1

Introduction to Biosafety

Environmental Health and Safety

Objectives

Have a good working knowledge of risk groups and

containment levels

Recognize the risks associated with biohazards in the

laboratory

Use safe practices when handling biohazards

Understand the strengths and limitations of autoclaves

and disinfectants

Know how to respond to accidental exposures and spills

of biohazardous material

www.mcgill.ca/ehs/laboratory/biosafety/manual

Regulations & Guidelines

Public Health Agency of Canada (PHAC) Guidelines

Canadian Food Inspection Agency (CFIA) Standards

Environment Canada – New Substances Notification

Transport Canada – Transport of Dangerous Goods

Workplace Hazardous Materials Information System

(WHMIS)

2

RegulationsP

H

A

C

Human Pathogens

C

F

I

A

Animal Pathogens

Canadian Biosafety Standards and Guidelines

Prions

C

F

I

A

CFIA Standards

Containment Standards for Facilities Handling Plant Pests

Containment Standards for Facilities Handling Aquatic Animal Pathogens (2010)

Environment Canada

New Substances Notification Regulations

(Organisms)

Reporting mandatory for manufactured/imported microorganisms if: Not on Domestic Substances List

Research & development exemption unless: Introduced into experimental field

study without containment

CLASS AND SYMBOL CHARACTERISTICS PRECAUTIONS

Class D Poisonous and

Infectious Material

Microbiological agents (e.g.,

bacteria, viruses, fungi and

their toxins) that may cause

illness or death

Wear the recommended protective

equipment and clothing

Work with these materials in

designated areas

Disinfect area after handling

Wash hands after handling

Division 3:

Biohazardous

Infectious

Materials

WHMIS (Workplace Hazardous

Materials Information System)

3

Where to find MSDS for infectious substances?

http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/index-eng.php

http://www.inspection.gc.ca/english/anima/disemala/disemalae.shtml

Transport of Dangerous GoodsDivision 6.2 Biological Substances

Transport Canada -TDG RegulationsDivision 6.2 Infectious Substances

Packaging and labeling of biological

agents or toxins transported to

another entity

Mandatory training for all who

transport (shipping and/or receiving)

4

Definition: Biohazards Biohazards

Bacteria

Viruses

Fungi

Parasites

Plants and algae

Toxins of biologicalorigin

Biohazards

Prions

Human tissues, blood and body fluids

Animals, animal tissues and carcasses

Cell lines

Nucleic acids

Genetically modified strains & organisms

Salmonella typhi

TetrodotoxinMould spores

Formaldehyde

Ethidium bromide

5

Consequences of Lapses

Personnel exposure

Contamination of research

Environmental release

Negative public perception

You or a colleague could be exposed...

Unvaccinated

student

Not handled by

victim

Genetically

modified strain

Stored in freezer,

not used for 5

years

You or a colleague could be exposed...continued

27 students, 1

teacher infected

Sheep anatomy

experiment

Lab disorganized,

unsanitary

Dean, party

secretary relieved

of duties

You or a colleague could be exposed...

Associate

Professor

Attenuated strain

Cause of death:

hemochromatosis

-induced iron

overload

6

...your research could be exposed...

Cell culture contamination with mycoplasma

…as well as the environment...

>600 animals

culled

Loss of £3.5

millions per day

Lasted >20 days

Laboratory-Acquired Infections

(LAI)

LAI: Routes of Transmission

Direct contact (e.g. splash)

Inhalation

Ingestion

Parenteral inoculation

Indirect contact

Vectors: animal/insect bites

7

LAI: Routes of Transmission

Inhalation - aerosols

Infectious airborne particles

Large droplets (100um) settle

Small droplets respirable

5-10 microns: upper

respiratory tract

<5 microns: lungs

LAI: Routes of Transmission

Inhalation - aerosols

Some aerosol generating procedures:

homogenizing, blending, grinding

vortexing, mixing, pipetting

centrifugation

opening snap-cap tubes

animal cage changing, necropsy

streaking agar plates

spills

LAI: U.S. and Canada 2000 - 2009

Under-reported (81 cases)

34 infections confirmed

3 deaths

Most common causes:

Inoculation (10)

Skin & mucous membrane contamination (3)

Unknown (21)

Public Health Agency of Canada 2009

What to do after accidental exposure?

