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Lab Safety Guide - Chapter 3: Biological Materials (pdf version)

The following covers safety, regulations, and other topics related to laboratory use of biological materials. Adapted from the NMSU Lab Safety Guide


The purpose of the Bloodborne Pathogens Standard is to limit occupational exposure to blood and other potentially infectious materials since exposure could result in transmission of bloodborne pathogens which could lead to disease or death.  (A copy of the regulations may be obtained by clicking here

  • This standard covers all employees who could be "reasonably anticipated"

    as the result performing their job duties to face contact with blood and other potentially infectious materials. OSHA has not attempted to list all occupations where exposures could occur. "Good Samaritan" acts such as assisting a co-worker with a nosebleed would not be considered occupational exposure

  • Infectious materials include:
    • semen, vaginal secretions, cerebrospinal fluid,synovial fluid, pleural fluid, pericardial fluid, peritoneal fluid, amniotic fluid, saliva in dental procedures, any body fluid visibly contaminated with blood and all body fluids in situations where it is difficult or impossible to differentiate between body fluids. They also include any unfixed tissue or organ other than intact skin from a human (living or dead) and human immunodeficiency virus (HIV)-containing cell or tissue cultures, organ cultures and HIV or hepatitis B (HBV)-containing culture or medium or other solutions as well as blood, organs or other tissues from experimental animals infected with HIV or HBV.
  • Since there is no population that is risk free for HIV or HBV infectivity, any employee who has occupational exposure to blood or other potentially infectious material will be included within the scope of this standard. Although a list is included below of a number of job classifications that may be associated with tasks that have occupational exposure to blood and other potentially infectious materials, the scope of this standard is in no way limited to employees in these jobs. The hazard of exposure to infectious materials affects employees in many types of employment and is not restricted to the health care industry.

    At the same time, employees in the following jobs are not automatically covered unless they have occupational exposure:

        Physicians, physician's assistants, nurses, nurse practitioners, and other health care employees in clinics and physicians' offices;

        Employees of clinical and diagnostic laboratories;

        Housekeepers in health care facilities;

        Personnel in hospital laundries or commercial laundries that service health care or public safety institutions;

        Tissue bank personnel;

        Employees in blood banks and plasma centers who collect, transport, and test blood;

        Freestanding clinic employees (e.g., hemodialysis clinics, urgent car clinics, health maintenance organization (HMO) clinics, and family planning clinics)

        Employees in clinics in industrial, educational, and correctional facilities (e.g., those who collect blood, and clean and dress wounds);

        Employees assigned to provide emergency first aid;

        Dentists, dental hygienists, dental assistants and dental laboratory technicians;

        Staff of institutions for the developmentally disabled;

        Hospice employees;

        Home health care workers;

        Staff of nursing homes and long-term care facilities;

        Employees of funeral homes and mortuaries;

        HIV and HBV research laboratory and production facility workers;

        Employees handling regulated waste;

        Medical equipment service and repair personnel;

        Emergency medical technicians, paramedics, and other emergency medical service providers; and

        Fire fighters, law enforcement personnel, and correctional officers (employees the private sector, and federal government, or a state or local government in a state that has an OSHA-approved state plan).

    Other Criteria

        Part-time, temporary, and health care workers known as "per diem" employees are covered by this standard.

        If an employee is trained in first aid and designated by the employer as responsible for rendering medical assistance as part of his/her job duties, that employee is covered by the standard. This definition does not cover "good Samaritan" acts which result in exposure to blood or other potentially infectious materials from assisting a fellow employee, although OSHA encourages employers to offer follow-up procedures in such cases.

        "Other Potentially Infectious Materials" (OPIM) coverage extends to blood and tissues of animals who are deliberately infected with HIV or HBV.

        Employees in the construction and maritime industries who have occupational exposure to blood or OPIM are covered by the standard.

        "Parenteral" includes human bites that break the skin, which are most likely to occur in violent situations such as may be encountered by prison personnel and police and in emergency rooms or psychiatric wards.

