Carbohydrate Fermentation Test

Carbohydrate Fermentation Test

Whenever a new micro-organism is discovered it becomes necessary to identify and catalogue it. For identification of this new microbe, it is first isolated and microscopically examined, and then further biochemically tested. Once these steps are carried out, we can identify this micro-organism. Biochemical tests are essential for the identification and classification of this micro-organism. Hence it is important to understand various biochemical tests and their functioning.

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There are various biochemical tests and we will go through these tests one by one in my upcoming post. The list of biochemical test that play an important role in microbiology are…

  1. Carbohydrate fermentation test.
  2. Sugar Mannitol fermentation test.
  3. IMViC test.
  4. Catalase
  5. Oxidase
  6. Huge and Leifson’s test
  7. Indole Production test
  8. Hydrogen sulphide production test
  9. Decarboxylation test
  10. Phenylalanine Deaminase test
  11. Urea Hydrolysis test
  12. Nitrate Reduction test
  13. Ammonia Production test
  14. Starch Hydrolysis test
  15. Casein Hydrolysis test
  16. Gelatine Hydrolysis test
  17. Lipid Hydrolysis test
  18. Dehydrogenase test
  19. Coagulase test
  20. Hemolysis production
  21. Triple Sugar Iron Agar test
  22. Litmus test
  23. Lysin Decarboxylation test

In this article, we are going to have a look on Carbohydrate fermentation test.


Carbohydrates are biological molecules which are made up of carbon, hydrogen and oxygen molecules. There are four types of carbohydrates and they are shown in the following figure.

Carbohydrates and its Types
Carbohydrates and its Types

Carbohydrates and its types

  • Monosaccharides
    Monosaccharides are the simple molecule of sugar. It is water-soluble and crystalline in nature. Examples are glucose, fructose, glyceraldehyde and galactose.
  • Disaccharides
    Disaccharides is a simple carbohydrate formed when two monosaccharide molecules are joined together and a water molecule is removed. Examples are lactose, sucrose and maltose.
  • Oligosaccharides
    Oligosaccharide is a polymer containing small number of monosaccharide.monosaccharide. Examples are fructo-oligosaccharide, galacto-oligosaccharide and mannan.
  • Polysaccharide
    Polysaccharide is complex carbohydrate containing the long chain of monosaccharides.Examples are starch, glycogen, cellulose and chitin.


Fermentation is a metabolic process in which bacteria or yeast convert substrate into a product like acid, gas or alcohol. The product formed in the fermentation process is of economic value.

Carbohydrate fermentation tests demonstrate fermentation of sugars like glucose, lactose or sucrose. The fermentation is noted by acid and gas production by bacterial cells.

Carbohydrate fermentation test


To determine fermentation of sugars (Glucose, Lactose and Sucrose) by bacterial cells.


1.Nutrient broth medium

a) Glucose broth medium

b) Sucrose broth medium

c) Lactose broth medium

2. Indicator – Phenol red or Andrade’s indicator.

3. Durham’s tube

4. Bacterial cell culture

The composition of Sugar Broth Media

  1. Peptone                                                 -1 gm
  2. Meat extract                                       -0.3 gm
  3. NaCl                                                       -0.5 gm
  4. Distil water                                         – 100 ml
  5. Indicator                                              -0.008 gm
  6. Sugar(glucose/lactose/sucrose) -0.5 gm


  1. Take three different tubes containing three different types of sugar broth and invert a Durham’s tube in it and screw cap the tubes.
  2. Further, the three sugar broth tubes are sterilized by autoclaving.
  3. After sterilization, the tubes are cooled down to room temperature and inoculated with a cell suspension in aseptic condition.
  4. The tubes are incubated at 37°C for 24 hours.
  5. After incubation, the tubes are examined for acid and gas production and results are noted down.


  1. The three sterile sugar broth tubes are inoculated with bacterial suspension and incubated for 24 hours. During incubation, the bacterial cells may or may not utilise sugar.
  2. If the cells utilize sugar, there is acid and gas production and this change is observed by a change in colour of broth.
  3.  First of all, in sugar broth phenol, red indicator is added this pH-sensitive indicator is added for determination of acid production. Initially, the broth tubes are red in colour and after incubation, if there is acid production, the colour of broth changes from red to yellow. If there is no change in colour of broth, then there is no acid production.
  4. Secondly, in sugar broth, a Durham’s tube is inverted to determine gas production. After incubation, if a gas bubble is observed then there is gas production.


Phenol red is a pH-sensitive indicator its pH range is at acidic pH it is yellow in colour and at alkaline pH, it is red in colour.


There are three possibilities and they are after incubation

  1. If the red colour of broth is observed then there is no fermentation.
  2. If the yellow colour of broth is observed then there is acid formation.
  3. If the yellow colour of a broth plus a gas accumulation in Durham’s tube is observed then there is acid plus gas formation.

Observation Diagram I

Observation Diagram II

Observation Diagram III


  • If there is no change in colour of a broth then the test is negative.
  • If the yellow colour of a broth is observed, fermentation is carried out resulting in acid production and it is indicated by the + sign
  • If the yellow colour of a broth along with gas bubble accumulation is observed then fermentation is carried out resulting in acid and gas production.


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