Projects incorporating microorganisms (including bacteria, viruses, viroids, prions, rickettsia, fungi and parasites), recombinant DNA (rDNA) technologies or human/animal fresh tissues, blood or bodily fluids may involve working with potentially hazardous biological agents and must complete a Potentially Hazardous Biological Agents Form (6A).

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 a risk assessment. A risk assessment defines the potential level of harm, injury or disease to plants, animals or humans that may occur when working with biological agents.

Experimentation with potentially hazardous biological agents is prohibited in a home environment. This means absolutely NO CULTURING may be done at home, but collection of samples may be done there.

ALL PHBAs must be properly disposed of by the Designated Supervisor or Qualified Science/Mentor at the end of experimentation in accordance with their biosafety level. Acceptable disposal methods for BSL-1 and BSL-2 organisms include:

• Autoclave at 121° for 20 minutes;
• Use of a 10% bleach solution (1:10 dilution of domestic bleach);
• Incineration;
• Alkaline hydrolysis;
• Biosafety pick-up; or
• Other manufacturer recommendations

Studies involving unknown microorganisms present a challenge because the presence, concentration and pathogenicity of possible agents are unknown. Usually, these studies involve the collection and culturing of microorganisms from the environment (i.e.: soil, household surfaces, skin, etc.).

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

• The organism is cultured in a plastic petri dish (or other standard, sterile, non-breakable container) and SEALED.
• The experiment involves only procedures in which the petri dish REMAINS SEALED throughout the experiment.
• The SEALED petri dish is disposed by the supervision of the Designated Supervisor.

If a culture container with unknown microorganisms is OPENED FOR ANY PURPOSE (except for disposal by the Designated Supervisor), it MUST be treated as a BSL-2 agent and conducted at a BSL-2 equipped lab.

The following are simple samples of projects with varying levels of biosafety requirements.

• Sample Form 6A for a project where the Qualified Scientist has determined the risk level of the study is a BSL-1.


• Sample Form 6A for a project where the Qualified Scientist has determined the risk level of the study is a BSL-2


• Sample Form 6A for a project that was done at a regulated research institution.

IMPORTANT: Please note that not all rules and guidelines are listed on this web site, please review more about CSEF rules regarding Potentially Hazardous Biological Agents and Tissue research to make sure you are in compliance and can compete in the CSEF competition.

BSL-4 risk group contains biological agents that usually produce very serious diseases that are often untreatable. Projects in the BSL-4 group are PROHIBITED.

BSL-3 risk group contains biological agents that usually cause serious disease or that can result in serious economic consequences. Projects in the BSL-3 group are PROHIBITED.

BSL-2 risk group contains biological agents that pose moderate risk to personnel and the environment. If exposure occurs in a lab situation, the risk of spread is limited and it rarely would cause infection that would lead to serious disease. These agents require a BioSafety Level 2 containment. Examples of BSL-2 organisms include: Mycobacterium, Streptococcus pneumoniae, Salmonella cholerasuis.

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

The following types of studies are not considered Potentially Hazardous Biological Agents and do not require prior SRC review, but MUST be included on the Risk Assessment Form (3):

• Studies involving baker's yeast and brewer's yeast, except in rDNA studies.


• Studies involving Lactobacillus (starter cultures for controlled fermentation), Bacillus thuringiensis (typically found in insecticides), nitrogen-fixing bacteria, oil-eating bacteria and algae-eating bacteria introduced into their natural environment. None of these studies are exempt if they are cultured in a Petri dish.


• Studies involving water or soil where the student is NOT purposely culturing bacteria.


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


• Studies of slime molds 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 rDNA studies.


• Studies involving protists and archaea and KNOWN nonpathogenic microorganisms.


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


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


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


• Studies with microbial fuel cells.

Studies involving fresh or frozen tissue, blood or body fluids obtained from humans and/or vertebrate animals may contain microorganisms and have the potential of causing disease and therefore, must complete a Potentially Hazardous Biological Agents Form (6A) AND a Human and Vertebrate Animal Tissue Form (6B).

The following types of tissue do not need to be treated as potentially hazardous biological agents and are thus exempt from prior SRC review and approval, but MUST be included on the Risk Assessment Form (3):

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


• Plant and non-primate ESTABLISHED cell lines and tissue culture collections (i.e.: obtained from the American Type Culture Collection); the source and/or catalog number of the cultures MUST be identified in the research plan.


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


• Hair, hooves, nails and feathers.


• Teeth that have been sterilized by a dentist to kill any blood-borne pathogens that may be present. the dentist who provided the teeth must submit a letter certifying the sterilization.


• Fossilized tissue or archaeological specimens.


• Prepared, fixed tissue samples.

The following is a simple sample of a project that involves the use of human and/or vertebrate animal tissue.

• Sample Form 6B were the blood sample is obtained from another study being done at a regulated research institution.