Working in microbiological and biomedical laboratories poses special risks with the involvement of infectious agents, recombinant DNA, synthetic nucleic acid molecules, and laboratory animals. Laboratory-associated infections (LAIs) are a reality, but with the implementation of National Biosafety Guidelines by the Centers for Disease Control (CDC) and the National Institutes of Health (NIH) LAIs have become infrequent. Strict adherence to standard microbiological practices and techniques as outlined in the Biosafety in Microbiological and Biomedical Laboratories manual (BMBL) and the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines) is paramount for biosafety and protection of laboratory personnel from LAIs. Understanding the principles of biosafety and adherence to the microbiological practices, containment, and facility safeguards contributes to a safer and healthier work environment for not only laboratory staff, but also adjacent personnel and the surrounding community.
To articulate the University’s objective of providing a safe, healthy and secure environment for all members of faculty and staff, student and visitors and to delineate responsibility for achieving it.
A. The University of Oklahoma’s role is to establish a process for compliance with the following documents:
• NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid
Molecules (NIH Guidelines), current edition;
• Biosafety in Microbiological and Biomedical Laboratories (BMBL), current edition.
• The Occupational Safety and Health Administration (OSHA) Bloodborne Pathogen Standard, 29 CRF 1910.1030
B. Definition of Biosafety
• Biosafety or biological safety encompasses all aspects of containment to prevent any exposure to and accidental release of infectious biological material. This also includes the containment of plants and animals.
C. NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules Overview
• Detail procedures and practices for the containment and safe conduct of various forms of research involving recombinant and synthetic nucleic acid molecules, including research involving genetically modified plants and animals, and human gene transfer research
• Institutions that receive funding from the National Institutes of Health must ensure all projects conducted at the institution are in compliance with the NIH Guidelines regardless of the funding source for each project. The rationale for this requirement is that for biosafety to be meaningful, it has to be observed by all investigators at an institution.
• The purpose of the NIH Guidelines is to specify the practices for constructing and handling:
1. recombinant nucleic acid molecules,
2. synthetic nucleic acid molecules, including those that are chemically or otherwise modified but can base pair with naturally occurring nucleic acid molecules, and
3. cells, organisms, and viruses containing such molecules.
D. NIH Definition of Recombinant and Synthetic Nucleic Acid Molecules are:
• Molecules that:
1. are constructed by joining nucleic acid molecules and
2. that can replicate in a living cell, i.e., recombinant nucleic acids;
• 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, i.e., synthetic nucleic acids, or
• Molecules that result from the replication of those molecules described in the first two points above.
E. Experiments covered by the NIH Guidelines
• Experiments that require IBC approval before initiation include those that involve:
1. risk Group 2, 3, 4, or Restricted Agents as host-vector systems,
2. cloning DNA from Risk Group 2, 3, 4, or Restricted Agents into nonpathogenic prokaryotic or lower eukaryotic host-vector systems,
3. the use of infectious DNA or RNA viruses, or defective DNA or RNA viruses in the presence of helper virus in tissue culture,
4. whole plants or animals, and
5. more than 10 liters of cultures
• Experiments that must be registered at the time of initiation include those that involve:
1. the formation of recombinant DNA molecules containing no more than 2/3 of the genome of any eukaryotic virus propagated in tissue culture,
2. recombinant DNA modified whole plants, and/or recombinant DNA modified organisms associate with whole plants, except those that fall under section III-A, III-B, III-C or III-D of the NIH Guidelines, and/or
3. the generation of transgenic rodents that require BSL-1 containment.
• Experiments exempt from the NIH Guidelines:
1. Experiments exempt from the NIH Guidelines, although requiring registration with the IBC, may be initiated immediately.
2. The Biosafety Officer will review the registration and confirm that the work is classified correctly according to the NIH Guidelines. Exempt experiments are those that:
a. use synthetic nucleic acids that can neither replicate nor generate nucleic acids capable of replicating in any living cell; are not designed to integrate into DNA, and do not produce a toxin that is lethal for vertebrates at an LD50 of <100 ng/kg body weight,
b. use rDNA molecules that are not in organisms or viruses,
c. consist entirely of DNA segments from a single non-chromosomal or viral DNA source, though one or more of the segments may be a synthetic equivalent,
d. consist entirely of DNA from a prokaryotic host including its indigenous plasmids or viruses when propagated only in that host (or a closely related strain of the same species), or when transferred to another host by well-established physiological means,
e. consist entirely of DNA from an eukaryotic host including its chloroplasts, mitochondria, or plasmids (but excluding viruses) when propagated only in that host (or a closely related strain of the same species),
f. consist entirely of DNA segments from different species that exchange DNA by known physiological processes, though one or more of the segments may be a synthetic equivalent,
g. do not present a significant risk to health or the environment as determined by the NIH Director, with the advice of the Recombinant DNA Advisory Committee (RAC), and following appropriate notice and opportunity for public comment;
h. contain less than one-half of any eukaryotic viral genome propagated in cell culture,
i. use E. coli K12, Saccharomyces cerevisiae, or Bacillus subtilis host-vector systems, unless genes from Risk Group 3 or 4 pathogens are cloned into these hosts, and
j. involve the purchase or transfer of transgenic rodents for experiments that require BSL1 containment.
F. Principal Investigator General Responsibilities
• Prior to the commencement of any project involving any use of such material, the Principal Investigator (P.I.) must perform the following steps:
1. Review the applicable guidelines and regulations and become familiar with the biological safety procedures and requirements. These guidelines and regulations include:
a. The National Institutes of Health (NIH) Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules;
b. The Centers for Disease Control and Prevention (CDC) and National Institutes of Health (NIH) publication entitled Biosafety in Microbiological and Biomedical Labs (BMBL) 5th edition; https://www.cdc.gov/biosafety/publications/bmbl5/
c. 32 CFR Part 626 Biological Defense Safety Program and 32 CFR Part 627 Biological Defense Safety Program, Technical Safety Requirements (DA Pamphlet 385-69); and
d. The Occupational Safety and Health Administration (OSHA) Bloodborne Pathogen Standard, 29 CRF 1910.1030; https://www.osha.gov/pls/oshaweb/owadisp.show_document?p_table=STANDARDS&p_id=10051
e. The CDC/United States Department to Agriculture (USDA) Select Agent program.
2. Perform a risk assessment of the agents and procedures to determine potential safety and environmental hazards.
3. Develop laboratory specific Standard Operating Procedures (SOPs) based on the risk assessment, guidelines, and regulations.
4. For all non-exempt recombinant DNA research; microorganism/virus work or work with human blood, tissue, xenograft or cell lines, complete and submit the appropriate Institutional Biosafety Committee (IBC) registration form(s):
1. Registration of Research with Recombinant or Synthetic Nucleic Acid Molecules Form
2. Infectious Agent Registration at http://apps.ouhsc.edu/IBCinventory/newbiologicalovt/
5. Do not initiate or modify any recombinant or synthetic nucleic acid molecule research which requires IBC approval prior to initiation until that research or the proposed modification thereof has been approved by the IBC and has met all other requirements of the NIH Guidelines;
6. Determine whether experiments are covered by Section III-E, Experiment that Require IBC Notice Simultaneous with Initiation, and ensure that the appropriate procedures are allowed;
7. Report any significant problems, violations of the NIH Guidelines, or any significant research related accidents and illnesses to the Biological Safety Officer, IBC and other appropriated authorities within 30 days.
8. Report any new information bearing on the NIH Guidelines to the IBC and to NIH/ OBA by email to: NIHGuidelines@od.nih.gov.
9. Be adequately trained in good microbiological techniques;
10. Adhere to IBC approved emergency plans for handling accidental spills and personnel contamination; and
11. Comply with shipping requirements for recombinant or synthetic nucleic acid molecules.
• Overview of Principal Investigator Responsibilities For Submissions
1) Make an initial determination of the required levels of physical and biological containment in accordance with the NIH guidelines http://osp.od.nih.gov/sites/default/files/resources/NIH_Guidelines.pdf
2) Select appropriate microbiological practices and laboratory techniques to be used for research;
3) Submit the initial research protocol and any subsequent changes (e.g., changes in the source of DNA or host-vector system), if covered under Sections III-A, III-B, III-C, III-D, or III-E (Experiments Covered by the NIH Guidelines), to the Institutional Biosafety Committee for review and approval/disapproval;
4) Remain in communication with the IBC throughout the length of the research project,
5) To submit IBC protocols for the University of Oklahoma, Norman campus, please visit our website at: http://compliance.ouhsc.edu/ibc/Home/Policies/Norman.aspx
6) To submit IBC protocols for OUHSC and Tulsa campuses, please visit our website at: http://compliance.ouhsc.edu/ibc/Home/Forms/OUHSC.aspx
• Responsibilities of the Principal Investigator Prior to Initiating Research
1. Protocols should be available to all laboratory personnel that describe the potential biohazards and the precautions to be taken.
2. Instruct and train laboratory staff in:
a. the practices and techniques required to ensure safety
b. the procedures for dealing with accidents
3. Inform laboratory personnel of the reasons and provisions for any precautionary medical practices advised or requested (e.g., vaccinations or serum collection).
• Responsibilities of the P.I. During the Length of the Research Project
1. Supervise the safety performance of the laboratory staff to ensure that the required safety practices and techniques are employed.
2. Investigate and report any significant problems pertaining to the operation and implementation of containment practices, procedures, violations of the NIH Guidelines, or any significant research-related accidents and illnesses in writing to the Safety Officer (where applicable), Animal Facility Director (where applicable), IBC, NIH OBA, and other appropriate authorities (if applicable) (reports to NIH OBA shall be sent to the Office of Science Policy, National Institutes of Health, preferably by e-mail to: NIHGuidelines@od.nih.gov.
3. Correct work errors and conditions that may result in the release of recombinant or synthetic nucleic acid molecule materials.
4. Ensure the integrity of the physical containment (e.g., biological safety cabinets) and the biological containment (e.g., purity and genotypic and phenotypic characteristics).
5. Comply with reporting requirements for human gene transfer experiments conducted in compliance with the NIH Guidelines (see Appendix M-I-C, Reporting Requirements).
6. Determine the need for IBC review before modifying recombinant or synthetic nucleic acid research already approved by the IBC. If help is required in determining this need, the P.I. may contact the IBC or the Biological Safety Officer.
7. Submit any subsequent changes (e.g., changes in the source of DNA or host-vector system) to the IBC for review and approval or disapproval.
8. Submit a new IBC protocol for review and approval at least every 3 years while the research is being conducted.
• OU Institution Biosafety Committee (IBC) Responsibilities
1. Reviewing recombinant or synthetic nucleic acid molecule research conducted at or sponsored by the institution for compliance with the NIH Guidelines and approving those research projects that are found to conform with the NIH Guidelines.
2. Notifying the P.I. of the results of the IBC’s review and approval.
3. Lowering containment levels for certain experiments as specified in experiments in which DNA from RG2, RG3, RG4 or Restricted Agents is Cloned into
Nonpathogenic Prokaryotic or Lower Eukaryotic Host-Vector Systems.
4. Setting containment levels as specified in experiments involving whole animals and experiments involving whole plants.
5. Periodically reviewing recombinant or synthetic nucleic acid molecule research conducted at the institution to ensure compliance with the NIH Guidelines.
6. Adopting emergency plans covering accidental spills and personnel contamination resulting from recombinant or synthetic nucleic acid molecule research.
7. Reporting any significant problems with or violations of the NIH Guidelines and any significant research-related accidents or illnesses to the appropriate institutional official and NIH Office of Science Policy (OSP) within 30 days, unless the Institutional Biosafety Committee determines that a report has already been filed by the Principal Investigator. reports to NIH OBA shall be sent to the Office of Science Policy, National Institutes of Health, preferably by e-mail to: NIHGuidelines@od.nih.gov., additional contact information is also available here and on the OSP website (www.od.osp.gov).
8. The Institutional Biosafety Committee may not authorize initiation of experiments which are not explicitly covered by the NIH Guidelines until NIH establishes the containment requirement.
9. Performing other functions as may be delegated to the IBC.
• Biological Safety Officer (BSO)
1. The institution shall appoint a Biological Safety Officer for the following:
1. If it engages in large-scale research or production activities involving viable organisms containing recombinant or synthetic nucleic acid molecules.
2. If it engages in recombinant or synthetic nucleic acid molecule research at BSL-3 or BSL-4. The Biological Safety Officer shall be a member of the Institutional Biosafety Committee.
2. The Biological Safety Officer's duties include, but are not be limited to:
1. Periodic inspections to ensure that laboratory standards are rigorously followed;
2. Reporting to the Institutional Biosafety Committee and the institution any significant problems, violations of the NIH Guidelines, and any significant research-related accidents or illnesses of which the Biological Safety Officer becomes aware unless the Biological Safety Officer determines that a report has already been filed by the Principal Investigator;
3. Developing emergency plans for handling accidental spills and personnel contamination and investigating laboratory accidents involving recombinant or synthetic nucleic acid molecule research;
4. Providing advice on laboratory security;
5. Providing technical advice to Principal Investigators and the Institutional Biosafety Committee on research safety procedures.
• Risk Assessment
1. A process used to identify the hazardous characteristics of a known infectious or potentially infectious agent or material, the activities that can result in a person’s exposure to an agent, the likelihood that such an exposure will cause a laboratory associated infection (LAI), and the probable consequences of such an infection.
2. Responsible parties include:
1. Directors and P.I.s of microbiological and biomedical laboratories,
2. Institutional Biosafety Committees (IBC),
3. Animal Care and Use Committees (IACUC),
4. biological safety professionals
5. animal veterinarians
3. The information identified by risk assessment will provide a guide for the selection of appropriate biosafety levels and microbiological practices, safety equipment, and facility safeguards that can prevent LAIs.
4. The safe handling of an infectious agent or biological toxin requires an objective evaluation of the hazard potential associated with each aspect of work activity.
5. Biological risk assessment is a subjective process requiring consideration of many hazardous characteristics of agents and procedures. Below is an outline of some of the considerations that should be evaluated before undertaking any work with any microorganisms, toxins, or recombinant or synthetic nucleic acid molecules.
1. Identify agent hazards and perform an initial risk assessment
1) Consider the risk group based upon the principal hazardous characteristics:
a) Capability to infect and cause disease in a susceptible human host,
b) severity of disease, and
c) the availability of preventive measures and effective treatments.
2) Make a preliminary determination of the biosafety level that best correlates with the initial risk assessment based on the identification and evaluation of the agent hazards.
3) Factors to be considered in determining the level of containment include:
a) agent virulence,
b) pathogenicity,
c) infectious dose,
d) environmental stability,
e) spread of infection,
f) communicability,
g) operations,
h) quantity,
i) availability of vaccine or treatment, and
j) gene product effects such as toxicity,
k) physiological activity,
l) and allergenicity.
m) Take into consideration the enhanced or decreased pathogenicity of recombinant organisms.
• Expression of a toxin for purification in a previously non-pathogenic strain of E. coli such as BL21 introduces a hazard that merits consideration of a higher containment level than the parent (wild-type) strain.
• Attenuated agents, made by either recombinant methods or those that have been demonstrated to have irreversibly lost known virulence factors, may be considered for a lower containment level than what would originally be chosen for the parent strain based upon the Risk Group assignment.
4) Remember that aerosol and droplet routes of agent transmission are also important considerations in specification of safety equipment and facility design that result in a given BSL level.
5) It is important to remember that any reduction of containment level, must first be approved by the Institutional Biosafety Committee (IBC).
2. Identify Laboratory Procedure Hazards
a) Complexity of a laboratory procedure can increase hazards.
b) Some situations where intended use of an agent increases need precautions
c) Hazards that need assessed are:
a) Agent concentration,
b) suspension volume,
c) equipment and procedures that generate small particle aerosols,
d) equipment and procedures that generate larger airborne particles (droplets), and
e) use of sharps.
3. Determine Biosafety Level & Include Appropriate Precautions
1) The selection of the appropriate biosafety level and the selection of any additional laboratory precautions require careful consideration of the previously covered topics.
2) It is also important to recognize that individuals in the laboratory may differ in their susceptibility to disease.
a) Pre-existing diseases, medications, compromised immunity, and pregnancy or breast-feeding that may increase exposure to infants from certain agents, are some of the conditions that may increase the risk of an individual for acquiring a LAI.
b) Consultation with an occupational physician knowledgeable in infectious diseases is advisable in these circumstances.
4. Evaluate Proficiency of Staff Regarding Safe Practices, Integrity of Safety Equipment
1) Protection of personnel, other persons associated with the laboratory, and the public will depend ultimately on the laboratory staff.
2) The laboratory director or P.I. should ensure that laboratory personnel have acquired the technical proficiency in the following:
a) Appropriate level of training,
experience in handling infectious agents,
b) proficiency in aseptic technique,
c) knowledge of operating all required equipment safely,
d) proficiency in sterile technique within a biological safety cabinet,
e) ability to respond to emergencies, and
f) willingness to accept responsibility for protecting one’s self and others.
3) The laboratory director or P.I. should ensure that the necessary safety equipment is available and operating properly.
5. Review Risk Assessment with a Biosafety Officer, Subject Matter Expert, & the IBC
a) All research that requires the use of BSL-2 containment or higher, involves the use of biological toxins, and/or involves the use of recombinant or synthetic nucleic acid molecules must be reviewed and approved by OUHSC IBC.
b) Consultation with an Institutional Biosafety Officer following the primary risk assessment, prior to submission of an IBC protocol is highly recommended.
c) Risk assessment is the basis for the safeguards developed by the CDC, NIH, and the microbiological and biomedical community to protect the health of laboratory workers and the public from risks associated with the use of hazardous biological agents in laboratories.
d) Experience shows that these established safe practices, equipment, and facility safeguards work in combination to prevent laboratory associated infections.
• Risk Groups & Biosafety Levels
1. Risk Groups
a. World Health Organization (WHO) has recommended an agent risk group classification for laboratory use that describes four general risk groups based on these principal characteristics and route of transmission of the natural disease.
b. The four groups address the risk to both the laboratory worker and the community.
c. NIH Guidelines established a comparable classification and assigned human etiological agents into four risk groups on the basis of hazard.
d. Risk Group classifications correlate with but do not equate to biosafety levels (Table 1).
e. A risk assessment will determine the degree of correlation between an agent’s risk group classification and biosafety level.
Table 1. Summary of Risk Group Classification
Risk Group Classification NIH Guidelines World Health Organization
Risk Group 1 Agents not associated with diseases in healthy adult humans No or low individual and community risk) A microorganism unlikely to cause human or animal disease
Risk Group 2 Agents associated with human disease that is rarely serious and for which preventive or therapeutic interventions are often available. (Moderate individual risk; low community risk) A pathogen that can cause human or animal disease but is unlikely to be a serious hazard to laboratory workers, the community, livestock or the environment. Laboratory exposures may cause serious infection, but effective treatment and preventive measures are available and the risk of spread of infection is limited.
Risk Group 3 Agents associated with serious or lethal human disease for which preventive or therapeutic interventions may be available (high individual risk but low community risk). (High individual risk; low community risk) A pathogen that usually causes serious human or animal disease but does not ordinarily spread from one infected individual to another. Effective treatment and preventive measures are available.
Risk Group 4 Agents likely to cause serious or lethal human disease for which preventive or therapeutic interventions are not usually available (high individual risk and high community risk). (High individual and community risk) A pathogen that usually causes serious human or animal disease and can be readily transmitted from one individual to another, directly or indirectly. Effective treatment and preventive measures are not usually available.
2. Biosafety Levels (BSL)
a. BSLs describe the facilities and work practices implemented when handling biological agents.
b. The CDC and NIH have established four biosafety levels which consist of combinations of laboratory practices and techniques, safety equipment including personal protective equipment, and laboratory facilities appropriate for the hazard posed by biological agents or recombinant/synthetic nucleic acid molecules.
c. BSLs should be differentiated from Risk Groups, as described in the NIH Guidelines and the World Health Organization Laboratory Biosafety Manual.
d. Risk groups are the result of a classification of microbiological agents based on their association with, and resulting severity of, disease in humans.
e. BMBL describes in detail the four BSLs covered in brief here.
1) Biosafety Level 1 (BSL-1) represents a basic level of containment that relies on standard microbiological practices with no special primary or secondary barriers recommended, other than a sink for hand washing, and is suitable for work involving well-characterized agents not known to consistently cause disease in immunocompetent adult humans, and present minimal potential hazard to laboratory personnel and the environment.
2) Biosafety Level 2 (BSL-2) practices, equipment, and facility design and construction are applicable to clinical, diagnostic, teaching, and other laboratories in which work is done with the broad spectrum of indigenous moderate-risk agents that are present in the community and associated with human disease of varying severity.
3) Biosafety Level 3 (BSL-3) practices, safety equipment, and facility design and construction are applicable to clinical, diagnostic, teaching, research, or production facilities in which work is done with indigenous or exotic agents with a potential for respiratory transmission, and which may cause serious and potentially lethal infection.
4) Biosafety Level 4 (BSL-4) practices, safety equipment, and facility design and construction are applicable for work with dangerous and exotic agents that pose a high individual risk of life-threatening disease, which may be transmitted via the aerosol route and for which there is no available vaccine or therapy At this time, the University of Oklahoma does not have a facility designed for work with organisms at BSL-4.
f. The risk group of an agent should be one factor considered in association with mode of transmission, procedural protocols, experience of staff, and other factors in determining the BSL in which the work will be conducted (Table 2).
3. Principles of Biosafety
a. A fundamental objective of any biosafety program is the containment of potentially harmful biological agents.
b. The term “containment” is used in describing safe methods, facilities, and equipment for managing infectious materials in the laboratory environment where they are being handled or maintained.
c. The purpose of containment is to reduce or eliminate exposure of laboratory workers, other persons, and the outside environment to potentially hazardous agents.
d. The use of vaccines may provide an increased level of personal protection.
e. The risk assessment of the work to be done with a specific agent will determine the appropriate combination of these elements.
• General Biosafety Laboratory Practices
a. Engineering controls such as biological safety cabinets (BSCs) should be examined and maintained or replaced on a regular schedule to ensure their effectiveness. It is imperative that Class I and II BSCs are tested and certified in place at the time of installation within the laboratory, at any time the cabinet is moved, and at least annually thereafter.
b. Gloves should be worn to protect hands from exposure to biological materials.
c. Glove selection should be based on an appropriate risk assessment and alternatives to latex gloves should be available.
d. Laboratory workers should change gloves when contaminated, integrity has been compromised, or when otherwise necessary.
e. Employees should wash their hands immediately or as soon as possible after removal of gloves or other personal protective equipment and after working with potentially hazardous materials or animals, blood, or other potentially infectious materials, and before leaving the laboratory.
f. Disposable gloves should not be washed or reused. Used gloves should be disposed of with other contaminated laboratory waste.
g. All personal protective equipment should be removed immediately upon leaving the work area or as soon as possible, if overtly contaminated, and placed in an appropriately designated area or container for storage, washing, decontamination, or disposal.
h. Policies for the safe handling of sharps, such as needles, scalpels, pipettes, and broken glassware should be developed and implemented. Whenever practical, P.I.s should adopt improved engineering and work practice controls that reduce risk of sharps injuries. Precautions, including those listed below, must always be taken with sharp items. These include:
1. Careful management of needles and other sharps are of primary importance. Needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal.
2. Sharps containers should be easily accessible to personnel and located in the area of use.
3. Immediately after use, contaminated sharps should be disposed in closable, puncture-resistant containers which are leak-proof on the sides and bottom and color-coded or labeled with the biohazard symbol.
