Fermentation Test – Principle, Procedure, Uses And Interpretation

Bacteria are a type of single cell microorganisms that have the ability to form organic compounds through the process of metabolizing carbohydrates called fermentation.

However, not all bacteria have this ability and thus, it is so important when it comes to identifying certain microorganisms. 

So, one method that many microbiologists use to classify and identify microorganisms is through the fermentation test.

Fermentation Test - Principle, Procedure, Uses And Interpretation

This test uses a purple broth base to study carbohydrate fermentation reactions with certain bacterias. 

So, here we are going to dive into the principle, procedure, uses and results interpretations of the fermentation test using purple broth.

Stick around to learn everything you need to know about this important classifying test! 


When carbohydrate fermentation is under way, the principle of the fermentation test states that the process will result in the acidification of the medium (in this case, the purple broth).

Purple broth contains peptone as well as the pH indicator known as bromocresol purple, hence why the name purple broth.

This can then be detected through a pH indicator dye to show if fermentation has taken place. 

If fermentation has indeed taken place, then the pH indicator dye should change color to reflect this.

For the fermentation test, it should turn yellow after fermentation or remain purple if fermentation has not taken place. 

However, not all microorganisms convert glucose to  pyruvate and so, the medium used for the fermentation test needs to contain a basal medium that features a single carbohydrate.

This carbohydrate can be anything from glucose, sucrose, lactose, the list goes on, but it needs to be added in a concentration between 0.5 to 1% as this will help against the depletion of the carbohydrate and potentially reversing the fermentation reaction.

The medium should also contain a PH indicator to detect the presence of acid to show that fermentation has happened.

A Durham tube can also be placed to capture the gas produced by metabolism and this can contribute to the identifying of different bacterial species. 


To start your own fermentation test, you will first need to isolate the medium and allow it to warm itself to room temperature. This should be done before inoculation. 

You will also need to inoculate the purple broth for at least 18 to 24 hours beforehand. This will leave you with a pure culture of your organism of choice. 

Now that everything is ready, it’s time to inoculate your control tube of purple broth. Place it parallel to the carbohydrate media. 

Now that inoculation is over, you will need to incubate the inoculated media for 3 to 5 days (or, up to 30 days depending on the microorganism).

The media should remain between the temperature of 95 to 98.60 degrees Fahrenheit (35 to 37 degrees Celscius) and be allowed to incubate aerobically.

Check on your media daily to see if there is any development in the color of your medium. This color change is vital to your results so take notes every day that passes during the incubation period. 

Then, you can gather up your results! 

Interpretation Of Results

If your medium has changed color from purple to yellow over the incubation period then this is a positive result.

It is a sign that carbohydrate fermentation has happened and as part of the reaction, acids have been produced which have reacted with the pH indicator in the medium, turning the color from purple to yellow.

This means that the microorganism used in this test is able to ferment carbohydrates.

Another sign of a positive result is the presence of air bubbles – this means that gas has been produced during the fermentation reaction, indicating that it has indeed taken place.

However, trapped bubbles can lead to false positive results so make sure that you release all the trapped bubbles in the Durham tube prior to your test. 

If your medium has not changed color (compare with your control tube just to double check) then this is a negative result.

This means that no carbohydrate fermentation reaction has taken place, thus no acids have been produced to change the color of your pH indicator. 

Uses Of The Fermentation Test

Like we said earlier, the fermentation test is highly important when it comes to identifying and classifying microorganisms including bacteria.

It plays an important role in separating certain bacteria from others and helps towards understanding their abilities further so they can be used to evoke certain reactions. 


And that is everything you need to know about the Fermentation test! 

This test is very important to classify certain bacteria and understand their abilities more, and luckily this is also a very easy test to carry out.

However, it is not without its limitations as it does take some time to carry out. Despite this, the fermentation test remains to be a popular way to define bacteria.

Jennifer Dawkins