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					     Safety in the
   Microbiology Lab
  An Introduction to Principles and
Practices at Biosafety Levels 1, 2 & 3
                 Pre-Test
Some Category A agents pose limited to
moderate risk to the laboratory worker
(BSL-2) while others (BSL-3) pose a greater
risk.
 – What does this tell you about the:
    1. ease or difficulty for bioterrorists to
    produce BSL-2 and BSL-3 agents?
    2. ease or difficulty to control BSL-2 and
    BSL-3 agents if used for bioterrorism?
           Pre-Test (con’t)
• Why is Anthrax (a BSL-2) agent
  considered a very likely biothreat agent?
• What microbiology clues would implicate
  tularemia as a bioterror event?
• How do microbiologists protect
  themselves form accidental exposure to
  pathogenic microorganisms?
     Learning Objectives
• By the end of the lesson the student will
  understand:
  – The need for and use of biosafety designations
  – Standard (or Good) Laboratory Practices
  – The basic principles and practices for working in
    Labs designated BSL 1, BSL 2 or BSL 3
  – CDC Priority Categories and the Select Agents
    Act
  – Examples of microorganisms designated by their
    Biosafety Level
Microorganism Categories
• How are microorganisms categorized?
  – By genetics to show how they are related
  – By tissues they infect to show how they
    cause disease
  – By pathogenicity and communicability
    (also known as their BioSafety Level)
Guidelines for Microorganism Use

• Besides federal law and regulations other
  guidelines exist for the use and control of
  microorganisms:
  – CDC/NIH Biosafety in Microbiological and
    Biomedical Laboratories (BMBL)
  – WHO (World Health Organization) Biosafety
    Manual
  – USDA (United States Department of Agriculture)
    protocols
Guidelines for Microorganism Use

• All the afore mentioned agencies use the same
  system to categorize microorganisms based on
  the organisms danger to the laboratory worker
  and other research personnel.

• The microbes are placed into 4 categories
  called : Biosafety Levels (BSL 1-4)
            BSL Labs
• Microbiology Laboratories are set up
  and maintained to meet a specific
  containment level. The designated
  level conveys information about
  infection potential and engineering
  controls implemented to protect
  workers.
Biosafety Levels for Infectious Agents

BSL                         Agents
 1    Not known to consistently cause disease in healthy
        adults
 2    Associated with human disease, hazard =
        percutaneous injury, ingestion, mucous
        membrane exposure
 3    Indigenous or exotic agents with potential for
        aerosol transmission; disease may have serious or
        lethal consequences
 4    Dangerous/exotic agents which pose high risk of life-
        threatening disease, aerosol-transmitted lab
        infections; or related agents with unknown risk of
        transmission
BSL = Containment
 Recommended Biosafety Level Practices*
BSL                       Practice
 1  Standard Microbiological Practices
 2  BSL-1 practice plus: Limited access, Biohazard
      warning signs, "Sharps" precautions, Biosafety
      manual defining any needed waste
      decontamination or medical surveillance policies
 3  BSL-2 practice plus: Controlled access,
      Decontamination of all waste, Decontamination
      of lab clothing before laundering,
      Baseline serum antibody analysis
 4  BSL-3 practices plus: Clothing change before
      entering, Shower on exit, All material
      decontaminated on exit from facility
     Engineering Controls by Biosafety Level
             Safety Equipment                          Facilities
BSL         (Primary Barriers)                    (Secondary Barriers)
 1    None required                        Open bench top & sink required
 2    Primary barriers = Class I or II     BSL-1 plus:
         BioSafety Cabinets; laboratory      • Autoclave available
         coats; gloves; face protection as
         needed
 3    Primary barriers = Class I or II     BSL-2 plus:
         BioSafety Cabinets; protective      • Self-closing, double-door access
         lab clothing; gloves;               • Exhausted air not recirculated
         respiratory protection as           • Negative airflow into
         needed                              laboratory
 4    Primary barriers = Class III         BSL-3 plus:
         BioSafety Cabinets or in            • Separate building or zone
         combination with full-body,         • Dedicated supply and exhaust,
         air-supplied, positive pressure     vacuum, and decon systems
         suit
CDC Categories of Diseases/Agents
• To further help in understanding which of
  the recognized agents are of highest risk,
  the CDC has also categorized micro-
  organisms and toxins based on their ability
  to be used as bioterrorist agents and on
  national security.

