Basic Biosafety BIO01-07

Overview

Institutional Biosafety Committees (IBCs) are typically assigned additional review responsibilities beyond the scope of the NIH Guidelines

At UNL the IBC reviews:

• Pathogens
• Transgenic organism use
• Bloodborne Pathogens and other Human Materials
• Select Agents and toxins
• Field Work (collection of animals and release of plant pathogens)
• In accordance with the NIH Guidelines, most work involving recombinant or synthetic nucleic acids must be reviewed by a local Institutional Biosafety Committee. Some experiments are also subject to review by the Office of Science Policy (OSP) or even the NIH Director.
• By policy, the scope of the IBC at UNL goes beyond recombinant and synthetic nucleic acid research and covers human, animal and plant pathogens; potentially infectious materials derived from humans, like blood and tissues; transgenic organism use; select agents; and some field work.
• Any person wishing to conduct work of this nature must submit a protocol to the IBC for review. Part of the IBC’s job is to aid the PI in assessing the risk of the work they want to perform and ensure that they have the proper facilities, procedures and engineering controls to conduct the work safely.

Overview

EHS Online Training

• Biosafety 100: Research Compliance
• Biosafety 101
• Biosafety 201
• Autoclave Operation

UNL Biosafety Guidelines

EHS SOPS

• Autoclave Operation and Use
• Spill and Exposure Response for Biohazardous Materials
• Biosafety Containment Levels
• Disposing of Biohazardous Materials Including Recombinant Nucleic Acids
• Chemical Disinfectants for Biohazardous Materials
• Preparing a Laboratory Biosafety Manual
• Biosafety Training
• Biosafety Cabinets
• Working in a Biosafety Cabinet

You should have taken the Biosafety 100: Research Compliance, Biosafety 101 and Biosafety 201 and Autoclave Operation EHS online training modules prior to taking this training. Being familiar with the materials listed will help you to establish a general understanding of the policies and procedures generally required to conduct work in a bio-lab in a safe and compliant manner. This understanding is key to completing bio audits in a competent and complete manner. You are not expected to be an expert in the world of biosafety, but having a general awareness will help you to recognize situations that you should discuss with the biosafety professional staff.

Nucleic Acids

Nucleic acids are biopolymers, or large biomolecules, essential for all known forms of life. Nucleic acids, which include DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), are made from monomers known as nucleotides.

Gene

A gene is a region of DNA that encodes a functional RNA or protein product, and is the molecular unit of heredity.

Plasmid

A plasmid is a small DNA molecule within a cell that is physically separated from a chromosomal DNA and can replicate independently.

Overview

Recombinant Nucleic Acid Experiments

Recombinant Nucleic Acid (r/NA) molecules are DNA or RNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in the natural genome.

Recombinant nucleic acid experiments involve taking a host plasmid, inserting a foreign gene of interest, and then using that plasmid to express the gene in a cell, microbe or organism.

This allows us to understand gene function in a living cell or organism. These techniques can also be used to allow us to visualize when and where a gene is expressed by including “reporter” genes such as GFP or luciferase.

This type of research is regulated under the NIH Guidelines when an institution receives funding for this type of research. The Guidelines state that the research must be reviewed and approved by a faculty committee of experts. This is the Institutional Biosafety Committee.

Overview

Synthetic Nucleic Acid & Gene Editing Experiments

Synthetic Nucleic Acid (s/NA) are nucleic acid molecules that are chemically or by other means synthesized or amplified, including those that are chemically or otherwise modified but can base pair with naturally occurring nucleic acid molecules. These molecules do not existing in a living organism in nature. However, these ”synthesized” or “unnatural” nucleic acids can be introduced into a living organism. In general, synthetic nucleic acid experiments are subject to the purview of the IBC.

One common example of synthetic nucleic acid experiments is RNA interference which uses short 22bp synthesized RNA fragments called “siRNA”. These fragments allow genes to be silenced or “turned off” temporarily. This graphic illustrates how this works.

Another example of synthetic nucleic acid experiments is CRISPR gene-editing. This includes the use of an enzyme from bacteria that utilizes a short synthetic piece of RNA as a guide to target a specific sequence in the host DNA. Once the location is found the enzyme binds the DNA and cuts it. After cutting, the natural DNA repair mechanism introduces a mutation that effective turns off the gene or another piece of DNA can be inserted at the site of the cut.

