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Bacteria: Fermentation of Carbohydrates Copy

Some bacteria, when grown in a sugar broth, will ferment the sugar as part of their normal metabolism. Possible types of carbohydrates that bacteria may use as an energy source are glucose, lactose, sucrose and cellulose. Many bacteria will produce an organic acid (such as lactic acid) and hydrogen and carbon dioxide gases when the carbohydrate is fermented. A fermentation tube can be used to detect acid and gas production. The test is performed by inverting a small tube (such as a Durham tube) inside a test tube which has been filled with the sugar solution, peptone (an acid indicator such as phenol red or bromthymol blue), and a 1% solution of the carbohydrate being tested. When the carbohydrate tube is sterilized, the air will be forced out of the Durham tube and the broth will be forced in during cooling, causing the tube to be completely filled. If the bacteria that are introduced into the tube are fermentors, the small Durham tube will collect some of the gas that is released during fermentation. If the product is an acid, then the indicator will change colors (both phenol and bromthymol blue will turn yellow in the presence of an acid).

Engaging Questions

  • Why is fermentation important?

  • What are the types of sugars?

  • What is a carbohydrate? Name some common sources of carbohydrates.

Teacher Goals

  • By using various carbohydrate fermentation techniques, one will be able to determine if a particular type of bacteria uses certain sugars as part of its normal metabolism.

Required Resources

  • The following materials will be needed to complete this lab experiment:

    • glucose fermentation tube
    • lactose fermentation tube
    • sucrose fermentation tube
    • MR-VP tube (Methyl red Voges-Proskauer tube)
    • slant of unknown bacteria

Steps

  1. Inoculate

    Using a sterile innoculation loop, inoculate the fermentation tubes with the unknown bacteria.

  2. Incubate

    Incubate the tubes at 35oC or at room temperature depending on the type of bacteria.  

  3. Examine

    Examine the tubes at the end of 24 hours and 48 hours for growth, color change, and gas production. 

    An acid production is indicated by a color change in the medium from red to yellow.  Gas production is indicated by a gas bubble in the Durham tube.  If no carbohydrate fermentation has ocurred, the color will remain the same or a deeper red will appear if it has become more alkaline.  This would be due to the break down of protein which could release ammonia and other organic compounds.
     
    Record your results.

  4. MR-VP Tube

    Transfer 2.0 ml of the culture to a clean test tube.

    To the remaining medium, add 4-5 drops of methyl red indicator. Methyl red is an indicator which will change colors if the pH is below 4.4. If neutral products are produced (acetone), methyl red will turn yellow, indicating a pH above 6.0.   Red is a positive test.
     
    To the original tube, add 1.0 ml of a-naphthol and 1.0 ml of potassium hydroxide solution.  Shake the tube with the caps off to expose the media to oxygen in order to oxidize the acetone to diacetyl.  This would be indicated by a pink-red color.
     
    Record results. 

Teacher Notes

  • Review with the class proper laboratory procedures for handling bacteria and using lab equipment. Circulate during the experiment to supervise and offer assistance where needed.

Learner Notes

  • Remember to use proper lab procedures. Clean your work station when finished. Thoroughly wash your hands.

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