Laboratory Investigations in Microbiology

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Chapter 18: Fermentation

Although respiration, be it aerobic or anaerobic, is the most efficient form of energy generation, not all bacteria can do respiration at all times. A less efficient alternative, called fermentation, is found in many bacteria. Some, such as Streptococcus, are entirely fermentative and do not have the ability to do respiration. Other bacteria choose fermentation when the electron acceptors of respiration are unavailable. Fermentation generates only ~10% of the energy that can be gained by respiration (per molecule of food used). In part, cells make up for this difference by fermenting significantly larger quantities of food than they would use in respiration alone.  The advantage of fermentation, however, is that no external electron acceptor (such as oxygen or nitrate) is needed. The most common electron acceptor in fermentation is pyruvate, produced through glycolysis. Thus, bacteria that break down glucose to pyruvate can turn around and immediately use this pyruvate as an electron acceptor. The bacteria keep only the ATP generated during glycolysis and the reduced form of pyruvate (often lactic acid) is lost to the cell as a waste product.

Fermentation is usually carried out in anaerobic environments, since facultative anaerobes will resort to respiration if oxygen is present. As bacteria ferment their food source, the fermentation products are released into the culture medium. Such fermentation products include organic acids (lactate, butyrate, acetate, formate, propionate), alcohols (ethanol, isopropanol, butanol, butanediol), and gases (CO2, H2). Because bacteria produce large quantities of fermentation byproducts, the culture medium is changed. By using a few simple indicators (such as phenol red, a pH indicator), we can detect the formation of acids, gases, and even butanediol. This forms the basis of the fermentation tests we will conduct today.

Glucose fermentation test (Atlas p. 52): Fermentation of glucose, one of the most easily used carbohydrates, is tested using a fermentation broth containing peptone (protein), glucose, phenol red (a pH indicator), and a Durham tube (an upside-down small glass tube inside the broth). When acid is produced, the color of the medium changes from red/orange (neutral) to yellow. This indicates a positive test for acid production. This test does not tell us which acids are made or how much acid is produced. An orange or red color indicates a negative test for fermentation

Some bacteria attack proteins in the medium if they cannot do fermentation. When they do, amino acids are broken down, releasing ammonium (see chapter 12). As a result, the medium turns magenta in color, indicating base production

When gas (CO2 or H2) is produced from fermentation, bubbles will be trapped inside the Durham tube. This indicates a positive test for gas production.

Production of alcohol (ethanol, e.g.) cannot be detected with this test.

Sucrose and lactose fermentation tests: These two tests work the same way as the glucose test. Instead of glucose, however, the disaccharides lactose or sucrose are used. Only bacteria that have the enzymes to hydrolyze these disaccharides first (producing glucose + one other monosaccharide) can ferment them! For these tests to be positive, bacteria must be able to do fermentation (see glucose test) AND have the enzyme to break down the disaccharide. A negative test in this case can have two explanations: the bacteria do not carry the disaccharidase necessary, or the bacteria do not carry out fermentation. Positive fermentation results are scored the same way as in the glucose test.

GLS combination test. Many bacterial identification systems available commercially use multiple test chambers/wells to test for the ability of a microbe to use or ferment a variety of sugars, and the overall pattern of sugar use can be used to identify the microbe. The GLS test is a scaled-down version using only 3 sugars (Glucose, Lactose, Sucrose), but even this milited combination allows for 5 different outcomes.  This allows a more rapid distinction among test microbes. The main limitation in this test is that it only detects Acid (A) so the outcome for each sugar is evaluated as either positive (A) or negative (-). No gas can be detected.

Voges Proskauer (VP) test (Atlas p. 82): Although traditional fermentatioVP-negative (left) and VP-positive (right) reactionsn tests can detect acids and gases, the precise fermentation pathway followed cannot be deduced. Some bacteria produce only lactic acid (homolactic fermentation), while others produce a mixture of acids and gases (mixed acid fermentation) or acids, gases, and butanediol (butanediol fermentation). To distinguish some of these possibilities, one can test for specific chemicals, such as acetoin (the precursor for butanediol). The Voges Proskauer test identifies bacteria that produce butanediol. The reagents (phenol + NaOH) react with acetoin, a butanediol precursor, to produce a red color. This indicates a positive VP test.  In a negative VP test, the reagents may be brown, yellow, or green in color.

Methyl Red (MR) test (Atlas p. 63): Negative MR test (left) and positive MR test (right)One can also determine the amount of acid produced using the MR test.  Methyl red is a pH indicator that turns from yellow (neutral) to red (acid) at a pH of ~ 4.4. bacteria that carry out Mixed acid fermentation produce many strong acids (lactic, acetic, formic) that lower the pH below 4.4, resulting in a red color when the methyl red is added. This indicates a positive MR test. Other bacteria produce weaker and/or fewer acids and therefore do not lower the pH of the medium as much, resulting in an orange or yellow color when methyl red is added. This indicates a negative MR test.  Please note that these color changes are opposite of those in the phenol red test!

Materials & Methods

Materials per lab group
Glucose, Lactose and Sucrose fermentation:
  1. Label 3 glucose fermentation broths with the names of the 3 microbes assigned to your group.
  2. Inoculate each tube with a loop full of culture broth. Take care not to agitate the tubes too much
  3. Repeat for the lactose and sucrose broths
  4. Bring tubes up front to be incubated

After 24 - 48 hours:

  1. Observe each tube for a color change. Evaluate and record data as A (acid), G (gas), AG (acid and gas), - (neutral/negative) or B (base/negative)
Voges Proskauer test:

Reagents: Barritt's Reagent A (α-Naphthol in ethanol) and B (Potassium hydroxide 30%)

  1. Label 3 MR-VP broths with the names of the microbes assigned to your group.
  2. Inoculate each broth with a loop full of culture broth
  3. Bring tubes up front to be incubated

After 24 - 48 hours:

  1. Pipette 1 ml of each MR-VP broth into a separate test tube
  2. Add 15 drops of Barritt's Reagent A to each 1 ml tube. Shake tubes vigorously
  3. Add 5 drops of Barritt's Reagent B to each 1 ml tube. Shake tubes vigorously
  4. Let tubes stand 15 minutes. Observe for a color change. Record data
Methyl Red test:

Reagents: Methyl Red Reagent (methyl red dye in ethanol)

  1. The same MR-VP broths are used as for the Voges-Proskauer test (see steps 1 - 3 above)

After 24 - 48 hours:

  1. Add 5 drops of methyl red reagent (Methyl red indicator in ethanol) to the remaining (after conducting the VP test) MR-VP broth. Observe for a color change
GLS test (demonstration plate)
  1. Examine the well plate for the GLS test. Each row of wells contains a different sugar (Glucose, Lactose, Sucrose) and was inoculated with the indicated microbe. For each microbe, evaluate the test result as either A (acid) or - (negative) for each well. Record the combination of the outcomes as either AAA, AA-, A-A, A-- or ---


© 2003 - 2019 Josť de Ondarza, Ph.D.