Environmental Health & Safety

Biological Safety Manual
Updated 01/05/2008

Foreword

The biosafety manual for Oregon State University has been adopted to accomplish the following goals: The biosafety manual provides university-wide safety guidelines, policies and procedures for the use and manipulation of biohazards. Although the implementation of these procedures is the responsibility of the Principal Investigator (PI), its success depends largely on the combined efforts of laboratory supervisors and employees. Planning for and implementation of biological safety must be part of every laboratory activity in which biohazardous materials are used. In general, the handling and manipulation of biological agents and toxins, as well as recombinant DNA molecules, requires the use of various precautionary measures depending on the materials involved. This manual will provide assistance in the evaluation, containment and control of biohazards. However, it is imperative that all parties involved or working with these materials seek additional advice and training when necessary. Environmental Health & Safety (EH&S) as well as the University Biological Safety Committee are available at OSU to assist in this endeavor.

Biological Safety and Biosafety Levels

Biological safety or biosafety is the application of knowledge, techniques and equipment to prevent personal, laboratory and environmental exposure to potentially infectious agents or biohazards. Biosafety defines the containment conditions under which infectious agents can be safely manipulated. The objective of containment is to confine biohazards and to reduce the potential exposure of the laboratory worker, persons outside of the laboratory, and the environment to potentially infectious agents. It can be accomplished through the following means:

Primary Containment:
Protection of personnel and the immediate laboratory environment through good microbiological technique (laboratory practice) and the use of appropriate safety equipment such as a biosafety cabinet.

Secondary Containment:
Protection of the environment external to the laboratory from exposure to infectious materials through a combination of facility design and operational practices.

Combinations of laboratory practices, containment equipment, and special laboratory design can be made to achieve different levels of physical containment. Currently four Biosafety Levels (1-4) define the level of containment necessary to protect personnel and the environment. A Biosafety Level 1 (BL-1) is the least restrictive, while Biosafety Level 4 (BL-4) requires a special containment laboratory or facility, which is not available at OSU. Since most of the research at OSU is conducted at Biosafety Levels 1 and 2 with the possibility of future experiments at BL-3, this manual will mainly focus on these three Biosafety Levels. For more information on Biosafety Level 4 requirements refer to the appropriate literature or contact the Biological Safety Officer. A summary of the different biosafety level requirements (BL-1, 2 and 3) can be found in Table 1. 

The most important element in maintaining a safe work environment is strict adherence to good microbiological and laboratory practices and techniques. Everybody working with infectious agents or potentially infected materials must be aware of the potential risks. In addition, they must be trained and proficient in the practices and techniques required for handling such material. It is the responsibility of the Principal Investigator or person in charge of the laboratory to provide or arrange for appropriate training of all personnel. 

Table 1. Summary of Biosafety Levels for Infectious Agents (BL-1 to BL-3)

Biosafety Level 1 (BL-1)

Agents:

Not known to cause disease in healthy adults

Practices:

Standard Microbiological Practices

Safety Equipment: (Primary Barriers)

None required

Facilities: (Secondary Barriers)

Open bench top sink required

Biosafety Level 2 (BL-2)

Agents:

Associated with human disease, hazard (exposure) = auto-inoculation, ingestion, mucous membrane exposure

Practices:

BL-1 practice plus: Limited access; biohazard warning signs; “Sharps” precautions; biosafety manual defining any needed waste decontamination or medical surveillance policies

Safety Equipment: (Primary Barriers)

Primary barriers = Class I or II Biological Safety Cabinets (BSCs) or other physical containment devices used for all manipulations of agents that cause splashes or aerosols of infectious materials; Personal Protective Equipment (PPE): laboratory coats, gloves, face and eye protection as needed

Facilities: (Secondary Barriers)

BL-1 plus:  Autoclave available

Biosafety Level 3 (BL-3)

Agents:

Indigenous or exotic agents with potential for aerosol transmission; disease may have serious or lethal consequences

Practices:

BL-2 practice plus: Controlled access; decontamination of all waste; decontamination of lab clothing before laundering; baseline serum

Safety Equipment: (Primary Barriers)

Primary barriers = Class I or II BSCs or other physical containment devices used for all manipulations of agents; PPE: protective lab clothing, gloves, face and eye protection, and respiratory protection as needed

Facilities: (Secondary Barriers)

BL-2 plus:  Physical separation from access corridors; self-closing, double door access; exhausted air not re-circulated, negative airflow into laboratory
 

Biohazard Definition

Infectious or etiologic (disease causing) agents, potentially infectious materials, certain toxins and other hazardous biological materials are included in the definition of a biohazard.

Biohazard
Biological agents and materials which are potentially hazardous to humans, animals and/or plants. Biohazardous agents may include but are not limited to:

Certain bacteria, fungi, viruses, rickettsiae, chlamydiae, parasites, recombinant products, allergens, cultured human or animal cells and the potentially infectious agents these cells may contain, viroids, prions and other infectious agents as outlined in laws, regulations, or guidelines.

Biohazards at OSU

Biological hazards can be found in the various research and support environments on campus. Current projects at OSU are dealing with infectious agents including human, animal and plant pathogens. While animal care facilities have to address zoonotic diseases, in the human health care environment, blood-borne pathogens pose the greatest risk. However, despite having biohazards on campus, OSU is a safe place to work, teach and learn. The information contained in this and other manuals will further increase our ability to maintain a safe and healthy work environment. 

Classification of Infectious Agents on the Basis of Hazard

Risk Groups

Worldwide there are several systems for classifying human and animal pathogens according to the hazard they present to an individual and the community. Although these classifications differ from each other, they all are based on the notion that some microorganisms are more hazardous than others. In general, the pathogenicity of the organism, mode of transmission, host range, availability of effective preventive measures and/or effective treatment are some of the criteria taken into consideration when classifying infectious agents. In the U.S., the most current classification is found in the NIH Guidelines for Research Involving Recombinant DNA Molecules. The human etiologic agents addressed in these guidelines are classified into four risk groups with Risk Group 1 (RG-1) of low or no hazard and Risk Group 4 (RG-4) representing highly infectious agents:

Table 2. Basis for the Classification of Biohazardous Agents by Risk Group

Risk Group

Risk to the individual and the community

Risk Group 1 (RG-1)

Agents that are not associated with disease in healthy adult humans.

 

Risk Group 2 (RG-2)

Agents that are associated with human disease which are rarely serious and for which preventive or therapeutic interventions are often available.
 

Risk Group 3 (RG-3)

Agents that are associated with serious or lethal human disease for which preventive or therapeutic interventions may be available (high individual risk but low community risk).
 

Risk Group 4 (RG-4)

Agents that are 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)

Examples of RG-1 agents include microorganisms like Escherichia coli-K12 or Saccharomyces cerevisiae. A comprehensive list of Risk Group 2, 3 and 4 agents as well as certain animal and plant pathogens can be found in Appendix C2. It is important to realize however, that none of the lists are inclusive. In addition, those agents not listed in Risk Groups ( ) 2, 3 and 4 are not automatically or implicitly classified in RG-1. Those unlisted agents need to be subjected to a risk assessment based on the known and potential properties of the agents and their relationship to agents that are listed.

       Risk Groups = Biosafety Level?

Determining the RG of a biological agent is part of the biosafety risk assessment and helps in assigning the correct biosafety level for containment. In general, RG-2 agents are handled at BL-2, and RG-3 agents at BL-3. However, the use of certain RG-2 agents in large quantities might require BL-3 conditions, while some RG-3 agents (such as the HIV virus) may be safely manipulated at a BL-2 under certain conditions. It is also true that some RG-2 agents can be handled at a BL-1 level. For more information refer to the section on risk assessment or contact the Biological Safety Officer.