Infection Control Plan    gototop.jpg (908 bytes)
    Requires employers to identify, in writing tasks and procedures as well as job classifications where occupational exposure to blood occurs-- without regard to personal protective clothing and equipment. It must also set forth the schedule for implementing other provisions of the standard and specify the procedure for evaluating circumstances surrounding exposure incidents. The plan must be accessible to employees and available to OSHA. Employers must review and update it at least annually--more often if necessary to accommodate workplace changes.  A model infection/exposure control plan can be found in the Appendices
Methods of Compliance   
    Mandates universal precautions, (treating body fluids/materials as if infectious) emphasizing engineering and work practice controls. The standard stresses hand washing and requires employers to provide facilities and ensure that employees use them following exposure to blood. It sets forth procedures to minimize needle sticks, minimize splashing and spraying of blood, ensure appropriate packaging of specimens and regulated wastes and decontaminate equipment or label it as contaminated before shipping to servicing facilities.
    Employers must provide, at no cost, and require employees to use appropriate personal protective equipment such as gloves, gowns, masks, mouthpieces and resuscitation bags and must clean, repair and replace these when necessary. Gloves are not necessarily required for routine phlebotomies in volunteer blood donation centers but must be made available to employees who want them.
    The standard requires a written schedule for cleaning, identifying the method of decontamination to be used, in addition to cleaning following contact with blood or other potentially infectious materials. It specifies methods for disposing of contaminated sharps and sets forth standards for containers for these items and other regulated waste. Further, the standard includes provisions for handling contaminated laundry to minimize exposures.
HIV and HBV Research Laboratories and Production Facilities   gototop.jpg (908 bytes)
    Calls for these facilities to follow standard microbiological practices and specifies additional practices intended to minimize exposures of employees working with concentrated viruses and reduce the risk of accidental exposure for other employees at the facility. These facilities must include required containment equipment and an autoclave for decontamination of regulated waste and must be constructed to limit risks and enable easy clean up. Additional training and experience requirements apply to workers in these facilities.

Hepatitis B Vaccination   

    Requires vaccinations to be made available to all employees who have occupational exposure to blood with 10 working days of assignment, at no cost,at a reasonable time and place, under the supervision of licensed physician/licensed healthcare professional and according to the latest recommendations of the U.S. Public Health Service (USPHS). Prescreening may not be required as a condition of receiving the vaccine. Employees must sign a declination form if they choose not to be vaccinated, but may later opt to receive the vaccine at no cost to the employee. Should booster doses later be recommended by the USPHS, employees must be offered them.

Post-Exposure Evaluation and Follow-up  

    Specifies procedures to be made available to all employees who have had an exposure incident plus any laboratory tests that must be conducted by an accredited laboratory at no cost the employee. Follow-up must include confidential medical evaluation documenting the circumstances of exposure,identifying and testing the source individual if feasible, testing the exposed employee's blood if he/she consents, post-exposure prophylaxis, counseling and evaluation of reported illnesses. Healthcare professionals must be provided specified information to facilitate the evaluation and their written opinion on the need for hepatitis B vaccine must be supplied to the employer. All diagnoses must remain confidential.

Hazard Communication    gototop.jpg (908 bytes)

    The Hazard Communication Standard (Right-to-know law) requires warning labels including the orange or orange-red biohazard symbol affixed to containers of regulated waste, refrigerators and freezers and other containers which are used to store or transport blood or other potentially infectious materials. Red bags or containers may be used instead of labeling.

    When a facility uses universal precautions in its handling of all specimens, labeling is not required within the facility. Likewise, when all laundry is handled with universal precautions, the laundry need not be labeled. Blood which has been tested and found free of HIV or HBV and released for clinical use, and regulated waste which has been decontaminated, need not be labeled. Signs must be used to identify restricted areas in HIV and HBV research laboratories and production facilities.

