STANDARD OPERATING PROCEDURE #APC018, Version 1

STANDARD OPERATING PROCEDURE – #APC018, Version 1

Title: Use of Liquid Nitrogen facilities on 5th Floor BSI, UCC

Effective Date:June 24th, 2009

Written By : Sharon McKenna

(Sharon McKenna)

Approvals:Declan Soden, General Manager CCRC & Eva Mc Ardle, Lab Manager APC.

Updated June 2009 by Sharon McKenna

PURPOSE

This standard operating procedure defines the correct procedures and practices for users ofthe Liquid Nitrogen Cryo-Storage facilities on the 5th Floor BSI (room no.5.20).

OVERVIEW

Liquid nitrogen (LN2) is inert, colorless, odorless, non-corrosive, non-flammable, tasteless, extremely cold, and has no warning properties. Special care must be taken by personnel who handle or work in areas where liquid nitrogen is used. The hazards associated with LN2include:

• Over-pressurization and explosion due to LN2 vaporizing to nitrogen gas (700x expansion ratio) in unvented containers (e.g. cryovial) and equipment.

• Severe burns caused by exposure to cold temperatures.

• A leakage of LN2can cause asphyxiation due to displacement of oxygen in the air in a confined work area.

Applicability

This Update provides hazard warning and safety precaution information to users of LN2. Laboratory personnel who work with or in areas where LN2is used must be familiar with these guidelines.

General precautions to be taken

Personal Protective Equipment

The following personal protective equipment must be usedwhen handling or using LN2:

• Water proof thermal insulated gloves (e.g., cryo gloves)
• Lab coats
• Full face shield

Hands must be protected with water proof thermal insulated gloves that can be quickly removed if LN2is spilled on them. Insulated gloves are not intended for submersing hands intoLN2.
Eyes are most sensitive to the extreme cold of LN2and its vapors. Over-pressurization may result in the explosion of improperly vented equipment. Full face shield must be utilized when handling LN2and when handling sealed containers that have been stored in LN2 (e.g., cryovails).
Body must be protected with pants, lab coats, and closed-toe shoes. /

Storage

Store and use LN2 only in well ventilated areas. Do not store in a confined space or non-ventilated areas (e.g., cold rooms).

Use only approved containers with lids to store and transport LN2. Lids must be vented to allow the off gassing of over-pressurized nitrogen gas

Never vapor-seal LN2 storage containers. Never plug, remove, or tamper with any pressure relief device. Under normal conditions, these containers are designed to periodically vent gas

Potential hazards with Liquid nitrogen used for cryostroage: Specific Areas for prEcaution:

1. Dispensing / RE filling/Transporting storage vessels (spillage)

Liquid nitrogen can cause cold burns with potentially severe tissue damage.

2. Thawing cryovials – avoiding explosion

An exploded cryovial can cause injury. In addition, with Risk Group 2 or 3 content this would constitute a biohazard spill which can contaminate equipment and expose users to infection

1. Dispensing / RE filling / transporting storage vessels

An Individual in each lab much be assigned to ensuring top up of Liquid Nitrogen.

Protective clothing must be worn as described.

Never attempt to re-fill cryo storage tank by pouring.

A LN2 dispenser has been provided on the 5th floor Lab for safe re-filling of cryo storage tank from a reservoir Dewar – this Dewar is re-filled by the supplier (see below)

Fill no more than half way up tank – so that at least upper storage boxes remain in gas phase.

Measure with LN2 measuring stick (Thermo- scientific - # 180143)

Re-Filling of Liquid Nitrogen Dewars in BSI

Arrange for the Liquid Nitrogen Supplier to fill the Dewars in the yard area under the BSI Extension where there is adequate space for dewars and trolleys.

The cylinders must be brought out to the yard via the rear access door from the lift and the back door to the yard.No access via the Foyer!

The gate from the back yard can be opened by the push bar from the inside.

If the gate is locked from the outside the key can be obtained from Shelley at Reception.