Rules, Regulations & Guidelines

The following is a brief summary of the regulatory authorities that either regulate or provide guidelines for the use of biological materials, infectious agents and recombinant DNA molecules.

  1. National Institute of Health (NIH): Guidelines for Research Involving Recombinant DNA Molecules. These guidelines address the safe conduct of research that involves construction and handling of recombinant DNA (rDNA) molecules and organisms containing them. In 1974, a recombinant DNA Advisory Committee (RAC) was established to determine appropriate biological and physical containment practices and procedures for experiments that potentially posed risks to human health and the environment. As a result of the committee’s activity, the initial version of the NIH Guidelines was published in 1976. It has been amended and revised many times since then. Included in the Guidelines is a requirement for the institution to establish an Institutional Biosafety Committee (IBC) with authority to approve or disapprove proposed rDNA research using the NIH Guidelines as a minimum standard. For more information, please refer to the Recombinant DNA Research section in this manual and the NIH Guidelines for Research Involving Recombinant DNA Molecules.
  2. Centers for Disease Control and Prevention (CDC) and the National Institute of Health (NIH) Guidelines on: Biosafety in Microbiological and Biomedical Laboratories (BMBL) 5 th edition, Feb 2007. In 1984, the CDC/NIH published the first edition of the BMBL. This document describes combinations of standard and special microbiological practices, safety equipment, and facilities that constitute Biosafety Levels 1-4, which are recommended for working with a variety of infectious agents in various laboratory settings. The BMBL has been revised several times and is commonly seen as the standard for biosafety. OSU is using the BMBL and the Biosafety Manual from Michigan State University as the basis for this biosafety manual.
  3. Occupational Safety and Health Administration: Blood-borne Pathogens Standard. In 19922, the Occupational Safety and Health Administration (OSHA) promulgated a rule to deal with the occupational health risk caused by exposure to human blood and other potentially infectious materials. OSHA’s rule includes a combination of engineering and work practice controls, personal protective clothing and equipment, training and medical follow-up of exposure incidents, vaccination, and other provisions.
  4. Department of Health and Human Services (HHS - 42CFR 73): Possession, Use, and Transfer of Select Agents and Toxins. In 2002, HHS published a set of rules that require facilities and institutions to be registered and approved in order to possess, use, or transfer certain biological agents and toxins. HHS requires OSU to comply with the BMBL (see above) and OSHA’s Laboratory Safety Standard 29 CFR 1910.1450. A copy of the most current list of restricted agents and toxins covered under this rule is included in Appendix A3.
  5. Packaging, shipment and transportation requirements for infectious substances, diagnostic specimens and biological products are addressed in the following rules and guidelines:
  6. Agencies
  7. Importation permits are required for certain infectious agents, biological materials and animals as outlined in US Public Health Service, 42 CFR Part 71, Foreign Quarantine. In addition, the Department of Agriculture (USDA) Animal and Plant Health Inspection Service (APHIS) requires permits for importation and transportation of controlled materials, certain organisms or vectors. This includes animal and plant pathogens, certain tissue cultures and live animals. APHIS also regulates the importation, interstate movement, or environmental release of genetically engineered organisms as regulated under 7 CFR Part 340.

Practices and Procedures

Routes of Infection

Working in a biological research environment like OSU, it is not unreasonable to expect that a laboratory person working with infectious materials is more likely to become infected than members of the general community. An infection occurs when disease-causing microorganisms enter the human body in sufficient numbers and by a particular route and overcome the body’s defense system. The following routes of infection have been reported for laboratory-acquired infections:
  1. Through the mouth
  1. Through the skin
  1. Through the eye
  1. Through the lungs
Most of the laboratory-acquired infections reported in the literature point to accidents during work with some type of infectious agent. These include spills, splashes and accidents involving needles or other sharp objects. The general laboratory procedures outlined in this manual address those issues and provide for guidance in handling infectious or potentially infectious materials. 

Administrative Controls

Biohazard Warning Sign

A biohazard label is required for all areas or equipment in which RG-2 or 3 agents are handled or stored or where BL-2 or 3 procedures are required. The appropriate place for posting the label is at the main entrance door(s) to laboratories and animal rooms, on equipment like refrigerators, incubators, transport containers, and/or lab benches. Labels can be obtained from the EH&S.

 

Training

Good microbiological and laboratory practices are essential for a safe work environment. Training and education on these practices and procedures needs to start already at the undergraduate level. In addition, all personnel working with RG-2 or 3 agents or at BL-2 or 3 should receive adequate laboratory specific training from the Principal Investigator (PI) or laboratory supervisor. Training should include at a minimum:

In addition, it is mandatory that all personnel working with select agents or at BL-2 or 3 or handling RG-2 or 3 agents attend the biosafety training offered by EH&S.

Blood-borne Pathogens Program In accordance with OR-OSHA requirements, OSU has established an Exposure Control Plan covering the potential exposure to blood-borne pathogens (e.g., HIV, Hepatitis B virus) found in human blood, serum and tissue as well as in other potentially infectious materials. Please refer to Section I in this manual for more information.

Recombinant DNA Program All research at OSU involving recombinant DNA, independent of the funding source, needs to be in compliance with the requirements of the NIH Guidelines for Research Involving Recombinant DNA Molecules and is subjected to BC (Biosafety Committee) approval. Please refer to Section H in this manual for more information.

Infectious Agents/rDNA Registration Form For all research at OSU involving RG-2 and 3 agents or BSL-2 and 3 procedures, or certain toxins, a registration form might need to be filed with EH&S prior to initiation of the project. The information provided in the registration document will be used for project review and emergency response. A copy of the Project Registration of Biohazard / Recombinant DNA Research is included in Appendix D.

CDC Select Agents Requirements The Centers for Disease Control and Prevention (CDC) mandates specific requirements for facilities which possess, use, or transfer certain infectious agents and toxins (HHS - 42CFR 73: Possession, Use, and Transfer of Select Agents and Toxins). A list of these restricted agents is included in Appendix A3. EH&S will function as the central unit handling all transfer requests of these agents by Principal Investigators. Please contact the Biosafety Officer for more information.

Biosafety Committee (BC) The BC was initially established to meet the requirements mandated in the NIH Guidelines for Research Involving Recombinant DNA Molecules. However, the BC is now involved in the oversight of all projects involving infectious agents (RG-2 and 3).

Biological Safety Officer The Biological Safety Officer is responsible for managing the day-to-day affairs of biological safety.

Environmental Health & Safety (EH&S) EH&S is the office responsible for overseeing safety procedures and practices at the University.

Engineering Controls

Biological Safety Cabinets (BSCs)

BSCs are designed to provide personnel, environmental and product protection when appropriate practices and procedures are followed. Three kinds of biological safety cabinets, designated as Class I, II and III have been developed to meet various research and clinical needs. Biological safety cabinets use high efficiency particulate air (HEPA) filters in their exhaust and/or supply systems. Biological safety cabinets must not be confused with other laminar flow devices or “clean benches”; in particular, horizontal flow cabinets which direct air towards the operator and should never be used for handling infectious, toxic or sensitizing materials.

Diagrams of BSC's can be found in the EH&S Local Ventilation Guide.

Laboratory personnel must be trained in the correct use and maintenance of biological safety cabinets to ensure that personnel and product protection (where applicable) are maintained. It is highly recommended that all users of BSCs attend one of the annual BSCs safety seminars offered by EH&S.  Before selecting any biosafety cabinet for purchase, contact the Biological Safety Officer for a work specific assessment and selection criteria.

