Advanced Biology Through Inquiry Teacher Guide

Lab Summary:

Students follow the growth and development of Wisconsin Fast Plants and determine if limiting cross-pollination to certain plants with a desired trait affects the frequency of that trait in the second generation.

Theory:

Students follow the growth and development of Wisconsin Fast Plants® (a variety of Brassica rapa) through two generations. These plants have a short life cycle, can be grown easily in the classroom, and offer an assortment of traits students can readily observe. Quantitative traits, such as height, trichome hairs, and number of true leaves, offer students an opportunity to apply statistics to describe the range and variation in these traits in a population. Unlike discrete traits, such as flower color, quantitative traits are continuous in a population and are typically represented in histograms (frequency graphs).

Method:

In the Initial Investigation, students measure the height and one additional quantitative trait of their choice. They then select a small proportion of plants to cross-pollinate and determine if the selection of plants based on a certain trait affects the frequency of the trait in the subsequent generation. The study of a real population, and the time invested in tending and growing two generations of plants, is invaluable in helping students develop true science process skills, especially the application of descriptive statistics to data. This lab is less about students obtaining a predicted set of results than it is about students engaging in the process of scientific inquiry. After they gain skills in growing the Fast Plants® and using statistics to make meaning of data, students can design and carry out an experiment of their own design.

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Lab Summary:

Students analyze the DNA and protein sequences of beta globin of five mammalian species to determine their evolutionary relatedness.

Theory:

In this activity, students employ the same tools used by research scientists all over the world: online databases that allow comparison of DNA and protein sequences across species. Students compare 100-nucleotide sequences of the Hemoglobin B gene (HBB) to determine the relatedness of five different mammals. HBB codes for the beta chain of the hemoglobin protein; this chain is also referred to as beta globin.

In just the first decade of the 21st century there has been an exponential increase in the number of species that have had their gene and protein sequences identified, and this information is readily available and searchable online. One such database is managed by the National Library of Medicine (NLM), part of the National Center of Biotechnology Information (NCBI). Using search tools such as BLAST® (Basic Local Alignment Search Tool), researchers can identify, from an unknown segment of DNA or a polypeptide, the precise genus and species from which it originated.

Method:

Importantly, BLAST can locate regions of genomes or proteomes similar to a given segment of interest, thus providing information about the “alignment” of species, that is, their evolutionary relatedness. Other databases can be accessed from NCBI to determine what is known about the structure of the protein, and embedded links provide easy access to the related scientific literature.

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Lab Summary:

Students work with a mathematical model and computer simulation to explore how inheritance patterns and gene frequencies change in a population.

Theory:

Students work with a mathematical model and computer simulation to explore how inheritance patterns and gene frequencies change in a population. The model lets students explore parameters that affect allele frequencies including population size, selection, and initial allele frequency.

Method:

Having students produce their own spreadsheet instead of using an existing one allows them to become more familiar with creating mathematical models and understanding the model’s limitations. This exercise will take two 45-min periods to complete and requires the teacher to be familiar with the creation and modification of the modeling spreadsheet. There are several excellent resources that can help:

• AP Biology Investigative Labs: An Inquiry-Based Approach, Teacher Manual. The College Board. 2012.
• A Biologist’s Guide to Mathematical Modeling in Ecology and Evolution by Sarah P. Otto and Troy Day, 2007, Princeton University Press.

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Lab Summary:

Students determine their phenotype for the PTC (phenylthiocarbamide) tasting trait and use class data to derive allele frequencies for a population.

Theory:

Students use class data for the PTC (phenylthiocarbamide) tasting trait to derive allele frequencies for a population. Based on their phenotype and predicted genotype, students use “allele cards” to simulate the next generation and determine if allele frequencies change over time in the absence of selection, non-random mating, mutations, and migration. Students may or may not observe genetic drift in the simulation due to the small population size of the class. Student-designed experiments focus on determining the effect of violating one of the Hardy–Weinberg conditions on allele frequencies in a gene pool.

Method:

The Initial Investigation is a teacher-directed activity. Be sure to review the directions of the investigation carefully so you are prepared to guide students through the steps and calculations.

Prior to the investigation, determine how you would like students to report their PTC phenotypes. Depending on class size, you may wish to just verbally poll the class. Alternatively, you can create a chart on the front board and ask students to come to the front and record a tally mark in the correct column. Another option is for a group leader to go to the front board to report the phenotypes for all members of that group.

