Potentially Hazardous Biological Agents (PHBA)

Does your project involve working with:

  • Microorganisms (bacteria, viruses, viroids, prions, rickettsia, fungi, mold, parasites)
  • Recombinant DNA (rDNA – this is usually in the form of DNA expressed in bacteria or yeast that the microorganism does not normally express, such as E. coli expressing human insulin; these are also known as genetically modified microorganisms)
  • Human or animal tissues (fresh or frozen, including blood and body fluids)

If you answered YES to any of these questions, please read the guidelines below for details about each topic, allowable projects, and any additional forms you may need to complete.

Also check out the Potentially Hazardous Biological Agents Biosafety Chart from ISEF

Microorganisms

  1. Potentially hazardous microorganisms are defined by ISEF as bacteria, viruses, viroids, prions, rickettsia, fungi, and parasites. Protists such as dinoflagellates, algae, and amoeba are not included in the ISEF potentially hazardous microorganism list and thus do not require PHBA forms or pre-approval.
  2. ELEMENTARY LEVEL STUDENTS ARE NOT PERMITTED to perform projects involving ISEF-defined microorganisms for North Carolina fairs. Elementary students, can, however, perform projects involving protists.
  3. Projects that involve culturing microorganisms CANNOT BE DONE AT HOME. This includes BSL1 organisms (defined below). These must be done in school laboratories or other approved institutions (hospital or clinic labs, university labs) in the presence of an approved supervisor (see below for definition). Culturing a microorganism involves placing it in an environmental situation in which you expect it to reproduce (grow). Examples of culturing projects:
    1. Swabbing doorknobs, skin, etc., and then streaking (rubbing) the swab on an agar plate or swirling it in a broth.
    2. Infecting plants with mold spores and allowing the mold to grow
  4. Approved supervisors may include (but are not limited to):
    1.  A parent (for non-culturing experiments done at home)
    2. The student's science teacher (for experiments conducted at school)
    3. A person possessing an MD, DDS, DMD, RN, NP, or other appropriate training to work in a hospital or clinic laboratory
    4. A person possessing or working towards a PhD or possessing the appropriate training to work in a university, commercial, or industrial laboratory
    5. A trained professional working at a water or sewer treatment facility
  5. Microorganism specimens meant to be cultured can be COLLECTED at home as long as they are immediately sealed and transported to an approved experimental location.
  6. Plant pathogens that are naturally occurring can be studied at home (e.g., viruses that cause leaf mottling), but these cannot be introduced to the plants by the student in a home environment.
  7. Studies involving MRSA (Methicillin-resistant Staphylococcus aureus) and VRE (Vancomycin-resistant Enterococcus spp.) are PROHIBITED.

Forms Required for Microorganism Studies

  1. These studies are exempt from prior SRC review and need no additional forms:
    1. Studies involving brewer's yeast or baker's yeast, unless they are used in a rDNA study (see below).
    2. Studies involving Lactobacillus spp., Bacillus thuringiensis, nitrogen-fixing and oil-eating bacteria, and slime mold and algae-eating bacteria introduced into their natural environments. These projects are not exempt if they are cultured.
    3. Mold growth studies on food, as long as the experiment is terminated at the first visual evidence of mold.
  2. These studies are exempt from prior SRC review and REQUIRE a completed Risk Assessment Form 3:
    1. Studies involving protists, archaebacteria, and other organisms not explicitly classified by ISEF as PHBA microorganisms
    2. Research using manure for composting, fuel production, or other experiments that do not culture microorganisms
    3. Water testing experiments using commercially available test kits that remain sealed and are properly disposed.
  3. Any studies that do not fall into these two broad categories REQUIRE a completed Risk Assessment Form 3 and a completed PHBA Risk Assessment Form 6A. These studies are also subject to prior SRC review (this MUST occur BEFORE any experimentation takes place).
  4. If the project is performed in a Regulated Research Institution setting (e.g., a university research laboratory or hospital laboratory), you must complete a Regulated Research Institution Form 1C.
  5. If the project requires supervision by a Qualified Scientist, you must complete a Qualified Scientist Form 2.

