THE UNIVERISTY OF NEWCASTLE - SCHOOL of BIOMEDICAL SCIENCES

THE UNIVERISTY OF NEWCASTLE - SCHOOL of BIOMEDICAL SCIENCES STANDARD OPERATING PROCEDURE PROCEDURE NO: SBS-GDP 016 MOD: 2nd Issue Page: 1 of 10 Procedure Type: General Discipline Procedure 1. Purpose: 1.1. To outline the procedures and potential hazards involved when working with Cryogenic Materials, specifically Liquid Nitrogen and Dry Ice. 2. Equipment: 2.1. Cryogloves 2.2. Full Face shield 2.3. Laboratory Coat / gown 2.4. Enclosed footwear 2.5. Suitable clothing covering legs and arms. 2.6. Forceps 3. Materials: 3.1. Liquid Nitrogen 3.2. Dry Ice 4. Set Up: 4.1. The safety equipment listed in step 2 is to be used at all times when handling any cryogenic fluid. 5. Method: 5.1. SAFETY HAZARDS The most common cryogenic liquid used in laboratories is Liquid Nitrogen. 5.1.1. Cold Burns: The main safety hazard involved when dealing with liquid nitrogen is from burns due to its very low temperature at -196 C. These burns cause a similar injury to high temperature thermal burns, and even brief contact may cause damage. Frostbite results in local freezing of body tissue, and can occur from actual contact with liquid nitrogen or merely when skin is exposed to the vapour. Eyes are most susceptible to damage from liquid nitrogen due to their high water content. WRITTEN BY REVIEWED BY CHECKED BY AUTHORISED BY NAME (signed) Sarah Cooper Lynn Herd Kelly Marquardt Alistair Sim DATE 13 th May 2007 30 th May 2007 10 th July 2007 Distributed To: GDP Master file / GDP Lab file

Page: 2 of 10 5.1.2. Inhalation of cold gases and vapours: Inhalation of cold gases and vapours may produce breathing discomfort and prolonged exposure can have a detrimental effect on the lungs. 5.1.3. Asphyxiation: Due to the rapid expansion of boiling nitrogen (1 volume of liquid creates 700 volumes of gas) asphyxiation may occur due to oxygen displacement. When the oxygen concentration is dangerously low, suffocation will occur and personnel can become unconscious without sensing any of the warning signs such as dizziness. Therefore liquid nitrogen must never be used or stored in confined spaces or poorly ventilated areas. 5.1.4. Explosion Risk: Cryogenic liquids always present an explosion risk due to the rapid expansion on boiling. Personnel should always be conscious of possible pressure build up in storage vessels. This explosion risk exists also for vials which have been removed from liquid nitrogen. See Handling Precautions 5.2.8 5.1.5. Splashing: Liquid nitrogen will boil and splash when it is transferred to a vessel of a higher temperature or has objects of a higher temperature inserted into it. Always pour and transfer liquid Nitrogen slowly and gently to prevent rapid boiling and splashing. 5.1.6. Mist: Atmospheric water vapour will condense in the close proximity of liquid nitrogen to create a mist. This may cause orientation problems in the event of a spill. 5.2. HANDLING PRECAUTIONS 5.2.1. Naked skin that contacts uninsulated objects may freeze to the cold surface and may tear when separating the skin from the surface. 5.2.2. Personal protection: 5.2.2.1. Protection of face A full face shield or goggles must be worn during all operations involving liquid nitrogen. 5.2.2.2. Protection of hands Cryogloves should be worn when working with both liquid nitrogen and dry ice and for carrying any cryogenic fluids. It is important that gloves are a loose fit and can be quickly removed should any liquid splash onto them. NEVER immerse hands even when gloved into liquid nitrogen. Thicker more protective gloves may be required for procedures involving filling of liquid nitrogen dewars from the storage tank. 5.2.2.3. Protective clothing clothing must minimize the forming of traps capable of holding liquid. The degree of body protection necessary will depend on the procedure to be carried out. Clothing should protect the body against splashes from liquid nitrogen. 5.2.2.4. Footwear laboratory approved enclosed shoes must be worn. 5.2.3. Stand as far away as possible from liquid nitrogen when transferring it to vessels of warmer temperatures, and when inserting warmer objects into liquid nitrogen. Always perform these operations slowly and make all attempts to minimise