Overview

Pathogen (pathogenic agent)

Any microbiological agent or biological toxin that is capable of causing disease in humans, animals or plants. Lab-adapted strains of microbes are not included under this definition; examples include K12-derived E. coli strains and S. cerevisiae.

Human Pathogen

Capable of causing disease in healthy human adults.

Opportunistic Pathogen

Capable of causing disease in hosts with compromised immune systems.

Zoonotic Agents

Capable of causing disease in humans and animals.

Biohazardardous waste

Waste that is contaminated with pathogenic agents and/or contains recombinant nucleic acid molecules and/or human tissues and fluids that fall under the OSHA Bloodborne Pathogens Standard.

Here are some important terms that you should be familiar with to help you distinguish biological hazards from non-hazards.

A pathogen is generally defined as a microbe or toxin that is capable of infecting and causing disease in humans, animals, or plants.

Human pathogens are further defined as being able to cause disease in healthy adults. There are some microorganisms that can cause disease in susceptible human hosts due to a compromised immune system, but not do not normally cause disease in healthy adults.

Opportunistic pathogens: An example of this is when a person is infected with HIV, they become more susceptible to infection by microbes that would not normally be able to cause disease such as yeast that live on our bodies as part of our normal flora. Such microorganisms are generally referred to opportunistic pathogens.

Zoonotic Agents are those pathogens that are able to infect and cause disease in both humans and animals. Salmonella is an example of this as well as Avian Influenza. These types of agents are restricted to BSL-2 or higher containment labs.

Biohazardous waste at UNL includes not only pathogen contaminated materials, but also materials that contain recombinant or synthetic nucleic acid molecules as well as all human cell lines, tissues, organs, fluids, etc.

To aid in risk assessment, the National Institute of Health (NIH) and World Health Organization (WHO) have developed a ranking mechanism to categorize microorganisms by:

1. its capability to infect and cause disease in a susceptible human or animal host,
2. its virulence as measured by the severity of disease, and
3. the availability of preventive measures and effective treatments for the disease.

These categories are called Risk Groups.

Risk groupings (RGs) are used to characterize the relative risk associated with a given agent based on potential routes of transmission, susceptibility of healthy adult population to infection, and disease morbidity and mortality characteristics.

Most of the labs you will inspect will have only Risk Group 1 or 2 agents, the exception to this is HIV which is classified as RG-3, but in a research lab setting can be safely worked with at BSL-2 with some procedural enhancements like double gloves and limited sharps use.

Many organizations have assigned Risk Groups (RG) to specific biological agents. While the assigned risk group may not be identical for any given agent across all organizations, all use a four-group system with the lowest risk agents assigned to Risk Group 1 and the highest risk agents assigned to Risk Group 4.

• RG4 agents are infectious by all routes of exposure, have extreme mortality/morbidity rates, treatment is usually not available, and agents in this group present a high community risk. Risk of death following infection with a RG 4 agent is high.
• RG3 agents are infectious by all routes of exposure (especially the AIRBORNE route) and have higher morbidity and mortality rates than Risk Group 2 agents. Treatment may/may not be available. They generally present a high individual risk, but much lower community risk.
• RG2 agents are generally transmitted via ingestion, through mucous membranes, and through the skin. The severity of disease is usually not as significant as agents in higher risk groups, and treatment is generally available. Mortality and morbidity is lower than high risk group classifications.
• RG1 agents are not associated with disease in healthy adults.

To simplify, think of the RG Classification like this:

• “RG1 – Don’t Drink it”;
• “RG2 – Don’t Touch it”;
• “RG3 – Don’t Breathe it”;
• and “RG4 – Don’t do it at UNL!”

A couple of important points to remember:

• RG classifications consider only a healthy adult population.
• Risk Groups do NOT account for agents that may have greater or lesser virulence acquired through genetic modification or passage.

There are currently no risk group classifications for animal pathogens or plant pathogens in the US, although some foreign governments have established these classifications. So the risk assessment for those agents is different than for human and zoonotic pathogens.

There can be strain variation among species. An example is E. coli where there are lab adapted strains that are non-pathogenic and other strains such as O157 that are pathogens and the difference between these is based on the genes expressed.

