Introduction to Lab Ex. 18: Fermentation of Carbohydrates Kligler Iron Agar (KIA)

Introduction to Lab Ex. 18 – Fermentation Of Carbohydrates: Kligler Iron Agar (KIA) Carbohydrates are good sources of energy for organisms. The most efficient metabolic process to harvest the energy from sugars is aerobic respiration. In the absence of aerobic conditions, organisms may be able to metabolize the sugars, though with minimal gain of ATP. These processes are characterized by the end products. The ability of cells to break down specific substrates is dependent on their ability to produce appropriate enzymes. Catabolic processes primarily include: aerobic respiration, fermentation

Fermentation is the catabolism of sugars in the absence of oxygen where the final electron acceptors is an organic molecule. Fermentation typically results in the formation of organic acids and alcohols that will accumulate in the medium. This will result in the lowering of the pH of the medium. With the inclusion of a pH indicator in the medium the lowering of the pH can become a visual reaction. Some bacteria characteristically produce gases during the fermentation process, which are visible as cracks in the agar or displacement of the agar in solid medium.

The ability to ferment specific sugars is dependant on the ability of the bacterium to produce the specific enzymes required for the transport and metabolism of that particular sugar. Thus fermentation of various sugars can be used to characterize bacteria.

Kligler Iron agar is a combination medium. While fermentation of various sugars can be tested to characterize different bacteria, the ability to ferment glucose and lactose are of special importance since these can be used to classify bacteria to various groups. Gram negative bacteria can be classified as lactose fermenters and non-lactose fermenters. To facilitate the testing of both sugars a combination medium is used. Kligler Iron agar also tests for the production of H2S by the bacteria. KIA contains the 2 sugars: glucose and lactose in a ratio of 1:10. The medium is used in the form of slants.

The abundance of lactose in the medium compared to glucose allows for differentiation of fermentation of one sugar against that of both sugars. Since lactose is a disaccharide (glucose and galactose) if lactose is fermented then glucose must be fermented too. Thus distinction is made between fermentation of one sugar (glucose) or both sugars (glucose and lactose). The reaction is made visible by the accumulation of various amounts of acids altering the pH of the medium to varying extents (either the slant alone – glucose alone fermented; or both slant and the butt of the agar changing color – due to increased amounts of acids produced by both sugars being fermented). The production of gas in these reactions is made visible by cracks or displacement of the agar in the medium. Production of H2S is made visible by the addition of ferrous salts that precipitate the H2S and form a black precipitate.

Possible reactions: Alk – alkaline; A- acid; G- gas; - no reaction; Butt alone yellow – Alk/A (Gas) – Glucose alone fermented, lactose not fermented Slant and Butt yellow – A/A (G) – Both glucose and lactose fermented Slant pink no change in butt – Alk/- - no fermentation Black coloration – H2S produced

Fermentation reactions have to be read within 18-24 hours before sugar reversion can occur.

Tube deamination glucose lactose H2S production amino acids fermentation fermentation (black color) 1 + – – – 2 + + – – 3 + + – + 4 + + + – 5 + + + +