Before directing students to acquire allele cards from the gene pool, you will need to assign some of the tasters to be homozygous in genotype (TT) and others to be heterozygous (Tt). For example, if you have 18 students who are tasters and five of these should be of the homozygous genotype, you can ask all tasters to raise their hands and then point to five students who have their hands raised and ask that they be homozygous.

You may need to help the class determine the total number of T and t alleles in the gene pool, which students are directed to do in the Initial Investigation. Each homozygous dominant individual has 2 T’s, so the number of T alleles for this genotype is 2 × number of persons of that genotype. Each heterozygote has 1 T and 1 t, so the T alleles and t alleles for this genotype are equal to the number of individuals with the genotype. Lastly, the number of t alleles from the homozygous recessive individuals is 2 × the number of person of that genotype.

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Lab Summary:

Students use a pH sensor to investigate the diffusion of hydrogen ions through a semipermeable membrane, comparing the rates of diffusion for two solutions that differ in their acidity.

Theory:

Students investigate the diffusion of ions through a semipermeable membrane using apple cider vinegar and a pH sensor. The vinegar is added to a dialysis bag to represent the intracellular solution of a model cell. Distilled water in a beaker represents extracellular fluid and students monitor the pH of the water over time to measure the rate of diffusion out of the model cell.

The color of the apple cider vinegar provides evidence of the semipermeable nature of the membrane; hydrogen ions easily diffuse through the membrane while pigments molecules do not. Student-designed experiments that test factors affecting the rate of diffusion can be accomplished with minimal materials and within the time frame of a single class period.

NOTE: Since hydrogen ions form bonds with water molecules, students are actually determining the rate of diffusion of hydronium ions: H3O+(aq).

Method:

Diffusion within biological systems is affected by several factors. The most notable is the concentration gradient across a membrane. A large concentration gradient across the membrane speeds up diffusion. Other factors affecting diffusion rates within biological systems include temperature, distance between solute and membrane, surface area-to-volume ratios, and solute size. All these factors can be manipulated in student-centered investigations.

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Lab Summary:

Students use a conductivity sensor to explain the results of different concentrations of salt water on plant tissue before they design an experiment to compare the water potential of different plant tissues.

Theory:

Students examine red onion tissue, looking for evidence of plasmolysis in the cells. Based upon their observations of plant tissue in three solutions with different (unknown) concentrations of salt, students determine which of the solutions has the highest salt concentration. They test their conclusions with a conductivity meter. Students then design an experiment that applies their understanding of water potential and the equation for water potential to compare that property in different plant tissues.

Method:

The concentrations of the unknown solutions do not need to be exactly the concentrations specified above. Prior to students performing the investigation, make wet mounts with each solution and observe the cells under the microscope. It is ideal if one solution (Solution “B”) shows significant plasmolysis, while another (Solution “A”) shows partial plasmolysis. The third can show little or no plasmolysis. If there is not enough difference in results, adjust the concentrations of the unknowns accordingly.

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Lab Summary:

Students use physical models of chromosomes to explore meiosis and genetic variation, and use cross over rates observed in Sordaria to calculate gene distance from the centromere.

Theory:

Students use physical models of chromosomes to explore the topics of meiosis and genetic variation. First, students use “paper chromosomes” to model the independent assortment and inheritance of fruit fly chromosomes. Fruit flies are a good candidate for a modeling activity since they have only 8 chromosomes, compared to 46 chromosomes in humans. Analyzing the variation in the phenotypes of five virtual offspring flies provides a springboard for students to connect the events of meiosis with its purpose of creating haploid cells and with the genetic variation that results from sexual reproduction.

Method:

Students then use “pop-bead chromosomes” to model the events of meiosis I and meiosis II and finally they determine the crossover rate between alleles that determine spore color in the fungus Sordaria fimicola. Spore color is a single gene trait with black being the wild-type form of the gene, symbolized by “+.” Tan color is the result of a mutation and its allele is symbolized by “tn.” Students determine the crossover rate by observing the arrangement of black and tan colored spores produced by a hybrid fungus (+ strain crossed with the tn strain). Using the crossover rate, students can determine the location of the gene for spore color relative to the chromosome centromere.

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Lab Summary:

After learning the technique for growing roots and preparing root tip squashes for microscope analysis, students observe the root tips for evidence of mitosis and statistically analyze the data.

Theory:

After learning the technique for growing roots and preparing root tip squashes for microscope analysis, students observe the root tips for evidence of mitosis. They compare the number of cells in mitosis to the number of cells in interphase. They apply the chi-square “test of independence” to compare their results with provided data. Following the initial investigation, students can move to independent inquiry and test a particular treatment to see if it affects the rate of mitosis in roots. Chi-square analysis can be applied to evaluate the significance of the results.

Method:

Many biology teachers are familiar with the chi-square “goodness-of-fit” test, which tests how well observed data fits with expected data. However, this investigation requires the less familiar chi-square “test of independence,” which tests whether two categories are independent of each other.

In many cases the goodness-of-fit test and the test of independence have similar outcomes for the same data set. For example, the chi-square value obtained from each method might indicate that the investigator should reject the null hypothesis. The value obtained from the test of independence will be more conservative in “treatment” situations—like the mitosis investigation—and is therefore a more valid statistical method to apply. (The value is less than what would be obtained for the goodness-of-fit test and makes it less likely that the null hypothesis is rejected.)

Rather than use the observed cells in the control group to calculate expected values for the treatment group, as would be done in a goodness-of-fit test, observed cells in both control and treatment groups are used to calculate expected values in a 2 × 2 contingency table for the test of independence.

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Lab Summary:

Students transform bacteria with a plasmid that contains an ampicillin resistance gene and a gfp gene that is regulated so only some transformed cells produce the green fluorescent protein.

Theory:

The discovery of bacteria transformation played an important role in understanding that DNA stores and transmits heritable information. Transformation remains important to biological research today. This lab is meant to help students solidify their understanding of the central dogma of molecular biology: the gene–protein connection.

Method:

The plasmid DNA used for the transformation contains both a gene providing resistance to the antibiotic ampicillin (ampR) and one that will cause bacteria to fluoresce (gfp). Students relate the change in the bacteria’s phenotype to the genes in the plasmid. They learn that not all genes are expressed and that the fluorescence due to the gfp gene depends on a factor called IPTG. Students apply the concept of an inducible promoter to explain the results of the investigation (differential gene expression). Students also learn to calculate the efficiency of the transformation.

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Lab Summary:

Students use a carbon dioxide gas sensor to investigate the rate of cellular respiration of germinating seeds.

Theory:

Students investigate the rate of cellular respiration of germinating seeds using a carbon dioxide gas sensor to measure one of the products of this process.

C
₆H
₁₂ O
₆ + 6O
₂ –> 6H
₂O  + 6CO
₂ + Energy (36 ATP)

Students design and conduct an experiment to investigate a factor that can affect the cellular respiration of germinating seeds or to measure respiration in other organisms, such as crickets. Investigations that vary the species, germination time, pH, temperature, and salinity work well.

Method:

Although the sensor reports oxygen concentration to a high resolution, its accuracy is ±1%, or 10,000 ppm. If students are measuring very small changes in oxygen concentration over a short period of time, they may see no change, or even a trend in the wrong direction. Often the change is insignificant despite the appearance of a trend on the data collection system. Students can try switching to a carbon dioxide gas sensor, measuring for a longer period of time, or increasing the size or number of organisms they are sampling to create a more observable change.

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Lab Summary:

Students use an oxygen gas sensor or pressure sensor to investigate the catalyzed decomposition of hydrogen peroxide by catalase.

Theory:

Enzymes are proteins that catalyze chemical reactions by reducing the reaction’s activation energy, This is important in many reactions. For example, to digest lactose (a sugar found in milk), lactase must be present. People born without the gene to produce the enzyme lactase cannot digest lactose.

Enzymes are hyper-specific—often an enzyme interacts with only one substrate. Like catalysts in other chemical reactions, enzymes are not consumed during the reaction but help turn the substrate into the final product. Notice in the following reaction that the enzyme is present before and after the reaction.

enzyme(l) + 2H
₂O
₂(l) → 2H
₂O(l) + O
₂(g) + enzyme(l)

Hydrogen peroxide (H
₂O
₂) is a byproduct of aerobic respiration in cells and is used in cell signaling and apoptosis. Hydrogen peroxide is highly reactive and can produce free radicals that damage nucleic acids, so cells must carefully regulate its concentration. To remove excess hydrogen peroxide, cells produce an enzyme (called catalase in animal cells and peroxidase

in plant cells) which breaks down H
₂O
₂ into oxygen (O
₂) and water (H
₂O), as shown above. This reaction proceeds spontaneously without the enzymes at a very slow rate. This uncatalyzed reaction will serve as the baseline, or control, in the initial investigation.

Method:

Students investigate the catalyzed decomposition of hydrogen peroxide by catalase or peroxidase according to the equation:

enzyme(l) + 2H
₂O
₂(l) → 2H
₂O(l) + O
₂(g) + enzyme(l)

Students can use either the oxygen gas sensor or pressure sensor to measure the oxygen gas produced as the reaction proceeds. Students design an experiment to investigate questions they devise about factors that can affect catalyzed reaction rates. Investigations that manipulate pH, temperature, enzyme concentration, or substrate concentration work well.

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Lab Summary:

Students use pedigree analysis and DNA analysis (electrophoresis) to confirm or refute the initial diagnosis of MELAS for two patients.

Theory:

Students use pedigree analysis and DNA analysis to confirm or refute the initial diagnosis of MELAS for two patients. MELAS and certain other genetic mitochondrial disorders are unusual in their inheritance: the mutation is passed along maternally only. This maternal inheritance, combined with the complexity in symptoms associated with the disorder, provides a case study that requires students to think critically and evaluate multiple lines of evidence.

Method:

The two investigations, “Pedigree Investigation” and “DNA Investigation,” both relate to the same two patients. Either investigation can be performed first or students can complete the investigations simultaneously. Based on the length of your class periods, your students’ prior knowledge, and your own pedagogical preference, you can determine how best to organize the investigations. In addition to the time required for the investigations, allow time for a discussion of the background information regarding the mitochondrial genome and MELAS. (Photocopy the supplement at the end of this document for students.)

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Lab Summary:

Students use a colorimeter to determine which extracellular fluid is hypertonic to a model cell and which solution is hypotonic.

Theory:

Students build models that help them understand how blood osmolarity and kidney function maintain homeostasis. Students determine which extracellular fluid is hypertonic to a model cell and which solution is hypotonic.

Method:

Diffusion within biological systems is affected by several factors. The most notable is the concentration gradient across a membrane. A large concentration gradient across the membrane speeds up diffusion. Other factors affecting diffusion rates within biological systems include temperature, distance between solute and membrane, surface area-to-volume ratios, and solute size. All these factors can be manipulated in student-centered investigations.

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Lab Summary:

Students use temperature probes to measure the effect of cell size on cell cooling rate using cubes of potato tissue.

Theory:

Students model the effect of cell size on cell cooling rate using cubes of potato tissue. Potato cubes of different sizes are placed in ice water and multiple temperature probes are used to simultaneously measure the interior temperature of each cube and the temperature of the ice bath. Students determine if and how the difference in cooling rates relates to the difference in surface-area-to-volume ratios (SA:V) of the cubes. Students then consider variables other than size that might affect the SA:V ratio and design an experiment that tests the effect of that variable on the cooling rate of a cell, using potato tissue to model the cell.

Method:

This lab provides a good opportunity for students to perform numerous trials for each condition of their student-designed experiment and calculate average cooling rates. The runs can be short (approximately 2 minutes), allowing students to run many trials during a single class period.

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Lab Summary:

Students use multiple temperature probes simultaneously to investigate the body’s ability to maintain homeostasis when subjected to a cold stimulus.

Theory:

Students investigate the body’s ability to maintain homeostasis with regard to body temperature by testing the body’s response to a cold stimulus. They use multiple temperature probes to simultaneously measure the surface temperature of the skin at two locations and relate the results to thermoregulation, which is controlled by the hypothalamus—the hypothalamus receives information from nerves which detect a stimulus in the external environment, interprets information, and then responds by sending signals via efferent nerves to multiple organ systems. Students should find that in response to the cold stimulus, the body adjusts blood flow to maintain homeostasis, keeping the body’s core temperature near 37 °C.

Method:

When working with human subjects, the data is rarely as “clean” as when working with enzymes, germinating seeds, or other biological samples. Expect results to vary among students and groups; however, all students and groups should see temperature changes that relate to a response to the ice water stimulus. Variability in data can lead to rich classroom discussions that focus students on graph analysis and using evidence to support their claims.

For student-designed experiments, encourage students to use a “large” sample size of test subjects and to acquire data from multiple trials that can be averaged.

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Lab Summary:

Students use electrophoresis to analyze DNA samples from a child and the child’s parents to determine if the child has inherited a mutation in the gene for hemoglobin B.

