EXERCISE. Proteins,Amino Acids, and Enzymes VII: Oxidase Test. Suggested Reading in Textbook. Pronunciation Guide. Materials per Student

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1 EXERCISE 30 Proteins,Amino Acids, SAFETY CONSIDERATIONS Be careful with the Bunsen burner flame. No mouth pipetting. The oxidase reagent is caustic. Avoid contact with eyes and skin. In case of contact, immediately flush eyes or skin with plenty of water for at least 15 minutes. Suggested Reading in Textbook 1. The Electron Transport Chain, section 9.5; see also figures Rapid Methods of Identification, section 36.2; see also table Materials per Student young 24-hour tryptic soy broth cultures of Alcaligenes faecalis (ATCC 8750), Escherichia coli (ATCC 25922), and Pseudomonas aeruginosa (ATCC 27853) tryptic soy agar plates tetramethyl-p-phenylenediamine dihydrochloride (oxidase reagent) Bunsen burner platinum or plastic loops wax pencil Pasteur pipette with pipettor Oxidase Disks or Dry Slides (Difco); Oxidase Strips (KEY Scientific Products); Spot Oxidase Reagent (Difco) wooden applicator sticks Whatman No. 2 filter paper Learning Objectives Each student should be able to 1. Understand the biochemistry underlying oxidase enzymes 2. Describe the experimental procedure that enables one to distinguish between groups of bacteria based on cytochrome oxidase activity 3. Give examples of oxidase-positive and oxidasenegative bacteria 4. Perform an oxidase test Pronunciation Guide Alcaligenes faecalis (al-kah-lij-e-neez fee-kal-iss) Escherichia coli (esh-er-i-ke-a KOH-lee) Pseudomonas aeruginosa (soo-do-mo-nas a-ruh-jin- OH-sah) Why Are the Above Bacteria Used in This Exercise? This exercise gives the student experience in performing the oxidase test. The oxidase test distinguishes between groups of bacteria based on cytochrome oxidase activity. Three bacteria will be used. Alcaligenes faecalis (L. faecium, of the dregs, of feces) is a gram-negative, aerobic rod (coccal rod or coccus) that possesses a strictly respiratory type of metabolism with oxygen as the terminal electron acceptor. It is thus oxidase positive. Escherichia coli is a facultatively anaerobic gram-negative rod that has both respiratory and fermentative types of metabolism and isoxidase negative. Pseudomonas aeruginosa is a gram-negative, aerobic rod having a strictly respiratory type of metabolism with oxygen as the terminal electron acceptor and thus is oxidase positive. 179

2 Medical Application The oxidase test is a useful procedure in the clinical laboratory because some gram-negative pathogenic species of bacteria (such as Neisseria gonorrhoeae, P. aeruginosa, and Vibrio species) are oxidase positive, in contrast to species in the family Enterobacteriaceae, which are oxidase negative. Principles Oxidase enzymes play an important role in the operation of the electron transport system during aerobic respiration. Cytochrome oxidase (aa 3 type) uses O 2 as an electron acceptor during the oxidation of reduced cytochrome c to form water and oxidized cytochrome c. The ability of bacteria to produce cytochrome oxidase can be determined by the addition of the oxidase test reagent or test strip (tetramethyl-p-phenylenediamine dihydrochloride or an Oxidase Disk, p-aminodimethylaniline) to colonies that have grown on a plate medium. Or, using a wooden applicator stick, a bacterial sample can either be rubbed on a Dry Slide Oxidase reaction area, on a KEY test strip, or filter paper moistened with the oxidase reagent. The light pink oxidase test reagent (Disk, strip, or Slide) serves as an artificial substrate, donating electrons to cytochrome oxidase and in the process becoming oxidized to a purple and then dark purple (figure 30.1) compound in the presence of free O 2 and the oxidase. The presence of this dark purple coloration represents a positive test. No color change or a light pink coloration on the colonies indicates the absence of oxidase and is a negative test. Procedure First Period 1. With a wax pencil, divide the bottom of a tryptic soy agar plate into three sections and label each with the name of the bacterium to be inoculated, your name, and date. 2. Using aseptic technique (see figure 14.3), make a single streak-line inoculation on the agar surface with the appropriate bacterium. 3. Incubate the plate in an inverted position for 24 to 47 hours at 35 C. Second Period 1. Add 2 to 3 drops of the oxidase reagent to the surface of the growth of several isolated colonies of each test bacterium or to some paste that has been transferred to a piece of filter paper. Using another colony, place an Oxidase Disk on it. Add a drop of sterile water. If Dry Slides or test strips are available, use a wooden applicator stick to transfer a sample to the slide, test strip, or filter paper moistened with oxidase reagent. Alternatively, drop a KEY oxidase test strip onto the surface of a slant culture and moisten it with water if necessary. 2. Observe the colony or sample for the presence or absence of a color change from pink to purple, and finally to dark purple. This color change will occur within 20 to 30 seconds. Color changes after 20 to 30 seconds are usually disregarded since the reagent begins to change color with time due to auto-oxidation. Oxidase-negative bacteria will not produce a color change or will produce a light pink color. 3. Based on your observations, determine and record in the report for exercise 30 whether or not each bacterium was capable of producing oxidase. HINTS AND PRECAUTIONS (1) Students should note the color change immediately following the addition of oxidase reagent. Color changes after 20 seconds are not valid. (2) Using Nichrome or other iron-containing inoculating devices may cause false-positive reactions. (3) If bacterial paste is transferred with an applicator stick, put the stick in a jar of disinfectant or a Biohazard bag immediately after use. 180 Biochemical Activities