Please refer to your Departments Health & Safety Statement guidelines for the safe use and handling of Liquid Nitrogen.

Consult the procedures below for Transporting Liquid Nitrogen in the BSI Lift.

Transporting Liquid Nitrogen in the BioSciences Institute Lift

Your Departmental Health & Safety Statement, Section 18.34.4 states that Cryogenic substances should not be carried in a passenger lift, with passengers present.

Users of Liquid Nitrogen in the BioSciences Institute must observe the following guidelines.

• Liquid Nitrogen Dewars must travel unaccompanied in the BSI lift. There is an asphyxiation risk from boil off in an enclosed space, especially if the lift breaks down. (One litre of liquid nitrogen evaporates to form 687 litres of nitrogen gas.)
• Two people must work together to transport liquid nitrogen via the lift.
• One person must be stationed on the relevant floor to receive the liquid nitrogen when the lift arrives –use the lift key switch as out lined below – to allow control of the lift and prevent access.
• The second person places the approved liquid nitrogen container in the lift, places the WARNING SIGN (A-frame, kept in the Reception area) immediately behind the lift door so it will bar entry to the lift, and then selects the floor to which the lift should stop and exits the lift before the doors close.
• The first person removes the liquid nitrogen and the sign when it arrives at the designated floor.
• The warning sign must be returned to the Reception Area immediately, before further work commences.

Controlled Use of the Lift in the BioSciences Institute for Transporting Liquid Nitrogen

These instructions are for the controlled operation of the BSI Lift for the transportation of Liquid Nitrogen Dewars. When the Lift is in ‘Remote Sending’ Mode it is not to be used for passenger transport. Users load their Liquid Nitrogen Dewar/s through the Ground Floor Rear (GR) Lift Doors and then walk to their required floor to call the lift to unload the Dewar/s. When the lift is being operated in this mode, the user has sole control of the lift and is responsible for its correct use.

1. Calling the Lift

Go to the Rear Lift Door. Ensure that the lift doors are fully closed.

Insert the SH2 key (Landing Remote Call Key Switch) into the Landing Remote Call Key Switch outside the lift.

Turn the key clockwise one quarter of a turn. The key will spring back once it is turned.

Lift Call is automatically registered for the Ground Rear Lift Doors.

Note: Once this key switch is activated all registered lift calls by other users will still be processed as normal, but no other calls will be accepted by new users. When all other lift calls are finished, the key holder will have full control of the lift.

2. Loading the Dewar/s into the Lift

The lift Doors will open on arrival at Ground Rear

Enter the lift and insert the SH3 key, Car Operating Panel (COP) key, into the top key switch - lift car override key switch–Special Cargo.

Turn the key clockwise. If this action is not completed within 20 seconds, the lift will return to normal operation.

Note: There is no need to register call on buttons or use a swipe card as the user now has control of the lift.

Load the Liquid Nitrogen Dewar/s. Leave override key switch, SH3, inserted in the Lift Car Operating Panel and ignore request from lift voice to select floor. Exit the lift.

Turn the SH2 key, in the landing remote call key switch, clockwise and hold in position to close the lift doors. Maintain pressure on SH2 key until the doors fully close. Remove the SH2 Key and bring it with you to the required floor.

Note: If pressure is not maintained doors will re-open. Please ensure doors are fully closed before leaving. Once doors are closed by the SH2 key they will not re-open at that floor. The user must go to next floor or required floor to call lift and re-open doors.

3. Unloading the Dewar/s on your Floor

Walk to the floor where you want to unload the Liquid Nitrogen.

Insert the Landing Key, SH2, into the landing remote call key switch.

Lift Call is automatically registered for that floor.

The Lift Doors will open on arrival at the floor. Unload the Dewar/s.

When the Dewar/s are unloaded turn the key, SH3, in the Lift Car Override Key Switch (Car Operating Panel, COP) anti-clockwise and remove it.

Exit lift

Remove the SH2 key from landing key switch. The Lift will now return to normal service.

4. Return the keys to BSI Reception once you have stored the Liquid Nitrogen in your Department/Centre.

Unattended liquid Nitrogen dewars.

