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Carbohydrate Fermentation Test


Materials Required:


  1. Phenol Red Carbohydrate Fermentation Broth.
  2. Bacterial culture.
  3. Inoculation loop.
  4. Incubator(370 C).



I. Preparation of Carbohydrate Fermentation Broth


  1. Weigh and dissolve trypticase, Sodium chloride, and Phenol red in 100 ml distilled water and transfer into conical flasks.
  2. Add 0.5% to 1% of desired carbohydrate into all flasks.
  3. Insert inverted Durham tubes into all tubes, the Durham tubes should be fully filled with broth.
  4. Sterilize at 1150 C for 15 minutes.
  5. Important: Do not overheat the Phenol red Carbohydrate fermentation broth. The overheating will result in breaking down of the molecules and form compounds with a characteristic color and flavour. The process is known as caramelization of sugar (the browning of sugar).
  6. Transfer the sugar into screw capped tubes or fermentation tubes and label properly.

Ingredients of the Fermentation Broth:


  1. Trypticase: 1g
  2. Carbohydrate: 0.5g
  3. Sodium Chloride: 0.5g
  4. Phenol red :  0.0189mg

         *Autoclave at 115o C for 15 minutes

II. Inoculation of Bacterial Culture into the Phenol Red Carbohydrate Broth


  1. Aseptically inoculate each labeled carbohydrate broth with bacterial culture.(keep uninoculated tubes as control tubes).
  2. Incubate the tubes at 18-24 hours at 37oC.
  3. Observe the reaction.



  1. After inoculation into a particular sugar, sterilize the loop in order to avoid cross contamination of the tube with other sugars.
  2. Keep uninoculated sugar tubes as control tubes.
  3. Do not use the tubes with Durham tubes that are partially filled or with bubbles.
  4. Over incubation will help the bacteria to degrade proteins and will result give false positive results.


Expected Results:


                                                                              Results of carbohydrate fermentation test


  1. Acid production: Changes the medium into yellow color- organism ferments the given carbohydrate and produce organic acids there by reducing the ph of the medium into acidic.
  2. Acid and Gas production: Changes the medium into yellow color-organism ferments the given Carbohydrate and produce organic acids and gas. Gas production can be etected by the presence of small bubbles in the inverted durham tubes.
  3. Absence of fermentation: The broth retains the red color. The organism cannot utilize the carbohydrate but the organism continues to grow in the medium using other energy sources in the medium.







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