SAMPLE QUESTIONS: You should be able to answer
these questions at the conclusion of this laboratory. REVISED: 08/04/99
Since EVERYTHING that a living organism does is the result of the activity of an ENZYME, the SUMMATION of the activities of all an organism's enzymes equals its BIOCHEMICAL FINGERPRINT. That is, an organism is the totality of its enzymes, so by determining which enzymes are present in an unknown organism one can DESCRIBE & IDENTIFY that organism. In this exercise you will test several bacteria for the presence of a variety of enzymes. For example, we know that some bacteria contain the enzymes that allow it to metabolize the sugar FRUCTOSE and to produce ACETIC ACID in the process. So we can ask the following question: "Does an unknown bacterium contain the enzymes for converting fructose to acetic acid"? Enzymes are detected by virtue of the CHEMICAL REACTION they carry out. However, it is necessary to have a means of measuring or "seeing" an enzyme's chemical reaction. One common measuring technique involves the formation of a COLORED CHEMICAL as a direct result of an enzyme's activity. There are chemicals (pH INDICATORS) that respond to the HYDROGEN ION, (H+), concentration by changing color.
Thus if an enzymatic reaction produces acids, which increases the H+ concentration, pH-indicators respond by CHANGING COLOR as the acidity changes. To answer the above question, one prepares a medium containing fructose (or other sugars you wish to test) and a pH INDICATOR that is RED at a neutral pH of 7.0, but which changes to BLUE at an acid pH of 5.0. This medium is then inoculated with the bacterium in question. If the color of the pH indicator changes from red to blue then an ACID HAS BEEN PRODUCED by the metabolism of the fructose by the bacterium. If another bacterium, inoculated into the same medium, does NOT cause the color to change you know that it does not have the same "fructose" enzymes as the first bacterium; i.e., "by your enzymes shall you be known".
This is like describing to the police the perpetrator of a crime you have witnessed as having "blue eyes". What you have really said is that the perpetrator "lacks the enzymes to make eye pigmentation". In this exercise you will add bacteria to medium containing the pH indicator PHENOL RED and several DIFFERENT SUGARS. In addition each tube will contain a smaller, inverted tube to trap any gas produced. Following incubation you will observe the tubes for a color change from red to yellow (acid) and for the production of gas (CO2) which will fill the inverted tube.
In addition you will test these bacteria for the presence of the enzymes that break down STARCH and HYDROGEN PEROXIDE. In the case of the former IODINE reacts with starch to produce an intense blue-black color. You will inoculate plates containing starch with the various bacteria, and following incubation you will flood the plates with iodine. Those bacterium producing STARCH-DIGESTING ENZYMES will have a CLEAR NON-STAINING AREA around their colonies.
The enzyme that breaks down hydrogen peroxide to WATER and OXYGEN is called CATALASE. To test for this enzyme you grow the bacteria on a plate of nutrient agar and then add a few drops of hydrogen peroxide to the colony. If catalase is present the solution will FOAM vigorously. This reaction is an excellent example of the #catalytic activity of enzymes discussed & demonstrated in lecture; i.e., the hydrogen peroxide remains intact until catalase (or a platinum catalyst) is added to lower the "ENERGY OF ACTIVATION" which allows a powerful reaction to occur. The reaction releases oxygen gas and considerable heat.
You will also test these bacteria for the enzymes that produce hydrogen sulfide, indole and digest urea. Hydrogen sulfide (H2S) is the rotten egg odor associated with spoiled eggs & rotting meat. H2S is a toxic gas that is formed from the breakdown of SULFUR-CONTAINING AMINO ACIDS. H2S reacts with IRON to produce a black precipitate (iron sulfide). To test for this ability you will inoculate medium containing iron and the sulfur amino acids and examine for the formation of a black precipitate in the medium following incubation.
Indole is a product of the breakdown of another amino acid, tryptophan by the enzyme TRYPTOPHANASE. To test for indole Kovacs reagent is added to the SIM medium following growth. If indole is present a red ring forms around the surface of the medium.
Urea is broken down by the enzyme UREASE into carbon dioxide and ammonia. Ammonia turns the medium alkaline; that is it raises the pH to above 7.0. In this test the bacteria are inoculated into urea broth which contains the pH indicator (phenol red) which changes from yellow to red/pink as the pH increases (Atlas pg. 79). After 24 to 48 hours of incubation the tubes are observed for a color change indicative of urea digestion.
Visit this site to see each of the tests you will perform in the next two weeks; Click on the particular test to see the results. Both of these sites 1 and 2 contains photos of many of the biochemical reactions you will perform as well as examples of differential media.
SAMPLE QUESTIONS: You should be able to answer
these questions at the conclusion of this laboratory. Copyright 
Dr. Ronald E. Hurlbert, 1998.
This material may be used for educational purposes only and may not be
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