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Basic Biosafety Safety in Experi


									   Basic Biosafety:
Safety in Experiments
  Environmental Health and Safety
          October 2009
  Environmental Health and Safety
EHS has been charged with ensuring the safety of
  this campus. We work to:
 Ensure that biological research is conducted in a
  safe fashion
 Ensure that biological research meets regulatory
 Support the Institutional Biological Safety
  Committee (IBSC)
     Biosafety means Practicing Safe
   Ask Questions BEFORE you start
   Plan safety into your experiment
   Biosafety manuals and UA manual – each lab should
    have these and provide training on their contents
   Good lab practices
   Hazard Communication – you must notify anyone who
    enters your area of risks
   Reduce risks to acceptable levels
   Concern for others and the environment – we have an
    obligation to protect the public and the environment
                   Biohazard Symbol
   Universal Symbol
   Communicates potential
   Typically red or orange
   Symbol should be defaced when
    hazard is no longer present
   Use sparingly, explicitly
       Use for cultures of pathogens
       Human blood, tissue
       Equipment used with above
       Storage areas of above
       Cages of infected animals
       Door into laboratory
              Risk Assessment
Risk assessments on lab procedures should be done
  before initiating them. They should answer the
 What are the hazards?
 What might happen?
 How likely is it to happen?
 How serious are the consequences if it happens?
 What are the possible exposures?
 How can I mitigate exposure?
 What is the WORST that can happen?
 Remember! Familiarity affects your perception of risk!
      Tools for Risk Assessment
   Risk Groups
   Biosafety Levels
   Guidance Documents
                  Risk Groups
   Based on transmissibility, invasiveness, virulence
    and lethality of the specific pathogen
   RG1 – not associated with disease
   RG2 – associated with disease that is rarely
    serious or for which there is treatment
   RG3 – serious or lethal human disease with
   RG4 – serious or lethal human disease with no
    treatment options
              Biosafety Levels
   Correlates to Risk Groups
   More commonly used on campus
   Describes containment practices, equipment,
    and facility design features recommended for
    safe handling of these organisms
                   Biosafety Level 1
   Class 1 agents are not associated with disease in healthy
    adult humans
   However, increased precautions may be appropriate
    when using these agents for transfection since foreign
    genes can be delivered even though disease is not
   Some examples include:
       E. coli nonpathogenic laboratory strains
       Saccharomyces cerevisiae
       Agrobacterium tumefaciens
       Baculovirus
       Duck hepatitis B virus
        Biosafety Level 1
“Good Microbiological Techniques”
   No eating, drinking, applying makeup, etc.
   No mouth pipetting
   Safety glasses worn
   Lab coats stay in lab
   Wash you hands
   Safe handling of sharps
   Decontaminate cultures and waste
   Laboratory access limited when work is in progress
                 Biosafety Level 2
   Class 2 agents are associated with human disease which
    is rarely serious and for which preventative or
    therapeutic interventions are often available.
   Some examples would include:
       E. coli, pathogenic strains
       Adenovirus
       Herpes simplex virus
       Chicken pox
       Moloney murine retrovirus, amphotropic
                Biosafety Level 2
   BSL 1 practices in effect
   Biohazard or restricted access sign on door
   Door closed – negative air pressure
   Limit/restrict access to laboratory
   Minimize aerosols
   Biosafety cabinet for aerosol control
   PPE required: gloves, lab coats, respirators in some
   High degree of precaution with sharps
   Decontaminate surfaces and equipment
                 Biosafety Level 3
   Class 3 agents are associated with serious or lethal
    human diseases for which preventative or therapeutic
    interventions may be available (high individual risk but
    low community risk)
       Francisella tularensis
       Human immunodeficiency virus
       Histoplasma capsulatum
       Mycobacterium tuberculosis
   Aerosol is a common route of transmission, and
    increases the risk potential for these agents
              Biosafety Level 4
   Class 4 agents are likely to cause serious or
    lethal human disease for which preventive or
    therapeutic interventions are not usually
    available (high individual risk and high
    community risk)
     Ebola virus
     Herpes B virus (Cercopithecine)

     Lassa fever virus
Risk    BSL Level      Examples                   Practices                 Facilities,
Group                                                                       Equipment

1       Basic          Basic Teaching,            Good Microbiological      None required; open
        BSL 1          Research                   Techniques (GMT)          benchwork,
                                                                            directional airflow

