1 Introduction to Micropipettes

Learning how to use micropipettes

Micropipettes are used to transfer small volumes of liquids, typically less than 1mL. They have been used for over 60 years in many fields of science including molecular biology, genetics, microbiology, and chemistry. As a budding scientist, it is important for you to learn and master the art of micropipetting. The scales on micropipettes are in microliters (1000 µl = 1 ml).  The three most commonly used sizes are p20, p200, and p1000. The size of the micropipette can be identified by the number on the round button on the plunger. The value is the maximum volume in microliters that can be transferred with that size pipette. They are used in conjunction with disposable sterile plastic tips.

Your instructor will demonstrate how to use the micropipettor using the process below:

1. Determine the volume required by twisting the thumbwheel dial to the required volume. Note that numbers are read from top to bottom. There may be a red line indicating a decimal point in the display. For example, 015 with a red line between the 1 and 5 would read 1.5 µL.
2. Attach (seat) a clean pipette tip by gently pushing the micropipette into the top of the tip. Be gentle removing the tip to avoid spilling the whole box.
3. Press the push button (plunger) on top to the first (soft) stop. Insert the disposable pipette tip (only the tip) into the liquid to be transferred. Slowly release the push button to suck up the liquid. Note—If you have pressed the push button too hard and gone to the harder blow-out setting you will pull up more than the desired volume.
4. Insert the pipette tip into the microfuge tube or other area where you need to move the material.
5. Press the push button past the first stop to the second (hard) stop to transfer (blow out) the liquid. Do not release the push button until you have removed the tip from the tube.
6. Eject the disposable tip into the appropriate waste container. Do not reuse tips, unless instructed, to avoid cross contamination.

Conducting an experiment to measure density of various liquids

As we learn how to use micropipettes to measure small volumes and an electronic balance to measure mass, we will also think through the scientific process to develop a hypothesis, differentiate between an independent and a dependent variable, graph and interpret results and make a conclusion about our hypothesis.  We will determine the densities of six different liquids which are:

1) water (H₂O), 2) vegetable oil (C₅₇H₁₁₀O₆), 3) ethanol (C₂H₆O), 4) 1-octanol (C₈H₁₈O), 5) 20% glucose (C₆H₁₂O₆), and 6) 20% sodium chloride (NaCl).

Exercise 1: Designing the experiment

1. Your instructor will show you 6 different liquids, talk with your lab group about these 6 liquids and develop a hypothesis about the density of these liquids. Which liquid do you think is least dense? Which liquid is most dense?
2. Write down a hypothesis to test for your experiment. Your hypothesis should be a statement you will test in an experiment. Here are two examples of hypotheses: 

Sodium chloride solution is more dense than water.

1mL of ethanol will weigh less than 1mL of water.

Exercise 2: Measuring mass of a liquid

Your instructor will provide you with one of the 6 liquids and 5 Eppendorf tubes. Labeled the tubes A, B, C, D and E, with the marker provided.

1. Weigh each tube on the balance and record the mass in the “Mass of empty tube” column of the data table. Enter the mass in grams to the nearest 0.01 g.

Name of liquid:

2. Use a micropipette to transfer the volume of your liquid into each tube. Each person in the group should try transfering the liquid at least once. This will require proper technique since many of the liquids will be viscous.  Hint: do not coat the outside of the pipette tip with the liquid and slowly push and release the plunger.  Close the lid of each Eppendorf tube after you have added the liquid. 

3. Weigh each filled tube on the balance and record the mass in the third column of the data table “Mass of filled tube”.

4. Subtract the mass of the empty tube from the mass of the filled tube to calculate the mass of the liquid.  Record in the 4th column of the table “Mass of liquid”.

Mass of liquid = mass of filled tube – mass of empty tube

Exercise 3: Calculating density of a liquid

Volume is a measure of how much space matter occupies, and it is used to calculate density ( d ),

d = m/v

where m is the mass of the object and v is the object’s volume. Predict whether the density of the tubes labeled A,B,C,D,E will be different based on the formula above. Do you expect the density of the liquid to be the same or different between tubes A – E?  Write your prediction down.

1. Graph the relationship between the volume and the mass of the liquid for each of the 5 tubes. Label the axes of the graph with the variable name and unit of measurement. The independent variable should be represented on the X-axis and the dependent variable on the Y-axis.   

Example: In an experiment testing the effect of sunlight on tomato plants, the amount of sunlight would be the independent variable and your measurement(s) of growth would be the dependent variable(s).