Within pipette calibration there are five widely used grades of pipettes, all of which have specific guidelines and requirements regarding use, testing, maintenance, and measurement. The five grades of pipettes include disposable/transfer, graduated/serological, single-channel, multichannel, and repeat pipette. From the most basic transfer pipette dropper to the advanced repeat dispensing pipettor, the manner in which the equipment is handled will impact the accuracy of the test results.
At Transcat, we pride ourselves on the compliance-oriented calibration service we provide our clients, but part of our expertise involves having in-depth knowledge of all the instruments we service. This post will define the various types of pipettes and how to use them to ensure your tests provide the most precise results with the smallest margin of error:
This is the most basic type of pipette; it is not a sophisticated piece of lab equipment and can be used for rough measurements only. However, when using a disposable pipette, it’s important to follow a standard pipetting technique. Always use a new pipette and dispose of it after testing, aspirate liquid at a 90-degree angle, dispense at a 45-degree angle, and touch off to make sure all the liquid is dispensed.
When using this type of pipette, the final volume is found by calculating the difference of the liquid level before and after liquid is dispensed, much like a burette. This is the standard technique for using a graduated pipette:
Hold pipette in solution, don’t touch the bottom.
Squeeze the bulb and attach to the top of the pipette.
Hold forefinger on top of pipette to control volume aspiration.
Subtract the amount needed into a separate beaker while staying eye level to assure proper measurement.
Measure solution from the bottom of the meniscuses, the crescent-shaped surface of the liquid that is visible in the pipette.
Subtract needed volume from the initial volume and find the volume needed to release to in order to get the desired amount.
A single-channel pipette is a non-disposable instrument, usually an air-displacement design which produces accurate measurement results with the use of one disposable tip. There are two common techniques associated with single-channel pipetting:
Forward Technique: This is the intended function and the most common technique for pipette measurements. To use this technique, press the plunger to first stop and slightly submerge the pipette tip into the liquid, aspirate your measured volume by releasing the plunger slowly to prevent bubbles. To dispense the liquid, place the tip against the side of the receptacle, then slowly press the plunger through the first stop to the final blow out position while ‘touching off’ the last drop from the tip.
Reverse Technique: When working with viscous solutions or solutions prone to bubbles we have the option to use a reverse pipetting technique, which will minimize interference from air bubbles. To use this technique, press plunger all the way through to the third stop position (all the way down), slightly submerge into liquid, and slowly release the plunger all the way back to the top and aspirate the liquid into the tip. Place the pipette tip against the wall of the receptacle and press the plunger to the first stop, holding the plunger in place and remove the tip from the receptacle. You will now have a sample of liquid that remains in the tip, but is not part of the measurement. You can then repeat the aspiration procedure and continue.
The technology and technique behind a multichannel pipette is similar to that of a single channel, except it takes more than one tip at a time. Since the liquid is aspirated at the same time from the same well into multiple channels, you must ensure the aspirated liquid levels are equivalent, then the liquid can be dispensed into your tubes or plate wells. The technique is similar to the single channel, but the outcome is much different:
Install of the proper tips to each channel and set the desired volume.
Hold the pipette in a vertical position, depress the plunger to the first stop.
Immerse the tip into the liquid, release the plunger back to the rest position.
Place the tip 45 degrees against the wall of the vessel receiving the liquid.
Depress the plunger to the first stop, wait one second, press the plunger to the second stop and expel all the liquid while ‘touching off’ the last drop.
Move the end of the tip away from the liquid and release the plunger to the rest position.
Repeat Pipette Dispenser
This type of pipette allows a technician to set and dispense a specific volume into multiple receptacles without having to aspirate in between dispenses. This multidispensing capability saves time and effort. The repeat pipette dispenser has a different design than the typical pipette. The difference is a filling and dispensing lever as opposed to a plunger. To use to a repeater pipette successfully, follow this technique:
Slide the filling lever down as far as it goes.
Raise the locking clamp upward.
Insert the syringe-type tip into the barrel so that it clicks into position, then close the lever.
Immerse the tip into the liquid at a 90 degree angle.
Slowly slide the filling lever upward to fill the tip completely.
Prime the tip by discarding the liquid from the first dispense.
The repeat pipette is now ready to operate.
Before beginning your liquid measurement and pipetting technique, it’s important that you evaluate the type of test you are conducting and determine which pipette type is the best fit. Using the wrong instrument or even using the right instrument incorrectly could significantly impact your results. Accuracy and repeatability are two of the most important aspects of testing with pipettes; however, technicians cannot expect precise results without expert knowledge of how to operate every piece of lab equipment.