Starch granules in fresh and processed foods are routinely identified by a variety of chemical and microscopic methods. In the teaching laboratory, the use of cross-polarized light in a microscope (monocular or binocular) quickly demonstrates the presence of starch granules by their characteristic birefringence, often called a Maltese cross. In the present study, we first identified raw potato starch granules by their birefringence, followed by three overlapping chemical methods to confirm the presence (or absence) of starch. First, Lugol's iodine solution, a starch indicator, immediately stained the granules black. Second, amylase digested the small starch granules within 30–60 minutes. Third, glucose test strips demonstrated the presence of glucose that resulted from the enzymatic breakdown of starch granules by amylase. Fourth, we also confirmed that the starch granules were broken down by the enzyme amylase by observing the “digested” product under crossed polarization and noting the disappearance of the birefringent effect. Although the demonstration of starch has long been a staple in teaching laboratories, we have combined the optical and chemical demonstrations of starch for the same lab, with each serving as a confirmation for the other.

## Introduction

Starch granules are a storage form of energy in plants and are found in fruits and vegetables. The starch granules are broken down into glucose to supply metabolic energy and also during ripening, which causes fruits like apples or bananas to sweeten (Tamarkin, 2015). Our digestive system also degrades starch by enzymatically digesting starchy foods, such as potatoes, into glucose and other sugars. We present here a laboratory experiment that combines techniques for identifying starch granules in potatoes (Hardin et al., 2012), along with the demonstration of their enzymatic breakdown into glucose via amylase. The starch granules will be observed and identified (1) optically (McMahon, 2004) by polarized light and (2) chemically by Lugol's iodine solution. The starch granules will then be broken down to glucose by amylase, the starch-digesting enzyme. The resulting product will be tested again for the presence or absence of starches, as well as for the presence of glucose. As a result, the laboratory experiments demonstrate that starch is built from glucose molecules.

## Lab Objectives

The objectives are to demonstrate the presence of starch and glucose using the following optical and chemical methods:

1. Identifying starch granules optically by using polarized light

2. Verifying the presence of starch chemically in the granules using Lugol's iodine

3. Verifying the presence of starch enzymatically by digesting the granules with amylase, a starch-digesting enzyme

4. Demonstrating that amylase enzymatically breaks the starch granules (polysaccharides) into glucose (monosaccharides) by using glucose test strips

## Materials Needed

This lab requires a few simple materials, listed in Table 1. The potato should be raw, but the variety does not matter; we have successfully used red, russet, fingerling, and Yukon varieties. The following equipment is needed to complete all experiments in this lab: a microscope, two slides, an incubator (used for enzymatic digestion of starch granules), a spatula, and a pair of pipette droppers. A cell phone camera can also be used to photograph starch granules under the microscope.

Table 1.
The materials needed for this lab, what they are used to accomplish, where they can be obtained, and cost.
MaterialUseWhere It Can Be ObtainedEstimated Cost
Potato Specimen Produce section of grocery store $1/potato Two polarizers Starch indicator – viewing Maltese cross in samples before but not after digestion Optics supplier (e.g., Edmund Optics, https://www.edmundoptics.com/f/Visible-Linear-Polarizing-Laminated-Film/12249/$12/pair of 2 inch × 2 inch sheets
Glucose test strips Detecting the presence (or absence) of glucose after (or before) digestion Biological classroom supplier (e.g., Carolina Biological Supply, https://www.carolina.com/other-diagnostic-instruments/urinary-glucose-test-strip-vial-of-100/695960.pr?question=glucose+test+strips$30 for 100 Lugol's iodine solution Starch indicator – dying starch granules Carolina Biological Supply (https://www.carolina.com/specialty-chemicals-d-l/lugol-solution-laboratory-grade-100-ml/872793.pr?question=lugol's$6.30 for 100 mL
Amylase Digest starch into glucose Digestive supplement capsules in pharmacy (https://www.pureformulas.com/amylase-formula-3-90-capsules-by-ness-enzymes.html$14 for 90 capsules NaCl and CaCl2 Buffering the amylase, providing ionic balance Brewing and vinting supplier or Amazon (https://www.amazon.com/LD-Carlson-6103B-Calcium-Chloride/dp/B00ZPVKG6C/$7.85 for 1 pound bag of CaCl2
MaterialUseWhere It Can Be ObtainedEstimated Cost
Potato Specimen Produce section of grocery store $1/potato Two polarizers Starch indicator – viewing Maltese cross in samples before but not after digestion Optics supplier (e.g., Edmund Optics, https://www.edmundoptics.com/f/Visible-Linear-Polarizing-Laminated-Film/12249/$12/pair of 2 inch × 2 inch sheets
Glucose test strips Detecting the presence (or absence) of glucose after (or before) digestion Biological classroom supplier (e.g., Carolina Biological Supply, https://www.carolina.com/other-diagnostic-instruments/urinary-glucose-test-strip-vial-of-100/695960.pr?question=glucose+test+strips$30 for 100 Lugol's iodine solution Starch indicator – dying starch granules Carolina Biological Supply (https://www.carolina.com/specialty-chemicals-d-l/lugol-solution-laboratory-grade-100-ml/872793.pr?question=lugol's$6.30 for 100 mL
Amylase Digest starch into glucose Digestive supplement capsules in pharmacy (https://www.pureformulas.com/amylase-formula-3-90-capsules-by-ness-enzymes.html$14 for 90 capsules NaCl and CaCl2 Buffering the amylase, providing ionic balance Brewing and vinting supplier or Amazon (https://www.amazon.com/LD-Carlson-6103B-Calcium-Chloride/dp/B00ZPVKG6C/$7.85 for 1 pound bag of CaCl2

## Lab Instructions

### 1. Bright-Field & Polarized Light

Each group will be given slices of potatoes. The recommended thickness is a few centimeters, so that the flesh can be easily scraped without being contaminated by the skin. Using a small spatula, gently scrape the flesh of each potato slice and quickly smear a thin coat on a slide. Place a drop of water over the smear and cover with a cover slip. Observe with both bright-field and polarized light. You will be able to clearly observe a “Maltese cross” (McMahon, 2004) in the potato starch granules under 40× and 10× magnification (do not use the 100× magnification). A sample image of starch granules under 10× and 40× magnification with both bright-field illumination and crossed polarized illumination is shown in Figure 1: the Maltese cross pattern, so named because of its resemblance to the stylized eight-pointed symbol of the Knights of Malta, can be seen in Figure 1C and 1D.

Figure 1.

Starch granules viewed under magnification with a classroom microscope. The magnification and illumination types are (A) 10× magnification under bright-field illumination, (B) 10× magnification under crossed polarized illumination, (C) 40× magnification under bright-field illumination, and (D) 40× magnification with crossed polarized illumination. These images were taken using the camera of a cell phone mounted to a classroom microscope.

Figure 1.

Starch granules viewed under magnification with a classroom microscope. The magnification and illumination types are (A) 10× magnification under bright-field illumination, (B) 10× magnification under crossed polarized illumination, (C) 40× magnification under bright-field illumination, and (D) 40× magnification with crossed polarized illumination. These images were taken using the camera of a cell phone mounted to a classroom microscope.

### 2. Lugol's Iodine

Using a small spatula, gently scrape the flesh of each potato slice and quickly smear a thin coat on a slide. Stain starch granules with Lugol's iodine solution. Place a cover slip over Lugol's iodine solution. Staining should occur in 15–20 seconds. Do not overstain: a single drop of the Lugol's iodine solution is adequate for staining purposes. Observe the staining with bright-field light. Do not use polarized light for this experiment because the Lugol's iodine stains the starch granules too darkly to see the Maltese crosses.

### 3. Amylase

You will need to prepare two solutions (control and experimental) for this part of the experiment. The control solution contains 140 mM NaCl and 10 mM CaCl2. The second solution contains the same ionic content along with amylase. For the latter, we used a single capsule (about 270 mg) of digestive supplement amylase, which was prepared by cutting the capsule open with a razor and stirring the contents into the solution.

Use two vials: (1) control and (2) experimental. Add 5 mL of control solution (140 mM NaCl and 10 mM CaCl2) to the control vial and add 5 mL of the amylase solution to the experimental vial. Then add 1 g of minced potato to the control and experimental vials. Stir for 15 seconds. The potato is minced by scraping 1 g of flesh from the interior of the potato and then grinding the flesh with mortar and pestle until it forms a smooth paste without any chunks in it.