2       Basic          Primary health services,   Level 1 plus protective   Open bench plus
        BSL 2          diagnostic, teaching,      clothing, biohazard       Biological Safety
                       public health              signage                   Cabinet (BSC) for
3       Containment    Special diagnostic,        Level 2 plus special      BSC and/or other
        BSL 3          research                   clothing, controlled      primary containment
                                                  access                    devices for all
4       Maximum        Dangerous pathogen unit    Level 3 plus airlock      Class III BSC or
        Containment                               entry, shower exit,       positive pressure
        BSL 3                                     special waste disposal    suits, double door
                                                                            autoclave, filtered air
             Recombinant DNA
   As an institution receiving research funds from the
    National Institutes of Health, we are subject to the
    NIH Guidelines for Research Involving Recombinant DNA
   All recombinant DNA experiments (rDNA) at the
    University of Alabama must be reviewed by the
    Institutional Biological Safety Committee
   All recombinant DNA work must be approved before
    work begins
   Protocols must be reviewed periodically
     Applicable Sections of the NIH
   III-D, IBSC review prior to initiation
     BSL2 or higher microbes as vectors
     Introduction of rDNA into BSL 2 or higher
     Large scale rDNA production (>10L)

   III-E, IBSC notification
     Most BSL1 research
     Many plant experiments
     Many transgenic rodent experiments
     Exempt from NIH Guidelines
            Section III-F
A common misconception is that low risk research is
  exempt from the Guidelines; in fact, exemptions are
  limited and specific.
 S. cerevisiae and some E. coli K-12 hosts
 Viral genome (<1/2) in tissue culture
 Sequencing and PCR
 DNA propagated solely in the species
 Gene transfer between species known to exchange
  DNA by physiological means
 Does not present risk, as determined by the NIH Director
    NIH Guidelines - Compliance
   “As a condition for NIH funding of rDNA research,
    institutions shall ensure that such research conducted at
    or sponsored by the institution, irrespective of the
    source of funding, shall comply with the NIH
   UA receives funding from NIH, so all research here
    must comply with the NIH Guidelines.
   Noncompliance may result in suspension, limitation, or
    termination of NIH funds at the institution.
Some Available References for Risk
   Assessment and Lab Safety

              Laboratory Biosafety
              Manual, 3rd edition

             World Health Organizatio
             Geneva 2004
        Pathogen Characteristics
We must think of the characteristics of a pathogen in order to
  determine how best to contain it.
 Ecotropic pathogens are less risk than amphotropic pathogens
  because amphotropic can infect humans.
 Some pathogens are carried by vectors (e.g., mosquitoes and
 Survivability in the environment varies greatly among pathogens,
  from minutes to years.
 Virulence can also vary greatly, even among different strains of
  the same microbe.
 Some pathogens produce toxins, many do not.
 Whether a pathogen stays local in the host or goes systemic
  (spreads to other organs) greatly affects virulence and host
         Routes of Transmission
   Fecal - oral
   Vector – e.g., mosquito
   Mucosal (splash)
   Cuts, scratches, bites
   Aerosol, inhalation **

