OHS026
Safe Work Procedure
Faculty/Division: Medicine
Document number
STGCL.SWP.9.1
School/ Divisional Unit: St George Clinical School
Initial Issue date
15/09/09
Current version
1.0
Current Version
Issue date
15/09/09
Next review date
September 2010
The Writing Safe Work Procedures Guideline (OHS027) should be consulted to assist in the completion of this form.
Safe Work Procedure Title and basic description
Title: Safe Work Procedure for adding & removing frozen ampoules from Liquid Nitrogen
Description: This Safe Work Procedure describes how to safely work with Liquid Nitrogen when adding samples to and removing
samples that have been stored in Liquid Nitrogen dewars and tanks. Liquid nitrogen is commonly used for storing material at
extremely low temperature (-196°C). The objective of this procedure is to ensure that the storage and retrieval of cryovials from liquid
nitrogen storage tanks is performed as safely as possible.
Associated risk assessment title and location: STGCL.RA.9.1
Describe the activity or process
Introduction for both adding / removing samples from Liquid Nitrogen: Liquid Nitrogen is classed as a Dangerous Good
(Class 2.2) with the main hazards being asphyxiation (suffocation) and Cold Burns (temperature -196°C). Before carrying out
this procedure, you must be familiar with the MSDS at a minimum. NOTE: Handling of Liquid Nitrogen is restricted to
Monday – Friday 08.00 – 18.00 because of the risk of asphyxiation.
Working with liquid nitrogen to remove materials from Liquid Nitrogen Tank
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Prior to going to the Liquid Nitrogen Lab, prepare a box with a lid in which you house a tray containing 370C water. The
box should be sturdy enough to withstand an ampoule exploding: suggest a thick polystyrene esky. The tray of water at
370C is needed to speed up defrosting of the cells and to limit damage of the cells during the thawing process.
It is important to be organized and to know the rack number, tray number, position and number of ampoules you require
prior to opening the Liquid Nitrogen Tank because cells thaw rapidly when they are taken out of Liquid Nitrogen. By being
prepared cell damage that is thought to occur in the –500C to 00C temperature range is minimised.
Go to the Liquid Nitrogen Lab but DO NOT ENTER THE LAB IF OXYGEN METER IS ALARMING.
Check that the Oxygen Meter is working correctly ie the oxygen meter should display a reading of ≥ 19.5%
Do not enter the Liquid Nitrogen if the ventilation is not working – check that the streamers placed at vents in the ceiling of
adjacent Labs are moving.
Before opening any liquid nitrogen tank/dewar, dress appropriately and wear the required personal protective
equipment (PPE) as follows:
Clothes Long sleeved clothing and trousers must be worn. Trouser legs should be worn outside footwear and should not
have cuffs.
Shoes must be solid and closed to prevent the possibility of splashes entering shoes.
Laboratory Gown
Cryogenic Gloves are required to protect the hands from extreme temperature. Note: Although gloves are worn, the
wearer must not immerse gloved hands in Liquid Nitrogen.
Neoprene (impervious) apron is required to prevent spilled liquid nitrogen becoming trapped in clothing when the liquid is
being poured or racks retrieved
Polycarbonate Full Face Shield is required to prevent injury to face / eyes from potential splashes and to protect against
injury from exploding vials
Ensure the door to the room housing the Liquid Nitrogen storage tanks is open.
Remove the lid from the Liquid Nitrogen Tank and place to the side on the lid of an unopened adjacent Liquid Nitrogen
Tank.
Using a step if the tank is tall, slowly and steadily lift the stack from Liquid Nitrogen Tank, draining the excess liquid
nitrogen back into the Tank to minimize boiling and spluttering.
Place the stack on a stable surface and then replace the lid back on the Liquid Nitrogen Tank.
Withdraw the tray and use a forceps to remove ampoule/s. Immediately place the ampoules in the tray containing 370C
water. Place the lid on the box which houses the tray/water/ampoules.
Return tray to stack, remove the lid from the Liquid Nitrogen Tank and slowly return stack.
Replace the lid of the Liquid Nitrogen Tank ensuring that it fits securely.
Return the PPE to the coat hooks / basket provided in the Liquid Nitrogen Lab for the next user.
