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Lab Module 8: Phenol-Red Carbohydrate Fermentation Broths Introduction Fermentation is a metabolic process performed by many types of bacteria. Fermentation allows the production of ATP without the need for atmospheric oxygen (O2). This process usually involves the following steps: Step One: Step Two: Step Three: Carbohydrate Glucose Glucose Pyruvate Pyruvate Fermentation By-products

There are many different kinds of fermentable carbohydrates. Not all bacteria can ferment all of these carbs. The ability (or inability) of a particular species to ferment a particular carbohydrate depends on the presence (or absence) of an enzyme system to convert that carb to glucose (Step One, above). Among those bacteria that can ferment a particular carbohydrate, there are also a variety of types of by-products. Lactobacillus (and human muscle cells) forms lactic acid as a fermentation by-product. Acetobacter forms acetic acid. Many bacteria form mixtures of products including liquids and gases. The particular types of by-products depends on the particular enzyme systems used to perform Step Three (above). One commonality among these forms of fermentation is the production of acids. Phenol-Red Carbohydrate Fermentation Broths are useful for helping to characterize bacteria based on their fermentation abilities. Each PR-Carb broth contains the following: Carbohydrate Each broth contains a single fermentable carbohydrate. There are other organic nutrients (such as amino acids) that are not fermentable but can be used for growth. This is a pH indicator that is RED at pH 7 or higher (alkaline) but turns YELLOW at low pH (acidic). The broth is initially pH neutral. So, if fermentation occurs, and acid by-products are formed, the solution will turn yellow.

Phenol-Red

Durhamn Tube This is a small test tube that is inverted (upside-down) in the broth. If gases are produced, they will be trapped in the Durham Tube and a gas bubble will form. Thus, two things can be determined from a PR-Carb broth. First, can the species ferment the carbohydrate or not? Second, if the species can ferment the carb, is it a gas-producing kind of fermentation? One word of caution: if PR-Carb broths are incubated for too long (more than two or three days), acids may be further metabolized and converted back to neutrality or even

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alkalinity. In these situations, a yellow broth will turn back to red. So PR-Carb broths need to be evaluated within a 2-3 days. DAY ONE ACTIVITIES (Performed by pairs of students) Inoculate one PR-Glucose, one PR-Lactose, and one PR-Sucrose set for each of the following (12 broths total per pair of students): a. Escherichia coli b. Proteus vulgaris c. Pseudomonas aeruginosa d. Staphylococcus aureus When you have completed your inoculations, combine your 12 broths into the same test tube rack with the broths from one or two other groups (this will save racks and incubator space). Incubate at 37C. DAY TWO ACTIVITIES The following data table contains the EXPECTED results. Complete the table by entering your OBSERVED results using the following symbols: A = Acid only AG= Acid with Gas - = Negative (no fermentation)

Glucose E. coli AG AG

Lactose V

Sucrose

P. vulgaris P. aeruginosa S. aureus

AG A

AG A

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QUESTIONS 1. Why does the formation of yellow color indicate fermentation?

2. What can go wrong during the culturing process to lead to erroneous results?

3. Even if everything is performed correctly, sometimes observed results do not match expectation. Give some reasons why this might happen.

4. Suppose you have a culture of unknown bacteria. Describe how you would use a PRCarb broth (or several) to help identify your unknown.

5. What does V mean? Can you observe V?