The purpose of this experiment is to distinguish species of bacteria by cultivating in different media and doing some tests. Another purpose of this lab is to learn the proper techniques of testing for fermentation of carbohydrates, production of indole, activity of urease, production of hydrogen sulfide, evidence of amylase activity, evidence of lipase activity, and evidence of protease activity. Different species of bacteria can be distinguished on the basis of the carbohydrates they do or do not utilize, as well as the nature of the products formed in the fermentation reaction. (Madigan and Thomas, 2009) Bacteria are able to ferment or breakdown simple carbohydrates to produce acidic, alcoholic, or gaseous end products. (Goldman 2009)
By testing which bacteria species will ferment which carbohydrate and what products are formed allows one to identify. (Madigan and Thomas, 2009) The medium for the test is a nutrient broth with the acid-base indicator bromocresol purple. The test tube contains a Durham tube to collect gas that will be released in the medium when the carbohydrate is fermented. (Goldman 2009) Some organisms will not ferment at all, some will produce acid products and no gas, and some will make both acid products and gas. (Davidson, 2010) After inoculation, the tubes are incubated at 37ï¿½C which is the optimal temperature for growth.
A negative result for the carbohydrate test is no change in pH(no colour change) or gas production. (Goldman 2009) A positive result is gas produced and a acid production (yellow). (Goldman 2009) The second test is the indole test. Indole is a by-product of the metabolic breakdown of the amino acid tryptophan by the enzyme tryptophanase. (Davidson, 2010) Another by-product of this degradation is pyruvate which is used as an energy source for the bacteria. (Madigan and Thomas, 2009)
The bacteria are grown on a medium containing tryptophan and the presence of indole is detected by adding the chemical indicator, Kovac’s reagent. The active ingredient in Kovac’s reagent is p-dimethylaminobenzaldehyde, which reacts with indole to produce a deep pink colour. The negative result for this reaction is no colur change which means there is no indole. A positive for the indole test is a red/ pink colour. (Goldman 2009) The third carbohydrate metabolism test is the urease test.
Proteus bacteria can split the urea molecule releasing carbon dioxide and ammonia. This reaction, mediated by the enzyme urease, can be seen if the culture medium is prepared with urea added as the substrate. (Davidson, 2010) Phenol red is also added as a pH indicator. When bacteria which generate urease are grown in this medium, degradation of urea with the release of ammonia can be detected as the pH becomes basic and the pH indicator color becomes reddish pink. (Goldman 2009) Therefore the positive result is a pink colour.
The first test of experiment 19 is the evidence of carbohydrase (amylase) activity. Another name for this test is the starch hydrolysis test. The test is used to find the presence of alpha-amylase. Alpha-amylase is an extracellular enzyme that acts on the 1,4-?-glucosidic bonds of starch. (Davidson, 2010) The two polysaccharides present in the starch polymer are amylose and amylopectin. Amylose absorbs iodine to produce a blue compound, while amylopectin produces a red-violet compound. (Madigan and Thomas, 2009)
The starch hydrolysis test is performed by inoculating streaks of bacteria on starch agar plate. After incubation, the plate is then flooded with Gram’s iodine solution. (Davidson, 2010) The presence of a colourless zone which is referred to as a halo surrounding the streaks of growth are an indication of a positive result which means starch hydrolysis took place. (Goldman 2009) The second test is the evidence of lipase activity. Hydrolysis of a lipid leads to the formation of glycerol and fatty acids. (Goldman 2009) The streak plate will appear cloudy or turbid. If the formation of the precipitate is seen the test is positive for lipolysis. (Davidson, 2010)
The third test is the evidence of protease activity. Casein is a nitrogenous protein found in the milk agar. (Madigan and Thomas, 2009) If a clear zone is surrounding the bacterial streak then the test is positive and the casein is hydrolyzed. The next test is the catalase test. Catalase is an endoenzyme. (Madigan and Thomas, 2009) Catalase acts on hydrogen peroxide, formed as an oxidative end product of aerobic respiration, catabolizing it into water and oxygen. (Madigan and Thomas, 2009) When bubbles of oxygen are observed when a hydrogen peroxide is added to a bacterial colony, the bacterial are considered catalase-producing microorganism. (Davidson, 2010)
Another endoenzyme is oxidase. In the oxidase test, a drop of tetramethyl-p-phenylenediamine reagent is placed on a piece of filter paper. Using a toothpick rub a small amount of bacterial growth from a culture plate into the drop and observe for the development of a purple colour. (Goldman 2009) The purple colour should form rapidly in about 10 seconds for the test to be declared positive. If the colour change happens slowly or takes more than a minute, a negative result should be recorded.
Escherichia coli is an facultive anaerobic, Gram negative rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms. (Madigan and Thomas, 2009) Pseudomonas aeruginosa is a common Gram- negative, aerobic, rod-shaped bacterium which can cause disease in animals. (Madigan and Thomas, 2009) Bacillus subtilis is a Gram-positive, catalase-positive bacterium commonly found in soil. Proteus vulgaris is a rod-shaped, Gram negative bacterium that inhabits the intestinal tracts of humans and animals. (Madigan and Thomas, 2009)
Materials and Methods
The protocol and materials used for the experiment were completed as written in the Department of Biology of the University of Waterloo Fall term 2010 Biology 140L Lab Manual. Experiment 18, Some Metabolic Activities of Bacteria, is found on pages 70 to 74. Experiment 19, Bacterial Enzymes, is found on pages 75 to 78. The only change made to the procedure is that the recording of urea broth tubes was not done on the urea broth tubes we prepared previously.