25/04/56. LTC Carmen A. Bell Chief, Laboratory Operations USAMC-AFRIMS

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1 Pipette Theory and Maintenance LTC Carmen A. Bell Chief, Laboratory Operations USAMC-AFRIMS References Pharma Express: 0/biotech13.shtml ThermoScientific: Wisconsin State Laboratory of Hygiene USAMC-AFRIMS SOP 2170, Rev 7 Biosafety in Microbiological and Biomedical Laboratories, 5th Edition (CDC/NIH)

2 References Connors, M. and Curtis, R. H. Pipetting error: A real problem with a simple solution. Part I, American Laboratory News (1999) 31(13):16-19 Fleming, D.O Laboratory biosafety practices. In Fleming, et al (eds.), Laboratory Safety: Principles and Practices, 2nd edition. ASM Press, Washington WHO Laboratory Biosafety Manual, 2nd edition revised Objectives Upon completion of this course, you will be able to: State the function of a pipette Identify pipette safety Identify the three types of pipettes List the parts of a pipette Use a pipette correctly Understand basics of pipette calibration

3 Pipette Function Instruments used to handle, measure and dispense liquids Uses a pressure gradient to draw a liquid Types of Pipettes (1 of 8) Six types Volumetric Measuring Mohr or Serological Glass/ Pasteur Air Displacement Positive Displacement

4 Types of Pipettes (2 of 8) Volumetric Measuring Air Displacement -Single channel (above right) -Multichannel (below right) Positive Displacement -Single channel (right) -Multichannel (left) Mohr Serological Types of Pipettes (3 of 8) Volumetric Transfers a specific volume Capacity of ml Used when precision is not important Measuring Straight tubes with a tapered end Capacity.1ml and 25 ml Clearly marked hash marks along one side of tube so multiple amounts can be dispensed Not very precise due to imperfections in the tube

5 Types of Pipettes (4 of 8 ) Mohr and Serological Mohr: the hash marks always end before the pipette tip Serological: the hash marks continue down into the tops and some are blow-out pipettes Blow-out pipettes are marked with a frosted band and two thin rings around the neck Pipettes have an open top and the user uses their thumb to create a vacuum and seals the liquid in the pipette Types of Pipettes (5 of 8) Glass/ Pasteur Glass tubes with a bulb on one end Calibrated to deliver or contain a specific amount, or have graduated markings to measure the volume of liquid they contain *Single use Precision is not critical

6 Types of Pipettes (6 of 8) Air Displacement Digital Piston Driven Contains a piston inside a cylinder that can be positions to specify a needed volume Expel the air first, draw up the liquid needed, then expel the liquid and the remaining air A volume of air is always held between the liquid and piston Very accurate Atmospheric pressure and type of liquid can affect the performance Types of Pipettes (7 of 8) Positive Displacement Similar to Air Displacement pipettes because they have a piston and cylinder Piston is always in direct contact with the liquid When depress, the piston moves down to the tip opening When released, the piston rises to the appropriate volume creating a partial vacuum that draws in the liquid Used with volatile and viscous liquids *Most accurate and costly

7 Types of Pipettes (8 or 8) Parts Plunger button Plunger rod Friction ring Tip ejector Digital Volume Indicator Piston Springs Seal, O-ring Shaft Shaft coupling Tip ejector Pipette Safety (1 of 4) Never mouth pipette Use rubber pumpette (bulb) Use plastic pipettes if available Safer than glass Do not allow the solution to be drawn into the bulb Leaks chemicals into the top where fingers may touch Do not insert the pipette into the bulb, insert the bulb onto the pipette Use proper ergomomics (body position)

8 Pipette Safety (2 of 4) When working with infectious agents, always use a Biological Safety Cabinet (BSC), or acceptable primary barrier Never expel infectious fluids Potential aerosols Mark to mark pipetting is best Never cause air bubbles to mix infectious fluids Discharge fluids close to other fluids, agar level or microwells Pipette Safety (3 of 4) Pipettes used for transferring infectious materials should always be plugged with cotton Syringes fitted with hypodermic needles MUST NOT be used for pipetting Avoid dropping material from the pipette on the work surface Place a disinfectant-dampened towel or other absorbent material on the work surface

