Primary and secondary containment Universit Ottawa by alicejenny

VIEWS: 14 PAGES: 111

									  Lois Sowden-Plunkett
        Sept, 2012

     Office of Risk Management
1. Challenge your
2. Establish good laboratory
3. Prevent contamination
   (you and your work)
4. Ensure research funding
   continues $$$            Because research is now
               interdisciplinary, it is now necessary
                   to retool yourself with new skills
                            and new understanding.



   A potentially infectious agent or hazardous
    biological material that presents a risk or
    potential risk to the health of humans,
    animals, plants, and the environment

viruses,                by virtue of
bacteria,               their replicative
                        properties, are
                        harmful to
biological toxins,      humans,
prions, and             animals, plants
other micro-organisms   and/or the
or genetic systems,     environment.
Recombinant DNA,
cultured cell lines,
tissues and
specimens from
human or animal
subjects, and blood
and other bodily
fluids are also
infectious unless
tested and proven
                       • found in your intestines
                       • aid in food digestion
Extensively used for
  recombinant DNA      • E.coli synthesize vitamins
                       B1,B2 & K.
                       • do not cause any harm if are
                       in limited number.
                       • deficiencies of these
                       vitamins cause many diseases
        DH5 alpha      • beware of your antibiotics
            BL 21
          XL1Blue      which can destroy your E.coli
                                           1,500 sick,
June 2, 2011                               470 have developed
                mutation of the E. coli
                                           a rare kidney failure
                 bacteria in Europe
World Health                               complication,
                                           18 dead

                                             unlabelled and/or
                                             unbranded ground
                        E-0157:H7              beef products
Sept 18, 2012
                (first reported in 1982)   available for sale from
                                             August 24 through
                                            September 16, 2012
   P. aeruginosa       Ubiquitous in the
   P. stutzeri
                        Opportunistic
   P. fluorescens
                        affects humans,
                         animals and plants

                        Survival out of host
                         (months on dry surfaces)

Risk groups          Characteristics
                                         Hepatitis B,C & HIV

                                         Don’t assume because you
                                          know the person there is no

         UPEI June 27, 2012              Don’t even assume you don’t
    300 Students at potential risk        have it.
   The testing involved a lancet        If it’s not tested, it’s not safe!
    and a receptacle for the blood.

   The lancets were single use       UNIVERSAL PRECAUTION
    only, but the receptacles were
    used by several students.
The combination of measures employed when handling
 biohazardous materials to:

  Protect personnel from exposure to infectious
  Prevent environmental release and contamination
It’s good for you,
            it’s good for the science and
                                     it’s the law!
Safety Principles:
1. Administrative controls

2. Engineering Controls

3. Practices and Procedures

4. Personal Protective Equipment

In practical terms, this means:
  Training, risk assessments, authorizations,
  certification of rooms and equipment,
  appropriated practices, health assessment
Measures employed to protect biohazardous
  materials, or critical relevant information,
  against theft or diversion by those who
   intend to pursue intentional misuse.
        (selected agents, U99, BSE)
   Physical barriers
             Buildings, doors, locks, key card access
   Psychological barriers
                          Security personnel, cameras
   Monitoring Activities
                 Patrols, monitoring by support staff
   Personnel Clearance
                Access to authorized personnel only

Biosurety is defined as the

combination of security, biosafety,

agent accountability, and personnel

reliability needed to prevent

unauthorized access.
To be safe and compliant
it is really quite easy, it’s
          all about:
1. Diligence

2. Knowing who is responsible

3. Knowing your risk

4. Practicing GMP

    (good microbiological practice)

          Senate                                                Board of Governors
     (Academic Issues)                                            (Governance /
                                                               Management Issues)

                                President & Vice-Chancellor
                                                                                                               B/G Ctte – Health Safety,
                                                                                         Administration Ctte       & Environment

