PROCEDURES FOR BIOHAZARD CONTROL

General Information

The most important element of containment of infectious materials is strict adherence to standard microbiological practices and techniques.  Persons working with infectious agents or infected materials must be aware of potential hazards and be trained and proficient in the practices and techniques for safely handling such material.

Each laboratory supervisor must develop or adopt safety and operational procedures to identify the hazards that will, or may likely, be encountered.  They must also specify practices and procedures designed to minimize or eliminate identified risks, as well as the procedures to be used in the event of an accidental exposure.  Personnel and students must be required to read and follow the established practices and procedures and must be advised of any special hazards present in the laboratory. 

Routes of Transmission

    Respiratory Route Infection       

A variety of agents infect by the respiratory route.  Aerosol generation and dissemination can be reduced by the following:

            1.        Properly operating laminar-flow biological safety cabinets for protection against
                       immediately generated aerosols.

            2.        Thorough decontamination of work surfaces before and after work and following
                       spills of biohazardous material.  This method is particularly effective in    
                       preventing secondary aerosols generated by agents resistant to drying.                

            3.        Use of absorbent materials on immediate work surfaces to contain splashes and
                       drips.

Infection by Ingestion

A variety of organisms used in the laboratory are enteric pathogens which use ingestion as the primary route of infection (intestinal parasites, Salmonella, agents of infectious hepatitis, polio virus, and enteropathogenic C. coli strains).   Infection by these organisms generally occurs in the following ways:
  1. Direct ingestion of the culture by mouth pipetting.  
       
  2. "Hand to mouth" infection whereby infectious materials are transmitted indirectly by the hand to the oral cavity. Activities such as smoking, eating, and drinking are therefore prohibitied in laboratories. Frequent handwashing with germicidal soap between activities is highly recommended.  

Needlesticks, Punctures, Contact with Non-intact Skin

            1.         Contact can be avoided by limiting the use of needles and syringes and by using
                        nonbreakable containers whenever possible.

            2.         Workers' hands must never come into direct contact with infectious agents.
                        Therefore, gloves should be worn and discarded appropriately before handling
                        other equipment or objects.

Exposure to Mucous Membranes

            1.         All manipulations capable of generating a splash or spray must be conducted
                        within a biological safety cabinet with the sash and seat properly adjusted to
                        afford protection of the eyes.

            2.         Manipulations which may create splashes that cannot be conducted within a
                        biological safety cabinet (e.g. disposal of disinfected liquid waste to the sanitary
                        sewer) must be performed while wearing eye protection or a full face shield.

    Operations and Equipment

        Aerosols

                    Exposure to airborne microorganisms can result in contamination of research
                    materials as well as infections of the laboratory workers.  In fact, aerosols are a
                    major source of laboratory acquired infections.  They are usually odorless and
                    often pass unnoticed because the small particles involved are invisible and
                    basically undetectable without mechanical aid.

        Pipetting

                    Mouth pipetting is prohibited in all situations.  Use one of the mechanical aids that
                    are commercially available.  Delivery must be accomplished with the tip of the
                    pipette resting against the container, allowing the fluid to flow down the surface
                    thereby minimizing aerosols.  In addition, the following practices must be observed:

                        1.     No infectious mixture should be prepared by bubbling air through the liquid
                                 with the pipette.

                        2.     No infectious material should be forcibly discharged from a pipette.

                        3.     Placing a disinfectant soaked towel over the immediate work surface is
                                 useful in minimizing aerosolization from accidental splashing.

        Use of Syringes and Needles

To reduce the risk of accidental injection, aerosol production or spills, the following practices must be observed:


                        1.    Restrict the use of needles and syringes for practices in which no alternative
                               is available, such as parenteral injection or aspiration of fluids from
                               laboratory animals.
                      
                        2.    Do not use a syringe and needle as a substitute for a pipette in making
                               dilutions of hazardous or infectious fluids.  Syringe-type pipettes with blunt-
                               ended delivery are preferable.

                        3.    Reusable or disposable syringes used with biohazardous materials should
                               be of the LEUR-LOK or equivalent type to assure that the needle cannot
                               separate during use.

                        4.   Used disposable needles must not be bent, sheared, broken, recapped, or
                               removed from disposable syringes.

                        5.    Disposable needles and syringes must be disposed as a single unit into
                               puncture-resistant leak-proof "sharps" containers.

                        6.    Syringes not associated with needles or which have not come into contact
                               with biohazardous material must still be disposed into red biohazard sharps containers. 
                               Syringes and needles must never be discarded into the regular waste stream.


                        7.    Never discard syringes and needles into pans containing pipettes or other
                               glassware which must be separated from syringes and needles.    

    Use of Centrifuges and Shakers

                    To reduce the opportunity for aerosol production of biological material when using
                    centrifuges and shakers, the following practices must be observed.