Biosafety Level Classifications for Microorganism Studies

Biosafety levels define the potential risk of harm, injury, or disease to plants, animals, and humans that may occur when working with microorganisms. This is important both to ensure that proper safety precautions are put in place and that the research is performed at the correct location with proper equipment and safeguards.

  1. Biosafety Level 1 (BSL-1): Low risk to personnel and environment. These organisms are unlikely to cause disease.
    1.  Examples: Escherichia coli K12, Agrobacterium tumefaciens (aka Rhizobium radiobacter), Micrococcus luteus, Neurospora crassa, Bacillus subtilis
    2. BSL-1 containment is found in most high school laboratories, water-testing facilities, and college microbiology class laboratories. Work can be done on the open bench. The supervisor can have general training in microbiology or a related science.
  2. Biosafety Level 2 (BSL-2): Moderate risk to personnel and environment. Disease, if it occurs, is mild and limited in spread. Effective treatments for disease are readily available.
    1. Examples: Mycobacterium spp., Streptococcus pneumonia, Salmonella choleraesuis, most viral species that cause colds (Rhinoviruses, some human Coronaviruses), and some species of Influenza.
    2. NOTE: New for 2012: Studies involving Klebsiella pneumonia must be performed in BSL-2 settings.
    3. Experiments involving human and animal waste, including sewage sludge. This does not include composted animal manure.
    4. BSL-2 containment is generally found in laboratories in hospitals, clinics, and universities. Access to the laboratory is generally restricted. Biological safety cabinets (Class 2, type A) must be available. Work should be supervised by a qualified scientist who understands the risks of working with the microorganisms involved.
  3. BSL-1 experiments can be conducted in a BSL-2 laboratory. However, BSL-2 experiments CANNOT be conducted in a BSL-1 laboratory.
  4. Biosafety Level 3 and Biosafety level 4 are reserved for organisms that cause serious diseases that are often fatal and/or untreatable. Studies involving these organisms are PROHIBITED.
  5. Experiments involving the culture of unknown microorganisms (e.g., swabbing doorknobs or skin and streaking agar plates) should be evaluated by your local SRC for biosafety level, but will most likely be considered BSL-2.
    1. An experiment involving unknown microorganisms may be considered BSL-1 if:
      1. The organism is cultured on SEALED plates (sealing can be done with Parafilm or by placing the plates in TWO 2-ply heavy-duty sealed plastic bags).
      2. PLATES REMAIN SEALED THROUGHOUT THE ENTIRE EXPERIMENT ONCE THEY ARE INOCULATED.
      3. Sealed plates are disposed of properly under the supervision of the Designated Supervisor

Safety Equipment and Disposal Procedures

  1. All PHBA must be disposed of properly at the end of experimentation in accordance with their biosafety level. Approved disposal methods include any of the following:
    1. Autoclaving at 121 °C for 20 minutes
    2. 10% bleach solution (ISEF does not specify a time, but 10 minutes of immersion/soaking in this solution is generally sufficient to disinfect)
    3. Incineration
    4. Alkaline hydrolysis
    5. Pickup by an approved biosafety removal group
    6. Other manufacturer-approved recommendations (e.g., for commercial water-testing kits)
    7. Other disposal methods must have been approved by the Institutional Review Board of the regulated research institution where the experimentation is performed. You may be required to submit documentation in this case.
  2. For BSL-1 studies, the following safety equipment and practices are required:
    1. Standard microbiological procedures, including aseptic technique.
    2. Lab coats are required, and disposable gloves are recommended.
  3. For BSL-2 studies, the following safety equipment and practices are required:
    1. An autoclave should be available for decontamination.
    2. Lab coats, disposable gloves, and face protection are required

Recombinant DNA (rDNA)