 Vice-President Academic        Vice- President Research                                                             Biosafety Committee
                                                              Vice- President Resource
       and Provost                  (PHAC and CFIA                                                                  ( Assoc. VP Research,
                                   Applicant Authority)                                                            Deans, Director ORM, &

                                 Assoc. V. P. Research
     Faculties - Deans            (Delegate of V.P R.              ORM - Director
                                     & Chair BSC)


       Chairs, Principal              BSO– Corp.                ORM- BSO – Corp.                               Corporate Governance
Investigators, & Professors –         (PHAC/ CFIA                ( Assist. Director
      (BMUC Holders)                 Lead Contact)            Radiation and Biosafety)                         Academic


                                                                  ORM – BSO-Op.
                                                                (Biosafety Specialist)
Your faculty, your colleague, PS,
 PRS, HR ...

    Federal & Provincial Agencies
    Funding Agencies
So let’s get a hand on this !

  Naturally occurring……..Manipulated
It’s as easy as: 1 -2-3-4 !
   Pathogencity
   Infectious dose
   Mode of transmission
   Survival outside host and host range
   Communicability
   Immunization
   Prophylaxis / Treatment
Class D, division 3 of WHMIS
(Poisonous and Infectious Material - Biohazardous
Infectious Material)

 Risk Group   Individual   Community          Implications

                                       Unlikely to cause disease in
     1          Low          Low       healthy workers or animals

                                       Rarely cause serious human
     2        Moderate      Limited         or animal disease

                                       May cause serious disease
     3          High         Low

                                       Likely to cause very serious
     4          High         High                 disease
Mammalian Cell Lines            Recombinant DNA
Untransformed mammalian          In vitro incorporation of
  cell lines - Risk Group 1       genetic material from one
   MCF-7 (Human breast           cell into another or from
    carcinoma cell line)          one organism to another
   NIH 3T3 (Mouse
    fibroblast cell line)        Level of risk depends on:
                                 • the source of DNA being
Transformed mammalian cell
  lines – Risk Group 2
                                 • the vector
   HeLa (Human - contains
     papovavirus)                • the host

Animal (may be infectious
  without your knowledge, or
  were intentionally injected
  with a pathogen
            PRIONS                              Toxins

Includes unconventional agents,       Endotoxins are part of the
  slow viruses and prions causing
                                        outer membrane of the cell
  progressive neurological diseases
  ex CJD, BSE, Scrapie
                                        wall of Gram-negative

Resistant to destruction                Escherichia coli, Salmonella,
                                            Shigella, Pseudomonas,
Precautions:                                Neisseria, Haemophilus
   Handle tissues as Risk Group 2            influenzae, Bordetella
    or higher                                   pertussis and Vibrio
   Handle formalin-fixed tissues                           cholerae.
    and paraffin-embedded blocks
    as if still infectious            Tetrodotoxin (TTX) is a potent
   Follow up-to-date disinfection      neurotoxin with no known
    protocols.                          antidote.
    CONTAINMENT LEVEL:                measures required for
                                       handling each organism
                                       safely in a laboratory
ROOM INTEGRITY                         setting

   Laboratory locations &            Specific to the risk group
    access control                     level and amounts being
   Air Handling and directional       used
    air flow
   Work Surfaces                     CL 1, 2, 3, or 4
   Agents Used
   Lab services (water, drains,
    gas, electrical and safety)

( certification, commissioning)
   First line of defence.
   Ensures protection of personnel and immediate
    environment from exposure to the infectious
   ‘Protective envelope’ that    encapsulates   the
    infectious agent or animal.
    Petri dish, vial
    Biological safety cabinets
    animal caging equipment
   Protects the environment
    external to the laboratory from

   Includes facility design and
    operational practices          Employs:
                                   1. Directional airflow
                                   2. Air and Drain filtration
                                   3. HEPA Filtration of lab air
                                   4. Pressure differentials
                                   5. Laboratory Design
                                   6. Operational Practices

Incubator                           Fumehood


                                4 BSC


                                Dead air
   Basic laboratory
   Requires no special design features
   Biosafety cabinets are not required
    and work may be performed on the
    open bench.
   Clinical, diagnostic, research and teaching
    facilities with level 2 agents.
   May require a class I or class II biological safety
   Emergency plan
   Access controlled
Containment Level 3

   Specialized design and construction including
    commissioning and annual certification
   Research projects reviewed by a specific panel
   Standard operating procedures enforced for the safety of
    the individual and proper operation of the lab.