Theory:

Many genetic disorders can be detected and diagnosed by a method employing polymerase chain reaction (PCR), restriction enzymes, and electrophoresis. Genetic tests often rely on digesting DNA with a restriction enzyme, which can cut within a normal gene sequence but not within the mutated sequence, resulting in different lengths of DNA. Electrophoresis separates DNA fragments based on the sizes of the fragments.

Method:

In this lab, students use electrophoresis to analyze DNA samples (previously treated with a restriction enzyme) from a child and the child’s parents to determine if the child has inherited a mutation in the gene for hemoglobin B (HBB). The mutation causes sickle cell anemia, a common form of sickle cell disease, in homozygous individuals.

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Lab Summary:

Students use a choice chamber to test the response of fruit flies to different stimuli and determine if there is a significant change in their behavior.

Theory:

Students test the response of fruit flies to different stimuli and determine if there is a significant change in their behavior. They do this by constructing a choice chamber from drinking straws and cotton swabs and exposing the flies in the chamber to two environments, one on each end of the straw. Students then conduct a chi-square test to determine if the flies display taxis and are indeed favoring one environment over another.

Method:

Wingless fruit flies are ideal for use inside the drinking straws. However, other small organisms, such as crickets or pill bugs, can be used. A choice chamber can be constructed from clear PVC or acrylic pipe 0.5–2 inches in diameter, plastic drinking bottles, or Petri dishes, to use with other organisms.

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Lab Summary:

Students use EcoChamber containers and a carbon dioxide gas sensor to estimate energy flow and carbon cycling within a variety of detritus-based ecosystems.

Theory:

Students set up a variety of simple detritus-based model systems to estimate energy flow and carbon cycling within an ecosystem. Students set up their ecosystem with a known detritivore, a known decomposer, or a combination of both detritivore and decomposer. The teacher provides students with data from two control systems to help interpret changes in the experimental systems.

Method:

Energy flux is estimated using gravimetric analysis and the carbon cycle is investigated using a carbon dioxide (CO2) gas sensor. By tracking the movement of energy within these systems, students gain an understanding of the laws of thermodynamics as they relate to energy transfers in ecosystems. Following the Initial Investigation, students design an experiment around an abiotic factor or a biotic component of the system to manipulate or they simply monitor decomposition for a longer time period.

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Lab Summary:

Students use an ethanol sensor to determine the ability of yeast to use different types of carbohydrates—sucrose and starch—for fermentation.

Theory:

Students determine the ability of yeast to use different types of carbohydrates—sucrose and starch—for fermentation. Yeast are facultative aerobes, carrying out both aerobic respiration and fermentation, depending on whether oxygen is readily available. When yeast ferment sugar, they produce ethanol and carbon dioxide and obtain ATP from glycolysis.

C
₆H
₁₂ O
₆ + 6O
₂ –> 2CO
₂ + 2C
₂H
₅OH

Since carbon dioxide is a product of both aerobic respiration and fermentation, this lab makes use of an ethanol sensor to measure a product formed only during fermentation. (Both processes can occur simultaneously, so measuring carbon dioxide concentration is not a direct measurement of the rate of fermentation.)

Method:

Best results are obtained using active dry yeast purchased in packets, as opposed to a jar of active yeast. Be sure the expiration date for the yeast has not passed.

The ethanol sensor probe contains a heating element. It can take up to 10 minutes for the sensor’s temperature to stabilize. The “warm-up” period should occur prior to calibration. (Refer to the product manual for calibration procedures.)

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Lab Summary:

Measure the rate of photosynthesis of a leaf using the carbon dioxide gas sensor.

Theory:

Plants and other producers utilize the sun’s energy to build complex organic compounds that serve as a source of energy for the organism. Chloroplasts are the site of this energy capture and biosynthesis. Chlorophyll and other pigment molecules play a vital role in the light reactions of photosynthesis, capturing energy that drives an electron transport chain and ATP synthesis. Ultimately, through light-independent reactions, the chemical energy is transferred and stored within molecules such as glucose. This entire process is summarized by the chemical equation:

6CO
₂ + 6H
₂O → C
₆H
₁₂O
₆ + 6O
₂

The uptake of carbon dioxide is an indication that photosynthesis is occurring within the chloroplasts of a leaf. The reactions that fix carbon into organic compounds depend on the products of the light reactions and are therefore dependent on the absorption of light by pigments.\ While sunlight is composed of many different wavelengths of light, those wavelengths are not equally available to a plant. This investigation compares the amount of photosynthesis that occurs when different colors of light are provided to a plant.