  1. Class I Biological Safety Cabinet

    This is a ventilated cabinet for personnel protection with an non-recirculated inward airflow away from the operator. This unit is fitted with a HEPA filter to protect the environment from discharged agents. A Class I BSC is suitable for work involving low to moderate risk agents, where there is a need for containment, but not for product protection (e.g., sterility).

  2. Class II Biological Safety Cabinet

    This is a ventilated cabinet for personnel, product and environmental protection which provides inward airflow and HEPA-filtered supply and exhaust air. The Class II cabinet has four designs depending on how much air is re-circulated and/or exhausted and if the BSC is hard-ducted to the ventilation system or not. Class II cabinets may be of use with low to moderate risk biological agents, minute quantities of toxic chemicals, and trace quantities of radionuclides; however, care must be exercised in selecting the correct Class II cabinet design for these purposes. 

  3. Class III Biological Safety Cabinet

    A Class III cabinet is a totally enclosed ventilated cabinet which is gas-tight, and maintained under negative air pressure (0.5 inches water gauge). The supply air is HEPA-filtered and the exhaust air has two HEPA filters in series. Work is performed in the cabinet by the use of attached rubber gloves.

Biological safety cabinets, when properly used in research and teaching activities involving the manipulation of biohazardous agents, are effective in containing and controlling particulates and aerosols and complement good laboratory practices and procedures. The correct location, installation, and certification of the biological safety cabinet is critical to its performance in containing infectious aerosols. All BSCs used for RG-2 or 3 and rDNA research must be inspected annually and certified by trained and accredited service personnel according to the NSF (National Sanitation Foundation) Standard 49. Inspection and re-certification is mandatory if the cabinet is relocated or after major repairs, filter changes etc. 

To request service or certification contact the EH&S. Fees for these services are charged to the Principal Investigator or Laboratory Director. 

CDC and NIH have published a guide on BSCs: Primary Containment for Biohazards: Selection, Installation and Use of Biological Safety Cabinets. Copies are available from the EH&S.

Safe and Effective Use of Biosafety Cabinets

  1. General:
  2. Operation:

Safety Equipment

    1. Safety Showers provide an immediate water drench of an affected person. Standards for location, design and maintenance of safety showers are outlined in the CHP.
    2. Eyewash Stations are required in all laboratories where injurious or corrosive chemicals are used or stored and where employees perform tasks that might result in splashes of potentially infectious materials. Standards for location, design and maintenance of emergency eyewash facilities are outlined in the CHP.
    3. Ventilation Controls are those controls intended to minimize employee exposure to hazardous chemicals and infectious or toxic substances by removing air contaminants from the work site. There are two main types of ventilation controls:
      • General (Dilution) Exhaust: a room or building-wide system which brings in air from outside and ventilates within. Laboratory air must be continually replaced, preventing the increase of air concentration of toxic substances during the work. General exhaust systems are inadequate for RG-3 agents or BL-3 work.
      • Local Exhaust or Filtration: a ventilated, enclosed work space intended to capture, contain and exhaust or filter harmful or dangerous fumes, vapors and particulate matter. In the case of hazardous chemicals this includes a fume hood. In the case of infectious agents BSCs should be used. For more information on ventilation requirements involving hazardous chemicals refer to the CHP.

Personal Protective Equipment (PPE)

PPE is used to protect personnel from contact with hazardous materials and infectious agents. Appropriate clothing may also protect the experiment from contamination. Personal protective devices and safety equipment must be provided to all employees under the appropriate circumstances and employees have the responsibility of properly using the equipment. The following PPE is recommended for regular use. 

Face Protection Splash goggles or safety glasses with solid side shields in combination with masks, or chin length face shields or other splatter guards are required for anticipated splashes, sprays or splatters of infectious or other hazardous materials to the face. Information on the availability of low cost prescription safety eyewear may be obtained by contacting EH&S. 

Laboratory Clothing This category includes laboratory coats, smocks, scrub suits, and gowns. Long-sleeved garments should be used to minimize the contamination of skin or street clothes. In circumstances where it is anticipated that splashes may occur, the garment must be resistant to liquid penetration to protect clothing from contamination. If the garment is not disposable, it must be capable of withstanding sterilization, in the event it becomes contaminated. At a minimum, a laboratory coat should be worn in all laboratories working at a BL-2. Additional criteria for selecting clothing are: comfort, appearance, closure types and location, antistatic properties and durability. Protective clothing must be removed and left in the laboratory before leaving for non-laboratory areas. Disposable clothing should be available for visitors, maintenance and service workers in the event it is required. All protective clothing should be either discarded in the laboratory or laundered by the university. Personnel must not take laboratory clothing home. 

Gloves Gloves must be selected based on the hazards involved and the activity to be conducted. Gloves must be worn when working with biohazards, toxic substances, hazardous chemicals and other physically hazardous agents. Temperature resistant gloves must be worn when handling hot material or dry ice. Delicate work requiring a high degree of precision dictates the use of thin walled gloves. Protection from contact with toxic or corrosive chemicals may also be required. For assistance in glove selection, contact EH&S.

Respirators For certain protocols and projects, additional PPE like respiratory protection may be required. Respirator selection is based on the hazard and the protection factor required. Personnel who require respiratory protection must contact EH&S for assistance in selection of proper equipment and training in its usage. Personnel wearing respirators need to be included in OSU’s Respiratory Protection Program.

Recommended Work Practices

Pipets and Pipet Aids

Mouth pipetting is strictly prohibited. Mechanical pipetting aids must be used. Confine pipetting of biohazardous or toxic fluids to a biosafety cabinet if possible. If pipetting is done on the open bench, use absorbent pads or paper on the bench. Use the following precautions:

Syringes and Needles

Syringes and hypodermic needles are dangerous objects that need to be handled with extreme caution to avoid accidental injection and aerosol generation. Generally, the use of syringes and needles should be restricted to procedures for which there is no alternative. Do not use a syringe and needle as a substitute for a pipette.

Use needle locking syringes or disposable syringe-needle units in which the needle is an integral part of the syringe.

When using syringes and needles with biohazardous or potentially infectious agents:

Needles should not be bent, sheared, replaced in the sheath or guard (capped), or removed from the syringe following use. If it is essential that a contaminated needle be recapped or removed from a syringe, the use of a mechanical device or the one-handed scoop method must be used. Always dispose of needle and syringe unit promptly into an approved sharps container.

Do not fill sharps containers more that 2/3 full. Contact EH&S for pickup (see Biohazardous Waste section).

Cryostats

Frozen sections of unfixed human tissue or animal tissue infected with an etiologic agent pose a risk because accidents can occur. Freezing tissue does not necessarily inactivate infectious agents. Freezing propellants under pressure should not be used for frozen sections as they may cause spattering of droplets of infectious material. Gloves should be worn during preparation of frozen sections. When working with biohazardous material in a cryostat, the following is recommended:

Centrifuge Equipment

Hazards associated with centrifuging include mechanical failure and the creation of aerosols. To minimize the risk of mechanical failure, centrifuges must be maintained and used according to the manufacturer’s instructions. Users should be properly trained and operating instructions including safety precautions should be prominently posted on the unit. 

Aerosols are created by practices such as filling centrifuge tubes, removing supernatant, and suspending sediment pellets. The greatest aerosol hazard is created if a tube breaks during centrifugation. To minimize the generation of aerosols when centrifuging biohazardous material, the following procedures should be followed:

Blenders, Ultrasonic Disrupters, Grinders and Lyophilizers

The use of any of these devices results in considerable aerosol production. Blending, cell-disrupting and grinding equipment should be used in a BSC when working with biohazardous materials.

Safety Blenders

Safety blenders, although expensive, are designed to prevent leakage from the bottom of the blender jar, provide a cooling jacket to avoid biological inactivation, and to withstand sterilization by autoclaving. If blender rotors are not leak-proof, they should be tested with sterile saline or dye solution prior to use with biohazardous material. The use of glass blender jars is not recommended because of the breakage potential. If they must be used, glass jars should be covered with a polypropylene jar to prevent spraying of glass and contents in the event the blender jar breaks. A towel moistened with disinfectant should be placed over the top of the blender during use. Before opening the blender jar, allow the unit to rest for at least one minute to allow the aerosol to settle. The device should be decontaminated promptly after use.

Lyophilizers and Ampoules

Depending on lyophilizer design, aerosol production may occur when material is loaded or removed from the lyophilizer unit. If possible, sample material should be loaded in a BSC. The vacuum pump exhaust should be filtered to remove any hazardous agents or, alternatively, the pump can be vented into a BSC. After lyophilization is completed, all surfaces of the unit that have been exposed to the agent should be disinfected. If the lyophilizer is equipped with a removable chamber, it should be closed off and moved to a BSC for unloading and decontamination. Handling of cultures should be minimized and vapor traps should be used wherever possible.

Opening ampoules containing liquid or lyophilized infectious culture material should be performed in a BSC to control the aerosol produced. Gloves must be worn. To open, nick the neck of the ampoule with a file, wrap it in disinfectant soaked towel, hold the ampoule upright and snap it open at the nick. Reconstitute the contents of the ampoule by slowly adding liquid to avoid making an aerosol of the dried material. Mix the container. Discard the towel and ampoule top and bottom as biohazardous waste.

Ampoules used to store biohazardous material in liquid nitrogen have exploded causing eye injuries and exposure to the infectious agent. The use of polypropylene tubes eliminates this hazard. These tubes are available dust free or pre-sterilized and are fitted with polyethylene caps with silicone washers. Heat seal able polypropylene tubes are also available.

Loop Sterilizers and Bunsen Burners

Sterilization of inoculating loops or needles in an open flame generates small particle aerosols which may contain viable microorganisms. The use of a shielded electric incinerator or hot bead sterilizers minimizes aerosol production during loop sterilization. Alternatively, disposable plastic loops and needles may be used for culture work where electric incinerators or gas flames are not available or recommended. 

Laundry

All personal protective clothing should be either discarded in the lab or laundered by the university at no cost to employees. Apparel contaminated with human blood or other potentially infectious materials should be handled as little as possible and needs to be collected in biohazard bags. Appropriate PPE must be worn by employees who handle contaminated laundry.

Housekeeping

Good housekeeping in laboratories is essential to reduce risks and protect the integrity of biological experiments. Routine housekeeping must be relied upon to provide work areas free of significant sources of contamination. Housekeeping procedures should be based on the highest degree of risk to which personnel and experimental integrity may be subjected.

Laboratory personnel are responsible for cleaning laboratory benches, equipment and areas that require specialized technical knowledge. Additional laboratory housekeeping concerns include:

Biohazard Spill Cleanup Procedures

Since spills of biological materials will happen, it is important to be prepared prior to dealing with the problem. Laboratories working with biohazards should have a basic biological spill kit ready to use at all times. For most instances the basic kit can be assembled with materials already used in the laboratory. Although it is preferable to have the content of the spill kit in one location, as long as the materials are easily accessible to everyone in the lab, prior assembly might not be necessary. Basic Biological Spill Kit
Disinfectant (e.g., bleach 1:10 dilution, prepared fresh) 
Absorbent Material (e.g., paper towels) 
Waste Container (e.g., biohazard bags, sharps containers) 
Personal Protective Equipment (e.g., lab coat, gloves, eye and face protection)
Mechanical Tools (e.g., forceps, dustpan and broom)
The following procedures are provided as a guideline to biohazardous spill cleanup and will need to be modified for specific situations. As with any emergency situation, stay calm, call 911 if necessary, and proceed with common sense. Call EH&S if further assistance is required, especially if the spill outgrows the resources in the laboratory. 

Spill Inside the Laboratory (BL-2, RG-2)

Clear spill area of all personnel. Wait for any aerosols to settle before entering spill area. Remove any contaminated clothing and place in biohazard bag for further processing by laundry (OSU or department). Don a disposable gown or lab coat, safety goggles and gloves.

Have a complete biological spill kit ready to go before you start the cleanup

Initiate cleanup with disinfectant as follows:

  • Cover spill with paper towels or other absorbent material containing disinfectant. 
  • Encircle the spill with disinfectant (if feasible and necessary), being careful to minimize aerosols. 
  • Decontaminate and remove all items within spill area. 
  • Remove broken glassware with forceps or broom and dustpan and dispose in sharps container. Do not pick up any contaminated sharp object with your hands. 
  • Remove paper towels and any other absorbent material and dispose in biohazard bags. 
  • Apply disinfectant to the spill area and allow for at least 10 minutes contact time to ensure germicidal action of disinfectant. 
  • Remove disinfectant with paper towels or other absorbent material and dispose of in biohazard bag. 
  • Wipe off any residual spilled material and reapply disinfectant before final cleanup 
  • Wipe equipment with equipment compatible disinfectant (e.g., non-corrosive). Rinse with water if necessary. 
  • Place disposable contaminated spill materials in biohazard bags for autoclaving. 
  • Place contaminated reusable items in biohazard bags, or heat resistant pans or containers with lids before autoclaving.·
  • Reopen area to general use only after spill cleanup and decontamination is complete. 
  • Inform all personnel and laboratory supervisor about the spill and successful cleanup as soon as possible. 

Spill Inside the Biological Safety Cabinet (BL-2, RG-2)

Have a complete biological spill kit ready to go before you start the cleanup

  • Wear lab coat, safety goggles and gloves during cleanup 
  • Allow cabinet to run during cleanup 
  • Soak up spilled material with disposable paper towels (work surface and drain basin) and apply disinfectant with a minimum of 10 minutes contact time. 
  • Wipe up spillage and disinfectant with disposable paper towels. 
  • Wipe the walls, work surface and any equipment in the cabinet with a disinfectant soaked paper towel. 
  • Discard contaminated disposable materials in biohazard bag(s) and autoclave before discarding as waste. 
  • Place contaminated reusable items in biohazard bags, or heat resistant pans or containers with lids before autoclaving and further cleanup 
  • Expose non-autoclavable materials to disinfectant, 10 minutes contact time, before removal from the BSC. 
  • Remove protective clothing used during cleanup and place in a biohazard bag for further processing by laundry (department responsibility). 
  • Run cabinet at least 10 minutes after cleanup and before resuming work. 
  • Inform all users of the BSC as well as the laboratory supervisor about the spill and successful cleanup as soon as possible. 

Spill Outside the Laboratory, During Transport on Campus.

  • Always transport biohazardous materials in an unbreakable well-sealed primary container placed inside a leak-proof, closed and unbreakable secondary container, labeled with the biohazard symbol (plastic cooler, bio-specimen pack, etc.).
  • Should a spill of RG-2 material occur in the public, contact EH&S. Do not attempt to clean up the spill without the proper personal protective equipment and spill cleanup material.
  • Should the spill occur inside a car, leave the vehicle, close all doors and windows, and contact EH&S for assistance.