3 Figure 30.1 Oxidase. Note the purple to dark purple color after the colonies have been added to filter paper moistened with oxidase reagent. Biochemistry within bacteria cytochrome 2 reduced cytochrome c + 2H + + 1/2O 2 2 oxidized cytochrome c + H 2 O oxidase Biochemistry on filter paper (disk/slide) H 3 C CH 3 H 3 C CH 3 N N + 2 oxidized cytochrome c reduced cytochrome c N N + H 3 C CH 3 H 3 C CH 3 Tetramethyl-p-phenylenediamine (reagent) Wurster's blue (dark purple) Proteins, Amino Acids, 181

4 Laboratory Report 30 Name: Date: Lab Section: Proteins, Amino Acids, 1. Complete the following table on the oxidase test. Color of Colonies after Adding Oxidase Production (+ or ) Bacterium Reagent Disk or Slide Reagent Disk or Slide A. faecalis E. coli P. aeruginosa 183

5 Review Questions 1. What metabolic property characterizes bacteria that possess oxidase activity? 2. What is the importance of cytochrome oxidase to bacteria that possess it? 3. Do anaerobic bacteria require oxidase? Explain your answer. 4. What is the function of the test reagent in the oxidase test? 5. The oxidase test is used to differentiate among which groups of bacteria? 6. Why should nichrome or other iron-containing inoculating devices not be used in the oxidase test? 7. Are there limitations to the oxidase test? 184 Biochemical Activities

