Carbohydrate Fermentation Test: Uses, Principle, Procedure, Results

The carbohydrate fermentation test is used to determine whether or not bacteria can ferment a specific carbohydrate. Carbohydrate fermentation patterns are useful in differentiating among bacterial groups or species.

It tests for the presence of acid and/or gas produced from carbohydrate fermentation. Basal medium containing a single carbohydrate source such as glucose, lactose, sucrose, or any other carbohydrate is used for this purpose.  A pH indicator (such as Andrade’s solution, bromocresol purple (BCP), bromothymol blue (BTB), or phenol red) is also present in the medium; which will detect the lowering of the pH of the medium due to acid production.  Small inverted tubes called Durham tube is also immersed in the medium to test for the production of the gas (hydrogen or carbon dioxide).

The term fermentation is often used to describe the breaking down or catabolism of a carbohydrate under anaerobic conditions. Therefore, bacteria capable of fermenting a carbohydrate are usually facultative anaerobes.

Uses of Carbohydrate Fermentation Test 

Carbohydrate fermentation patterns can be used to differentiate among bacterial groups or species.

  1. All members of Enterobacteriaceae family are glucose fermenters (they can metabolize glucose anaerobically).
  2. Maltose fermentation differentiates Proteus vulgaris (positive) from Proteus mirabilis (negative).
  3. Both Neisseria gonorrhoeae (gonococci) and Neisseria meningitides (meningococci) ferments glucose, but only meningococci ferments maltose.
  4. Rapid carbohydrate utilization tests (RCUTs) can be performed to identify Corynebacterium diphtheriae and other Corynebacterium species.

You may like “Differences between Neisseria gonorrhoeae (gonococci) and Neisseria meningitides (meningococci)”


When microorganisms ferment carbohydrate an acid or acid with gas are produced. Depending upon the organisms involved and the substrate being fermented, the end products may vary. Common end-products of bacterial fermentation include lactic acid, formic acid, acetic acid, butyric acid, butyl alcohol, acetone, ethyl alcohol, carbon dioxide, and hydrogen.
The production of the acid lowers the pH of the test medium, which is detected by the color change of the pH indicator. Color change only occurs when a sufficient amount of acid is produced, as bacteria may utilize the peptone producing alkaline by-products.

Common pH Indicators for Carbohydrate Fermentation Media (Source: ASMCUE)

Phenol red is commonly used as a pH indicator in carbohydrate fermentation tests. Other pH indicators such as bromocresol/bromocresol purple (BCP), bromothymol/bromothymol blue (BTB), and Andrade’s can be used.

Durham tubes are inserted upside down in the test tubes to detect gas production. If the test organisms produce gas, the gas displaces the media present inside the tube and gets trapped producing a visible air bubble.

Based on the characteristics reactions observed, bacteria can be classified as:

  • Fermenter with acid production only
  • Fermenter with acid and gas production
  • Non-fermenter

Test Procedure

Phenol red carbohydrate broth is commonly used in carbohydrate fermentation tests. The carbohydrate sources can vary based on your test requirements.

Common broth media are:

  1. Phenol red glucose broth
  2. Phenol red lactose broth
  3. Phenol red maltose broth
  4. Phenol red mannitol broth
  5. Phenol red sucrose broth

Preparation and Composition of the Media 

Get specific phenol red carbohydrate test media from the commercial suppliers or phenol red broth base and add specific carbohydrate source based on your test requirements, or you can prepare media mixing the following ingredients.

Composition of Phenol Red Carbohydrate Broth

  • Trypticase or protease peptone No. 3: 10 g
  • Sodium chloride (NaCl): 5 g
  • Beef extract (optional): 1 g
  • Phenol red (7.2 ml of 0.25% phenol red solution): 0.018 g
  • Carbohydrate source: 10 g

A. Preparation of the media 

  • Prepare broth media by mixing all ingredients in 1000 mL of distilled/deionized water and heating gently to dissolve it (Note: Use a single carbohydrate source based on your requirements).
  • Fill 13 x 100 mm test tubes with 4-5 ml of phenol red carbohydrate broth.
  • Insert a Durham tube to detect gas production.
  • Autoclave the prepared test media (at 121°C for 15 minutes) to sterilize. The sterilization process will also drive the broth into the inverted Durham tube. (Note: When using arabinose, lactose, maltose, salicin, sucrose, trehalose, or xylose, autoclave at 121°C for only 3 minutes as these carbohydrates are subject to breakdown by autoclaving)

The prepared broth media will be a light red color and the final pH should be 7.4 ± 0.2.

Alternatively, prepare phenol red broth base, heat sterilize and cool to 45°C. Prepare specific carbohydrate solution separately, and filter the solution using membrane filter (pore size: 0.45 μm). Add carbohydrate solution to the broth base and mix it. The preferred carbohydrate concentration is 1%.

B.Inoculation and Incubation

  • Aseptically inoculate each test tube with the test microorganism using an inoculating needle or loop. Alternatively, inoculate each test tube with 1-2 drops of an 18- to 24-hour brain-heart infusion broth culture of the desired organism.
  • Incubate tubes at 35-37°C for 18-24 hours. Longer incubation periods may be required to confirm a negative result.

C.Interpretation of the results 

Image source: ASMCUE
  1. Acid production
    1. Positive: After incubation, the liquid in the tube turns yellow (indicated by the change in the color of the phenol red indicator). It indicates that there is a drop in the pH because of the production of the acid by the fermentation of the carbohydrate (sugar) present in the media.
      NOTE:*If you are using other pH indicators please refer to Table 1 for their corresponding colors in particular pH.
    2. Negative: The tube containing medium will remain red, indicating the bacteria cannot ferment that particular carbohydrate source present in the media.
  2. Gas production
    1. Positive: A bubble (small or big depending on the amount of gas produced) will be seen in the inverted Durham tube.
    2. Negative: There won’t be any bubble in the inverted Durham tube i.e. bacteria do not produce gas from the fermentation of that particular carbohydrate present in the media i.e. anaerogenic organisms.

References and further readings

  1. Carbohydrate Fermentation Protocol by Microbe Library

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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