                        1.     All tubes must be capped.

                        2.     Biohazardous agents must be centrifuged in an enclosed centrifuge with a
                                sealed rotor.  Safety cups and rotors with covers and O-rings are both
                                effective at minimizing aerosol production.

                        3.     Decanting from centrifuge tubes must be performed in a biological safety
                                cabinet.

                        4.     When mixing broth cultures utilizing a Vortex or similar mixer, avoid
                                wetting the plug or cap.

                        5.    As an additional safety measure, centrifuges and shakers should not be
                               permitted in corridor areas and should be housed within laboratory or
                               common equipment spaces.

Note: Items for centrifugation should always be balanced to avoid excessive vibration, which can result in failure of the unit as well as considerable aerosolization.

        Opening Culture Plates, Tubes, Bottles, and Ampules

                Aerosols are produced when contaminated plugs or screw caps are removed from
                tubes and bottles.  Employing good, sterile technique when opening tubes, bottles
                and culture plates will minimize the potential for aerosolizing the culture.

                Opening ampules is also potentially hazardous after the seal has been broken because
                air rushing in causes the dry contents to be dispersed.

                    1.     After scoring the ampule with a file, wrap it in cotton that has been wet with
                            disinfectant.   Wear gloves.

                    2.     If a disinfectant may damage the culture, use a biological safety cabinet and
                            the following procedure:

                                After scoring the ampule with a file, apply a hot, glass rod to the mark.
                                The glass will crack, allowing air to enter the ampule and equalize the
                                pressure.   After a few seconds, wrap the ampule in a few layers of
                                tissue, and break it along the crack.

                                The tissues and ampule neck must be discarded appropriately.

                                Employing good, sterile techniques when opening tubes, bottles and culture
                                plates will minimize the potential for aerosolizing the culture.  Also, it is
                                recommended that a culture plate be open so that the lid is between you
                                and the culture medium.

      Blenders, Ultrasonic Disintegrators, Grinders, Mortars and Pestles, and Homogenizers

            Blenders, disintegrators, grinders and homogenizers release considerable aerosols
            during their operation.

         1.    Blending, grinding, and homogenizing must be performed within a biological
                safety cabinet.

         2.    Disinfectant-soaked absorbent material can be placed over the blender during
                operation to further reduce the production of aerosols. 

    Water Baths and Warburg Baths

            It is recommended that water baths and Warburg baths used to inactivate, incubate or
            test biohazardous materials, contain a disinfectant such as Clorox (2.9 ml/3.8L of water)
            or a phenolic detergent (29 ml/3.9L of water).  Water should be changed at frequent
            intervals.

    Laboratory Vacuum Lines

            When a laboratory vacuum is used to manipulate biohazardous materials, a trap
            containing a suitable disinfectant must be employed to ensure that the building vacuum
            lines do not become contaminated.  Clorox, added such that the final concentration
            will equal 10%, is a suitable agent.  An inline filter must also be present between the
            trap and vacuum line. Empty all traps frequently and whenever more than 3/4 full.

    Contaminated Glassware (flasks, beakers, reusable pipettes, etc.)

            Contaminated glassware and similar materials which will be used again must be
            disinfected before washing.

    Labeling

            Storage vessels containing biohazardous agents must be labeled to provide identification
            of their content.

            Equipment used for the manipulation or storage of biohazardous material must be
            labeled with a biohazard sticker and a description of content (e.g. human cell lines).

    Contaminated Materials

            Contaminated materials that are transferred from work sites to decontamination and
            disposal staging areas shall be properly labeled with the individual's name and
            transported in a manner that prevents accidental spills.

    Containers

            Nonbreakable impermeable closed containers must be used during transport of
            biohazardous material through a building corridor or between buildings. 

    Personal Protective Equipment (PPE)

            Gloves and adequate protective clothing such as a fully fastened laboratory coat
            must be worn as a minimal form of protection against exposure to biological agents.
            When additional risks are present, other types of PPE may be necessary (such as
            faceshields or goggles to protect against splashing, etc.)  PPE must not be worn
            outside the laboratory nor to public eating areas.

    Refrigerators, Deep Freezers and Dry Ice Chests Used to Store Biological Material

            1.     Refrigerators, deep freezers, and dry ice chests must be checked, defrosted
                    and disinfected periodically.  Remove any samples which may have broken
                    during storage.

            2.     Equipment containing potentially biohazardous material must be locked at all
                    times when stored outside of the laboratory in a hallway or common equipment
                    area.   Such equipment must also be labeled with the materials scientific name,
                    and the name and telephone number of a contact individual, in the event of an
                    equipment failure or emergency.

            3.    If flammable solutions must be refrigerated, they can only be stored in refrigerators
                    approved for flammable liquid storage.   

View Recommended Procedures for Preventing the Production of Aerosols for additional information.      


University of the Sciences in Philadelphia • 600 South Forty-third Street • Philadelphia, PA 19104-4495 • phone: 215-596-8800 • email: safety@usp.edu