Exoenzymes, Carbohydrate Fermentation, Nitrate Reduction and Urea Hydrolysis Lecture Notes

Exoenzymes, Carbohydrate Fermentation, Nitrate Reduction and Urea Hydrolysis Lecture Notes

I.) EXOENZYMES AND THE MAJOR FOOD GROUPS

EXTRACELLULAR ENZYMES are EXOENZYMES

Polysaccharides, proteins and many lipids are too large to be brought into the cell directly. They are often HYDROLYZED into smaller molecules and these then are transported.

Polysaccharides are broken down into monosaccharides and disaccharides; proteins are broken down into amino acids and lipids are hydrolyed into fatty acids and glycerol.

STARCH is a branched polymer of glucose. The enzyme amylase breaks starch into disaccharide maltose subunits. Maltose can then be further hydrolyzed into glucose.

To do the STARCH TEST pour iodine onto the starch plate. Starch reacts with iodine to form a blue-black complex, therefore you will be looking for a clear zone around and beneath the bacterial growth.

GELATIN is partially hydrolyzed COLLAGEN, a major connective tissue protein found in the skin, bone and cartilage of humans and other animals. Below 25^oC gelatin is a solid unless it has been completely hydrolyzed by gelatinase. Above 25^oC gelatin is liquid and therefore test tubes removed from the 37^oC incubator will always be liquified. The results can only be evaluated after the tubes have been placed in the refrigerator for a while.

II.) CARBOHYDRATE FERMENTATION

When carbohydrates are fermented a number of organic and inorganic waste products are produced. These waste products include acids such as lactic acid and acetic acid, neutral products such as alcohol and acetyl methyl carbinol, and gases such as carbon dioxide, hydrogen, and methane.

It is easy to detect the presence of acids by adding pH indicator dyes to the bacteriological growth medium. The fermentation tubes which we are using contain PHENOL RED in addition to the specific sugar. Phenol red is red at pH 7. It becomes orange and then yellow as the pH drops; at alkaline pH this indicator is reddish-purple.

Different bacterial species differ in their ability to ferment particular sugars. Therefore by testing a variety of carbohydrates on an organism one may determine a fermentation pattern which can be useful in identifying a microorganism.

Strict aerobic organisms will produce very little if any acid (the CO₂ generated in the Kreb's cycle will contribute some slight acidity) and their growth will be confined to the surface of the semi-soft agar. Also there are proteins in the medium which some bacteria can catabolize to produce alkaline products like ammonia.

III.) NITRATE REDUCTION

Some microorganisms are capable of ANAEROBIC RESPIRATION in which an inorganic molecule other than oxygen serves as the terminal electron acceptor in the electron transport chain. NITRATE and NITRITE frequently serve such a role. Some organisms stop after reducing NO₃ ---> NO₂ + H₂O; others are capable of carrying the reductions further by reducing NO₂ to ammonia (NH₃) or nitrogen (N₂).

The test directly measures the production of nitrite from nitrate either by the organism (a positive test) or by zinc (a negative test). The test works on the basis of a chemical reaction between nitrite, sulfanilic acid and alpha-napthylamine which forms a bright red compound. If no color develops after the addition of zinc the test is positive. In this case, neither nitrate nor nitrite would be present in the broth and the bacteria therefore must have reduced them away.

IV.) UREA HYDROLYSIS

Some microbes are able to break down urea into ammonia and carbon dioxide. The ammonia causes the pH to rise and this can be detected if there is a pH indicator present. The medium we use has phenol red in it, therefore a positive urease test would be when the medium turns purple-red.