1. Ask for help

2. Serious injury: dial

3. Needlesticks and cuts: wash with soap and

water

4. Splashes: flush eyes, mouth, nose

911 and Security

Downtown: 514-398-3000Macdonald Campus: 514-398-7777MNI: 55-555

8

What to do after accidental exposure?

5. If required, seek medical attention:

Student Health Services

Downtown

M-F 9:00 – 16:00 Brown Bldg, Rm. 3300

514-398-6017

Macdonald Campus

M/W/Th 9:00 -15:30 Centennial Centre, Rm. CC1-124

T/F 13:00-15:30

514-398-7565

Nearest hospital or clinic

What to do after accidental exposure?

6. Notify supervisor

7. Complete Accident, Incident & Occupational

Disease Report Page 1: ‘victim’

Page 2: supervisor/safety representative

Submit to EHS (MNI Rm 778 if working at MNI)

8. Monitor for symptoms - most pathogens

have an incubation period

Accidental ExposureBloodborne pathogens

www.mcgill.ca/ehs/laboratory/ohs/bloodborne-pathogens

9

Risk Groups & Containment Levels Risk Groups

Categorization of relative hazards of infective

organisms (4 levels)

Risk classification:

Pathogenicity

Infectious dose

Mode of transmission

Host range

Effective prevention, e.g. vaccine

Effective treatment, e.g. antibiotic, antiviral PEP

Risk Groups

Risk Group 1

• Low individual risk

• Low community risk

• Unlikely to infect healthy humans & animals

Standard molecular biology E. coli K12, Lactobacillus spp., Bacillus subtilis,

Micrococcus spp., Aerococcus spp.

Risk Groups

Risk Group 2

• Moderate individual risk

• Low community risk

• Treatment/prevention available

Listeria spp., Salmonella spp., Leishmania spp., Toxoplasma spp., Ascaris

spp., enteropathogenic E. coli, cell lines

10

Risk Groups

Risk Group 3

• High individual risk

• Low community risk

• Cause serious disease, treatment/prevention usually available

Hantavirus, Bacillus anthracis, Yersinia pestis, Histoplasma capsulatum,

cultures of Hepatitis B or HIV

Risk Groups

Risk Group 4

• High individual risk

• High community risk

• Cause serious or lethal disease by casual contact

• Treatment/prevention notusually available

Marburg virus, Ebola virus, Herpesvirus simiae, Crimean-Congo hemorrhagic

fever

Saccharomyces cerevisiae

Clostridium difficile

Bovine Spongiform

Encephalopathy

Containment Levels

Minimum requirements for safe handling (4 levels) Operational practices

Safe work practices

Engineering, technical, physical: Location & access

Surface finishes & casework HVAC

Containment perimeter (windows, autoclave location, etc.)

Services (water, drains, gas, electricity, equipment)

11

Risk Groups vs Containment levels

Risk groups agent-specific

Containment levels based on:

Risk groups

Potential for aerosol generation

Quantity

Concentration

In vitro vs in vivo

RG and CL not always the same

Containment Level 1

Containment Level 1

Physical requirements:

Basic design features

Operational requirements:

Work may be done on open bench top

BSC not required, may be used for sterility

Good microbiological practices (good hygiene)

Containment Level 2

12

Containment Level 2

Additional physical requirements, e.g.

Limited access, signage, lockable doors

Resistant, non-absorptive surfaces (for disinfection)

Containment of aerosols, e.g.:

BSC

Centrifuges with sealed rotors or safety cups

Minimize environmental contamination:

Handwashing sinks

Decontamination facilities (autoclaves)

Containment Level 2+

Level 2 physical, Level 3 operational

Mandatory Standard Operational Procedure (SOP)

Entry, exit protocols

Personal protective equipment

Experimental procedures

Effective use of BSC

Safe use of lab equipment

Emergency procedures

Decontamination and waste disposal

Specific training required, must be documented

Containment Levels 3 and 4

Level 3

Respiratory protection

HEPA filtration of lab exhaust

Strictly controlled access

Level 4

Isolated facility with sealed perimeter

Positive pressure suits or Class 3 BSC

Effluent sterilization system

Risk Assessment

Biohazards

Contamination of research

Contamination of the

environment

Public perception

Laboratory associated infection

13

Human Blood and Body Fluids

May contain bloodborne pathogens e.g.

