Phenol Red Carbohydrate Fermentation Test

In this phenol red carbohydrate fermentation test post we have briefly explained phenol red carbohydrate fermentation test principle, objectives, requirements, carbohydrate fermentation test, uses and limitations of phenol red test.

Phenol Red Test Definition

What does phenol red test for? Phenol red broth test is a differential medium. It evaluates an organism’s ability to ferment glucose as well as convert pyruvic acid, the end product of glycolysis, into gaseous by-products. Carbohydrate fermentation test is a typical test for identifying Gram-negative enteric bacteria that are all glucose fermenters but only some of which generate gas.

Phenol red broth test medium, like MSA, contains phenol red, a pH indicator. Acidic by-products are created when an organism is capable of digesting the sugar glucose, and the pH indicator turns yellow. Proteus mirabilis and Shigella dysenteriae (Left), as well as Escherichia coli (Right), are capable of fermenting glucose. Non-fermenter Pseudomonas aeruginosa (Center).

Pyruvate is the end product of glycolysis. Gas is produced by organisms that can convert pyruvate to formic acid and formic acid to H2 and CO2 using the enzyme formic hydrogen lyase. This gas is trapped in the Durham tube and appears at the tube’s top as a bubble. Proteus mirabilis (far right) and Escherichia coli (far left) are both gas generators. Shigella dysenteriae ferments glucose but does not create gas (far left).

Phenol Red Test Principle

Peptone, phenol red (a pH indicator), and the carbohydrate to be evaluated are all included in the Phenol Red Fermentation medium. Phenol red is yellow at pH 6.8 and red at pH > 7.4, therefore if a bacterium ferments a sugar to acid, the result will be a yellow colour.

An inverted Durham tube is also found in phenol red tubes. If bacteria create a gas during fermentation, the Durham tube will capture the gas and a bubble will appear. In some cases, an orange tint appears in the tube; this indicates a pH greater than 6.8 and should not be deemed positive.

Be wary of tubes that generate acid at first but then turn red or pink as nitrogenous chemicals are formed. This type of reaction is characteristic of oxidative, not fermentative metabolism.

Phenol Red Test Essentials


Caesin Peptone: 10.0 gm

Phenol Red: 8.0 mg

Sodium Chloride: 5.0 gm

Demineralized water: 1000ml


Phenol red broth is a multipurpose differential test medium that is commonly used to distinguish gram-negative enteric bacteria. Peptone, phenol red (a pH indicator), a Durham tube, and one carbohydrate are all included (glucose, lactose, or sucrose).

Phenol red is a pH indicator that becomes yellow when the pH is below 6.8 and fuchsia when the pH is over 7.4. If the organism can use the carbohydrate, an acid by-product is produced, turning the media yellow.

If the organism cannot use the carbohydrate but can use the peptone, the by-product is ammonia, which elevates the pH of the media and causes it to turn fuchsia. A gas by-product may be created when the organism is able to utilise the glucose.

An air bubble will be trapped inside the Durham tube if this is the case. Gas will not be created if the organism is unable to use the carbohydrate, and no air bubble will occur.

Phenol Red Test Procedure

Using an inoculating needle or loop, aseptically inoculate each test tube with the test microorganism. Alternatively, inoculate each test tube with 1-2 drops of the target organism’s 18- to 24-hour brain-heart infusion broth culture. Incubate tubes for 18-24 hours at 35-37°C. Look for any colour changes or gas development.

Phenol Red Carbohydrate Fermentation Test

Phenol Red Broth Test Results, Image Source :

Phenol Red Test Results

Acid production

Positive: The liquid in the tube becomes yellow after incubation (indicated by the change in the colour of the phenol red indicator).

Negative: The medium-filled tube will remain red, suggesting that the bacteria are unable to ferment the carbohydrate source in the media.

Gas Production

Positive: A bubble in the inverted Durham tube it may be little or large depending on the amount of gas produced.

Negative: No bubble in the inverted Durham tube, indicating that the bacterium does not create gas from the fermentation of the carbohydrate present in the media, indicates that the organism is anaerogenic.

Quality Control

Phenol red broth base was made, packaged, and processed in compliance with current Good Manufacturing Practice laws for each lot number.

Control organism testing should be carried out in compliance with laboratory quality control protocols.

Control organisms with a positive and negative reaction to each carbohydrate examined should be chosen. Sample results should not be published if quality control results are abnormal.

Phenol Red Test Applications

  1. For microorganism differentiation, it is advised that the fermentation reaction of carbohydrates be determined using carbohydrate fermentation test.
  2. It’s excellent for recognizing Gram-negative bacteria, notably those belonging to the Enterobacteriaceae family.

Phenol Red Test Limitations

  1. Carbohydrate fermentation test is not meant to be used in humans to diagnose sickness or other disorders.
  2. Some strains may grow badly or fail to thrive on this medium as a result of nutritional differences. An acid reaction could occur if some carbs are added to the basal medium.

We hope phenol red carbohydrate fermentation test helped you a lot. You can learn more microbial biochemical analysis at

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