General Introduction to

Biosafety

General Introduction to

Biosafety

Robert Heckert, DVM, PhD, CBSP

Robert Heckert Consulting, LLC

www.safevet.com

Robert Heckert, DVM, PhD, CBSP

Robert Heckert Consulting, LLC

www.safevet.com

COURSE OUTLINE

Introduction Laboratory Associated Infections Blood-borne Pathogens Classification of Biohazards Infection/Biohazard Control Spill Response Biomedical Waste Regulations

BIOSAFETY

INTRODUCTION

WHAT IS A BIOHAZARD?

A potential hazard to humans, animals or the environment caused by a biological organism, or by material produced by such an organism

Examples:Viruses, bacteria, fungi, and parasites and their product. Blood and body fluids, as well as tissues from humans and animals.Transformed cell lines and certain types of nucleic acids .

WHAT IS BIOSAFETY?

Measures employed when handling biohazardous materials to avoid infecting oneself, others or the environment.

Achieved through; Administrative Controls

Engineering Controls Personal Protective Equipment Practices and Procedures

WHY ARE WE CONCERNED?

Potential for acquiring a laboratory-associated infection (LAI)

Contamination of the environment Contamination of research Public perception

LABORATORY ASSOCIATED INFECTIONS

LABORATORY ASSOCIATED INFECTIONS

Route of Transmission

Susceptible Host

Infection Source

Percutaneous inoculation Inhalation of aerosols Contact of mucous

membranes Ingestion

Cultures and stocks Research animals Specimens Items contaminated

with above

Immune system Vaccination status Age

Only 20% causative or defined event 80% of which are caused by human factors 20% are caused by equipment failure

Top 4 accidents resulting in infection Spillages & splashes Needle and syringe Sharp object, broken glass Bite or scratch from animals or ectoparasites

http://www.weizmann.ac.il/safety/bio2.html

LAIS

LAIS

WHO WHAT WHERE WHEN HOW

Researcher SARS Taiwan December 2003

Microbiologist West Nile Virus

United States August 2002 Laceration

Laboratory Worker

Meningococcal Disease

United States 2000 Aerosol?

Laboratory worker

Vaccinia virus United States 2004 Many ways

BLOOD-BORNE PATHOGENS

BLOODBORNE PATHOGENS (BBP) Sources

Blood Semen Vaginal Secretions Other Bodily Fluids

CerebrospinalAmnioticSynovial

Tissue Cultures Organ Cultures Infected Experimental

Animals

RISK OF EXPOSURE

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

SPECIFIC EXAMPLES OF BBPS

Hepatitis B

Hepatitis C

HIV

ISSUES TO CONSIDER

Symptoms Mode of transmission Incubation period Survival outside host Communicability Immunization Prophylaxis / Treatment

IF AN EXPOSURE OCCURS

Initiate first aid Notify your supervisor / designated person Report to hospital emergency department or

local Health Services Report incident to regulators (if necessary)

UNIVERSAL PRECAUTIONS

Minimum standard of practice for preventing the transmission of BBP includes:

Education Hand washing Wearing protective barriers Use safe work practices

If samples cannot be guaranteed non-infective …… treat as infectious!

BIOHAZARD CLASSIFICATION

BIOHAZARD CLASSIFICATION Conventional Agents Recombinant DNA Tissue Culture Animal Work Anatomical Specimens Unconventional Agents

BIOHAZARD CLASSIFICATION

Organisms are categorized into a group base on the particular characteristics of each organism, such as Pathogenicity

Infectious dose Mode of transmission Host Range Availability of effective preventive measures Availability of effective treatment

BIOHAZARD CLASSIFICATION

Organisms are categorized based on the measures required for handling each organism safely in a laboratory setting, such as Engineering Requirements

Operational Requirements Technical Requirements Physical Requirements

Risk Grou

p

Individual Risk

Community Risk

Containment

Level

Examples

1 Low Low Level 1 E.coli, B. subtilis, S. aureus, Trichoderma reesei

2 Moderate

Limited Level 2 Streptococcus & Salmonella spp, Measles, Adenoviruses, Hepatitis A, B & C, Influenza

3 High Low Level 3 Bacillus anthracis, Mycobacterium tuberculosis, HIV, Yellow fever virus

4 High High Level 4 Lassa virus, Ebola virus, Marburg virus

Unlikely to cause disease in healthy

workers or animals

Rarely cause serious human or animal

disease

May cause serious disease

Likely to cause very serious disease

CONVENTIONAL AGENTS

RECOMBINANT DNA

Genetic Engineering = in vitro incorporation of genetic material from one cell into another The level of risk depends on source of DNA, vector and host. The biosafety community may be able to assist the investigator in this determination.