For safety reasons, John Ring, UCC Safety Officer has requested that Liquid Nitrogen Dewars are not left unattended. Please arrange with your supplier to contact you when he arrives on site. The dewars should then be brought outside for filling. In the event of you not being available please give the supplier an alternative contact number.

For additional queries on transport please contact:

Myriam Cotter,Chief Technical Officer,

BioSciences Institute Technical Co-ordinator,

Telephone: +353 21 4901391

Fax: +353 21 4901377Web: bsi.ucc.ie

2. Thawing cryovials – avoiding explosion

Liquid nitrogen has a 700x expansion ratio which may create physical hazards and injuries from the explosion of an unvented cryovials

An exploded cryovial with Risk Group 2 or 3 content would constitute a biohazard spill which can contaminate equipment and expose users to infection.

2.1 use the correct storage vessels

When storing samples in the liquid phase, use internally threaded cryovials with a silicone gasket.

Do not over tighten the caps prior to freezing as this will distort the gasket.

Vials should be certified for use in liquid nitrogen. Avoid storage of cryovials in the liquid phase of liquid nitrogen – if they are in Liquid phase – move to gas phase before opening – see below.

The 2 vials on the lefthave male capswith silicon Orings.

The 2 vials on the righthave femalecapswith no O-rings.

An investigation in the University of Edinburgh found their incidents of exploding vials were all

of a similar design, having female caps, with a fine thread and no sealing ring. No incidents were reported using vials with a male cap, a thickerthread and a sealing ring (see illustration). NB this does not mean it won’t happen with these as the rings can fail to seal if distorted by overtightening.

All precautions out-lined below should be adhered to for all cryo vials.

2.2 Sample Storage Precautions

Laboratory personnel must use extreme caution when preserving samples in LN2. LN2storage consists of a liquid phase and a gaseous phase as illustrated below. If cryovials are immersed in the liquid phase, LN2can still enter the closed screw-top cryovials with o-rings during storage. The cryovial may then explode when it is removed from storage due to the vaporization and expansion (700x expansion ratio) of the liquid nitrogen inside the cryovial.

cryovials should be approved by the manufacturer. Liquid can still enter closed screw-top cryovials with o-rings and explode when removed from storage.

2.2 Allow trapped liquid to vaporise gradually

Before thawing a cryo vial from the Liguid nitrogen (LN) – move it into the gas phase of the nitrogen – ie top box of the LN for 24 hours (orover night)this will allow any gas to escape before taking it out to thaw (you must therefore be planning in advance). The top storage box should be left free for this purpose. The cane can be taken into a safety hood (TC hood) or preferably behind a protective screen for this transfer. A screen is provided at the sink in the tissue culture room for this purpose. If anyone in in the vicinity – e.g at the microscope – ask them to step away for a couple of minutes.

Following period in Gas phase - remove top box to a tissue culture hood and lift out required vial using tweezers. Release any potential pressure behind safety screen (in hood/safety cabinet) before thawing.

Cells can then be thawed in a water bath (with lid) or bio-container in the safety hood (this extra level of protection should be considered particularly where the frozen material is a biohazard (e.g. contains a virus or other biohazard).

Protective equipment should not be removed until the vials have reached room temperature.

First Aid

Frostbite Exposure: In the event of frostbite from skin contact with LN2follow the procedures below:

Remove any clothing that may restrict circulation to the frozen area.

Do not rub frozen parts, as tissue damage may result.

Place the affected area in a warm water bath / or sprinkle with warm water that has a temperature not in excess of 105°F (40°C). Never use dry heat.

Cover burn with a large sterile dressing.

If frozen area is extensive or deeply frozen, take the casualty to hospital.

