LAB SAFETY

Biological Safety

Missouri S&T Biological Hygiene Plan
(Revised 9/3/2010)

The Missouri University of Science and Technology is committed to providing a safe learning and working environment for our students and employees.  As part of this commitment, this biological hygiene plan has been developed to establish a standard to provide this safe environment.  The goals of the University's biological safety program are to:

• protect staff and students from exposure to infectious agents
• prevent environmental contamination
• protect experimental material
• comply with all laws and regulations

This plan provides university-wide safety guidelines for those working with biohazards.  It outlines general policies and procedures for using and disposing of infectious or potentially infectious materials.  Principal investigators or laboratory supervisors must call EHS at (573) 341-4305 if they are uncertain how to categorize, handle, store, treat or discard biologically derived material.

I.    Responsibilities and Accountability

A.  The Chancellor's Office

The  Chancellor's office has ultimate responsibility for biological safety within the University and must, along with other officials, provide support for biological safety.

B.  Biosafety Committee

The biosafety committee is responsible for developing biosafety policies applicable to Missouri S&T activities to include work practices and is made up of the members of the current Institutional Biosafety Committee (IBC).  Other responsibilities include: reviewing and approving new research proposals in accordance with Center for Disease Control/National Institutes of Health (CDC/NIH) guidelines, setting required containment levels for research projects (generally to follow CDC and NIH guidelines), investigating significant violations of Missouri S&T biosafety procedures or policies.  If appropriate, the committee will recommend disciplinary action to the proper Missouri S&T officials.

C.  Environmental Health and Safety Department

EHS monitors compliance with university safety policies and procedures regarding potentially infectious and biohazardous material and assists PI's in the selection of laboratory practices, equipment and controls. Technical guidance is also provided to all personnel on matters related to laboratory safety, as well as training to promote techniques for the safe handling and disposal of biohazardous material. 

D.  Department Chairs

Department Chairs are responsible for the implementation of safe practices and procedures in their departments.

E.  Principal Investigators (PIs)

PIs are responsible for identifying potentially infectious and biohazardous materials and carrying out specific control procedures within their own laboratories. This responsibility may not be shifted to inexperienced or untrained personnel. PIs are also responsible for the instruction of students and staff in the potential hazards of biologically derived materials.

F.  Employees

Employees must comply with safety guidelines and procedure required for the task(s) performed. They must report unsafe conditions to the PI, supervisor or EHS and seek guidance when they are uncertain how to handle, store or dispose of any hazardous or biohazardous material.

II.    Biohazards and Potentially Infectious Material

A.  Definition:

Biohazards are infectious agents or biologically derived materials that present a risk or potential risk to the health of humans or animals, either directly through infections or indirectly through damage to the environment. Infectious agents have the ability to replicate and give rise to the potential of large populations in nature when small numbers are released from a controlled situation.

B.  Categories of Biohazards or Potentially Infectious Materials

1.      Human, animal and plant pathogens:

• bacteria, including those with drug resistance plasmids
• fungi
• viruses, including oncogenic viruses
• parasites

2.      All human blood, blood products, tissues and certain body fluids.
3.      Cultured cells (all human and certain animal) and potentially infectious agents these cells may contain.
4.      Allergens
5.      Toxins
6.      Certain recombinant products
7.      Clinical specimens
8.      Infected animals and animal tissues

C.  Recombinant DNA (rDNA)

Generation of rDNA

Experiments involving the generation of rDNA may require registration and approval by the IBC committee. The National Institutes of Health (NIH) Guidelines for research involving recombinant DNA is an excellent source for information.

Human Gene Therapy and Transgenic Plants and Animals

All protocols involving the generation of rDNA for human gene therapy must be approved by the IBC committee prior to submittal to outside agencies or initiation of experimentation. The creation of transgenic plants and animals requires approval as well.

D.  Other Potentially Hazardous Biological Materials

Human blood, blood products, body fluids and tissues

Biosafety Level 2 practices and procedures must be followed when handling human blood, blood products, body fluids and tissues because of the infectious agents they may contain. Biosafety Level 2 practices are consistent with the concept of Universal Precautions which requires all specimens of human blood or other potentially infectious materials to be treated as if they are infectious. In 1991, the Occupational Safety and Health Administration (OSHA) promulgated a standard to eliminate or minimize occupational exposure to Hepatitis B Virus (HBV), Human Immunodeficiency Virus (HIV), and other bloodborne pathogens. The federal regulation mandates a combination of engineering controls and work practice controls, training, Hepatitis B vaccination, and other provisions to help control the health risk to employees resulting from occupational exposure to human blood and other potentially infectious materials.

An exposure control plan must be completed prior to work using human blood, blood products, body fluids or tissues. This plan is available in Appendix A of this manual. The complete plan must be available in the laboratory for all workers.

