Working with a Pipette

Lab 1 - Working With A Pipette

Introduction

Pipettes are essential tools used to measures specific volumes of solutions accurately and effectively. The user-friendly design of pipettes allows biologists, engineers, and chemists to easily move a solution from one vessel to another, making sure that the volume of solution needed is accurate. Pipettes are meant to be used with disposable tips that are designed for specific pipettes based on the maximum volume of solution that pipette can handle. Tips are changed in order to maintain sterility, although they can be reused if extracting from the same solution. One of the most important aspects of pipettes is making sure they are properly calibrated so that they are indeed picking up the amount of working fluid that they are supposed to.

The objectives of this lab report were to become competent in using a pipette by properly extracting/disposing solutions and to understand the importance of using an accurate pipette by analyzing the errors associated with pipettes. This laboratory procedure was divided into three parts, which allowed for learning to properly use a pipette and measuring volumes of working fluid in the correct manner. It is expected that errors will occur throughout the process, which will lead to inconsistent results and indications of improper use.



Materials and Methods
Lab 1 was performed in three sections in the following manner, with Part 1 focusing on the mechanical use of the pipette and Parts 2 and 3 focusing on the measurement of fluid using the pipette: 

• Part 1 was conducted using a 100 μl Eppendorf micropipettor. The top knob on the pipette was rotated in order to set the volume of solution needed. A disposable tip was then attached to the end of the pipette in order to extract the solution. The plunger on the top of the pipette was pressed and held at the first stop point, as pushing it any further would contaminate the pipette and any solution to be sued after. The tip of the pipette was then inserted into the red food coloring solution, perpendicular to the surface of the solution to prevent air from forming bubbles inside the tip and reducing the amount of solution retrieved, and the plunger was subsequently released. The solution inside the pipette was then moved into a beaker, where the plunger was pushed to the first stop point to evacuate the fluid from the tip AND then to second stop point to "send a puff of air to purge the system completely of fluid". This was performed multiple times until the process for transporting solution with a pipette was understood and performed correctly.
• Part 2 was conducted using a 100 μl Eppendorf micropipettor. A saran wrap was used as a final destination for the samples of food coloring that would be retrieved. 20 μl of red,  25 μl of yellow, 10 μl of blue, and 15 μl of green food coloring were placed on top of each other, in order to form a single drop.  The volume of the combined food coloring drops was recorded, as well as the color of the drop itself. The pipette was then reset to retrieve the recorded volume to determine whether the pipette measured the volumes correctly. 
• Part 3 was performed using a 1000 μl micropipettor. Two test tubes were also used to contain two food coloring mixes. The first mix contained 200 μl and 300 μl of red and blue food colorings, respectively; the second test consisted of  250 μl of green and 200 μl of yellow food coloring. The final color and volume in each tube was recorded.The 1000 μl micropipettor was adjusted to the total volume in each test tube, to make sure the pipette was calibrated to the right volumes. Calibration was verified by determining if the micropipettor was able to extract all of the food coloring in each test tube.

Results and Discussion
Since Parts 2 and 3 depended on whether or not we can properly and effectively use the pipette, it was imperative that we mastered Part 1 of this lab. Many trials were performed in order to perfect the use of the pipette, which involved adjusting the angle of extraction and the use of the first and second stop positions. Figure 1 below shows the results of the three experimenters successfully using the pipette.

Figure 1. (Left to Right) Sebastian Arbelaez, Jose Castano, and Jabari Lee using a micropipettor

Part 2 heavily relied on the accuracy of the pipette to determine the amount of food coloring extracted. It was recorded that 70 μl of food coloring were used, which led to it black-colored appearance, but it was observed that the pipette left more that 25% of the food coloring still on the saran wrap, as can be appreciated in Figure 2, shown below.

Figure 2. Food coloring solution before (left) and after (right) extraction

A source of error observed by all three experimenters was that the level of fluid dropped after the plunger was completely released, indicating that air was getting into the disposable tip from the top. This means that the seal between the pipette and the disposable tip was not airtight, leading to fluid being pushed out of the tip. Other sources of error that may contribute to the inaccuracy of the pipette in retrieving the solution are:

• the improper use of the pipette (which would include not holding the pipette perpendicular to the surface of the solution and using the plunger incorrectly),
• an improper pipette calibration,
• residue of solution left in the pipette tip (leading to extra solution being added to the final drop), 
• and using the pipette too close to the saran wrap (this would cause the saran wrap to cover the tip, preventing the fluid from being extracted). 

Due to these factors of error, calibration of the pipette and the use thereof are crucial for obtaining accurate measurements during the procedure, and therefore, more accurate results in the end.


Figure 3. Food coloring solution Test Tube 1 (left) and Test Tube 2 (right).

Part 3 followed the same practice of Part 2, with the biggest differences being the use of a larger pipette and greater volumes to be retrieved. Figure 3 depicts the final solutions in test tubes 1and 2 before the pipette was reset in order to extract shown amount of solution from each test tube. 

It was observed that this pipette was calibrated much better than the 100 μl pipette used in Part 2; the 1000 μl pipette picked up nearly all of the fluid in both test tubes 1 and 2. It was also observed the colors within test tubes 1 and 2 were dark blue and green, respectively. Because the 1000 μl pipette performed the task of recollecting nearly all of the fluid in both cases, it was determined that the error was minimal, and that the use of the pipette was properly executed.