Consistent and accurate manipulation of liquids is essential for many biology experiments. Teaching these skills is challenging in the laboratory. I have developed Training Tips, a homemade teaching tool to train students to use micropipettes in the classroom and laboratory. Training Tips provide obvious visual targets that enable inexperienced students to know immediately if they are pipetting accurately.
Pipetting is the principle technique used by bench scientists to move microliters of liquid accurately. The proper and consistent execution of this technique is essential for numerous scientific disciplines (Guzman, 2001; White et al., 2014). In particular, molecular biology protocols rely upon precise ratios of reagents (Lessard, 2013; Matsumura, 2015). Small deviations from these precise ratios can have a variety of detrimental consequences, all of which lead to decreased productivity and efficiency.
Determining if you are pipetting small volumes correctly is challenging to check by eye. For instance, 1 μl (microliter) looks significantly different depending upon the shape or size of the tip that is used, and even for experienced bench scientists, visually assessing the difference between 800 μl and 850 μl is difficult. Many pipette tips have gradations marked at significant volumes, for example 20 μl and 100 μl. However, the broadness and transparency of these gradations render such markings only minimally helpful as a training tool. Given these challenges, it is critical that we develop tools to aid in teaching inexperienced scientists the foundational skill of pipetting so they can focus on and are better prepared to develop more advanced techniques critical to their scientific training.
To help teach students and inexperienced bench scientists to use micropipettes, I have developed Training Tips, micropipette tips that are clearly marked and labeled. The markings provide a visual target for beginner bench scientists to use as a guide for how far up the tip different volumes of liquid should reach. Students experience how depression and release of the plunger feels when the mark is hit versus when it is not. This instant feedback is helpful for novice bench scientists, and provides critical, individual feedback impractical for single instructors to give a large group.
Training Tips are homemade sets of marked pipette tips that guide the trainee as they learn to use micropipettes. In our lab, we most often pipette between 0.5 μl and 1000 μl, so I have developed three sets of training tips used with either the P1000, P200/20, or P10 pipette. To craft Training Tips, pipette a volume of water into the pipette tip and mark the meniscus with a black marker. Mark each tip at only a single volume. Making the lines on Training Tips as close to parallel with the base of the tip is important. Wrapping tape slightly above and below the meniscus, coloring the exposed region, and removing the tape can generate neater lines but increases how long it takes to make each tip. On the P1000 tips, 1000 μl through 200 μl are marked at 50 μl intervals. For P200/20 tips, from 200 μl to 20 μl are marked at 10 μl intervals. In addition, the P200/20 tips are also marked at 15, 10, 7.5, 5, 2, and 1 μl (Figure 1A).
For P10 tips, between 10 μl and 1 μl were marked at 1 μl intervals. Depending upon the training situation, more or fewer tips may be appropriate. On one side and the top of each tip, I have labeled what volume the mark designates (Figure 1B). The black marker lines on Training Tips are easier to see than the clear gradations provided on standard tips. In addition, most standard tips provide only one or two gradations, limiting the students' ability to check their accuracy. Training Tips are marked at finer gradations and at a single site, thus students can clearly see how far the liquid should come up the tip for the indicated volume. A key on the top of each pipette tip box indicates the tip housed in each well (Figure 2). The key helps students quickly find the appropriate tip, and makes it more likely for students to return the tip to the slot that it came from. This helps maintain tip order when multiple students are using one box.
When the trainee pipettes accurately, liquid will rise to the mark on the side of the tip, but not past it. Training Tips allow students to see immediately if they are drawing the correct amount of liquid into the pipette. If students push past the first stop while depressing the pipette plunger, or do not allow the liquid to slowly enter the tip, the liquid will not be drawn equal to the marked line and/or bubbles will form. The marked lines draw students' attention to the pipette tip, and it is more likely they will notice bubbles and adjust their technique. In addition, if students use the incorrect pipette—for instance, P200 to pipette 5 μl—the liquid will not reach the mark. Training Tips do not require a balance for students to determine if they have pipetted accurately; however, a balance is still needed for pipette calibration and may be useful when assessing student progress. The marking strategy is applicable to any brand of pipette tip and takes roughly one to two hours per set to make. An entire set is not needed for every student in a class, and since Training Tips should not be used for experiments and are only a training tool, they can be reused multiple times with H20. Thus the time commitment required to make Training Tips is not onerous. To further streamline the process of generating Training Tips, one could focus on pipette tips commonly used in a class or particular exercise. The only materials needed to make Training Tips are pipette tips, a fine-tipped black marker, and H20, thus the cost is minimal.
In our lab, we have found that allowing students to practice with Training Tips helps them learn good pipetting technique and focuses their attention on the amount of liquid being drawn into the pipette. We have found that depending upon the color of the tip and the volume of liquid being pipetted, pipetting colored liquid can provide an even more dramatic visual benchmark to see if they are pipetting correctly. Importantly, Training Tips quickly show students if they are not pipetting correctly and has thus made students seek assistance much sooner. The implementation of this training method has made students focus more intently on learning to pipette. In turn, although training time has not increased, student proficiency has improved.