9 Pipette Safety (4 of 4) Place contaminated pipettes horizontally in an autoclave bag on a tray Vertical positioning may cause aerosoles Discard contaminated Pasteur pipettes and broken glass in a biohazardous sharps container. Dispose of when 2/3 full Pans or sharps containers should be kept inside the BSC General Guidelines (1 of 2) Check your pipette to ensure it is clear of dust and dirt Wipe, if needed with 70% ethanol Set the volume within the range specified Hold the pipette so the griippy finger rest rest on the index finger For accuracy, the pipette, tip and liquid should be at the same temperature Use recommended tips

10 General Guidelines(2 of 2) Tips are single use They should NOT BE cleaned and reused Avoid turning the pipette on its side to prevent contamination Avoid contamination to or from fingers by using the tip ejector and gloves Store pipettes in an upright position when not being used *Pre-rinse (1-3 times) the tip with liquid to improve accuracy, especially with positive displacement tips *Check calibration regularly, depending on the frequency of use and on the application, but at least once a year Maintenance Keep pipette clean Free of dust particles Wipe the surface with a sponge moistened with disinfectant Store in an upright position Clean Piston, Piston Spring and O-rings (70% ethanol) Lubricate Piston, Piston Spring and O-rings (grease)

11 Calibration (1 of 3) Three parts Operator Equipment Environment Calibration The pipette is held in the calibration room at least 2 hours before testing Check for maximum and minimum volume or 10% of the maximum volume (whichever is higher) Perform a series of ten pipettings of both volumes Calculate the accuracy and precision *Adjust pipette if needed

12 Calibration Accurate and Precise Precise But not Accurate Accurate But not Precise Calibration Operator

13 Pipetting Technique (1 of 9) Optimizing volume range Micrometer setting Tip immersion angle Tip immersion depth Pre-rinsing Aspiration rate Dispensing technique Consistency **Errors from poor technique can range from 0.1 % to 5% or more Pipetting Technique (2 of 9) Optimizing volume range Assures accuracy and precision Normal Range µL µL µL Optimizing Range µL µL µL Optimizing volume range typically improves accuracy up to 1%

14 Pipetting Technique (3 of 9) Micrometer Setting Ensure the volume is set correctly *Correct settings improve accuracy up to 0.2% Pipetting Technique (4 of 9) Angle Aspirate at 90 degrees Dispense at 45 degrees *Improves accuracy up to 0.5% Incorrect immersion angle Correct immersion angle

15 Pipetting Technique (5 of 9) Tip Immersion Depth *Improves accuracy up to 5% Pipette size Immersion depth 2 and 10 µl 1 mm 20 and 100 µl 2-3 mm 200 and 1000 µl 3-6 mm 5000 µl and 10 ml 6-10 mm Pipetting Technique (6 of 9) Pre-rinsing Pre-rinsing tip with same liquid that is being dispensed Aspirate with tip, and then dispense back into reservoir or to waste Provides identical contract surfaces for all aliquots * Two rinses provides up to 0.2 % greater accuracy

16 Pipetting Technique (7 of 9) Aspiration Rate Effects Maintain smooth, controlled aspiration rate Aspirating too quickly causes Splashing into the piston and seal damage Introduction of aerosols/ sample crosscontamination * Controlled aspiration improve accuracy 0.2% -5% Pipetting Technique (8 of 9) Dispensing Technique Thin-wall tips provide maximum droplet dispensing Along side-wall techniques *Correct dispensing technique improves accuracy 0.5%-1%

17 Pipetting Technique (9 of 9) Consistency Pipetting rhythm Pressure on plunger Speed and smoothness Use electronic pipettes for best consistency Less user dependent * Consistency in pipetting can improve accuracy up to 1% Calibration Equipment

18 Pipetting Direction Technique (1 of 9) Forward Reverse Used for pipetting solutions with a high viscosity or a tendency to foam Only possible with air displacement pipettes Repetitive Used for repeated pipetting of the same volume. Only used with electronic and repeator pipettes Pipetting Direction Technique (2 of 9) Forward Press the button to the first stop Place the tip in the solution and slowly release the button Dispense the liquid into the receiving vessel or fluid (pressing to the first stop, then press to the second stop) Release the button

19 Pipetting Direction Technique (3 of 9) Forward Pipetting Technique Pipetting Direction Technique (4 of 9) Reverse Press the operating button Place the tip in the solution and slowly release the button Dispense the liquid into the receiving vessel or fluid. The volume is equal to the set volume. Hold the button in this position. Some liquid should remain The remaining liquid can be pipetted back into the original solution or disposed of Release the operating button

