Download Lab Module 8: Phenol-Red Carbohydrate Fermentation Broths

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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.
Phenol-Red
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.
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 37°C.
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
Lactose
Sucrose
E. coli
AG
AG
V
P. vulgaris
AG
-
AG
P. aeruginosa
-
-
-
S. aureus
A
A
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”?