**Aerosol transmission tends to be the most hazardous
  and hardest to contain. We will give these some
  additional consideration
      Some Common Activities That
           Generate Aerosols
   Vortexing
   Pipetting
   Sonication
   Electroporation
   Popping tube caps
   Flame sterilizing tools
   Flow cytometry
   Centrifugation
   Infected animals
   Examples of Infectious Doses
It is the low infectious doses that make some
   diseases so dangerous to human life.
 M. Tuberculosis              10 cells
 Salmonella typhi             100,000 cells
 Listeria                     < 1,000 cells
 Cryptosporidium              <10 cells
 Rotavirus                    10-100 i.u.
 Vibrio cholera               1,000,000 cells
           Host Susceptibility
   Age
   Immune Competence
   Medication
   Nutritional Status
   Pregnancy
   Metabolic Disorders
   Malignancy
           Some Other Hazards
   Bloodborne Pathogens – for those working with
    human blood or tissue
   Mixed Hazards – for those who use chemical
    and biological materials combined
   Physical Sharps Hazard – for needles and
    broken glass
          Bloodborne Pathogens
   Universal Precautions – handle human blood,
    body fluids and tissues as if they harbor
    pathogens like HIV, Hep B, etc
   Use BSL 2 containment:
     Protect against cuts, needle stick injuries
     Use a BSC for aerosol control
     Hepatitis B vaccine recommended
     Wear gloves, lab coat, safety glasses, etc
     Decontaminate spills with approved disinfectant
     Mixed Hazards: Chemical and
     Biological – Some Lab Practices
   Door closed: negative air pressure
   Limit/Restrict access to lab
   Use of signs/labels
   Minimize aerosol production
   Containment: fume hoods for chemicals
   PPE required: gloves, lab coats, safety glasses,
    respirators in some cases
   High degree of caution with sharps
   Decontaminate surfaces and equipment
      What are considered Sharps?
   Hypodermic needles and syringes, IV needles and
    tubing, blades, etc., are regulated as medical waste
   Glassware exposed to an infectious agent must be
    managed as a sharp until it has been autoclaved.
   Sharps containers must be red in color and display the
    International Biohazard Symbol, say Medical Waste, or
    be labeled as Infectious Waste
   Sharps containers should be puncture proof. Sharps
    may not be disposed of in red bags.
        Some Protective Equipment
   Respiratory Protection
   Ventilation Devices
     Fume Hoods
     Laminar Flow Hoods

     Biological Safety Cabinets
          Respiratory Protection
   Surgical or dust mask for large particles
   Chemical masks for vapors, acids
   Filtering facepiece, air purifying respirator for

   Must be FIT TESTED by EHS for respirator
            Ventilation Devices
   Fume Hood
   Laminar Flow Hoods
   Biological Safety Cabinet
Fume Hoods
        Fume hoods protect
         workers from chemical
        Handle hazardous
         chemicals in fume hoods
         whenever possible.
Laminar Flow Hoods
            These hoods are only
             appropriate for very few
             purposes and they are
             often misused.
            Don’t work with any
             hazardous in a laminar
             flow hood.
            They protect the working
             surface only, NOT the
Biological Safety Cabinets (BSC)
                   BSCs work to protect
                    workers and material
                    from microbes.
                   Avoid working with
                    volatile chemicals in a
            Proper Use of a BSC
   Turn it on!
   Maintain constant air curtain – minimize movements
    in/out, traffic
   No Bunsen Burners
   Avoid clutter, keep grille clean
   Disinfect working surface and interior
   Certify performance annually (EHS does this!)
   Avoid extended use of UV lamps – use them with care.
 Disposal of Potentially Hazardous
 Chemical Disinfection
 Autoclave
   Aim for total destruction of target organism
     Autoclave sterilization
     Bleach - effective against microbial agents of
      diseases at a concentration of 0.1 percent
     Detergents

     Alcohol (70%)
    Factors Affecting Bleach Efficacy
   Amount of organic matter
   Exposure to light
   Age of bleach solution
   Concentration

We recommend that you make a fresh solution of bleach at least
   monthly and store it in a opaque container that has a good seal.
   Date it so you know when to toss it. It is common for bleach to
   50% or more of its efficacy when in storage for just one month.
Bleach itself is a hazard – it burns the skin and it is corrosive.
Do not autoclave a solution that has been treated with bleach. It is
   hard on the autoclave and can cause chlorine gas to be released.
Can I autoclave something then put
       it into regular trash?
   The short answer is YES – on some things:
   So what can I autoclave and dispose?
     Medical Waste
     Class 1 Agents

