Exercise X. IMViC Tests

Transcription

1 Exercise X IMViC Tests IMViC tests are used to study the physiological characteristics of bacteria from the Family Enterobacteriaceae, especially Escherichia and Enterobacter. The biochemical characteristics of bacteria can be used to demonstrate their metabolic diversity. Bacteria exhibit a very large range of metabolic capabilities, partially explaining why these organisms can be found in virtually every environmental habitat. Not only are bacteria capable of obtaining energy by a variety of pathways, some of which are unique to bacteria, they are also capable of utilizing a very large number of different metabolites. The reactions determine if certain carbohydrates are used (glucose, lactose, sucrose) and whether or not the sugars can be fermented with the production of acids and gas, or only acid. ph indicators are used in the media to determine whether acids are produced. Methyl red will be red at a ph of 4.4 and below 6.0, and turns yellow at a ph of 6.0 or above. Brom thymol blue will change from yellow to green to blue as the ph moves from 6.0 to 7.6. The IMViC series consists of four definitive tests: indole production, the methyl red test, the Voges-Proskauer test, and the citrate utilization test. Since bacterial organisms are similar in morphological and cultural characteristics, four biochemical tests are used to differentiate between these bacteria. The IMViC tests determine specialized physiological properties of the bacteria. They are especially useful in differentiating Gram negative intestinal bacteria, particularly Escherichia coli and the Enterobacter Klebsiella group (coliform bacteria). E. coli is found in the normal flora of the intestinal tract of animals and humans. It has long been realized that contamination of water with large numbers of E. coli is a great indicator of human fecal contamination. Enterobacter aerogenes is widely distributed in nature, especially on plants and plant products. Indole Production Indole production is the I portion of the IMViC tests used to characterize enteric bacteria. Tryptophan is an essential amino acid that can undergo oxidation by way of the enzymatic activities of some bacteria. Conversion of tryptophan into metabolic products is mediated by the enzyme tryptophanase. The ability to hydrolyze tryptophan with the production of indole is not a characteristic of all bacteria and therefore serves as a biochemical marker. Some bacteria, including E. coli, are capable of degrading the amino acid tryptophan to indole. This ability to produce indole from tryptophan has been utilized as a method of separating the genus Escherichia from the genus Enterobacter. The amino acid tryptophan can be broken down by the enzyme tryptophanase to indole, pyruvic acid, and ammonia, as end products. Indole is produced when the amino acid tryptophan is hydrolyzed to 70

2 pyruvic acid (used for energy metabolism) and indole (which is excreted). The presence of tryptophanase is a significant distinction between E. coli, which produces it and is therefore indole positive, and the other enteric organisms. Tryptophanase differentiates the indole-positive E. coli from the indole-negative Enterobacter aerogenes and Serratia marcescens, which may be positive or negative. Indole is produced in tryptone broth by the enzyme systems of certain bacteria. Tryptone broth is rich in the amino acid tryptophan, which can be used by some bacteria as a source of carbon and nitrogen as well as energy. As these enzymes increase in the tryptone broth, they attack the tryptophan and degrade it into indole, pyruvic acid, and ammonia. A tube of tryptone broth is inoculated and incubated. After incubation, Kovac s reagent is added to the tube. Kovac s reagent contains amyl alcohol, HCl, and paradimethylaminobenzaldehyde, which react with the indole to form a red ring on the top of the broth. A red color indicates a positive test for indole production and a yellow or green color indicates that tryptophan was not hydrolyzed and is a negative test for indole production. Methyl Red Test The methyl red test is the M portion of the IMViC tests used to characterize enteric bacteria. The methyl red test is used to identify enteric bacteria based on the pattern of glucose metabolism. All enterics initially produce pyruvic acid from glucose metabolism. Some enterics subsequently use the mixed acid pathway to metabolize pyruvic acid to other acids, such as lactic, acetic, and formic acids. These bacteria are called methyl red positive and include Escherichia coli. Other enterics subsequently use the butylene glycol pathway to metabolize pyruvic acid to neutral end products. These bacteria are methyl red negative and include Enterobacter aerogenes and Serratia marcescens. Glucose is the major substrate oxidized by all enteric bacteria for energy production. The end products of this process will vary depending on the specific enzymatic pathway present in the bacteria. The ph indicator methyl red detects the presence of large concentrations of acid end products. Enterics initially produce organic acid end products during the early incubation period. Enterics that subsequently metabolize pyruvic acid to other acids lower the ph of the medium to The low acid ph is stabilized and maintained by E. coli at the end of incubation. The low ph inhibits the growth of the bacteria. At this ph, methyl red turns red. A red color represents a positive test. During the later incubation period, Enterobacter aerogenes and Serratia marcescens enzymatically convert these acids to non-acidic end products such as 2-3butanediol and acetoin (acetylmethylcarbinol), resulting in an elevated ph of approximately 6.0. The high ph does not inhibit their growth, and when the glucose is exhausted, these bacteria attack the peptone, causing the ph to rise above 6.3. Enterics that subsequently metabolize pyruvic acid to neutral end products lower the ph of the medium to only 6.0. At this ph, methyl red is yellow. A yellow color represents a negative test. 71

