Safe Handling of Infectious Agents
Primary and Continuous Cell Cultures
Cell cultures, in general, present
few biohazards in the laboratory, as evidenced by their extremely
wide usage and the rare cases of transmitted infections to
laboratory personnel. Primary cell cultures initiated with
tissues from infected humans or animals are recognized hazards.
Continuous cell cultures present no real documented risk
in the laboratory unless they are carelessly contaminated
with an infectious agent. All continuous cell lines should
be regularly monitored for contamination with infectious agents,
and it should be emphasized that all nutrient media or other
reagents that may contain ingredients of biologic origin must
be treated as though they contain potentially infectious agents.
The Seven Basic Rules of Biosafety
The most common means of exposure can be essentially eliminated
as occupational hazards by following the seven basic rules
of biosafety:
- Do not mouth pipette.
- Manipulate infectious fluids carefully to avoid spills
and the production of aerosols and droplets.
- Restrict the use of needles and syringes to those procedures
for which there are no alternatives; use needles, syringes,
and other "sharps" carefully to avoid self-inoculation;
and dispose of "sharps" in leak- and puncture-resistant
containers.
- Use protective laboratory coats and gloves.
- Wash hands following all laboratory activities, following
the removal of gloves, and immediately following contact
with infectious materials at the end of an operational at
the end of the day.
- Decontaminate work surfaces before and after use, and
immediately after spills.
- Do not eat, drink, store food, or apply cosmetics in the
laboratory.
Labeling of Specimens With the Laboratory
Some form of labeling
is necessary to maintain the identity of specimens in the
laboratory and to ensure that the analytical results obtained
are properly recorded and reported. In addition, it is the
practice in many cases that special hazard warning labels
be affixed to specimens that are known to be hazardous (e.g.,
specimens obtained from patients known to be infected with
hepatitis B virus (HBV) or human immunodeficiency virus (HIV).
The need for such special labeling is concerned more with
ethical or regulatory issues (e.g., workers' right-to-know)
than with laboratory safety. All clinical material must
be considered to be infectious, and must be handled with exactly
the same precautions as are used for processing specimens
with hazard warning labels.
Containment Equipment
Introduction
The risk of exposure of laboratory personnel
can be minimized by the use of carefully selected safety equipment.
A primary objective of containment is to control aerosols,
but in a broader sense safety equipment should serve effectively
to isolate the worker from the toxic or infectious material
being processed. In many situations, however, the need is
just the reverse: i.e., to protect the product or the work
from contamination originating with the worker or the environment.
Finally, there is often the need to protect both the worker
and the product, as in handling cell cultures.
Biological Safety Cabinets
Most laboratory procedures generate aerosols
that may spread infectious material in the work area and pose
a risk of infection to the worker. Biological safety cabinets
are used extensively to prevents the escape of aerosols or
droplets and to protect materials from airborne contamination.
There are three major types of this very useful safety device,
referred to as Class I, Class II, and Class III. These instruments
are distinct from horizontal or vertical laminar flow "clean
benches," which should never be used for handling infectious,
toxic, or sensitizing material.
The type used at California State University, Fullerton is
the Class II biological safety cabinet, which provides protection
to the worker, the environment, and the products. The airflow
velocity at the face of the work opening is at least 75 linear
feet per minute (lfpm), and both the supply and the exhaust
air are HEPA-filtered. These cabinets are partial containment
devices, which, if used in conjunction with good laboratory
practices, can dramatically reduce the risk of exposure of
operators to infectious aerosols and droplets.
It is emphasized that biological safety cabinets are not
chemical fume hoods. Some of the air (30 to 70 percent) drawn
in through the work opening of these cabinets is recirculated
within the cabinet. Accordingly, users should be aware of
the possible buildup of hazardous concentrations within the
cabinet if toxic, flammable, or explosive materials are used.
In addition, users of Class IIA type cabinets should know
that nonparticulate toxic, flammable, or explosive materials
are not removed by HEPA filters, and are thus discharged back
into the laboratory room.
The operational efficiency of each biological safety cabinet
should be specifically tested and the system certified before
the instrument is placed in operation after installation,
and subsequently on an annual basis. Recertification is also
required if the unit is relocated or if maintenance that may
affect performance is done. Maintenance work on biological
safety cabinets should be performed by trained service personnel
only. In addition, cabinet users should understand the operation
of the equipment, its limitations, and the proper procedures
to be followed. Laboratory directors are responsible for providing
such training.
Clothing, Masks, and Face Shields
Laboratory coats, gowns, safety glasses, face
shields, masks, and gloves offer some personal protection
and are often used in combination with other safety devices
such as biological safety cabinets. Special laboratory clothing
protects street wear from contamination. It should not be
worn outside of the laboratory. Each of these items has a
particular use in protecting the worker and should be used
when circumstances require. Gloves are especially important
when handling any potentially infectious material such as
blood or other biological specimens. Safety glasses, face
shields, and masks may protect mucous membranes of the eye,
nose, and mouth from splash or droplet hazards during operations
performed outside of a biological safety cabinet.
Waste Handling
The primary responsibility
for the safe handling and disposal of infectious waste resides
with the generator of the waste. This responsibility extends
to the ultimate point of disposal even when there are other
parties involved in handling the waste. No waste management
program is functional unless all appropriate personnel are
cognizant of the aims of the program and trained in the procedures
for handling the waste. Training should be a continuing process.
Persons who generate laboratory waste are responsible for
preparing the waste so that potential occupational exposures
and environmental contamination are minimized. Cell culture
and infectious wastes need to be segregated by the generator
from all other waste streams.
Containment and Treatment
After use, disposable Petri plates, flasks
and other containment growth containers must be placed in
autoclavable bags which are clearly marked with the biohazard
symbol. Autoclave bags should be placed inside leak proof
containers while they are being filled. When full, bags should
be lightly closed with tape or a rubber band, then
autoclaved. Place waste bags in metal pans to catch any leakage
that occurs during autoclaving. Autoclave on the "liquids"
cycle for 45 minutes at 1210 C (2500 F) and at least 15 pounds
of pressure. The autoclaved bags must then be taken directly
outside to the trash dumpsters for immediate disposal.
Liquids should be treated with 0.01% to 0.05% (final concentration)
bleach. Household bleach solutions are 5-7% sodium hypochlorite,
so diluting them 100 fold in the liquid to be treated is sufficient
to destroy most cultures. For example, add 1 ml of bleach
for every 100 ml of culture. Leave the solution for at least
ten minutes for maximum effectiveness. This solution can then
be safely dumped in the laboratory drain, but it must be flushed
with copious amounts of water to flush the solution out of
the drain trap.
Large volumes of cultures, or cultures which are pathogenic
or toxic, must be inactivated by autoclaving. Place culture
containers in secondary containers whenever a chance of spillage
may occur. Autoclave on the "liquids" cycle at the
parameters described above.
DO NOT LET FULL BAGS (AUTOCLAVED OR NOT) OR OLD CULTURES
SIT AROUND. TAKE CARE OF THEM IMMEDIATELY!
If you have any questions about cell cultures, or any other
infectious agents, please contact the Research & Instructional
Safety Office in MH557.
Most of this information is taken directly from Biosafety
in the Laboratory, National Academy Press, Washington, DC.
1989.