Most students are unfamiliar with μl volumes before working with micropipettes. Training Tips provide visual benchmarks for the difference between volumes that, even to the trained eye, appear very similar (Figure 1A and B). Such small differences in volume can have dramatic effects on experimental success and reproducibility. For volumes of liquid that can be accurately pipetted with multiple tip types, it enables students to see how high the liquid should rise in each tip. For instance, pipetting 5 μl will look and feel different if you use a P10 as opposed to a P20 (Figure 3A). Training Tips allow students to see what a specific volume of liquid should look like under these different circumstances.
Training Tips have worked well to train both first-year and upperclass college students, as well as nontraditional students, to pipette. Given the obvious visual cues Training Tips provide, they could also be used to teach high school students and middle school students. The scope of the exercise, number of replicates, volumes used, and assessment activities should be modified depending upon the students receiving instruction.
Viscosity and surface tensions of liquids can severely affect pipetting. Unfortunately, many such liquids are solvents and/or leave residue in tips, thus using Training Tips with these liquids is less efficient, especially in a classroom setting. However, Training Tips can still be used as a guide by being held next to a tip after pipetting a more viscous liquid, and will help students gain an appreciation for how viscosity affects pipetting.
Pipetting accuracy can be assessed in a number of ways and will depend upon the students and lab infrastructure. To assess middle school and high school students, visual observation of pipetting using Training Tips may be appropriate. For more advanced students, accuracy can be assessed in a number of ways. Students can weigh pipetted water using an analytical balance of sufficient precision and determine how accurately they pipetted. Alternatively, students can make dilutions of a dye of known absorbance and test the accuracy of the dilutions using a spectrophotometer. Both of these assessments require additional infrastructure, and instructors should implement these methodologies as appropriate.
Micropipettes are an essential tool in many fields and learning to use them is a fundamental part of a scientist's training. I have developed Training Tips, a homemade tool to teach pipetting to novice bench scientists. Training Tips provide obvious visual marks by which one can monitor pipetting accuracy and are a cheap, convenient tool to guide micropipette instruction in the classroom and laboratory.
Tips for Accurate Pipetting
Provided below are general tips for accurate pipetting. Depending upon the brand of pipette tip and pipette being used, recommended techniques may vary slightly. Always check with manufacturer for best practices, and check with your instructor for recommendations on how to handle the pipette.
Pipettes have particular ranges of volumes in which they are accurate. For a given volume you should choose a pipette in which the desired volume falls within the pipette's range. (The closer to the middle of the range the better. For instance, to pipette 100 μl, you would use a P200 as opposed to a P1000.)
Make sure to press the tip firmly onto the pipette, because a loose tip will not draw the proper volume of liquid.
Slowly depress the plunger before submerging the tip into the liquid.
Submerge the tip 3 to 4 mm into the liquid. The more you submerge, the more liquid may stick to the outside of the tip. (Make sure the tip remains submerged as liquid is brought into the pipette.)
Slowly release the plunger of the pipette after the tip has been submerged. Slowly releasing the plunger will limit the amount of air bubbles you draw into the tip.
Hold the pipette vertically when moving and dispensing liquid.
Hold the tube you are pipetting into at an angle (~45 degrees) and pipette either into the liquid in the tube or onto the side of the tube.
Training Tips Exercise
Display proper pipetting technique.
Correctly identify which pipette is appropriate for different volumes.
Accurately pipette μl volumes of liquid.
Exercise: Details of the exercise may vary depending upon the number of students and lab infrastructure available. (It is important to have accurately calibrated pipettes to perform this exercise.)
After reviewing Tips for Accurate Pipetting, obtain a set of Training Tips, pipettes, 1.5 ml tubes, and 20 ml H2O.
Set the P1000 to 1000 μl and firmly insert the P1000 tip marked at 1000 μl. Depress the plunger and lower the pipette such that 3–4 mm of the tip is submerged. Slowly release the plunger, drawing the H2O into the pipette. If performed correctly, the H2O will reach but not go past the marked line. Depress the plunger to dispense the H2O into the 1.5 ml tube. (Do not have someone else hold the 1.5 ml tube for you. Either hold the tube yourself or use a tube rack.)
Using the P1000 with the 1000 μl marked tip, remove the H2O from the 1.5 ml tube and dispense back into the 20 ml of H2O. (All of the H2O should be removed from the 1.5 ml tube and expelled from the tip.)
Repeat steps 2 and 3 with the other P1000 Training Tips, taking note of the changes in volumes between tips. (Make sure to replace the Training Tips in the correct slots in the box.)
Repeat steps 2 and 3 with the P200, P20, and P10, using the appropriate Training Tips.
Using Training Tips, pipette 1 μl of H2O into a 1.5 ml tube. In a separate tube, using standard tips, pipette 1 μl of H2O into a different 1.5 ml tube. Compare the volumes of liquids in the tubes.
Repeat step 6 with all P10 and P20 training tips.
Assessment: Using a non-labeled pipette tip, pipette 10 μl, 100 μl, 200 μl, or 1000 μl of H2O into clean 1.5 ml tubes. Weigh these tubes using an analytical balance to determine how accurately you have pipetted. Continue until you can consistently pipette these volumes accurately.
Which pipette should be used to most accurately pipette 1 μl, 10 μl, 100 μl, and 1000 μl.
True/False: When pipetting liquid, you should release the plunger quickly to draw up liquid.
Multiple choice: When pipetting you should
submerge the tip 3–4 mm.
press the tip firmly into the pipette.
hold the pipette vertically when dispensing liquid.
all of the above.