THE UNIVERSITY OF NEWCASTLE - DISCIPLINE OF MEDICAL BIOCHEMISTRY Page: 1 of 9 1. Risk Assessment: This risk assessment has been prepared using the National Safety Council of Australia Risk Score Calculator and risks have been determined to be as stated. This 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. Operation of Pipettes TASK PERFORMED HAZARDS 1. Repetitive Strain Injury (RSI) 2. Exposure to biohazards/hazardous substances RISK ASSESSMENT 1. The risk of developing muscle strain that leads to RSI is present whenever pipettes are used, however, risk of injury increases as the period of use increases. The risk of developing a permanent injury due to excessive or incorrect use of pipettes is possible. 2. The risk of exposure to biohazards and hazardous substances while using a pipette is present whenever these substances are being handled. The probability of serious illness occurring as a result of exposure is minimal, provided these substances are handled in safe environment and personal protective equipment is used. RISK CONTROL 1. See Safety Precautions (point 4.5) in this GEP. 2. Draw and dispense volumes slowly and with care to avoid splashing. Ensure that biohazards and hazardous substances are only pipetted in appropriate environments such as in biohazard hoods or fume cupboards. Always wear gloves (and safety glasses where necessary) when pipetting any substance. Read Material Safety Data Sheets before handling WRITTEN BY CHECKED BY NAME (signed) Ingo Lehmann Lynn Herd DATE 24 th March 2003 5 th May 2003 Distributed To: GEP Master File / GEP Lab File
Page: 2 of 9 any unfamiliar chemicals. 2. Calibration: 2.1 Frequency All pipettes should be calibrated at three-month intervals, or after major cleaning. 2.2 Method 2.2.1 Allow the temperature of the pipettes, tips, thermometer, a beaker of water and an empty beaker to equilibrate for one hour. They should be placed as close as possible to the balance being used. 2.2.2 Set the volume of the pipettes as per the following table. Pipette Set volume to Permitted Range µl 0.1-2.5 µl 0.8µL 0.55-1.05 0.5-10 µl 2µL 1.75-2.25 2-20µL 4µL 3.9-4.1 10-100µL 20µL 19.7-20.3 20-200 µl 40µL 39.6-40.4 100-1000 µl 200µL 198.7-201.3 500-5000 µl 2mL 1990.0-2010.0 2.2.3 With a pipette tip attached to the pipette, the desired volume is pipetted and weighed 10 times. 2.2.4 The mean of this weighing is converted into μl using the following formula: Volume = Weight/Density of water (at the temperature specified) Temp o C Density Temp o C Density Temp o C x 15 1.0020 20.5 1.0030 26 1.0043 15.5 1.0020 21 1.0031 26.5 1.0044 16 1.0021 21.5 1.0032 27 1.0045 16.5 1.0022 22 1.0033 27.5 1.0047 17 1.0023 22.5 1.0034 28 1.0048 17.5 1.0024 23 1.0035 28.5 1.0050
Page: 3 of 9 18 1.0025 23.5 1.0036 29 1.0051 18.5 1.0026 24 1.0038 29.5 1.0052 19 1.0027 24.5 1.0039 30 1.0054 19.5 1.0028 25 1.0040 20 1.0029 25.5 1.0041 2.2.5 If all the volumes are within the allowed limits then calibration is complete. If not, the pipette must be recalibrated, as follows: - Hold the pipette vertically and insert side D of the wrench horizontally into the alignment opening in the pipette grip. - The wrench is then rotated into a vertical position. - When the setting ring is rotated (either in + or in - direction), the piston stroke of the pipette is altered. One rotation is equal to the following: Pipette Δvolume/rotation 0.1-2.5 µl 0.1 µl 0.5-10 µl 0,5 µl 2-20µL 1 µl 10-100µL 5 µl 20-200 µl 10 µl 100-1000 µl 50 µl 500-5000 µl 250 µl 2.2.6 Remove the wrench and move the setting ring backwards and forwards until the counter and the stroke system lock together. 2.2.7 After the adjustment has been made, repeat steps 2.2.2-2.2.6 until correct calibration is achieved. 2.3 Records All calibration measurements and adjustments must be recorded on the Calibration Record Sheet and filed in the appropriate section of the Equipment Records folder when completed.