Use of animals

The use of animals in research requires compliance with the “Animal Welfare Act" and any state or local regulations covering the care or use of animals. Facilities for laboratory animals used for studies of infectious or non-infectious disease should be physically separated from clinical laboratories.

Vertebrate animal biosafety level criteria must be adhered to where appropriate. All animal protocols involving the use of rDNA; infectious or transmissible agents; human blood, body fluids or tissues; toxins; carcinogenic, mutagenic, teratogenic chemicals; or physically hazardous chemicals (reactive, explosive, etc.) must be submitted to the Biosafety Committee, EHS and the Institutional Animal Care and Use Committee (IACUC) for review and approval.

Tissue culture / Cell lines

When cell cultures are known to contain an etiologic agent or an oncogenic virus, the cell line can be classified as the same level as that recommended for the agent. The Centers for Disease Control (CDC) and OSHA recommend that all cell lines of human origin be handled at Biosafety Level 2.

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 1 cell lines and handled at Biosafety Level 1. Primate cell lines derived from lymphoid or tumor tissue, all cell lines exposed to or transformed by a primate oncogenic virus, all clinical material, all primate tissue, all cell lines new to the laboratory and all virus and mycoplasma-containing primate cell lines are classified as Class 2 and should be handled at Biosafety Level 2. Studies involving suspensions of HIV prepared from T cell lines must be handled at Biosafety Level 3.

Biological agents / Biohazard classifications

Biological agents are classified according to risk as follows:

Class 1
Agents of no or minimal hazard under ordinary conditions of handling.

Class 2
Agents of ordinary potential hazard. This class includes agents which may produce disease of varying degrees of severity from accidental inoculation or injection or other means of cutaneous penetration but which are contained by ordinary laboratory techniques.

Class 3
Agents involving special hazard or agents derived from outside the United States which requires a federal permit for importation unless they are specified for higher classification. This class includes pathogens which require special conditions for containment.

Class 4
Agents that require the most stringent conditions for their containment because they are extremely hazardous to laboratory personnel or may cause serious epidemic disease. This class includes Class 3 agents from outside the United States when they are employed in entomological experiments or when other entomological experiments are conducted in the same laboratory area.

Class 5
Foreign animal pathogens that are excluded from the United Sates by law or whose entry is restricted by USDA administrative policy.

Note: Federally licensed vaccines containing live bacteria or viruses are not subject to these classifications. Classifications are applicable to cultures of the strains used for vaccine production, or further passages of the vaccine strains.

III.      Principals of Biosafety

A.  Containment

The term “containment" is used in describing safe methods for managing infectious agents in the laboratory environment where they are being handled or maintained. The purpose of containment is to reduce or eliminate exposure of laboratory workers, other people and the outside environment to potentially hazardous agents. The three elements of contamination include: laboratory practice and technique, safety equipment, and facility design.

Primary Containment: the protection of personnel and the immediate laboratory environment from exposure to infectious agents, is provided by good microbiological technique and the use of appropriate safety equipment. The use of vaccines may provide an increase level of personal protection.

Secondary Containment: the protection of the environment external to the laboratory from exposure to infectious materials, is provided by a combination of facility design and operational practices. The risk assessment of the work to be done with a specific agent will determine the appropriate combination of these elements.

Laboratory Practice and Technique: Adherence to standard microbiological practices and techniques is the most important element of containment. Persons working with infectious agents or infected materials must be aware of potential hazards and must be trained and proficient in the techniques required to handle the material safely. The PI is responsible for arranging for the appropriate training through EHS.

Standard operating procedures should be developed for each lab, detailing specific hazards that might be encountered, and what practices and procedures should be used to minimize or eliminate risks. A scientist that is trained and knowledgeable in the appropriate lab techniques and safety procedures associated with the research must direct the lab activities.

When standard laboratory practices are not sufficient to control the hazards associated with a particular agent or procedure, additional measures will be needed. The PI is responsible for selecting additional safety practices in keeping with the hazard associated with the agent or procedure. Appropriate facility design and engineering features, safety equipment and management practices must supplement lab personnel safety practices and techniques.

Safety Equipment (Primary Barriers): Safety equipment includes biological safety cabinets, enclosed containers and other engineering controls designed to remove or minimize exposures to hazardous biological materials.

Safety equipment may also include items for personal protection such as personal protective clothing, respirators, face shields, safety glasses or goggles. Personal protective equipment (PPE) is often used in combination with other safety equipment when working with biohazardous materials. Often, personal protective clothing may form the primary barrier between personnel and the infectious materials.

