RED DEER COLLEGE, SCIENCE DEPARTMENT

Biosafety Containment Level 2

Standard Operating Procedures

10/7/2011

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Contents

HANDLING OF PATHOGENS IN A CONTAINMENT LEVEL 2 LABORATORY ............................. 3

1. INTENT................................................................................................................................................ 3

2. DEFINITIONS ...................................................................................................................................... 3

3. ROLES AND RESPONSIBILITIES ......................................................................................................... 4

3.1 Faculty members/Laboratory Supervisors ..................................................................... 4

3.2 Waste Generators ............................................................................................................... 4

3.3 Staff/Students .................................................................................................................... 4

4. LABORATORY BIOSAFETY GUIDELINES ........................................................................................... 5

4.1 Working in a Biological Containment Level 2 Laboratory ............................................... 5

4.2 Guidelines of CL2 Laboratories ............................................................................................ 5

5. PROCEDURES....................................................................................................................................... 6

5.1 Personal Protective Equipment (PPE) ............................................................................... 6

5.2 Working in a Biological Safety Cabinet (BSC) .................................................................... 6

5.3 Avoiding the Production of Aerosols ................................................................................... 7

5.4 Decontamination And Disposal Of Biohazardous Waste .................................................. 8

5.5 Spill Procedures ...................................................................................................................... 9

6. EXPOSURE CONTROL PLAN ............................................................................................................ 10

APPENDICES ................................................................................................ 11

Application Form for a Biosafety Operating Permit ....................................................................... 12

Biosafety Resume for Authorized Workers ...................................................................................... 13

Inventory ................................................................................................................................................ 14

Guidelines for Cleaning Equipment or Area Destined for Release .............................................. 15

Equipment / Area Release Form ....................................................................................................... 16

Culture Tracking Form ........................................................................................................................ 17

Autoclave Efficiency Monitoring ........................................................................................................ 18

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HANDLING OF PATHOGENS IN A CONTAINMENT LEVEL 2 LABORATORY

1. INTENT

This Biosafety Standard Operating Procedure (SOP) applies to all faculty, staff, and students

at Red Deer College. The standards and practices listed in this SOP apply to all containment

level 2 biological laboratories. It was developed to ensure that work with biohazardous

materials is conducted in a safe manner reflecting best practices and adheres to the

Laboratory Biosafety Guidelines published by the Public Health Agency of Canada.

2. DEFINITIONS

Containment Level 2 (CL2) Laboratory

A laboratory that handles Risk Group 2 agents. The primary exposure hazards associated with

organisms requiring the CL2 are through the ingestion, inoculation and mucous membrane

route. Level 2 agents are not generally transmitted by airborne routes, but care must be

taken to avoid the production of aerosols or splashes. Primary containment devices such as

Biological Safety Cabinets are to be used, as well as appropriate Person Protective

Equipment. All persons entering the facility must be informed of the hazards present.

Notifiable Biological Substances: Genetically modified micro-organisms or genetic

combinations which are not known to occur naturally

Permit Holder: A Principal Investigator or Faculty Member or Laboratory Supervisor who is authorized to work with Biological Substances and/or Biohazardous Materials by the Biosafety Advisory Committee

Risk Group 1 Organisms

Any biological agent that is unlikely to cause disease in healthy workers or animals. These

organisms are referred to as low individual and community risk as they are unlikely to cause

disease in healthy workers or animals.

Risk Group 2 Organisms

Any pathogen that can cause human 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 preventive measures are available, and the risk of spread is limited. They are referred to

as moderate individual risk, low community risk.

* For a list of organisms and their Risk Group go to http://lois-laws.justice.gc.ca/eng/acts/H-

5.67/page-20.html#h-24

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3. ROLES AND RESPONSIBILITIES

3.1 Faculty members/Laboratory Supervisors

Faculty members and lab supervisors are responsible for ensuring that:

their work areas are compliant at all times with this SOP, RDC Biosafety and Health,

Safety and Wellness policies, and applicable federal and provincial health, safety, and

environmental regulations.

they register and/or obtain the required permits for any activities that use Risk Group

2 substances, biohazardous material, and notifiable substances

staff and students have been given adequate safety orientation, site specific training

for the work assigned, and instruction on the potential hazards associated with

working with biological agents

staff and students are competently supervised and have reviewed the department

policies and procedures for safety

staff and students have access to any hazard information on the substances being used

staff and students are provided with the appropriate safety equipment and personal

protective equipment necessary to protect their health and safety, and that they know

how to use and maintain the equipment

biohazardous agents have been disposed of properly

3.2 Waste Generators

Waste generators are any persons who generate biohazardous waste as a result of work

conducted, including laboratory supervisors, faculty members, technologists, and students.

