Bacterial Metabolism and Enzymatic Growth

Bacteria differ in the substances from which they derive energy and the enzymes that they produce. Enzymes are biological catalysts; they are substances produced by the body to control the rate of reactions within the body. Particular enzymes, exoenzymes, are produced in large quantities and are excreted into the cell’s environment where they speed up the catabolism of insoluble polymers for nutrients (Madigan, 2009). Endoenzymes, unlike exoenzymes are produced in small quantities and remain within the cell (Kocholaty et al, 1938).

The purpose of these experiments is to determine the enzymatic and metabolic activities of a variety of bacteria in varying environments. The environments in which the bacteria are observed contain the presence of indole, urease, hydrogen sulfide, and carbohydrates lactose, sucrose, and glucose. The species of tester bacteria cultures for metabolic activities are Escherichia coli (Ec), Pseudomonas aeruoginosa (Pa), Bacillus subtilis (Bs), and Proteus vulgaris (Pv). The enzymatic activities of bacteria are observed in the presence of the enzymes amylase, lipase, and protease. The bacteria used are Escherichia coli, Pseudomonas aeruoginosa, and Bacillus subtilis.

The metabolism of the aforementioned carbohydrates results in a change of acidity, evident by a colour change in the presence of the bromocresol purple indicator. Each carbohydrate broth is inoculated with two loopfuls of each bacterium. If the broth undergoes metabolism and becomes yellow in colour, it is acidic. The broth is basic if, after metabolism, it becomes dark blue in colour. The broth will remain purple if it is neutral after metabolism. The metabolism of particular carbohydrates by some bacteria may be accompanied by gas formation which will be collected in an inverted tube (Madigan et al, 2009).

Indole is a compound derived from the amino acid tryptophan. A broth of tryptone is inoculated with two loopfuls of each bacterium. If the bacterium is one which converts tryptophan to indole, a red surface layer appears upon addition of dimethyl-aminobenzaldehyde, more commonly known as Kovac’s reagent. Otherwise, the colour of the broth will remain completely yellow (Madigan et al, 2009). In the urease test, a urea broth, yellow before incubation, is inoculated with two loopfuls of each bacterium. If the broth, after inoculation, increases in acidity, the colour changes to pink with the presence of a phenol red indicator. If no colour change has occurred, the urea has not been broken down, meaning that the bacterium does not produce urease (Madigan et al, 2009).

Bacteria that produce hydrogen sulfide are found in the intestines of animals. They are known as “enteric” bacteria – bacteria that are gram-negative, facultative anaerobes with a rod-shaped morphology. To test for the formation of hydrogen sulfide, a straight inoculation is made with an inoculating needle into a SIM agar butt. If hydrogen sulfide is produced by the bacteria, the SIM medium will blacken. If hydrogen sulfide is not produced by the bacterium, the SIM medium may become paler, as some motion of the bacteria may have occurred, however the colour of the medium will remain a pale yellow (Madigan et al, 2009).

Enzymatic activity on different agars is observed by the physical properties of and around the bacterial colonies. The three species of bacteria, previously mentioned, were spotted onto three different agar mediums, starch, tween 80, and milk and incubated for 48-72 hours at 37 C. If, on the starch agar, there was a clearing around the bacterial colony, the bacterium is one which hydrolyzes starch. To better see the clearing, Gram’s iodine solution was added to the agar plate and any starch present was stained.

Lipolysis activity, which is the breakdown of fat, is evident if the area around which the bacterium was spotted on the tween 80 agar is cloudy. Proteolysis, the breakdown of protein, like starch hydrolysis, results in a clearing surrounding the bacterial colony. To determine the bacterium’s production of catalase, hydrogen peroxide was added to each colony. Catalase, if present, will result in the formation of oxygen bubbles. The lack of oxygen bubbles indicates a lack of catalase. The presence of oxidase was determined by the combination of Kovac’s reagent, which acts as an artificial electron acceptor, and undergoes oxidation by cytochrome c. A positive reaction is one in which the bacteria darken. A colour change will not occur if oxidase is not present.

The expected results for each bacterium in each medium can be found in table 1 and table 2 in the results section. The ability to identify bacteria allows the ability to confirm the infection of such bacteria and distinguish between two similar bacteria. For example, salmonella and edwardsiella are similar in morphology and react similarly in various tests. The two bacteria may be differentiated with an indole test; edwardsiella produces indole and tests positive whereas salmonella does not.

Materials and Procedure The materials involved, and the procedure by which these experiments were performed, are as stated in the experiments 18 and 19, outlined in the University of Waterloo Fall 2009 Biol 140L Lab Manual, pp69-77, titled Some Metabolic Activities of Bacteria and Bacterial Enzymes. No changes were made to the outline procedure. Department of Biology 2009 Fundamentals of Microbiology Lab Manual. University of Waterloo, Waterloo. pp22-30. Results Table 1: Expected results for metabolic activity Below are the expected results for carbohydrate fermentation, the conversion of tryptophan to indole, the production of urease, and the production of hydrogen sulfide in a SIM media by four species of bacteria: Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Proteus vulgaris.

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