Recommended Procedures for Preventing the Production of Aerosols
Whenever infectious aerosols may be generated during work with materials,
work must be conducted in a biological safety cabinet, preferably Class II, or
other physical containment device to protect yourself from an exposure.
Aerosols and droplets can be created simply by vortexing a test tube or
opening a microcentrifuge tube, and these droplets can be invisible to the
naked eye. Other procedures that may generate aerosols include; pipetting,
centrifugation, tissue culture, using needles, sonication, opening containers of
infectious material whose internal pressures may be different from ambient
pressures, vigorous shaking or mixing, blending of tissues, inoculation of
animals intranasally and harvesting infected tissues from animals or eggs.
Aerosols are a serious hazard because they are ubiquitous in laboratory
procedures, are usually undetected, are extremely pervasive, placing the
laboratory worker carrying out the procedure and other persons in the
laboratory at risk of infection.
Air sampling studies have shown that most of the common
manipulations of bacterial and viral cultures in research laboratories
release aerosols of viable organisms. This must be considered when
evaluating a need for the use of the biological safety cabinet or other
physical containment device.
A biological safety cabinet or other physical containment device must
also be used whenever high concentrations of large volumes of
infectious agents are used. However, such materials may be centrifuged in
the open laboratory if sealed rotor heads or centrifuge safety cups are used,
and if these rotors or safety cups are opened only in a biological safety
cabinet.
Centrifuge Operation

Hazards associated with centrifuging include mechanical failure and the
creation of aerosols.
To minimize the risk of mechanical failure,
centrifuges must be maintained and used according to the manufacturer’s
instructions. Users should be properly trained and operating instructions
that include safety precautions should be prominently posted on the unit.

Aerosols are created by practices such as filling centrifuge tubes,
removing plugs or caps from tubes after centrifugation, removing
supernatant, and re-suspending sedimented pellets. The greatest aerosol
hazard is created if a tube breaks during centrifugation. Also, if a
microorganism is not contained within a centrifuge, aerosols can be
produced during the high spin process. To minimize the generation of
aerosols when centrifuging biohazardous material, follow the procedures
below:
 Use sealed tubes and safety buckets that seal with O-rings. Before
use, inspect tubes, o-rings and buckets for cracks, chips, erosion,
bits of broken glass, etc. Do not use aluminum foil to cap
centrifuge tubes because it may detach or rupture during
centrifugation.
 Fill and open centrifuge tubes, rotors and accessories in a
biological safety cabinet. Avoid overfilling of centrifuge tubes so
that closures do not become wet. After tubes are filled and sealed,
wipe them down with a disinfectant.
 Add disinfectant to the space between the tube and the bucket to
disinfect material in the event of breakage during centrifugation.
 Always balance buckets, tubes and rotors properly before
centrifugation.
 Do not decant or pour off supernatant. Use a vacuum system with
appropriate in-line reservoirs and filters.
 Work in a biological safety cabinet when re-suspending sedimented
material. Use a swirling rotary motion rather than shaking. If
shaking is necessary, wait a few minutes to permit the aerosol to
settle before opening the tube.

Small low-speed centrifuges may be placed in a biological safety cabinet
during use to contain aerosols.

High-speed centrifuges pose additional hazards. Take precautions to filter
the exhaust air from vacuum lines; avoid metal fatigue resulting in
disintegration of rotors; and use proper cleaning techniques and centrifuge
components. Follow manufacturer’s recommendations meticulously to
avoid metal fatigue, distortion and corrosion.

Avoid the use of celluloid (cellulose nitrate) tubes with biohazardous
materials. Celluloid centrifuge tubes are highly flammable and prone to
shrinkage with age. They distort on boiling and can be highly explosive in
an autoclave. If celluloid tubes must be used, an appropriate chemical
disinfectant must be used to decontaminate them.
Mixing Operations

Sonicators, shakers and homogenizers can generate aerosols during
operation. Minimize the risks when mixing by taking the following steps:
 Operate mixing equipment in biological safety cabinets.
 Use sealed vessels during mixing and allow the vessels to settle
before opening.
 Open mixing vessels inside of biological safety cabinets.
 Check the condition of the mixing equipment routinely for
deterioration.
 Disinfect all exposed surfaces before and after use.
Vacuum and Aspirating Equipment

Minimize the risk of generating aerosols during vacuum and aspiration
operations by taking the following steps:
 Use non-breakable equipment (i.e., not glass).
 Ensure that vacuum equipment is fitted with a HEPA filter.
 Place a disinfectant in the overflow flask of aspirating equipment.
Needles and Syringes

Minimize the risk of generating aerosols while using needles and syringes
by taking the following steps:
 Perform all operations with needles and syringes in a biological
safety cabinet.
 Fill syringes carefully. Avoid frothing or introducing air bubbles.
 Do not bend, remove, or recap needles by hand. [Contact EHRS if
re-capping is unavoidable. It is required to document that no
other alternative is feasible. If approved, then either a safety device
for re-capping must be used (e.g., self-sheathing or retractable
needles) or a one-handed “scoop the cap” technique must be used.
Re-capping is prohibited when working in the Vivarium.)
 Dispose used and non- contaminated needles and syringes
properly, as contaminated sharps.
 Use blunt-end needles for removal or introduction of fluids through
small apertures in equipment.
Pipettes

Minimize the risk while using pipettes by taking the following steps:
 Use cotton-plugged pipettes.
 Keep pipettes upright while in use and between steps to prevent
contamination of the mechanical suction device and work surface.
 Gently expel the contents of the pipette close to the surface of the
liquid or allow to flow down the side of the container.
 Do not mix contents of a container by alternating suction and
blowing with a pipette.
 Use TD pipettes as opposed to TC pipettes. That way you do not
need to expel the last drops from the pipette to get an accurate
measurement.
 Submerge used non-disposable pipette horizontally in disinfectant
solution. Dropping them in vertically may force out any remaining
liquid.
Transfer Loops

Minimize the risk while using transfer loops by taking the following steps:
 When disinfecting the loop between inoculations, hold the loop
close to, but not in, the flame until the material has charred, then
fully insert it into the flame.
 Substitute an enclosed micro-incinerator for an open flame burner.
If aerosols may be generated, work must be performed in a Class II biosafety
cabinet or other physical containment device.