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1.17: Starch Hydrolysis

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    Learning Objectives
    • Explain what the starch hydrolysis test is and how it works.
    • Explain how starch hydrolysis relates to the amylase gene and the enzyme amylase.
    • State the chemical reaction that occurs when bacteria hydrolyze starch.
    • Successfully conduct the starch hydrolysis test.
    • Interpret the results of the starch hydrolysis test.
    • State why starch hydrolysis is a beneficial characteristic for bacteria.
    • Tell that starch hydrolysis is a characteristic that only some bacterial species have and that is therefore useful for species identification and characterization.

     

    Starch Hydrolysis Test

    Starch is a long carbohydrate molecule made of hundreds of glucose molecules bonded together into a very long chain. There is a lot of energy in starch! If bacteria are able to break down starch, this is an advantage because that means they can access the sugars in starch and the energy in those sugars (as well as the carbon).

    reaction catalyzed by amylase

    Figure 1: Above is the chemical reaction catalyzed by the enzyme amylase. The long polysaccharide starch is broken down (hydrolyzed) by the enzyme amylase. The products are glucose molecules (a monosaccharide) and maltose (a disaccharide). These smaller sugars are small enough that a bacterial cell could transport them inside. Then once inside the bacterial cell, these sugars can be used by the bacteria as a source of energy and carbon.

     

    Some species of bacteria can break down starch and some species of bacteria cannot break down starch. Whether a bacterial species can break down starch or not is based on whether they have a gene (a segment of DNA) that codes for the enzyme amylase. Amylase is an enzyme that breaks down starch into smaller sugars.

    Starch is too large to pass through the plasma membrane of a cell and must be split into individual glucose molecules. Bacteria that can produce the exoenzyme amylase are able to hydrolyze starch by secreting these enzymes into the environment around them. The enzyme amylase is secreted out of the cells (amylase is considered an exoenzyme, meaning it is an enzyme secreted outside of the cells ["exo-" means "outside"]) into the surrounding media, catalyzing the breakdown of starch into smaller sugars. These smaller sugars can then be absorbed by the cells and the cells can use the sugars as a source of energy and carbon.

     

    amylase production from amylase gene; amylase is excreted outside of cells

    Figure 2: Diagram summarizing how a bacterial cell produces and secretes amylase. The information for building amylase is found in the cell's DNA. The amylase gene is expressed (RNA is created from the information in the gene (the DNA) and from the RNA, ribosomes build the amylase proteins [DNA-->RNA-->protein]). Then amylase is secreted outside of the cell (amylase is an exoenzyme). Amylase can then hydrolyze starch if it is available in the bacterial cell's environment.

     

    Since only some species of bacteria are capable of breaking down starch (aka starch hydrolysis) testing if bacteria can break down starch or not is a way we can differentiate one bacterial species from another. Therefore, examining starch hydrolysis of a bacterial species is useful for identifying and characterizing bacterial species. Starch can be added to petri plate media in order to determine if bacteria are capable of breaking down the starch.

    Starch inside of a petri plate is clear and cannot be seen without using iodine. Iodine reacts with starch, producing a black or bluish-black color. As starch is hydrolyzed by bacterial amylase and is converted to sugars, there will be less and less starch to react with the iodine. Strong amylase producers may convert all of the starch in the agar to sugars, while weak amylase producers may convert the starch surrounding the growth areas only.

    After bacteria are allowed to grow, iodine is added to the petri plate to detect the presence and absence of starch.

    • Where starch is on the petri plate will appear blue-black. Iodine reacts with starch to produce blue-black coloration when starch is present.
    • Where starch isn't on the petri plate will appear clear or yellowish. Iodine will not produce a blue-black color where starch is absent. These regions therefore appear a clear or yellowish color on the petri plate when starch is not present.

    Since the entire petri plate contained starch at the start of the test, clear halos surrounding bacterial growth indicates that the bacterial species was able to hydrolyze the starch resulting in the clear zone without starch (we would say that this bacterial species is starch hydrolysis positive). If the growth does not exhibit clear zones surrounding it, the starch is intact throughout the plate (no starch hydrolysis or we say that the bacterial species is starch hydrolysis negative).

     

    starch hydrolysis test interpretation

    Figure 3: A starch plate with different bacterial species growing on it after iodine has been added to the petri plate. The black regions are where starch is still present since iodine reacts with starch to create the black color. The clear regions are where starch is no longer present (where starch has been hydrolyzed). The bacterial growth may look light-colored on the plate. It is easy to mistake the bacterial growth from a clear zone that indicates starch hydrolysis. The key here is that the clear zone will surround the bacterial growth if starch hydrolysis occurred. Be aware that some species of bacteria hydrolyze starch at such a rapid rate that they may have consumed starch around other bacterial species on the same plate when those other species do not hydrolyze starch.

     

    Laboratory Instructions

    Inoculating Starch Plates

    1. Obtain a starch agar petri plate.
    2. Write on the bottom of the starch agar petri plate to separate the plate into four sections. Label as shown above.
    3. Aseptically make a single line streak of the corresponding bacterial species in each of the sections on the petri plate. The "control" region will remain empty (no bacterial streak).
    4. Invert the petri plate and incubate at either 25º C or 37º C for 24-28 hours.

     

    Adding Iodine to Visualize Location of Starch

     

    starch hydrolysis plate setup

    Figure 4: Draw on the bottom of the starch plate the lines and the labels shown above. The bacterial species names have been abbreviated for simplicity. Their full names are Escherichia coli, Bacillus subtilis, and Proteus vulgaris. The control will be used for comparison of how the plate should look when no starch hydrolysis occurs since no bacteria is being streaked in this region.

    1. After the starch plate has incubated and bacteria is growing on the surface, placing the agar plate on a white piece of paper or white background since this will really help you to distinguish whether or not clear zones occurred.
    2. Cover the agar and growth with iodine.
    3. Examine the petri plate. Species that hydrolyze starch will have a region around the bacterial growth that is yellowish or clear around the growth (the growth may appear light in color - look for a clear zone surrounding the growth [the growth is raised on the plate]). When starch is not hydrolyzed, the growth may appear lighter in color, but there is no clear zone surrounding the growth (unless the clear zone is extending from a bacterial species that does hydrolyze starch.

     

    Results & Questions

     

    Bacterial Species Result for Starch Hydrolysis (+ or -) Does this species produce amylase? Does this species have the amylase gene?
    Escherichia coli      
    Bacillus subtilis      
    Proteus vulgaris      
    1. Complete the table above to indicate the results of this experiment.
    2. What does it mean, in the starch hydrolysis test, when there is a clear zone surrounding bacterial growth?
    3. What does it mean, in the starch hydrolysis test, when there is no clear zone surrounding bacterial growth?
    4. What is hydrolysis?
    5. What is starch hydrolysis?
    6. What is amylase?
    7. Give the chemical reaction catalyzed by amylase.
    8. Why is it an advantage for a bacterial species to be able to hydrolyze starch?
    9. Do all bacteria hydrolyze starch? Explain your answer.
    10. Fill in the blank. Whether a species of bacteria is capable of producing amylase is dependent on _________.
    11. The starch hydrolysis test is useful for identifying and characterizing bacteria. Why?

     

    This page titled 1.17: Starch Hydrolysis is shared under a CC BY-NC-SA 4.0 license and was authored, remixed, and/or curated by Rosanna Hartline.

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