Facility Design (Secondary Barriers): The design of a facility is important in providing a barrier to protect people working inside and outside the laboratory, and to protect people or animals in the community from infectious agents which may accidentally be released from the lab. Facilities must be designed around the lab's function and recommended biosafety level for the agent being manipulated.

The recommended secondary barrier(s) will depend on the risk of transmission of specific agents. Exposure risks for most laboratory work in Biosafety Levels 1 and 2 would be direct contact with the agents, or inadvertent contact exposures through contaminated work environments. Secondary barriers in these laboratories include separation of the lab work area from public access, availability of decontamination facility (e.g. autoclave) and hand washing facilities.

As the risk of aerosol transmission increases, higher levels of primary containment and multiple secondary barriers may become necessary to prevent infectious agents from escaping into the environment. Specialized ventilation systems to assure directional airflow, air treatment systems to decontaminate or remove agents from exhaust air, controlled access zones, airlocks at lab entrances, or separate buildings or modules for isolation of the lab are some design features that could be utilized.

B.  Biosafety Levels

There are four biosafety levels which consist of combinations of laboratory practices and techniques, safety equipment, and lab facilities. Each combination is specifically appropriate for the operations performed, the documented or suspected routes of transmission or the infectious agents, and for the laboratory function or research. The biosafety level recommendation for an organism is the level at which that organism can be handled safely.

Biosafety Level 1 -  is appropriate for work done with defined and characterized strains of viable microorganisms not known to cause disease in healthy adults. It is 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. Access to the laboratory can be limited or restricted when experiments or work with cultures and specimens is in progress. Work surfaces are to be decontaminated at least once a day and after any spill of viable material. All procedures should be performed to carefully minimize the creation of splashes or aerosols. All cultures, stocks, and other regulated wastes should be decontaminated prior to disposal by an approved decontamination method (e.g. autoclave).

Biosafety Level 2 -  is applicable to work done with a broad spectrum if indigenous moderate-risk agents associated with human disease of varying severity. Agents can be used safely on the open bench, provided the potential for producing splashes or aerosols is low. The primary hazards to personnel working with these agents are accidental percutaneous or mucous membrane exposures or ingestion of infectious materials. Procedures with high aerosol or splash potential must be conducted in primary containment equipment such as biosafety cabinets. Primary barriers such as splash shields, face protection, gowns and gloves should be used and secondary barriers such as hand washing and waste decontamination facilities must be available and used. Access to the laboratory is limited or restricted while work is ongoing. Lab personnel should have specific training in handling pathogenic agents and are directed by scientists. Precaution must always be taken with any contaminated sharp item. Needles and syringes or other sharp instruments should be restricted for use only when there is no alternative. Plastic ware should be substituted for glassware whenever possible. Laboratory equipment should be decontaminated with an appropriate disinfectant on a routine basis.

Biosafety Level 3 -  applies to work done with indigenous or exotic agents with the potential for respiratory transmission and which could cause serious and potentially lethal infections. Primary hazards to personnel include autoinoculation, ingestion, and exposure to infectious aerosols. Greater emphasis is placed on primary and secondary barriers to protect personnel in adjoining areas, the community and the environment. All laboratory manipulations should be performed in a biological safety cabinet or other enclosed equipment. Secondary barriers include controlled access and a specialized ventilation system that minimizes releases of infectious aerosols. All potentially contaminated waste materials must be decontaminated before disposal or reuse. Protective laboratory clothing such as solid front or wrap around gowns, scrub suits, or coveralls must be worn inside the lab only. Reusable lab clothing must be decontaminated before being laundered. Windows in the lab are closed and sealed. A sink for hand washing should be available near the lab exit, preferably with foot, elbow, or automatically operated.

Biosafety Level 4 -  applies to work with dangerous and exotic agents that pose a high individual risk of life-threatening disease, which may be transmitted by aerosol contamination and for which there is no available vaccine or therapy. Agents with close or identical antigenic relationship to BSL4 agents should also be handled at this level. The primary hazards to workers include respiratory exposure to infectious aerosols, mucous membrane exposure to infectious droplets and autoinoculation. All manipulations of materials pose a high risk of exposure and infections to personnel, the community and the environment. Isolation of aerosols is accomplished by working in a class III biological safety cabinet or full-body, air-supplied positive pressure suit. The facility is generally a separate building or completely isolated zone within a complex with specialized ventilation and waste management systems to prevent release of viable agents to the environment.

IV.      Practices and Procedures

A.  Administrative Controls

Biohazard warning signs and postings

Each laboratory must have a room sign providing safety information to visitors and service personnel. Room signs must contain designations for all laboratory hazards in use within the lab (carcinogens, teratogens, etc.). All laboratories that contain biohazardous material must be posted with a biohazard sign. The sign must be red/orange in color with a biohazard symbol and lettering in black. All areas in the lab that contains biohazardous material must be posted “No eating, smoking, and applying cosmetics allowed in this area."

