Standing Operating Procedure: Cryogenic Substances
Physical Properties
This standard operating procedure (SOP) is intended to provide general guidance on working safely with
cryogenic liquids and dry ice. Cryogenic liquids are liquefied gases that are kept in their liquid state at very
low temperatures [below -153°C (-243°F)] and at normal temperature and pressure will be gases. Liquid
nitrogen (LN2) which has a boiling point of -196°C (-321°F) is a commonly used cryogen in Marywood
University (MU) laboratories. It provides a fluid media that is useful for researchers to preserve their sample
materials for experimental procedures. Liquid nitrogen is inert, colorless and odorless when vaporized to a
gas. As the gas boils off, it condenses moisture in surrounding air creating a highly visible fog that is
mistaken for gas (which is invisible). Although not a cryogen, solid/frozen carbon dioxide or dry ice converts
directly to carbon dioxide gas at -78°C (-109°F) is also commonly used in MU laboratories. Dry ice as well as
cryogens can be hazardous to personnel if not handled properly.
Applicability
This procedure applies to all University Faculty, Staff, and Students who work with liquid Nitrogen or frozen
carbon dioxide.
Responsibilities
Cocciardi & Associates, Inc. provides technical assistance for lab personnel about the safe handling, storage
and disposal of cryogenic liquids and training as needed.
The Chemical Hygiene Officer and Science Department Laboratory Manager ensure that cryogenic liquids are
properly managed and disposed in accordance with this procedure. Training for applicable personnel will
occur before use of cryogenic liquids and when changes are made to this procedure. Records of training are
retained by the Laboratory Manager.
The Laboratory Manager and Chemical Hygiene Officer must approve of any deviation from this SOP.
The Principle Investigator (PI) must ensure that prior to conducting any work with liquid nitrogen,
designated personnel must complete training to his/her laboratory personnel specific to the hazards involved
in working with this substance, work area decontamination, and emergency procedures. The PI must provide
his/her laboratory personnel with a copy of this SOP and a copy of the SDS provided by the manufacturer.
Potential Hazards
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Oxygen Deficiency and Asphyxiation: As liquid nitrogen evaporates into a gas, it expands 700
times its liquid volume. This gas is heavier than air and results an oxygen deficient environment that
has the potential to cause asphyxiation.
Adhesion: The cold surface of equipment (even non-metallic) containing LN2 can cause skin to stick
to the surface. Skin will tear in an attempt to remove it from the surface.
Cold Burns and Frostbite: Unprotected skin may freeze onto surfaces cooled by liquid nitrogen.
Prolonged skin exposure may result in cold burns, frostbite and blood clots. Prolonged inhalation of
cold vapor may cause serious lung damage. Splashes can cause instant freezing of the eye tissues and
permanent damage.
Pressure and Explosions: As LN2 vaporized inside a sealed containers (i.e. cryovials) rapid pressure
changes occur from large liquid-to gas ratios. All cryogens can condense moisture from air which can
freeze and block the opening of the storage vessel. This results in a buildup of trapped gases in the
container and can lead to an explosion. For example, cryovials immersed in liquid nitrogen may
explode when removing from the Dewar.
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Condensation of liquid oxygen: Using liquid nitrogen as a cold trap coolant is prohibited at MU.
This may cause condensation of liquid oxygen which is highly explosive with many organic materials.
Boiling and Splashing: LN2 can boil violently and splash when first added to a warm container.
Embrittlement: Many materials become brittle and fail due to contact with extreme low
temperatures (i.e. drains). Use materials specifically designed for liquid nitrogen (i.e. cryovials rated
for LN2, Dewars, cryoclaw/tongs, and transfer vessels dipper).
Safe Handling Procedures
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Be familiar with the hazards associated with cryogenic use.
Check oxygen monitors and alarms before and during cryogen use.
Only work with cryogenic liquids in a well-ventilated area to avoid oxygens depletion.
Always wear safety goggles and face shield.
Always wear appropriate a lab coat and loose cryogenic gloves: do not leave skin exposed. Do not
wear metal jewelry or watches.
Examine Dewar/container for signs of defect. Never use one with defects.
Cold cryogenic liquids may alter the physical characteristics of many materials, making them
brittle and lead to material failure. Use containers and equipment (cryovials, cryogenic gloves,
Dewars, cryoclaw/tongs, transfer vessel dipper) rated for LN2 use.
Schedule large Dewar fills during normal business hours, when staff are available to assist.
All cryogenic systems and Dewars must have pressure relief valves to release excessive pressure
and bursting discs and loose fitting lids on Dewar flasks.
Do not put your head inside an enclosed space containing LN 2.
Do not use hollow rods or tubes a dipsticks. When a warn tube is inserted into a cryogen, liquid
will spurt from the top of the tube.
Use only transfer tubes designed for use with the Dewar container. Damaged transfer tubes
should be replaced. Do not handle transfer tubes with your bare hands as the fitting is not
insulated.
When transferring to a secondary container (Dewar or cryovial) do not fill the container to more
than 80% of the capacity (60% if the temperature is likely to be above 30°C) to protect against
thermal expansion.
