Bacterial Enzymes Microbiology Lab Exercise #6 Dr. Carol G. Leggett Name ta Enzymes are very important in everyday life; they catalyze anabolic and catabolic reactions. In order for media to test for the presence of the enzymes urease, catalase, gelatinase, phenylalaine deaminase, an enzyme to work, it must bind a specific reactant or substrate. In this lab, you will be using substrate lysine decarboxylase, and ornithine decarboxylase. These different media will reveal if your unknown bacterium produces specific enzymes, and the results will help to elucidate the genus of your unknown urea into ammonia and carbon dioxide. Phenol red is used in the media as a pH indicator. If urea is The purpose of urea medium is to determine if your unknown produces urease which can break down broken down into ammonia and carbon dioxide, the ammonia will increase the pH (more alkaline) which through the process of elimination. results in a color change in the media. (Orange-pink to dark-pink) Urea chart Positive or negative Number of your unknown Color of the urea medium before culture Color of urea medium after culture 22 21 pos ive unuta negative urca test Photo courtesy of: http://t2.gstatic.com/images?q=tbn:ANd9GcRZt2GDYMUASMRDYFVIWCWRMESQGB01-0qNtGeP CZCF8b RepUq1xg 1 Catalase Test sm6 JIM hydrogen peroxide and releases oxygen gas (O2). Hydrogen peroxide will be dropped onto the The purpose of the catalase test is to see if your unknown produces catalase, which breaks down bacterium that has been smeared onto a clean glass slide. This test should not be performed while the unknown is growing on a blood-containing medium because this enzyme is found in red blood cells and 9V 916 29 mysn3 0297 er 101 297 of sibem inimo bns 925lyxodroosb sniay! 1 / sns pite 29ubong muit91 6d Goonie to 220001q art dguont the test may present a false positive. Purpose: to determine if your unknown produces catalase. Procedure: al mism 5970 to szoquent od 1. bras sintomims in 5970 1. Draw a circle on a clean glass slide with your wax pencil. 2. With a sterilized and cooled inoculating loop, pick up a small amount of the unknown culture from the nutrient agar slant. Smear the culture directly onto the center of your wax circle. The smear should be about the size of a pea. 3. Place one drop of hydrogen peroxide onto the smear. Observe the fluid over the smear for the appearance of gas bubbles. Record the results in the chart. Discard the slide in the beaker of disinfectant solution at the front desk. Bubbles Positive or Negative Results of Catalase Test for Your Unknown Bacterium CATALASE TEST Photo courtesy of: http://nhscience.lonestar.edu/biol/wellmeyer/media/catalaseResults2.jpg Phenylalanine Deaminase (PDase) The process removes the amine group (deamination) from the amino acid. Phenylalanine is added into This test determines if the unknown bacteria can produce an enzyme that breaks down phenylalanine. the medium and if the bacterium can produce PDase the phenylalanine is degraded into phenylpyruvic acid and free ammonia. This reaction can be observed by adding ferric (iron containing compounds) ions which react with the newly produced acid to form a green compound. Purpose:- To view the activity of the PDase and to determine if the unknown produces it. Procedure: 1. Inoculate your unknown onto the surface of a phenylalanine agar slant. 2. Incubate the new cultures at 37 degrees Celsius until the next lab. 3. Examine the tubes for heavy growth. If it is adequate, run a few drops of 10% ferric chloride solution down the surface of each slant. 4. Observe the tubes for development of a green color. PDase (+ or -) Unknown Color phanylalaning agar Negative Positive Photo courtesy of: http://delrio.dcccd.edu/reynolds/microbiology/2421/lab manual/atlas/biol2420photoatlas 038.htm 3 Ornithine and Lysine Decarboxylase Identifying members of the Enterobacteriaceae, a group that contains many medically 19 SAT important bacteria, is often difficult. It is especially difficult to separate members of the generasman Klebsiella, Enterobacter and Citrobacter. Measuring the ability of these bacteria to decarboxylate certain amino acids has proven a useful taxonomic tool. The amino acids that have proven to be most useful are Under anaerobic conditions, specific decarboxylase enzymes are able to remove the carboxyl group from these amino acids and thus change the pH of the media to make it more alkaline. A pH indicator in the media, such as brom cresol purple, can detect this shift and provide an easy way to measure these reactions. To create suitable anaerobic environments the bacteria are grown in broth tubes of the appropriate media, which have been capped after inoculation with mineral oil. lysine, arginine and ornithine. Uninoculated Pos. Neg. Neg. Purpose: To determine if your unknown bacterium produces decarboxylase for lysine or ornithine. Procedure: 1. Inoculate a tube of ornithine and lysine decarboxylase medium using a sterile loop. 2. Add 3-4 drops of mineral oil to the top of each tube to create an anaerobic environment. 3. Place in the incubator until the next lab. Positive or Negative Results Ornithine Decarboxylase Lysine Decarboxylase Facts courtesy of people.uncw.edu/.../rkswww/B10425Lab/Decarboxylasa%20Reactions.htm Photo courtesy of: http://www.vetmed.wisc.edu/pbs/courses/bacılabmanual/c4decarboxylase.html 4 Gelatinase This is a differential medium test to determine whether or not your unknown produces gelatinase (an animals. When placed in an environment that is below 32 degrees Celsius, gelatin solidifies. If your exoenzyme) which breaks down gelatin. Gelatin is a protein that comes from the connective tissues of unknown produces the enzyme known as gelatinase the medium will not solidify but rather stay in the liquid state. positiis relatia acgativet gelatnases Facts courtesy of:http://www.austincc.edu/microbugz/gelatinase test.php Photo courtesy of: http://www.mesacc.edu/viohnson/labtools/Obiochem/gelatin.jpg Purpose: To determine if your unknown bacteria produces the exoenzyme gelatinase Procedure: 1. Use a sterile needle stab to inoculate the gelatin medium. 2. Place in the incubator until the next lab session. 3. Next lab, place your gelatin tube in the refrigerator for 10 minutes. 4. After the 10 minutes, tilt your tube to see if the medium is a liquid or a solid. 5. If the tube is still a liquid, it is positive for gelatinase. Results for Gelatinase Solid or Liquid Positive or Negative 5
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