Laboratories which house microorganisms for research are similarly categorized, according to design. Containment laboratories are assigned a “biosafety level” (BSL), ranging from 1-4, with BSL-1 laboratories designed with the lowest level of containment. Unlike risk group classifications which are specific to microorganisms affecting humans, laboratory containment levels are used with any type of microorganism regardless of whether it is a human, animal, or plant pathogen. The containment level progressively increases with increasing risks that the organism presents to humans and the environment and considering the type of work to be conducted with the organism.

Microorganisms that present little to no risk to humans or the environment are generally conducted in biosafety level one containment facilities, with consideration given to the laboratory techniques to be conducted. Microorganisms that present grave risk to human health or the environment are worked with in biosafety level 3 or 4 facilities. These organisms are often lethal to a susceptible host and are easily transmitted between members of the host population.

Notice that each containment level builds from the next lower containment. Detailed BSL-1 laboratory practices are discussed later. Note: Organism “Risk Group” and laboratory “Biosafety Level” are not synonymous. It is not accurate to refer to an organism as a “BSL-1 organism.” However, the two concepts do compliment each other. Risk groups correlate to Biosafety Levels, but they are not always equivalent. If a PI has a question regarding microorganism risk group or appropriate biosafety level, please refer them to the BSO.

Overview

Disinfectant containers are not appropriately labeled (name, made/mfg. date and expiration date). Expired disinfectants are in use.

Use of appropriate disinfectants in biological laboratories is an important safety issue. When considering this checklist item make sure the chemical is used for disinfection purposes before citing this. Some labs will keep bleach and 70% alcohol solutions for general cleaning or other purposes that are not related to disinfection of biohazardous materials used in research.

For this item we are primarily looking at:

1. Appropriate labeling of disinfectants to ensure that the disinfectants are easily identifiable in the lab to researchers and others; and
2. That the disinfectants will be effective when used (that is, they are not expired).

Cite this item if disinfectant bottles are not appropriately labeled as described next or if you identify an expired bottle of disinfectant. Be sure to list the name and manufacturer of the disinfectant when recording this deficiency in EHSA.

Verifying appropriate disinfectant container labels is the minimum expectation for auditors, if you have concerns about the appropriateness of the disinfectant used, please discuss your concerns with the BSO, but do not cite that in the inspection report unless directed to do so by the BSO. The IBC approves disinfectants for a lab to use based on the risk assessment of the agents in use and the work performed. A change to disinfectants must be approved by the BSO.

Citations and Related Resources

Citations

BMBL 6th Ed. Section IV BSL-1 A-14

Decontaminate work surfaces after completion of work and after any spill or splash of potentially infectious material with appropriate disinfectant.

Per the EHS SOP Chemical Disinfectants for Biohazardous Materials it is essential that containers of disinfectants used to decontaminate biohazardous materials be labeled to ensure that the disinfectants are: 1) made at the appropriate concentration; 2) have not expired. For these reasons, auditors need to be familiar with common disinfectants and check durable containers of disinfectants for appropriate labeling.

Appropriate labeling consists of the chemical name; including the concentration, if applicable (some disinfectants are used full strength, others are used in a diluted solution), the date the solution was made or if it is used undiluted, the manufactured date or received date*, and the date of expiration (shelf life).

*The received date is the date the chemical was acquired by the lab. While the manufactured date is preferred as it is more accurate for shelf-life, the date the chemical was acquired by the lab is acceptable. If only the received date is indicated on a commercial bleach bottle, it is recommended the auditor add an expiration date of 1 year post receiving and let lab occupants know that concentrated bleach solutions expire after 1 year.

The shelf life of disinfectants varies widely with some working solutions only being good for 24 hours, while others can be good for 6 months or more. If lab workers have questions about this, always refer them to the EHS SOP Chemical Disinfectants for Biohazardous Materials, which provides guidance about selecting an appropriate disinfectant as well as disinfectant shelf life, making diluted solutions, and disposal of expired disinfectants.

Bleach solutions will be most common in labs and 5-6000ppm is equivalent to a 10% solution made from bleach with 5-6% sodium hypochlorite. 8000ppm is a 10% solution made from 8% sodium hypochlorite. 10000ppm would be a 20% solution of 5-6% sodium hypochlorite.

Purchased 70% EtOH or IPA solutions likely will not have an expiration date and shouldn’t be cited for not having one.