Biosafety levels

In general, Class 1 agents are handled at Biosafety Level 1, Class 2 at BSL 2, etc. The levels are in ascending order by degree of protection provided to personnel, the environment and the community.

B.  Engineering Controls

Biological safety cabinets (BSCs)

BSCs are designed to contain aerosols generated during work with infectious material through the use of laminar airflow and high efficiency particulate air (HEPA) filtration. There are three types used in microbiological laboratories, Class I, II, and III. Open-fronted Class I and II cabinets are partial containment devices, which provide a primary barrier offering greater levels of protection to laboratory personnel and to the environment when used in combination with good microbiological techniques.

The Class 1 BSC is suitable for work with low to moderate risk materials. This level of protection will not protect the product from “dirty" room air, but it will protect personnel and the environment from contaminants within the cabinet.

The Class 2 BSC will protect the product, personnel and the environment. Material being manipulated within the cabinet will be protected from external contamination. There are three basic types: Type A, Type B and 100% Exhaust. The major difference is found in the percent of air that is exhausted or recirculated and the manner in which exhaust air is removed from the work area.

The gas-tight Class 3 BSC or glove box will provide the highest attainable level of protection to personnel, environment and the product. It is the only cabinetry that provides a total physical barrier between the product and personnel. It is for high risk biological agents and is used when absolute containment is required.

Please note that laminar flow clean benches should not be used with biohazardous or chemically hazardous material. Clean benches provide product protection but do not protect personnel or the environment.

Safety equipment

Gloves, coats, gowns, shoe covers, boots, respirators, face shields, safety glasses or goggles are all examples of personal protective equipment (PPE). PPE is generally used with BSCs and other devices that contain the biohazardous material, animals, or agents. Other safety equipment such as safety centrifuge cups and safety blenders are enclosed containers designed to prevent the release of aerosols during centrifugation or homogenization of material.

C.  Recommended Work Practices

Pipettes and pipetting aids

Pipettes are used for volumetric measurements and transfer of fluid that may contain infectious, toxic, or radioactive agents. Laboratory associated infections have occurred from oral aspiration of infectious materials, mouth transfer from a contaminated finger and inhalation of aerosols. A pipette may become a hazardous piece of equipment if improperly used. The following safe pipetting techniques will minimize the potential for exposure to hazardous materials:

• Never mouth pipette, use pipetting aid.
• Use a biosafety cabinet when working with biohazardous or toxic fluids.
• Always use cotton plugged pipettes when working with biohazardous or toxic materials, even when using pipetting aids.
• Do not prepare biohazardous materials by bubbling expiratory air through a liquid with a pipette.
• Do not forcibly expel biohazardous material out of a pipette.
• Use "to deliver" pipettes and not those requiring "blowout".
• Never mix biohazardous or toxic material by suction and expulsion through a pipette. Do not discharge material from a pipette at a height. Whenever, possible allow the discharge to run down the container wall.
• Place contaminated, reusable pipettes horizontally in a pan containing enough liquid disinfectant to completely cover them. Do not place pipettes vertically into a cylinder. Autoclave the pan and pipettes as a unit before processing them for reuse.
• Discard contaminated disposable pipettes in an appropriate sharps container.
• Pans or sharps containers for use with contaminated pipettes should be placed inside the biosafety cabinet.

Syringes and needles

Syringes and hypodermic needles are dangerous instruments and use should be restricted to procedures for which there is no alternative. Blunt cannulas should be used as alternatives to needles wherever possible. Never use syringes or needles as a substitute for pipettes. Use disposable needle locking syringe devices whenever possible. When using syringes and needles with biohazardous or potentially infectious agents: work in a biosafety cabinet, wear gloves, fill the syringe carefully to minimize air bubbles, expel the air, liquid, and bubbles from the syringe vertically into a cotton pledget moistened with disinfectant. Do not use a syringe to mix infectious fluid forcefully. Do not contaminate the needle hub when filling the syringe to avoid contamination of fingers. Wrap the needle and stopper in a cotton pledget moistened with disinfectant when removing a needle from a rubber-stoppered bottle.

Bending, recapping, clipping or removal of needles from syringes is strictly prohibited. Use a separate pan of disinfectant for reusable syringes and needles. Do not place them in pans containing pipettes or other glassware. Used disposable needles and syringes must be placed in an appropriate sharps disposal container.