   Personnel – additional training and supervision.
                           Canadian Centre for Human
                                 and Animal Health in
                                      Winnipeg, Man.

   Design specifications are
    extremely stringent
   The worker is completely isolated
    from infectious material.
   personnel security clearance and
    qualifications scrutinized
Only 20% can identify the cause or event

      80% are caused by human errors
      20% are caused by equipment failure
Types of accidents causing LAIs
    Spills and sprays
                                           Vaccinia virus
    Needles
    Sharp objects and broken glass
    Bites or scratches from animals


              Attenuated – Lab Adapted Strains
           Laboratory-Acquired Infection With an
         Attenuated Yersinia pestis Strain—Chicago,
                       Illinois, 2009
    • Agent characteristics & biological material

    • Personnel supervising & personnel using the material

    • Environment: laboratory, facility, community

    • Experimental protocols & lab practices equipment

    • Equipment
      Agent characteristics & biological material

   Is this a material you have used before?
   Do you know the source and whether it has been tested for
    which agents and to prove it is non-replicating
   What are the characteristics of the material upon receipt?
   What are the implications of the manipulations you are
   Is this a material for which LAI have been reported or are
    materials of concern ( gov’t, society, etc)?
                 SOURCE OF INFECTION
                 • Microorganisms
                 • Cells and tissues
                 • Blood and body fluids
                 • Any items contaminated with the above

Associated                  SOURCE
                      HOST      ROUTE

                                     ROUTE OF TRANSMISSION
     SUSCEPTIBLE HOST                • Percutaneous inoculations
     • Immune system                 • Inhalation of aerosols
     • Vaccination status            • Contact of mucous membranes
     • Age                           • Ingestion
Vaginal Secretions
Tissue Cultures
Organ Cultures
Infected Experimental Animals
Other Bodily Fluids:
     Cerebrospinal, Amniotic, Synovial
 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
                                          Recognized Resource
                                           Designed, Research
                                            And Maintained By
ONLY IN CANADA                                 PHAC And CFIA

       Personnel supervising & personnel using the material

• your knowledge and experience
• the level of mentorship available
• New User Registration Form &
Biosafety Health Assessment Forms

   • records your knowledge level
   • what you work with
   • health status
                                       Helps identify risk
                                              factors and
                                        potential for LAIs
   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
         the individual is well trained

   Ensure PPE is removed before
    leaving the lab
           - yes they are mandatory

   Double gloving a good practice                Herpes simplex virus (HSV)
   Gloves should not be reused
   Gloves should be changed frequently
   Glove selection: latex, nitrile, rubber & vinyl
   Use the correct donning and doffing technique
        Lab Coats/Gowns
   Protect street clothing from spills
   Offer additional body protection

   Periodic cleaning required
                                                Echo virus type 9

Eyewear – need I say more !

Eye glasses, goggles, facemask

                                          Epstein-Barr Virus Infections
Facemask – prevent

How many agents do you know
that have flu-like symptoms.

