinets, ventilation)
• 0.2 micron (physically removes particulates )
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4. Standard Operating Procedures
Waste Disposal -Autoclaves
sterilization is a process which
results in the total destruction of all
living and viable organisms (with a
probability of 1 in 1 million that 1
organism survived)
sterilization is achieved through
autoclaves
autoclaves utilize both pressure and
high temperatures to provide an
effective way of sterilizing items
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4. Standard Operating Procedures
Waste Disposal -Autoclaves
Items that CAN be autoclaved:
cultures and stocks of infectious material
culture dishes and related devices
discarded live and attenuated vaccines
contaminated solid items (petri dishes,
eppendorf tips, pipettes, gloves, paper towels)
Items that CANNOT be autoclaved:
chemical or radioactive waste
certain kinds of plastics
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4. Standard Operating Procedures
Waste Disposal -Autoclaves
Items waiting for autoclaving are placed in
specially designated biohazard waste
containers containing autoclave bags
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4. Standard Operating Procedures
Waste Disposal -Autoclaves
PREPARATION OF WASTE:
Use only approved autoclave bags.
Do not overfill autoclave bags
Separate material for re-use from that which will be disposed and dry
from liquid material .
If outside of bag is contaminated, double bag.
The autoclave bags must not be tightly closed when placed inside the
autoclave.
Steam must penetrate inside the containers and circulate freely in
order for complete sterilization to occur
All flasks containing biological material should be capped with
aluminum foil.
Effectiveness of decontamination from a steam autoclave depends
on appropriately loading items into the autoclave.
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4. Standard Operating Procedures
Waste Disposal -Autoclaves
after materials have been autoclaved, the bags must be tightly
sealed and placed inside unmarked green or black regular waste
garbage bags
garbage bags are then sealed by laboratory personnel and placed
in a designated area away from the biohazard area for regular
garbage disposal by caretaking staff
Disposal
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4. Standard Operating Procedures
Waste Disposal
Treated waste (through autoclaving or other effective
method of sterilization) is no longer considered
biohazardous and can be disposed in the regular waste
stream
ALL biological waste must be treated prior to
disposal (including level 1 agents).
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Biological Safety Training – Certificate Holder and User Training
4. Standard Operating Procedures
Waste Disposal –External
Contractor
Any waste that cannot be treated (i.e. sharps, tissues)
remains biohazardous waste and must be disposed off site
using a licensed waste disposal contractor.
Contaminated materials destined for disposal must be placed
in clearly marked red or orange biohazard bags and sealed.
If using an external contractor to dispose of non autoclaved
waste materials, please contact the Biological Safety Officer
to assist in registration of waste materials with the Ontario
Ministry of the Environment
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Continue on to:
Large Scale Work
Return to SOP Main Menu
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Biological Safety Training – Certificate Holder and User Training
4. Standard Operating Procedures
Large Scale or High Concentrations
Containment levels based on laboratory scale operations:
increase in containment may be required if working
with high concentrations or shifting to manipulations
involving >10 litres
increase in the amount of potentially infectious
material may require additional safety precautions
and/or increase in containment
risk assessment should determine whether there is an
increase in aerosol generation
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