Safe and effective use of biosafety cabinets

Make sure your BSC is certified annually after initial installation and certification. Check the magnehelic gauge regularly for an indication of a problem. Understand how you cabinet works. Such things as rapidly moving your arms in and out of the cabinet, people walking rapidly behind you or open lab doors may disrupt the airflow pattern and reduce the effectiveness of the cabinet. Minimize the storage of materials in and around the cabinet and always leave the BSC running while in use. Turn it on several minutes prior to beginning work if it is not running.

Before beginning work, wipe the cabinet surface with 70% alcohol. Wipe off each item you need for your procedures and place in the cabinet. DO NOT place objects over the front air intake grille and DO NOT block the rear exhaust grille. Segregate contaminated materials from clean and work from "clean to dirty".  Place a pan with disinfectant and/or sharps container inside the BSC for pipette discard, DO NOT use a vertical canister outside the cabinet. It is not necessary to flame items in the cabinet. It creates turbulence in the airflow and compromises sterility and the heat can damage the filters. If you use a piece of equipment that disrupts the airflow or causes turbulence (centrifuge), place the equipment in the back 1/3 of the cabinet and stop all other work while the equipment is running. Always protect the building vacuum system from biohazards by placing a cartridge filter between the vacuum trap and the source valve in the cabinet. Clean up all spills in the cabinet immediately and wait 10 minutes before resuming work. When all work is finished, remove all materials and wipe all interior surfaces with 70% alcohol. Remove lab coats and wash hands thoroughly before leaving laboratory.

Centrifuge equipment

Mechanical failure and the creation of aerosols are the hazards associated with centrifuges. Centrifuges must be operated and maintained according to the manufacturer's instructions to minimize this risk. Only properly trained personnel should operate centrifuges. To help minimize the generation of aerosols during centrifugation, the following procedures should be followed:

• Use sealed tubes and safety buckets that seal with O-rings and inspect the tubes, rings and buckets for cracks, chips, erosions, etc. prior to each use.
• Fill and open tubes, rotors and accessories in a BSC. Do not overfill centrifuge tubes so that closures are wet and after the tubes are filled and sealed, wipe them down with disinfectant.
• Place disinfectant into the space between the tube and the bucket to disinfect any material should a tube rupture. Always balance tubes before centrifugation.
• Do not decant or pour off supernatant. Use a vacuum system with in-line reservoirs and filters. Work in a BSC when re-suspending sedimented material. Use a swirling rotary motion. If shaking is necessary, wait before opening to allow the aerosol to settle.
• Small low-speed centrifuges may be placed in the BSC during use to reduce the aerosols escape. Avoid using celluloid tubes with biohazardous materials. They are highly flammable and prone to shrinkage with age and may not be autoclaved.

Personal protective equipment (PPE)

PPE is used to protect personnel from contact with hazardous materials and infectious agents. Proper PPE can also protect the experiment from contamination. The following PPE is recommended for regular use:

Face Protection
Goggles or safety glasses with solid side shields in combination with masks or other splatter guards are required for anticipated splashes, sprays or splatters of hazardous material. Do not wear contact lenses in lab.

Laboratory Clothing
Laboratory coats, smocks, scrub suits, and gowns are appropriate for protective clothing. Long sleeved garments should be used to minimize the contamination of skin or street clothes and to reduce shedding of microorganisms from the arms. If splashes may occur, the lab clothing must be resistant to liquid penetration to protect clothing from contamination and if not disposable, must be capable of withstanding sterilization. Protective clothing must be removed and left in the lab, it should never be worn outside the lab. Disposables should be available for visitors, maintenance and service workers in the event it is required. All protective clothing must be discarded in the lab or laundered by the facility. Personnel should not launder clothing at home.

Gloves
Gloves should be selected based on the hazards involved and the activity to be conducted. Gloves must be worn when working with biohazards, toxics, and other physically hazardous agents. Temperature resistant gloves should be worn when working with hot material or dry ice. The glove when working with hazardous material should overlap the lower sleeve and cuff of the lab coat/garment. A long sleeved glove or disposable arm-shield may be worn for further protection. Double gloving may be appropriate in some instances. If a spill occurs, hands will be protected after the contaminated outer gloves are removed. Gloves must be disposed of when contaminated, and removed when work with infectious materials is complete. Do not wear gloves outside the lab and do not wash or reuse disposable gloves.

Respirators
Respirator selection is based on the hazard and the protection factor required. Missouri S&T has a respirator program and anyone wishing to wear respirators should contact EHS x4305 for proper fit and selection of appropriate respirator.

Blenders, ultrasonic disruptors, grinders and lyonphilizers

Using any of these devices can generate considerable amounts of aerosols. This equipment should be used in a BSC when working with hazardous materials.

