Proper Pipetting Methods

Care and Use of a Pipette

Air displacement pipettes are laboratory tools used to transfer specific sample fluid volumes from one container to another.  If done correctly, this transfer will be accurate and reproducible. There are several factors that contribute to maintaining an accurate & reproducible volume transfer of fluid.  These include proper care of the pipette, routine calibrations and development of a skill level that is associated with correct technique.

Proper Procedure – Traditional Method

1. If you are using an adjustable pipette, select the desire volume to be transferred.  This is done by rotating the volume selector wheel to the desired volume.  Always go higher then the desired volume then, slowly turn the wheel down to the appropriate volume.  Some pipettes have locking mechanisms.  These need to be unlocked prior to selecting the transfer volume then locked after selection.  Note: Do not use an adjustable pipette outside its recommended range.
2. Select the proper tip for the pipette.  To attach, firmly press the shaft of the pipette into the large open end of the tip with light force to ensure a good seal.  Do not repetitively bang the pipette shaft into the tip.  This may cause damage to the shaft over time.
3. Place either your index or thumb over the top of the plunger and press down to the first stop.  Hold the plunger in this position.
4. Place the pipette tip into the sample at the proper immersion depth and relax the pressure on the plunger.  Be sure that the pipette is either vertical or within 20° of vertical.  This will allow the piston to rise, aspirating sample fluid into the pipette tip.  Do not remove your thumb from the plunger or let the plunger rise up quickly.   This will result in dispensing incorrect samples volumes.

5. Once the plunger reaches the starting position, pause for a second or two to ensure that the aspiration of the sample fluid is complete.  Be sure there are no bubbles in the tip.
6. Remove the pipette tip from the sample volume, and then wipe any excess fluid from the exterior of the tip with a lint free tissue being careful not to touch the bottom of the tip.
7. To dispense, touch the pipette tip to the side of the receiving tube and then press the plunger down slow and steadily pass the first stop all the way to the bottom of the piston stroke. Wait 1-2 seconds to allow complete dispensing of the sample fluid.
8. Remove the pipette tip from the wall of the receiving tube and then release the plunger.
9. Press the ejector button to remove the pipette tip or carefully pull the tip off with your fingers and disposed of the tip in the proper biohazard &/or sharps container according to the safety requirements of your facility.

Reverse Pipette Method

1. If you are using an adjustable pipette, select the desire volume to be transferred.  This is done by rotating the volume selector wheel to the desired volume.  Always go higher then the desired volume then, slowly turn the wheel down to the appropriate volume.  Some pipettes have locking mechanisms.  These need to be unlocked prior to selecting the transfer volume then locked after selection.  Note: Do not use an adjustable pipette outside its recommended range.
2. Select the proper tip for the pipette.  To attach, firmly press the shaft of the pipette into the large open end of the tip with light force to ensure a good seal.  Do not repetitively bang the pipette shaft into the tip.  This may cause damage to the shaft over time.
3. Place either your index or thumb over the top of the plunger and press down through the first stop to the second stop.  Hold the plunger in this position.
4. Place the pipette tip into the sample at the proper immersion depth and relax the pressure on the plunger.  Be sure that the pipette is either vertical or within 20° of vertical.  This will allow the piston to rise, aspirating sample fluid into the pipette tip.  Do not remove your thumb from the plunger or let the plunger rise up quickly.   This will result in dispensing incorrect samples volumes.

5. Once the plunger reaches the starting position, pause for a second or two to ensure that the aspiration of the sample fluid is complete.  Be sure there are no bubbles in the tip.
6. Remove the pipette tip from the sample volume, and then wipe any excess fluid from the exterior of the tip with a lint free tissue being careful not to touch the bottom of the tip.
7. To dispense, touch the pipette tip to the side of the receiving tube and then press the plunger down slow and steadily to the first stop. Wait 1-2 seconds to allow complete dispensing of the sample fluid.
8. Remove the pipette tip from the wall of the receiving tube without dispensing the remaining sample fluid and then release the plunger.
9. Press the ejector button to remove the pipette tip or carefully pull the tip off with your fingers and disposed of the tip in the proper biohazard &/or sharps container according to the safety requirements of your facility.

 Helpful Hints for Accurate Precise Measurements 

1.      Pipettes should be stored in an upright position

2.      Pre-wet the pipette tip three times prior to delivering the specified sample volume. 

3.      Always use the properly prescribed tip for the selected pipette to ensure a good seal.  Check the tip of the pipette shaft for scratches, hairline fractures or other damage that may compromise a tight seal between the pipette and the tip.

4.      Develop a steady aspiration and dispensing cadence that emphases speed and skill.

5.      Never invert or lay the pipette on its side while there is liquid in the tip.

6.      Allow all sample fluids and equipment to reach room temperature prior to pipetting.

7.      Do not use adjustable pipettes outside recommended ranges.

8.      Calibrate pipettes every six months, more frequently if the lab is either clinical or GMP in nature.

9.      Prime the piston by depressing the plunger a few times prior to using the pipette for the first time each day of use.

10.   When pipetting liquids that do not have the density or vapor pressure similar to water, consider using positive displacement pipettes.

11.   Leaks in the seal will adversely affect the reliability of the pipette. To verify that there is no leak, adjust the volume setting to the maximum and then aspirate with water.  Keep the pipette in a vertical position for 20 sec and look for a drop of water at the bottom of the tip.  If a drop appears then there is a leak.  If there is no drop, immerse the same water filled tip in water.  The level of water within the tip should remain the same.  If the level drops, then there is a leak.