They are responsible for:

collecting the waste in accordance with RDC’s disposal procedures (see 5.4.3)

ensuring that waste is properly segregated, identified, and labelled for disposal

keeping an inventory of the waste

3.3 Staff/Students

Staff and students working in labs are responsible for ensuring that they:

conduct their work in a safe and responsible manner in order to protect their health

and safety as well as that of others who may be affected by their acts or negligence

are familiar with the potential hazards and this SOP as it relates to working with

biohazardous agents

promptly report any known accidents/incidents, spills, or unsafe conditions to their

supervisor

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4. LABORATORY BIOSAFETY GUIDELINES

4.1 Working in a Biological Containment Level 2 Laboratory

The most important element of containment is strict adherence to standard microbiological

practices and techniques. Persons working with infectious agents or infected materials shall

be aware of potential hazards and shall be trained and proficient in the practices and

techniques required for safely handling such material.

A CL2 laboratory is suitable for work involving agents of moderate potential hazard to

personnel and the environment. Working in a CL2 laboratory involves:

working under the supervision of personnel that have training in handling pathogenic

agents

having controlled and limited access to the laboratory

taking extreme precautions with contaminated sharp items

special procedures when there is a possibility of infectious aerosols or splashes being

created

Persons who are at increased risk of acquiring infection, or for whom infection may have

serious consequences are not allowed in the laboratory. The laboratory instructor/supervisor

has the final responsibility for assessing each circumstance and determining who may enter or

work in the laboratory.

4.2 Guidelines of CL2 Laboratories

all guidelines of general laboratory safety must be followed (see Biology Laboratory

Safety Procedures)

all personnel must be trained and understand the hazards

appropriate personal protective equipment must be worn

personal items (e.g. coats, purses, backpacks) must remain outside the laboratory

doors to the laboratory must be closed at all times, and locked when the lab is not

occupied

hands must be washed after removing gloves and before leaving the lab

work surfaces must be disinfected before work begins and after it is completed

all mobile equipment must be decontaminated before being removed from the area

aerosol production is to be minimized

all contaminated or infectious material must be decontaminated before leaving the

area

use of cell phones is prohibited

access to laboratories is limited to authorized personnel only

hazard warning signs must be posted

efficacy monitoring of autoclaves using biological indicators must be done regularly

and records of those results and cycle logs must be kept on file

all accidents/incidents, spills, or unsafe conditions must be reported in writing

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leak proof containers are to be used for the transport of infectious materials within

the facility

in order to release a laboratory from CL2, complete decontamination of work surfaces,

fridges, incubators, and equipment must be completed

5. PROCEDURES

5.1 Personal Protective Equipment (PPE)

a lab coat, long pants, and shoes that cover the entire foot are to be worn at all times

while working in the laboratory

gloves must be worn at all times while working with biohazardous materials. Under no

circumstance should they be worn outside the lab, and must be disposed of in the

appropriate biohazard waste container

lab coats must remain in the lab. Under no circumstance should they be worn outside

the lab, until they have been properly decontaminated

5.2 Working in a Biological Safety Cabinet (BSC)

Biological Safety Cabinets (BSCs) are among the most effective and the most commonly used

primary containment devices in laboratories working with infectious agents. They provide

protection to the worker as well as to the work being performed.

All procedures must be performed carefully to minimize the creation of aerosols. Procedures

that create aerosols must be conducted in a Biological Safety Cabinet.

Operation of a BSC:

Before using the cabinet:

if the room is occupied, do not turn on the UV light. If the UV light is used, ensure

that it is turned off before continuing.

disinfect work surfaces with Oxivir Tb.

place required items inside the cabinet

turn on the blower and allow to run 5 minutes

use of an open flame in the BSC should be kept to a minimum.

After completion of work:

allow the blower to run for 5 minutes in order to purge the BSC

remove and decontaminate materials and equipment

disinfect work surfaces

turn off the blower and fluorescent light, and turn on the UV light if room is not

occupied

For more detailed instructions on using a BSC, refer to Operation and Maintenance Manual

(located in drawer 1430-31).