     Class 2 Agents that can’t be aerosolized to class 3
             Autoclavable Waste
   ADEM regulations have specific prohibitions on the
    regular trash disposal of all items bearing either an
    international symbol or any wording indicating that the
    items contain infectious waste, biohazardous waste, or
    medical waste.
   In order to dispose of treated medical waste as regular
    trash the autoclaved bag must not be red or orange nor
    contain any wording or symbols indicating that it
    contained medical waste.
   The state prohibits using an orange/red bag for
    autoclaving, then placing it into a black trash bag for
    How do I handle the collection of
   Many times the materials are
    biohazardous/medical waste until they are
    autoclaved – then they can be regular trash. If I
    can’t trash the red bags, how do I handle these?
       You should acquire outer secondary containers (ex:
        trash receptacle) and affix a biohazard symbol to the
        exterior surface. Use a black autoclave bag inside
        the secondary container. This allows the material to
        be clearly identified in the lab and still allows
        disposal of bagged material in regular trash stream.
                Autoclave Guidelines
   Equipment should continuously monitor and record temperature and
    pressure during the entire length of each cycle.
   If not so equipped, temp sensitive tape should be affixed to each bag or
   Equivalent tests can be approved by ADEM
   Effectiveness must be evaluated under a full load at least once every 40 hours
    of operation
   In any routine monitoring of autoclave performance, biological indicators or
    thermocouples should be places at the center of each load.
   Sterilizers used for waste treatment shall not be used for sterilization of
    equipment, food, or other related items.
   Each bag must be exposed to a minimum of:
        250 degrees Fahrenheit
        15 pounds of pressure
        At least 30 minutes time AT THESE CONDITIONS
         Autoclave Recordkeeping
   A written log or other means of documentation as
    approved by ADEM shall be maintained for each unit
    and shall contain the following:
       Date, time, duration, and operator of each cycle
       Approximate weight/volume of medical waste treated during
        each cycle
       Temperature and pressure maintained during each cycle
       Method utilized for confirmation of temperature and
       Dates and results of calibration and maintenance
   Written log must be maintained for three years
Purpose of biosecurity measures is to protect the
  community, prevent theft, and comply with federal and
  state regulations
CDC guidelines for even BSL 1 labs:
  “Access to the laboratory is limited or restricted at the
  discretion of the laboratory director when experiments
  or work with cultures or specimens is in progress.”
This means that everyone entering your lab should have
  approval to be there!
    Some things you can do to secure
               your lab:
   Know the people in your area - question unfamiliar
   Maintain inventory of materials and equipment
   Safeguard hazardous materials – unauthorized persons
    should not be able to access your inventory
   Limit access - everyone entering lab has approval to be
   Lock lab whenever it is unattended – do not prop lab
    doors open
   It is up to you to protect yourself, your co-
    workers, the community, and the environment!
   Good lab practices are fundamental
   Communicate about hazardous materials
   When in doubt, ASK!!!!
             How to Reach Us
   Environmental Health and Safety
       15 Research Drive
       Box 870178
       Tuscaloosa, AL 35487

   Marcy Huey
                       Last Step!
   The final step is to take a brief quiz. This will test your
    knowledge and understanding of this material.
   Print the quiz, complete it and the information sheet,
    and send them (email, fax or campus mail) :
                      Marcy Huey
                      Box 870178
                      Fax: 348-7773

   We will send you a certificate for training upon grading.
    You must get a 90% for a passing grade.
                       Quiz – page 1
1.   Name “3” things you would want to know about a pathogen that would
     help you determine an appropriate means of containment:

2.   Name “3” things that could make you more susceptible to a pathogen:

3.   Name three routes of transmission for an infectious agent:

4.   List three references to aid you in Risk Assessment and Lab Safety:

5.   Name two common laboratory activities that could generate an aerosol:
                             Quiz – page 2
6.    If you handle human blood or body fluids/tissues, what containment level should you use?

7.    List the minimum conditions a bag must be exposed to in an autoclave in meet ADEM requirements
      for Medical Waste treatment:

8.    The efficacy of bleach for disinfection depends on: (Mark all that apply)
      a. Concentration                                     d. The amount of organic material present
      b. The brand                              e. The container in which it was stored
      c. The age of the solution

9.    Which of the following can go into regular trash streams?
      a. Class 4 biological agents             c. Class 2 agents that can aerosolize to Class 3
      b. Select Agents regulated by the CDC d. Class 2 agents after being autoclaved according to
      ADEM regs

10.   Name three things you can do to protect your lab area:
                          Information Sheet
   Name: ________________________________________________________
   Date:__________________________________________________________
   Department: ___________________________________________________
   Supervisor: ____________________________________________________
   Building/Room: _________________________________________________
   Phone: ________________________________________________________
   Campus Mailing Address: _________________________________________
   Email: ________________________________________________________

   Certification Statement: I hereby certify that I have completed the training for Basic Biosafety and that I am
    submitting my quiz answers for review.

   Signature: _______________________________________
   Print: ___________________________________________
   Date: ___________________________________________

   Fax this form and quiz to Marcy Huey at 348-7773, email or send through
    campus mail to Box 870178. You should receive your certificate within 2 weeks.

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