6 31. Proteins, Amino Acids, and Enzymes VIII: Urease Activity EXERCISE 31 Proteins,Amino Acids, and Enzymes VIII: Urease Activity SAFETY CONSIDERATIONS Be careful with the Bunsen burner flame. Keep all culture tubes upright in a test-tube rack or in a can. Materials per Student 24- to 48-hour tryptic soy agar slants of Escherichia coli (ATCC 11229), Klebsiella pneumoniae (ATCC e13883), Proteus vulgaris (ATCC 13315), and Salmonella cholerae-suis (ATCC 29631) 5 urea broth tubes Bunsen burner test-tube rack inoculating loop incubator set at 35 C urea disks (Difco) or urease test tablets (KEY Scientific Products) 4 sterile test tubes wax pencil sterile forceps Learning Objectives Each student should be able to 1. Understand the biochemical process of urea hydrolysis 2. Determine the ability of bacteria to degrade urea by means of the enzyme urease 3. Tell when the urease test is used 4. Perform a urease test Suggested Reading in Textbook 1. Pseudomonas and the Enterobacteriaceae, section 22.3; see also figure 22.8 and tables 22.6, Pronunciation Guide Escherichia coli (esh-er-i-ke-a KOH-lee) Klebsiella pneumoniae (kleb-se-el-lah nu-mo-ne-ah) Proteus vulgaris (PRO-tee-us vul-ga-ris) Salmonella cholerae-suis (sal-mon-el-ah coler-ah SU-is) Why Are the Above Bacteria Used in This Exercise? In this exercise, the student will perform a urease test to determine the ability of bacteria to degrade urea by means of the enzyme urease. The authors have chosen two ureasepositive bacteria (Klebsiella pneumoniae and Proteus vulgaris) and two urease-negative bacteria (Escherichia coli and Salmonella cholerae-suis). Medical Application In the clinical laboratory, members of the genus Proteus can be distinguished from other enteric nonlactose-fermenting bacteria (Salmonella, Shigella) by their fast urease activity. P. mirabilis is a major cause of human urinary tract infections. Principles Some bacteria are able to produce an enzyme called urease that attacks the nitrogen and carbon bond in amide compounds such as urea, forming the end products ammonia, CO 2, and water (figure 31.1). Urease activity (the urease test) is detected by growing bacteria in a medium containing urea and using a ph indicator such as phenol red (see appendix E). When urea is hydrolyzed, ammonia accumulates in the medium and makes it alkaline. This increase in ph causes the indicator to change from orange-red to deep pink or purplish red (cerise) and is a positive test for urea hydrolysis. Failure of a deep pink color to develop is a negative test. Procedure First Period 1. Label each of the urea broth tubes with the name of the bacterium to be inoculated, your name, and date. 185

7 31. Proteins, Amino Acids, and Enzymes VIII: Urease Activity Figure 31.1 Urea Hydrolysis. (a) Uninoculated control. (b) Weakly positive reaction (delayed positive). (c) Very rapid positive reaction. (d) Negative reaction. Biochemistry within bacteria H 2 N C H 2 N Urea O + 2H 2 O Water urease CO 2 + H 2 O + 2NH 3 Carbon dioxide Water Ammonia Biochemistry within tubes Ammonia + phenol red deep pink (a) (b) (c) (d) 2. Using aseptic technique (see figure 14.3), inoculate each tube with the appropriate bacterium by means of a loop inoculation. 3. Incubate the tubes for 24 to 48 hours at 35 C. Urea Disks or Tablets 1. Add 0.5 ml (about 20 drops) of sterile distilled water to four sterile test tubes for the Difco disk or 1 ml distilled water for the KEY tablet. 2. Transfer one or two loopfuls of bacterial paste to each tube. Label with your name and date. 3. Using sterile forceps, add one urea or urease disk tablet to each tube. 4. Incubate up to 4 hours at 35 C. Check for a color change each hour. (The KEY test may be incubated up to 24 hours if necessary.) Second Period 1. Examine all of the urea broth cultures and urea disk or urease tablet tubes to determine their color (figures 31.1 and 31.2). Figure 31.2 KEY for Urea. After incubation, a pink to red color constitutes a positive test (tube on the left). If the original straw color persists, the test is negative (tube on the right). 2. Based on your observations, determine and record in the report for exercise 31 whether each bacterium was capable of hydrolyzing urea. HINTS AND PRECAUTIONS Some bacteria have a delayed urease reaction that may require an incubation period longer than 48 hours. 186 Biochemical Activities

8 31. Proteins, Amino Acids, and Enzymes VIII: Urease Activity Laboratory Report 31 Name: Date: Lab Section: Proteins, Amino Acids, and Enzymes VIII: Urease Activity 1. Complete the following table on urease activity. Color of Bacterium Urea Broth Disks Urea Hydrolysis (+ or ) E. coli K. pneumoniae P. vulgaris S. cholerae-suis 187

9 31. Proteins, Amino Acids, and Enzymes VIII: Urease Activity Review Questions 1. Explain the biochemistry of the urease reaction. 2. What is the purpose of the phenol red in the urea broth medium? 3. When would you use the urease test? 4. Why does the urea disk change color? 5. What is the main advantage of the urea disk over the broth tubes with respect to the detection of urease? 6. What is in urea broth? 7. What color is cerise? 188 Biochemical Activities