Potentially Hazardous Biological Agents

Students are permitted to do research projects with potentially hazardous biological agents meeting the conditions and rules described below which were designed to protect students and to ensure adherence to international biosafety regulations and guidelines.

When dealing with potentially hazardous biological agents, it is the responsibility of the student and all of the adults involved in a research project to conduct and document a risk assessment to define the potential level of harm, injury or disease to plants, animals and humans that may occur when working with biological agents. The risk assessment determines a biosafety level which in turn determines if the project can proceed, and if so, the laboratory facilities, equipment, training, and supervision required.

All projects involving microorganisms, recombinant DNA technologies and human or animal fresh/frozen tissues, blood or body fluids must adhere to the rules below AND, depending on the study, to the additional rules in Section A or B.

Information on Biosafety Levels (BSL) is contained at the end of this document.

Rules for ALL Studies with Potentially Hazardous Biological Agents (PHBA)

1. Prior review and approval is required for the use of potentially hazardous microorganisms (including bacteria, viruses, viroids, prions, rickettsia, fungi, and parasites), recombinant DNA (rDNA) technologies or human or animal fresh/frozen tissues, blood, or body fluids.

2. The Science Fair Leadership Committee (SFLC) must approve all research before experimentation begins. The initial risk assessment determined by the student researcher and adults supervising the project must be confirmed by the SFLC.

3. Experimentation involving the culturing of potentially hazardous biological agents, even BSL-1 organisms, is prohibited in a home environment. However, specimens may be collected at home as long as they are immediately transported to a laboratory with the BSL containment determined by the SFLC.

4. Research determined to be at Biosafety Level 1 (BSL-1) must be conducted in a BSL-1 or higher laboratory. The research must be supervised by a trained Designated Supervisor or a Qualified Scientist. The student must be properly trained in standard microbiological practices.

5. Research determined to be a Biosafety Level 2 (BSL-2) must be conducted in a laboratory rated BSL-2 or above (commonly limited to a Regulated Research Institution). The research must be reviewed and approved by the Institutional Biosafety Committee (IBC) if the Regulated Research Institution requires the review. The research must be supervised by a Qualified Scientist. For a high school BSL-2 laboratory, the SFLC must review and approve. The research must be supervised by a Qualified Scientist.

6. Students are prohibited from designing or participating in BSL- 3 or BSL-4 Research.

7. Laboratory studies designed to culture known clinically significant multidrug resistant organisms (MDROs) are not allowed.

8. Insertion of antibiotic resistance markers for the clonal selection of bioengineered organisms is not permitted.

9. Extreme caution must be exercised when selecting and sub-culturing antibiotic-resistant organisms. Studies using such organisms, including BSL-1 organisms that may have originally been exempt from prior SFLC approval, require at least BSL-2 containment.

10. The culturing of human or animal waste, including sewage sludge, is considered a BSL-2 study.

11. Naturally-occurring plant pathogens may be studied (not cultured) at home, but may not be introduced into a home/garden environment.

12. All potentially hazardous biological agents must be properly disposed of at the end of experimentation in accordance with their biosafety level. For BSL 1 or BSL 2 organisms: Autoclave at 121 degrees Celsius for 20 minutes, use of a 10% bleach solution (1:10 dilution of domestic bleach), for a minimum of 30 minutes, incineration, alkaline hydrolysis, biosafety pick-up and other manufacturer recommendations are acceptable.

13. Any proposed changes in the Research Plan by the student after SFLC approval must undergo subsequent SFLC review and approval before such changes are made and before experimentation resumes.

A. Additional Rules for Projects Involving Unknown Microorganisms

Studies involving unknown microorganisms present a challenge because the presence, concentration and pathogenicity of possible agents are unknown. In science fair projects, these studies typically involve the collection and culturing of microorganisms from the environment (e.g. soil, household surfaces, skin.)

1. Research with unknown microorganisms can be treated as a BSL-1 study under the following conditions:

• Organisms are cultured in a plastic petri dish (or other standard non-breakable container) and sealed.

• Experiment involves only procedures in which the petri dish remains sealed throughout the experiment (e.g., counting presence of organisms or colonies).

• The sealed petri dish is disposed of via autoclaving or disinfection under the supervision of the Designated Supervisor.

2. If a culture container with unknown microorganisms is opened for any purpose, (except for disinfection for disposal), it must be treated as a BSL-2 study and involve BSL-2 laboratory precautions.