Information and Training  

    Mandates training within 90 days of effective date, initially upon assignment and annually--employees who have received appropriate training within the past year need only receive additional training in items not previously covered. Training must include making accessible a copy of the regulatory text of the standard and explanation of its contents, general discussion on bloodborne diseases and their transmission, exposure control plan, engineering and work practice controls, personal protective equipment, hepatitis B vaccine, response to emergencies involving blood, how to handle exposure incidents, the post-exposure evaluation and follow-up program, signs/labels/color-coding. There must be opportunity for questions and answers, and the trainer must be knowledgeable in the subject matter. Laboratory and production facility workers must receive additional specialized initial training.

Recordkeeping    gototop.jpg (908 bytes)

    Calls for medical records to be kept for each employee with occupational exposure for the duration of employment plus 30 years, must be confidential and must include name and social security number; hepatitis B vaccination status (including dates); results of any examinations, medical testing and follow-up procedures; a copy of the healthcare professional's written opinion; and a copy of information provided to the healthcare professional. Training records must be maintained for three years and must include dates, contents of the training program or a summary, trainer's name and qualifications, name s and job titles of all persons attending the sessions. Medical records must be made available to the subject employee, anyone with written consent of the employee, OSHA and NIOSH--they are not available to the employer. Disposal of records must be in accord with OSHA's standard covering access to records.


    Effective Date: March 6, 1992. Exposure Control Plan: May 5, 1992.

    Information and Training Requirements and Recordkeeping: June 4, 1992.

    And the following other provisions took effect on July 6, 1992: engineering and work practice controls, personal protective equipment, housekeeping, special provisions covering HIV and HBV research laboratories and production facilities, hepatitis B vaccination and post-exposure evaluation and follow-up and labels.

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The general hazards associated with microorganisms are

1. exposure of people to the toxic byproducts of the organisms

2. infection of people or other living beings (which might later function as disease carriers) with hazardous microorganisms which initiate disease (pathogens).

The Center for Disease Control and National Institutes of Health (CDC/NIH) provide guidance for selection of an appropriate biosafety level and specific information on laboratory hazards associated with a particular agent or animal study and recommendations regarding practical safeguards that can significantly reduce the risk of laboratory-associated diseases. Selection of a biosafety level depends upon a number of factors including: the virulence, pathogenicity, biological stability, route of spread, and communicability of the agent; the nature or function of the laboratory; the procedures and manipulations involving the agent; the endemicity of the agent; and the availability of effective vaccines or therapeutic measures.

Risk assessments and biosafety levels recommended presuppose a population of immunocompetent individuals. Persons with altered immunocompetence may be at a increased risk when exposed to infectious agents. The laboratory director/supervisor is responsible for appropriate risk assessment, designation of biosafety level, and supervisor for providing appropriate training of personnel and assuring utilization of appropriate proactive, containment equipment, and facilities for infectious agents or infected animals.

General criteria for determining the laboratory Biosafety Level (BSL) are taken verbatim from CDC/NIH guidelines. For more specific information on agents, practices and techniques, safety equipment and laboratory facilities see Biosafety in Microbiological and Biomedical Laboratories.

Biosafety Levels

Biosafety Level 1 practices, safety equipment, and facilities are appropriate for undergraduate and secondary educational training and teaching laboratories, and for other facilities in which work is done with defined and characterized strains of viable microorganisms not known to cause disease in healthy adult humans. Bacillus subtillis, Naegleria gruberi, and infectious canine hepatitis virus are representative of those microorganisms meeting these criteria. Many agents not ordinarily associated with disease processes in humans, are, however, opportunistic pathogens and may cause infection in the young, the aged, and immunodeficient or immunosuppressed individuals. Vaccine strains which have undergone multiple in vivo passages should not be considered a virulent simply because they are vaccine strains.

Biosafety Level 1 represents a basic level of containment that relies on standard microbiological practices with no special primary or secondary barriers recommended, other than a sink for handwashing. 

