Carbohydrate fermentation test

 

Most microorganisms obtain their energy through a series of orderly and integrated enzymatic reactions leading to the biooxidation of a substrate, frequently a carbohydrate.  The major pathways by which this is accomplished are aerobic/anaerobic cellular respiration and fermentation.  Fermentation is an anaerobic metabolic process in which the final electron acceptor is an organic substrate instead of oxygen.  The actual end products depend on the factors like organism involved, nature of substrate, temperature of incubation and acidity produced. The most commonly produced end product is lactic acid even though many other substances are also produced as a result of different fermentation pathways.  In fermentation, substrates such as carbohydrates and alcohols undergo anaerobic dissimilation and produce an organic acid (lactic, formic or acetic acid) that may be accompanied by gases such as hydrogen or carbon dioxide.  Many bacteria are facultative anaerobes and are able to carry out fermentation.

Aim

To determine the ability of microorganisms to degrade and ferment carbohydrates with the production of acid and gas.

Principle

Fermentative degradation under anaerobic conditions is carried out in a fermentation broth tube containing a Durham tube, an inverted inner vial for the detection of gas production.  A typical carbohydrate fermentation medium contains (1) nutrient broth ingredients for the support of growth of all organisms (2) a specific carbohydrate that serves as the substrate for determining the organism’s fermentative capabilities and (3) the pH indicator phenol red, which is red at the neutral pH (7) and changes to yellow at a slightly acidic pH of 6.8, indicating that slight amounts of acid will cause a color change.  Following incubation with organisms, carbohydrates that have been fermented with the production of acidic wastes will cause phenol red to turn yellow, thereby indicating a positive reaction.  In some cases, acid production is accompanied by the evolution of gas that will be visible as a bubble in the inverted tube.  Cultures that are not capable of fermenting a carbohydrate substrate will not change the indicator, and the tubes will appear red; there will not be a concomitant evolution of  gas.  This is a negative reaction.     

Requirements

24 to 48 hr broth culture of species Bacillus, Streptococcus, Staphylococcus, Pseudomonas and E.coli.

Phenol red dextrose, maltose, mannitol, sucrose and lactose broths.

Test tubes, Durham tubes, bunsen burner, inoculating loop, glass marking pencil etc.

 Procedure

1. Using sterile techniques, inoculate each experimental organism into the appropriately labeled fermentation tubes using inoculation loop. Do not shake the tubes as it may cause the air bubbles to get trapped in Durham tube. 2.      Incubate all tubes for 24 hrs at 37^O C.

 Result

Microorganisms able to ferment the given sugars changed the color of the medium into yellow indicating the acid production.  In some cases, it was accompanied by gas bubble formation in the Durham tubes.Microorganisms unable to ferment the given sugars did not change the color of the medium indicating a negative reaction. The detailed result is given in table- 1.

 

 Table 1. Production of acid or acid/gas in fermentation tubes by different bacterial species

 

Bacterial species	Carbohydrate
	Glucose	Maltose	Mannitol	Sucrose	Lactose
Bacillus	AG	A	__	A	__
Streptococcus	AG	AG	__	AG	AG
Staphylococcus	A	A	__	A	A
Pseudomonas	__	__	__	A	__
E. coli	AG	AG	AG	V	AG

Note: A = acid only (broth turned yellow)

       AG = acid and gas (broth turned yellow + bubble trapped in Durham tube)

       V = variable

        _ = no change

§    Phenol red dextrose/maltose/mannitol/sucrose/lactose broth

            Trypticase                                                                    10.0 g

            dextrose/maltose/mannitol/sucrose/lactose                 5.0 g

            Sodium chloride                                                          5.0 g

            Phenol red                                                                   0.018 g

            Distilled water                                                            1 litre

            pH                                                                               7.3

 (Note: Autoclave at 12 lb pressure for 15 minutes)