B. Additional Rules for Projects with Tissues and Body Fluids, including Blood and Blood Products

Studies involving fresh/frozen tissue, blood or body fluids obtained from humans and/or vertebrates may contain microorganisms and have the potential of causing disease. Therefore, a proper risk assessment is required.

1. Research involving human and/or non-human primate established cell lines and tissue culture collections (e.g., obtained from the American Type Culture Collection) must be considered a BSL-1 or BSL-2 level organism as indicated by source information and treated accordingly. The source and/or catalogue number of the cultures must be identified in the Research Plan/Project Summary.

2. If tissues are obtained from an animal that was euthanised for a purpose other than the student’s project, it may be considered a tissue study.

3. If the animal was euthanised solely for the student’s project, the study must be considered a vertebrate animal project and is subject to the vertebrate animal rules for studies. (See vertebrate animal rules.)

4. The collection and examination of fresh/frozen tissue and/or body fluids, (see Rule 7 for additional rules and restrictions for blood or blood products) from a non-infectious source with little likelihood of microorganisms present must be considered biosafety Level 1 studies and must be conducted in a BSL-1 laboratory or higher and must be supervised by a Qualified Scientist or trained Designated Supervisor.

5. The collection and examination of fresh/frozen tissues or body fluids or meat, meat byproducts, pasteurised milk or eggs NOT obtained from food stores, restaurants, or packing houses may contain microorganisms. Because of the increased risk from unknown potentially hazardous agents, these studies must be considered biosafety level 2 studies conducted in a BSL-2 laboratory under the supervision of a Qualified Scientist.

6. Human breast milk of unknown origin, unless certified free of HIV and Hepatitis C, and domestic unpasteurised animal milk are considered BSL-2.

7. All studies involving human or wild animal blood or blood products should be considered at a minimum a Biosafety level 2 study and must be conducted in a BSL-2 laboratory under the supervision of a Qualified Scientist. Known BSL-3 or BSL-4 blood is prohibited. Studies involving domestic animal blood may be considered a BSL-1 level study. All blood must be handled in accordance with standards and guidelines set forth in the OSHA, 29CFR, Subpart Z. Any tissue or instruments with the potential of containing blood-borne pathogens (e.g. blood, blood products, tissues that release blood when compressed, blood contaminated instruments) must be properly disposed of after experimentation.

8. Studies of human body fluids, where the sample can be identified with a specific person, must have SFLC review and approval, and informed consent.

9. Any study involving the collection and examination of body fluids which may contain biological agents belonging to BSL-3 or -4 is prohibited.

10. A project involving a student researcher using their own body fluids (if not cultured)

• can be considered a BSL-1 study

• may be conducted in a home setting

• must have SFLC review if the body fluid is serving as a measure of an effect of an experimental procedure on the student researcher (e.g. student manipulates diet and takes a blood or urine sample). An example of a project not needing SFLC review would be collecting urine to serve as a deer repellent.

• must receive prior SFLC review and approval prior to experimentation.

11. Studies involving embryonic human stem cells are not permitted.

Exempt Studies (no SFLC pre-approval required)

The following types of studies are exempt from requiring SFLC pre-approval as listed below, but may be subject to additional rules dependent upon the design of the project. Student researchers and adult sponsors are required to refer to sections A and B of this section to review additional rules for projects that involve unknown organisms, recombinant DNA (rDNA) technologies, tissues, fluids, blood, or blood products before deciding upon a final biosafety level (BSL) designation for projects.

1. The following types of studies are exempt from prior SFLC review, but require a Risk Assessment:

• Studies involving protists and archaea.

• Research using manure for composting, fuel production, or other non-culturing experiments.

• Commercially-available colour change coliform water test kits. These kits must remain sealed and must be properly disposed of.

• Studies involving decomposition of vertebrate organisms (such as in forensic projects).

• Studies with microbial fuel cells.

• Studies with microbial fuel cells in which the device is sealed during experimentation and disposed of properly at the conclusion of the study

2. The following types of studies involve BSL-1 organisms and are exempt from prior SFLC review and require no additional forms:

• Studies involving fermentation of baker’s yeast and brewer’s yeast, except in rDNA studies

• Studies involving Lactobacillus, Bacillus thuringiensis, nitrogen-fixing, oil-eating bacteria, and algae-eating bacteria introduced into their natural environment. (Not exempt if cultured in a petri dish environment.)

• Studies involving water or soil microbes not concentrated in media conducive to their microbial growth.

• Studies of mould growth on food items if the experiment is terminated at the first evidence of mould.

• Studies of slime moulds and edible mushrooms.