                                          Influenza Virus

   Closed toe and heel shoes
   No sandals!
                                Streptococci bacteria
Minimum standard of practice for preventing the
 transmission of BBP includes:
   Hand washing
   Use safe work practice
   Wearing appropriate protective equipment

   If samples cannot be guaranteed non-infective
              …… treat as infectious!
            Environment: laboratory, facility, community

   Regulatory                                          Contain your
  RequirementS                                          biohazard

  PHAC, CFIA, EC,                                          (Primary and
Fed/Prov/Munipcial)                                         secondary

       Lab Design

                                                       Control Access
                                                       (Physical controls:
                                                       lab and inventory)
     • Experimental Protocols & Lab Practices Equipment

Experimental Protocols
• have to be researched thoroughly
• designed with safety in mind as well as research
• engage the supervisor
• protocols on-line:

                          Remember once you start the protocol
                                       you are in research mode,
                      so you better have thought of safety first!
Lab Practices
      Good Lab Practices can save your life !

So adopt then from the start RG 1 or 2 or 3
The practices should change and the level of

consciousness you exhibit should as well.

                                            Set up
                                            Leaving the lab
 What material is presently being used and/or
                               What samples are
   Location                   critical to save if
                               storage fails, and
   Expiry date                 have I identified
   Use log book

   MSDS’s

 Mandatory

  • Infectious substances
      •Diagnostic samples

                              Yes there is
                             more training
                            you have to go

                             IT’S THE LAW
   Transportation of Dangerous Goods Act: Class
    6.2 (Infectious Substances)
   PHAC/CFIA restrictions
   Ensure:
    Proper classification
    Proper packaging
    Proper labeling
    Proper documentation
    Import/Export Permits
   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
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 ORM
At Shipping & Receiving
   Verify shipment is yours, and expected.

   Inspect the integrity of the outer container, to identify if
    possible damage may have been incurred.
   If no damage suspected, transfer to lab using appropriate
   Open outer packaging (note this may require the use of a
    biological safety cabinet if the risk group requires it.)
                   Package appears damaged
    thank goodness for tdg my life just got a hole lot easier !
   If damage and breakage possible, get your spill kit for your
    biological sample ( Isn’t it handy that you did a risk

   transfer the package into a secondary container lined with
    absorbent paper (absorbent side up), close lid of container

   Transfer to a cart with 4 sides for transfer to lab.
•Open in an appropriate fashion; inspecting for leakage,
breaks etc

•Decontaminate all the areas potentially contaminated.

•Dispose of sample in the appropriate manner

•Package must be sterilized, defaced prior to disposal.

   REPORTING: If sample breach containment, inform your
              supervisor, ORM (x. 3153), and PS
Before starting any manipulations
Before leaving the lab
Whenever the integrity of your gloves is
 questioned or your 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
                                         – ABC
 Aseptic technique is a set of specific practices and procedures
performed under carefully controlled conditions with the goal of
           minimizing contamination by pathogens.

            S      Space and work flow?
                   Clean, aseptic, or sterile technique?
                   Routine, aseptic or surgical hand hygiene?
                   Instruments and supplies?
            I      Personal protective equipment?
            P      Trash: sharps, infectious waste, radioactive
            T      waste, pathology or routine waste?
                      a·sep·sis /āsepsis/
                      ·                                                  :
                          The absence of bacteria, viruses, and other microorganisms.

                      · The exclusion of bacteria and other microorganisms, typically
                      during surgery,
   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

    containersharps containers
   Mouth pipetting is prohibited.
   Never force fluids out.
   To avoid splashes, discharge the liquid down the receiving
    container wall.

   Never mix material by suction and expulsion.
   Reusable pipettes should be placed horizontally in a
    disinfectant-filled pan.
   Sterilization in an open flame may create
    aerosols which may contain viable

   Shorter handles minimize vibrations

   Disposable plastic loops are good alternatives

            Flaming produces
             aerosols so why
                  do it?
• Equipment

                 Use it &
                maintain it
                 your life
              depends on it !
   Before use
     Check centrifuge rotors &
      tubes for cracks
     Avoid Overfilling
     Place caps or stoppers properly
     Balance loads
     Use sealed buckets (safety cups) or sealed rotors

