THE UNIVERSITY OF NEWCASTLE- DISCIPLINE OF MEDICAL BIOCHEMISTRY. STANDARD OPERATING PROCEDURE PROCEDURE NO: GLP 022 MOD: 1st Issue Page: 1 of 8

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1 THE UNIVERSITY OF NEWCASTLE- DISCIPLINE OF MEDICAL BIOCHEMISTRY STANDARD OPERATING PROCEDURE PROCEDURE NO: GLP 022 MOD: 1st Issue Page: 1 of 8 Procedure Type: General Laboratory Procedure 1. Risk Assessment: Risk Assessment is to be used as a general guide and as such, cannot accommodate all the varying factors that may be encountered when using this equipment. Therefore, personnel are requested to conduct their own Risk Assessment before using this equipment to include any extra hazards introduced by the task performed. TASK PERFORMED Adding and Removing Vials from the Liquid Nitrogen Storage System using the Lifting Device. HAZARDS 1. Cold burns Liquid Nitrogen: -196 o C. The main safety hazard involved when dealing with liquid nitrogen is from burns, due to its very low temperature. 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. 2. Inhalation of cold gases and vapours may produce breathing discomfort and prolonged exposure can lead to serious illness 3. Asphyxiation Replacement of oxygen in air by Nitrogen. 4. Splashing of Liquid nitrogen. 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. 5. Injury can result from stretching/reaching when using the lifting device. 6. Infectious cells. WRITTEN BY CHECKED BY AUTHORISED BY NAME (signed) Ashley Carter Petrina Guthrie / Lynn Herd Phillip Dickson DATE 24 th May th February nd February 2007 Distributed To: GLP Master File / GLP Lab File

2 Page: 2 of 8 RISK ASSESSMENT 1. The risk from cold burns is moderate during this procedure. All risk controls must be followed to effectively control this risk. 2. The risk from inhalation of cold gases and vapours is moderately low during this procedure. Although such gases are present, following the risk controls should reduce exposure to them. 3. The risk from asphyxiation is low with exhaust fan in place and doors open. 4. The risk of splashes from liquid nitrogen is controlled by good work practices and training. The risk is moderate. 5. The risk from manual handling stretching/reaching is present due to the dimensions of the tank, the racks and the lifting device each require a degree of reaching, stretching and lifting which can generally be accommodated by the average person provided due care is taken. There is, however, the potential risk of injury to persons of short or lighter build attempting to retrieve towers/racks from the tank, even when using the lifting device. 6. The risk from infectious cells is only present if vials have thawed or tubes have broken. Wearing appropriate PPE, and handling vials using forceps, reduces this risk to low. RISK CONTROL 1. The use of the Liquid Nitrogen lifting device must be carried out by persons trained in the appropriate method and procedures. Training must be provided by personnel experienced in the procedure. 2. Two persons are required to be present for this procedure. 3. Ensure storage vessel is functioning properly and in good condition. 4. Regularly remove any ice build-up around the lid underside and check that the lid seal is in good order. 5. Keep the storage container-lid locked at all times when not being used. 6. Although some laboratory work is carried out in this room at times the use of the room for other purposes should be limited, and at no time should gas bottles or any other sources of heat be stored or used in this room. 7. Cold vapours or gases may be emitted from the filler unit during this procedure. If you find that you are inhaling cold vapours, vacate the area, and obtain assistance. 8. The buildup of cold vapours should be controlled by ensuring there is good ventilation to the room. Put in place the two portable barriers with the DO NOT ENTER signs across both doorways to ensure good ventilation and prevent unauthorized access.

3 Page: 3 of 8 9. The risk of asphyxiation from liquid nitrogen is increased when in a confined space. Do not travel in the lift with the liquid nitrogen unit. 10. Work efficiently to minimize the time spent with the lid open and close the lid immediately after job is completed. 11. Work away from the tank as much as possible and avoid standing with head over the opening. 12. Since the storage area on Level 2 Life Sciences has been fitted with an exhaust fan, risk of asphyxiation is reduced. 13. Splashing of liquid nitrogen should be avoided. A minor spill of liquid nitrogen onto the floor or bench is not uncommon and will quickly evaporate. 14. Work practices should be designed to reduce the risk of splashing. The highest likelihood of splashing occurring is during the lowering of the racks into liquid nitrogen. Ensure that this is carried out in a slow, controlled manner. 15. If splashing occurs onto clothing or gloves, quickly remove affected item. 16. Manual handling hazards can be controlled by appropriate training and planning of lift. 17. Where a person s stature precludes them from safely operating the lifting device, assistance should be sought from other laboratory members. 18. When working with infectious cells, which are in no danger of thawing, additional precautions should not be necessary. 19. If broken or thawed vials are encounter, treat cells as potentially infectious and dispose into bag for autoclaving. 20. Appropriate PPE must be used during this procedure. Blue cryogenic gloves should be used when materials which have been in contact with liquid nitrogen. Laboratory gown and full face mask must be worn by both persons present during the procedure. All PPE should be easily removed in the event of becoming wet by liquid nitrogen. 21. Clothing which covers both arms and legs (eg. Long-sleeved shirt and long pants) should be worn when working with liquid nitrogen. 22. Lab gown should be worn when accessing PC2 laboratory areas, but should be removed when accessing public areas. 2. Purpose: 2.1. To safely use the Liquid Nitrogen Storage System and the Lifting Device to store and retrieve vials from the cell storage tank.