  1. All rDNA studies must be pre-approved by the SRC.
  2.  Forms required for rDNA studies must take into account the microorganism used as well as the DNA being introduced into the organism.
  3. No rDNA projects can be conducted at home.
  4. There are a few rDNA studies that can be performed in a BSL-1 environment. These include:
    1. BSL-1 microorganisms (E. coli and Saccharomyces cerevisiae) and BSL-1 vector systems (these should be evaluated by the SRC).
    2. BSL-1 rDNA studies must be supervised by a Qualified Scientist or trained Designated Supervisor.
  5. rDNA projects that involve the introduction of a BSL-2 gene (e.g., a disease factor from a BSL-2 organism) into a BSL-1 host microorganism must be conducted at BSL-2.
  6. BSL-2 studies must be conducted at regulated research institutions.
  7. The following rDNA studies are expressly PROHIBITED:
    1. rDNA studies involving the introduction of oncogenes (cancer-causing genes) or other human, plant, or animal toxins.
    2. Studies intended to genetically engineer bacteria with multiple antibiotic resistance genes.
      1. Note: Some commercial plasmid subcloning and expression vectors code for more than one antibiotic resistance gene, so care must be taken in planning a student's experiment.
  8. All rDNA studies are subject to the same biosafety level rules and equipment and disposal procedures as were outlined in the Microorganisms section.
  9. All rDNA projects require the completion of a Risk Assessment Form 3 and a PHBA Risk Assessment Form 6A. They may also require a Regulated Research Institution Form 1C and/or a Qualified Scientist Form 2 as outlined in the Microorganisms section above.

Tissue, Body Fluid, and Blood Studies

  1. All studies involving the use of tissue, body fluid, or blood samples are subject to the Biosafety guidelines outlined in the Microorganisms section above.
    1. BSL-1 studies involve the collection of tissue and body fluids, not including blood or blood products, when the source has little risk of microorganism contamination.
      1. NOTE: New for 2012: Self-sampling of capillary blood for analysis (e.g. for a glucometer reading) can be conducted in a home (BSL-1) setting.
    2.  BSL-2 studies involve the collection of tissue and body fluids that may be contaminated with microorganisms, regardless of whether the microorganisms are classified as BSL-1 or BSL-2.
    3. Studies involving blood or blood products from domestic animals that have little risk of microorganism contamination can be considered BSL-1.
    4. Studies involving blood or blood products from humans or wild animals or domestic animals that have a risk of microorganism contamination are also classified as BSL-2.
      1. If the tissues come from animals believed to be contaminated with BSL-3 or BSL-4 organisms, the study is PROHIBITED.
    5. Studies involving human breast milk (unless it is certified free of Hepatitis C and HIV) and unpasteurized animal milk are classified as BSL-2.
    6. Studies involving human samples, WHEN THE SAMPLES CAN BE IDENTIFIED WITH A SPECIFIC PERSON, must have IRB review and informed consent. They must also include a completed Human Participants Form 4 and Consent Forms [see sample] for all human participates. These rules do not apply if the samples come solely from the student performing the study.
    7. Studies involving embryonic human stem cells must be conducted at a regulated research institution, and the project must be reviewed and approved by the ESCRO (Embryonic Stem Cell Research Oversight Committee).
  2. Most types of studies involving these materials require the completion of a Risk Assessment Form 3 and a Human and Vertebrate Animal Tissue Form 6B. Additionally, these tissue studies should complete a PHBA Risk Assessment Form 6A. The exceptions to these guidelines are listed in the next point.
  3. Some tissue studies do not need to be treated as PHBAs. These types of studies DO NOT REQUIRE ANY ADDITIONAL FORMS. These include:
    1. Plant tissue
    2. Established cell and tissue cultures. The source of the cultures should be included in the research plan.
    3. Meat, meat by-products, pasteurized milk, or eggs obtained from commercial stores, restaurants, or packing houses.
    4. Hair
    5. Teeth that have been sterilized via chemical disinfection or autoclaving at 121°C for 20 minutes
    6. Fossilized tissue or archaeological specimens
    7. Prepared, fixed tissue
  4. If the work was performed at a regulated research institution and/or in the presence of a Qualified Scientist, the Regulated Research Institution Form 1C and/or the Qualified Scientist Form 2 must be completed.