General Guidelines and Policies

Biological Risk Assessment

The assessment of risk is an essential element of safety in the laboratory. For most situations, guidelines, rules and regulations have clearly defined the procedures and practices to be followed in order to achieve safety in the work place. However, the newly isolated agent or toxin, or the new procedure never before employed need further evaluation. Questions concerning the appropriate safety equipment, training and waste disposal need to be addressed as well as safe procedures and practices. Something is considered safe if the risk associated with it is judged to be acceptable. However, since individual judgment involves both personal and social values, opinions on what is safe or not can vary significantly. In order to find a common ground for an acceptable risk assessment, the “rule of reason” needs to be applied. The following factors should be considered for the determination of reasonableness:

  1. Custom of usage (or prevailing professional practice): Many laboratory procedures involve the maintenance of sterility and cleanliness. These procedures are commonly considered safe, since adverse effects would have been obvious over time. However, because a procedure has been used for many years does not necessarily imply that it is safe. The best example is mouth-pipetting, which was used for centuries and finally considered a very dangerous procedure and habit.

  2. Best available practice, highest practicable protection, and lowest practicable exposure: It should be common practice in the microbiological laboratory to use the best available procedures with the highest level of protection. This not only provides for a safe work environment but also fosters excellence in scientific conduct.

  3. Degree of necessity or benefit: The common question to ask is, are the benefits worth the risk? There is no need to use a human pathogen causing severe gastroenteritis in a teaching laboratory when principal microbiological practices can be taught with an organism that is not considered to be infectious.

  4. No detectable adverse effects: This can be a very weak criterion since it involves uncertainty or even ignorance.

  5. Principal knowledge: A lot of times, existing procedures are modified, involving the same or similar toxic chemicals or agents. For that reason, similar safety procedures should be applied. If new agents are isolated, we need to ask what we know about the close relatives. Many agents of known etiologic character are already categorized in risk groups allowing for the selection of the appropriate biosafety level. New isolates from infected animals or humans with known infectious relatives warrant at a minimum the same level of protection. 

Taking the above mentioned factors, as well as others, into consideration will allow for a reasonable approach to a new challenge. EH&S is available to assist in this process and should be contacted for questions concerning radiation, chemical and biological safety. Once a risk assessment is completed, the results should be communicated to everyone involved in the process. If necessary, written standard operating procedures (SOPs) should be established and distributed.

Guidelines for Working with Tissue Culture/Cell Lines

When cell cultures are known to contain an etiologic agent or an oncogenic virus, the cell line can be classified at the same RG level as that recommended for the agent.

The Centers for Disease Control and Prevention (CDC) and OSHA recommend that all cell lines of human origin be handled at BL-2. All personnel working with or handling these materials need to be included in OSU’s Exposure Control Plan.

Cell lines which are non-primate or are of normal primate origin, which do not harbor a primate virus, which are not contaminated with bacteria, mycoplasma or fungi and which are well established may be considered Class I cell lines and handled at a Biosafety Level 1. Appropriate tests should confirm this assessment.

Primate cell lines derived from lymphoid or tumor tissue, all cell lines exposed to or transformed by a primate oncogenic virus, all clinical material (e.g., samples of human tissues and fluids obtained after surgical resection or autopsy), all primate tissue, all cell lines new to the laboratory (until shown to be free of all adventitious agents) and all virus and mycoplasma-containing primate cell lines are classified as RG-2 and should be handled at a Biosafety Level 2. Studies involving suspensions of HIV prepared from T-cell lines must be handled at BL-3.

Guidelines for Preventing the Transmission of Tuberculosis

Since 1985, the incidence of tuberculosis in the United States has been increasing steadily, reversing a 30 year downward trend. Recently, drug resistant strains of Mycobacterium tuberculosis have become a serious concern. Outbreaks of tuberculosis, including drug resistant strains, have occurred in healthcare environments. Several hundred employees have become infected after workplace exposure to tuberculosis, requiring medical treatment. A number of healthcare workers have died.

In October 1994, CDC published its Guidelines for Preventing the Transmission of Tuberculosis in Health-Care Facilities. The guidelines contain specific information on ventilation requirements, respiratory protection, medical surveillance and training for those personnel who are considered at risk for exposure to tuberculosis. For more information, contact the OSU Biosafety Officer.

Investigators intending to work with Mycobacterium sp. in the laboratory must register with EH&S by using the Infectious Agent/rDNA Registration Form. Propagation and/or manipulation of Mycobacterium tuberculosis and M. bovis cultures in the laboratory or animal room must be performed at BL-3 and require BC approval.

Guidelines for Clinical Laboratories

Clinical laboratories receive clinical specimens with requests for a variety of diagnostic services. The infectious nature of this material is largely unknown. In most circumstances, the initial processing of clinical specimens and identification of microbial isolates can be done safely at BL-2.  

A primary barrier, such as a biological safety cabinet, should be used: 

All laboratory personnel who handle human source materials are included in the Blood-borne Pathogens Program as outlined in OSU’s Exposure Control Plan. “Universal Precautions” need to be followed when handling human blood, blood products, body fluids or tissues.

The segregation of clinical laboratory functions and restricting access to specific areas is the responsibility of the laboratory director. It is also the director’s responsibility to establish standard, written procedures that address the potential hazards and the required precautions to be implemented. A copy of the Exposure Control Plan must be available in all laboratories. Additional recommendations specific for clinical laboratories may be obtained from the National Committee for Clinical Laboratory Standards (NCCLS).

Guidelines for Experiments with Wild Rodents

Field Research Practices for OSU

These guidelines are based on practices used by the Centers for Disease Control and Prevention (CDC) personnel in areas known to contain rodents carrying agents that cause hantavirus pulmonary syndrome (HPS). These guidelines have been modified for fieldwork in areas of low or undefined risk. They are intended to provide information about work practices useful in protecting against HPS.

EH&S is available to assist in adapting these guidelines to specific fieldwork projects. Contact the EH&S if you have comments or suggestions about the guidelines. 

Background: 

Rodents are the known main reservoirs of Hantaviruses, while other small mammals can be infected as well. Although current evidence indicates that the rodents harboring Hantaviruses are most prevalent in rural settings, suburban or urban areas cannot be excluded as being potentially affected. 

Infection is believed to result from inhalation, inoculation through broken skin, or rodent bites. Persons have been infected after only a few minutes of exposure to laboratory rodents infected with a similar virus. 

People who frequently handle or are exposed to rodents (e.g., mammalogists, field biologists, pest-control workers) are at higher risk for HPS than the general public. The nature of their work brings them into closer and more frequent contact with rodents and their excretions. The likelihood of infection with HPS appears to be low, but the fatality rate for some of the virus strains is high (~60%). At present, there is no effective vaccine to prevent infection, and diagnosis and treatment are difficult. Enhanced precautions are warranted to protect against possible infection. 

It is the responsibility of each individual to take appropriate protective measures. It is the responsibility of the supervisor to ensure training and access to appropriate protective measures. 

