Phenol Red Broth Test: Carbohydrate Fermentation

Fermentation media are used to differentiate organisms based on their ability to ferment carbohydrates incorporated into the basal medium. Phenol Red Broth Medium with various added carbohydrates serves as a differential medium by aiding in differentiation of various species and genera by their ability to ferment the specific carbohydrate, with the production of acid or acid and gas. Moreover, the carbohydrate source can vary based on test requirements.

Principle

Carbohydrate fermentation is the process microorganisms use to produce energy. Most microorganisms convert glucose to pyruvate during glycolysis; however, some organisms use alternate pathways. A fermentation medium consists of a basal medium containing a single carbohydrate (glucose, lactose, sucrose, mannitol etc.) for fermentation. However, the medium may contain various color indicators. In addition to a color indicator to detect the production of acid from fermentation, a Durham tube is placed in each tube to capture gas produced by metabolism. The carbohydrate fermentation patterns shown by different organisms are useful in differentiating among bacterial groups or species.

Media

Phenol Red Broth is a general-purpose differential test medium typically used to differentiate gram negative enteric bacteria.  It contains peptone, phenol red (a pH indicator), a Durham tube, and one carbohydrate (glucose, lactose, or sucrose). Phenol red is a pH indicator which turns yellow below a pH of 6.8 and fuchsia above a pH of 7.4. If the organism is able to utilize the carbohydrate, an acid by-product is created, which turns the media yellow.  If the organism is unable to utilize the carbohydrate but does use the peptone, the by-product is ammonia, which raises the pH of the media and turns it fuchsia. When the organism is able to use the carbohydrate, a gas by-product may be produced. If it is, an air bubble will be trapped inside the Durham tube.  If the organism is unable to utilize the carbohydrate, gas will not be produced, and no air bubble will be formed.

Materials required for carbohydrate fermentation are:

1. Phenol Red Carbohydrate Fermentation Broth.
2. Bacterial culture.
3. Inoculation loop.
4. Incubator(370 C).

Phenol Red Broth is a general-purpose differential test medium typically used to differentiate gram negative enteric bacteria.  It contains peptone, phenol red (a pH indicator), a Durham tube, and one carbohydrate.  

Phenol red is a pH indicator which turns yellow below a pH of 6.8 and fuchsia above a pH of 7.4. If the organism is able to utilize the carbohydrate, an acid by-product is created, which turns the media yellow.  If the organism is unable to utilize the carbohydrate but does use the peptone, the by-product is ammonia, which raises the pH of the media and turns it fuchsia.  When the organism is able to use the carbohydrate, a gas by-product may be produced. If it is, an air bubble will be trapped inside the Durham tube.  If the organism is unable to utilize the carbohydrate, gas will not be produced, and no air bubble will be formed. 

Conclusion

To detect acidic and gaseous end products is used the Phenol Red Carbohydrate Fermentation Broth which is a liquid broth medium that contains a Single Carbohydrate (glucose, sucrose, or lactose), peptones, and phenol red pH indicator. Durham tubes are also present to trap gas. Carbohydrate fermentation produces acid and gas. A yellow broth means it is positive for carb. fermentation(Acid/A). Furthermore, an air bubble in the Durham tube of the yellow broth means it is also positive for gas formation. If the media is the same color as the negative control (neutral) or is pinker than the control(Alkaline/K), it is negative for the carbohydrate fermentation. After completing the labs and incubating the test tubes over a day, results shows the different end products that were formed depending on each individual organism. As glucose is converted into pyruvate through the process of glycolysis, pyruvate itself then gets converted into other end products. Most of the results from the end products were fairly even, some of them had formed gas shown in the Durham’s tube while others didn’t form gas at all.