4.6.2 General Safe Work Precautions
1. All workers should routinely use appropriate barrier precautions to
prevent skin and mucous membrane exposure when contact with
human blood or other body fluids is anticipated.
2. Eating, drinking, smoking, applying cosmetics or lip balm, and handling
contact lenses are prohibited.
3. Gloves shall be worn when touching blood and body fluids, mucous
membranes, or non-intact skin, for handling items or surfaces soiled with
blood or body fluids, and for performing venipuncture and other
vascular access procedures. If a glove is torn or damaged during use, it
shall be removed and a new glove used as promptly as safety permits.
Disposable gloves shall not be washed or disinfected for reuse. Washing
with surfactants may enhance penetration of liquids through undetected
holes in the glove. Disinfecting agents may cause deterioration of the
glove material.
4. Masks and protective eyewear or face shields shall be worn during
procedures that are likely to generate droplets of blood or other body
fluids to prevent exposure of mucous membranes of the mouth, nose,
and eyes.
5. Long sleeved lab coats completely buttoned or aprons shall be worn
during procedures that are likely to generate splashes of blood or other
body fluids. Protective clothing should be removed before leaving the
area.
6. Hands and other skin surfaces shall be washed immediately and
thoroughly if contaminated with blood or other body fluids. Hands shall
be washed immediately after gloves are removed since no barrier is
100% effective.
7. Workers shall take precautions to prevent injuries caused by needles,
scalpels, and other sharp instruments or devices during procedures,
when cleaning used instruments, during disposal of used needles, and
when handling sharp instruments after procedures. Needles and
syringes should beused only in those situations when there is no
alternative. To prevent needlestick injuries, needles should not be
recapped, purposely bent or broken by hand, removed from disposable
syringes, or otherwise manipulated by hand. After they are used,
disposable syringes and needles, scalpel blades, and other sharp items
shall be placed in puncture-resistant containers for disposal. The
puncture-resistant container shall be located as close to the use area
as practical.
8. Workers who have exudative lesions, weeping dermatitis, cuts, open
wounds or other breaks in the skin shall either refrain from all direct
contact with blood and other body fluids until the condition resolves,
or utilize protective barriers to reduce the risk of exposure.
9. Pregnant workers shall be especially familiar with and strictly adhere
to precautions to minimize the risk of perinatal transmission of
bloodborne pathogens.
4.7 Biosafety Signs and Labels
The Public Health Agency of Canada and the Medical Research Council (MRC)
require that warning signs and/or symbols be used to inform personnel and
visitors of the potential of hazards in the workplace. Specifically, with regard
to biohazards, the universal biohazard warning sign must be used to "signify
the actual or potential presence of a biohazard and to identify equipment,
containers, rooms, materials, experimental animals or combinations thereof,
which contain and/or are contaminated with, viable hazardous agents."
1. TRU requires that the universal biohazard symbol be used to
designate the presence of agents/substances that are believed to be
biohazardous.
2. All laboratories and work areas utilizing and/or storing biohazardous
substances must have the appropriate biohazardous caution sign
posted prominently. If infectious agents are used, a biohazard sign
must be located outside the laboratory door to indicate the nature
of the hazard, the biohazard level, special provisions for entry and
contact information for the Principal Investigator and/or other
responsible person(s).
3. Principal Investigators/Supervisors are responsible for ensuring that
all hazard signs are current and accurate. Notify the Biosafety
Officer if changes are necessary in laboratory door signage and/or
equipment labeling.
4.8 Access/Security Controls
Doors must be locked when laboratories are unoccupied and only authorized
persons are permitted to enter laboratory working areas. Children under the
age of 14 years must not be permitted to enter laboratory working areas.
4.9 Cell Culture
All new cell lines introduced into TRU must be registered with the Biosafety
Officer.
Storage and retrieval of frozen cell cultures from liquid nitrogen require
appropriate personal protective equipment. There are three major risks
associated with liquid nitrogen (-196 °C): frostbite, asphyxiation and
exposure. Gloves shall be worn that are thick enough to provide insulation,
but flexible enough to allow manipulation of ampoules.