General Practices: 
  1. Practice good personal hygiene at all times. Wash your hands with soap and water or with a disinfectant wipe before eating, drinking, smoking, or applying lip balm, sunscreen or cosmetics. 
  2. Workers in potentially high-risk settings should receive a thorough orientation about Hantavirus transmission and the symptoms of the disease. They should be given detailed guidance on prevention measures and trained to safely perform the required activities. EH&S has appropriate training materials available.
  3. Workers who develop febrile or respiratory illness within 45 days of potential exposure should immediately seek medical attention and inform the physician of the occupational risk of Hantavirus infection. If appropriate blood samples may be submitted for Hantavirus antibody testing. 
  4. Hantaviruses are lipid-enveloped viruses and are susceptible to most disinfectants, like dilute hypochlorite solutions (1:10 bleach solution), 70% alcohol, detergents, phenolics, or most general-purpose household disinfectants. The survival time of the virus in the environment in liquids, aerosols, or dried states is not known. In the field, carry a spray bottle of disinfectant or handwipes containing alcohol or detergent. 
  5. Workers should wear rubber, plastic or latex gloves when handling rodents or traps contaminated by rodents or whenever the worker has broken skin. If non-disposable rubber gloves are used, wash gloved hands in a disinfectant before removing the gloves. Thoroughly wash hands with soap and water after removing gloves. If this is not possible, then rinse gloves with water or use a disinfectant wipe; wash your hands thoroughly at end of the work period. Never wash or reuse disposable gloves.
  6. Workers may need to wear respiratory protection when handling field-caught rodents or contaminated traps or when disturbing rodent burrows and nests. Contact EH&S for an evaluation of your work practices and for information about OSU’s Respiratory Protection Program. 
  7. Until the infectivity of Hantaviruses is better understood, respirators should be used to minimize exposure to airborne particles of rodent excreta during procedures that generate aerosols. The proper use of respirators will provide protection against airborne particles of rodent excreta, which is the presumed cause of most Hantavirus infections. Individual workers should consult EH&S for an analysis of  their work practices, risks of exposure, and personal fitness to determine their need for respiratory protection. The recommended respiratory protection against Hantavirus is a half-face or full-face air-purifying (negative-pressure) respirator with HEPA filters or a powered air-purifying respirator (PAPR) equipped with HEPA filters.
  8. Disinfect all traps contaminated by rodent urine or feces or in which a rodent was captured. If this is not done until the end of the trapping run, wear a respirator whenever handling contaminated traps, and transport empty traps in closed plastic bags. 
  9. In populated areas, dispose of dead rodents by placing the carcasses in a plastic bag containing enough disinfectant to thoroughly wet the carcasses. Seal the bag and dispose of in the landfill. 
  10. Personnel performing procedures with a high risk of contacting animal body fluids or creating aerosols, such as removing organs or obtaining blood from wild caught rodents, should contact EH&S for detailed safety precautions. 
  11. Do not enter enclosed spaces or buildings visibly contaminated with rodents or rodent droppings without the proper protection. Contact the facility manager or EH&S for assistance. 
Specific Practices: 
  1. Visual Survey of Area, Walking, Hiking
  2. Setting Trap Lines
  3. Recovery and Transport of Traps Holding Live Animals
  4. Handling Live Rodents
  5. Field Dissection
  6. Clean Up
  7. Additional Precautions

Use of Animals in Research, Teaching, and Service

The use of animals in research, teaching, and outreach activities is subject to state and federal laws and guidelines. OSU’s policy specifies that:

The IACUC should be contacted for questions regarding the use of animals for teaching and research.

Principal Investigators planning to use animals for any OSU-related activity must submit proper animal use forms to the IACUC for review prior to the start of the project, regardless of the source of funding for the project.  Completed forms will include descriptions of experimental protocols, plans for animal care, available facilities, and information on the use of hazardous materials including infectious agents.

All areas housing research animals need to have appropriate hazard warnings posted on the main entrance door if the project involves any of the hazards listed above. It is the responsibility of the Principal Investigator to establish this form listing all relevant entry, animal care and emergency procedures. EH&S as well as the appropriate animal Facility Supervisor will assist the PI in this process.

Use of Human Subjects and Materials

Federal and University regulations and policies require that all research involving human subjects or materials be reviewed and approved before initiation by the University’s Institutional Review Board (IRB) to protect the rights and welfare of human subjects. 

Prescribed by the National Research Act of 1974 (PL 93-348) and endorsed by the University, the IRB reviews applications for research involving human subjects. Reviews are performed by the IRB in accordance with the U.S. Department of Health and Human Services (HHS) regulations for the Protection of Human Research Subjects (45 CFR 46, as amended).

It is the responsibility of the Project Investigator to assure that all research involving human subjects is reviewed and approved by the IRB prior to initiation. All personnel with a reasonable anticipated risk of exposure to bloodborne pathogens through the contact with human blood or other human materials must be included in OSU’s Bloodborne Pathogen Program. 

For more information or for consultation, contact the IRB at (541) 737-3437, or via Email at IRB@oregonstate.edu.

Transportation of Biological Materials On and Off Campus

All biological materials should be transported in a way that maintains the integrity of the material during normal transport conditions, as well as prevents any accidental release and endangerment of the public and the environment.

Transportation in-between buildings or locations on and off campus roads:

Diagnostic and clinical specimens, infectious materials and recombinant DNA molecules need to be packaged in a sealed, leakproof primary container (e.g., glass tube), which is securely positioned in a secondary leakproof and closable container (e.g., cooler, ice chest) containing a clearly visible biohazard symbol on the outside. A list of contents as well as emergency information (e.g., PI phone number) needs to be accompanying the material (e.g., attached to the cooler in a plastic pouch). The use of private cars for the transportation of such materials on or off campus is highly discouraged. University vehicles are available upon request through the individual departments. In case of an emergency (e.g., car accident), make all police and safety personnel aware of the presence of biohazardous materials and contact EH&S.

Transportation and shipment via carrier off campus:

The shipment of diagnostic and clinical specimens, biological products, infectious agents and recombinant DNA molecules is regulated by national and international transportation rules. This includes specific procedures for the correct packing and packaging of these materials, necessary documentation and labeling and permits. For more information about specific shipment requirements, contact EH&S.

Decontamination

Methods of Decontamination

Decontamination is defined as the reduction of microorganisms to an acceptable level. Methods applied to reach this goal can vary and most often include disinfection or sterilization. Generally speaking, disinfection is used when the acceptable level of microorganisms is defined as being below the level necessary to cause disease. This means, that viable microorganisms are still present. In contrast, sterilization is defined as the complete killing of all organisms present. Depending on the circumstances and tasks, decontamination of a surface (e.g., lab bench) is accomplished with a disinfectant, while decontamination of biomedical waste is done by sterilization in an autoclave. 

In order to select the proper method and tools, it is important to consider, for example, the following aspects:

Physical and chemical means of decontamination fall into four main categories:

Disinfection is normally accomplished by applying liquid chemicals or wet heat during boiling or pasteurization. To sterilize, vapors and gases (e.g., ethylene oxide), radiation, and wet heat (steam sterilization in an autoclave) are used. Some liquid chemicals are also applied for sterilization, if used in the right concentration and incubation time. The following paragraphs will focus on some of these methods.

Heat

In order to kill microbial agents, heat can be applied in dry or wet form. The advantage of wet heat is a better heat transfer to and into the cell resulting in overall shorter exposure time and lower temperature. Steam sterilization uses pressurized steam at 121-132º C (250-270ºF) for 30 or 40 minutes. This type of heat kills all microbial cells including spores, which are normally heat resistant. In order to accomplish the same effect with dry heat in an oven, the temperature needs to be increased to 160-170º C (320-338º F) for periods of 2 to 4 hours.