Safety blenders are designed to prevent leakage from the bottom of the blender jar and provide a cooling jacket to avoid biological inactivation and to withstand sterilization by autoclave. If blender rotors are not leak-proof, they should be tested with sterile saline or dye solution prior to use. The use of glass blender jars is not recommended because of the possibility of breakage during use. If they must be used, cover them with a polypropylene jar to prevent spraying of glass and contents in case the jar breaks. A towel moistened with disinfectant should be placed over the top of the blender during use. Allow the blender to rest for at least one minute to allow aerosols to settle after each use. Decontaminate the device after each use.

Lyophilizers and ampoules Depending on design, aerosol production may occur when material is loaded or removed from the lyophilizer unit. If possible, load the sample 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 complete, all surfaces of the unit should be disinfected. If the equipment has a removable chamber, it should be closed off and moved to a BSC for unloading and decontamination.

Opening ampoules containing liquid or lyophilized culture material should be performed in a BSC to control the aerosol produced. Gloves must be worn while working with ampoules. To open, nick the neck with a file, wrap in a 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 aerosolization of the dried material. Mix the contents without bubbling and discard the towel and ampoule top and bottom as infectious waste. Ampoules used to store biohazardous material in liquid nitrogen have exploded causing eye injuries. The use of polypropylene tubes eliminates this hazard.

Loop sterilizers and bunsen burners

Small particle aerosols can be produced which may contain viable microorganisms during the sterilization of inoculating loops or needles in an open flame. The use of a shielded electric incinerator minimizes aerosol production. Disposable plastic loops and needles may also be used and are semiquantitative and can be used for counting bacteria. Continuous flame gas burners should not be used in BSCs as they can produce turbulence, which disturbs the protective airflow patterns of the cabinet.

Laundry

All PPE must be cleaned, laundered or disposed of by the employer at no cost to the employee. Apparel should be handled as little as possible and decontaminated before being sent to the laundry for cleaning. Employees who handle contaminated PPE, should also be wearing PPE.

Housekeeping

Good housekeeping 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 should be based on the degree of risk for personnel and for the experiment.

Personnel are responsible for cleaning the bench tops, equipment and areas that require specialized technical knowledge. Keep the lab free of clutter and access to emergency showers/eye wash stations and fire extinguishers should not be blocked. Properly handle all chemicals and wastes according to Missouri S&T policy. Pay attention to physical hazards and make sure aisle space is sufficient. Properly secure all gas cylinders. Do not store chemicals in hoods.

Biohazard spill response procedures

1. Inside the BSC:

• Wear lab coat, safety glasses and gloves during cleanup. Make sure the cabinet is kept running during the cleanup. Apply disinfectant and allow a minimum of 20 minutes contact time. Wipe up any spills with a disposable disinfectant soaked cloth. Wipe the walls and work surfaces and any equipment in the cabinet with a disinfectant soaked cloth. Autoclave contaminated material prior to disposal. Expose non-autoclaved materials to disinfectant for a minimum of 20 minutes contact time before removing from the cabinet. Remove and autoclave protective clothing used during cleanup. Run the cabinet for 10 minutes after the cleanup before resuming work or shutting the cabinet off.  

2. In the lab, outside the BSC:

• Clear the area of personnel and wait for aerosol to settle before re-entering the spill area. Remove any contaminated clothing and place in a biohazard bag to be autoclaved. Wear a disposable gown and safety glasses for the clean up. Using the proper disinfectant
• Soak paper towels in disinfectant and place over the spill.
• Encircle the spill with additional disinfectant being careful to minimize aerosolization but assure adequate contact.
• Decontaminate all items in the spill area.
• Allow 20 minutes contact time.
• Wipe equipment with 1:10 bleach solution followed by water and then 70% alcohol.
• Place contaminated materials in biohazard bags and autoclave.
   

3. Inside the Centrifuge

• Clear the area of personnel and wait 30 minutes for aerosol to settle before attempting to clean up. Wear lab coat and safety glasses and gloves during clean up. Remove rotors and buckets to nearest BSC for clean up. Thoroughly disinfect inside the centrifuge. Remove contaminated debris, place in biohazard bag and autoclave.
  
  

4. Outside the lab during transport

• Transport biohazardous material in an unbreakable well-sealed primary container placed inside of a second, unbreakable, container with a lid and label with biohazard symbol. Should a spill occur in a public area, do not attempt a clean up without the proper PPE. Wear gloves and place paper towel, preferably soaked in disinfectant, directly on the spill. Surround the spill with disinfectant if available to minimize aerosols. Call EHS to assist in the cleanup, 341-4305.

Decontamination

Decontamination is a means of treatment that renders a medical device, instrument, or environmental surface safe to handle. A decontamination procedure can range from sterilization to simple cleaning with soap and water.

Sterilization is a physical or chemical procedure to destroy all microbial life, including highly resistant bacterial endo-spores.