When ampoules are submerged in liquid nitrogen, a high-pressure differential
results between the outside and the inside of the ampoule. If the ampoule is
not perfectly sealed, the pressure differential may result in inspiration of
liquid nitrogen, which may cause the ampoule to explode violently when
thawed. Wear eye protection, a face shield and earplugs are recommended
Biological safety cabinets shall be kept clean and free of unnecessary
equipment and material to ensure proper functioning of the cabinet.
Liquid waste shall be decontaminated by chemical disinfectant (e.g., 10%
hypochlorite). Vacuum collection flasks for liquid waste shall be kept outside
the cabinet in a secure place and should contain an appropriate disinfectant
.The collection flask shall also have a back-up trap to protect the central
vacuum line. Decanting shall be done to minimize splashing.
All flasks should be properly labelled.
Decontamination of the biological safety cabinet shall be done with a liberal
spray with supplied disinfectant followed by a wipe with 70% ethanol at the
end of the procedure.
Solid waste shall be placed in biological waste bags and the bags then sealed
for autoclaving. Biohazard disposal containers with lids shall be used for
primary disposal.
Glass pipets shalld be placed in a pipet container with an appropriate
disinfectant. Plastic disposable pipets must be disposed of in an appropriate
container.
Contaminated sharps shall be placed in the yellow biohazardous sharps
containers
4.10 Personal Protective Equipment (PPE)
The type and extent of clothing and equipment to be selected for any
particular procedure depend on the research operations and levels of risk
associated with them. At a minimum, a lab coat, closed-toe shoes, and gloves
must be worn in any microbiology laboratory. Lab coats, closed-toe shoes,
and gloves prevent biohazardous materials from contact with the skin,
including areas where there might be cuts, abrasions, or dermatitis. The legs
are a vulnerable area if uncovered, so it is inappropriate to wear skirts or
shorts. Closed- toe shoes protect the feet from spills as well as injuries from
dropped sharps. Soles must be non slip to avoid slips and falls,
4.10.1 Lab Coats
The lab coat protects street clothing from contamination and prevents
possible cross-contamination from any normal flora present on the skin.
Lab coats must be worn by all personnel, including visitors, trainees and
others entering orworking in the laboratory. Coats must be properly
fastened. If contaminated, lab coats shall be decontaminated by autoclaving
before being placed in the laundry. If decontamination is not possible, any
contaminated coat shall be placed in the biohazard waste container.
Lab coats are stored in area separate from street clothes.
4.10.2 Gloves
Appropriate gloves must be worn for all procedures that might involve direct
or accidental skin contact with biohazardous materials. Latex or vinyl gloves
offer a high level of dexterity and a higher level of sensitivity; however, they
don’t offer a great deal of protection from needle sticks, animal bites or
sharps. All gloves will eventually permeate and shall therefore be changed
periodically. If gloves become contaminated or torn, remove immediately
and wash hands with soap.
Some procedures may require double gloving.
Gloves should overwrap the cuff and lower sleeve of the lab coat if double
gloving is practiced
Gloves must be removed prior to leaving the laboratory and decontaminated
with other laboratory wastes before disposal. The following chart lists
preferential gloves for different products.
TYPE
ADVANTAGES
Natural rubber
latex
Low cost, good physical
properties, dexterity
Natural rubber
blends
Low cost, dexterity,
generally better chemical
resistance than natural
rubber.
Low cost, very good
physical properties, average
chemical resistance.
Polyvinyl
chloride (PVC)
DISADVANTAGES
FOR USE WITH:
Poor against oils, greases,
organic solvents, ethidium
bromide. May cause allergic
reactions.
Physical properties often
inferior to natural rubber.
May cause allergic reaction.
Bases, acids, alcohols, dilute
aqueous solutions.
Fair vs. aldehydes, ketones.
Plasticizers can be stripped.
Strong acids and bases, salts,
aqueous solutions, alcohols, o
greases and petroleum produc
Bases, acids, alcohols, dilute
aqueous solutions.
Fair vs. aldehydes, ketones.
Neoprene
Average cost, average
chemical resistance,
average physical properties,
high tensile strength, high
heat resistance.
Low cost, excellent physical
properties, dexterity
Poor vs. chlorinated
hydrocarbons
Oxidizing acids, alcohols, anilin
phenol, glycol ethers, solvents
oils, mild corrosives
Poor vs. chlorinated organic
solvents
Good resistance to polar
organics, high resistance to
gas and water vapour
Resists broad range of
organics, good physical
properties.
Expensive, poor vs.
hydrocarbons, chlorinated
solvents
Very expensive. Water
sensitive, poor vs. light
alcohols, acids and bases.
Oils, greases, xylene, aliphatic
hydrocarbons, perchloroethyle
trichloroethane, ethidium
bromide. Fair vs. toluene.
Glycol ethers, ketones, esters,
aldehydes, polar organic solve
Fluroelastomer
(Vitron®)
Good resistance to organic
and aromatic solvents.
Flexible.
Norfoil, Silver
Shield™, 4H™
Excellent chemical
resistance.
Extremely expensive. Poor
physical properties. Poor vs.
some ketones, esters,
amines
Poor fit, stiff, easily
punctures, poor grip.
Nitrile
Butyl
Polyvinyl
alcohol (PVA)
Aliphatic and aromatic
hydrocarbons, chlorinated
solvents, ketones (except
acetone), esters, ethers
Aromatics and aliphatic
hydrocarbons, chlorinated
solvents, oils, lubricants, mine
acids, alcohols.
Use for Hazmat work. Good fo
range of solvents, acids and ba
5. BIOLOGICAL SAFETY CABINETS (BSCs)
Protection of the respiratory system is of major concern in any biological
safety program because infectious organisms can readily enter the human
body through the respiratory tract. Engineering controls (biological safety
cabinets) are the primary barrier for inhalation of biohazard and should be
use whenever possible. Respirators should only be used as secondary
means of control. Any individual needing respiratory protection will be
required to participate in the Respiratory Protection Program. The BSO will
contact the Dean of Science who will ensure respirators will be made to
individuals who require such.
A biological safety cabinet is a ventilated cabinet that uses a combination of
HEPA (high efficiency particulate air) filtration, laminar air flow and
containment to provide personnel, product and environmental protection
from particulates or aerosols involving biohazardous materials. It is
distinguished from a chemical fume hood by the presence of HEPA filtration
and the laminar nature of the air flow.
The following is a basic description of the two classes of biological safety
cabinets, their capabilities and the limitations of each class.
5.1 Class I Biological Safety Cabinet
This is a ventilated cabinet which provides partial protection to the worker
and environment but no protection to the work. These cabinets have
unrecirculated airflow away from the operator that is discharged to the
atmosphere after filtration through a HEPA filter. Chemical carcinogens
and low level of radioactive materials and volatile solvents can be used in a
Class I safety cabinet.
5.2 Class II Biological Safety Cabinet
This is a ventilated cabinet that provides personnel, product and
environmental protection. These cabinets have an inward airflow and
HEPA-filtered supply and exhaust air.
A class II biological safety cabinet shall be used whenever there is a risk of
creating potentially infectious aerosols or droplets. Examples of processes
that may create aerosols are: centrifuging,
grinding, blending, vigorous shaking or mixing, opening containers of
infectious materials that are at other than ambient pressure. Use of a
centrifuge in the open laboratory may be carried out safely only if the
samples have sealed heads or centrifuge safety caps are used. However, it
is strongly recommended that centrifuging gets done in a contaminant
hood at all times.
Class II BSCs are the most commonly used BSC on campus. These cabinets
provide personnel, environmental and product protection. (See Figure 3).
Only BSCs that have hard ducts to the outside and provide a face velocity
of 80 to
125 feet per minute should be used when working with volatile chemicals.
Note: these cabinets are not designed to prevent ignition of volatile
flammable chemicals
5.3 Working in a Biological Safety Cabinet
•
Turn the cabinet on for at least 10-15 minutes prior to use, if the
cabinet is not always operating.
•
Disinfect work surface with suitable disinfectant followed by 70%
alcohol.
•
Consider the materials necessary for the work to be conducted in
the cabinet.
•
Place all required materials on an absorbent pad to avoid aerosol
generation
•
Place items into the cabinet so they can be used efficiently without
unnecessary disruption of the air flow, working with materials
from the clean to the dirty side.
•
Wear appropriate personal protective equipment. At a minimum,
this will include a buttoned laboratory coat and gloves.
•
Delay manipulation of materials for approximately one minute
after placing the hands/arms inside the cabinet.
•
Minimize the frequency of hand movements in and out of the
cabinet.
•
If it is required to move out of the BSC pull hands slowly and
horizonatally from the cabinet. Allow time for the air flow
disruption to diminish before putting hands back into the BSC
•
Do not disturb the airflow by covering any grillwork with materials.
•
Work at a moderate pace to prevent the air flow disruption that
occurs with rapid movements.
•
Wipe the bottom and side of the hood surfaces with disinfectant
when work is completed.
•
Leave the hood running for several minutes following the
procedures before turning off the blower
NOTE: BE VERY CAREFUL WHEN USING SMALL PIECES OF MATERIAL SUCH
AS KIMWIPES IN THE HOOD. THESE CAN BE BLOWN INTO THE HOOD AND
DISRUPT THE MOTOR OPERATIONS.
** THE BSC IN S365C IS T USE FOR STERILITY ONLY. FOR USE WITH
POTENTIAL PATHOGENS USE THE BSC IN S367. NO CHEMICAL USE IS
ALLOWED IN EITHER OF THESE BSCs.
THE BSC IN S363 MAY BE USED WHEN VOLATILE CHEMICALS ARE
REQUIRED TO BE USED IN THE BSC
5.4 Certification of BSC
All biological safety cabinets must be certified on installation, when the
filters are changed, before and after a move or transfer and annually.
Cabinets must not be moved without first undergoing a decontamination
process. The Biosafety Officer must approve any modifications to any
biological safety cabinets. Cabinets must undergo certification following
modification.
The certification process is arranged for annually by the BSO.
If problems are encountered in operating a biological safety cabinet, do not
continue to use it; contact the BSO immediately.
The certification of the biological safety cabinets is essential to their safe
and effective use.
6. LABORATORY EQUIPMENT SAFE OPERATING PROCEDURES
6.1 Sonicators
When used with infectious agents sonicators can release significant
amounts of hazardous aerosols, and shall be operated inside a biological
safety cabinet whenever possible.
Sonicators are devices commonly used for disrupting cells and mixing
samples. Vortexers are also used for mixing samples. The following
safety measures should be used with a sonicator to reduce the chance of
aerosol formation.
Safe Operating Procedures include:
•
Loosely cap all samples.
•
Make sure there is enough water in the sonicator.
•
Avoid prolonged sonication.
•
Inspect all glassware to be used in the sonicator. Do not use
chipped or cracked glassware.
•
Routinely replace the sonicator liquid.
•
Avoid sonicating volatile compounds.
•
When possible, use secondary containment (container within
container within the sonicator).
•
Perform sonicating in isolated rooms and areas.
•
Make sure you have adequate ear protection.
•
Allow aerosols to settle for at least one minute before opening
containers.
6.2 Centrifuges
Safe use of centrifuges requires proper maintenance and operation. Failed
mechanical parts or improper operation can result in release of projectiles,
hazardous chemicals and biohazardous aerosols. Maintenance and repairs
must be performed only by trained, qualified personnel.
Centrifuges are a source of potential biological contamination due to
the rapid speeds and relatively high pressure exerted by such devices.
The following safety measures shall be used when using any centrifuge:
Safe Operating Procedure includes:
•
Prior to starting, make sure the centrifuge is clean. Do not
operate with any material spills in either the body or the rotor.
•
Ensure that the interlocking device prevents the lid from being
opened when the rotor is in motion and the centrifuge from
starting when the lid is open.
•
Make sure the centrifuge is level. If a portable model, make
sure it is secure on the bench top before starting.
•
Inspect all equipment to be placed in centrifuge for cracks or
weak areas. Ensure that the tube material provides the necessary
chemical resistance and speed rating.
•
Use the lowest speed and time setting that will accomplish the
job.
•
Avoid over-filling tubes.
•
Balance all loads.
•
Do not open the lid until it comes to a complete stop.
•
Wait for at least one minute before opening the lid to remove
your sample.
•
Should a spill occur, disinfect immediately and dry completely
before the next run.
•
Periodically inspect centrifuge. Check seal around top, baskets,
rotors and wiring.
•
Avoid use of volatile materials when possible.
•
Plastic centrifuge tubes with seal-forming screw tops should be
used whenever possible.
•
Centrifuges should not be placed into a biological safety cabinet
if the motor produces strong air current because the air
turbulence generated may disrupt the laminar airflow.