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Biosafety Level 2 practices, equipment, and facilities are applicable to clinical, diagnostic, teaching and other facilities in which work is done with the broad spectrum of indigenous moderate-risk agents present in the community and associated with human disease of varying severity. With good microbiological techniques, these agents can be used safely in activities conducted on the open bench, provided the potential for producing splashes or aerosols is low. Hepatitis B virus, the salmonellae, and Toxoplasma spp. are representative of microorganism assigned to this contaminated level. Biosafety Level 2 is appropriate when work is done with any human-derived blood, body fluids, or tissues where the presence of an infectious agent may be unknown. (Laboratory personnel working with human-derived materials should refer to the Bloodborne Pathogen Standard 187 for specific, required precautions).

Primary hazards to personnel working with these agents relate to accidental percutaneous or mucous membrane exposures, or ingestion of infectious materials. Extreme precaution with contaminated needles or sharp instruments must be emphasized. Even though organisms routinely manipulated at BSL2 are not known to be transmissible by the aerosol route, procedures with aerosol or high splash potential that my increase the risk of such personnel exposure must be conducted in primary containment equipment, or devices such as a biosafety cabinet or safety centrifuge cups. Other primary barriers should be used as appropriate, such as splash shields, face protection, gowns, and gloves.

Secondary barriers such as handwashing and waste decontamination facilities must be available to reduce potential environmental contamination.  

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Biosafety Level 3 practices, safety equipment, and facilities are applicable to clinical, diagnostic, teaching, research, or production facilities in which work is done with indigenous or exotic agents with a potential for respiratory transmission, and which may cause serious and potentially lethal infection. Mycobacterium tuberculosis, St. Louis encephalitis virus, and Coxiella burnetii are representative of microorganisms assigned to this level. Primary hazards to personnel working with these agents relate to autoinoculation, ingestion, and exposure to infectious aerosols.

At BSL 3, more emphasis is placed on primary and secondary barriers to protect personnel in contiguous areas, the community, and the environment from exposure to potentially infectious aerosols. For example, all laboratory manipulations should be performed in a biosafety cabinet or other enclosed equipment, such as a gas-tight aerosol generation chamber. Secondary barriers for this level include controlled access to the laboratory and a specialized ventilation system that minimizes the release of infectious aerosols from the laboratory.  

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Biosafety Level 4 practices, safety equipment, and facilities are applicable for work with dangerous and exotic agents which pose a high individual risk of life-threatening disease, which may be transmitted via the aerosol route, and for which there is no available vaccine or therapy. Additionally, agents with a close or identical antigenic relationship to BSL 4 agents should also be handled at this level. When sufficient data are obtained, work with these agents may continue at this level or at a lower level. Viruses such as Marburg or Congo-Crimean hemorrhagic fever are manipulated at BSL 4.

The primary hazards to personnel working with BSL 4 agents are respiratory exposure to infectious aerosols, mucous membrane exposure to infectious droplets, and autoinoculation. All manipulations of potentially infectious diagnostic materials, isolates, and naturally or experimentally infected animals pose a high risk of exposure and infection to laboratory personnel, the community, and the environment.

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Laboratory Biosafety Level Criteria  

The essential elements of the four biosafety levels for activities involving infectious microorganisms and laboratory animals are summarized in Tables 3.1 and 3.2. The levels are designated in ascending order, by degree of protection provided to personnel, the environment, and the community.

Table 3.1 Summary of recommended biosafety levels for infectious agents.




Safety Equipment (Primary Barriers)


(Secondary Barriers)


Not known to cause disease in healthy adults Standard Microbiological Practices None required Open bench top Sink required


Associated with human disease.

Hazard: percutaneous exposure, ingestion, mucous membrane exposure.