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5.3 Avoiding the Production of Aerosols

5.3.1 Centrifuge Operation

If a microorganism is not contained within a centrifuge, aerosols can be produced during the

centrifugation process. This could occur with uncapped samples, or when a leak, spill or

breakage of the tube occurs. Minimize the risks when centrifuging by following the guidelines

below:

use unbreakable tubes

avoid overfilling the tubes

use caps or stoppers on centrifuge tubes

use outer, sealable safety cups that can be loaded and unloaded in a BSC

decontaminate the outside of the cups or buckets before and after use and inspect

seals regularly for deterioration

ensure that the centrifuge is properly balanced

do not open the lid during or immediately after operation

allow the centrifuge to come to a complete stop before opening

5.3.2 Mixing Operations

Sonicators, shakers and homogenizers can generate aerosols during operation. Minimize the

risks when mixing by following the guidelines below:

use sealed vessels during mixing and allow the vessels to settle before opening

open mixing vessels inside a BSC

check the condition of the mixing equipment routinely for deterioration

disinfect all exposed surfaces before and after use

5.3.3. Vacuum and Aspirating Equipment

Minimize the risk of generating aerosols during vacuum and aspiration operations by following

the guidelines below:

use non-breakable equipment (i.e. do not use glass)

ensure that vacuum equipment is fitted with a HEPA filter

place a disinfectant in the overflow flask of the aspirating equipment

5.3.4 Needles and Syringes

Minimize risk while using needles and syringes by following the guidelines below:

perform all operations with needles and syringes in a BSC

fill syringes carefully to avoid frothing or introducing air bubbles

use blunt-end needles for removal or introduction of fluids through small apertures in

equipment

dispose of needles and syringes into appropriate sharps container

5.3.5 Pipettes

Minimize risk while using pipettes by following the guidelines below:

use cotton-plugged pipettes

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keep pipettes upright while in use and between steps to prevent contamination of the

mechanical suction device and work surface

gently expel the contents of the pipette close to the surface of the liquid to allow to

flow down the side of the container

do not mix contents of a container by alternating suction and expulsion with the

pipette

“to deliver” (TD) pipettes are recommended vs. “to contain” (TC) pipettes so the last

drops do not need to be expelled for an accurate measurement

TC pipettes are to be used in the BSC due to the potential for aerosol production

submerge used non-disposable pipettes horizontally in disinfectant solution to avoid

forcing out the any liquid remaining in the pipette

5.3.6 Transfer Loop

Minimize risk while using transfer loops by following the guidelines below:

when disinfecting the loop between inoculations, hold the loop close to, but not in the

flame until the material has charred and then fully insert into the flame

substitute an enclosed micro-incinerator for an open flame burner or disposable loops

where possible

5.4 Decontamination And Disposal Of Biohazardous Waste

5.4.1 Chemical Disinfection

Chemical disinfectants are used for the decontamination of surfaces and equipment that

cannot be autoclaved, any item for which heat treatment is not feasible, and for cleanup of

spills of infectious materials. Oxivir Tb may be used for disinfection.

5.4.2 Autoclave

Infectious laboratory wastes (Petri dishes, pipettes, culture tubes, glassware) can be

effectively decontaminated in an autoclave.

Operation of an Autoclave

turn the autoclave on and allow to warm up for ½ hour

place items in the chamber. Bags must be unsealed and must be placed in leak proof

autoclavable trays

place an indicator strip in appropriate area of the autoclave chamber

close door

choose the appropriate cycle depending on the items and the size of the load

press start

when the cycle is finished, open the door carefully to avoid escaping steam

wearing heat resistant gloves, remove items and place “decontaminated “ labels on

any items to be placed for general garbage pick-up

Efficiency testing must be performed on the autoclave on an on-going basis. See the

Autoclave Efficiency Monitoring form in the appendix.

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For more detailed information on the operation of the autoclave refer to Operator Manual

(located in instructors’ desk, room 1430).

5.4.3 Disposal

All biohazardous wastes must be disposed of in the appropriate labelled containers.

contaminated sharps (needles and syringes, broken glass, microscope slides)

must be placed in rigid, puncture resistant, leak-proof, closable sharps

containers labelled with WHMIS approved biohazard symbol

contaminated disposable non-sharps (Petri plates, culture tubes, gloves, paper

towels) must be placed in bags labelled with WHMIS approved biohazard symbol

contaminated non-disposable items (instruments, glassware) must be placed

into appropriately labelled containers

After proper disposal, contaminated items must be autoclaved or otherwise

decontaminated in a timely manner.