TISSUE CULTURE

Have the potential to contain pathogenic organisms

In general;

Human & non-human primate, and mycoplasma-containing cell lines Level 2

Others Level 1

A detailed risk assessment should be undertaken when using a new cell

line.

ANIMAL WORK

Animals can harbour infectious organisms (naturally or introduced)

Level dependent on type of work being conducted.

Special Animal Care training is required for all personnel working with animals.

All work involving animal use must receive prior approval from the Animal Care Committee

ANATOMICAL SPECIMENS

All specimens should be considered infectious due to potential presence of infectious agents

Important to consider the type of specimen blood, organs, tissues Spinal sample, brain tissue From infectious patient

In general Level 2 but it depends on the nature of the work.

UNCONVENTIONAL PATHOGENS

TSE prion diseases; lethal transmissible neurodegenerative conditions Creutzfeld-Jakob disease, Variant C-J Disease, Mad

Cow Disease, Scrapie, Chronic Wasting Disease

Resistant to destruction by procedures that normally inactivate viruses.

Contact regulatory authorities to assess requirements (containment, procedures, waste disposal, etc.)

CONTAINMENT LEVEL 2

Clinical, diagnostic, research and teaching facilities with level 2 agents.

Requires a class I or class II biological safety cabinet if any potential for aerosol or splash exists.

An emergency plan for handling spills must be developed.

Access should be controlled.

Specialized design and construction primary barriers to protect the individual secondary barriers to protect the environment

All staff must undergo special training on the agents being used, PPE, equipment, waste management as well as practices and procedures above and beyond the scope of this course.

CONTAINMENT LEVEL 3

CONTAINMENT LEVEL 4

Only few level 4 facilities in the world Design specifications are extremely stringent,

worker is completely isolated from infectious material.

BIOLOGICAL SAFETY CABINETS

Effective means of primary physical containment for biological agents, especially when aerosols are generated.

HEPA filters remove particles (min 0.3 microns) with 99.97% efficiency.

There are 3 main classes of cabinets (I, II, III) which provide various levels of protection.

BIOLOGICAL SAFETY CABINETS

Laminar flow hoodsVertical or horizontal laminar flowHEPA filtered air (intake only)Product protection only

Biological Safety CabinetHEPA filtered laminar air flowExhaust Personnel, environment & product protection

VS

WORKING SAFELY IN A BSC

Before using the cabinet: Ensure BSC is certified Turn off UV lamp; turn on fluorescent lamp Disinfect work surfaces with appropriate

disinfectant Place essential items inside cabinet Allow the blower to run for 5-10 min before work

While using the cabinet: Ensure material and equipment is placed near the

back of the hood, especially aerosol-generating equipment. 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 Open flame in BSC’s is controversial

WORKING SAFELY IN A BSC

WORKING SAFELY IN A BSC

After using the cabinet: 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

WORKING SAFELY IN A BSC

Maintenance: Before and after each use - Work surfaces wiped

down Weekly - UV lamp should be wiped clean Monthly - All vertical surfaces wiped down Annually - UV lamp intensity verified

- Decontamination with formaldehyde gas - Certification

WORKING SAFELY IN A BSC

PERSONAL PROTECTIVE EQUIPMENT (PPE)

PPE can become an important line of defence (last line of defense)

Responsibility of both the user and the supervisor to ensure that PPE is worn

PPE

Criteria for consideration Routes of exposure that need to be blocked Degree of protection offered Ease of use

Only effective if correctly selected, fitted, used and cared for, and the individual is trained

Ensure PPE is removed before leaving the lab

PPE

Footwear Closed toed shoes should always be worn. Booties are

worn in some higher containment labs and animal facilities.

Lab Coats/Gowns Long-sleeved, knee length with snaps Elastic cuffs Back-closing gowns Periodic cleaning required

PPE

Gloves Latex, nitrile & vinyl for work with biological agents.