Use only a transfer vessel dipper when transferring of cryogenic liquids from a large Dewar.
Extreme care should be used when pouring to avoid splashing.
Do not lower warm experiments into Dewars of LN 2.
Immediately re-cap any container to prevent atmospheric moisture from entering and forming
an ice plug in the opening.
Use care in transporting cryogens; do not use fragile containers. Use hand truck of the lowest
shelf of a cart for the transport of cryogens. When available, use service elevators for transferring
unsealed containers of cryogens. Do not accompany unsealed containers of cryogenic liquid in
elevators.
Cryovial Safety
Cryovials are commonly used at MU for the cryogenic storage of biological materials. Liquid nitrogen can leak
into the vials. If this occurs, the LN2 may rapidly expand its liquid volume as the vial warms resulting in a vial
explosion. This may cause serious physical injury and may also expose you to infection from the vial
contents.
Safety Guidelines for Cryovial Safety
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Samples for freezing and storage should be placed in appropriate containers such as polypropylene
cryovials and not glass or polystyrene which may crack
All biological samples in cryovials should be stored in the nitrogen vapor phase if possible
When storing samples in the liquid phase, use internally threaded cryovials with a silicone gasket
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Do not over tighten the caps prior to freezing as this will distort the gasket
Vials should be certified for use in liquid nitrogen
Wear appropriate protective clothing including cryogenic gloves, lab coat, goggles and face shield
during the entire transfer process
Storage
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Store cryogens in well-ventilated area equipped with an oxygen monitor and alarm to prevent
oxygen deficiency. Alarms will sound if oxygen levels fall below 19.5%.
Use only approved storage vessels.
Never adjust, block or plug a pressure relief valve. The vendor is required to check the pressure
relief valve before filling the Dewar.
Avoid contact of moisture with storage containers to prevent ice plugs in relief devices.
Periodically check container neck for ice plugs
Keep all heat sources away from cryogenic liquids
Do not use cryogens of dry ice in walk=in cold rooms, because they may not have sufficient air
exchange and could become oxygen deficient.
Always push Dewars if they need to be moved. Never pull on Dewars. They are heavy and can tip and
injure you. Large Dewars can lead to ergonomic injuries (i.e. crushing of hands/feet, back/neck
injuries)
Personal Protective Equipment
Hand protection
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Use cryogenic cloves which are specifically designed for freezers below -80°C and for handling
containers/vials stored at these temperatures.
Wear loose fitting cryogenic gloves (blue) since they can be thrown off quickly if a cryogen leaks or
piece of dry ice is spilled into them.
Rubber gloves should not be used because thy will harden instantly and you may not be able to
remove them.
A thin gas barrier forms between the skin and the cryogenic liquid when it is spilled on skin. This will
initially be protective unless it hits the skin under force. This barrier is very cold and can burn.
Use non-metallic tongs to add or remove materials from cryogenic liquids.
Eye Protection
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Face Shields and goggles provide the best protection for the eyes and face. Safety glasses will not
protect your face, and cold liquids can hit your face and run under the glasses into your eyes. Safety
goggles alone will keep the liquid out of your eyes but leave the face exposed.
When filling Dewars or transferring cryogenic liquids from one container to another, face shields
must be worn.
Avoid working with cryogens overhead, as a spill will more likely result in serious injury.
Clothing
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Lab coats are required when handling cryogenics.
Closed toe shoes are required when handling cryogenic liquids or dry ice. Leather will shed the
spilled liquid. Cuffless pants should cover the shoe top. Avoid wearing sneakers. They are typically
made with absorbent materials which could draw liquid toward your skin.
Long sleeve shirts made of non-absorbent material are best when working with cryogens.
Injuries/First Aid
 In case of exposure to cryogens or dry ice, remove any clothing that is not frozen to the skin.
Comment [WDL1]:
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If skin/eye comes into contact with a cryogen, run the area under cool or warm water for fifteen
minutes. Never use hot or cold water. Never use dry heat. The re-warming or thawing of the affected
area(s) should be done gradually. It may take up to sixty minutes to thaw the affected area(s) and
bring back the natural color of the skin.
If a finger is burned, do not put it in your mouth. This could burn the mouth or tongue.
Do not rub a burned area: rubbing can cause further tissue damage.
Always seek medical attention for frostbite injuries.
Seek medical attention as soon as possible when cryogens contact your skin.
Immediately after exposure, the frozen skin may appear waxy and yellow and the burn may not be
painful. While the skin defrosts it may painfully swell and blister.
Disposal/Spill Requirements
 Large spills require immediate evacuation and activation of the Hazmat team by calling security at x.
6242.
References:
1. University of Oxford Policy Statement S4/03: Liquid Nitrogen
2. National Institute of Health, Protocol for Use and Maintenance of Oxygen Monitoring
Devices (revised February 2014)
3. UC Santa Cruz, Liquid Nitrogen SOP
4. UC Berkeley, Cryogenic Liquids Fact Sheet (revised March 14, 2013)
5. https://www.osha.gov/.../OSHAquickfacts-lab-safety-cryogens-dryice.pdf
6. UCSF, Chemical Safety Update, Cryogenic liquids