5.5 Spill Procedures

5.5.1 Inside a BSC

remove gloves inside the BSC and leave the area for at least 5 minutes

wear lab coat, safety glasses, and gloves during clean-up

place contaminated reusable items in autoclave bags or trays

cover spill with absorbent paper towels and soak with disinfectant (Oxivir Tb). Allow

at least 10 minutes contact time

place towels and any other disposable items in autoclave bag

wipe down work surfaces, walls, and equipment in the BSC with disinfectant (Oxivir

Tb)

allow the BSC to run 10 minutes before resuming work or turning it off

5.5.2 Outside a BSC

clear area of all personnel, and wait at least 30 minutes for the aerosol to settle

remove contaminated clothing and shoes and place in a biohazard bag

put on a lab coat, safety glasses, and gloves

cover spill with absorbent paper towels and soak with disinfectant (Oxivir Tb). Allow

at least 10 minutes contact time

decontaminate all items within the spill area

place paper towels and any other disposable items in an autoclave bag

5.5.3 Reporting

all spills must be reported on an Incident/Injury Report Form. This form can be found

on the Red Deer College website, www.rdc.ab.ca, Log in to the Loop, click on Quick

Access and then Forms Index.

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6. EXPOSURE CONTROL PLAN

The Permit Holder shall develop a written Exposure Control Plan (ECP) for situations where

any student or worker is required to handle, use, or produce hazardous or infectious

materials. This plan shall:

identify any student or worker at the place of employment who could be exposed to

hazards in the workplace

describe the ways in which material can enter the body and the risks associated with

that entry

describe the signs and symptoms of any illness that may arise from exposure

describe (referencing this SOP) the safety control measures and their limitations,

including:

o administrative safety procedures

o procedural safety controls and training

o engineering safety controls and training

o PPE (include training & maintenance)

o immunization in accordance with the University Immunization Policy and

o the use of Universal Precautions when handling blood and body fluids.

describe (referencing this SOP) emergency response procedures for:

o spills or leaks of hazardous materials

o when a worker has been or believes they have been exposed to a hazardous

material

describe (referencing this SOP) methods of cleaning, disinfecting, or disposing of

clothing, PPE or other equipment if contaminated with hazardous materials, and

indicate who is responsible for carrying out those activities

describe the training that will be provided to students or workers and the means by

which this training will be provided

require the investigation and documentation of any work-related exposure incident,

including the route of exposure and the circumstances under which the exposure

occurred

require the investigation of any occurrence of an occupationally acquired illness or an

occupationally transmitted infectious disease in order to identify the route of

exposure and to implement measures to prevent further illnesses

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APPENDICES

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Application Form for a Biosafety Operating Permit

Name of Applicant:

Position:

Department:

Office Phone #:

E-mail :

Project Description:

Attach a brief summary of the experimental procedures including an inventory of the names of any biohazardous materials or notifiable biological substances used indicating their Risk Group Level.

Identify what species the agent could infect, comment on its virulence, hosts, vectors, routes of infection, etc.

State what potential impact the biohazardous materials or notifiable biological substances may have on the health and safety of personnel, the public and the environment. Develop a written Exposure Control Plan for situations where a student or worker is required to handle, use, or produce biohazardous or infectious materials.

( ) Check this box to confirm that you will be working in an appropriate containment facility and using the appropriate microbiological procedures as set forth in the "Laboratory Biosafety Guidelines" (Health Canada). If NOT applicable, check ( ).

Attach any permit (import/export) restrictions or conditions associated with this work.

Work Location and Containment Level:

Room #s:

Outdoor locations identified if applicable:

Agent Storage Locations (i.e. freezers etc):

Containment Level Assigned to area: Level 1 ( ) Level 2 ( ) Level 3 ( ) Level 4 ( )

Do you have a BSC: Yes ( ) No ( ) Other Containment Required: Yes ( ) No ( )

Cabinet Model Number: Serial Number: Date Last Tested:

Is there controlled access to the work area?: Yes ( ) No ( )

Authorized Workers: Attach a resume form for each worker and permit holder.

I accept responsibility for the accuracy of the information in this application and ensure that

this project will be performed in accordance with the RDC Biosafety Standard Operating

Procedures.