Liquid Chemicals Used as Disinfectants

The appropriate liquid disinfectant should be chosen after carefully assessing the biohazardous agent and the type of material to be decontaminated. Liquid disinfectants are preferably used for solid surfaces and equipment. They vary greatly in their efficiency, depending on the chemical constituents and the agents involved. Variables to remember when disinfecting:

Table 3. Resistance to Chemical Disinfectants

Least resistant

 

 

Most Resistant

  
Examples
LIPID OR MEDIUM-SIZE VIRUSES Herpes simplex virus
Cytomegalovirus
Respiratory syncytial virus
Hepatitis B virus
Human Immunodeficiency virus
VEGETATIVE BACTERIA Pseudomonas aeruginosa
Staphylococcus aureus
Salmonella choleraesuis
FUNGI Trichophyton sp.
Cryptococcus sp.
Candida sp.
NONLIPID OR SMALL VIRUSES Poliovirus
Coxsackievirus
Rhinovirus
MYCOBACTERIA Mycobacterium tuberculosis;
M. bovis
BACTERIAL SPORES Bacillus subtilis
Clostridium sporogenes

There are many different liquid disinfectants available under a variety of trade names. In general, these can be categorized as halogens, acids or alkalies, heavy metal salts, quaternary ammonium compounds, aldehydes, ketones, alcohols, and amines. Unfortunately, the most effective disinfectants are often very aggressive (corrosive) and toxic. Some of the more common ones are discussed below:

Alcohols: Ethyl or isopropyl alcohol in concentration of 70% to 90% are good general-use disinfectants. However, they evaporate fast and therefore have limited exposure time. They are less active against non-lipid viruses and ineffective against bacterial spores. Concentrations above 90% are less effective.

Formalin:  Formalin is 37% solution of formaldehyde in water. Dilution of formalin to 5% results in an effective disinfectant. Formaldehyde is a human carcinogen and creates respiratory problems at low levels of concentration.

Glutaraldehyde:  This compound although chemically related to formaldehyde, is more effective against all types of bacteria, fungi, and viruses. Vapors of glutaraldehydes are irritating to the eyes, nasal passages and upper respiratory tract. They should be used always in accordance with the instructions on the label and the appropriate personal protective equipment.

Phenol and Phenol Derivatives:  Phenol based disinfectants come in various concentrations ranging mostly from 5% to 10 %. These derivatives including phenol have an odor, which can be somewhat unpleasant. Phenol itself is toxic and appropriate personal protective equipment is necessary during application. The phenolic disinfectants are used frequently to disinfect contaminated surfaces (e.g., walls, floors, bench tops). They effectively kill bacteria including Mycobacterium tuberculosis, fungi and lipid-containing viruses. They are not active against spores or non-lipid viruses. 

Quaternary Ammonium Compounds (Quats):  Quats are cationic detergents with strong surface activity. They are acceptable for general-use disinfectants and are active against Gram-positive bacteria and lipid-containing viruses. They are less active against Gram-negative bacteria and are not active against non-lipid-containing viruses. Quats are easily inactivated by organic materials, anionic detergents or salts of metals found in water. If Quats are mixed with phenols, they are very effective disinfectants as well as cleaners. Quats are relatively nontoxic and can be used for decontamination of food equipment and for general cleaning.

Halogens (Chlorine and Iodine): 

Chlorine-containing solutions have broad spectrum activity. Sodium hypochlorite is the most common base for chlorine disinfectants. Common household bleach (5% available chlorine) can be diluted 1/10 to 1/100 with water to yield a satisfactory disinfectant solution. Diluted solutions may be kept for extended periods if kept in a closed container and protected from light. However, it is recommended to use freshly prepared solutions for spill clean-up purposes. Chlorine-containing disinfectants are inactivated by excess organic materials. They are also strong oxidizers and very corrosive. Always use appropriate personal protective equipment when using these compounds. At high concentrations and extended contact time, hypochlorite solutions are considered cold sterilants since they inactivate bacterial spores.

Iodine has similar properties to chlorine. Iodophors (organically bound iodine) are recommended disinfectants. They are most often used as antiseptics and in surgical soaps and are relatively nontoxic to humans.

Vapors and Gases:

A variety of vapors and gases possess germicidal properties. The most commonly used are formaldehyde and ethylene oxide. Applied in closed systems under controlled conditions (e.g., humidity) these gases achieve sterility. 

Formaldehyde gas is primarily used in the decontamination of spaces or biological containment equipment like biological safety cabinets. Formaldehyde is a toxic substance and a suspected human carcinogen. Considerable caution must be exercised in handling, storing, and using formaldehyde.

Ethylene oxide is used in gas sterilizers under controlled conditions. Ethylene oxide is also a human carcinogen and monitoring is necessary during its use.

Radiation:

Gamma and X-ray are two principal types of ionizing radiation used in sterilization. Their application is mainly centered on the sterilization of prepackaged medical devices. 

Ultraviolet (UV) radiation is a practical method for inactivating viruses, mycoplasma, bacteria and fungi. UV radiation is successfully used in the destruction of airborne microorganisms. UV light sterilizing capabilities are limited on surfaces because of its lack of penetrating power.

For a summary of properties and applications of disinfectants refer to Appendix C1.

Biohazardous Waste

At Oregon State University, the term biohazardous waste is used to describe different types of waste that might include infectious agents. Currently, the following waste categories are all considered to be biohazardous waste: The CDC/NIH Biosafety Guidelines cover contaminated waste that is potentially infectious or hazardous for humans and animals. The same is true for the NIH Guidelines on recombinant DNA which also cover contaminated waste potentially infectious or hazardous for plants.

General Labeling, Packaging and Disposal Procedures

Currently, biohazardous waste is to be decontaminated before leaving OSU. Most of the waste can be autoclaved prior to disposal, while some waste maybe incinerated. The responsibility for decontamination and proper disposal of biohazardous waste lies with the producing facility (e.g., laboratory and department). EH&S assists only in the disposal of sharps.

All biohazardous waste needs to be packaged, contained and located in a way that protects and prevents the waste from release at any time at the producing facility prior to ultimate disposal. If storage is necessary, putrefaction and the release of infectious agents in the air must be prevented. 

No biohazardous waste can be stored for more than 90 days at OSU.

If not stated otherwise (see below), most biohazardous waste will be disposed of in biohazard bags. OSU requires the use of orange or red biohazard bags that include the biohazard symbol and a built-in heat indicator. Bags that meet these requirements are available through Scientific supply sources. All waste disposed of in these bags is to be autoclaved until the waste is decontaminated. The built-in heat indicator will turn dark. For specific autoclave procedures, please contact EH&S.

All autoclaves used for the decontamination of biohazardous waste will be tested by EH&S at least on an annual basis. Please contact our office for more information. After successful autoclaving (decontamination), all biohazard bags need to be bagged in opaque (black) plastic non-biohazard bags that are leakproof. These opaque bags can be put in the regular garbage for pick up by custodial services. Biohazardous waste that has been successfully decontaminated by autoclaving is no longer considered hazardous.  

Since autoclaves are an integral part of OSU’s biohazardous waste treatment procedure, proper operation and maintenance is very important. All users of autoclaves need to be trained in the proper operating procedures either through the laboratory supervisor or Principal Investigator or whoever was put in charge by the department. Maintenance and repair of autoclaves used for the decontamination of biohazardous waste are the responsibility of the individual departments. If the department chooses to not use autoclaves for their biohazardous waste treatment, alternative procedures (e.g., outside biomedical waste hauler) need to be established. For more information contact EH&S.

Waste Specific Procedures for BL-1 and 2

Cultures, Stocks and Related Materials

Cultures and stocks of infectious agents and associated biologicals (as defined above), shall be placed in biohazard bags and decontaminated by autoclaving. Double or triple bagging may be required to avoid rupture or puncture of the bags.

Bulk Liquid Waste, Blood and Blood Products

All liquid waste from humans or animals such as blood, blood products and certain body fluids, not known to contain infectious agents, can be disposed of directly by flushing down a sanitary sewer. However, due to coagulation, flushing of large quantities of blood is impractical. Contact EH&S for additional information on disposal of large volumes of blood. All other liquid biohazardous waste needs to be autoclaved prior to disposal or treated with a disinfectant.

