Carbohydrate Fermentation Test

Objective:

To determine the ability of microorganism to ferment the given carbohydrates with acid or acid and gas end products. 

Principle:

Fermentation is the metabolic process to breaking down or catabolism of the sugars under anaerobic conditions. The result of this process is production of energy source ATP by substrate level phosphorylation, in the absence of aerobic respiration electron transport chain. Microorganisms utilize the carbohydrates by their enzymatic reactions and produce organic acids, acid with gas and alcohol. The types and proportion of the products depend on bacterial species and carbohydrates.

 Formation of acids in sugar broths can be detected by using the pH indicator phenol red which is red at neutral pH and changes to yellow at acidic conditions, slight amount of acid can cause color change. Gas formation can be detected by using an inverted ‘Durham’s tube’.

Materials required:

-24 Hours old bacterial cultures (E.coli. Pseudomonas sp. Klebsiella sp. and Salmonella sp.)

-Peptone broth. Nutrient broth with pH indicator (Phenol red)

-Test tubes and Durham tube

Procedure:

- Prepare and sterilize the tubes containing 5 ml of peptone broth/nutrient broth for each sugar lactose, sucrose and glucose with pH indicator.

- Carefully insert the Durham tube in the broth tube in inverted position without air bubble.

- Incubate the tubes at 37°C for 24 - 48 hours

Result:

-E.coli and Klebsiella sp., shows both yellow color changes in the medium and bubble formation inside Durham’s tubes in all the sugars.

-Salmonella sp. shows yellow color changes in the medium and gas bubble formation in glucose, only yellow color change in sucrose and no reaction in lactose sugar.

-Pseudomonas sp. shows yellow color change in glucose, no reaction in sucrose and lactose.

Interpretation:

If the medium changes colorless to yellow color and gas bubble formation in Durham’s tube that indicates acid production and gas production. In some cases the gas does not evolved during fermentation. If no changes in the medium it indicates the sugars not degraded by the organisms.

Carbohydrate Fermentation Medium Composition:

Peptone     10.0 g

Sodium chloride   5.0 g

Sugar    1 g

Phenol red   5 ml

Distilled water    1000ml

pH   - 7.00

Sugar fermentation by different bacterial species