6.3 Vacuum
If there is a vacuum system serving multiple areas, care should be taken that
there are filters in the system, and that there is an overflow trap containing
an appropriate disinfectant to prevent entry of contaminated material into
the piping system and pumps. It is often best to use either a stand-alone
pump-type vacuum system, or to use a water siphon vacuum system that is
attached to a faucet (provided that measures are taken to prevent backsiphonage).
6.4 Gas Chromatograph
Gas chromatography (GC) procedures involve the use of compressed gas
cylinders and may involve the use of flammable solvents and toxic
chemicals. Be familiar with the use and handling of compressed gas
cylinders, and with hazardous properties, precautionary measures, and
handling instructions for any hazardous materials being used. Refer to
MSDSs (found on-line – use the MSDS icon on the desktop) or other
reliable reference material. The following guidelines will assist in the safe
operation of GCs:
•
Wear proper eye protection. GC columns are fragile and
breakage could result in small projectiles during handling. As
well, samples are prepared in various hazardous solvents that
could damage the eyes upon contact.
•
When cutting a GC column, be sure that the cut is made away
from the body.
•
Ensure that GC column cutters are capped or otherwise stored
to prevent injury when not in use.
•
Discard small pieces of GC columns as sharps waste.
•
Ensure that the oven is allowed to cool before installing or
removing a column or injector or prior to performing
maintenance.
•
Ensure that gases are turned off prior to removing or installing a
column.
•
Test for leaks after the installation of the column and whenever
a leak is suspected. Use a technique that will not damage or
sacrifice the integrity of the instrument.
•
Electron capture detectors (ECD) have a radioactive source and
therefore need to be registered as part of the University’s
Radiation Safety program. Contact Ron Smith ext.5544 for more
information about Canadian Nuclear Safety Commission (CNSC)
requirements.
•
Ensure that the instrument and gases are turned off and the
power cord disconnected prior to performing maintenance.
6.5 Ultraviolet lamps
Exposure to ultraviolet light (UV) may result in serious and painful injury to
the eyes or skin depending on the wavelength and intensity of the light and
the duration of exposure.
•
Label all UV light sources conspicuously with the following
warning (or equivalent): “Warning – this device produces
potentially harmful UV light. Protect eyes and skin from
exposure.”
•
Ensure that the UV light source is shielded.
•
Ensure that appropriate PPE is worn and is sufficient to protect
the eyes and skin. PPE shall include at least UV resistant face
shield, gloves, and lab coat.
•
Shielding the equipment or the work area may be warranted.
6.6 Bunsen burners
Bunsen burners are used for heating (fixing cells onto slides) and
sterilization of loops and picks. Allow material to dry before sterilizing a loop
or pick to avoid splattering of material and creating possible aerosol. Start
flaming at top of loop or pick and move down toward the end. Do not use
Bunsen burners in BSCs.
6.7 Pipetting aids
Pipetting aids minimze the risk of creating aerosols. Dispensing liquid and
subsequent mixing with a glass pipet can create aerosols. Use filtered pipets
and pipets aidsand filtered pipet tips for micropipettors. Discharge liquids
as close to the wall of the receiving vessel as possible or to the surface of
media to avoid the creation of aerosols. Avoid forecfully aspirating oe
expelling liquid from a pipette.
6.8 Standard Operation for Autoclaves In S365
I. ASSOCIATED RISKS
Autoclaves are sterilizers using high pressure and high temperature steam. The potential
safety risks for the operators are:
•
•
Heat burns -from hot materials and autoclave chamber walls and door
Steam burns -from residual steam coming out from autoclave and materials on
completion of cycle
•
•
•
Hot fluid scalds- from boiling liquids and spillage in autoclave.
Hand and arm injuries when closing the door.
Body injury if there is an explosion
II. Equipment to protect against scalds and burns:
•
•
Heat-insulating gloves that provide complete coverage of hands and forearms.
Closed-toed footwear.
III. OPERATOR INSTRUCTIONS Training
All operators must have successfully completed an authorized training session on the
safe operating procedures of this autoclave. This requirement applies to both new and
experienced personnel. A list of authorized users will be kept with the cycle records.