3 The medium contains peptone, dextrose (glucose), and dipotassium phosphate. Dextrose is a significant ingredient that determines what types of end products an organism forms from degrading glucose. The ability to produce a large amount of acid in dextrose broth is used as a means to differentiate E. coli from Enterobacter aerogenes. When E. coli is grown in dextrose broth, the bacteria degrade the dextrose to organic acids such as lactic, formic, and acetic acids. If the ph of the medium drops to , Enterobacter aerogenes and Serratia marcescens utilize dextrose to produce ethylene glycol and ethanol, both relatively neutral products, so that the ph of the medium is approximately After incubation, the ph of the medium is determined by the addition of the ph indicator methyl red. The methyl red is a red color between ph , indicating a positive test, and yellow between ph indicating a negative test. In between 5.2 and 6.0 the medium is orange which indicates an inconclusive or a negative test. Citrate Utilization Citrate utilization is the C portion of the IMViC tests, which are used for characterizing enteric bacteria. Citrate is an organic molecule that can be utilized by bacteria that produce the enzyme citrase. In the absence of fermentable glucose or lactose, some bacteria are capable of using citrate as a carbon source for their energy. Citrate is acted upon by the enzyme citrase, which produces oxaloacetic acid and acetate. These products are enzymatically converted to pyruvic acid and carbon dioxide. The medium becomes alkaline when the CO 2 combines with sodium and H 2 0 to form sodium carbonate, an alkaline product. Simmon s citrate agar is used to determine whether an organism produces citrase. It contains citrate as the only carbon source, ammonium phosphate salt as a nitrogen source, and the ph indicator brom thymol blue, which is green at the initial ph of 6.9 and blue at a ph of 7.6 and above. The bacteria convert ammonium phosphate to ammonia and ammonium hydroxide, both of which tend to alkalinize the agar. The ability of certain bacteria to use citrate as a carbon source is a means of differentiating certain Gram negative bacteria, particularly Escherichia coli and Enterobacter aerogenes. Enterobacter aerogenes, but not E. coli, utilizes citrate as the sole carbon source and grows on this medium, changing the color of the medium from green to blue. Although E. coli possesses the enzyme that catalyzes the cleavage of citrate, the citrate cannot be utilized because E. coli lacks the transport system or the enzyme citrate permease that would permit the citrate to enter the cell. Occasionally citrate positive bacteria will grow on a Simmon s citrate slant without producing a color change. In most cases this is due to incomplete incubation. In the absence of color change, growth on the slant indicates citrate is being utilized, and is 72