Sharps

All sharps must be placed in a rigid, puncture resistant, closable and leak proof container, which is labeled with the word “Sharps” and the biohazard symbol. Food containers (e.g., empty coffee cans) are not permissible as sharps containers. All sharps must be handled with extreme caution. The clipping, breaking, and recapping of needles is not recommended. Sharps containers should not be filled more than 2/3. After use, the container needs to be closed and labeled with an OSU Hazardous Materials Label. To comply with the 90 day storage limit, contact EH&S for pick-up as soon as possible. 

Contaminated Solid Waste

Contaminated solid waste includes cloth, plastic and paper items that have been exposed to agents infectious or hazardous to humans, animals, or plants. These contaminated items shall be placed in biohazard bags and decontaminated by autoclaving. Double or triple bagging may be required to avoid rupture or puncture of the bags. Contaminated Pasteur pipettes are considered sharps and need to be disposed of in a sharps container.

Waste Specific Procedures for Biosafety Level 3 (BL-3)

Pathological Waste

Animal Waste

Human Waste

Department or Facility Specific Waste Procedures

Decontamination of Biohazardous Waste by Autoclaving

Autoclave Procedures:

Recombinant DNA Research

As a condition for funding of recombinant DNA research, OSU must ensure that research conducted at or sponsored by OSU, irrespective of the source of funding, complies with the most current (NIH) Guidelines for Research Involving Recombinant DNA Molecules. At OSU, the responsibility for ensuring that recombinant DNA activities comply with all applicable guidelines rests with the institution and the Biosafety Committee (BC) acting on its behalf.

Before experiments involving recombinant DNA begin, the Principal Investigator (PI) must submit a Project Registration of Biohazard / Recombinant DNA Research form to the BC.

All recombinant DNA research proposals require the PI to make an initial determination of the required level of physical and biological containment. For that reason, the NIH has developed six categories (III-A to III-F) addressing different types of rDNA research.

If the proposed research falls within section III-A of the NIH Guidelines, the experiment is considered a “Major Action”. This includes experiments involving human gene transfer experiments. As a result, the experiment cannot be initiated without submission of relevant information to the Office of Recombinant DNA Activities at NIH. In addition, the proposal has to be published in the Federal Register for 15 days, it needs to be reviewed by the RAC, and specific approval by the NIH has to be obtained. The containment conditions for such an experiment will be recommended by the RAC and set by the NIH at the time of approval. The proposal requires BC approval before initiation.

If the proposed research falls within section III-B, the research cannot be initiated without submission of relevant information on the proposed experiment to NIH/ORDA (For exceptions see the guidelines). Experiments covered in III-B include the cloning of toxic molecules. The containment conditions for such experiments will be determined by NIH/ORDA in consultation with ad hoc experts. Such experiments require Biosafety Committee approval before initiation. Please refer to the guidelines for more specifics.

In section III-C, experiments with human subjects are covered. These experiments require BC and IRB (Institutional Review Board) approval and NIH/ORDA registration before initiation.

Section III-D, the next category, covers whole animal or plant experiments as well as projects involving DNA from Risk Group 2, 3 or 4 agents. Prior to the initiation of an experiment that falls into Section III-D, the PI must submit a Registration Document for Recombinant DNA Research to the Biosafety Committee. The BC reviews and approves all experiments in this category prior to their initiation. 

Section III-E experiments require that the filing of a Registration Document for Recombinant DNA Research with the BC at the time the experiment is initiated. The IBC reviews and approves all such proposals, but Biosafety Committee review and approval prior to initiation of the experiment is not required. 

Section III-F experiments are exempt and a registration with the BC is not required.

For every recombinant DNA research proposal (except for exempt experiments, such as in section III-F), the following information must be submitted to the BC as part of the Registration Document for Recombinant DNA Research:

Description of the proposed research

The descriptions must provide sufficient information about the experiments so that reference to other documents is not required. 

Accident, Spill and Disposal Procedures

A spill contingency plan must be described and implemented. This plan must provide for the containment as well as the safe clean up and decontamination of any spilled recombinant DNA material. Disposal methods must also be documented (refer to OSU’s Waste Disposal Guide).

Precautionary Medical Practices

Describe the reasons for using any medical monitoring of your personnel (e.g., immunization, baseline serum sampling). This description should include the specific test used, frequency, and actions to be taken upon receipt of test results.

Petitions

See the appropriate sections of the NIH Guidelines if you wish to petition NIH for exemption.

Compliance Statement

A compliance statement, reproduced below, must appear on each Registration Document. The Principal Investigator (PI) in charge of the recombinant DNA project must then sign and date the document. 

“I agree to fully comply with the NIH requirements pertaining to the shipment, transfer, and accident reporting for recombinant DNA materials. I agree to abide by all provisions of the most current NIH Guidelines. I have carefully reviewed and accept the responsibilities for Principal Investigators described in the NIH Guidelines. The information above is accurate and complete.” 

Responsibility of Principal Investigator (PI) for Recombinant DNA Research

The Principal Investigator is responsible for full compliance with the NIH Guidelines in the conduct of recombinant DNA research. Please refer to the most recent edition of the NIH Guidelines for Research Involving Recombinant DNA Molecules for more information.

General Responsibilities

As part of this general responsibility, the Principal Investigator shall:
  1. Initiate or modify no recombinant DNA research which requires Biosafety Committee approval prior to initiation until that research or the proposed modification thereof has been approved by the Biosafety Committee and has met all other requirements of the NIH Guidelines;
  2. Determine whether experiments are covered by Section III-E, Experiments that Require Biosafety Committee Notice Simultaneous with Initiation, and that the appropriate procedures are followed;
  3. Report any significant problems, violations of the NIH Guidelines, or any significant research-related accidents and illnesses to the Biological Safety Officer, the Biosafety Committee, NIH/ORDA, and other appropriate authorities (if applicable) within 30 days (reports to NIH/ORDA shall be sent to the Office of Recombinant DNA Activities, National Institutes of Health/MSC 7010, 6000 Executive Boulevard, Suite 302, Bethesda, Maryland 20892-7010, (301) 496-9838;
  4. Report any new information bearing on the NIH Guidelines to the Institutional Biosafety Committee and to NIH/ORDA (reports to NIH/ORDA shall be sent to the Office of Recombinant DNA Activities, National Institutes of Health/MSC 7010, 6000 Executive Boulevard, Suite 302, Bethesda, Maryland 20892-7010, (301) 496-9838);
  5. Be adequately trained in good microbiological techniques;
  6. Adhere to Institutional Biosafety Committee-approved emergency plans for handling accidental spills and personnel contamination; and
  7. Comply with shipping requirements for recombinant DNA molecules. Contact EH&S for more information.

Submissions by the Principal Investigator to the NIH/ORDA

The Principal Investigator shall:
  1. Submit information to NIH/ORDA for certification of new host-vector systems;
  2. Petition NIH/ORDA, with notice to the Institutional Biosafety Committee, for proposed exemptions to the NIH Guidelines;
  3. Petition NIH/ORDA, with concurrence of the Institutional Biosafety Committee, for approval to conduct experiments specified in Sections III-A-1, Major Actions Under the NIH Guidelines, and III-B, Experiments that Require NIH/ORDA and Institutional Biosafety Committee Approval Before Initiation;
  4. Petition NIH/ORDA for determination of containment for experiments requiring case-by-case review; and