- Phenol Red Carbohydrate Fermentation Broth.
- Bacterial culture.
- Inoculation loop.
- Incubator(370 C).
I. Preparation of Carbohydrate Fermentation Broth
- Weigh and dissolve trypticase, Sodium chloride, and Phenol red in 100 ml distilled water and transfer into conical flasks.
- Add 0.5% to 1% of desired carbohydrate into all flasks.
- Insert inverted Durham tubes into all tubes, the Durham tubes should be fully filled with broth.
- Sterilize at 1150 C for 15 minutes.
- 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).
- Transfer the sugar into screw capped tubes or fermentation tubes and label properly.
Ingredients of the Fermentation Broth:
- Trypticase: 1g
- Carbohydrate: 0.5g
- Sodium Chloride: 0.5g
- Phenol red : 0.0189mg
*Autoclave at 115o C for 15 minutes
II. Inoculation of Bacterial Culture into the Phenol Red Carbohydrate Broth
- Aseptically inoculate each labeled carbohydrate broth with bacterial culture.(keep uninoculated tubes as control tubes).
- Incubate the tubes at 18-24 hours at 37oC.
- Observe the reaction.
- After inoculation into a particular sugar, sterilize the loop in order to avoid cross contamination of the tube with other sugars.
- Keep uninoculated sugar tubes as control tubes.
- Do not use the tubes with Durham tubes that are partially filled or with bubbles.
- Over incubation will help the bacteria to degrade proteins and will result give false positive results.
Results of carbohydrate fermentation test
- 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.
- 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.
- 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.