4 evidence of a positive reaction. Look for growth as well as a color change, because only citrate-positive bacteria will grow on a medium that contains citrate as the only carbon source. Be careful not to confuse between growth and a heavy inoculation that was made. When the enzyme citrase metabolizes citrate to produce alkaline end products, they raise the ph of the medium to 7.6, causing the brom thymol blue to turn blue. The presence of growth and a blue color represent a positive test for citrate utilization. The absence of growth and a green color represent a negative test for citrate utilization. Materials Needed for the First Lab Period culture of Escherichia coli culture of Enterobacter aerogenes 2 sterile tubes of tryptone broth 2 sterile tubes of dextrose broth 2 sterile citrate slants Bunsen burner striker inoculating loop inoculating needle test tube rack wax pencil goggles gloves spray bottle of disinfectant paper towels Procedure 1: Indole Test 1. Obtain a culture of Escherichia coli. 2. Obtain a sterile tube of tryptone broth. 3. Using a wax pencil label the tryptone broth tube with the initials of the bacteria and the initials of someone in the group. 4. Using an inoculating loop and aseptic technique, transfer a loopful of bacteria into the tryptone broth. 5. Be sure to shake the loop in the broth and touch it to the side of the tube to remove excess broth. 6. Repeat the procedure using Enterobacter aerogenes. 7. Place both tubes in a test tube rack and place them in the incubator. 73

5 Procedure 2: Methyl Red Test 1. Obtain a culture of Escherichia coli. 2. Obtain a sterile tube of dextrose broth. 3. Using a wax pencil label the dextrose broth with the initials of the bacteria and the initials of someone in the group. 4. Using an inoculating loop and aseptic technique, transfer a loopful of bacteria into the dextrose broth. 5. Be sure to shake the loop in the broth and touch the loop to the side of the tube to remove excess broth. 6. Repeat the procedure with Enterobacter aerogenes. 7. Place both tubes in a test tube rack and place them in the incubator. Procedure 3: Citrate Utilization 1. Obtain a culture of Escherichia coli. 2. Obtain a sterile citrate slant. 3. Using a wax pencil label the citrate slant with the initials of the bacteria and the initials of someone in the group. 4. Using an inoculating needle and aseptic technique, obtain a sample of the bacteria. 5. Insert the needle into the butt of the citrate and as the needle is pulled out of the butt, streak the slant in a zigzag pattern. 6. Repeat the procedure with Enterobacter aerogenes. 7. Place both tubes in a test tube rack and place them in the incubator. Clean-up Procedures 1. Return the Bunsen burner, inoculating loop, inoculating needle, and other material to the appropriate place. 2. Spray and wipe the table top with disinfectant. Second Lab Period Materials Needed Kovac s reagent methyl red test tube rack gloves spray bottle of disinfectant paper towels 74

6 Procedure 1: Indole Test 1. Obtain the inoculated tryptone broth tubes from the incubator 2. Add 10 drops of Kovac s reagent to the tube inoculated with Escherichia coli. 3. Allow the tube to stand for 5 minutes. 4. Repeat the procedure with the tube inoculated with Enterobacter aerogenes. 5. A red ring will form at the top of the broth if it positive for indole production. 6. A yellow or green ring will form at the top of the broth if it is negative for indole production. Procedure 2: Methyl Red Test 1. Obtain the inoculated dextrose broth tubes from the incubator. 2. Add 5 drops of Methyl red to the tube inoculated with Escherichia coli. 3. Roll the tube between your hands to mix. 4. Repeat the procedure with the tube inoculated with Enterobacter aerogenes. 5. A red color indicates a positive test for acid production. 6. A yellow color indicates a negative test for acid production. 7. An orange color indicates an inconclusive or negative test for acid production. Procedure 3: Citrate Utilization 1. Obtain the inoculated Simmon s citrate slants from the incubator. 2. Observe the slant inoculated with Escherichia coli. 3. Observe the slant inoculated with Enterobacter aerogenes. 4. A blue color of the agar indicates a positive test for citrate utilization. 5. A green color of the agar indicates a negative test for citrate utilization. Clean-up Procedures 1. Place the inoculated tryptone broth tubes, dextrose broth tubes, and citrate slants in the waste bin. 2. Spray and wipe the table top with disinfectant. 75

7 76

8 77