• This resulted in the Select Agents Act
  defining Category A, Category B, and
  Category C agents
        Select Agents Act
• Code of Federal Regulations
  – 42 CFR Parts 72 and 73
  – 42 CFR 1003
• Defines microbial agents and toxins
  based on pathogenicity, communicability
  and potential misuse
           Biological Agent
“Biological agent means any microorganism
  (including, but not limited to, bacteria, viruses,
  fungi, rickettsiae, or protozoa), or infectious
  substance, or any naturally occurring,
  bioengineered, or synthesized component of any
  such microorganism or infectious substance,
  capable of causing death, disease, or other
  biological malfunction in a human, an animal, a
  plant, or another living organism; deterioration of
  food, water, equipment, supplies, or material of
  any kind; or deleterious alteration of the
  environment.”
                          42 CFR 73.1 Definitions
                     Toxin
“Toxin means the toxic material or product of plants,
  animals, microorganisms (including, but not
  limited to, bacteria, viruses, fungi, rickettsiae, or
  protozoa), or infectious substances, or a
  recombinant or synthesized molecule, whatever
  their origin and method of production, and
  includes any poisonous substance or biological
  product that may be engineered as a result of
  biotechnology, produced by a living organism; or
  any poisonous isomer or biological product,
  homolog, or derivative of such a substance.”

                                 42 CFR 73.1 Definitions
     What are the Select Agents?
• Abrin                     •   Diacetoxyscirpenol
• Bacillus anthracis        •   Ebola viruses
• Cercopithecine            •   Lassa fever virus
  herpesvirus 1 (Herpes B   •   Marburg virus
  virus)                    •   Monkeypox virus
• Coccidioides posadasii    •   Ricin
• Conotoxins                •   Rickettsia
• Crimean-Congo                 prowazekii
  haemorrhagic fever
  virus
      What are the Select Agents?
• Rickettsia rickettsii   • Tetrodotoxin
• Saxitoxin               • Tick-borne
• Shiga-like ribosome       encephalitis
  inactivating              complex viruses
  proteins                • Variola major virus
• South American            (Smallpox virus)
  Haemorrhagic            • Variola minor
  Fever viruses             virus
                          • Yersinia pestis
           However…
• Select Agent category does NOT define
  biosafety level
• Select Agent category does NOT convey
  engineering controls or personal
  protective equipment
• Select Agent category ONLY relates
  terrorism use risk
        Bioterrorism Agents:
          Laboratory Risk
Agent               BSL     Laboratory
 Risk
B. anthracis         2    low
Y. pestis            2    medium
Brucella spp.       2/3   high
F. tularensis       2/3   high
Botulinum toxin      2    medium
Smallpox Virus       4    high
Viral Hemorrhagic    4    high
  Fever Viruses
       Category A Definition
High-priority agents include organisms that
pose a risk to national security because they:
  can be easily disseminated or transmitted from
  person to person;
  result in high mortality rates and have the
  potential for major public health impact;
  might cause public panic and social disruption;
  and
  require special action for public health
  preparedness.
      Category A Disease/Agents
•   Anthrax (Bacillus anthracis)
•   Botulism (Clostridium botulinum toxin)
•   Plague (Yersinia pestis)
•   Smallpox (Variola major)
•   Tularemia (Francisella tularensis)
•   Viral hemorrhagic fevers (filoviruses [e.g.,
    Ebola, Marburg] and arenaviruses [e.g.,
    Lassa, Machupo])
Category A Agents                                        Biosafety
                                                         Level
Anthrax (Bacillus anthracis)                             BSL-2*
Botulism (Clostridium botulinum                          BSL-2*
toxin)
Plague (Yersinia pestis)                                 BSL-2*
Smallpox (variola major)                                 BSL-4
Tularemia (Francisella tularensis)                       BSL-2*
Viral hemorrhagic fevers (filoviruses                    BSL-4
[e.g., Ebola, Marburg] and
arenaviruses [e.g., Lassa, Machupo])
 * BSL-2 when working with clinical specimens, BSL-3 under certain
 circumstances such as culturing and concentrating, or potential for aerosols.
     Category B Definition
Second highest priority agents include
those that:
  are moderately easy to disseminate;
  result in moderate morbidity rates and low
  mortality rates; and
  require specific enhancements of CDC's
  diagnostic capacity and enhanced disease
  surveillance.
    Category B Disease/Agents
• Brucellosis (Brucella species)
• Epsilon toxin of Clostridium perfringens
• Food safety threats (e.g., Salmonella species,
  Escherichia coli O157:H7, Shigella)
• Glanders (Burkholderia mallei)
• Melioidosis (Burkholderia pseudomallei)
• Psittacosis (Chlamydia psittaci)
• Q fever (Coxiella burnetii)