Before Autoclaving can be done Ensure you do the Following Before turning on
Autoclave
•
•
•
•
•
Before turning on the autoclave, drain the blue generator by :
o opening the tap(labeled #1) at the bottom of the generator
o opening the tap (labeled #2) on the side of the autoclave facing the wall
to the lab
o allow the water to drain for 5 minutes
Close the taps that were opened to drain the generator
Turn on the power supply switch on the wall beside the autoclave (labeled
power switch)
Turn on the water supply lever labeled water supply, (it is on when it is parallel
with the water supply pipe and off when it is perpendicular to the pipe)
Allow the pressure in the jacket to come up to 15 pounds before starting the
autoclave (about 30 minutes)
Material Preparation
•
•
•
Ensure that the material is able to be autoclaved
 samples containing solvents or substances that may emit toxic fumes should
not be autoclaved.
Glassware must be inspected for cracks prior to autoclaving.
Prepare and package material suitably:
 Loose dry materials must be wrapped or bagged in steam-penetrable
paper or loosely covered with aluminum foil. Wrapping too tightly will
impede steam penetration, decreasing efficiency of the process.
 All containers must be covered by a loosened lid or steam-penetrable
bung.
 Containers of liquid must be a maximum of 2/3 full, with lids loosened.
 Glassware must be heat-resistant borosilicate.
 Plastics must be heat-resistant eg: polycarbonate (PC), PTFE ("Teflon")
and most polypropylene (PP) items.
 Items or containers must be tagged with autoclave tape to verify
sterilization.
 Loosen all lids to prevent pressure buildup.
 Add water to containers as appropriate.
•
•
Place items in containers to secure and contain spills:
 Items should be placed in a stainless steel or autoclavable plastic
container for their stability and ease of handling.
 Place containers of liquid, bags of agar plates, or other materials that may
boil over or leak, into a secondary pan in the autoclave.
 The pan must be large enough to contain a total spill of the contents.
 Bags must not be tightly sealed as steam cannot penetrate.
 Remove all labels from glassware prior to autoclaving
Biohazard materials must be labelled as such and secured in containment vessels
or autoclavable bags and processed as soon as possible according to
requirements for the handling of infectious or biohazard materials.
Loading Autoclave
•
•
•
•
•
Wear heat-insulating gloves, and closed toed shoes.
Place material in autoclave. Do not mix incompatible materials.
Do not overload; leave sufficient room for steam circulation. If necessary, place
the container on its side to maximize steam penetration and avoid entrapment
of air.
Close and latch door firmly by raising the lid gently till it clicks into place.
Do Not Let it Slam at the top as it will break the switch located there
Operating Autoclave
•
•
•
•
•
Choose appropriate cycle ( e.g. liquid, dry unwrapped or wrapped etc) for the
material.
Set appropriate temperature for the cycle (if necessary, usually all loads are
processed at 121° C
Press the start button
Do not attempt to open the door while autoclave is operating.
The manuals for operation of the autoclave are located in the cupboard adjacent
to small autoclave and under behind door panel of large autoclave
Unloading Autoclave
•
•
•
•
•
•
Wear heat-insulating gloves and closed toed shoes.
Ensure the load is complete
Wear gloves and stand back from the door as a precaution, carefully crack door
open no more than 1 inch (2.5 cm) to release residual steam and allow pressure
within liquids and containers to normalize.
Allow sterilized material to stand for 10 minutes in the chamber. This will allow
steam to clear and trapped air to escape from hot liquids, reducing risk to
operator.
Do not agitate containers of super-heated liquids or remove caps before
unloading.
After removal from the autoclave, place liquid agar in the water bath in the
media area that should be turned on before starting the load. This will allow the
media t cool to a temp ideal for pouring
Maintenance and repair
• No person shall operate the autoclave unless the autoclave is in good repair
• Users are not to make repairs
• Report possible malfunctions to Supervisor or Micro Lab Technician
IV. CONTINGENCY PLAN
Equipment malfunction
•
•
•
If the autoclave does not operate exactly as expected, do not attempt to fix the
problem. A notice shall be placed on the autoclave indicating that it is not to be
used until the problem is diagnosed and corrected.
Record the problem in the autoclave log book. Contact _Carolynne or Shannon
to report the problem.
Repair of autoclaves shall be performed by qualified persons only.