PROCEDURES FOR BIOHAZARD CONTROL
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:|
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
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
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
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
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.
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
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
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
disintegrators, grinders and homogenizers release considerable aerosols
during their operation.
1. Blending, grinding, and homogenizing must be performed within a biological
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
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.
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 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.
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
1. Refrigerators, deep freezers, and dry ice chests must be checked, defrosted
and disinfected periodically. Remove any samples which may have broken
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: firstname.lastname@example.org|