Method:

In this lab, students test the effect of light color on the rate of photosynthesis. Given the equation for photosynthesis, students can determine that either a carbon dioxide gas sensor or an oxygen gas sensor would be appropriate equipment for determining photosynthetic rate.

6CO
₂ + 6H
₂O → C
₆H
₁₂O
₆ + 6O
₂

For this investigation, a carbon dioxide gas sensor is used to determine the rate of uptake of CO
₂ by spinach leaves. Students first test the change in carbon dioxide concentration caused by photosynthesis occurring in the leaves under red light. These results are then compared to the rate of photosynthesis under green light.

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Lab Summary:

Students analyze spinach pigments and chloroplasts using paper chromatography, a colorimeter, and a spectrometer to understand how plants capture light for photosynthesis.

Theory:

Students extract pigments from spinach leaves for analysis using chromatography and colorimetry. Paper chromatography separates the pigments present in the extract so they can be identified. Analysis of the extract with a colorimeter allows students to determine the relative absorbance of four different colors of light (blue, green, orange, and red). They relate the chromatography results to the colorimeter measurements to refine their understanding of how plants capture light for photosynthesis. If available, a spectrometer allows students to view the full absorbance spectrum for spinach leaves.

The colorimeter is used again in Part 2 to measure photosynthesis using DPIP (2,6-dichloropheno-lindophenol), an electron acceptor that experiences a color change if photosynthesis is occurring. For student-designed experiments, students can analyze factors such as the effect of different light sources, distance from a light source, and comparison of pigments in the leaves of different plants using chromatography, DPIP analysis, or both.

Method:

A large number of chromatograms need to be created to have sufficient quantities of isolated pigments for analysis. (This is one of the suggested inquiry options.) One option is to have all students contribute their chromatograms to this purpose after they record their observations in the Initial Investigation.

Students should cut carefully along the border lines between pigments to cut strips of chromatography paper that have only one pigment per strip. Discard the sections of paper that do not contain pigments. Place all of the chlorophyll a strips into a beaker with ethanol or chromatography solvent. Place all of the chlorophyll b strips into a different beaker, and do the same for the carotenoids and xanothophylls. Over time, the pigments will dissolve out of the paper and into the solvent. Cutting the strips into smaller pieces, or crumpling the strips, can accelerate the process.

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Lab Summary:

Students use a low pressure sensor (barometer) and a weather sensor to investigate the rate of transpiration in plants under normal and humid conditions.

Theory:

Students investigate the rate of transpiration in plants under normal and humid conditions and have an opportunity to conduct inquiry experiments of their own design to test additional factors. In the Initial Investigation, students create a potometer using a pressure sensor and they monitor microclimate conditions using a weather sensor. The potometer measures transpiration by detecting the change in pressure due to the evaporation of water from the leaves of a plant sample.

The Initial Investigation provides students with sample data from a “whole-plant” transpiration experiment to introduce students to this alternate method of measuring transpiration. For experiments of their own design, students can use either the potometer method or whole-plant method to measure how certain factors affect the rate of transpiration.

Method:

• In order for transpiration to occur, it is critical that there is an unbroken water column that extends from the water in the tubing to the water in the xylem of the plant. Any air bubbles in the tubing or at the cut surface of the stem will affect the data. This is why the tubing and plant stem must remain submerged in the tub of water during the setup.
• The assembly is difficult for one person to coordinate. Students should work in pairs to complete the procedures.
• The stem of the plant sample must fit tightly in the opening of the tubing. If students collect plant samples from the schoolyard or home, they should take the tubing outside with them to test for a good fit of the stem in the tubing. When using plants obtained this way, students do not need to immerse the plant samples in water in the field. They can cut the sample from a plant in the schoolyard and then cut the stem again (as specified) during the procedure of the Initial Investigation.
• To ensure an airtight seal, a small piece of paraffin film can be wrapped around the tubing edge where the plant stem is inserted. Alternatively, a dab of petroleum jelly can be applied at the edge of the tubing. However, the paraffin film or jelly is not sufficient to provide a seal if there is a gap between the stem and tubing. Avoid getting jelly in the tubing as it is difficult to remove.

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