HBV, HCV, Treponema (Syphillis), HIV

Use universal precautions

HBV vaccination

Cell Cultures

Risks based on:

Source of tissue

Primary vs. characterized cell line

ATCC screens for bacteria, fungi,

mycoplasma only

Presence of pathogens: Naturally

Via contamination, recombination or

transfection

Cell Lines

May contain viral DNA sequences:

HEK 293: Adenovirus-5

COS-1, COS-7, SVEC4-10: Simian virus-40

Daudi: Epstein-Barr virus

HS-5: Human papillomavirus-16

HeLa: Human papillomavirus

RWPE-1: Human papillomavirus

Cell Cultures

Contaminants can affect:

Growth of cells

Cell metabolism

Cell morphology and swelling

Genome structure

Recombination

Yield of virus production

…

Pauwels et al. 2007

14

Recombinant Organisms

Case-by-case assessment based on

Risks associated with recipient & donor

Function of gene expressed in recombinant

Host range alteration

Replication capacity of recombinant

Capability to revert to wild type

Viability of host-vector system outside lab

Viral Vectors

Principles of Biosafety, PCIH 2004

Risk Group Virus

1Adeno-Associated virus

Baculovirus (insect cells)

2

Adenovirus

Herpesvirus (EBV)

Poxvirus (vaccinia, fowlpox)

2+ or 3Lentivirus (HIV, SIV)

Flavivirus

Animals

Risks based on type of work

being conducted

Animals can harbour infectious

organisms (natural or introduced)

Animal Use Protocols

Occupational Health Program

You are transfecting a containment level 1 cell line

with a containment level 2 vector.

What is the containment level of the transfected

cell line?

15

You are culturing small volumes of recombinant

E. coli K-12 expressing constitutively a

mammalian oncogene for injection in mice.

What factors increase the risk of this project?

Definition: Biosecurity

Prevention of theft, misuse, intentional release

Specific for facility

Includes:

physical protection (e.g. card access, locks)

personnel suitability/reliability

pathogen accountability (e.g. inventory & tracking)

incident reporting, emergency response

Definition: Biosafety

Measures employed when handling

biohazardous materials to avoid infecting

oneself, others or the environment

Engineering controls

Reduce or eliminate

hazards at source

First and best strategy

Administrative controls

Engineering Controls

Handwashing facilities

Autoclave for waste decontamination

Cleanable surfaces

Adequate lighting

Biological safety cabinets

Examples:

16

Engineering Controls

Filters for flasks, pipette tips & vacuum lines

Plasticware substituted for glassware

Gasketed blenders, homogenizers

Sealed centrifuge rotors, safety cups

Microincinerators………

Examples:

Administrative Controls

Limited access

Safety manual available and use encouraged

Insect/rodent control

Record-keeping

Medical surveillance

Training

Spill response plan

Inventory

Examples:

Administrative Controls

Inventory

Description (cell lines, organisms, vectors, etc.)

Supplier name, if applicable

Quantity and volume

Location

Date in storage

Date out of storage

Initials

Occupational illnesses in the US

BioWarfare Program (1943-1969)

Era YearsCase by million man

hours of lab work

Pre-BSC 1943-50 35

BSC 1951-59 9.1

Vaccines & BSC’s 1960-69 2

Wedum AG. J Amer Biol Safty Assn 1996;1:7-25

17

Standard Microbiological Practices

Eating, drinking, applying cosmetics,

inserting and removing contact

lenses, storing food and utensils not

permitted

Minimize splashes and aerosols

Mouth pipetting prohibited

Standard Microbiological Practices

Cover open wounds, cuts, scratches with

waterproof dressing

Work on plastic-backed absorbent mat

Transport biohazards in leak-proof containers

Use 4-sided cart

Decontaminate work surfaces daily

Decontaminate waste

Standard Microbiological Practices

Handwashing

Effective in preventing infection

When to wash?