Exam gloves should not be reused, change frequently. Utility gloves can be disinfected and reused if they show no sign of degradation.

Consider tensile characteristics, length of cuff.

Double gloving.

BSO can provide assistance finding an alternative for people with allergies.

PPE

Eye & Face Protection Goggles, safety glasses to protect the eyes Full face shield to protect facial skin.

Respirators Only personnel who have been fit-tested and trained

should wear respirators.

PRACTICES AND PROCEDURES General Safety Guidelines Good Microbiological Practice Handwashing Suspicious Packages Specific Procedures

Centrifuges Needles & Syringes and other sharps Pipettes Blenders, Grinders, Sonicators & Lyophilizers Inoculation Loops Cryostats

GENERAL LABORATORY SAFETY GUIDELINES

Mostly common sense, but you must understand the hazards you face in the laboratory and be adequately trained to deal with them.

Basic must be known for all labs.

b i o s a f e t y

GOOD MICROBIOLOGICAL PRACTICE (GMP)

Basic code of practice that should be applied to all types of work involving microorganisms.

Objectives: prevent contamination of laboratory workers and

the environment prevent contamination of the experiment/samples

Application of aseptic technique, minimization of aerosols, contamination control, personal protection, emergency response

HANDWASHING

One of the single effective means of preventing infections if done properly and frequently

When to wash? Before starting any manipulations Before leaving the lab When hands are obviously soiled Before and after completing any task in a BSC Every time gloves are removed Before contact with one’s face or mouth At the end of the day

SAFE USE OF CENTRIFUGES

Before use Stress lines? Overfilled? Balanced? Caps or stoppers properly in place? Run conditions achieved?

Use sealable buckets (safety cups) or sealed rotors

After run Centrifuge completely stopped? Spills or leaks? Allow aerosols to settle (30 min) or open in a BSC.

NEEDLES AND SYRINGES

Avoid use whenever possible Use a BSC for all operations with infectious

material Fill syringes carefully Shield needles when withdrawing from

stoppers Do not bend, shear or recap needles. Dispose of all used needles/syringes in yellow

sharps containers

PIPETTES

Mouth pipetting is prohibited. Never force fluids out. To avoid splashes, allow discharge to run

down dispense the receiving container wall. Never mix material by suction and expulsion. Reusable pipettes should be placed

horizontally in a disinfectant filled pan.

BLENDERS, GRINDERS, SONICATORS, AND LYOPHILIZERS

• Operate in a BSC whenever possible. Allow aerosols to settle for 5 minutes before opening.

• Safety Blender Do not use glass blender jars Decontaminate immediately after use

• Lyophilizers Use glassware designed for vacuum work, ensure

there is no damage before using All surfaces should be disinfected after use Use vapour traps whenever possible

INOCULATION LOOPS

Sterilization 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

CRYOSTATS

Wear gloves during preparation of frozen sections and heavy gloves when accessing the cryostat.

Decontaminate frequently (100 or 70% Ethanol)

SPILL RESPONSE

SPILLS

Spill response will vary depending on: What was spilled? How much was spilled? Where was the spill? What is the potential for release to the

environment?

Spills should be cleaned up immediately (unless an aerosol was generated), to ensure proper decontamination.

Ensure appropriate PPE is worn and clean-up equipment is readily available.

SPILLS-GENERAL CLEAN-UP

Cover spill area with absorbent material Soak the spill area with an appropriate disinfectant (i.e.

10% bleach) Pour disinfectant from the outside of the absorbent

material towards the inside Ensure any broken glass is picked up (with forceps!)

and placed in a sharps container Leave on for 20 to 30 minutes Wipe up with absorbent material Waste should be disposed in appropriate biohazard

bags and where possible autoclaved

SPILLS-SPECIAL CASES

Within a Centrifuge Within a BSC Open Areas (lab, during transport) A spill response plan should be prepared

BEFORE the spill occurs

SPILLS

All users of biological materials should be familiar with the spill clean-up procedures.

All spills are to be reported ASAP to the lab supervisor and the BSO.

Additional assistance is available from: Your departmental safety officer

Local community services (if available)

BIOMEDICAL WASTE

DECONTAMINATION, DISINFECTION, AND STERILIZATION

Decontamination: Free of contamination, the destruction of microorganisms to a lower level such that it removes danger of infection to individuals.