Page: 3 of 10 splashing and violent boiling. Allow items to be inserted in liquid nitrogen to cool in the evolved vapour before immersing. Wherever possible, cool objects prior to contact with liquid nitrogen. 5.2.4. Do not put gloved hands into liquid nitrogen. Gloves are designed to protect hands from the cold vapours and DO NOT protect against liquid saturation of fabric. 5.2.5. Objects such as tongs often become brittle when immersed in liquid nitrogen and are easily broken. 5.2.6. It is preferable to freeze cryovials containing cells at -80 C first, before storing them in Liquid Nitrogen. This two-step process will help to prevent cell-damage and loss of viability caused by rapid temperature adjustment. 5.2.7. To put new cryovials into the Storage tank, remove the lid from the tank and place it upside down on a flat surface. 5.2.7.1. Canes: Extract the container required and remove the desired cane. Replace the container, slowly. Slide off the cane cover (if it has one) then use forceps to insert the new vial into the desired position. Replace the slide cover, extract the container from the tank and insert the cane into the container. Slowly lower the container back into the liquid nitrogen tank. 5.2.7.2. Freezer boxes: Carefully raise the required tower and allow liquid nitrogen to drain off the tower into the dewar. Remove the security pin and then remove the desired freezer box. Replace the security pin and slowly lower tower back into dewar before actioning freezer box. Remove/add tubes as required using forceps, and then repeat above procedure to return box to tower. 5.2.7.3. Ensure that the lid is replaced properly. Be sure to add all new additions to the Liquid Nitrogen Logbook. 5.2.8. When cryovials have been removed from liquid nitrogen they should be treated as an explosion risk. It is always a risk that some liquid nitrogen may have entered the vial, and as it heats up, the nitrogen expands and can cause the vial to explode. Always handle cryovials with forceps, never in the hand, and place the cryovial in a lidded secondary container, or lidded waterbath as it is brought up to temperature. 5.2.9. Smaller Liquid nitrogen tank may be used when required for experiments. Small quantities of liquid Nitrogen may be extracted from this vessel by pouring it into a foam esky. Seek assistance in lifting if the container is full. Remove only the minimum amount of liquid nitrogen required. 5.2.10. Never pour surplus liquid nitrogen back into the container or down the sink. Ensure that the remainder is left to evaporate in a secure, well-ventilated area. 5.3. STORAGE AND TRANSPORT

Page: 4 of 10 5.3.1. Use only specially designed containers for storing and transporting cryogenic fluids. Liquefied gases at atmospheric pressure should always be contained in vacuum-jacketed vessels. 5.3.2. Appropriate signage should be displayed on the outside of these vessels indicating the cold hazard including W010 in ISO 7010. (see appendix) 5.3.3. Ensure that storage containers are checked regularly for ice build up in and around the vents. 5.3.4. Use only the stopper/lid provided with the storage cylinder. 5.3.5. Use a trolley to move heavy cryogenic containers around. Ensure that the trolley is stable and will hold the containers securely in position. 5.3.6. DO NOT travel in a lift with liquid nitrogen. 5.3.6.1. If lifts must be used to transport vessels from one floor to another, then there should be one person present to place the vessel into the lift, together with a large DO NOT ENTER sign (see appendix), so that other persons will not enter the lift from another level. 5.3.6.2. A second person should be waiting on the floor to which the lift is sent to receive the liquid nitrogen vessel and remove it from the lift. 5.3.6.3. Where possible use a dedicated goods lift. 5.4. DRY ICE Dry Ice (solid carbon dioxide) is not strictly a cryogenic substance, but is commonly used in the lab and does have hazards, similar to those of cryogenic materials. (The temperature of Dry Ice is -78 o C). 5.4.1. Do not lower your face too close to the dry ice as the lack of oxygen present may cause suffocation. 5.4.2. Never handle dry ice with the bare hands as a severe burn will result; use gloves or tongs. 5.4.3. Use an esky (foam or plastic) to transport and store dry ice. 5.4.4. To dispose of Dry Ice, leave the labeled esky in a well-ventilated area to evaporate. 5.5. EMERGENCY PROCEDURES The best way to deal with a liquid nitrogen spill is to provide the area with maximum ventilation and allow the spill to evaporate. However, be aware of the probability of rapid oxygen displacement and close the area off to personnel accordingly. In the event of a Liquid Nitrogen spill: 1 Assess the suffocation risks and order an evacuation if necessary. 2 Administer first aid where required.