Carry the box containing the ampoule/s & tray to the Biological Safety Cabinet. Remove the lid from the box to allow
the sample to thaw. Swab sample with 70% (w/v) Ethanol and place in the Biosafety Cabinet for use.
Update the records maintained by your department to include information regarding removal of the particular vials from
Liquid Nitrogen Tank / Dewar.
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Page 1 of 3
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007
Working with liquid nitrogen to add samples to a Liquid Nitrogen Tank / Dewar
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To prevent Liquid Nitrogen from entering the vials and causing an explosion when the sample is removed from Liquid
Nitrogen, the lids of the cryovials must be tightly screwed on prior to immersing the samples in Liquid Nitrogen.
Samples to be added to Liquid Nitrogen are generally be frozen to -80 to help protect cell viability and to minimize boiling
and spluttering when immersing samples in Liquid nitrogen.
It is important to be organized and to know what rack number, tray number, position and number of ampoules you require
prior to opening the Liquid Nitrogen Tank to eliminate unnecessary exposure to Liquid Nitrogen.
Go to the Liquid Nitrogen Lab but DO NOT ENTER THE LAB IF OXYGEN METER IS ALARMING.
Check that the Oxygen Meter is working correctly ie the oxygen meter should display a reading of ≥ 19.5%
Do not enter the Liquid Nitrogen if the ventilation is not working. To ensure ventilation is working, check that streamers
placed at the vents in the ceiling of adjacent Labs are moving.
Before opening any liquid nitrogen tank/dewar, dress appropriately and wear the required personal protective
equipment (PPE) as follows:
Clothes Long sleeved clothing and trousers must be worn. Trouser legs should be worn outside footwear and should not
have cuffs.
Shoes must be solid and closed to prevent the possibility of splashes entering shoes.
Laboratory Gown
Cryogenic Gloves are required to protect the hands from extreme temperature. Note: Although gloves are worn, the
wearer must not immerse gloved hands in Liquid Nitrogen.
Neoprene (impervious) apron is required to prevent spilled liquid nitrogen becoming trapped in clothing when the liquid
is being poured or racks retrieved
Polycarbonate Full Face Shield is required to prevent injury to face / eyes from potential splashes and to protect against
injury from exploding vials
Ensure the door to the room housing the Liquid Nitrogen storage tanks is open.
Remove the lid from the Liquid Nitrogen Tank and place to the side on the lid of an unopened adjacent Liquid Nitrogen
Tank.
Using a step if the tank is tall, slowly and steadily lift the stack from Liquid Nitrogen Tank, draining the excess liquid
nitrogen back into the Tank to minimize boiling and spluttering.
Place the stack on a stable surface and then replace the lid back on the Liquid Nitrogen Tank.
Withdraw the tray and use a forceps to add ampoule/s to the tray.
Return tray to stack, remove the lid from the Liquid Nitrogen Tank and slowly return stack.
Replace the lid of the Liquid Nitrogen Tank ensuring that it fits securely.
Return the PPE to the coat hooks / basket provided in the Liquid Nitrogen Lab for the next user.
Record the details of the samples store, the specific location of these samples etc as required by your department.
List all resources required including plant,
chemicals, personal protective clothing and
equipment, etc
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Clothes Long sleeved clothing and trousers must be worn. Trouser legs should be worn outside footwear and should not
have cuffs.
Shoes must be solid and closed to prevent the possibility of splashes entering shoes.
Laboratory Gown
Cryogenic Gloves are required to protect the hands from extreme temperature. Note: Although gloves are worn, the
wearer must not immerse gloved hands in Liquid Nitrogen.
Neoprene (impervious) apron is required to prevent spilled liquid nitrogen becoming trapped in clothing when the liquid is
being poured or racks retrieved
Polycarbonate Full Face Shield is required to prevent injury to face / eyes from potential splashes and to protect against
injury from exploding vials
Tongs or tweezers must be used to hold cryovials. Ampoules recovered from liquid nitrogen must not be held by
hand.
Oxygen Meter
Mechanical Ventilation
A box with a lid in which you house a tray containing 370C water. The box should be sturdy enough to withstand an
ampoule exploding: suggest a thick polystyrene esky.
Step if Liquid NitrogenTank is tall.