Sterilization: The complete destruction of all viable microorganisms.

Disinfection: Use of agents (physical or chemical) to destroy harmful organisms on inanimate objects (not necessarily all organisms)

DECONTAMINATION: PHYSICAL

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)

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 (petrie dishes, eppendorf

tips, pipettes, gloves, paper towels)

AUTOCLAVES

Items that CAN NOT be autoclaved: chemicals (flammables, oxidizers, phenols, acids,

alkalides) chemotherapeutic or radioactive waste Bleach (or other chlorinated products) certain kinds of plastics

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 All flasks containing biological material should be

capped with aluminum foil Ensure items are labeled with contact information

SAFE USE OF AUTOCLAVES

Many autoclaves are now run by dedicated staff, however, if you are operating an autoclave; Learn how to use it! Ensure PPE is worn Recognize acceptable material and packaging Proper loading and unloading

All users/operators must take the autoclave training

DECONTAMINATION: CHEMICAL

Generally for disinfection rather than sterilization

Choice depends on; Type of material to be disinfected Organic load Chemical characteristics

Most common are chlorine compounds and alcohols (broad range)

WHAT TO USE FOR MY AGENT?

Vegetative bacteria (E.coli, Staph)

2% domestic bleach 75% Ethanol Quaternary ammonia 6% formulated Hydrogen peroxide

Mycobacteria and fungi

10% domestic bleach 75% Ethanol Phenolic compounds 6% formulated Hydrogen peroxide

Spore forming bacteria (Bacillus)

10% domestic bleach Gluteraldehyde Formaldehyde 6% formulated Hydrogen peroxide

VirusesEnveloped (HIV, Herpes) 2% domestic bleach 75% Ethanol Quaternary ammonia 6% formulated Hydrogen

peroxide*

Non enveloped (Hepatitis, Adenovirus)

10% domestic bleach 6% formulated Hydrogen

peroxide* Gluteraldehyde Formaldehyde

WASTE MANAGEMENT

Discarded biological material from teaching, clinical and research laboratories and operations is biomedical waste. Biomedical waste includes but is not limited to; Animal waste Biological laboratory waste Human anatomical waste Human blood and body fluid waste Sharps

WASTE MANAGEMENT

All biological waste should be decontaminated prior to disposal (including level 1 agents).

Treated waste is no longer considered ‘biomedical’ (i.e. microbiological waste, blood and bodily fluid waste) and can be disposed in the regular waste stream.

Any waste that cannot be treated (i.e. sharps, carcasses, tissues and body parts) remains biomedical waste and must be incinerated off site.

WASTE DISPOSAL

Biomedical Waste (untreated)

WASTE DISPOSAL

Biomedical Waste (treated)

With H2O 1:10

In compliance with Sewer use by-laws*

SPECIAL WASTE

EtBr Toxins Recombinant DNA Contact local authorities

REGULATIONS

KEY REGULATED ACTIVITIES

May be restrictions on pathogen use Waste disposal (air, land, sewer) Transportation Storage Information

INVENTORY

What material is presently being used and/or stored Location Expiry date Use log book for remaining amount MSDS’s

Mandatory

SHIPPING AND RECEIVING

Transportation of Dangerous Goods Act: Class 6.2 of (Infectious Substances)

Local country restrictions Ensure;

Proper classification Proper packaging Proper labeling Proper documentation Import/Export Permits

TRANSPORTATION OF DANGEROUS GOODS

Pre-approved Authorized Individuals Lead time (International Regulations….) Appropriate Scheduling (Holidays, Weekends) Transportation within the building Between lab to lab Colleague to Colleague Between Institutions

TRANSPORTATION

Important Considerations: does material need to be transported at all packaging requirements means and route of transportation regulatory requirements

Between lab transfers - 4 sided cart, sealed primary container, secondary container, low traffic route.

Off Campus transfers – consult your BSO

THE BOTTOM LINE

If you are not careful and diligent with biological agents you risk: Infecting yourself, others or the environment Contaminating your research Being in violation of local/national/international

regulations

Thank you!

Tina Preseau & Rita Toussaint-Archambault

University of Ottawa