Do not remove sticking clothing until completely thawed

Group responsibility (training/supervision) for tissue culture training and Safe use of Liquid nitrogen: As of June 09

Baukje Elzinga – CCRC (TC)

Sharon McKenna – CCRC

See also:UCCDEPARTMENT SAFETY STATEMENT DOCUMENT NO. 2: Requirements for the Control of Hazards and Risks SECTION 18.34.0 - Cryogenic Technology

(attached below)
UCC generic guidlines

DEPARTMENT SAFETY STATEMENT DOCUMENT NO. 2: Requirements for the Control of Hazards and Risks SECTION 18.34.0 - Cryogenic Technology Rev.2 AppSc\Doc 2\Sect 18.34 1 of 2

* INTERPRETATION - "Department" means College/School/Department/Centre/Unit as relevant.

18.34.1 INTRODUCTION

Cryogenics may be defined as the science of very low temperatures. To distinguish it from refrigeration, temperatures below -73.30C (-1000F) are considered being a broad definition of cryogenic technology. The most common application is the liquefaction of gases principally of helium, hydrogen, nitrogen, fluorine, argon, oxygen and methane.

18.34.2. HAZARDS

Cryogenic fluids require special handling requirements to avoid the main hazards associated with their use. The principal hazards related to their usage re: fire, explosions, burns, toxicity, asphyxiation, high pressure, embrittlement of equipment of materials, oxidation and chemical reactivity.

Hydrogen, methane are obvious flammability hazards, however, oxygen greatly increases the flammability of ordinary combustibles and can cause non-combustible material to burn readily. Liquefied inert gases (such as nitrogen and helium) or very cold metal surfaces can condense oxygen from the atmosphere, causing oxygen enrichment.

The high-pressure gas hazard is always present when cryogenic fluids are used because of the large expansion ratio from liquid to gas on evaporation.

Equipment must be carefully selected for cryogenic service because of the changes in their properties at very low temperatures - normally ductile materials may become extremely brittle. Methods of joining materials must also receive careful consideration, because of different rates of contraction and embrittlement of sealant. Chemical reactivity between the fluid and apparatus must be studied also.

Personnel hazards exist in several areas. Exposure of personnel to the above hazards must be avoided, but of prime concern is bodily contact with the extremely low temperatures involved. Brief contact with cryogenic materials can cause burns similar to thermal burns from high temperature contact. Prolonged contact with these temperatures will cause embrittlement of the affected parts because of the high water content of the body. The eyes are especially vulnerable, so eye protection is mandatory.

While a number of gases in the cryogenic range are not toxic, they are capable for causing asphyxiation by displacing oxygen. Even oxygen may have harmful physiological effects on prolonged breathing of the pure gas.

18.34.3. RISKS

The risks associated with the aforementioned hazards are to be assessed by the Department (using the appropriate technical input where necessary). These assessments shall be carried out using:

(i) Check lists provided (where applicable) and

(ii) Departmental Hazard Identification/Risk Assessment Work Sheets contained in Document No.3 i.e. Departmental Hazard Risk Assessment (D.H.R.A.).

ARRANGEMENTS AND CONTROLS REQUIRED

These hazards and risks will be minimised by the following arrangements and controls:

Personnel must be thoroughly instructed and trained in the nature of the hazards and precautions against them, including emergency procedures, operating equipment, safety devices, the properties of the materials used and personal protective equipment required.

If contact with a cryogenic fluid is possible, eye protection (or full-face protection is preferred), an impervious coat, trousers without turn-ups and high top shoes or boots must be worn. Watches and jewellery are not permitted, i.e. anything capable of trapping a cryogenic fluid close to the skin. Gloves may be worn as desired, but if they are necessary to handle containers or cold metal, they should be insulated, impervious and large enough to be tossed off the hand in case of a spill.

Care is required in the transport and storage of cryogenic substances. Slow evaporation is bound to take place leading to contamination of the atmosphere, unless there is good ventilation. Cryogenic substances should not be carried in a passenger lift, with passengers present.

Instructions for the storage of cryogens should comply with supplier's recommendations.

Low boiling point gases are protected in their vacuum vessel by a guard jacket of liquid nitrogen which must be replenished regularly. If the supply of liquid nitrogen fails on a helium storage vessel, explosion may occur.