Page: 4 of 9 3. Monitoring: Not applicable 4. General Operation: 4.1 The delivery volume is set using the operating button on the top of the pipette. Ensure that the desired volume clicks into place and that the digits are completely visible on the handle display. Do not set volumes outside the pipette's specified volume range. 4.2 The pipette is fitted with a tip ejector system on the top of the tip holder. Direct the tip towards a suitable waste receptacle and press on the ejector mechanism, disposing of the tip safely. 4.3 Forward Pipetting Technique 4.3.1 Depress the operating button to the first stop. 4.3.2 Lower the tip under the surface of the liquid and slowly release the operating button. Withdraw the tip from the liquid, touching it against the side of the container to remove excess liquid. 4.3.3 Deliver the liquid by gently depressing the operating button through the first stop all the way to the second stop. This action will empty the tip. 4.3.4 Release the operating button to the ready position. Replace tip if necessary and continue with pipetting. 4.4 Reverse Pipetting Technique 4.4.1 The reverse technique is suitable for pipetting liquids having high viscosity, a tendency to foam easily or for very small volumes. NB Performing a serial dilution is more accurate than a straight dilution when very small volumes are involved (less than 10μL). For example, it would be more accurate to perform a serial dilution involving two dilutions of 1/10 than a straight dilution of 1/100. 4.4.2 Depress the operating button all the way to the second stop. 4.4.3 Lower the tips under the surface of the liquid and slowly release the operating button. This action will fill the tip. Withdraw the tip from the liquid touching it against the side of the container to remove excess liquid. 4.4.4 Deliver the preset volume by gently depressing the operating button to the first stop. Some liquid will remain in the tip and should not be included in the delivery. It should either be discarded or pipetted back into the container.
Page: 5 of 9 4.5 SAFETY PRECAUTIONS - excessive pipette use can lead to RSI (Repetitive Strain Injury). Therefore the following guidelines should be noted: 4.5.1 Wrists should be held in a straight, neutral position - it may be necessary to incline the sample holders or solution flasks, rather than the wrists. 4.5.2 The workload should be shared between right and left hands. 4.5.3 Elbows should be held as close as possible to the users' side. 4.5.4 Reaching may be reduced by using shorter pipettes; shallower waste containers for used tips and shallower solution containers. 4.5.5 Items should be positioned on the bench and as close as possible to the worker to minimise twisting of the neck and torso. 4.5.6 The chair should provide adequate lower back and thigh support. 4.5.7 Regular breaks should be taken - even very short breaks help. 4.5.8 Work processes should be evaluated to spread pipetting throughout the day. 4.5.9 Pipetting should be rotated among several staff. 4.5.10 Pipetting tasks should be automated wherever possible. 5. Cleaning: 5.1 Frequency Before use - A routine cleanliness check should be performed and the pipette cleaned if required. 5.2 Method 5.2.1 Check for dust and dirt on the outside surfaces of the pipette. Particularly the tip cone. If necessary clean the pipettes in a soap solution or sterilize them using 60 % isopropanol. 5.2.2 The lower half of an Eppendorf pipette may be sterilised by autoclave if required. No special preparations are needed, though a steam sterilisation bag may be used if required. Autoclave at 121 o C for a minimum of 20 min. Allow the autoclaved parts to dry at room temperature. Do not reassemble the pipette until it has cooled down completely. Then the pipette should be calibrated before use. It is recommended that 0.5-1000µL pipettes be calibrated after every 25 sterilisation cycles and 1-10mL pipettes after every 10th cycle.
Page: 6 of 9 6. Maintenance: 6.1 Frequency If the pipette is used daily then it should be cleaned and calibrated every 3 months. 6.2 Method 6.2.1 Obtain a copy of the instruction manual for the pipette from the Instruction Manuals File to use as a guide and disassemble the pipette as follows: 6.2.2 Hold down the ejection button on the side of the pipette and pull off the ejection sleeve using force. 6.2.3 Unscrew the pipette lower part from the grip by turning it to the left. 6.2.4 Press the piston holder together and remove it. 6.2.5 Remove the piston with a spring and replace if necessary. 6.2.6 After cleaning reassemble the parts and calibrate pipette according to step 2.1. 6.3 Records Records of date and details of maintenance are to be entered on the Maintenance Record Sheet (Illustration 9.2) and completed sheets filed in the appropriate section of the Equipment Records folder. 7. Instruction Manual: 7.1 See Attachments 10.1 and 10.1 8. Repairs: 8.1 Check ERN for Service Company and warranty details. 8.2 Contact the University Workshop for advice and repairs. 9. Illustrations: 9.1 Pipette (Eppendorf pipette) Calibration Record Sheet 9.2 Pipette (Ependorf pipette) Maintenance Record Sheet 10. Attachments: 10.1 Instruction Manual for Eppendorf Pipettes, Part A 10.2 Instruction Manual for Eppendorf Pipettes, Part B
Page: 7 of 9 11. Change History: 11.1 Issue Number: 1st Issue Date Issued: 5 th May 2003 11.2 Issue Number: Date Issued: Reason for Change:
Page: 8 of 9 ILLUSTRATION 9.1 CALIBRATION RECORD SHEET PIPETTE ( EPPENDORF PIPETTE) ERN: INITIALS: DATE: Acceptable Weight Range: WATER TEMP 1 WATER TEMP 2 AVERAGE TEMP CONVERSION FACTOR WEIGHT CONVERTED MASS ACCEPTABLE? Y/N 1 2 3 4 5 6 7 8 9 10
Page: 9 of 9 ILLUSTRATION 9.2 MAINTENANCE RECORD SHEET PIPETTE (EPPENDORF PIPETTE) ERN: Date Maintenance Performed Signed