4. Used disposable needles and syringes must be carefully placed in conveniently located puncture-resistant containers used for sharps disposal.
i. Non-disposable sharps must be placed in a hard-walled container for transport to a processing area for decontamination, preferably by autoclaving.
j. Safe needle devices should be used whenever such a device is available for the task requiring the use of a needle/syringe.
k. Broken glassware must not be handled directly. Instead, it must be removed using a brush and dustpan, tongs, or forceps.
l. Plasticware should be substituted for glassware whenever possible.
m. An integrated pest management program should be in effect.
n. Eating, drinking, smoking, applying cosmetics or lip balm, and handling contact lenses are prohibited in areas where work with biohazardous materials is performed
o. Food and drink must not be stored in refrigerators, freezers, or cabinets where biohazardous materials are stored.
p. All procedures involving biohazardous materials should be performed in such a manner as to minimize splashing, spraying, and aerosolization of these substances.
q. Mouth pipetting/suctioning is prohibited.
r. All cultures, stocks, other potentially infectious materials, and other regulated wastes must be decontaminated before disposal by an approved decontamination method, such as autoclaving.
s. Materials to be decontaminated outside of the immediate laboratory must be placed in a durable, leak-proof container and closed for transport from the laboratory.
t. Biomedical waste that will be shipped off-campus with a vendor must be packed in accordance with the procedures described in the University of Oklahoma Laboratory Safety Manual.
u. Biological hazard (biohazard) tags or labels should be used to identify the actual or potential presence of a biological hazard and to identify equipment, containers, rooms, experimental animals, or combinations thereof, that contain or are contaminated with hazardous biological agents.
v. Any significant research-related accidents and illnesses should be reported immediately to your supervisor and the IBC through the Institutional Biosafety Officer immediately.
1) Examples of situations that need to be reported include any spills or accidents in BSL-2 laboratories resulting in an overt exposure, spills of high-risk recombinant materials occurring outside of a biosafety cabinet, spills or accidents occurring in BSL-3 laboratories resulting in an overt or potential exposure, skin punctures with needles containing recombinant DNA, and the escape or improper disposition of a transgenic animal.
• Biosafety Level 1 (BSL-1)
1. In addition to the general biosafety laboratory procedures identified above, the following standard and special practices, safety equipment and facilities apply to agents and recombinant and synthetic nucleic acid molecules assigned to BSL-1.
1) Access to the laboratory should be limited or restricted at the discretion of the laboratory director when experiments are in progress. The Principal Investigator (P.I.) must enforce the institutional policies that control access to the laboratory.
2) Work surfaces should be decontaminated at least once a day and after any spill of viable or potentially infectious material with an appropriate disinfectant.
3) A sign incorporating the universal biohazard symbol must be posted at the entrance to the laboratory when infectious agents or organisms containing recombinant or synthetic nucleic acid molecules are present. The sign must include the name of the agent(s) in use and the name and phone number of the P.I. or other responsible personnel.
4) The P.I. must ensure that laboratory personnel receive appropriate training regarding their duties, the necessary precautions to prevent exposures, and exposure evaluation procedures.
5) Personnel must receive annual updates or additional training when procedural or policy changes occur.
6) Personal health status may impact an individual’s susceptibility to infection, ability to receive immunizations or prophylactic interventions. Therefore, all laboratory personnel and particularly women of child-bearing age should be provided with information regarding immune competence and conditions that may predispose them to infection.
1) Individuals having these conditions should be encouraged to self-identify to Employee Health for appropriate counseling and guidance.
7) Laboratory coats, gowns, or uniforms are recommended to be worn to prevent contamination of personal clothing.
8) The laboratory should be designed so that it can be easily cleaned. Carpets and rugs in laboratories are not appropriate.
9) Bench tops must be impervious to water and are resistant to moderate heat and the organic solvents, acids, alkalis, and other chemicals (such as those used to decontaminate the work surface and equipment).
10) Chairs used in laboratory work must be covered with a non-porous material that can be easily cleaned and decontaminated with an appropriate disinfectant.
11) Laboratory windows that open to the exterior should be fitted with screens.
• Biosafety Level 2 (BSL-2)
1. The vast majority of research performed at the University of Oklahoma campuses is conducted under BSL-2 containment. Research involving human blood, body fluids, cells, or unfixed tissue, which is covered by the CDC BSL-2 requirements, the NIH Guidelines and the OSHA Bloodborne Pathogen Standard must be conducted under BSL-2 containment. In addition to the general biosafety requirements, the BSL-2 requirements are as follows:
1) Access to the laboratory should be limited or restricted when work is being conducted. A sign incorporating the universal biohazard symbol must be posted at the entrance to the laboratory when infectious agents or recombinant/synthetic nucleic acid molecules are in use. Posted information must include:
1) the laboratory’s biosafety level; for recombinant organisms; the supervisor’s name (or other responsible personnel) and telephone number;
2) required procedures for entering and exiting the laboratory.
3) For work with organisms containing recombinant or synthetic nucleic acid molecules, the agent must also be posted.
b. The P.I. must ensure that laboratory personnel receive appropriate training regarding their duties, the necessary precautions to prevent exposures, and exposure evaluation procedures.
c. Personnel must receive annual updates or additional training when procedural or policy changes occur.
d. P.I. must ensure that laboratory personnel demonstrate proficiency in standard and special microbiological practices before working with BSL-2 agents. This training must be documented in the IBC protocol.
e. Laboratory personnel must be provided medical surveillance and offered appropriate immunizations for agents handled or potentially present in the laboratory
f. All persons entering the laboratory must be advised of the potential hazards and meet specific entry/exit requirements.
g. A laboratory specific, as well as possibly project specific, IBC protocol must be submitted to and approved by the University of Oklahoma Institutional Biosafety Committee. The approved protocol will serve as the Standard Operating Procedure for the laboratory/project and must be made available and accessible to all laboratory personnel.
h. Hypodermic needles and syringes should only be used for parenteral injection and aspiration of fluids from laboratory animals or diaphragm bottles.
i. Only needle-locking syringes or disposable syringe-needle unites, e.g. the needle is integral to the syringe, may be used for the injection or aspiration of fluids containing microorganisms or viruses.
j. Spills involving infectious materials and recombinant or synthetic nucleic acid molecules must be contained, decontaminated, and cleaned up by staff properly trained and equipped to work with infectious material.
k. All spills or potential exposures to potentially infectious material or recombinant/synthetic nucleic acid molecules must be reported to the principal investigator and the Institutional Biosafety Officer/Institutional Biosafety Committee.
l. Work surfaces should be decontaminated after completion of work or after any spill or splash of potentially infectious material or recombinant/synthetic nucleic acid molecules with an appropriate disinfectant that is effective against the agent(s) of concern.
m. Potentially infectious materials must be placed in a durable, leak proof container during collection, handling, processing, storage, or transport within a facility.
n. Laboratory equipment should be routinely decontaminated and decontaminated after spills, splashes, or other potential contamination.
o. Equipment must be decontaminated before repair, maintenance, or removal from the laboratory.
p. Incidents that may result in exposure to infectious materials or recombinant/synthetic nucleic acid molecules must be immediately evaluated and treated according to procedures described in the laboratory biosafety safety manual/SOPs. All such incidents must be reported to the P.I. and to the Institutional Biosafety Officer/Institutional Biosafety Committee. Exposures to recombinant or synthetic nucleic acid molecules will require a report to NIH. Medical evaluation, surveillance, and treatment through Employee Health or the nearest emergency room should be provided and appropriate records maintained.
q. Animals and plants not involved in the work being performed are not permitted in the lab.
r. All procedures involving the manipulation of infectious materials or recombinant/synthetic nucleic acid molecules that may generate an aerosol should be conducted within a biological safety cabinet (BSC) or other physical containment devices. Properly maintained BSCs (preferably Class II), other appropriate personal protective equipment, or other physical containment devices must be used whenever:
1) Procedures with a potential for creating infectious aerosols or splashes are conducted. These may include pipetting, centrifuging, grinding, blending, shaking, mixing, sonicating, opening containers of infectious materials, inoculating animals intranasally, and harvesting infected tissues from animals or embryonate eggs.
2) High concentrations or large volumes of infectious agents are used. Such materials may be centrifuged in the open laboratory using sealed rotor heads or centrifuge safety cups are used, and if these rotors or safety cups are opened only in a BSC.
s. Protective laboratory coats, gowns, smocks, or uniforms designated for lab use must be worn when working with hazardous materials. This protective clothing should be removed and left in the laboratory before leaving for non-laboratory areas (e.g., cafeteria, library, administrative offices).
t. All protective clothing should be either disposed properly or deposited for laundering by the institution. Laboratory protective clothing should not be taken home.
u. Eye and face protection (goggles, mask, face shield or other splatter guards) should be used for anticipated splashes or sprays of infectious or other hazardous materials when the microorganisms must be manipulated outside the BSC or containment device.
v. Gloves must be worn to protect hands from exposure to hazardous material.
a) Wearing two pairs of gloves may be appropriate.
b) Gloves must not be worn outside the laboratory.
c) Gloves should be disposed with other contaminated laboratory waste.
w. Laboratories must have a sink for handwashing. The sink may be manually, hands-free, or automatically operated. It should be located near the exit door.
x. The laboratory should be designed so that it can be easily cleaned and decontaminated. Carpets and rugs in laboratories are not permitted.
y. Chairs and other furniture used in laboratory work should be covered with a non-porous material that can be easily cleaned and decontaminated with appropriate disinfectant.
z. Vacuum lines should be protected with High Efficiency Particulate Air (HEPA) filters, or their equivalent. Filters must be replaced as needed. Liquid disinfectant traps may be required.
aa. An eyewash station must be readily available.
bb. A method for decontaminating all laboratory wastes should be available in the facility (e.g., autoclave, chemical disinfection, incineration, or other validated decontamination method).
• Biosafety Level 3 (BSL-3)
1. BSL-3 containment places higher emphasis on primary and secondary barriers to protect personnel in contiguous areas, the community, and the environment from exposure to potentially infectious aerosols.
2. Laboratory personnel must receive specific training in handling pathogenic and potentially lethal agents, and must be supervised by scientists competent in handling infectious agents and associated procedures.
3. All procedures involving the manipulation of infectious materials must be conducted within BSCs or other physical containment devices, or by personnel wearing appropriate personal protective equipment.
4. Oversight of the University of Oklahoma BSL-3 facility is provided by the Institutional Biosafety Committee and the Director of the BSL-3 laboratory.
5. The laboratory is available for use by all OU investigators, but the Principal Investigator and laboratory users must first be trained on the BSL-3 specific SOPs by the BSL-3 Director, and the principal investigator must obtain IBC approval prior to beginning the project.
a. To obtain more detailed information, please contact the Biosafety Officer at IBC@ouhsc.edu.
• Biosafety Level 4 (BSL-4)
1. Biosafety Level 4 (BSL4) is required for work with dangerous and exotic agents which pose a high individual risk of aerosol-transmitted laboratory infections and life-threatening disease.
2. The University of Oklahoma does not currently maintain a facility designed for work with organisms at BSL4.
BSL Agents Practices Primary Barriers and Safety Equipment Facility (Secondary Barriers)
1 Not known to consistently cause diseases in healthy adults Standard Microbiological Practices ? None required
? PPE: Lab coats and gloves; eye and face protection as needed Laboratory bench and sink
2 Agents associated with human disease:
? Routes of transmission include percutaneous injury, ingestion, mucous membrane exposure BSL-1 practices plus:
? Limited access
? Biohazard warning signs
? “sharps precautions
? Biosafety manual defining any needed waste decontamination or medical evaluation program Primary barriers:
? Class I or II BSCs or other physical containment devices used for all manipulations of agents that cause splashes or aerosol of infectious materials
? PPE: Lab coats;
gloves; face protection as needed BSL-2 plus:
? Autoclave available
3 Indigenous or exotic agents with potential for aerosol.
Diseases may have serious or lethal consequences BSL-2 practices plus:
? Controlled access
? Decontamination of all waste
? Decontamination of laboratory clothing before laundering
? Baseline serum Primary barriers:
? Class I or II BSCs or other physical containment devices used for all open manipulation of agents
? PPE:
Protective laboratory clothing; gloves; respiratory protection as needed BSL-2 plus:
? Physical separation from access corridors
? Self-closing, double door access
? Exhaust air not recirculated
? Negative airflow into laboratory
Table 2. Summary of Recommended Biosafety Levels
• Animal Biosafety Levels
1. Four standard biosafety levels are also described for activities involving infectious disease work with commonly used experimental animals. These four combinations of practices, safety equipment, and facilities are designated Animal Biosafety Levels 1, 2, 3, and 4, and provide increasing levels of protection to personnel and the environment.
2. One additional biosafety level, designated BSL-3-Agriculture (or BSL 3-Ag) addresses activities involving large or loose-housed animals and/or studies involving agents designated as High Consequence Pathogens by the USDA. BSL 3-Ag laboratories are designed so that the laboratory facility itself acts as a primary barrier to prevent release of infectious agents into the environment (Table 3).
3. Currently OU maintains facilities for ABSL-1 and ABSL-2 containment.
a. Work that requires ABSL-2 containment must be registered with and approved by both the Institutional Biosafety Committee and the Institutional Animal Care and Use Committee.
b. In conjunction with the IACUC and Comparative Medicine, the IBC has set standard SOPs for working at ABSL-1 and ABSL-2 containment. In addition, it is important to note that the IACUC and the IBC have developed a specific SOP, IACUC SOP 306: Working with Human Cells in Animals, that requires the use of ABSL-2 containment for any research that involves the administration of primary human cells or human cell lines to animals.
• Animal Biosafety level 1 (ABSL-1)
1. Suitable for work in animals involving well-characterized agents that are not known to cause disease in immunocompetent adult humans, and present minimal potential hazard to personnel and the environment.
2. ABSL-1 facilities should be separated from the general traffic patterns of the building and restricted as appropriate. Special containment equipment or facility design may be required as determined by appropriate risk assessment.
3. Personnel must have specific training in animal facility procedures and must be supervised by an individual with adequate knowledge of potential hazards and experimental animal procedures.
4. The following standard practices, safety equipment, and facility requirements apply to ABSL-1.
a. Standard Microbiological Practices:
1) The animal facility director establishes policies procedures and protocols for emergency situations. Prior to any project’s initiation each protocol must be submitted for review and approval by the Institutional Animal care and Use committee (IACUC) and the Institutional Biosafety Committee (IBC). Any special practices are approved at this time.
2) A safety manual specific to the animal facility is prepared or adopted in consultation with the animal facility director and appropriate safety professionals. The safety manual must be available and accessible.
3) Personnel are advised of potential hazards and are required to read and follow instructions on practices and procedures.
4) The supervisor must ensure that animal care, laboratory and support personnel receive appropriate training regarding their duties, animal husbandry procedures, potential hazards, manipulations of infectious agents, necessary precautions to prevent exposures, and hazard/exposure evaluation procedures (physical hazards, splashes, aerosolization, etc.).
5) Personnel must receive annual updates and additional training when procedures or policies change.
6) Records are maintained for all hazard evaluations, employee training sessions and staff attendance.
7) All employees working with animals must participate in the medical surveillance program through Employee Health, and must complete and submit the Animal Handler’s Questionnaire to Employee Health.
8) Personal health status may impact an individual’s susceptibility to infection, ability to receive immunizations or prophylactic interventions. Therefore, all personnel and particularly women of child-bearing age should be provided information regarding immune competence and conditions that may predispose them to infection.
9) Individuals having these conditions should be encouraged to self-identify to the institution’s healthcare provider for appropriate counseling and guidance.
10) Personnel using respirators must be enrolled in an appropriately constituted respiratory protection program.
11) A sign incorporating safety information must be posted at the entrance to the areas where infectious materials and/or animals are housed or are manipulated.
a) The sign must include the animal biosafety level, general occupational health requirements, personal protective equipment requirements, the supervisor’s name (or other responsible personnel), telephone number, and required procedures for entering and exiting the animal areas.
b) Identification of specific infectious agents is recommended when more than one agent is being used within an animal room. Security-sensitive agent information should be posted in accordance with the institutional policy.
12) Access to the animal room is limited. Only those persons required for program or support purposes are authorized to enter the facility. All persons including facility personnel, service workers, and visitors are advised of the potential hazards (natural or research pathogens, allergens, etc.) and are instructed on the appropriate safeguards.
13) Protective laboratory coats, gowns, or uniforms are recommended to prevent contamination of personal clothing.
a) Gloves are worn to prevent skin contact with contaminated, infectious and hazardous materials, and when handling animals.
b) Gloves and personal protective equipment should be removed in a manner that minimizes transfer of infectious materials outside of the areas where infectious materials and/or animals are housed or are manipulated.
c) Persons must wash their hands after removing gloves, and before leaving the area where infectious materials and/or animals are housed or are manipulated.
d) Eye and face and respiratory protection should be used in rooms containing infected animals, as dictated by the risk assessment.
14) Eating, drinking, smoking, handling contact lenses, applying cosmetics and storing food for human consumptions must not be permitted in laboratory areas.
a) Food must be stored outside of the laboratory in cabinets or refrigerators designed and used for this purpose
15) All procedures are carefully performed to minimize the creation of aerosols or splatters of infectious materials and waste.
16) Mouth pipetting is prohibited. Mechanical pipetting devices must be used.
17) Policies for the safe handling of sharps, such as needles, scalpels, pipettes, and broken glassware must be developed and implemented. When applicable, laboratory supervisors should adopt improved engineering and work practice controls that reduce the risk of sharps injuries. Precautions, including those listed below, must always be taken with sharp items. These include:
a) Use of needles and syringes or other sharp instruments in the animal facility is limited to situations where there is no alternative for such procedures as parenteral injection, blood collection, or aspiration of fluids from laboratory animals and diaphragm bottles
b) Disposable needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal. Used disposable needles must be carefully placed in puncture-resistant containers used for sharps disposal. Sharps containers should be located as close to the work site as possible.
c) Non-disposable sharps must be placed in a hard-walled container for transport to a processing area for decontamination, preferably by autoclaving.
18) Broken glassware must not be handled directly. Instead, it must be removed using a brush and dustpan, tongs, or forceps.
a) Plastic ware should be substituted for glassware whenever possible.
19) Equipment containing sharp edges and corners should be avoided.
20) Equipment and work surfaces are routinely decontaminated with an appropriate disinfectant after work with an infectious agent, and after any spills, splashes, or other overt contamination.
21) Animals and plants not associated with the work being performed must not be permitted in the areas where infectious materials and/ or animals are housed or are manipulated.
22) An effective integrated pest management program is required.
23) All wastes from the animal room (including animal tissues, carcasses, and bedding) are transported from the animal room in leak-proof, covered containers for appropriate disposal in compliance with applicable institutional, local and state requirements.
a) Decontaminate all potentially infectious materials before disposal using an effective method.
b. Special Practices
1) None required.
c. Safety Equipment (Primary Barriers and Personal Protective Equipment)
1) A risk assessment should determine the appropriate type of personal protective equipment to be utilized.
2) Special containment devices or equipment may not be required as determined by appropriate risk assessment.
3) Protective laboratory coats, gowns, or uniforms may be required to prevent contamination of personal clothing.
a) Protective outer clothing is not worn outside areas where infectious materials and/or animals are housed or manipulated.
b) Gowns and uniforms are not worn outside the facility.
4) Protective eyewear is worn when conducting procedures that have the potential to create splashes of microorganisms or other hazardous materials.
5) Persons who wear contact lenses should also wear eye protection when entering areas with potentially high concentrations or airborne particulates.
6) Persons having contact with NHPs must assess risk of mucous membrane exposure and wear protective equipment (e.g., masks, goggles, face shields, etc.) as appropriate for the task to be performed.
7) Gloves are worn to protect hands from exposure to hazardous materials. A risk assessment should be performed to identify the appropriate glove for the task and alternatives to latex gloves should be available.
a) Change gloves when contaminated, glove integrity is compromised, or when otherwise necessary.
b) Gloves must not be worn outside the animal rooms.
c) Gloves and personal protective equipment should be removed in a manner that prevents transfer of infectious materials. Do not wash or reuse disposable gloves.
d) Dispose of used gloves with other contaminated waste.
8) Persons must wash their hands after handling animals and before leaving the areas where infectious materials and/or animals are housed or are manipulated. Hand washing should occur after the removal of gloves.
• Animal Biosafety Level 2 (ABSL-2)
1. Animal Biosafety Level 2 builds upon the practices, procedures, containment equipment, and facility requirements of ABSL-1.
2. ABSL-2 is suitable for work involving laboratory animals infected with agents associated with human disease and pose moderate hazards to personnel and the environment. It also addresses hazards from ingestion as well as from percutaneous and mucous membrane exposure.
3. ABSL-2 requires that:
a. access to the animal facility is restricted;
b. personnel must have specific training in animal facility procedures, the handling of infected animals and the manipulation of pathogenic agents;
c. personnel must be supervised by individuals with adequate knowledge of potential hazards, microbiological agents, animal manipulations and husbandry procedures; and
d. BSCs or other physical containment equipment is used when procedures involve the manipulation of infectious materials, or where aerosols or splashes may be created.
e. Appropriate personal protective equipment must be utilized to reduce exposure to infectious agents, animals, and contaminated equipment. Implementation of employee occupational health programs should be considered.
4. The following standard and special practices, safety equipment, and facility requirements apply to ABSL-2:
a. Standard Microbiological Practices
1) The animal facility director establishes and enforces policies, procedures, and protocols for institutional policies and emergencies. Each organization must assure that worker safety and health concerns are addressed as part of the animal protocol review. Prior to beginning a study, animal protocols must also be reviewed and approved by the IACUC and the Institutional Biosafety Committee.
2) A safety manual specific to the animal facility is prepared or adopted in consultation with the animal facility director and appropriate safety professionals. The safety manual must be available and accessible. Personnel are advised of potential hazards, and are required to read and follow instructions on practices and procedures. Consideration should be given to specific biohazards unique to the animal species and protocol in use.
3) The supervisor must ensure that animal care, laboratory, and support personnel receive appropriate training regarding their duties, animal husbandry procedure, potential hazards, manipulations of infectious agents, necessary precautions to prevent hazard or exposures, and hazard/exposure evaluation procedures (physical hazards, splashes, aerosolization, etc.).
4) Personnel must receive annual updates or additional training when procedures or policies change. Records are maintained for all hazard evaluations, employee training sessions and staff attendance.
5) All employees working with animals must participate in the medical surveillance program through Employee Health, and must complete and submit the Animal Handler’s Questionnaire to Employee Health.
a) Personal health status may impact an individual’s susceptibility to infection, ability to receive immunizations or prophylactic interventions. Therefore, all personnel and particularly women of child-bearing age should be provided information regarding immune competence and conditions that may predispose them to infection.
b) Individuals having these conditions should be encouraged to self-identify to the institution’s healthcare provider for appropriate counseling and guidance. Personnel using respirators must be enrolled in an appropriately constituted respiratory protection program.
6) A sign incorporating the universal biohazard symbol must be posted at the entrance to areas where infectious materials and/ or animals are housed or are manipulated when infectious agents are present.
a) The sign must include the animal biosafety level, general occupational health requirements, personal protective equipment requirements, the supervisor’s name (or names of other responsible personnel), telephone number, and required procedures for entering and exiting the animal areas.
b) Identification of all infectious agents is necessary when more than one agent is being used within an animal room. Security-sensitive agent information and occupational health requirements should be posted in accordance with the institutional policy.
7) Advance consideration should be given to emergency and disaster recovery plans, as a contingency for man-made or natural disasters.
8) Access to the animal room is limited. Only those persons required for program or support purposes are authorized to enter the animal facility and the areas where infectious materials and/or animals are housed or manipulated. All persons including facility personnel, service workers, and visitors are advised of the potential hazards (physical, naturally occurring, or research pathogens, allergens, etc.) and are instructed on the appropriate safeguards.
9) Protective laboratory coats, gowns, or uniforms are recommended to prevent contamination of personal clothing.
a) Gloves are worn to prevent skin contact with contaminated, infectious and hazardous materials and when handling animals.
b) Gloves and personal protective equipment should be removed in a manner that prevents transfer of infectious materials outside of the areas where infectious materials and/or animals are housed or are manipulated.
c) Persons must wash their hands after removing gloves, and before leaving the areas where infectious materials and/or animals are housed or are manipulated.
d) Eye, face and respiratory protection should be used in rooms containing infected animals, as dictated by the risk assessment.
10) Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human consumption must not be permitted in laboratory areas.
a) Food must be stored outside of the laboratory in cabinets or refrigerators designated and used for this purpose.
11) All procedures are carefully performed to minimize the creation of aerosols or splatters of infectious materials and waste.
12) Mouth pipetting is prohibited. Mechanical pipetting devices must be used.