Signature of Applicant: _______________________________ Date: __________________

Approval of the Biosafety Officer: ______________________ Date: __________________

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Biosafety Resume for Authorized Workers

(PLEASE PRINT OR TYPE ALL OF THE FOLLOWING INFORMATION)

Personal Information:

Name: ________________________________ Department: _________________________

E-mail: ______________________________ __ Room: _____________

Phone: ________________________________

Education and Experience:

Degree(s), Diploma(s) & Position: ___________________________________________________

_________________________________________________________________________________

Number of years worked with biohazardous materials or notifiable biological substances:

_____

Training:

List any formal Biosafety courses taken (Date, Length, Location):_________________________

_____________________________________________________________________________________

Please verify the following:

have attended the RDC biosafety course

principles and practices of biological exposure/infection controls are followed

have read the Laboratory Biosafety Guidelines (Health Canada) or watched the Lab Biosafety 101 DVD

know the biological effects of exposure (signs & symptoms) and the ways it can occur

information (or PSDS) is available on the biohazardous materials or notifiable biological substances used understand the function/use and maintenance of biosafety cabinets and personal protective equipment (PPE) understand the emergency procedures that are in place for exposure, accidents, incidents, and spills with a written recording and follow up system

will not remove Risk Group 2 organisms from the Containment Level 2 labs

have read the Red Deer College Biosafety Policy and the SOP on Level 2 Containment

understand the Right to Know the hazards in the workplace under the Health and Safety Legislation

understand the Right to Refuse unsafe acts under the Health and Safety Legislation

I have read the Biosafety Standard Operating Procedures and understand and accept my duties

and responsibilities in accordance with the Biosafety Standard Operating Procedures.

Signature of Authorized Worker: ________________________________________

Date: _______________________________________

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Inventory

Biological Material or Agent Host Ranges Containment/ Risk Group Level

2.

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Guidelines for Cleaning Equipment or Area Destined for Release

Objective

The objective of the Equipment or Area Release guidelines are to protect the health and

safety of all staff, students, and the public at large from being exposed unnecessarily to

equipment or areas that may contain hazardous biological substances. It is hoped that the

person requesting service in an area or on a piece of equipment will take the time to evaluate

the associated risks from the point of view of the service technician who will be completing

the work.

Biological Substances

The minimum for cleaning any area or equipment contaminated with biological agents is for

the technical staff in the laboratory to wipe down the area or equipment with a disinfectant

that is effective on that biological substance (Oxivir TB). Equipment shall be autoclaved if

this is a feasible option.

Personal Protective Equipment and Precautions

Personal protective equipment and precautions shall be taken in handling equipment or upon

entering a certain area. Ask technical staff what you should wear when handling or entering

the designated area to work. Always protect your eyes and hands. Wash your hands and tools

upon completion of the work.

Consultation

The Office of Health, Safety and Wellness will provide consultation if there is any unresolved

safety concerns.

Examples

Equipment that may require decontamination and the use of personal protective equipment

are:

biosafety cabinets

fume hoods

autoclaves

incubators

refrigerators

water baths

spectrophotometers

vortex mixers

sonicators

shaker units

centrifuges

pipette pumps

lab coats

Areas that may require decontamination and the use of personal protective equipment are:

laboratories

prep rooms

fume hood /BSC discharge areas

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Equipment / Area Release Form

Supervisors, Technologists, and Faculty members releasing equipment or areas for service

work, relocation, disposal, or resale which may contain biohazardous residue within or on the

surface are required to complete an Equipment /Area Release Form. This form may be

attached to the equipment or given to the service employee.

Room #: __________________________

Description of Area /Equipment: ___________________________________________________

Model #: _________________________

Serial #: (attach list if necessary) __________________________________________________

Destination:

( ) Service ( ) Relocation ( ) Disposal ( ) Area turnover

Comments: ______________________________________________________________________

I declare that the equipment /area specified above has been cleaned and decontaminated so

that it does not present any hazard associated with biohazardous substances.

Signature: __________________________________________

Date: ______________________________________________

Phone #: ___________________________________________

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Culture Tracking Form

Culture:

Date Acquired:

Subculture Date Batch # Purpose Initials Disposal Date Initials

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Autoclave Efficiency Monitoring

Daily: Clean sediment screens; blow down boiler; Bowie-Dick Test

If Bowie Dick Test fails, perform Vacuum Leak Test

Weekly: Biological Control testing

Bowie-Dick Test (SMART

Pack)

Biological (Biosign

Biological Indicators)

Bowie-Dick Test (SMART

Pack)

Biological (Biosign

Biological Indicators)

Date Date