BSL - I practice plus:

•Limited access

•Biohazard warning sign



•Biosafety manual defining any needed waste decontamination or medical surveillance policies

Primary barriers = Class I or II BSCs or other physical containment devices used for all manipulations of agent that cause splashes or aerosols of infectious materials. PPE: laboratory coats; gloves; face protection as needed BSL - 1 Plus:

Autoclave available


Indigenous or exotic agents with potential for aerosol transmission; disease may have serious or lethal consequences BSL - 2 practice plus:

•Controlled access

•Decontamination of all waste

•Decontamination of lab clothing before laundering

•Baseline serum

Primary barriers - Class I or II BSCs or other physical containment devices used for all manipulations of agents. PPE: protective lab clothing; gloves; respiratory protection as needed BSL - 2 plus:

•Physical separation from access corridors

•Self-closing, double door access

•Exhausted air not recirculated

•Negative airflow into laboratory


Dangerous/exotic agents which pose high risk of life threatening disease, aerosol-transmitted lab infections, or related agents with unknown risk of transmission BSL - 3 practices plus:

•Clothing change before entering

•Shower on exit •All material decontaminated on exit from facility

Primary barriers = All procedures conducted in Class III BSCs or Class I or II BSCs in combination with full body, air-supplied, positive pressure suit BSL - 3 plus:

•Separate building or isolated zone

•Dedicated supply/exhaust, vacuum, and decon systems

•Other requirements outlined in the text

BSL = Biosafety level BSC = Biosafety cabinet PPE = Personal protective equipment

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Table 3.2 Summary of recommended biosafety levels for activities in which experimentally or naturally infected vertebrate animals are used.   

ABSL Agents Practices Safety Equipment

(Primary Barriers)


(Secondary Barriers)

1 Not known to cause disease in healthy human adults Standard animal care and management practices, including appropriate medical surveillance programs As required for normal care of each species. Standard animal facility

• non recirculation of exhaust air

• directional air flow recommended

2 Associated with human disease.


percutaneous exposure, ingestion, mucous membrane exposure.

ABSL -1 practices plus:

• limited access

• biohazard warning signs

• sharps precautions

• biosafety manual

• decontamination of all infectious wastes and of animal cages prior to washing

ABSL -1 plus primary barriers: containment equipment appropriate for animal species. ppe: laboratory coats, gloves, face and respiratory protection as needed ASBL -1 plus:

• autoclave available

• handwashing sink available in the animal room.

3 Indigenous or exotic agents with potential for aerosol transmission; disease may have serious health effects. ABSL -2 practices plus:

• controlled access

• decontamination of clothing before laundering

• cages decontaminated before bedding removed

• disinfectant foot bath as needed

ABSL -2, equipment plus:

• containment equipment for housing animals and cage dumping activities

• Class I or II BSCs available for manipulative procedures (inoculation, necropsy) that may create infectious aerosols. ppe: appropriate respiratory protection

ABSL -2 facility plus:

• physical separation from access corridors

• self-closing, double door access

• sealed penetrations, sealed windows

• autoclave available in facility

4 Dangerous exotic agents which pose high risk of life threatening disease;

aerosol transmission or related agents with unknown risk of transmission.

ABSL -3 practices plus:

• entrance through change room where personal clothing is removed and laboratory clothing is put on; shower on exiting;

• all wastes are decontaminated before removal from the facility

ABSL -3 equipment plus:

• Maximum containment equipment (i.e.., Class III BSC or partial containment equipment in combination with full body, air-supplied positive-pressure personnel suit) used for all procedures and activities

ABSL -3 facility plus:

• separate building or isolated zone.

•Dedicated supply/exhaust, vacuum, and decon systems

• other requirement outlined in the text.

ABSL = Animal Biosafety Level BSC = Biosafety Cabinet PPE = Personal Protective Equipment

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Biohazard Symbol.145(E)(4)

    wpe7.jpg (6069 bytes)The biological hazard warning shall be used to signify the actual or potential presence of a biohazard and to identify equipment, containers, rooms, materials, experimental animals, or combinations thereof, which contain, or are contaminated with, viable hazardous agents.

    The symbol design for biological hazard tags shall conform
    to the design shown below, in the following colors:

    "BIOLOGICAL HAZARD" -- Florescent orange or orange-red, or predominantly so, with lettering or symbols in a contrasting color.