13) Policies for the safe handling of sharps, such as needles, scalpels, pipettes, and broken glassware must be developed and implemented. When applicable, laboratory supervisors should adopt improved engineering and work practice controls that reduce the risk of sharps injuries. Precautions must always be taken with sharp items. These include:
a) The use of needles and syringes or other sharp instruments in the animal facility is limited to situations where there is no alternative such as parenteral injection, blood collection, aspiration of fluids from laboratory animals and diaphragm bottles.
b) Disposable needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal. Used, disposable needles must be carefully placed in puncture-resistant containers used for sharps disposal. Sharps containers should be located as close to the work site as possible.
c) Non-disposable sharps must be placed in a hard-walled container for transport to a processing area for decontamination, preferably by autoclaving.
14) Broken glassware must not be handled directly; it should be removed using a brush and dustpan, tongs, or forceps.
a) Plastic ware should be substituted for glassware whenever possible.
15) Use of equipment with sharp edges and corners should be avoided.
16) Equipment and work surfaces are routinely decontaminated with an appropriate disinfectant after work with an infectious agent, and after any spills, splashes, or other overt contamination.
17) Animals and plants not associated with the work being performed must not be permitted in the areas where infectious materials and/ or animals are housed or manipulated.
18) An effective integrated pest management program is required.
19) All wastes from the animal room (including animal tissues, carcasses, and bedding) are transported from the animal room in leak-proof containers for appropriate disposal in compliance with applicable institutional, local and state requirements.
b. Special Practices
1) Animal care staff, laboratory and routine support personnel must be provided a medical surveillance program as dictated by the risk assessment and administered appropriate immunizations for agents handled or potentially present, before entry into animal rooms.
2) When appropriate, a base line serum sample should be stored.
3) Procedures involving a high potential for generating aerosols should be conducted within a biosafety cabinet or other physical containment device.
a) When a procedure cannot be performed within a biosafety cabinet, a combination of personal protective equipment and other containment devices must be used.
b) Restraint devices and practices that reduce the risk of exposure during animal manipulations (e.g., physical restraint devices, chemical restraint medications) should be used whenever possible.
4) Decontamination by an appropriate method (e.g. autoclave, chemical disinfection, or other approved decontamination methods) is necessary for all potentially infectious materials and animal waste before movement outside the areas where infectious materials and/or animals are housed or are manipulated.
5) This includes potentially infectious animal tissues, carcasses, contaminated bedding, unused feed, sharps, and other refuse.
6) A method for decontaminating routine husbandry equipment, sensitive electronic and medical equipment should be identified and implemented.
7) Materials to be decontaminated outside of the immediate areas where infectious materials and/or animals are housed or are manipulated must be placed in a durable, leak proof, covered container and secured for transport.
a) The outer surface of the container is disinfected prior to moving materials.
b) The transport container must have a universal biohazard label.
c) Develop and implement an appropriate waste disposal program in compliance with applicable institutional, local and state requirements.
d) Autoclaving of content prior to incineration is recommended.
8) Equipment, cages, and racks should be handled in a manner that minimizes contamination of other areas.
a) Equipment must be decontaminated before repair, maintenance, or removal from the areas where infectious materials and/or animals are housed or are manipulated.
9) Spills involving infectious materials must be contained, decontaminated, and cleaned up by staff properly trained and equipped to work with infectious material.
10) Incidents that may result in exposure to infectious materials must be immediately evaluated and treated according to procedures described in the safety manual.
a) All such incidents must be reported to the animal facility supervisor or personnel designated by the institution.
b) Medical evaluation, surveillance, and treatment should be provided as appropriate and records maintained.
c. Safety Equipment (Primary Barriers and Personal Protective Equipment)
1) Properly maintained BSCs, personal protective equipment (e.g., gloves, lab coats, face shields, respirators, etc.) and/or other physical containment devices or equipment, are used whenever conducting procedures with a potential for creating aerosols, splashes, or other potential exposures to hazardous materials.
2) These include necropsy of infected animals, harvesting of tissues or fluids from infected animals or eggs, and intranasal inoculation of animals.
3) When indicated by risk assessment, animals are housed in primary biosafety containment equipment appropriate for the animal species, such as solid wall and bottom cages covered with filter bonnets for rodents or other equivalent primary containment systems for larger animal cages.
4) A risk assessment should determine the appropriate type of personal protective equipment to be utilized.
a) Scrubs and uniforms are removed before leaving the animal facility.
b) Reusable clothing is appropriately contained and decontaminated before being laundered.
c) Laboratory and protective clothing should never be taken home.
d) Gowns, uniforms, laboratory coats and personal protective equipment are worn while in the areas where infectious materials and/or animals are housed or manipulated and removed prior to exiting.
e) Disposable personal protective equipment and other contaminated waste are appropriately contained and decontaminated prior to disposal.
5) Eye and face protection (mask, goggles, face shield or other splatter guard) are used for manipulations or activities that may result in splashes or sprays from infectious or other hazardous materials and when the animal or microorganisms must be handled outside the BSC or containment device.
a) Eye and face protection must be disposed of with other contaminated laboratory waste or decontaminated before reuse.
b) Persons who wear contact lenses should also wear eye protection when entering areas with potentially high concentrations or airborne particulates.
c) Persons having contact with NHPs should assess risk of mucous membrane exposure and wear protective equipment (e.g., masks, goggles, face shields) appropriate for the task to be performed. Respiratory protection is worn based upon risk assessment.
6) Gloves are worn to protect hands from exposure to hazardous materials. A risk assessment should be performed to identify the appropriate glove for the task and alternatives to latex gloves should be available.
a) Gloves are changed when contaminated, glove integrity is compromised, or when otherwise necessary.
b) Gloves must not be worn outside the animal rooms.
c) Gloves and personal protective equipment should be removed in a manner that prevents transfer of infectious materials.
d) Do not wash or reuse disposable gloves. Dispose of used gloves with other contaminated waste.
e) Persons must wash their hands after handling animals and before leaving the areas where infectious materials and/or animals are housed or are manipulated. Hand washing should occur after the removal of gloves.
BSL Agents Practices Primary Barriers and Safety Equipment Facility (Secondary Barriers)
1 Not known to consistently cause diseases in healthy adults Standard animal care and management practices, including appropriate medical surveillance programs As required for normal care of each species
? PPE: Lab coats and gloves; eye and face protection as needed Standard animal facility:
? No recirculation of exhaust air
? Directional air flow recommended
? Hand washing sink is available
2 Agents associated with human disease
Hazard: percutaneous injury, ingestion, mucous membrane exposure ABSL-1 practices plus:
? Limited access
? Biohazard warning signs
? “sharps” precautions
? Biosafety manual defining any needed waste
? Decontamination of all infectious wastes and animal cages prior to washing ABSL-1 equipment plus primary barriers:
? Containment equipment appropriate for animal special
? If available, use of class I, II, or III BSCs or other engineering containment devices used for all manipulations that cause splashes or aerosols
? PPE: Lab coats and gloves; eye and face protection as needed BSL-2 plus:
? Autoclave available
? Hand washing sink available
? Mechanical cage washer recommended
? Negative airflow into animal and procedure rooms recommended
Table 3. Recommended Animal Biosafety Levels
• Guidelines for Work with Biological Toxins
1. Each laboratory worker must be trained in the theory and practice of the biological toxins to be used, with special emphasis on the nature of the practical hazards associated with laboratory operations.
a. This includes how to handle transfers of liquids containing toxin, where to place waste solutions and contaminated materials or equipment, and how to decontaminate work areas after routine operations, as well as after accidental spills.
b. The worker must be reliable and sufficiently adept at all required manipulations before being provided with toxin.
2. A risk assessment should be conducted to develop safe operating procedures before undertaking laboratory operations with toxins. If toxins and infectious agents are used together, then both must be considered when containment equipment is selected and safety procedures are developed. Likewise, animal safety practices must be considered for toxin work involving animals.
3. Each laboratory that uses toxins should develop standard operating procedures specific for the toxin(s) to be used.
4. An inventory control system should be in place to account for toxin use and disposition.
a. If toxins are stored in the laboratory, containers should be sealed, labeled, and secured to ensure restricted access; refrigerators and other storage containers should be clearly labeled and provide contact information for trained, responsible laboratory staff.
5. Laboratory work with toxins should be done only in designated rooms with controlled access and at pre-determined bench areas.
a. When toxins are in use, the room should be clearly posted: “Toxins in Use-Authorized Personnel Only.”
b. Unrelated and nonessential work should be restricted from areas where stock solutions of toxin or organisms producing toxin are used.
c. Visitors or other untrained personnel granted laboratory access must be monitored and protected from inadvertently handling laboratory equipment used to manipulate the toxin or organism.
6. Routine operations with dilute toxin solutions should be conducted under BSL-2 conditions with the aid of personal protective equipment and a well-maintained BSC or comparable engineering controls.
a. Engineering controls should be selected according to the risk assessment for each specific toxin operation.
b. A certified BSC or chemical fume hood will suffice for routine operations with most protein toxins.
c. Low molecular weight toxin solutions, or work involving volatile chemicals or radionucleotides combined with toxin solutions, may require the use of a charcoal-based hood filter in addition to HEPA filtration.
d. All work with toxins should be conducted within the operationally effective zone of the hood or BSC, and each user should verify the inward airflow before initiating work.
7. When using an open-fronted fume hood or BSC, workers should wear suitable laboratory PPE to protect the hands and arms, such as laboratory coats, smocks, or coveralls and disposable gloves.
a. When working with toxins that pose direct percutaneous hazards, special care must be taken to select gloves that are impervious to the toxin and the diluents or solvents employed.
b. When conducting liquid transfers and other operations that pose a potential splash or droplet hazard in an open-fronted hood or BSC, safety glasses and disposable N-95 respirator and/or a face shield, should be worn.
c. Toxin should be removed from the hood or BSC only after the exterior of the closed primary container has been decontaminated and placed in a clean secondary container.
d. Toxin solutions, especially concentrated stock solutions, should be transported in leak/spill-proof secondary containers.
e. The interior of the hood or BSC should be decontaminated periodically, for example, at the end of a series of related experiments. Until thoroughly decontaminated, the hood or BSC should be posted to indicate that toxins remain in use, and access should remain restricted.
8. Selected operations with toxins may require modified BSL-3 practices and procedures.
9. The determination to use BSL-3 should be made in consultation with the Institutional Biosafety Committee and is based upon a risk assessment that considers the variables of each specific laboratory operation, especially the toxin under study, the physical state of the toxin (solution or dry form), the total amount of toxin used relative to the estimated human lethal dose, the volume of the material manipulated, the methodology, and any human or equipment performance limitations.
10. Emphasis must be placed on evaluating and modifying experimental procedures to eliminate the possibility of inadvertent generation of toxin aerosols.
11. Pressurized tubes or other containers holding toxins should be opened in a BSC, chemical fume hood, or other ventilated enclosure.
a. Operations that expose toxin solutions to vacuum or pressure, for example sterilization of toxin solutions by membrane filtration, should always be handled in this manner, and the operator should also use appropriate respiratory protection.
b. If vacuum lines are used with toxin, they should be protected with a HEPA filter to prevent entry of toxins into the line.
c. Centrifugation of cultures or materials potentially containing toxins should only be performed using sealed, thick-walled tubes in safety centrifuge cups or sealed rotors.
d. The outside surfaces of containers and rotors should be routinely cleaned before each use to prevent contamination that may generate an aerosol.
e. After centrifugation, the entire rotor assembly is taken from the centrifuge to a BSC to open it and remove its tubes.
12. Accidental needle-sticks or mechanical injury from “sharps” such as glass or metal implements pose a well-known risk to laboratory workers, and the consequences may be catastrophic for operations involving toxins in amounts that exceed a human lethal dose.
a. Only workers trained and experienced in handling animals should be permitted to conduct operations involving injection of toxin solutions using hollow-bore needles.
b. Discarded needles/syringes and other sharps should be placed directly into properly labeled, puncture-resistant sharps containers, and decontaminated as soon as is practical.
c. Glassware should be replaced with plastic for handling toxin solutions wherever practical to minimize the risk of cuts or abrasions from contaminated surfaces.
d. Thin-walled glass equipment should be completely avoided. Glass Pasteur pipettes are particularly dangerous for transferring toxin solutions and should be replaced with disposable plastic pipettes.
e. Glass chromatography columns under pressure must be enclosed within a plastic water jacket or other secondary container.