           (continued on next slide)
Category B Disease/Agents (cont.)
• Ricin toxin from Ricinus communis (castor beans)
• Staphylococcal enterotoxin B
• Typhus fever (Rickettsia prowazekii)
• Viral encephalitis (alphaviruses [e.g., Venezuelan
  equine encephalitis, eastern equine encephalitis,
  western equine encephalitis])
• Water safety threats (e.g., Vibrio cholerae,
  Cryptosporidium parvum)
Category B Agents                                        Biosafety
                                                         Level
Brucellosis (Brucella species)                           BSL-2*

Epsilon toxin of Clostridium                             BSL-2
perfringens
Food safety threats (e.g., Salmonella species,           BSL-2
Escherichia coli O157:H7, Shigella)•Glanders             BSL-2*
(Burkholderia mallei)•Melioidosis (Burkholderia
pseudomallei)
Psittacosis (Chlamydia psittaci)                         BSL-2*

Q fever (Coxiella burnetii)                              BSL-2*
 * BSL-2 when working with clinical specimens, BSL-3 under certain
 circumstances such as culturing and concentrating, or potential for aerosols.
Category B Agents (con’t) Biosafety
                                                         Level
Ricin toxin from Ricinus communis                        Not
(castor beans)                                           Applicable

Staphylococcal enterotoxin B                             BSL-2

Typhus fever (Rickettsia prowazekii)                     BSL-2*
Viral encephalitis (alphaviruses [e.g.,                  BSL-3 (VEE)
Venezuelan equine encephalitis, eastern                  BSL-2 (EEE,
equine encephalitis, western equine                      WEE)
encephalitis])
Water safety threats (e.g., Vibrio                       BSL-2
cholerae, Cryptosporidium parvum)
 * BSL-2 when working with clinical specimens, BSL-3 under certain
 circumstances such as culturing and concentrating, or potential for aerosols.
       Category C Definition
Third highest priority agents include
emerging pathogens that could be engineered
for mass dissemination in the future because
of:
    availability;
    ease of production and dissemination; and
    potential for high morbidity and mortality
    rates and major health impact.
    Category C Disease/Agents
•   Nipah virus
•   Hantaviruses
•   Tickborne hemorrhagic fever viruses
•   Tickborne encephalitis viruses
•   Yellow fever virus
•   Multi-drug-resistant Mycobacterium
    tuberculosis
    Category C Agents                                       Biosafety
                                                            Level
    Hantavirus                                              BSL-3**

    Nipah virus                                             BSL-3**

    Tickborne hemorrhagic fever viruses                     BSL-4

    Tickborne encephalitis viruses                          BSL-3, BSL-4

    Yellow fever virus                                      BSL-3

    Multi-drug-resistant Mycobacterium                      BSL-2*
       tuberculosis
* BSL-2 when working with clinical specimens,         ** BSL-4  under certain
BSL-3 under certain circumstances such as culturing   circumstances or require BSL-4
and concentrating, or potential for aerosols.         trained personnel
                Post Test
Some Category A agents pose limited to
moderate risk to the laboratory worker
(BSL-2) while others (BSL-3) pose a greater
risk.
 – What does this tell you about the:
    1. ease or difficulty for bioterrorists to
    produce BSL-2 and BSL-3 agents?
    2. ease or difficulty to control BSL-2 and
    BSL-3 agents if used for bioterrorism?
           Post Test (con’t)
• Why is Anthrax (a BSL-2) agent
  considered a very likely biothreat agent?
• What microbiology clues would implicate
  tularemia as a bioterror event?
• How do microbiologists protect
  themselves form accidental exposure to
  pathogenic microorganisms?

				
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