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Page 2 of 3
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007
List potential hazards and risk controls including
specific precautions required
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Hazard: Thermal Burns / frostbite due to low temperature (-196°C)
Risk Control: Long sleeved clothing and trousers must be worn. Trouser legs should be worn outside footwear and should
not have cuffs, Fully enclosed footwear. Use appropriate personal protective equipment (PPE) - lab gown, Latex gloves,
cryogenic gloves (Cuffs of Lab Gown must be outside the cryogenic gloves to prevent Liquid Nitrogen entering and coming
in contact with skin), neoprene (impervious) apron, solid enclosed shoes and a full-face shield
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Hazard: Asphyxiation( Suffocation): Nitrogen gas displaces oxygen
Risk Control: perform work with Liquid Nitrogen in well-ventilated area. Oxygen Meter monitors the level of oxygen in the
air and this meter will alarm if oxygen level decreases. Failure of the ventilation system or tank rupture can cause the
nitrogen gas concentration to rise so that symptoms of asphyxiation could occur to persons entering the room or in the
area around the room. If the oxygen meter alarm sounds you must leave the vicinity immediately and notify the
manager of this area or another senior Lab person of the issue. (See emergency procedure located under alarm
panel for further information).
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Hazard: Storage vials have been known to explode unexpectedly due to extreme temperature changes (liquid rapidly
converted into gaseous nitrogen) when removed from liquid nitrogen (– 1960C) to room temperature (230C). This occurs
due to excessive pressure build-up within the vial from escaping nitrogen gas as a result of vials initially being placed
within liquid nitrogen with loose caps or lids, thereby allowing liquid nitrogen to enter the vial.
Risk Control: when initially freezing the ampoules use approved cryogenic storage vials. Cryovial lids must be tightly
screwed on to avoid the liquid nitrogen from entering the ampoule. Samples should also be placed in a –800C freezer
prior to transfer into liquid nitrogen for storage to minimize boiling and spluttering of Liquid Nitrogen and to maintain cell
viability.
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Hazard: Manual Handling when lifting stacks from Tanks
Risk Control: Use step if Tank is tall. Attend Manual Handling Training
List emergency shutdown instructions
If there is a failure of the ventilation system or if a tank/ valve ruptures, the concentration of nitrogen gas will rise and anyone in the
room or general area could suffer asphyxiation (suffocate). In such an instance the oxygen monitor will alarm. As the largest Tank
holds 230L of Liquid Nitrogen if there was an incident involving this tank, the gas generated would fill this volume:
230L x 682 = 156, 860L = 156 m³. As the volume of the Room is only 3.1m (L) x 2m (w) x 2.6m (h) = 16.12 m3 it is critical that:
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If the oxygen meter alarm sounds you must leave the vicinity immediately, close the door to this room (if
possible) and notify the manager of this area or another senior Lab person of the issue.
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If the alarm is caused by a tank/ valve rupture, everyone must evacuate Level 3 and remain at the assembly point
(BBQ Area) until instructed otherwise by emergency personnel. When Oxygen concentration has returned to
normal, the Oxygen alarm will cease.
AND
Dial 666 immediately to report the incident so that emergency services can be notified and respond.
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List clean up and waste disposal requirements
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If there is a spill of Liquid Nitrogen, the liquid will heat up to room temperature and will dissipate as Nitrogen Gas. If the
Oxygen Alarm is triggered, please leave the area immediately and notify the manager of the area or another senior Lab
person
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If a cryogenic vial explodes on removal from liquid nitrogen, this is a similar hazard to a biohazard spill. Treat the
contaminated area with a 1% Hypochlorite solution.
List legislation, standards and codes of practice used
in the development of the SWP
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Liquid nitrogen MSDS from supplier
AS 1894 1997 – The storage and handling of non-flammable cryogenic and refrigerated liquids
OHS Regulation 2001
Supervisory approval, training, and review
Supervisor: Prof Michael Grimm
Signature:
Plant custodian: Prof Michael Grimm
Signature
List competency required – qualifications, certificates, licencing, training - eg course or instruction: All personnel who handle samples
in Liquid Nitrogen must be trained in this Safe Work Procedure and must be familiar with the MSDS for Liquid Nitrogen.
SWP review date: September 2010
Responsibility for SWP review: Sinead O’Reilly
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Page 3 of 3
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007