13. Experiments should be planned to eliminate or minimize work with dry toxin (e.g. freeze-dried preparations).
a. Unavoidable operations with dry toxin should only be undertaken with appropriate respiratory protection and engineering controls.
b. Dry toxin can be manipulated using a Class III BSC, or with the use of secondary containment such as a disposable glove bag or glove box within a hood or Class II BSC.
c. “Static-free” disposable gloves should be worn when working with dry forms of toxins that are subject to spread by electrostatic dispersal.
d. In specialized laboratories, the intentional, controlled generation of aerosols from toxin solutions may be undertaken to test antidotes or vaccines in experimental animals. These are extremely hazardous operations that should only be conducted after extensive validation of equipment and personnel, using non-toxic simulants.
1) Aerosol exposure of animals should be done in a certified Class III BSC or hood line.
2) While removing exposed animals from the hood line, and for required animal handling during the first 24 hours after exposure, workers should take additional precautions, including wearing protective clothing (e.g., disposable Tyvek suit) and appropriate respiratory protection.
3) To minimize the risk of dry toxin generating a secondary aerosol, areas of animal skin or fur exposed to aerosols should be gently wiped with a damp cloth containing water or buffered cleaning solution before the animals are returned to holding areas.
4) For high-risk operations involving dry forms of toxins, intentional aerosol formation, or the use of hollow-bore needles in conjunction with amounts of toxin estimated to be lethal for humans, consideration should be given to requiring the presence of at least two knowledgeable individuals at all times in the laboratory.
14. Procedures for toxin inactivation should be established and followed
a. Depending upon the toxin, contaminated materials and toxin waste solutions can be inactivated by incineration or extensive autoclaving, or by soaking in suitable decontamination solutions
b. All disposable material used for toxin work should be placed in secondary containers, autoclaved and disposed of as toxic waste.
c. Contaminated or potentially contaminated protective clothing and equipment should be decontaminated using suitable chemical methods or autoclaving before removal from the laboratory for disposal, cleaning or repair.
d. If decontamination is impracticable, materials should be disposed of as hazardous or biohazardous waste, in consultation with the EHSO.
15. In the event of a spill, avoid splashes or generating aerosols during cleanup by covering the spill with paper towels or other disposable, absorbent material.
a. Apply an appropriate decontamination solution to the spill, beginning at the perimeter and working towards the center, and allow sufficient contact time to completely inactivate the toxin.
• Working with Human Blood or Other Potentially Infectious Agents/Diseases
1. Definition of Infectious Disease:
a. Infectious diseases are caused by pathogenic microorganisms, such as bacteria, viruses, parasites or fungi; the diseases can be spread, directly or indirectly, from one person to another. Zoonotic diseases are infectious diseases of animals that can cause disease when transmitted to humans.
2. Departments with employees who have reasonably anticipated eye, skin, mucous membrane or parenteral contact with human blood or other potentially infectious materials must follow the University of Oklahoma Exposure Control Plan in compliance with the OSHA Bloodborne Pathogen Standard.
3. Human body fluids:
a. such as semen, vaginal secretions, pericardial fluid, cerebrospinal fluid, synovial fluid, pleural fluid, peritoneal fluid, amniotic fluid, saliva in dental procedures, body fluid that is visibly contaminated with blood and all body fluids in situations where it is difficult or impossible to differentiate between body fluids.
b. Other body such any unfixed tissue or organ.
c. (other than intact skin) from a human living or dead culture, human organ tissue, HIV or hepatitis B virus (HBV) containing culture medium or other solutions and blood, organs, or other tissues from experimental animals infected with HIV, HBV or other bloodborne pathogens infectious to men.
4. Special Procedures for Human Primary Cells/Cell Lines
a. Only established human cell lines and human cell strains which are characterized (tested by antigenic screening for viral or agent markers, co-cultivation with indicator cells allowing contaminants to grow, or molecular technology such as polymerase chain reaction or nucleic acid hybridization) to be free of bloodborne pathogens (including HIV, HBV, Epstein-Barr virus, Herpes virus and papilloma members of the Papovavirus group, etc.) and documented as such may be excluded from the requirements of the OSHA Bloodborne Pathogen Standard.
b. Cell lines/strains that are procured from commercial vendors or other sources with documented testing to be free of human bloodborne pathogens and which have been protected from contamination may be excluded from the requirements of the OSHA Bloodborne Pathogen Standard.
c. Use of human tissue/cells/cell lines in the laboratory setting requires the approval of the University of Oklahoma Institutional Review Board.
5. Universal precautions should be observed which dictates that all human blood and other potentially infectious materials should be treated as infectious for HBV, HIV, and other bloodborne pathogens.
6. Engineering and work practice controls should be utilized first to minimize employee exposure. Where occupational exposure remains after the institution of engineering controls, PPE should also be used as follows:
a. Gloves should be worn when it can be reasonably anticipated that the employee may have hand contact with blood or other potentially infectious materials, mucous membranes, and non-intact skin such as during phlebotomies and when handling or touching contaminated items.
b. Masks in combination with eye protection devices such as goggles or face shields should be worn whenever splashes, spray, spatter, or droplets of blood or other potentially infectious material may be generated and eye, nose, or mouth contamination can be reasonably anticipated.
c. Gowns, aprons, lab coats, surgical caps or hoods, and/or shoe covers should be worn when gross contamination can be reasonably anticipated.
a. The type and characteristics of this protective clothing will depend upon the task and degree of exposure anticipated.
7. Handwashing facilities should be readily accessible to employees.
a. Personnel in work areas that do not have handwashing facilities readily accessible should be provided with an appropriate hand cleanser in conjunction with clean cloth or paper towels or antiseptic towelettes.
b. Employees should wash their hands with soap and running water as soon as feasible after using antiseptic hand cleansers or towelettes.
8. All garments should be removed as soon as possible if penetrated by blood or other potentially infectious material.
9. Removed PPE should be placed in a designated area or container for storage, washing, decontamination, or disposal.
a. Contaminated PPE should be placed in a designated container labeled with the biohazard symbol.
b. PPE should be cleaned, laundered and/or disposed in a proper manner.
1) Contaminated disposable PPE should be placed in a biohazard bag until it can be sterilized/autoclaved.
2) Contaminated launderable PPE should be placed in a container labeled with the biohazard symbol until sent to an appropriate laundry in accordance with the University of Oklahoma Exposure Control Plan.
3) Employees should wash their hands immediately or as soon as feasible after removal of gloves or other PPE.
10. Employees should wash their hands or other skin with soap and water or flush mucous membranes with water immediately or as soon as feasible following contact of such body areas with blood or other potentially infectious materials.
11. Safe needle devices should be used where possible or appropriate.
12. Contaminated needles or other contaminated sharps should not be bent, recapped, or removed.
a. If needles must be recapped, a mechanical means or a one-handed technique should be used.
13. Immediately or as soon as possible after use, contaminated sharps should be placed in appropriate containers, even if the sharps are reusable and will be reprocessed.
a. These containers should be:
1) puncture resistant,
2) labeled with the biohazard symbol or color-coded,
3) leak-proof on the sides and bottom, and
4) not be allowed to overfill (a good guideline is to dispose when approximately two-thirds full).
b. Other guidelines for selection of sharps containers should consider issues such as lids that lock tight for safe disposal, a container that is specifically constructed for the method of sterilization that will be used (if sharps containers are not specifically constructed to be autoclaved, the resulting mass of melted plastic is extremely hazardous due to the needles that often protrude), and a clear top that would allow inspection.
14. Eating, drinking, smoking, applying cosmetics or lip balm and handling contact lenses is prohibited in areas where there is a reasonable likelihood of occupational exposure.
15. Food or drink should not be kept in areas where blood or other potentially infectious materials are present or stored.
16. Procedures which minimize spraying, splashing, spattering, and generation of droplets of infectious material shall be used whenever possible.
17. No mouth pipetting should occur.
18. Biohazard labels should be affixed to all containers of regulated waste, refrigerators, freezers and other containers that hold or are contaminated with blood or other potentially infectious material.
a. Red bags or containers may be substituted for labels.
19. Specimens of blood or other potentially infectious materials should be placed in a container which prevents leakage during collection, storage, transport, or shipping.
a. This container should be red or labeled with the biohazard symbol and closed prior to being stored, transported, or shipped.
b. If contamination outside this primary container occurs or is likely to occur, it should be placed in a second red or similarly labeled container which prevents leakage during handling processing, storage, transport, or shipping.
20. Equipment which has been in contact with blood or other potentially infected material should be examined prior to servicing or shipping and should be decontaminated as necessary.
a. Where complete decontamination cannot occur prior to servicing, a readily observable biohazard label should be attached to the equipment stating which portions of the equipment remain contaminated, and
b. the employee requesting the service or repair is responsible for ensuring that information is conveyed to all affected employees, service representatives such as the University Biomed Shop and/or the manufacturer prior to handling, servicing, or shipping so that appropriate precautions can be taken.
21. Contaminated work surfaces should be decontaminated after completion of procedures, immediately or as soon as feasible after any spill of blood or other potentially infectious material, and at the end of the work shift if the surface has become contaminated since the last cleaning.
22. Broken glassware which may be contaminated should not be picked up directly with the hands but by mechanical means such as a brush and dustpan, tongs or forceps.
23. All employees with occupational exposure should receive bloodborne pathogen training at the time of assignment to tasks where occupational exposure may take place, when changes affect employees' occupational exposure and at least annually thereafter.
24. The hepatitis B vaccine should be made available to all employees who have occupational exposure to blood or other potentially infectious materials.
25. If an employee sustains an exposure incident (such as a stick with a contaminated needle/scalpel/dental wire or a splash of potentially infectious material in the eye, mouth, mucous membrane, or non-intact skin), the exposed person should immediately:
a. clean the wound with soap; flush mucous membranes with water or normal saline solution;
b. notify his/her supervisor, designated coordinator, or other designated individual;
c. proceed for treatment within 1-2 hours of the exposure (see the University of Oklahoma Infectious Diseases Policy for current recommended treatment locations); and
d. if possible, for laboratory exposures, bring a sample of the source material to the treatment facility for testing.
• Plant Biosafety
1. Physical and biological containment suitable to the greenhouse conduct of experiments involving recombinant or synthetic nucleic acid molecule research involving plants that are of a size, number, or have growth requirements that preclude the use of general biosafety containment conditions, such as mosses, liverworts, macroscopic algae, and vascular plants including terrestrial crops, forest, and ornamental species should follow the procedures found in the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid, Appendix P.
2. General Definitions
a. Plant-associated microorganisms include viroids, virusoids, viruses, bacteria, fungi, protozoans, certain small algae, and microorganisms that have a benign or beneficial association with plants, such as certain Rhizobium species and microorganisms known to cause plant diseases.
b. Plant-associated small animals include those arthropods that are in obligate association with plants, are plant pests, are plant pollinators, or transmit plant disease agents, as well as other small animals such as nematodes for which tests of biological properties necessitate the use of plants.
c. Microorganisms associated with such small animals (e.g., pathogens or symbionts) are included.
d. The term "greenhouse" refers to a structure with walls, a roof, and a floor designed and used principally for growing plants in a controlled and protected environment.
e. The walls and roof are usually constructed of transparent or translucent material to allow passage of sunlight for plant growth.
f. The term "greenhouse facility" includes the actual greenhouse rooms or compartments for growing plants, including all immediately contiguous hallways and head-house areas, and is considered part of the confinement area.
3. Plant Biosafety Level 1 (BL1-P)
a. Access to the greenhouse shall be limited or restricted, at the discretion of the greenhouse Director, when experiments are in progress.
b. Prior to entering the greenhouse, personnel shall be required to read and follow instructions on BL1-P greenhouse practices and procedures. All procedures shall be performed in accordance with accepted greenhouse practices that are appropriate to the experimental organism.
c. A record shall be kept of experiments currently in progress in the greenhouse facility.
d. Experimental organisms shall be rendered biologically inactive by appropriate methods before disposal outside of the greenhouse facility.
e. A program shall be implemented to control undesired species (e.g., weed, rodent, or arthropod pests and pathogens), by methods appropriate to the organisms and in accordance with applicable state and Federal laws.
f. Arthropods and other motile microorganisms shall be housed in appropriate cages.
a) If microorganisms (e.g., flying arthropods or nematodes) are released within the greenhouse, precautions shall be taken to minimize escape from the greenhouse facility.
g. Experiments involving other organisms that require a containment level lower than BL1-P may be conducted in the greenhouse concurrently with experiments that require BL1-P containment, provided that all work is conducted in accordance with BL1-P greenhouse practices.
h. The greenhouse floor may be composed of gravel or other porous material.
