The Scientific Method: Observation

How do I start?

Observation is the key tool of a scientist. Observation helps a researcher to identify promising aspects of natural phenomena that are worth knowing about. The scientist is specifically looking for causal relationships in nature that (taken together with other knowledge) will help to explain in the broadest terms how natural systems work. The outcome of any observational process will be the formulation of one or more research questions (RQ’s). RQ’s are (at some level) guesses about the biological principles operating in a given situation. 

Today’s exercise will be about the observation of Beta vulgaris L., the common garden beet. It contains a reddish pigment called betacyanin. Betacyanin is found deep in the cells of the beetroot in a cellular organelle called the vacuole. 

• Examine the beet that you have available at your table. Cut some cylinders of beet tissue using the cork borer. What was the result? Can you think of other types of treatments that might also cause betacyanin to leak from the root? Try out as many of these ideas as you have time to do. Make notes about your observations. As a hint to get started, we have available in the lab some solutions of ethanol in water. You may observe that certain treatments will cause betacyanin to leak from the beetroot, whereas others will not. You may also note different rates of leakage with specific treatments.
• As you pose your RQ’s, remember that you are looking for ideas that lead to an understanding of causal relationships in nature. Put another way, you are trying to learn something about why betacyanin normally stays in the root by considering the results of treatments that will cause it to leak out!
• Avoid posing RQ’s such as, “Will blows with a sledge hammer cause betacyanin to leak from the beetroot?” Only RQ’s with sound reasoning behind them (what scientists call the rationale) are worth pursuing. A much better question might be, “Will ethanol cause betacyanin to leak from the beetroot faster than water and why?” 

Visual estimates of betacyanin leakage from beetroots, although useful, are only the first step in our investigation. We also need to quantitatively measure how much of this pigment is present in a given sample. Our method for doing this is spectrophotometry, a technique that estimates how much betacyanin is present by determining how much light it absorbs. Before we can do quantitative measurements with the spectrophotometer, we first need to know what wavelength of visible light betacyanin absorbs most strongly. This wavelength is called the λ_max. 

Directions for Measuring Betacyanin with a Spectrophotometer and Determining λ_max

1. Turn on your spectrophotometer using the left hand knob on the front.
2. Wait 10 minutes
3. Get two cuvettes. Label one tube “S” for sample, and the other “B” for blank.
4. Put 5 ml of ethanol in the “B” tube and 5 ml of betacyanin solution in the “S” tube.
5. Using the left knob adjust the needle until it reads infinity on the lower scale. This will be toward the left of the bottom scale on the display.
6. Set the wavelength knob to 500 nm.
7. Get the “B” tube and place it in the cuvette holder. Using the right hand knob, adjust the absorbance to zero. This will be towards the right of the display.
8. Remove the “B” tube and put in the “S” tube. Record in your notebook the indicated absorbance on the lower scale.
9. Take out the “S” tube and recheck that the needle still is on infinity (step 5 above).
10. Adjust the wavelength to 510 nm.
11. Repeat steps 7-9 until you reach 650 nm.
12. Plot the results on graph paper with absorbance (dependent variable) on the “Y” axis and wavelength (independent variable) on the “X” axis.
13. Identify the wavelength that exhibits the highest absorbance. This is λ_max for betacyanin.