   Before leaving: ensure centrifuge achieves run conditions

   After run
     Centrifuge has to be completely stopped before opening the
     Check for spills or leaks before removing samples. Clean spills
     Allow aerosols to settle (30 min) or open in a BSC
   Operate in a BSC whenever possible. Allow aerosols to
    settle for 5 minutes before opening.
   Decontaminate after use
     Do not use glass blender jars
     Use safety blenders which can be autoclaved
Lyophilizers (used for dehydration process)
     Use glassware designed for vacuum work, ensure there
      is no damage before using
     Use vapour traps whenever possible
Cryostats, Nitrogen Storage
  Vessels, - 80 °C Freezers

Wear gloves during preparation
  of frozen sections and heavy
  gloves when accessing the
  cryostat. Decontaminate
contain HEPA filters which
remove particles (min 0.3
microns) from supply and
exhaust air with 99.97%
efficiency .
Vertical or horizontal laminar

HEPA filtered supply air only

Provide product protection
 HEPA filtered supply and               Biological
 exhaust air                             Safety
 Personnel and environment

  3 Class II + III
Before using the cabinet:
  Ensure BSC is certified
  Disinfect work surfaces with appropriate
  Turn off UV lamp; turn on fluorescent lamp
  Place essential items inside cabinet be prepared to
   work from Clean to Dirty
  Allow the blower to run for 5-10 min before work
Hand movements when
entering, within and
exiting BSC must be
slow and deliberate to
prevent disturbing air

          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.
After using the cabinet:
  Leave blower on at least 5 minutes to purge
  Remove and decontaminate equipment and
  Disinfect cabinet surfaces
  Turn off blower and fluorescent lamp, turn on UV
   Before and after each use - Wipe down work surfaces
   Weekly     - Clean UV lamp
   Monthly - Wipe down all vertical surfaces
   Annually – Verify UV lamp intensity
                - Decontamination with formaldehyde gas
                 (managed by ORM x 3153)
   Certification – is required if the cabinet was moved that
   as filter seal could have been breached
   (contact ORM x 3153)
   Decontamination:
   The destruction of microorganisms to a lower level
    such that it removes danger of infection to
   Sterilization:
   The complete destruction of all viable
   Disinfection:
   Use of agents (physical or chemical) to destroy
    harmful organisms on inanimate objects
   Heat:
    Autoclaving (most practical and
    Incineration (for disposal of sharps and
   Irradiation:
    UV light (wavelength of 253 nm is germicidal)
    Gamma (disrupts DNA and RNA)
   Filtration
    HEPA (biological safety cabinets, ventilation)
Items that CAN be autoclaved:

   Culture dishes and related devices
   Cultures and stocks of infectious material
   Discarded live and attenuated vaccines
   Contaminated solid items (petri dishes,
    eppendorf tips, pipettes, gloves, paper towels)
Items that CANNOT be autoclaved:
   chemicals (flammables, oxidizers, phenols, acids,
   chemotherapeutic or radioactive waste
   bleach (or other chlorinated products)
   certain kinds of plastics
   Sharps (not at the University of Ottawa)
   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 autoclave training
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
   Generally for disinfection rather than
   Choice depends on:
    Type of material to be disinfected
    Organic load
    Chemical characteristics
   Most common are chlorine compounds and
    alcohols (broad range)
Vegetative bacteria (E.coli,           Viruses
                                       Enveloped (HIV, Herpes)
   2% domestic bleach
   75% Ethanol                           2% domestic bleach
   Quaternary ammonia                    75% Ethanol
   6% formulated Hydrogen peroxide       Quaternary ammonia
                                          6% formulated Hydrogen peroxide*
Mycobacteria and fungi
   10% domestic bleach                Non enveloped (Hepatitis,
   75% Ethanol                            Adenovirus)
   Phenolic compounds
                                          10% domestic bleach
                                           6% formulated Hydrogen peroxide*
Spore forming bacteria                 
    (Bacillus)                            Glutaraldehyde
   10% domestic bleach                   Formaldehyde
    Glutaraldehyde
    Formaldehyde
    6% formulated Hydrogen peroxide
Discarded biological material from teaching, clinical
and research laboratories and operations is
 biomedical waste.
Includes but is not limited to:
        Animal waste
        Biological laboratory waste
        Human anatomical waste
        Human blood and body fluid waste
        Sharps
   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 of in the regular waste