1) At a minimum, impervious (e.g., concrete) walkways are recommended.
2) Windows and other openings in the walls and roof of the greenhouse facility may be open for ventilation as needed for proper operation and do not require any special barrier to contain or exclude pollen, microorganisms, or small flying animals (e.g., arthropods and birds); however, screens are recommended.
4. Plant Biosafety Level 2 (BL2-P)
a. Access to the greenhouse shall be limited or restricted, at the discretion of the Greenhouse Director, to individuals directly involved with the experiments when they are in progress.
b. Personnel shall be required to read and follow instructions on BL2-P practices and procedures. All procedures shall be conducted in accordance with accepted greenhouse practices that are appropriate to the experimental organisms.
c. A record shall be kept of experimental plants, microorganisms, or small animals that are brought into or removed from the greenhouse facility.
d. A record shall be kept of experiments currently in progress in the greenhouse facility.
e. The Principal Investigator shall report any greenhouse accident involving the inadvertent release or spill of microorganisms to the Greenhouse Director, Institutional Biosafety Committee, NIH/OBA and other appropriate authorities immediately (if applicable).
1) Reports to the NIH/OBA shall be sent to the Office of Biotechnology Activities, National Institutes of Health, 6705 Rockledge Drive, Suite 750, MSC 7985, Bethesda, MD 20892-7985 (20817 for non-USPS mail), 301-496-9838, 301-496-9839 (fax).
2) Documentation of any such accident shall be prepared and maintained.
f. Experimental organisms shall be rendered biologically inactive by appropriate methods before disposal outside of the greenhouse facility.
g. Decontamination of run-off water is not necessarily required. If part of the greenhouse is composed of gravel or similar material, appropriate treatments should be made periodically to eliminate, or render inactive, any organisms potentially entrapped by the gravel.
h. A program shall be implemented to control undesired species (e.g., weed, rodent, or arthropod pests and pathogens) by methods appropriate to the organisms and in accordance with applicable state and Federal laws.
1) Biosafety Level 2 – Plants (BL2- P) NIH Guidelines for Research Involving Recombinant DNA Molecules Appendix P-II-B2) Arthropods and other motile microorganisms shall be housed in appropriate cages.
2) If microorganisms (e.g., flying arthropods or nematodes) are released within the greenhouse, precautions shall be taken to minimize escape from the greenhouse facility.
i. Experiments involving other organisms that require a containment level lower than BL2-P may be conducted in the greenhouse concurrently with experiments that require BL2-P containment provided that all work is conducted in accordance with BL2-P greenhouse practices.
j. A sign shall be posted indicating that a restricted experiment is in progress. The sign shall indicate the following:
1) the name of the responsible individual,
2) the plants in use, and
3) any special requirements for using the area.
4) If organisms are used that have a recognized potential for causing serious detrimental impacts on managed or natural ecosystems, their presence shall be indicated on a sign posted on the greenhouse access doors.
k. If there is a risk to human health, a sign shall be posted incorporating the universal biosafety symbol.
l. Materials containing experimental microorganisms, which are brought into or removed from the greenhouse facility in a viable or intact state, shall be transferred in a closed non-breakable container.
m. A greenhouse practices manual shall be prepared or adopted. This manual shall:
1) advise personnel of the potential consequences if such practices are not followed, and
2) outline contingency plans to be implemented in the event of the unintentional release of organisms.
n. The greenhouse floor should be composed of an impervious material.
1) Concrete is recommended, but gravel or other porous material under benches is acceptable unless propagules of experimental organisms are readily disseminated through soil.
2) Soil beds are acceptable unless propagules of experimental organisms are readily disseminated through soil.
3) Windows and other openings in the walls and roof of the greenhouse facility may be open for ventilation as needed for proper operation and do not require any special barrier to exclude pollen or microorganisms; however, screens are required to exclude small flying animals (e.g., arthropods and birds).
o. An autoclave shall be available for the treatment of contaminated greenhouse materials.
p. If intake fans are used in the ventilation system, measures shall be taken to minimize the ingress of arthropods.
1) Louvers or fans shall be constructed such that they can only be opened when the fan is in operation.
q. BL2-P greenhouse containment requirements may be satisfied by using a growth chamber or growth room within a building provided that the external physical structure limits access and escape of microorganisms and macroorganisms in a manner that satisfies the intent of the foregoing clauses.
• Arthropod Safety
1. Arthropod Containment Level 1 (ACL-1)
a. suitable for work with uninfected arthropod vectors or those infected with a non-pathogen including arthropods that are already present in the local geographic region regardless of whether there is active vector borne disease transmission in the locale, and exotic arthropods that upon escape would be inviable or become only temporarily established in areas not having active vector borne disease transmission.
b. The insectary area is separated from areas that are used for general traffic within the building.
1) Door openings, whether covered by rigid panels, glass, screens, plastic sheets or cloth, minimize escape and entrance of arthropods.
2) Windows, if present, effectively prevent escape of the smallest arthropods contained within.
c. Furniture and incubators containing arthropods are located in such a way that accidental contact and release is minimized.
1) This may be achieved by locating arthropods out of the flow of general traffic, avoiding hallways, or placing them in closets.
2) The area is maintained to allow detection of escaped arthropods.
3) For example, materials unrelated to arthropod rearing and experimentation (e.g., plants, unused containers, clutter) that provide breeding sites and harborages are minimized.
d. Accidental sources of arthropods from within the insectary are eliminated. This may be accomplished by cleaning work surfaces after a spill of materials, including soil or water that might contain viable eggs. Pools of water are mopped up immediately.
e. Practices should be in place such that arthropods do not escape by inadvertent disposal in primary containers. Cages and other culture containers are appropriately cleaned to prevent arthropod survival and escape (e.g. heated to, or chilled below, the lethal temperature).
f. Cages used to hold arthropods effectively prevent escape of all stages.
1) Screened mesh, if used, is durable and of a size appropriate to prevent escape.
2) Non-breakable cages are recommended. Bags, rearing trays and so on effectively prevent leakage and escape.
g. Living arthropods are not to be disposed of.
1) All wastes from the insectary (including arthropod carcasses, and rearing medium) are transported from the insectary in leak-proof, sealed containers for appropriate disposal as biomedical waste.
2) All stages of arthropods are killed before disposal.
3) Autoclaving or incineration of material infected with a non-pathogen is recommended.
h. Arthropods are identified and labeled adequately.
1) Labels giving species, strain/origin, date of collection, responsible investigator, and so on are firmly attached to the container (and cover if removable).
2) Vessels containing stages with limited mobility (e.g., eggs, pupae, hibernating adults) are securely stored.
i. Personnel take appropriate precautions to prevent transport or dissemination of arthropods from the insectary on their persons or via the sewer.
j. A program to prevent the entrance of wild arthropods (e.g., houseflies, cockroaches, spiders) and rodents effectively precludes predation, contamination, and possible inadvertent infection.
k. Investigators assess whether escapes are occurring. An effective arthropod trapping program is recommended to monitor the escape prevention program.
l. Harborage and breeding areas are reduced as appropriate. Furniture and racks are minimized and can be easily moved to permit cleaning and location of escaped arthropods.
m. Syringes that re-sheath the needle, needle-less systems, and other safe devices are used when appropriate.
n. Plastic-ware is substituted for glassware whenever possible.
o. Signage must be present so that persons entering the area are aware of the presence of arthropod vectors.
p. Animals not necessary for culture of the arthropods are not accessible to the arthropods.
q. Animals used as hosts or blood sources may be housed within the insectary but are adequately protected from access by escaped arthropods.
r. Arthropods fed on host animals are prevented from accidental transfer to host cages.
1) When handling/removing animals after exposure to arthropods, precautions must be taken to prevent arthropod escape through screens, covers, and by flying.
2) Host animals are inspected closely (e.g., concealment in fur, ears, crevices), and the primary container is sufficiently robust to prevent escape during feeding.
3) The blood source is considered as a source of inadvertent arthropod infection and transmission.
a) Measures are implemented to prevent such an event.
b) Use of sterile blood or blood from sources known to be pathogen-free is recommended.
c) In contrast, use of blood from animals or humans whose disease status is uncertain is to be avoided.
s. White laboratory coats, gowns, and/or uniforms are worn at all times in the insectary when handling blood and vertebrate animals.
1) Gloves are worn when handling host animals or blood used to feed the arthropods.
2) Personal protective equipment is worn as appropriate e.g., respirators for arthropod-associated allergies, particle masks, head covers.
t. Escaped arthropods are killed or collected and properly disposed of.
u. The insectary director and the OU IBC must be notified promptly of accidental release of vectors.
2. Arthropod Containment Level 2 (ACL-2)
a. Arthropod Containment Level 2 (ACL-2) must be practiced if working with exotic and indigenous arthropods infected with BSL-2 agents associated with animal and/or human disease, or that are suspected of being infected with such agents.
b. Uninfected genetically modified arthropod vectors also fall under this level provided the modification has no, or only negative effects on viability, survivorship, host range, or vector capacity.
c. ACL-2 builds upon the practices, procedures, containment equipment, and facility requirements of ACL-1, but is more stringent in the physical containment, restriction of access, disposal, and facilities design.
d. In addition to ACL-1 procedures, ACL-2 procedures must also include the following:
1) The insectary is separated from areas that are open to unrestricted personnel traffic within the building.
2) It is recommended that this be accomplished by at least two self-closing doors that prevent passage of the arthropods.
3) Increased levels of physical isolation are recommended, e.g., separate buildings, wings, suites.
4) Recommended entrance to the insectary is via a double-door vestibule that prevents flying and crawling arthropod escape.
a) For example, the two contiguous doors must not be opened simultaneously. Internal doors may open outwards or be sliding, but are self-closing, and are kept closed when arthropods are present. Additional barriers (e.g., screened partitions, hanging curtains, and air curtains) are highly recommended.
5) Infected arthropods are prevented from release into the laboratory area. This may be accomplished by secure glove boxes, biosafety cabinets, custom handling trays etc.
a) These may vary from BSL recommendations insofar as necessary to safely contain both the arthropod and any agent.
b) Such modifications should be made only in consultation with experts in handling the specific types of infected arthropods and biosafety experts.
c) A dedicated area for handling infected material is recommended.
d) This is preferably a separate cubicle, walk-in incubator, or screen room.
e) Uninfected arthropods are isolated from infected arthropods.
6) The area is designed and maintained to enhance detection of escaped arthropods. Investigators assess whether escapes are occurring by instituting an effective arthropod trapping program to monitor the escape prevention program.
a) Oviposition traps, ground-level flea traps, oil-filled channels surrounding tick colonies, light traps for mosquitoes, etc., on are recommended.
b) When exotic arthropods are used, exterior monitoring is performed.
c) Records of exterior captures are maintained.
7) Windows are not recommended, but if present cannot be opened and are well sealed.
a) Windows must be resistant to breakage (e.g., double paned or wire reinforced).
8) If a central vacuum system is installed, each service outlet is fitted with suitable barriers/filters to prevent arthropod escape.
a) Filters are installed to permit decontamination and servicing.
b) Other vacuum devices are appropriately filtered to prevent transfer and exhausting of arthropods.
9) The insectary is designed, constructed, and maintained to facilitate cleaning and housekeeping.
a) The interior walls are light-colored so that a loose arthropod can be easily located, recaptured, or killed.
b) Gloss finishes, ideally resistant to chemical disinfectants and fumigants, are recommended.
c) Floors are light colored, smooth and uncovered.
d) Ceilings are as low as possible to simplify detection and capture of flying insects.
10) Floor drains are modified to prevent accidental release of arthropods and agents.
a) If present, traps must be filled with an appropriate chemical treatment to prevent survival of all arthropod stages (e.g., mosquito larvae).
11) Internal facility appurtenances (e.g., light fixtures, pipes, ducting) are minimal since these provide hiding places for loose arthropods.
a) Penetrations of walls, floors, and ceilings are minimal and sealed/caulked.
b) Ideally, light fixtures are flush with the ceiling, sealed, and accessed from above.