20 Pipetting Direction Technique (5 of 9) Reverse Pipetting Technique Pipetting Direction Technique (6 of 9) Repetitive Press the operating button to the second stop Place the tip onto the solution and slowly release the button. Withdraw the tip from the liquid, touching against the side of the resevoir Dispense the liquid into the receiving vessel by pressing the operating button to the first stop. Hold the button in position. Some liquid will remain in the tip. Do not dispense it. Continue pipetting by repeating the above two steps

21 Pipetting Direction Technique (7 of 9) Repetitive Pipetting Technique Pipetting Direction Technique (8 of 9) Whole Blood Use first two steps of the forward technique to fill the tip with blood. Wipe the top with a dry clean cloth Place the tip into the blood and press the operating button to the first stop. Make sure the tip is below the surface Release the button slowly to fill the tip with blood Press the button to the first stop and release slowly. Repeat this process until the wall of the tip is clear Press the operating button to the second stop and completely empty the tip. Remove the tip by sliding it along the wall of the vessel Release the button

22 Pipetting Direction Technique (9 of 9) Whole Blood Technique Pipetting Recommendations Solution/ Compound Aqueous solution Viscous solution Nucleotide solutions Radioactive compounds Acid/ Alkalis Toxic samples Volatile compounds Examples Pipette Tip Technique Comments Buffers, diluted salt solution Protein and nucleic solutions, glycerol, Tween 20/40/60/80 Genomic DNA, PCR Products Carbonate, Y- thymidine H 2 So 4, HCL, NaOH Methanol, Hexane Air Displacement Standard Forward Air Displacement Positive Displacement Air Displacement Positive Displacement Air Displacement Positive Displacement Filter Positive Displacement Filter or wide orifice Positive Displacement Filter Positive Displacement Reverse Forward Forward Air Displacement Filter Forward Positive Displacement Air Displacement Positive Displacement Positive Displacement Pipette slowly to avoid bubble formation For genomic DNA wide orifice should be used to avoid mechanical shearing Filter Positive Displacement Forward Pipette rapidly to avoid evaporation. Carbon filter tips prevent vapors from going into the Pipette

23 Contamination Prevention Pipette-to-Sample (contaminated pipette/tips) Use sterilized tips or sterilized filter tips Change tips after pipetting each sample Sample-to-Pipette (samples or aerosole in cone) Keep the pipette vertical Release the push-button slowly Use filter tips or positive displacement pipettes Store the pipette vertically Sample-to-Sample (sample A mixed with B) Change the tip after each sample If you suspect contamination, autoclave or clean the pipette Parts of the Pipette Plunger button Plunger rod Friction ring Tip ejector Digital Volume Indicator Piston Springs Seal, O-ring Shaft Shaft coupling Tip ejector

24 Calibration Environment Factors affecting the accuracy of Air Displacement Pipettes (1 of 2) Temperature The most important factor in pipetting accuracy is the liquid temperature Density Density is the mass/volume ratio of the liquid. It varies according to the temperature and air pressure Altitude Altitude affects the accuracy through the air pressure

25 Factors affecting the accuracy of Air Displacement Pipettes (2 of 2) Why/ When Calibrate? Calibrate Determine the difference between the dispensed volume and selected volume Occurs upon receipt and periodically during the lifetime of use (at least annually) Part of Quality Control Checks accuracy and precision Ensures integrity of data Produces quality liquid handling Produces quality results

26 Calibration The pipette is held in the calibration room at least 2 hours before testing Check for maximum and minimum volume or 10% of the maximum volume (whichever is higher) Perform a series of ten pipettings of both volumes Calculate the accuracy and precision *Adjust pipette if needed Calibration Accurate and Precise Precise But not Accurate Accurate But not Precise

27 Calibration Methods Gravimetric Most commonly used Based on simple principle Colorimetric (photometric) Uses light to measure volume of diluted dye Calibration Methods Gravimetric Uses a certain mass of water with a known specific gravity to predict the volume Uses an enclosed calibrated analytical balance with an accuracy of g Air and Water Temperature Requires a stringently controlled environment Air Pressure (Barometer) Humidity

28 Calibration Methods Gravimetric (con t) Requires highly skilled technician Requires an understanding of statistics Calibration Methods Colorimetric This method involves the analysis of volumes of diluted dye in a cell of known path length Less prone to environmental influences Requires standardized consumables Dye must be very carefully manufactured

29 Calibration Methods Adjustments Manual Pipettes Adjustment is done at the lower volume Use the service tool provided with the pipette Turn the service tool clockwise to increase the volume or counter clockwise to decrease the volume After adjusting, recalibrate Electronic Pipettes Follow specific manufacturers instructions Questions

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