Dip the first glucose test strip into the control solution and dip the second glucose test strip into the experimental solution for three seconds each. Place the glucose test strips (enzyme side up) on the absorbent liner and wait about 60 seconds for color to appear. The starting time is called “time zero.”

Incubate both the control and experimental vials at 38°C for 45 minutes, using either the water bath or the air incubator. Shake vials every 10 minutes. At the end of the incubation period (45 minutes), use two new glucose test strips. Dip the first test strip into the control vial for three seconds and the second into the experimental vial for three seconds each. Place the glucose test strips (enzyme side up) on an absorbent liner and wait about 60 seconds for color to appear.

Compare the colors of the test strips at time zero to those after 45 minutes of incubation. Quantitate the glucose concentrations by comparing the colors on the test strips to those on the glucose chart.

### 4. Starch Granules: Bright-Field & Polarized Light

Using separate pipettes, draw a small sample from the bottom of the control vial and experimental vial, both at time zero and after 45 minutes. Place samples on microscope slides. Make sure that the two samples do not mix with each other. Observe the granules under both bright-field and polarized light.

### 5. Starch Granules: Response to Lugol's Solution after Incubation

Repeat experiment 4. Add Lugol's solution to the control and experimental solutions. Observe the number of large, intermediate, and small starch granules in the control and experimental solutions under bright-field light. Compare the results to those of experiment 4. Once again, do not use polarized light, because the stained granules are too dark to observe structural details.

## Additional Notes to Teachers

In keeping with Bloom's taxonomy, this lab was designed so that it can be adapted to different academic levels, ranging from high school to college, based on the collection, quantitation, and analysis of data and the synthesis of conclusions. The lab is also modular so that the teacher can select some or all of the experiments, based on time, lab resources, and skill levels of students.

Slight changes to the lab procedure can make it more suitable to different academic levels. For example, small starch granules are digested more rapidly than large granules. As a result, small granules will disappear while the larger granules will remain. Students could quantitate the difference in amylase breakdown rates between small (length ≤15 µm), intermediate, and large (length ≥45 µm) granules by making multiple observations (e.g., at 0, 15, 30, and 45 minutes incubation). In addition, conducting the same experiment at both room temperature and in an incubator (38°C) will demonstrate that the higher temperature accelerates digestion. A sample worksheet for this lab (as presented in a college sophomore-level cell biology class) and student assessment questions are provided in the  Appendix.

We thank Miranda Brown for help in preparing specimens for this experiment, and Rakshak Adhikari for his advice and work in its early stages.

## References

References
Hardin, J., Bertoni, G. & Kleinsmith, L.J. (
2012
).
Becker's World of the Cell
, 8th ed. (p.
88
).
Boston, MA
:
Benjamin Cummings
.
McMahon, K.A. (
2004
).
Practical botany – the Maltese cross
. In M.A. O'Donnell (Ed.),
Tested Studies for Laboratory Teaching
, vol.
25
(pp.
352
357
).
Proceedings of the 25th Workshop/Conference of the Association for Biology Laboratory Education
.
Tamarkin, D.A. (
2015
).
Exploring carbohydrates with bananas
.
American Biology Teacher
,
77
,
620
623
.

### Appendix: Sample Lab Worksheet

1A. Draw and describe the appearances of potato smears under bright-field and polarized light. Use the same slide for both optical techniques.

1B. Place a drop of Lugol's iodine on the potato smears. Draw and describe the appearances.

2. Chemical analysis: vials and glucose test strips. Each vial contains 5 mL of ionic solutions (NaCl and CaCl2). One vial is prepared with an enzyme (amylase) which will digest the small starch granules in the minced potato; the other vial serves as the control.

3A. Using the same vials as in question 2 (above), draw and describe the appearances of minced potato under bright-field and polarized light at 0 and 45 minutes incubation times in the control and enzyme (amylase) solutions. Use the same slide for both optical techniques.

3B. Place a drop of Lugol's iodine on the incubated solutions in 3A

4. Based on optical and chemical techniques, how do the experiments demonstrate the presence of starch and the evidence that starch can be broken down to glucose?