Part 1: Overall pipette function, Volume selection and use: Technique and accuracy.
15-20 minutes
Use Pipette Technique PowerPoint
Note: The PowerPoint includes photos of proper use and technique for students to reference
Pipette Technique:
- Making sure you have good technique when using appropriate tools is one way you can increase your accuracy. The same way you would shoot free throws in practice to ensure you are ready for the big game, in lab work you want to make sure your skills are polished and ready! Practice, practice, practice…
- Have students practice the soft and hard stops on the micropipette. Set the volume to 25 ul and practice, paying special attention to how far the students must move their thumb to reach the soft and hard stop. Now move to the 50 ul volume and repeat. Note the difference in the distance your thumb must move in order to draw up and expel the different volumes.
- Set pipette to 25 ul, practice drawing up a sample. Depress to the soft stop, place tip of the pipette into the solution, being careful not to touch the sides or the bottom of the container, release your thumb allowing the plunger to move back up, drawing the solution into the pipette tip. Note the amount of liquid in the tip, and the absence of any air bubbles.
- Over the waxed paper, expel the sample. Depress the plunger all the way to the hard stop. Hold the hard stop as you bring your hand and the tip of the pipette above the small sphere of fluid on the waxed paper before releasing the plunger. If you release the plunger before removing the tip from the fluid, you will draw the fluid back into the pipette.
- Repeat this process multiple times so you can become comfortable with the proper technique. If you find you have air bubbles in the bottom of the pipette tip, expel the sample back into the cup and try again. Make sure you are using proper technique!
- Change the volume to 50 ul and repeat the process above in an area at least 2 squares away from your first sphere. Note the difference in the size of the sphere relative to your first sample. This sphere should be approximately twice as large since you are selecting to use twice the volume.
- Repeat these steps as many times as needed to become comfortable with proper technique.
- Using your pipette combine two spheres of sample, one 25 ul and one 50 ul, by placing the tip of the pipette into one sphere and carefully dragging the sphere toward the other sample until the two spheres merge.
- Adjust the volume of the pipet to 75 ul, try to draw up all of the sample in this now merged sphere. If you accurately used your pipette with proper technique, you should be able to take in all of the sample without having bubbles at the end of your pipette tip.
- Continue to practice this skill, changing volume on the pipette and combining bubbles to draw up larger samples until you feel sufficiently comfortable.
- NOTE: This is also a possible lab practicum assessment possibility, have students repeat this process in your process to ensure the proper technique is being use.
Part 2: The right tool for the job?
20-30 minutes
Scenario: You are in a lab setting with very limited access to lab equipment. You have been tasked with the job of pipetting 2 ml of sample onto a surface. You only have two tools to use, a micropipette, with variable volume selections, and a scale. Your job is to figure out what setting to use in order to pipette the 2 ml of sample with both accuracy and precision.
- Step 1: What should your volume setting be?
- You know that your pipette can choose sample between 20 and 200 ul. You know that 1000 ul = 1 ml so…. How many ul would you need to measure total? (1000 ul (2 ml) = 2000 ul)
- How can you measure that total using this one instrument?
- Possible options:
- 10 samples of 200 ul
- 20 samples of 100 ul
- 16 samples of 125 ul
- 40 samples of 50 ul
- 80 samples of 25 ul
- You decide that you will test 3 sample sizes to determine the precision of your equipment. (Note: Not all equipment is precise at all volume.)
- Step 2: You have no other way to check your overall measurement in terms of volume but you know 1 ml of water = 1g, so you can use the scale!!!
- Your total value should be 2 g of sample when weighed using the scale. (Note: the scale has been recently calibrated and you know for certain is quite precise and accurate.)
- Place the waxed paper onto the scale, tare or zero your scale if it is registering any weight with only the waxed paper.
- Now pipette the appropriate number of samples onto the waxed paper. As a good scientist you are going to take this opportunity to make sure your technique is flawless! So you will work to make sure you are being precise with your sample placement by not allowing the sample bubbles to touch one another.
- Note the weight in g for this trial of your data.
- Step 3: Calculate your accuracy using the % error formula. This is one way to use a mathematical model to see how far away you are from “the bullseye”.
- %error = 100 (measured weight – expected weight) / expected weight
- The smaller % Error the better!
- Compare your % errors for each of the sample volumes you completed in Step 2. Make an assessment about which volume selection you feel you can pipette with the most accuracy.
- Step 4: Check your precision or % CV (Coefficient of Variation). This is one way to use a mathematical model to see how far away your shots are from one another.
- Using the volume you felt would be the best suite in this case, check your pipette’s precision. We now have to see if you have repeatable data!
- Repeat the process of Step 2, 10 more times, remembering to record the overall weight at the end of each trial.
- This should not take more than 2 minutes per trial when choosing the larger volumes.
- If using waxed paper make sure you are using a new piece of waxed paper for each trial so you are not inadvertently creating error with water that has soaked into the paper.
- Use the data you collected to complete a statistical analysis.
- Input the trial measurements into a Microsoft Excel file in one column.
- At the bottom of the column type in the following formula:
- =STDEV(A2:A13)/AVERAGE(A2:A13)*100
- Adjust the cell tags if necessary.
- You are simply using Microsoft Excel’s short cut for determining the standard deviation for a set of data. This is an example of a mathematical model that we can use to assess our data.
- The closer your calculated value is to a 0 value, the more precise you are in your measurements.
- Repeat this process with the other volumes you selected from Step 2.
PART 3: Analysis and Decision time:
20-30 minutes
- Using your mathematical models determine what is the optimal volume setting for this particular task.
- Why would having a variable pipette be a good thing? How does it affect your overall % error if you are completing less samples for one total volume?
- What are some things that could affect your overall accuracy in this lab?
- How could you control these variables?
- What are some things that could affect your overall precision in this lab?
- How could you control these variables?
- Why is it important for scientists to ensure their tools are correctly calibrated?
- In major seed development labs, where precision and accuracy can be vitally important, most pipetting is automated and controlled via computer. How would this increase the accuracy and precision of their labs? How would that impact the quality of their overall product?
Note: Some students have difficulties steadying their hand when using pipettes. As precision in sample placement is part of proper technique, it is important to go slow and provide plenty of time for student to practice before any lab practicums or skills assessments. One possible aid to students is to place both elbows on the table while grasping the pipette in both hands during use. This technique allows for more stability by providing support for the arms and wrists.