It was realized during the early development of genetic engineering techniques that potential risks to the safety of laboratory personnel, the general public, and the environment were possible. Meetings of scientists and administrators from governmental agencies, educational institutions and industrial laboratories led to the development of safety regulations and guidelines. The guidelines are now published and issued by the National Institute of Health (NIH).*

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Institutional Biosafety Committee

The NIH guidelines direct the establishment of a local Institutional Biosafety Committee (IBC) to "review, approve, and oversee" genetic engineering research projects on this campus. The IBC is composed of laboratory scientists, others with experience and expertise in recombinant DNA technology, and members of the community. This committee has the responsibility to assess the safety of recombinant DNA experiments and potential risks to public health or environment caused by such experiments. This group is committed to adherence to the NIH guidelines, and if necessary and appropriate, the development of any special procedures, or physical or biological barriers which enhance the safety practices presented in those guidelines. The committee, the principal investigators, the laboratory technical personnel, and the administration of New Mexico State University will take precautions to ensure that genetic engineering research at New Mexico State University is carefully monitored and controlled according to the NIH guidelines in order to ensure safety to all concerned.

Administrative procedures to facilitate IBC project review are established with the Office of Sponsored Programs. An assessment of the required containment levels and adequacy of local facilities will be made considering proposed procedures, laboratory practices, and the training and expertise of personnel involved in the project. The review will also evaluate emergency plans covering accidental spills and personnel contamination resulting from the research. The principal investigator and the institution will be notified in writing of the results of the review.

Liaison will be maintained between the committee and the principal investigator for the duration of the project. The IBC will maintain a current inventory of principal investigators, technicians and students engaged in recombinant DNA projects, including a description of the research and where it is being conducted. This will be accomplished by means of an administrative form which will be completed semi-annually by those concerned. This form shall also be completed by personnel involved before any new research may commence and the materials for this research (organisms, plasmids, or viruses) may be received by the laboratory.

Principal investigators

It is understood that the principal investigators, regardless of the funding source for their research, are responsible for complying fully with the NIH guidelines pertaining to recombinant DNA research. They should notify the IBC of all projects involving recombinant DNA. It is important that they be aware of potential hazards of their research program and that appropriate safety precautions be taken. This includes the safety training of laboratory associates and technicians. Principal investigators are also responsible for reporting hazards, spills and accidents that may occur to the institution Safety Office (Environmental Health and Safety).

NIH guidelines

The NIH guidelines provide standards for evaluating the conceivable danger of particular experiments involving recombinant DNA molecules by providing containment, or safeguard, levels which are dependent on the assessed possible dangers of the experiment. In the absence of evidence of any hazard actually occurring, these standards are based on relevant current knowledge. Certain experiments which have potential for extreme hazard are prohibited.

The safeguards in the NIH guidelines require the use of procedures and physical containment systems to protect laboratory workers and the environment from exposure to potentially harmful organisms. These requirements include procedures, equipment, special features of laboratory and building construction, and appropriate training of workers. The systems are grouped into four "biosafety levels" of containment -- BL1, BL2, BL3, and BL4. Each category provides a level of containment more restrictive than the one preceding it. The level of containment chosen is based on an assessment of the degree of hazard involved considering the combinations of vectors and recombinant DNA hosts and the potential of escape and survival of the host-vector combination.

The NIH guidelines are available on microfiche at the Government Documents section of the NMSU Branson Library or, a copy may be "checked out" from the IBC chair for the purpose of duplication by principal investigators for their own files.

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All laboratories must segregate ordinary autoclaved waste (Biosafety Level 1) from infectious waste (Biosafety Level 2). Waste generated from laboratories designated as Biosafety Level 3 or 4 must be handled with individual special consideration. New Mexico Solid Waste Management Regulations regarding landfill disposal of biohazard waste applies without regard to the quantity of infectious waste produced by each laboratory. The NMSU Procedure for Laboratory Microbiological Wastes, Appendix IV, must be posted or distributed within each laboratory generating autoclaved microbiological wastes.

Procedures for disposal of preserved biological wastes can be found in NMSU Procedures for Biological Wastes, Appendix IV. Fresh biological material can be disposed by via landfill and is the responsibility of the laboratory director/supervisor.

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