Updated 21 December 04
Enzymes are proteins produced by cells and are the most important mediators of cellular activities. Each enzyme is specific for one or a few substrate molecules and facilitates the reaction that produces a specific product from the substrate. Each kind of cell has a variety of different enzymes and activities. Many of the most important activities occur in each type of cell. However, some activities are not present in the cells of some species because they do not produce the necessary enzyme. If the substrate for a certain enzyme is presented to the cells of a given species it is often possible to discover if that enzyme activity is present in that species. The presence or absence of an enzymatic activity can be the basis for differential tests to help the microbiologist distinguish between different species of bacteria (see specific experiments in lab books for details).
Enzymes are often identified by using the name of the substrate with the suffix -ase. Thus, amylase is an enzyme that assists in the degradation of amylose (starch), gelatinase aids in the degradation of gelatin (protein), and deoxyribonuclease (DNAse) aids in the degradation of DNA. Bacteria producing these enzymes are able to utilize the degradation products (glucose, peptides, amino acids, nucloetides) from these substrates for energy or building blocks for growth.
Information about cell morphology, staining reactions, colony morphology, and biochemical characteristics is used to distinguish between bacteria. Diagnostic keys are often constructed to compare the characteristics of unknown organisms to a wide variety of known organisms. Information about known organisms is collected into "Bergy's Manual of Determinative Bacteriology". In practice a dichotomous key listing increasingly specific phenotypic traits allows one to assign a name to an organism by a stepwise elimination of other organisms.
Go to Identibacter Interactus and try identifying an unknown. With sufficient background information on the tests, the organisms available, and practice you should become very efficient (use a minimum of tests).
Gelatinase catalyzes the hydrolysis of the protein gelatin (collagen). Gelatin in solution liquefies above 25 degrees C. At room temperature or below it becomes a solid (gel). When bacteria that produce gelatinase are grown in gelatin medium, the enzyme digests the gelatin and the medium cannot solidify even at cold temperatures. See Gelatin in Identibacter Interactus.
1. Inoculate nutrient broth-gelatin tubes by stabbing straight down into the medium with a loopful of an individual species. Leave one tube uninoculated as a control.
2. Incubate the tubes at 37 degrees C.
3. After 24 hours examine the tubes. All should be liquid. Place all the tubes in the refrigerator for 30 minutes. Check to see if any tubes are still liquid. Reincubate the solid tubes and recheck after 48 hours and 7 days as necessary.
4. Record which species are positive or negative for gelatinase. If positive, record the time it took for the positive results.
The extracellular enzyme alpha amylase catalyzes the breakdown of starch to maltose. The smaller maltose molecules can enter the cell to be used for energy. Iodine interacts with starch and makes it dark blue. If starch has been removed from a culture medium by the action of the bacterial enzyme no blue color appears in the presence of iodine. If iodine stains the starch medium blue in the presence of growing bacterial colonies then no amylase is produced by that species. See Starch in Identibacter Interactus.
1. Inoculate starch agar plates by making a short streak with each of several species in different sectors.
2. Incubate at 37 degrees C. Check in 5-7 days for the presence of starch around the streaks by flooding the area with iodine solution.
3. Record the results for each species as + or - for amylase.
Catalase is an enzyme that catalyzes the breakdown of hydrogen peroxide to water and oxygen gas. Most aerobic microorganisms produce catalase. The presence of catalase is important in the prevention of toxic wastes that would kill the cell. The presence of catalase is indicated when hydrogen peroxide is added to a colony or a loopful of bacteria and bubbles of oxygen are released from the surface. See Catalase in Identibacter Interactus.
1. Aseptically pick up some bacterial cells from an agar culture with an inoculating loop.
2. Put a drop of hydrogen peroxide onto the mass of cells adhering to the loop.
3. Observe for the release of bubbles. Flame the loop when finished.
4. Record your observations as + or - for catalase.
The oxidase test indicates the presence of cytochrome oxidase and aids in differentiation among members of the genera Neisseria and Pseudomonas, which are strict aerobes and oxidase-positive, and Enterobacteriaceae, which are oxidase-negative. The test reagent, p-aminodimethylaniline oxalate is added to colonies. This light pink reagent is oxidized to a blackish compound in the presence of oxidase and free oxygen. The development of maroon to black coloration on colonies is a positive test. No color change, or a pink coloration on colonies, is a negative test.
1. Add 2 drops or the liquid test reagent to colonies. Alternatively, cells from a colony may be spread on filter paper impregnated with p-aminodimethylaniline oxalate solution.
2. Observe for a color change to very dark colonies or smear and record + or - for oxidase production.
How do you account for any differences in reaction between the different test organisms?
Cite references where used.