Chapter 11 Methods

The Methods give readers a general idea of procedures you used to test your hypothesis.

These are the questions your should try to answer in this section.

Briefly, how did you do your experiment? What are the essential details that someone else would need to know to repeat it?
If appropriate, where and how were the organisms collected and maintained? If you used materials extracted or obtained from organisms, what was the source?
If you used an unusual method to create the independent variable, describe how you did it. If you used commonly available methods, state them without explanation.
What volume and concentration of key reagents (drugs added, volumes injected, etc.) were used in the experiment?
What statistical tests did you use? What groups were compared?

11.1 Be Careful With Details

Many of our students writing their first lab report have trouble writing this section. It is hard to judge how much information is needed. There should be enough detailed information provided so that someone who is not directly involved in the project (but works in a lab setting) could repeat the experiments.

At the same time, we do not want them to just recopy the lab manual or protocol or add useless details. Examples of TOO much detail are “…I used a paint brush to place a red dot on each grasshopper…” or “…we graphed all twelve of our data points on an X-Y graph…”

You want to summarize your methods, use diagrams, and find other ways to make your reader understand how you did your studies. Look at articles you have read; what did they keep or leave out?

11.2 Other Tips

The Methods section describes something you already did in the past, so should be written in past tense. Usually it is written without saying “we” or “I”, although this is becoming less important in some fields.

Usually the methods you use are well established so you do not need to cite a source for the procedures. The exception is if you change a well-known method because of something another study found or reported; in this case it is appropriate to cite the source for the facts that made you change your methods from what is done normally.

11.3 Examples of Poorly Written Methods Sections

11.3.1 Example 1

This particular example has too much detail. It reads just like our laboratory manual, and in fact most of it was copied directly. So it is both poorly written AND plagiarized.

In order to test how differing temperatures affect chloroplast activity in spinach leaves we first collected enriched chloroplast samples. In order to do this we removed the large central veins from 4 medium-sized spinach leaves and washed them with cold water. We then added the leaves and 10 ml of 400 mM sucrose in Tricine buffer (grinding buffer) to a mortar and pestle and grinded the spinach until it became a thick paste. Following this, we added an additional 10 ml of grinding buffer to the paste and strained it through three layers of cheesecloth into the chilled beaker. Next, we transferred the liquid to a chilled 50 ml centrifuge tube marked “A”, and spun it in a centrifuge at 200 x G for 3 minutes at 4°C. After, we poured the supernatant out of the tube marked “A” into the second chilled 50 ml centrifuge tube marked “B”. We then centrifuged the 50 ml tube “B” at 1,000 x G for 10 minutes at 4°C. Next, we poured the supernatant, into a clean 50 ml tube marked “C”. Finally, we add 20 ml of grinding buffer to the pellet at the bottom of tube “B” and suspend it in solution. In a previous experiment we determined that “sample of pellet B” contains the enriched chloroplasts because it had the highest chloroplast to debree ratio. As a result, the chloroplasts from “sample of pellet B” are what we used in our samples for our chloroplast activity assay. We started the chloroplast activity assay by numbering 13 test tubes “blank” and “1-12”. We added 8 mL of water, 2 mL of reaction buffer, and 100 microliters of enriched chloroplasts to the “blank tube”. In tubes 1-12 we added 6 mL of water, 2 mL of DCIP, 2 mL of reaction buffer, and 100 microliters of enriched chloroplasts. Next, we mixed each tube and added their contents to individual cuvettes. Following this, we used the blank tube to blank the spectrophotometer. We then recorded the absorbance of each sample. These absorbance values served as our baseline measurements (0 minutes). We then place samples 1-3 in ice water (0°C), samples 4-6 in room temperature water (23°C), samples 7-9 in 40°C water, and samples 10-12 in 55°C water. We recorded the absorbance of each sample every ten minutes for a duration or thirty minutes. We then calculated the percent change for every absorbance measurement using the formula % change = ((absorbance at time zero - absorbance at time N) / absorbance at time 0) x 100. Following this, we plotted the change in absorbance against time for each temperature group. Finally, we used a one way ANOVA test to determine if there was a statistically significant difference in chloroplast activity between any of the temperature groups.

This example also has numerous misspellings and poor word choices. Some examples:

grinded (should be ground)
“Finally we add 20 mL” (should be in past tense)
“a duration or thirty minutes” (should be “of”)
The statistical analysis is incomplete. The text reads: “Finally, we used a one way ANOVA test to determine if there was a statistically significant difference in chloroplast activity between any of the temperature groups.” ANOVA can tell us if any of the groups are different from each other, but it cannot tell us which groups differ. The author needs to include results of a post-hoc test.

11.3.2 Example 2

This is an example of not providing enough detail. This is the entire methods section, not an excerpt.

For three dissected frog legs, electrical probes were applied to the gastrocnemius muscle, and the muscle was attached with string at one end to a force sensor. The electrical probes and force sensor were used to collect data on the threshold for producing notable muscle contractions in each frog’s gastrocnemius under various voltage stimuli. Once each muscle’s threshold had been determined as a control, each gastrocnemius was injected with Lidocaine, and the new thresholds were determined. Each muscle was also rinsed in Ringer’s solution to prevent changes in threshold resulting from deficient salts or variables other than the sodium ion concentration. After each gastrocnemius’s thresholds were found, a t-test with a P value of 0.05 was performed to determine if a significant change had occurred.

What is missing? What needs revision?

What equipment is being used? What voltage is being applied? How is threshold determined?
How much lidocaine is being injected, and at what concentration?
The statistical analysis is not the right one to use, and it is not described correctly. The data should be analyzed using a paired t-test, and the P value should be the alpha value (P value is calculated for the dataset; alpha is the pre-determined point where data are considered significant.) What groups were compared?

