BIOLOGICAL SAFETY FACT SHEET

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Northwestern UniversityVice President for Research Chemical and Biological Safety Committee Office for Research Safety 

BIOSAFETY LEVEL 2 CONTAINMENT  

A. Principles Of Biosafety Containment. "Containment" refers to a reliable plan for managing infectious agents in the lab environment where they are being handled or maintained. The purpose of containment is to reduce or eliminate exposure of lab workers, other persons, and the outside environment to potentially hazardous agents.  

Biosafety Level 2 (BL2) practices, equipment, and facilities are applicable to clinical, diagnostic, teaching and other facilities in which work is performed with the broad spectrum of indigenous moderate-risk agents present in the community and associated with human disease of varying severity. With good microbiological techniques, such agents can be used safely in activities conducted on the open bench, provided the potential for producing splashes or aerosols is low. Hepatitis B virus, the salmonellae, and Toxoplasma spp. are representative of microorganisms assigned to this containment level.  

B. Applicability Of Biosafety Level 2. The BL2 requirements apply to research work:  

• studying known infectious agents classified as requiring BL2 precautions by the CDC/NIH in Biosafety in Microbiological and Biomedical Laboratories (BMBL), and 
• involving the handling of vertebrate animals experimentally or naturally infected with agents classified as requiring BL2 controls by BMBL. 

The biological hazard exists in the potential for autoinoculation, ingestion, or mucous membrane exposure by a worker handling the agents or infected animals.  

In addition, BL2 requirements are prescribed for the handling of human blood, blood products, and other potentially infectious human-derived materials. The following materials are considered potentially infectious:  

• Human blood, blood components, and blood products 
• Semen 
• Vaginal secretions 
• Cerebrospinal fluid 
• Synovial fluid 
• Pleural fluid 
• Peritoneal fluid 
• Amniotic fluid 
• Saliva in dental procedures 
• Any body fluid visibly contaminated with blood 
• All body fluids in situations where it is difficult or impossible to differentiate between body fluids 
• Any unfixed tissue or organs from a human (living or dead) 
• HIV-containing cell or tissue cultures, organ cultures, and HIV- or HBV-containing culture medium or other solutions 
• Blood, organs, or other tissues from experimental animals infected with HIV or HBV 
• Human albumin 
• Human tissue culture cell lines (even those that are established). 

Note: Human albumin and established human cell lines are exempt from the requirements of the Bloodborne Pathogens Standard if they can be characterized as free of contamination from human hepatitis viruses, human immunodeficiency viruses, and other recognized bloodborne pathogens. The standard states that the final determination that human or other animal cell lines in culture are free of bloodborne pathogens must be made by a biological safety professional or other qualified scientist with background and experience to review such potential contamination and risk. The professional would be expected to comment, in writing, on the test methods and molecular technology applied to a cell line sample to identify or screen for latent viruses capable of infecting humans.  

Documentation that given cell lines in use in a lab are not classified as "other potentially infectious materials" should be available in the lab's Safety Desk Book. Documentation may be provided by the cell line distributor or vendor (e.g., ATCC) at the point of origin but these records do not generally account for potential contamination during shipping. To meet the requirements of the law, the principal investigator would need to document that the package was protected from environmental contamination during transport and arrived at the lab undamaged.  

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SPILLS  

A biological spill shall be followed by prompt action to contain and clean-up the spill. When a spill occurs, warn everyone in the area and call for assistance as needed. Assess the degree of risk involved in the spill based on:  

• the volume of material spilled 
• the potential concentration of organisms in the material spilled 
• the hazard of the organisms involved 
• the route of infection of the organisms 
• the diseases caused by the organisms. 

Spills of biological agents can contaminate areas and lead to infection of lab workers. Thus, prevention of exposure is the primary goal in spill containment and clean-up, exactly as in chemical spills. In evaluating the risks of spill response, consider the potential for generation of aerosols or droplets.  

If an accident is expected to generate droplets or aerosols in the laboratory room atmosphere, the room shall be evacuated immediately. Doors shall be closed and clothing decontaminated. Call ORS to supervise the clean-up. In general, a 30-minute wait is sufficient for the droplets to settle and aerosols to be reduced by air changes. Longer waiting periods may be imposed depending on the situation and the ventilation system in this area. Lab workers and/or ORS will exercise judgment as to the need for outside emergency help in evacuation.  

If a spill of a biological agent occurs in a public area, evacuation of the area shall be immediate. The principal investigator will be responsible for designating the extent of evacuation until ORS or emergency personnel arrive. Remember that prevention of exposure to hazardous aerosols is of primary importance.  

Anyone cleaning a spill wears personal protective equipment (for example, lab coat, shoe covers, gloves, safety glasses, and, possibly, respiratory protection) to prevent exposure to organisms. An air-purifying negative-pressure respirator with HEPA filter cartridges is generally adequate protection against inhalation of most biological agents. Only personnel trained and cleared through ORS are permitted to wear respirators.  

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PROPER WORK AND HANDLING PRACTICES  

A. Standard Practices. 

1. Access to the lab is limited or restricted by the supervisor when work with infectious agents is in progress. 
2. Work surfaces are decontaminated once a day and after any spill of infectious material. 
3. All infectious wastes are chemically decontaminated or autoclaved before disposal. 
4. Lab workers wash their hands immediately after removing gloves, after handling agents, and before leaving the lab. 
5. All procedures are performed carefully to minimize the creation of aerosols. 
6. Eating, drinking, smoking, gum chewing, or applying of cosmetics are prohibited in the work area. 
7. Food storage is prohibited in the work area. 
8. Food is stored in cabinets or refrigerators designated for such use only. 
9. Mechanical pipetting devices are used; mouth pipetting is prohibited. 
10. An insect and rodent control program is in effect (can be verified by contacting Facilities Management). 

B. Special Practices. 

1. Lab doors are kept closed when experiments are in progress. 
2. Contaminated materials that are to be transported in public areas (hallways, etc.) are placed in leakproof, closed, and labeled or color-coded containers before removal. 
3. Access to the lab is restricted. Persons who are at increased risk of acquiring infection or for whom infection may be unusually hazardous are not permitted. The supervisor has final responsibility regarding entry and work in the lab. Only persons who are advised of the potential hazard and who meet entry requirements, such as immunization, may enter the lab. 
4. If the infectious agents used require special entry provisions, such as immunization, a hazard warning sign is posted on access doors to the lab. This includes the universal biohazards symbol, infectious agent, principal investigator's name and phone number, and special entry requirements. 
5. Animals unrelated to research are prohibited in the lab. 
6. Hypodermic needles and syringes are used only for injection and aspiration of fluids. Needle-locking syringes or disposable units are used. 
7. Used needles and syringes are immediately placed in a puncture-resistant container and chemically decontaminated or autoclaved before discard or reuse. This statement is not true for syringes used with human products. See page 18, section B. Special Practices And Primary Barriers. 
8. Extreme caution is used when handling needles and syringes to avoid autoinoculation and generation of aerosols during use. 
9. Spills and accidents resulting in overt exposures are reported to the supervisor, ORS, Division of Safety and Loss Prevention, and University Police. 

C. Primary Barriers. 

1. Lab workers wear clothing that protects street clothing. This includes at least one of the following options: lab coats, solid-front gown, smocks, or uniforms. 
2. Lab clothing is worn only inside the lab. 
3. Lab clothing that is contaminated is either autoclaved or disinfected with 10% bleach solution prior to laundering. 
4. Lab workers wear gloves when handling infectious materials. 
5. Gloves are carefully removed and changed when visibly contaminated. 
6. Persons coming into indirect contact with potentially infectious materials wear gloves if they have lesions or dermatitis on their hands. 
7. Eye protection is worn when splashes, spray, spatter, or droplets of potentially infectious materials may occur. 
8. Face shields, or surgical masks and eye protection, are worn when splashes, spray, spatter, or droplets of potentially infectious materials may occur. 

D. Containment Equipment. The following procedures are conducted only in a biological safety cabinet:  

1. Centrifuging, grinding, blending, vigorous shaking or mixing, sonic disruption, opening infectious-material containers for which internal pressures may differ from ambient pressures, inoculating animals intranasally, and harvesting infected tissue from animals or eggs 

Exception: Materials may be centrifuged in the open lab if sealed heads or centrifuge safety cups are used and if the centrifuge tubes are opened only in a biological safety cabinet.  

2. Use of high concentrations or large volumes of infectious agents. 

Lab workers are to be trained in the proper use of biological safety cabinets, with an emphasis on activities that may disrupt the inward flow of air through the work opening. Staff are aware that the activities that can cause escape of aerosols include:  

• repeated insertion and withdrawal of worker's arms 
• opening and closing lab doors or isolation cubicle 
• improper placement or operation of equipment or materials in the cabinet 
• brisk walking past the cabinet during use. 

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ADDITIONAL PRACTICES SPECIFIC TO THE USE OF HUMAN BLOOD, BLOOD PRODUCTS OR OTHER POTENTIALLY INFECTIOUS MATERIALS  

Based on 29 CFR 1910.1030, Bloodborne pathogens.  

A. Standard Practices. The requirements of the bloodborne pathogens program are discussed in your department's Exposure Control Plan. A copy of the plan should be available in your departmental Safety Desk Book. The plan relates the general elements of the program and the department-specific protocols that apply in your lab.  

Any lab worker who may be exposed to human blood or other human products shall:  

• be included in the department's exposure determination 
• practice universal precautions 
• have the opportunity to receive Hepatitis B vaccination 
• sign a waiver or consent form for vaccination 
• be offered postexposure evaluation and follow-up (PEEFU) 
• be trained to understand the hazards of bloodborne pathogens and how to protect against these hazards. 

B. Special Practices And Primary Barriers. 

1. When working with human blood, blood products, and other potentially infectious materials, lab workers are prohibited from bending or shearing needles. Needles shall not be replaced in the guards or removed from the syringe. 

Note: This is a difference in procedure from standard BL2 practices. When handling BL2 agents, needles are required to be decontaminated before being bent, sheared, replaced, or removed. For needles contaminated by human products, this type of handling is never permitted.  

2. Lab workers are aware of the difference in protocol regarding handling of needles used in contact with human products. 
3. Waste materials from operations involving human blood, blood products, and other potentially infectious materials are autoclaved or treated with 10% bleach solution before being discarded in regular trash. 
4. Spilled material is treated with 10% bleach solution and wiped up with paper towels by lab workers wearing gloves. 
5. All paper towels, gloves, and other supplies used in cleaning the spill are disinfected with bleach. 
6. Bleached materials are not autoclaved due to corrosion of the steel equipment by potential generation of chlorine gas. 
7. Spills on skin are immediately washed with soap and water. 
8. Contaminated clothing is immediately autoclaved or treated with 10% bleach solution. 
9. Contaminated clothing is not sent for laundering unless it has been autoclaved or treated with 10% bleach solution. 

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RECOMBINANT DNA SAFETY PROGRAM  

Please refer to the University's Recombinant DNA Safety Program document for further, detailed information regarding safe RDNA procedures.  

Research involving recombinant DNA shall comply with the National Institute of Health's Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines) as published in the Federal Register. The recombinant DNA guidelines are applicable to all recombinant DNA research conducted at or sponsored by an institution that receives any support for recombinant DNA research from NIH. The purpose of the NIH Guidelines is to specify practices for constructing and handling recombinant DNA molecules, and organisms and viruses containing recombinant DNA molecules.  

NIH defines Recombinant DNA molecules as:  

• molecules that are constructed outside living cells by joining natural or synthetic DNA segments to DNA molecules that can replicate in a living cell 
• molecules that result from the replication of those described above. 

Synthetic DNA segments which are likely to yield a potentially harmful polynucleotide or polypeptide (e.g., a toxin or a pharmacologically active agent) are considered as equivalent to their natural DNA counterpart. If the synthetic DNA segment is not expressed in vivo as a biologically active polynucleotide or polypeptide product, it is exempt from the NIH guidelines. Genomic DNA of plants and bacteria that have acquired a transposable element, even if the latter was donated from a recombinant vector no longer present, are not subject to the NIH guidelines unless the transposon itself contains recombinant DNA. 

Synthetic DNA segments which are likely to yield a potentially harmful polynucleotide or polypeptide (e.g., a toxin or a pharmacologically active agent) are considered as equivalent to their natural DNA counterpart. If the synthetic DNA segment is not expressed in vivo as a biologically active polynucleotide or polypeptide product, it is exempt from the NIH guidelines. Genomic DNA of plants and bacteria that have acquired a transposable element, even if the latter was donated from a recombinant vector no longer present, are not subject to the NIH guidelines unless the transposon itself contains recombinant DNA. 

Synthetic DNA segments which are likely to yield a potentially harmful polynucleotide or polypeptide (e.g., a toxin or a pharmacologically active agent) are considered as equivalent to their natural DNA counterpart. If the synthetic DNA segment is not expressed in vivo as a biologically active polynucleotide or polypeptide product, it is exempt from the NIH guidelines. Genomic DNA of plants and bacteria that have acquired a transposable element, even if the latter was donated from a recombinant vector no longer present, are not subject to the NIH guidelines unless the transposon itself contains recombinant DNA.