When centrifuging biohazards What is the recommended wait time before opening?

Almost all procedures in the lab can create aerosols.  Solid or liquid aerosol particles are suspended in air by breaking surface tension or imparting energy to a substance. Heavier, larger particles fall out of the air quickly and create a risk of surface contamination increasing the direct contact of mucous membranes and open skin via self-inoculation by touching these surfaces.  Smaller aerosols are higher risk as they can remain suspended in air currents which move them to new locations away from where they were originally generated.  Close to 80% of lab acquired infections have an unknown route of exposure pointing to smaller aerosols.

Procedures that Create Aerosols

Centrifugation

Dissemination of aerosols through high-speed revolutions due to open tubes, tubes that crack, snap-cap tubes not sealed, expired tubes, overfilled tubes, and no secondary containment. 

Containment with safety buckets and safety rotors are required for biohazardous material processing.  Ensure appropriate balancing of the centrifuge. Always check the vessels prior to centrifuging:  do not overfill, ensure they are not expired, and the integrity. Waiting at least 1-5 minutes after the centrifuge stops prior to opening is highly recommended and with higher risk materials it is required.

Cell Sorting

Cell sorters use high pressure to separate cells into a single stream and further separate specially labelled cells.  In the event the instrument becomes clogged, or a deflection occurs, this can cause a significant aerosol cloud to erupt from the instrument.

Companies are working to help confine aerosols within the sorter when in use, however some may require containment in a biosafety cabinet. 

Documentation of procedures for sorting BSL-2 materials is required to use the flow cytometry core at MSU.

Spills

Dropped flasks of biohazard material create aerosols of different sizes and require evacuation of the laboratory if it occurs outside of the biosafety cabinet. After aerosols are given appropriate time to settle, EHS trained personnel can assess the situation and perform the clean up procedure. Contact EHS to inform them of the spill and clean up procedure. If the spill is too large for the kit, contact EHS immediately for assistance.

Vortexing, Blending, Homogenizing

Energy directed at the material through these means, presents a significant increase in aerosol creation risk.  It is recommended that all three of these procedures be done within a biosafety cabinet when biohazardous materials are used. Contact EHS Biosafety Team for information on specialty cabinets or other information.

Centrifuges are used in many aspects of laboratory research to separate or concentrate particles in a liquid medium. Tubes/bottles of samples are placed in balanced positions in rotors (the mobile part of a centrifuge); when loaded rotors are rapidly spun by a centrifuge, constituents in the samples will sediment differently according to their physical properties, and the sample’s density and viscosity. Some centrifuges are refrigerated to reduce the frictional heat created by this process.

Centrifuge types:

• Microfuge or microcentrifuge – Small benchtop centrifuge that accommodates small tubes with capacities of 250 microliters to 2.0 milliliters; spins up to 15,000 rpm.
• Benchtop – Counter-top models typically spin from 10,000 to 20,000 rpm.
• High speed centrifuge – Usually floor model; spins at 26,000 to 30,000 rpm.
• Ultracentrifuge – Usually floor model; spins up to 100,000 to 150,000 rpm.

Rotors for centrifuges typically are made of dense, heavy material to create momentum when spinning, and thus require less energy input to keep spinning. Rotors are often stored in refrigeration to keep them at or near centrifuge refrigeration temperatures.

Different makes and models of centrifuges use different rotors, and each model comes with a table or graph that relates centrifugal force to rotational speed (rpm) for each rotor or swing bucket it can use.  

Things to consider:

• DO NOT EXCEED THE PARAMETERS ON THESE TABLES/GRAPHS WHEN SETTING CENTRIFUGATION CONDITIONS.  
• ONLY USE THE SPECIFIC ROTORS AND BUCKETS LISTED ON THESE TABLES/GRAPHS WITH A PARTICULAR CENTRIFUGE; using a rotor that is not designed for a centrifuge is hazardous. It also could ruin both rotor and centrifuge.
• A given centrifuge typically offers several rotor or bucket types/sizes for flexibility in choosing centrifugation conditions, and accommodating various sample containers.

The centrifugal force (expressed in number of gravities, or # xg) generated is proportional to the rotation rate of the rotor (rpm) and the distance between the rotor center and the sample tube. In lab write-ups, always record the centrifugal force used (# of gravities), and the duration of time elapsed while that force was applied; this is because centrifugal force is the only transferable unit among different centrifuges.

4.20.2 Centrifuge Hazards

Physical hazards:

• Injury or death can occur if centrifuge mechanical failure leads to loss of integrity and explosive breakup during operation.  Damage leading to failures can be caused by 1) metal stress or fatigue reaching a critical state, 2) rotor corrosion or structural damage (i.e., forced loading of rotor onto a centrifuge spindle such that the alignment fittings are disabled and rotor is unmoored), 3) grossly imbalanced sample load in rotor, 4) attempted use of a rotor not designed for the unit.  
• Back injury can occur from leaning over to lift heavy rotors.
• Crush injuries can occur from dropping a heavy rotor on hands/feet.

Preventative Measures:

• Ensure that centrifuges and rotors receive regular preventive maintenance from a qualified service provider.
• DO NOT USE A DAMAGED ROTOR; report it to a supervisor.
• When operational problems appear (vibration, etc.), take centrifuges out of service immediately until repairs can be made.
• Keep rotors clean and free of sample residues to prevent corrosion; corrosive damage can progress over time and eventually cause structural failure. Have your service provider monitor rotors for this damage on a regular basis.
• Pay attention to your posture and plan your actions when lifting, carrying or placing heavy rotors.

Exposure hazards due to aerosolization of biohazardous materials:

Centrifuges can be used with biohazardous agents only if they are equipped with solid covers and have safety interlocks that prevent opening until the centrifuge has come to a complete stop.

Per their risk assessments, some biohazardous materials require that respiratory protection is available in case of a spill when centrifuging. If you are uncertain, check the agent’s SDS or risk assessment, or consult with EHS to determine if respiratory protection is required for spill situations.

What are the rules for using a centrifuge?

Which if the following is a good work practice when centrifuging biohazards?

What principles must be observed to avoid damage to the centrifuge or the specimen and danger to personnel?

Which laboratory must have an effluent decontamination?