Disinfection eliminates almost all pathogenic non-spore forming microorganisms but not necessarily all microbial forms on inanimate objects. The kinds and numbers of organisms, the amount of organic matter, the object being disinfected and the chemical exposure time, temperature and concentration influence effectiveness.

Antisepsis is the application of a liquid antimicrobial chemical to skin or living tissue to inhibit or destroy microorganisms. It includes swabbing an injection site on a person or animal and hand washing with a germicidal solution. Some chemicals may be utilized as either a disinfectant or an antiseptic but adequacy for one application does not guarantee adequacy for the other. Manufacturers' recommendations for use should always be followed.

1. General Procedure
    
   a)  All infectious material and all contaminated equipment or apparatus should be decontaminated before being washed, stored or discarded. Autoclaving is the preferred method. Each individual working with biohazardous material should be responsible for its proper handling.
   b)  Biohazardous materials should not be placed in autoclaves overnight in anticipation of autoclaving the next day.
   c)  Autoclaves should not be operated unattended or by untrained personnel.
   d)  Dry hypochlorites, or any other strong oxidizing material, must not be autoclaved with organic materials such as paper, cloth or oil due to explosion risk.
   
   
2. Methods

There are four main categories of physical and chemical means of decontamination: heat, liquid disinfection, vapors and gases and radiation. Each category is discussed below.

a.  Heat

1. Wet heat is the most dependable method of sterilization. Autoclaving (saturated steam under pressure of approximately 15 psi to achieve a chamber temperature of at least 250 degrees F for a set period of time) is the most convenient method for rapidly achieving destruction of all forms of microbial life. Material to be sterilized must come in contact with steam, so prevention of entrapment of air is critical to achieving sterility. Chemical indicators such as autoclave tape must be used with each load autoclaved. Autoclave tape alone is not an adequate monitor however and autoclave sterility monitoring should be conducted on a regular basis using appropriate biological indicators.
2. Dry heat is less efficient than wet and requires longer times and/or higher temperatures to achieve sterilization. It works well for the destruction of viable organisms on impermeable non-organic surfaces such as glass.  It is not reliable in the presence of shallow layers of organic or inorganic materials that may act as insulation. Glassware can be sterilized at 160-170 degrees C for periods of 2-4 hours. Dry heat sterilizers should be monitored on a regular basis using appropriate indicators.
3. Incineration is another effective means of decontamination and has the advantage of reducing the volume of material for final disposal.

b.  Liquid disinfection
The most practical use of liquid disinfectants is for surface decontamination and if used in sufficient concentration, for liquid wastes prior to final disposal in the sanitary sewer. Liquid disinfectants must be shown to be effective against the organism present in the waste prior to use.

c.  Vapors and Gases
Vapors and gases are primarily used to decontaminate biological safety cabinets and associated systems, bulky or stationary equipment not suited to liquid decontamination methods, and rooms, buildings and associated air-handling systems. Glutaraldehyde and formaldehyde vapor, ethylene oxide gas, peracetic acid and hydrogen peroxide vapor are choices available. When used in closed systems and under controlled conditions of temperature and humidity, excellent disinfection can be obtained. However, great care must be exercised with these compounds due to the hazardous nature of some.

d.  Radiation
Although ionizing radiation will destroy microorganisms, it is not a very practical tool for laboratory use. Nonionizing radiation in the form of ultraviolet radiation (UV) is used for inactivating viruses, bacteria and fungi. It will destroy airborne organisms and inactive organisms on exposed surfaces or in the presence of products of unstable composition that cannot be treated by conventional means.

The low penetrating power of UV limits its usefulness as microorganisms inside dust or soil particles will be protected from its action. UV may be used in air locks, animal holding areas, ventilated cabinets and laboratory rooms to reduce levels of airborne microorganisms and maintain good air hygiene. Proper shielding should be maintained as UV can cause burns to the eyes and skin. UV lamps that are used for space decontamination should be interlocked with the general room or cabinet lighting, so that turning on room lights shut off the UV lamp. UV lamps must be properly maintained. The lamps intensity or destructive power decreases over time and should be checked yearly with a UV meter.

Infectious waste management

Blood and blood products, pathological waste, culture and stocks of infectious agents and associated biologicals, contaminated animals carcasses, body parts and bedding, and sharps are the categories of infectious waste.  Body waste products such as vomitus, urine, and feces without blood are not regulated waste and as such, may be discarded as regular trash.

Handling
All infectious waste from Missouri S&T laboratories must be autoclaved by the generator prior to disposal in appropriate waste bags with labels. An outside contractor will pick up autoclaved waste for final disposal. EHS or the biosafety committee must approve treatment other than autoclaving.