11.3.3 Example 3

This author has focused on the wrong details, and left out essential information. Again, this is the entire methods section, not an excerpt. Spelling and grammatical errors have not been corrected.

The materials that were used was soil with either nitrogen or without nitrogen, as well as pots, water, field pea seas, and time. The seeds were planted in their respective pots, either with or without nitrogen. The seeds were then planted in their pots and left to grow over two weeks, with one member of the group going to the greenhouse every other day in order to water the plants and check on their growth. After the two weeks were over, the plants were collected and the roots/shoots were taken out of their pots, cut apart, and measured using a ruler (cm). The data was recorded and the waste was disposed of.

What is missing? What needs to be revised?

The first sentence is not needed; we do not need a list of materials.
The second and third sentences need to be combined and revised to be clearer.
What does high and low nitrogen mean? (There was no option to use nitrogen-free soil.)
What size pots, and how many field pea seeds per pot?
How exactly were plants cut apart? If they were measured with a ruler, why cut them apart?
What data values were recorded, and how were they analyzed?

11.4 Examples of Well-Written Methods Sections

11.4.1 Example 1

Four, 10 x 10 cm x 3 cm deep black plastic nursery containers were filled with vermiculite, then pre-wetted by soaking from the bottom until the vermiculite was saturated. Containers were allowed to drain, then each was flushed with 50 mL of distilled water. Two of 4 containers were watered with 50mL of 1x MiracleGro plant food prepared according to manufacturer directions. The other two containers were watered with 50 mL of distilled water. Two of the containers (one container with plant food and one with water only) were then labeled to denote that they were to be treated with salt water, while the other two were labeled to receive tap water. Approximately 100 buckwheat seeds were planted in each of the four containers, and covered with 1 cm of pre-soaked vermiculite. All four containers were placed in the greenhouse to ensure similar humidity, temperature and light exposure. The four containers were watered every other day for 2 weeks with either 50 mL of 3% NaCl in water or 50 mL of tap water.

After 2 weeks, 5 individual seedlings from each container were gently uprooted (keeping the entirety of the plant intact). The length of the entire sprout was measured in centimeters and weighed in grams, then the sprout was cut at the top of the root, where it meets the stem, and the lengths of the root and shoot were measured individually. Finally, weights of the roots and shoots were measured for each seedling. Root:shoot length ratios and weight ratios were calculated for each individual seedling, then the average ratios for the control and treatment groups were calculated. A one-tailed, two-sample t-test was used to compare the average overall lengths and weights, and root:shoot ratios.

What Is Particularly Good?

The author clearly explains how the test conditions were created, and how they standardized the four containers.
The study treatments are explained without too much unneeded detail.
How the data were collected and analyzed is clear.
None of the sentences could be removed without leaving out an important part of the methods.

11.4.2 Example 2

Three frog legs were used for this experiment. Each leg served as its own control and experimental replicate. Legs were skinned, and the inner thigh muscles were separated to reveal the sciatic nerve that would be stimulated. The gastrocnemius muscle was then freed from the attachment at the ankle, and suspended by a string from an AMI-200 force transducer connected to an iWorx TA201 data interface. Raw data on force generated by leg muscle contractions was recorded, processed, and displayed using the iWorx LabScribe program.

To determine baseline, a stimulus was applied to the sciatic nerve of each frog leg, starting at 0.0 V and increasing by 0.1 V intervals until a threshold voltage for contraction was reached. This threshold voltage plus another 0.1 V was the final stimulation voltage for that particular leg.

To measure control contractile activity, each leg was stimulated through the sciatic nerve with the final stimulation voltage three times, and the contraction amplitude in volts and total time in seconds were recorded. Then the muscle was injected with 0.1 mL of 25mM lidocaine (a sodium channel blocker), and allowed to sit 3-5 mins. Each leg was stimulated again in the same way as the controls, and the contraction amplitude and contraction time recorded again. Each set of 3 values for amplitude and time was averaged. Then a one-tailed paired t-test was used to analyze the data, with an alpha value of 0.05 for significant difference between control and lidocaine-treated measurements

What Is Particularly Good?

This author included important information about the equipment they used. The specific brands and types is not always important, but it can be useful in certain situations.
The author also clearly explained how they determined their final stimulus voltage, how the data were collected and summarized, and how they analyzed the results.

11.4.3 Example 3

This methods section is somewhat different because it is describing an animal behavioral study. It shows you that the description of the methods needs to be tailored to the type of study. There is not one formula that fits every experiment.

Our testing tank was a 5-gallon freshwater tank with a dome-shaped, translucent white shelter that filled about 1/3 of the available tank space. The shelter had multiple openings where a fish could enter and exit. The shelter was positioned so that the fish could go in and out of it freely, and was able to see out of the shelter when using it. The rest of the tank space was open, so fish had space to swim outside of the shelter.

For the control, we placed a male betta into the testing tank, allowed it to acclimate 5 minutes, then recorded the number of seconds it spent inside the shelter in a five minute period. Next we added a second male betta (the rival) contained in a glass beaker to the tank. The test fish could see the rival, but was physically separated from it. We recorded the number of seconds the test fish spent using the shelter in the next five minute period.

Four male Betta fish were used to run twelve trials. Each male was the test fish three times, using each of the other three males its rival once. We used a paired t-test to determine whether there was a significant difference between the time spent in shelter when there was another male present compared to when there was not another male present.

What Is Particularly Good?

This methods section still answers the main question: what does a reader need to know to understand how this study was done?
The description has fewer specific numbers, but they are not needed in this case.

One change that could improve this example would be to remove the personal references like “we” and “our.”