Before starting any manipulations

When hands are obviously soiled

Whenever gloves are removed

Whenever leaving the lab

Use blunt-end needles if possible

Do not bend, shear, recap

needles

Discard immediately after use

Separate contaminated and non-

contaminated

Use puncture-proof waste

containers

Standard Microbiological Practices

Safe use of sharps

18

CFU recovered from operator’s gloves

Run Before pouringAfter pouring into

centrifuge tubes

1 0 48 000

2 0 48 000

3 0 7900

4 0 8900

Average 0 28 000

Suspension poured contained 109/ml. Flavobacterium (Burnett et al. 2005)

Standard Microbiological Practices

Safe pipetting

Use mechanical pipetting devices

Avoid mixing by vigorous suction & expulsion

Discharge liquid down side of container

Deliver as close as possible to contents

Never blow out last drop

Personal Protective Equipment

No shorts, open shoes, etc.

Use personal protective equipment

Fastened lab coat

Gloves

Eye protection

Respirators only if fit-tested

Contact EHS

McGill PPE Policy

19

Sterilization & Disinfection Definitions

Sterilization

Destruction of all microorganisms, including spores

Disinfection

Destruction of microorganisms – may not kill spores

Decontamination

Destruction or reduction of pathogens to safe level

Microbial Resistance

Prions

Bacterial spores, protozoan cysts (Bacillus subtilis, Clostridium sporogenes)

Mycobacteria(Tubercle bacterium)

Non-lipid or small viruses (non-enveloped)(Poliovirus, Coxsackievirus, Rhinovirus)

Fungi(Trichophyton, Cryptococcus, Candida, Aspergillus)

Vegetative bacteria(P. aeruginosa, S. aureus, S. choleraesuis)

Lipid or medium-size viruses (enveloped)(Herpes simplex, HBV, HIV, CMV)

Sterilization & DisinfectionPhysical Methods

Heat: dry, moist

UV light

Infrared radiation (small metal & glass items)

Microwaves (biowaste, liquids, nonmetallics)

Filtration (heat- or chemical- sensitive liquids)

Gamma irradiation (chemical-, heat-, pressure-

sensitive items)

20

Alcohols (70-85%)

Aldehydes (glutaraldehyde, formaldehyde)

Phenolic compounds (HG7)

Halogens (iodine, chlorine)

Quaternary ammonium compounds (“Quats”)

Oxidizing agents (hydrogen peroxide, ethylene oxide gas)

Sterilization & DisinfectionChemical Agents

Number and nature of microbes

Type of item to be treated

Purpose (disinfection vs sterilization)

Interactions with other chemicals

Amount of soil/organic matter

Selection CriteriaChemical Agents

Required contact time

Toxicity to cultures, humans, etc

pH, temperature, hardness of dilution water

Cost

Selection CriteriaChemical Agents

Organic Load

DisinfectantLog reduction of Listeria monocytogenes

TSB* Serum Milk

70% Ethanol 3 1 1

Sodium hypochlorite 5 1 1

Glutaraldehyde 3 3 1

Gauggel et al. 2004

Ex: Blood, sputum, milk, bedding, feed, manure

Proteins physically protect and stabilize many

microorganisms

*TSB: trypticase soy broth (soybean casein)

21

Contact Time

Extending contact time may increase

effectiveness

DisinfectantLog reduction of Mycobacteria spp.

1 min 10 min 20 min

70% Ethanol 1 4 5

Sodium hypochlorite 5 5 5

Glutaraldehyde 1 2.5 5

Gauggel et al. 2004

www.mcgill.ca/ehs/biosafety/manual/

Autoclaves Autoclaves

Hazards:

Steam

Heat

High chamber pressure

Potential exposure to

biohazards

22

Autoclaves

Mode of action: coagulation of

proteins

Effective conditions: Gravity displacement:121oC/15 psi, 15-90

mins

Pre-vacuum: 132oC/27 psi for 4-20 mins

Steam must contact material

For heat and moisture-stable material

Autoclaves

Gravity: Longer sterilization time, lower temp and pressure

than pre-vac

For dry materials (tubes, tips, pipettes, etc.) and

liquids

Pre-vacuum

Creates vacuum to pull steam into chamber, removes

air pockets

Shorter cycle, higher temp and pressure than gravity

Not all items can withstand temp or pressure

Autoclave

Do not autoclave:

Chemicals (solvents,

corrosives)

Radioactive materials

Bleach and other

chlorinated products

Plastic and glass not rated

for autoclave use

Primary & Secondary Containers

Use only approved

autoclave bags

Use secondary

containers to contain

potential spillage

Ensure that they are

autoclavable

23

Primary & Secondary Containers

Best:

Polypropylene (PP) and polycarbonate (PC)

Borosilicate glass (Pyrex)

Stainless steel

Poor:

Polystyrene (PS), polyethylene (PE) and high

density polyethylene plastics (HDPE)

Regular glassware.

Solids:

pack for steam penetration, do not close bags/containers tightly

Do not overpack autoclave bags (max 75%)

Liquids: Loosen screw caps or use self-venting

caps to avoid rupture

Do not fill more than 2/3 full

Cap open containers loosely with foil

Packing & Loading

Clean drain screen

Avoid crowding or stacking

Items must not touch top or sides

Do not place items on autoclave floor

- use racks or secondary trays

Know which cycle to use (e.g. liquid

vs dry)

Packing & Loading

Wear insulated gloves or mitts

Ensure chamber pressure gauge

reads 0 psi

“Crack” open door, wait 5-10 mins

Liquids

Wear rubber apron in addition

Beware of bubbling (explosion hazard)

Remove load, allow to cool before

handling

Unloading

24

Temperature Sensitive Tape

Indicates autoclaving temperature

reached

Markings appear at ~121°C

Markings do not prove successful

sterilization of contents

Biological Indicators

Use biological indicators, e.g.

Geobacillus stearothermophilus,

regularly (weekly) to prove that

effective cycle in use

Indicator spores killed after 15

mins at 121oC

Contact EHS for more information Virginia Tech Environmental

Health & Safety 2009

Spill Kit

Disposable protective clothing

Absorbent paper

Autoclavable container or bags

(if using autoclave)

Appropriate disinfectant (fresh)

Autoclavable (or disposable)

forceps, dustpan

Spill Response

Don’t panic

Remove contaminated clothing

Assess degree of contamination

& action needed

Allow aerosols to settle

25

Spill Response

Don protective clothing

Cover spill with disinfectant-

soaked towel

Gently pour disinfectant

around perimeter

Work disinfectant toward

centre

Let sit 30 minutes

Spill Response

Wipe down walls, equipment,

etc.

Pick up broken sharps with

forceps, place in sharps

container

Pick up (forceps, dustpan)

absorbent materials and dispose

appropriately

Spill in BSC

Leave BSC running

Follow spill response procedure

If material has gone through grill:

Pour disinfectant through grill

Allow sufficient contact time

Open drain valve, collect liquid for

disposal

Video

26

Biohazards ApplicationsMcGill Biohazards Policy*

``Prior to beginning work with

biohazardous materials, the responsible

person must complete and submit an

Application to Use Biohazardous

Materials to Environmental Health &

Safety for review and approval.``

*University Laboratory Safety Committee September 24, 2007

Biohazards Applications

Completed by PI/lab supervisor prior to

starting project

Required for research, teaching, diagnostic

activities using biohazards

Required for all activities, including Level 1

Submit to EHS for approval (~1 week)

Biohazards ApplicationsGeneral requirements for Master’s and

Doctoral theses*

“Research involving..........microorganisms, living cells, other biohazards .......... must have had the appropriate compliance certification. Copies of any certificates of compliance must be provided to the Thesis Office at the time of submission”

*Graduate and Postdoctoral Studies: http://www.mcgill.ca/gps/thesis/guidelines/general-requirements

Environmental Health & Safety

Contacts

Telephone: 514-398-4563

Fax: 514-398-8047

Email: ehs@mcgill.ca

Website: www.mcgill.ca/ehs