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Appendix 3: Introduction to Micropipetting

  • Page ID
    16061
  • When scientists need to transfer small volumes of liquid, they use a piece of equipment known as a micropipettor (or pipettor). There are a few different types of micropipettors based on the minimum and maximum volumes of liquid they are designed to transfer. Today you will learn how to use a micropipettor to a) transfer 1 ml (1000 µl) volumes of liquid in the preparation of serial dilutions, and 2) transfer 0.1 ml (100 µl) of these dilutions onto agar plates.

    A. How to set the micropipettor for the appropriate volume:

    The picture of the micropipettor shown below is similar to the one you will be using. As you can see, this micropipettor is designed to transfer volumes between 100 µl -1000 µl (this type of micropipettor is sometimes called a P1000). The display window reads “1000” when the pipettor is set to its maximum volume (1000 µl or 1 ml). If we were using this same pipettor to transfer 500 µl (or 0.5 ml) the window would read 500.

    pipette.png

    Figure 1: P1000 micropipettor

    Similarly, the pipettor shown below (a P200) is designed to transfer volumes between 20- 200 µl (0.02- 0.2 ml) It is set at 50 µl —to pipet 100 µl you would set the display to read ‘100’.

    pipettor2.png

    Figure 2: P200 micropipettor

    The volume is set by turning the knob at the top of the pipettor until the correct volume is reached.

    Note

    Never try to turn the knob above the minimum or maximum volumes or you may damage the micropipettor!

    B. How to attach a pipet tip:

    After setting the appropriate volume, you will be attaching a pipet tip to the narrow end of the micropipettor. The P1000 uses blue tips; P200’s use clear or yellow tips. The tips have been sterilized in the autoclave so as not to introduce any contaminating microbes into your sample. To pick up a tip:

    1. Open the box of tips and insert the end of the pipettor into the end of a pipet tip.

    2. Press down gently to ensure that the tip stays attached to the micropipettor.

    3. Close the box to prevent the other tips from getting contaminated.

    C. To transfer liquid using the pipettor:

    Note

    Follow these steps very carefully!

    1. With your thumb, depress the plunger on the top of the pipettor until you first feel resistance.

    Note

    It is important not to go past the first stop. If you do, you will picking up too much liquid!

    2. While holding the plunger in the depressed position, place the pipet tip into the liquid you will be transferring.

    Note

    If you depress the liquid after placing the tip in the liquid you will be blowing air into the liquid!

    3. Slowly release the plunger to allow the tip to pick up the liquid.

    Note

    Be sure to keep the tip in the liquid the entire time so that you don’t get air bubbles.

    4. Place the tip into the tube (or plate) where you will be transferring the liquid to.

    5. Slowly press the plunger down again until all the liquid leaves the pipet tip.

    6. Withdraw the tip from the tube before releasing your thumb from the plunger.

    7. Use the tip ejector to discard the tip in the container as instructed by your professor.

    Note

    Pipet tips NEVER get disposed of in the regular garbage.

    Other important things to remember:

    1. It is important not to let the pipet tip touch anything other than the liquid you are transferring.

    2. Always keep the pipettor in the upright position when in use. If you tilt it too much the liquid in the tip will make contact with the pipettor and will no longer be sterile (this could also damage the pipettor).

    Practice:

    Set your P200 pipettor to 100 µl and practice pipetting by transferring the liquid onto a piece of parafilm. Let your lab partners try as well. All droplets should be the same size (and the same size as your instructor’s). Try transferring different volumes as instructed until you feel comfortable using the pipettor.