carbohydrate fermentation test

CARBOHYDRATE FERMENTATION TEST

AIM:

To perform a carbohydrate fermentation test to a given bacterial culture.

PRINCIPLE:

 The ability of microorganisms to ferment carbohydrates and the types of products formed are very useful in identification. A given carbohydrate may be fermented to a number of different end products depending upon the microorganism involved. These end products (alcohols, acids, gases, or other organic molecules) are characteristic of the particular microorganisms. For example, if fermenting bacteria are grown in a liquid culture medium containing the carbohydrate glucose, they may produce organic acids as by-products of the fermentation. These acids are released into the medium and lower its pH. If a pH indicator such as phenol red or bromcresol purple is included in the medium, the acid production will change the medium from its original color to yellow. Gases produced during the fermentation process can be detected by using a small, inverted tube, called a Durham tube (named after Herbert Edward Durham, English bacteriologist, 1866–1945), within the liquid culture medium. After adding the proper amount of broth, Durham tubes are inserted into each culture tube. During autoclaving, the air is expelled from the Durham tubes, and they become filled with the medium. If gas is produced, the liquid medium inside the Durham tube will be displaced, entrapping the gas in the form of a bubble.

MATERIALS REQUIRED:

24-hour trypticase soy broth bacterial cultures, phenol red (or bromo cresol purple) dextrose, lactose, and sucrose peptone broths with Durham tubes, Bunsen burner, inoculating loop and forceps, test-tube rack, incubator set at 35°C, trypticase agar base tubes, Inoculating needle and forceps, 100-ml beakers containing 70% ethanol,1-ml pipette and pipettor,

 PROCEDURE:

Phenol Red Carbohydrate Broth is commonly used to perform this test. The carbohydrate source can varies based on the 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

Media Preparation 

Composition of Phenol Red Carbohydrate Broth

§  Trypticase or proteose 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 

1. The media was prepared by mixing all ingredients in 1000 mL of distilled water (The preferred carbohydrate concentration is 1%) and heating gently to dissolve it.

2. The test tubes were filled with 4-5 ml of phenol red carbohydrate broth.

3. A Durham tube was inserted in to the media tube to detect gas production.

4. The prepared test media was autoclaved (at 121°C for 15 minutes) to sterilize and the broth pH was adjusted to 7.

B. Inoculation and Incubation

1. Aseptically inoculate each test tube with the test microorganism using an inoculating needle or loop.

2. The tubes were incubated at 35-37°C for 18-24 hours. Longer incubation periods may be required to confirm a negative result.

C. Interpretation of the results 

Acid production:

1.      Positive: After incubation the liquid in the tube turns yellow. It indicates that there is drop in the pH because of the production of the acid by the fermentation of the carbohydrate (sugar) present in the media.

2.      Negative: The tube containing medium will remain red, indicating the bacteria cannot ferment that particular carbohydrate source present in the media.

Gas Production

3.      Positive: A bubble (small or big depending up the amount of gas produced) will be seen in the inverted Durham tube.

4.      Negative: There won’t be any bubble in the inverted Durham tube i.e. bacteria does not produce gas from the fermentation of that particular carbohydrate present in the media i.e. anaerogenic organism.