CARBOHYDRATES FERMENTATION TEST

CARBOHYDRATE FERMENTATION TEST

AIM

            To test the ability of the microorganisms to utilize and digest several Carbohydrate sugars.

PRINCIPLE   

        Carbohydrates are biological molecules which are made up of carbon, hydrogen and oxygen molecules. There are three types of Carbohydrates based on their structure and number of carbon atoms present, they are Monosaccharides (simple sugar containing 3-7 carbon atoms), Disaccharides (comprising of two monosaccharides linked together by the glycosidic bond) and Polysaccharides (containing 8 or more monosaccharide molecules). Carbohydrate fermentation is the process by which the microorganism utilizes the Carbohydrate sugars to produce energy in the form of ATP, the ultimate energy source of the organism.  The purpose of the Carbohydrate fermentation test is to check whether the bacteria can ferment a specific carbohydrate or not. Carbohydrate fermentation patterns are useful in differentiating among bacterial groups or species particularly Enterobacteriaceae family.

           Fermentation of the Carbohydrates by bacteria under the anaerobic condition is carried out in the fermentation tube, which comprises of Durham tube for the detection of the Gas production. A fermentation medium Carbohydrate broth is composed of a specific Carbohydrate (Glucose, Sucrose, or Cellulose) along with a pH indicator (Phenol red, Bromocresol purple, Bromothymol blue or Andrade’s indicator). When the bacteria ferment Carbohydrates, various Organic acids and other products (Lactic acid, Formic acid, Acetic acid, Butyric acid, Butyl alcohol, Acetone, Ethyl alcohol, CO₂ and Hydrogen) are produced. The production of the acid lowers the pH (Acidic pH - below pH of 6.8) of the test Carbohydrate broth, which is detected by the color change from Red to Orange or Yellow because of the pH indicator. Durham tubes are inserted upside down in the test tubes to detect Gas production. If the test organism produces gas, the gas displaces the media present inside the tube and gets trapped producing a visible air bubble. Color change into Yellow only occurs when a sufficient amount of acid is produced, as bacteria may utilize the Peptone producing alkaline by-products. Based on the characteristics reactions observed, bacteria can be classified as: (i) Fermenter with acid production only, (ii) Fermenter with acid and gas production and (iii) Non-fermenter.

 MATERIALS REQUIRED

1. Sterile Test tubes
2. Carbohydrate broth with Phenol Red (Peptone – 3.10 g/L; NaCl – 5 g/L; Beef extract – 1 g/L; Phenol Red – 0.0189 mg/L; Carbohydrate source – 10 g/L)
3. Inoculation loop
4. Incubator
5. Durham's tube 

PROCEDURE

a)     Prepare the Carbohydrate broth by mixing all the ingredients and dissolve it.

b)     Fill the test tubes with 6 to 8 ml of Carbohydrate broth.

c)     Insert a Durham tube in inverted position to detect the Gas production.

d)   Autoclave the prepared test media (at 121 °C for 15 minutes) to sterilize. Do not overheat the Carbohydrate fermentation broth. The overheating will result in breaking down the molecules (pH indicator) and form compounds with a characteristic color and flavor. The process is known as the Caramelization of sugar (the browning of sugar).

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

f)      Transfer the Carbohydrate broth into Screw capped tubes or Fermentation tubes and label.

g)     Aseptically inoculate each test tube with the test bacterial culture using an Inoculation loop.

h)    Incubate tubes at 37 °C for 24 hours. Longer incubation periods may be required to confirm a negative result.

i)      After incubation, examine the tubes for acid and gas production.

OBSERVATION AND RESULTS

·       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. The Gas production can be detected 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.

A – Acid production; A/K – Gas production; Control – Absence of fermentation

Figure – 1: Carbohydrate fermentation test