Phenol Red Fermentation Test: What It Is And How It Works

Phenol Red is one of the most commonly used Methods of using this as a pH indicator in biology as well as medicine.

It can help to identify E. coli strains within the realm of microbiological culture.

Phenol Red Fermentation Test: What It Is And How It Works

One of its clinical diagnostic applications is the investigative nature of gastric and duodenal ulcers as well as its assessment of tear function in ophthalmology.

Today we will explore the phenol red fermentation test, its procedure and how you can interpret the data.

What Is Phenol Red?

Phenol red is a compound in the phenol phenolsulfonphthalein group.

It is present as a sulfonated hydroquinone When present in aqueous Solutions. When we take alkaline Solutions, the dye will form a hydrophilic dianion.

In particular, Phenol red broth can be used as a general purpose differential test medium that can detect gram-negative enteric bacteria.

Phenol red indicates the pH by turning yellow below a pH of 6.8 or fuschia above a pH of 7.4.

Also, so if the organism is able to utilize the specific carbohydrate, this turns the media yellow as an acid by-product is created.

Another thing to note is that If the organism is unable to utilize the carbohydrate but still uses the peptone, the final by-product will be ammonia which raises the pH and turns it the color fuchsia.

Similar to fermentation in the gut, when the organism uses the carbohydrate, a gas by-product will be produced; in this instance an air bubble will be trapped inside the tube.

Though, no not every organism will be able to utilize the carbohydrate and so gas will not be produced, and no air bubbles form.

Why Are Fermentation Tests Used?

Fermentation tests and their Media are utilized to differentiate organisms that are based on their ability for fermentation of certain carbohydrates incorporated into the basal medium.

When we consider phenol red; when added with various carbohydrates it serves as a differential medium.

It does this by aging in differentiation with the production of acid for the production of acid and gas.

Depending on the test will depend on the carbohydrate. however the common broth Media use with this test are :

  • Phenol red glucose broth
  • phenol red maltose broth
  • phenol red sucrose broth
  • phenol red lactose broth
  • phenol red mannitol broth

What Is Carbohydrate Fermentation?

Carbohydrates are synonymous with fermentation, and even within our diet and digestive system, many forms of carbohydrate will ferment in the gut.

Carbohydrate fermentation is essentially the process of microorganisms in which they use this to energy.

Many microorganisms convert glucose to pyruvate during the process of glycolysis, which is one of the energy pathways used for creating energy for humans.

However, certain organisms use alternate pathways to create energy.

One thing to note is that a fermentation medium will consist of a basal medium that contains a single form of carbohydrate, i.e. glucose or lactose.

They will use this for fermentation. However the media may contain color indicators, and a color indicator can detect the production of acid via fermentation.

To achieve this, a tube is placed in each tube to trap gas produced by this metabolic process.

Carbohydrate fermentation and their patterns which are shown by the various organisms can be useful in differentiating amongst different groups or species of bacteria.

How To Carry Out The Fermentation Test

How To Carry Out The Fermentation Test

The first step is to aseptically inoculate each of the test tubes used in the test with the test microorganism.

You can use an inoculating needle or loop to achieve this.

The best results will be to inoculate each test tube with a round one to two drops of an 18 to 24-hour brain heart infusion broth culture.

Next comes the incubation period. you want to incubate tubes at around 35 to 37 degrees C for 18 to 24-hours.

Finally you want to check for any color changes or the formation of a gas.

Interpretation Of The Results

The two things you’re looking out for are acid production or gas production and we will look at each individually now.

Acid Production

You will either receive a positive or negative acid production result. For a positive result the liquid in the Tube will turn yellow after the incubation period.

This is noticeable by the change in the color of the phenol red indicator.

What this represents is a drop-in the total PH and this is down to the production of the acids via the fermentation of the carbohydrate that is present in the media.

In contrast to this, a negative test is also possible and the tube containing the median will simply remain red.

This indicates that bacteria did not comment on that particular carbohydrate source that is present in the media.

Gas Production

As mentioned, fermentation of carbohydrates typically has a by-product of releasing gas.

For a positive gas production result, a bubble will be seen in the inverted Durham tube.

Depending on the amount of gas produced will depend on how big the bubble is, and a small bubble represents a small amount of gas produced, whilst a big bubble represents a large amount of gas being produced.

There may also be a negative gas production, and as you have probably already guessed; no bubbles will be produced in the inverted on tube, which simply indicates that bacteria does not produce gas from the fermentation of the carbohydrate of choice that is present in the media.


As with any testing, they’re always going to be limitations.

One such limitation is that due to nutritional variation, some of the strains may group poorly or fail to grow on a particular medium and this is down to some of the strains.

In addition to this, some carbohydrates may result in an acid reaction when added to the basal medium.

To prevent false positives and to ensure accuracy, using inoculated control tubes in parallel with the fermentation tests will act as a strong control.

One final thing to note is that phenol red broth should not be intended for use in any diagnosis of disease or other conditions found in the human body, and it’s strictly for testing purposes only.

Final Thoughts

This test Is extremely useful as it can help to identify the fermentation reaction of a number of carbohydrates for the differentiation of microorganisms.

Along with this it is also useful for identifying gram negative bacilli; and especially the gram-negative enterobacteriaceae.

Interpreting the results is quite simple and if you follow the steps above, you will be able to identify the fermentation of the specific carbohydrate that is present in the media, whether gas has been produced and whether there is any acid production.

Jennifer Dawkins

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