Using a pipette is one of the first skills you'll learn in a biology or chemistry lab class. It might seem easy, but it's important to get it right because you'll use a pipette in many of your experiments, so if you consistently employ bad technique, it could ruin many of your results. There are three kinds of pipettes typically used in labs: Pasteur pipettes, volumetric pipettes and micropipettes. Volumetric pipettes are more common in chemistry laboratories, while micropipettes and Pasteur pipettes are indispensable in molecular biology and biochemistry labs.
Using a Volumetric or Pasteur Pipet
Look at your volumetric pipettes. Notice a number and a line or mark on the side of each. The number indicates the number of milliliters the pipette holds or dispenses when the pipette is filled all the way to the line or the mark. Volumetric pipettes are calibrated to have a very high level of accuracy, so when you dispense a certain volume with a volumetric pipette, you can report that volume in your notes with up to two figures after the decimal point (e.g. 5.00 mL).
Notice that your volumetric pipette is long and narrow except for a swollen reservoir in the middle, typically not far below the fill mark. When you suck fluid into the pipette with the rubber bulb, the fluid level rises more slowly in the reservoir than in the tube above or below it.
Add some water to the beaker so you can use it for practice. Place the rubber bulb (which looks a little like a turkey baster) at the top of the pipette and squeeze it to empty it of air. Then, with the tip of the pipette submerged in the water, gently relax the bulb to draw water up into the pipette.
Allow the level of the fluid in the pipette to rise a couple centimeters above the line or mark on the side. While you are drawing up fluid make sure the the tip of the pipette always remains beneath the surface of the fluid. Do not allow the fluid to rise up into the bulb itself.
Remove the bulb and quickly cap the open top of the pipette with your finger. By tilting your finger to one side, allow a little air into the pipette so the fluid drains out until the bottom of the meniscus (the curve-shaped depression in the top of the fluid) reaches the fill mark or line.
Remove the pipette from the reagent solution and transfer it to the receiving beaker or flask. (If you're just practicing with water in a beaker, you can use the same beaker as reagent and receiving vessel.) Allow the pipette to drain into the receiving beaker or flask.
Take out your Pasteur pipettes if you have or are using them and examine them. Pasteur pipettes are not designed to measure out a specific volume; you can use them to add drops of a reagent or an indeterminate amount of a reagent, but do not use them if you need to know exactly how much reagent you are adding — for that, you should use a volumetric pipette or micropipette.
Fit a rubber bulb on the top of the Pasteur pipette. Squeeze the bulb to eject air from the pipette and submerge the tip in the reagent solution (or water in a beaker for practice).
Gently relax the rubber bulb to suck fluid up into the Pasteur pipette. Do not allow fluid to rise all the way into the rubber bulb.
Transfer the Pasteur pipette to the receiving beaker or flask and squeeze the bulb gently to eject drops of the solution into the receiving flask.
Rinse volumetric pipettes and Pasteur pipettes following their use. Pasteur pipettes are often used as disposables, especially in biology labs where they may be contaminated with biological material; follow your lab's guidelines on how to work with or dispose of these items.
Never, ever, draw fluid up into a Pasteur or volumetric pipette by sucking on it with your mouth. Some chemists and biologists did this "back in the day," and predictably enough it sometimes caused serious accidents.
Examine your micropipette. At the top it has a plunger that you can push in to empty the micropipette; next to the plunger is an ejector you can use to eject the plastic tip from the end of the micropipette. Along the side, it has a volume adjustment wheel you can use to adjust the volume the pipette will take up or contain.
Look at the volume dial along the side of the micropipette. Micropipettes measure volumes in microliters. Determine what the volume is set to at present and adjust that volume with the volume adjustment wheel to reach the appropriate or desired volume.
Insert the end of the micropipette shaft into one of the plastic tips in your plastic tip box. Do not handle the plastic tip with your fingers.
Depress the plunger with your thumb until you reach the first stop.
Insert the plastic tip of the pipette just below the surface of the fluid or water in your beaker.
Release your thumb pressure on the plunger, slowly and gently, drawing fluid into the plastic tip of the micropipette. Once the plunger has traveled all the way out, remove the pipette tip from the solution.
Transfer the pipette to the receiving vessel/beaker/microfuge tube and place the tip just below the surface of the fluid in the receiving vessel. Do not submerge it completely.
Depress the plunger slowly and gently to expel all the fluid in the micropipette tip. This time, continue to apply pressure past the first stop until you reach the second stop.
Remove the pipette tip from the solution. Then release your thumb pressure on the plunger of the pipette.
Follow your lab's protocol for disposing of the micropipette tips.
- California State University Dominguez Hills: Helpful Hints on the Use of a Volumetric Pipet
- "Biochemical Techniques, Laboratory Manual"; Aaron Coleman, et al.; 2010
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