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

                             *in compliance with
                             sewer use by-laws

          with H2O (1:10)
Biomedical Waste (untreated)
Mitigates the risk of:
  1. Personnel exposure
  2. Contamination
       a) sample
       b) environment      It’s you or them,
                          make your decision!
                          It’s you or it’s them, make a choice!

          What are we talking about ?
   Are you

   Did you

       I told you that
      risk assessment
       would come in
                  ◦ What is the risk?
                  ◦ What is the route of exposure?
                  ◦ Are aerosols still suspended?
                  ◦ Is the risk contained?

REMEMBER – if the risk was
 inhalation, there may not be
any evidence of an exposure
             having occurred.
   Inform all those in the vicinity.

   Restrict access and resuspending or relocation
    of particles.

   Vacate area for 30 minutes before re-entering.

   Report, sign area, seek medical assistance.
Spill response will vary depending on:
  what, where, how much, when, who

   Cover spill area with absorbent material
   Soak the spill area with an appropriate
    disinfectant (i.e. 10% bleach, Virox)
   Pour disinfectant from the outside of the
    absorbent material towards the inside
   Leave on for 20 to 30 minutes
   Pick up any broken glass (with forceps!) and
    place in a SHARPS container
   Wipe up with absorbent material
   Waste should be disposed in appropriate
    biohazardous waste container
UO template is available:


Don’t forget it must be tailored to be lab specific.
              All potential exposures
               immediately to:
               Your      supervisor /PI
               ORM         x 5892
               5411(through Protection
               Occupational Health,
No Excuses!    Disability and Leave Form
                      Purchasing & Receiving of Biological
                              PHAC, CFIA, Environment
                              Inventory Records

                      Transportation/Transfer

                              Transport Canada- TDG

          Funding Agencies , Contracts (MTA)
Require compliance to the established biosafety program
(based on federal, provincial, municipal and international
    Project Spec Form ( Project Review)
              New User Registration Form

Streamlining them, On-line migration
But they must be complete in that they must be detailed
                  EFFORT = RESULTS
     Delays are a result of incomplete submissions
   Project specific form (Tricouncil requires biosafety review)

   Describe what you are doing as it relates to your awarded

   What agents you will use ---- NO SHORT CUTS! It has to be
    what is described in your grant proposal or MTA

                         ASK YOURSELF

    Would my answers pass a Funding Agency AUDIT ?
   Biohazardous Material User Registration Form

   It incorporates: training, experience,
    proposed work details

   Biosafey Health Assessment Form ( it is for
    your own safety)
“How soon do you need it?” “You want it when?”

In order to facilitate a quick turnaround, provide:
     reference to which grant
     copies of MSDS’s
     reference documents
  REMEMBER: Suppliers may need PHAC / CFIA
  Importation permits or letters of compliance.
                                                    Time factors:
*Restrictions may exist
                                                     complete
 Material Transfer Agreement conditions            submission
 Existing Import Permit conditions                  gov’t turn
*Permits required                                   around (2-4 wk)
     (PHAC, CFIA (animal, plant, aquatic), DFAIT)
                                                     Lead time
*Facility certification                              Grant cycle
*Transportation of Dangerous Goods
*International Holidays

Biohazardous Materials
User Registration
Biosafety Health
Assessment Survey
  UOttawa webpage



Programs (left column)

                                                  You haven’t finished
                                                    training until you
                                                    take the training

                                                   It’s for your own
 Access to test on Virtual Campus is granted by
   Biosafety Compliance Specialist, requires
providing: Name, email, employee/student #
Lois Sowden-Plunkett
Ext. 3058

Pierre Laflamme
Ext. 3153

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