• Studies involving E. coli k-12 (and other strains of E. coli used solely as a food source for C. elegans) that are performed at school and are not subject to additional rules for recombinant DNA studies or use of antibiotic resistant organisms.

Exempt Tissues (no SFLC pre-approval required)

1) The following types of tissue do not need to be treated as potentially hazardous biological agents:

• Plant tissue (except those known to be toxic or hazardous)

• Plant and non-primate established cell lines and tissue culture collections (e.g., obtained from the American Type Culture Collection). The source and/or catalogue number of the cultures must be identified in the Research Plan.

• Human capillary/blood collection (i.e. finger stick) of the student researcher to themself; blood collection from any other human participants must be reviewed and approved by the SFLC

• Fresh or frozen meat, meat by-products, pasteurised milk or eggs obtained from food stores, restaurants, or packing houses.

• Hair, hooves, nails and feathers.

• Teeth that have been sterilised to kill any blood- borne pathogen that may be present.

• Fossilised tissue or archeological specimens.

• Prepared fixed tissue.

Potentially Hazardous Biological Agents Risk Assessment

Risk assessment defines the potential level of harm, injury or disease to plants, animals and humans that may occur when working with biological agents. The end result of a risk assessment is the assignment of a biosafety level which then determines the laboratory facilities, equipment, training, and supervision required.

Risk assessment involves:

1. Assignment of the biological agent to a risk group

2. Studies involving a known microorganism must begin with an initial assignment of the microorganism to a biosafety level risk group based on information available through a literature search.

3. The study of unknown microorganisms and the use of fresh tissues relies on the expertise of the supervising adult(s).

4. Determination of the level of biological containment available to the student researcher to conduct the experimentation. (See “Levels of Biological Containment” for details.)

5. Assessment of the experience and expertise of the adult(s) supervising the student.

6. Assignment of a biosafety level for the study based on risk group of biological agent, level of biological containment available and the expertise of the Qualified Scientist or Designated Supervisor who will be supervising the project

Classification of Biological Agents

Risk Groups

Biological agents, plant or animal, are classified according to biosafety level risk groups. These classifications presume ordinary circumstances in the research laboratory, or growth of agents in small volumes for diagnostic and experimental purposes.

BSL-1 risk group contains biological agents that pose low risk to personnel and the environment. These agents are highly unlikely to cause disease in healthy laboratory workers, animals or plants. The agents require Biosafety Level 1 containment. Examples of BSL-1 organisms are: Agrobacterium tumifaciens, Micrococcus leuteus, Neurospora crassa, Bacillus subtilis.

BSL-2 risk group contains biological agents that pose moderate risk to personnel and the environment. If exposure occurs in a laboratory situation, the risk of spread is limited and it rarely would cause infection that would lead to serious disease. Effective treatment and preventive measures are available in the event that an infection occurs. The agents require Biosafety Level 2 containment. Examples of BSL-2 organisms are: Mycobacterium, Streptococcus pneumonia, Salmonella choleraesuis.

BSL-3 risk group contains biological agents that usually cause serious disease (human, animal or plant) or that can result in serious economic consequences. Projects in the BSL-3 group are prohibited.

BSL-4 risk group contains biological agents that usually produce very serious disease (human, animal or plant) that is often untreatable. Projects in the BSL-4 group are prohibited.

Levels of Biological Containment

There are four levels of biological containment (Biosafety Level 1–4). Each level has guidelines for laboratory facilities, safety equipment and laboratory practices and techniques.

BSL-1 containment is normally found in water-testing laboratories, in high schools, and in colleges teaching introductory microbiology classes. Work is done on an open bench or in an appropriate biosafety hood. Standard microbiological practices are used when working in the laboratory. Decontamination can be achieved by treating with chemical disinfectants or by steam autoclaving. Lab coats and gloves are required. The laboratory work is supervised by an individual with general training in microbiology or a related science.

BSL-2 containment is designed to maximise safety when working with agents of moderate risk to humans and the environment. Access to the laboratory is restricted. Biological safety cabinets (Class 2, type A, BSC) must be available. An autoclave should be readily available for decontaminating waste materials. Lab coats and gloves are required; eye protection and face shields must also be worn as needed. The laboratory work must be supervised by a scientist who understands the risk associated with working with the agents involved.

BSL-3 containment is required for infectious agents that may cause serious or potentially lethal diseases as a result of exposure by inhalation. Projects in the BSL-3 group are prohibited.

BSL-4 containment is required for dangerous/exotic agents that pose high risk of life-threatening disease. Projects in the BSL-4 group are prohibited.