12) Ventilation is appropriate for arthropod maintenance, but does not compromise containment of the agent or arthropod.
a) Examples include: exhaust air is discharged to the outside without being recirculated to other rooms; appropriate filter/barriers are installed to prevent escape of arthropods; the direction of airflow in the insectary is inward; a progressively negative pressure gradient is maintained as distance from the main entrance increases; fans located in the vestibule and internal corridor can be used to help prevent escape of flying arthropods; air curtains are located in vestibules and doorways.
13) An autoclave is available conveniently located to rooms containing arthropods within the insectary building.
14) The facility has a hand-washing sink with hot water and with suitable plumbing to prevent arthropod escape.
15) Illumination is appropriate for arthropod maintenance but does not compromise arthropod containment, impede vision, or adversely influence the safety of procedures within the insectary.
a) Lighted (or dark) openings that attract escaped arthropods are avoided.
16) Furniture and incubators containing arthropods are located in such a way that accidental contact and release by laboratorians, custodians, and service persons is unlikely.
a) This may be achieved by locating arthropods in dedicated rooms, closets, incubators located out of the traffic flow or similar measures.
17) Cages used to hold arthropods are non-breakable and screened with mesh of a size to prevent escape.
a) Containers are preferably autoclavable or disposable.
b) Openings designed to prevent escape during removal and introduction of arthropods are recommended.
18) Equipment and supplies not required for operation of the insectary should not be located in the insectary.
a) All supplies for insect maintenance that must be kept within the insectary are located in a designated area and not on open shelves.
b) It is recommended that a closed storage room, cabinets with tight-fitting doors or drawers be used.
c) Doors and drawers are opened only for access.
d) Insect diet should be kept in sealed containers.
e) Equipment in which water is stored or might accumulate (e.g., humidifiers) is screened to prevent arthropod access, or contains chemicals to prevent arthropod survival.
19) All equipment must be appropriately decontaminated and disinfested before transfer between rooms within the insectary, and before removal from the insectary.
20) Before leaving the insectary and after handling cultures and infected arthropods, personnel wash their hands, taking care not to disperse viable life stages into the drainage system. No infected material is disposed of through the sewer.
21) In addition to cleaning cages and culture containers to prevent arthropod escape, containers are disinfected chemically and/or autoclaved if used for infected material.
a) Autoclaving or incineration of primary containers is recommended for containers holding uninfected material.
b) Equipment and work surfaces in the insectary are routinely decontaminated with an effective chemical or by radiation (e.g., heat) after actual or potential contact with an infectious agent, and especially after overt spills and splashes of viable materials (including soil or water that might contain infectious agents or eggs).
c) Infected arthropods are autoclaved or incinerated.
22) Signage is required to ensure persons entering the area are aware of the presence of arthropod vectors.
a) If infected material is present, a BSL-2 biohazard sign is posted on the entrance to the insectary listing all species handled within and is updated whenever new species are introduced or pathogenic infectious agents are present.
b) The hazard warning sign identifies the arthropod species, agent(s) known or suspected to be present, lists the name and telephone number of the responsible person(s), and indicates any special requirements for entering the insectary (e.g., the need for immunizations or respirators).
23) Laboratory personnel are advised of special hazards and are required to follow instructions on practices and procedures contained in the safety manual.
a) Adherence to established safety procedures and policies is made a condition of employment and is part of the annual performance review of every employee.
b) Personnel receive annual updates and additional training as necessary for procedural or policy changes.
c) Records of all training are maintained.
24) An appropriate medical surveillance program is in place.
a) All personnel receive appropriate immunizations or tests for the agents handled or likely to be present.
b) Personnel are aware of the symptoms of infection and the procedure to follow in reporting these.
c) In general, persons who may be at increased risk of acquiring infection, or for whom infection may be unusually hazardous (e.g., immunocompromised), are not allowed in the insectary unless special personal protection procedures are in place to eliminate extra risk.
25) Routine access is limited to trained persons and accompanied guests.
a) Service persons are made aware of the hazards present and the consequences of arthropod release and contact with agents that may be present.
26) All infectious and potentially infectious samples are collected, labeled, transported, and processed in a manner that contains and prevents transmission of the agent(s).
a) Transfer of arthropods between manipulation and holding areas is in non-breakable secure containers.
27) Loose arthropods must be killed and disposed of, or recaptured and returned to the container from which they escaped.
a) Infected arthropods must not be killed with bare hands, and must be transferred using filtered mechanical or vacuum aspirators.
28) All procedures are carefully designed and performed to prevent arthropod escape.
a) A release procedure is developed and posted.
b) This includes contacts and immediate mitigating actions.
c) Accidents that result in release of infected arthropods from primary containment vessels, or that result in overt exposure to infectious material must be reported immediately to the insectary director who is responsible for ensuring that appropriate and documented action is taken to mitigate the release, and to the OU IBC.
d) Location, number, and type of material are prominently posted until the source is eliminated. Follow-up medical evaluation, surveillance, and treatment are provided as appropriate, and written records are maintained.
29) Appropriate face/eye and respiratory protection are worn by all personnel entering the insectary.
30) Gloves are worn when handling potentially infected arthropods, blood, and associated equipment and when contact with potentially infectious material is unavoidable.
31) White laboratory coats, gowns, and/or uniforms are worn at all times in the insectary when handling blood, vertebrate animals, and infected materials.
32) Clothing should minimize the area of exposed skin (e.g., skirts, shorts, open-toed shoes, sandals, tee shirts are inadvisable), since this can increase the risk of attracting and being bitten by a loose arthropod.
33) A safety manual is prepared, approved by the IBC, and adopted.
a) The manual contains emergency procedures, standard operating procedures, waste disposal and other information necessary to inform personnel of the methods for safe maintenance and operation of the insectary.
34) The facility is evaluated annually for compliance to the ACL-2 level. The principle investigator or insectary director inspects the facility annually to ensure that alterations and maintenance have not compromised the containment characteristics.
• Animal Safety
1. Zoonotic Diseases
a. A zoonotic disease is defined as one that is communicable from animals to humans. Strategies for staying healthy and reducing the risk of exposure are as follows.
b. Wash your hands frequently - the most common method of contracting a zoonotic infection is by placing infectious material in your mouth, nose, or eyes.
c. Wear personal protective clothing and equipment and do not take unlaundered protective clothing home.
d. Follow recommended safe work practices.
e. Notify your supervisor immediately if an exposure incident occurs, even if it seems minor, then seek medical attention.
1) First-aid kits are readily available in all buildings housing animals, then report to a medical facility such as Employee Health.
f. Tell your personal physician that you work with animals. Many zoonotic diseases have flu-like symptoms: your physician needs this information for making an accurate diagnosis.
2. Injury
a. Environmental factors, as well as factors intrinsic to the animal, can lead to greater risk for injury.
1) Animals respond to sounds and smells, sometimes undetectable to humans, which can frighten the animal.
2) Animals may have a flight zone or a particular sign of distress that animal handlers should be aware of to reduce risk.
3) Inappropriate handling can induce discomfort, pain and distress, provoking an animal to inflict injury on its handler.
4) Animals, especially non-human primates, may grab or get caught in loose clothing, long hair, etc., or may spit or throw feces.
5) Guidelines for preventing injury include:
a) Know the animal’s flight zone and signs of distress.
b) Use proper handling techniques.
c) Minimize the use of sharps and glass and ensure proper disposal or same.
d) Determine the potential risk and wear appropriate protective equipment for the hazard, which may include leather gloves, latex/nitrile gloves, face shield, etc.
e) If you must lift heavy objects, contact the EHSO for safe lifting procedures and training.
f) Minimize the amount of time a floor is allowed to remain wet, and use slip-resistant footwear, mats and signage whenever wet floors cannot be avoided.
6) If a bite or a scratch is sustained, it is important that medical care be sought, even if the injury seems trivial, due to the potential for disease transmission, which could include the rabies virus, hantavirus, cat-scratch fever, tularemia, rat-bite fever, Staphylococcus infection, or other intentionally-administered research-related viral or microbial agents. Procedures to follow in the event of a bite or scratch are:
a) While wearing gloves, carefully express the wound and apply gentle pressure around the wound to encourage bleeding.
b) Rinse the wound under warm running water for 15 minutes and continue massaging the site.
c) If the injury involves a macaque or any animal known to be infected with a zoonotic disease, you may wash the wound and surrounding area with povidone-iodine solution for 5 minutes.
d) Care should be taken when using iodine, as prolonged skin exposure to iodine can increase wound healing time and can cause tissue damage.
e) For all other wounds, or if you are concerned about using iodine, wash the wound and surrounding area with soap and water for 5 minutes.
f) Continue to rinse periodically. If normal saline is available, rinse with normal saline.
g) Pat the injury dry using sterile gauze pads.
h) Cover the wound with a pad and secure it with gauze and tape.
i) Seek medical attention.
3. Animal Allergies/Asthma
a. Animals and animal products such as dander, hair, scales, fur, saliva, and body wastes contain powerful allergens that can cause both respiratory and skin disorders.
1) Sources of exposure to animal allergens vary with animal species.
2) For example, allergens have been found in the urine of rats; the urine, saliva, and pelts of guinea pigs; rabbit pelts, cat saliva and dander; dog dander; and horse serum and dander.
3) Inhalation is one way for animal allergens to enter the body.
a) After a period of time (often several months, but occasionally many years), one may inhale sufficient quantities of allergens to become sensitized - that is, develop symptoms when exposed again, even to tiny amounts of the allergen.
b) Other routes of exposures may come from animal bites or scratches.
b. In sensitized persons, reactions often occur soon after exposure to the animal or animal product, but they may be delayed for 2 to 8 hours or more.
1) Symptoms vary from mild reactions such as sneezing and runny nose to more serious reactions such as cough, chest tightness, wheezing, or shortness of breath.
c. The National Institute for Occupational Safety and Health (NIOSH) recommends several measures to reduce exposures to animal allergens in the workplace and prevent animal induced asthma and allergies, including the following:
1) Provide training to educate workers about animal allergies and steps for risk reduction.
2) Perform animal manipulations within ventilated hoods or safety cabinets when possible.
3) Avoid wearing street clothes while working with animals. Leave work clothes at the workplace to avoid potential exposure problems for family members.
4) Keep cages and animal areas clean. Take particular care to control exposures during cleaning (minimize dust and aerosols, etc.).
5) Reduce skin contact with animal products such as dander, serum, and urine by using gloves, lab coats, and approved particulate respirators with face-shields where appropriate.
6) Provide health monitoring and appropriate counseling and medical follow-up for workers who have become sensitized or have developed allergy symptoms.
• Institutional Animal Care and Use Committee (IACUC) Requirements
1. The University of Oklahoma IACUC is responsible for the overview of animal research at the University of Oklahoma and has determined that annual training in both animal safety and animal handling procedures is mandatory for all OUHSC, OU-Tulsa and OU Norman campus personnel working with animals.
2. Anyone who has a concern about the care, tending, and/or use of research animals should report the matter directly to either the Animal Resources Director, the IACUC chair, or any IACUC committee member. These concerns, and any action that results, will be presented to the IACUC chair and then to the entire IACUC committee.
3. Each facility or program that uses animals will be inspected by the IACUC at least once every six months.
References:
1. Biosafety in the Laboratory: Prudent Practices for the Handling and Disposal of Infectious Materials, National Research Council, National Academy Press, 1989.
2. CDC/NIH Biosafety in Microbiological and Biomedical Laboratories, U.S. Department of Health and Human Services Public Health Services, Centers for Disease Control and Prevention and National Institutes of Health, HHS Publication No. (CDC) 93-8395, 4th. Edition, May, 1999, and 5th. Edition, Feb., 2007.
3. Centers for Disease Control and Prevention/National Institutes of Health, U. S. Government Printing Office, 2009.
4. NIOSH ALERT: Preventing Asthma in Animal Handlers, DHHS (NIOSH) Publication No. 97 116, January 1998.
5. NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules, March 2016.
6. Occupational Health and Safety in the Care and Use of Research Animals, National Research Council, National Academy Press, 1997.
7. OSHA Bloodborne Pathogens Standard (29 CFR 1910.1030) the University of Oklahoma Infectious Diseases Policy.