Page: 5 of 10 3 Ventilate the area well. 5.6. FIRST AID 5.6.1. Cold contact burns 5.6.2. Tissue damage does not normally occur immediately cryogenic liquid touches warm flesh. Initially, the blood supply to the tissue acts as a heat source and creates a heat-insulating gas film at the liquid/flesh interface. This time delay is usually sufficient for severe injury to be avoided even when a large volume of fluid is involved, provided that appropriate action is taken. 5.6.3. If liquid nitrogen is splashed on the body, the affected area should be immediately flushed with cold water, and any clothing that is saturated with the fluid should be removed. It is especially important to remove gloves or footwear quickly if liquid is splashed into them, as large volumes can be trapped, thereby enhancing the risk of tissue damage by prolonged contact. (AS2243.2:2006 Safety in Laboratories Chemical aspects) 5.6.4. When working with liquid nitrogen ensure you know where the closest safety shower is situated. 5.6.5. If, despite initial treatment, cold contact burns are inflicted, the over-riding first-aid considerations should be to get the casualty to hospital quickly. Professional medical attention should always be sought for cold contact burns because of the risk of complications. 5.6.6. DO NOT rub or massage the affected area 5.6.7. DO NOT expose the affected area to a radiant heat source 5.6.8. DO NOT allow the casualty to smoke, eat or drink until medical advice is obtained. 5.6.9. First aid and general treatment for cryogenic burns are virtually the same as those specified for frostbite in first aid manuals. (AS2243.2:2006 Safety in Laboratories Chemical aspects) 5.6.10. Fill out or witness an accident form 5.6.11. Asphyxiation The rescuer of an asphyxiated victim should be careful not to take excessive risks and become another casualty. Take the casualty to a well-ventilated area. Have someone else contact a first aid officer immediately. Apply artificial respiration if breathing has stopped. Apply external cardiac compression if circulation has failed. Seek professional medical attention. Fill out or witness an accident form

Page: 6 of 10 5.7. LIQUID NITROGEN/DRY ICE ORDERING PROCEDURES 5.7.1. Liquid Nitrogen can be obtained from an on-campus supply tank, situated near the Biological Sciences greenhouse. A biological sciences staff member fills all transport tanks daily at approximately 9:30am. Unauthorised staff are not permitted to fill tanks themselves. Alternatively, Medical Stores personnel fill and return tanks to the Stores dock twice weekly. To use this service, ensure that the liquid nitrogen transport tank is in the store by Monday or Wednesday afternoon. The tanks will be filled and returned to the Store dock on Tuesday and Thursday mornings respectively. The transport tanks are no longer delivered to the labs and must be collected from the stores dock by lab staff. NB: Use a trolley to transport heavy tanks and do not attempt to lift excessive weights. 5.7.2. To order dry ice, contact the medical stores staff at least a day in advance with the details. The dry ice delivery will require a cost centre number (appropriation number). See the Research Support Unit. 5.8. OTHER CRYOGENIC MATERIALS If you intend to use cryogenic materials other than liquid nitrogen or dry ice, such as helium, hydrogen, argon, oxygen, methane and liquefied natural gas, consult the CCH Laboratory Safety Manual (the chapter called 'Safe Handling of Cryogenic fluids' in the Chemical Safety section) and the Australian Standard AS2243.2 Safety in Laboratories Chemical aspects for detailed safety information. 6. Maintenance: 6.1. If any part of this procedure changes or requires updating consult the Professional Officer. 7. Shutdown: 7.1. If you were required to read this GDP, sign the Requested Reading Record Sheet (Illustration 5.2) of GDP 001 Induction Procedure. 8. Safety Precautions: 8.1. Ensure that all safety instructions are understood and adhered to.

Page: 7 of 10 9. Illustrations: 9.1. Liquid Nitrogen Emergency Measures and First aid sign see attached 9.2. Safety signage W010 in ISO 7010 9.3. Do Not Enter sign for use when transporting liquid nitrogen in lifts. 10. Check List: N/A 11. References: 11.1. CCH Laboratory Safety Manual 11.2. AS2243.2 Safety in Laboratories Chemical Aspects 12. Change History: 12.1. Issue Number: 1st Issue Date Issued: 12.2. Issue Number: 2 nd Issue Date Issued: 26 th September Reason for Change: Review of procedure. Change of first aid procedures to those recommended in the Australian Standards.

LIQUID NITROGEN EMERGENCY MEASURES AND FIRST AID AS/NZS 2243.2:2006 If liquid nitrogen is splashed on the body, the affected area should be immediately flushed with cold water, and any clothing that is saturated with the fluid should be removed. It is especially important to remove gloves or footwear quickly if liquid is splashed into them, as large volumes can be trapped. If, despite initial treatment, cold contact burns are inflicted, the over-riding first-aid considerations should be to get the casualty to hospital quickly. Professional medical attention should always be sought for cold contact burns because of the risk of complications. (a) DO NOT rub or massage the affected area. (b) DO NOT expose the affected area to a radiant heat source. (c) DO NOT allow the casualty to smoke, eat or drink until medical advice is obtained. Nearest safety shower: in lab

Low Temperature / Freezing Conditions Liquid Nitrogen

DO NOT ENTER HAZARDOUS MATERIAL! VACATE LIFT UNTIL TANK REMOVED.