The primary responsibility for identifying and disposing of infectious materials rests with the principal investigator or lab supervisor. The responsibility may not be shifted to inexperienced or untrained personnel.

Used sharps must be segregated into nonbreakable, leak-proof, impervious to moisture, rigid, tightly lidded, puncture resistance, red containers labeled with the biohazard symbol.

Fluids in volumes greater than 20 cc that are discarded as infectious waste must be segregated in containers that are leak-proof, impervious to moisture, break resistant, tightly lidded, red and marked with biohazard symbol. Volumes less than 20 cc may be decontaminated then flushed into the sanitary sewer. Accompany pouring of this waste with plenty of water. The empty container may be disposed of with other infectious waste or autoclaved and washed if reusable.

Other infectious waste must be discarded directly into containers or plastic autoclave bags which are clearly identifiable from general waste. Containers must be marked with the biohazard symbol. Autoclave bags must be red/orange in color and also marked with the biohazard symbol. These bags may only be used for infectious waste.

Decontaminated infectious waste must be put into the proper containers provided by Missouri S&T's disposal company.

Mixed Waste

Contact EHS for infectious waste contaminated with radioactive material or wastes substantially contaminated with toxic /carcinogenic compounds for disposal instructions.

Storage

Waste may be stored in small quantities in biohazard bags on the bench top until full or for the duration of the semester, then autoclaved.

Importation/Exportation of Etiologic Agents

Importation of infectious materials, etiologic agents and vectors that may contain them is governed by federal regulation. In general, an import permit is required for any infectious agent known to cause disease in man. Please contact EHS with any questions concerning permit requirements.

* Thanks to Northeastern University and Jack Price for allowing the generous use of the university's biological safety plan contents in the make up of this plan.

Appendix A
Exposure Control Plan

Hypoallergenic gloves, glove liners and other alternatives should be readily available to employees that have an allergy to latex, chemicals, and/or powder. Department supervisors and principal investigators are responsible for ensuring all work areas and employees have appropriate PPE available. Employees should be trained in the proper use of PPE for their job requirements.

All PPE should be inspected periodically and repaired or replaced as needed. Reusable PPE must be cleaned, laundered and decontaminated as needed as well.

Employees must follow all procedures concerning PPE, including:

1. Any garments penetrated by blood or other infectious materials must be removed as soon as feasible.
2. All PPE must be removed prior to leaving the lab.
3. Gloves must be worn whenever employees anticipate hand contact with potentially infectious materials.
4. Disposable gloves should be replaced as soon as practical after contamination or it they are torn, punctured or otherwise fail to function as an exposure barrier.
5. Masks and eye protection must be used whenever splashes or sprays may generate droplets of infectious materials.
6. Protective clothing must be worn whenever potential exposure to the body is anticipated.

Housekeeping

A written schedule for cleaning and decontamination should be initiated and include the locations to be cleaned, cleansers and disinfectants to be used and any special instructions.

As part of the housekeeping program, all equipment and surfaces should be cleaned and decontaminated after contact with blood or other infectious materials. All pails, bins, cans and other receptacles intended for use should be routinely inspected, cleaned and decontaminated.

Hepatitis B Vaccination, Post-exposure evaluation and Follow-up

Hepatitis B Vaccination Program
Presently, all employees bear the cost of the Hepatitis B vaccine and must seek this protection individually. Hepatitis B vaccines are recommended for all personnel working with blood products.

Labels and Signs

Obvious warnings of possible exposure to bloodborne pathogens are biohazard labels. A comprehensive biohazard warning labeling program includes using labels, or when appropriate, red "color-coded" containers. The following items must be labeled: containers of regulated waste, refrigerators/freezers containing blood or other infectious materials, sharps containers, contaminated equipment.

Information and Training

All employees whose jobs could potentially expose them to bloodborne pathogens must receive an initial orientation and annual refresher training. Well-informed employees are important in minimizing exposure.

EHS is responsible for facilitating the training process and for the development of the educational program.

Training topics that will be covered in this program include:

• The Bloodborne Pathogens Standard
• The epidemiology and symptoms of diseases
• The modes of transmission
• The Exposure Control Plan and where to find it
• Methods to recognize tasks and activities that may involve exposure
• Review of use and limitations of methods that prevent or reduce exposure
• Selection of appropriate and proper use of PPE
• Visual warning signs including labels, signs, and "color-coded" containers
• Information on Hepatitis B vaccine
• Procedure to follow if an exposure incident occurs in an emergency or otherwise, including reporting

Training presentations will use several training techniques including the following: lecture presentations, videotape programs, training manuals, self assessment tests, or on-site evaluations.

EHS will maintain records or training sessions in its office.

For more information please reference the following website by the Center for Disease Control:
http://www.cdc.gov/OD/ohs/biosfty.htm