4 Page: 4 of 8 3. Equipment: 3.1. Liquid Nitrogen Storage Tank and the attached Lifting Device Keys to the liquid nitrogen storage room LS2.19 and for the lid of the storage tank Thermal gloves Face shields Long sleeved laboratory coat Liquid nitrogen transport vessel with lid and security rod Tweezers Items to record position of vials to be removed or added. 4. Materials: 4.1. Frozen cells in vials that have been suitably identified Items to record location of vials added or removed. 5. Safety Precautions: 5.1. Operators must be trained and have read and fully understood the Risk Assessment and Control sections at the head of this procedure. Note that this risk assessment covers the majority of hazards to be encountered in performing this procedure; however operators are advised to include any extra hazards introduced by this task. 6. Method: 6.1. Determine from the diagram attached to the front of the storage tank the relative position of the tower(s) in which the cells are located or are to be placed Unlock and open the lid to the storage tank and, using the tweezers, carefully raise both handles on top of the selected tower to approx. 45 to accommodate the lifting device hook Remove the lifting device foot control with attached cable from its storage position and place on the floor close to and in front of the tank Holding the hook by the stem move it into position above the selected tower Again holding the hook by the stem, depress the foot pedal to release the brake and pull the hook down to the raised tower handles Securely insert the hook under both handles and, with the brake pedal depressed, lift by the stem of the hook, using both hands, and raise the tower to the required height so that it is suspended freely above the tank. Release the foot/brake pedal and provided the tower is hanging freely and/or the hook is under tension the tower will remain in position allowing the user to release their hands.

5 Page: 5 of Pull the security pin up through the top of the tower; locate the box required (all boxes are numbered from the bottom of the tower up) and remove it carefully. Tilt the box to drain any excess liquid nitrogen back into the tank, taking care not to pour across glove, and place on the bench. Drop the security pin back down into position so that no boxes can fall into tank If the job will take longer than a few minutes, it is desirable to lower the tower back into liquid nitrogen tank using the foot/brake pedal as previously described If the removed box will be out on the bench for an extended time, it is desirable to maintain its temperature. The recommended method is to obtain an esky lid or similar which can usually be found in the same room, place the box into the esky lid and pour in approx. 1½ cm of liquid nitrogen from the smaller storage dewar Using tweezers remove or add vials in the desired positions and, where applicable, place into the transport vessel, which should contain some liquid nitrogen. Check the labeling on the vials as they are removed and record the locations. Also record locations when adding vials to the box Without further delay, if job completed, replace lid on the box in correct alignment with corner flats, return it to the tower, replace security pin and return tower to the correct position in the storage tank If other towers are to be removed the procedure is the same as above, otherwise, unhook the handles and return the hook all the way to its uppermost position then release the foot pedal. Replace pedal to its storage position and close the storage tank lid, then lock it. 7. Maintenance: 7.1. The Liquid Nitrogen Storage Tank is filled twice weekly to keep the level within half a box height down to no more than six boxes down. Refer to GLP 035 Filling Liquid Nitrogen Filler Tank. 8. Shutdown: 8.1. When completed, remove the DO NOT ENTER barriers and lock the door when leaving the room Any vials being taken back to the lab should be carried in the transport vessel with liquid nitrogen and the lid held in place with the security rod. Liquid Nitrogen should not be transported in a lift in case of lift failure and subsequent nitrogen displacement in an enclosed space Record the details of vials removed or added into the Liquid Nitrogen database

6 Page: 6 of Change History: 11.1 Issue Number: 1st Issue Date Issued: 6 th February Issue Number: Date Issued: Reason for Change: