Laboratory Exercises in Microbiology - part 5 pptx

Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition IV. Biochemical Activities of Bacteria 30. Proteins, Amino Acids, and Enzymes VII: Oxidase Test © The McGraw−Hill Companies, 2002 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 Test Strips (KEY Scientific Products); SpotTest 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 oxidase- negative bacteria 4. Perform an oxidase test Suggested Reading in Textbook 1. The Electron Transport Chain, section 9.5; see also figures 9.13–9.15. 2. Rapid Methods of Identification, section 36.2; see also table 36.3. 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. fae- cium, of the dregs, of feces) is a gram-negative, aerobic rod (coccal rod or coccus) that possesses a strictly respi- ratory 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 metabo- lism 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 EXERCISE Proteins,Amino Acids, and Enzymes VII: Oxidase Test 30 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. Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition IV. Biochemical Activities of Bacteria 30. Proteins, Amino Acids, and Enzymes VII: Oxidase Test © The McGraw−Hill Companies, 2002 Medical Application The oxidase test is a useful procedure in the clinical labora- tory because some gram-negative pathogenic species of bac- teria (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 opera- tion of the electron transport system during aerobic res- piration. Cytochrome oxidase (aa 3 type) uses O 2 as an electron acceptor during the oxidation of reduced cy- tochrome c to form water and oxidized cytochrome c. The ability of bacteria to produce cytochrome ox- idase can be determined by the addition of the oxidase test reagent or test strip (tetramethyl-p-phenylenedi- amine dihydrochloride or an Oxidase Disk, p-amino- dimethylaniline) 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 cy- tochrome oxidase and in the process becoming oxi- dized 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 col- oration on the colonies indicates the absence of oxi- dase 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. 180 Biochemical Activities of Bacteria 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 trans- ferred with an applicator stick, put the stick in a jar of disinfectant or a Biohazard bag immediately after use. Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition IV. Biochemical Activities of Bacteria 30. Proteins, Amino Acids, and Enzymes VII: Oxidase Test © The McGraw−Hill Companies, 2002 Proteins, Amino Acids, and Enzymes VII: Oxidase Test 181 Figure 30.1 Oxidase Test. Note the purple to dark purple color after the colonies have been added to filter paper moistened with oxidase reagent. 2 reduced cytochrome c + 2H + + 2 oxidized cytochrome c + H 2 O 1 / 2 O 2 2 oxidized cytochrome c + + 2 reduced cytochrome c N N Wurster's blue (dark purple) Biochemistry within bacteria Biochemistry on filter paper (disk/slide) cytochrome oxidase H 3 CCH 3 H 3 CCH 3 H 3 CCH 3 H 3 CCH 3 N + N + Tetramethyl-p-phenylenediamine (reagent) Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition IV. Biochemical Activities of Bacteria 30. Proteins, Amino Acids, and Enzymes VII: Oxidase Test © The McGraw−Hill Companies, 2002 183 Name: ——————————————————————— Date: ———————————————————————— Lab Section: ————————————————————— Laboratory Report 30 Proteins, Amino Acids, and Enzymes VII: Oxidase Test 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 ____________ ____________ ____________ ____________ Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition IV. Biochemical Activities of Bacteria 30. Proteins, Amino Acids, and Enzymes VII: Oxidase Test © The McGraw−Hill Companies, 2002 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 of Bacteria Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition IV. Biochemical Activities of Bacteria 31. Proteins, Amino Acids, and Enzymes VIII: Urease Activity © The McGraw−Hill Companies, 2002 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, 22.7. 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 de- termine the ability of bacteria to degrade urea by means of the enzyme urease. The authors have chosen two urease- positive bacteria (Klebsiella pneumoniae and Proteus vul- garis) 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 prod- ucts 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 EXERCISE Proteins,Amino Acids, and Enzymes VIII: Urease Activity 31 SAFETY CONSIDERATIONS Be careful with the Bunsen burner flame. Keep all cul- ture tubes upright in a test-tube rack or in a can. Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition IV. Biochemical Activities of Bacteria 31. Proteins, Amino Acids, and Enzymes VIII: Urease Activity © The McGraw−Hill Companies, 2002 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). 2. Based on your observations, determine and record in the report for exercise 31 whether each bacterium was capable of hydrolyzing urea. 186 Biochemical Activities of Bacteria HINTS AND PRECAUTIONS Some bacteria have a delayed urease reaction that may require an incubation period longer than 48 hours. Figure 31.1 Urea Hydrolysis. (a) Uninoculated control. (b) Weakly positive reaction (delayed positive). (c) Very rapid positive reaction. (d) Negative reaction. Ammonia + phenol red Biochemistry within tubes Biochemistry within bacteria Water urease CO 2 + H 2 O + 2NH 3 Carbon dioxide Ammonia WaterUrea C deep pink (c)(b)(a) (d) H 2 N O + 2H 2 O H 2 N Figure 31.2 KEY Test 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). Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition IV. Biochemical Activities of Bacteria 31. Proteins, Amino Acids, and Enzymes VIII: Urease Activity © The McGraw−Hill Companies, 2002 187 Name: ——————————————————————— Date: ———————————————————————— Lab Section: ————————————————————— Laboratory Report 31 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 ______________ ____________ _____________________ Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition IV. Biochemical Activities of Bacteria 31. Proteins, Amino Acids, and Enzymes VIII: Urease Activity © The McGraw−Hill Companies, 2002 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 of Bacteria Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition IV. Biochemical Activities of Bacteria 32. Proteins, Amino Acids, & Enzymes IX: Lysine & Ornithine Decarboxylase Test © The McGraw−Hill Companies, 2002 Materials per Student 24- to 48-hour tryptic soy broth cultures of Enterobacter aerogenes (ATCC 13048), Citrobacter freundii (ATCC 8090), Klebsiella pneumoniae (ATCC e13883), and Proteus vulgaris (ATCC 13315) 4 Moeller’s lysine decarboxylase broth with lysine (LDC) 4 lysine iron agar slants (LIA) 4 Moeller’s ornithine decarboxylase broth with ornithine (ODC) 1 Moeller’s lysine decarboxylase broth without lysine (DC), which will serve as the control 1 Moeller’s ornithine decarboxylase broth without ornithine (OD), which will serve as the control Pasteur pipettes with pipettor inoculating loop test-tube rack sterile distilled water sterile mineral oil incubator set at 35°C 8 sterile test tubes ornithine, lysine, and decarboxylase KEY Rapid Substrate Tablets and strips (KEY Scientific Products, 1402 Chisholm Trail, Suite D, Round Rock, TX 78681; 800–843–1539; www.keyscientific.com) Bunsen burner ninhydrin in chloroform (Dissolve 50 mg ninhydrin in 0.4 ml of dimethylsulfoxide [DMSO], then add 25 ml of chloroform to the DMSO solution.) 10% KOH Learning Objectives Each student should be able to 1. Understand the biochemical process of decarboxylation 2. Tell why decarboxylases are important to some bacteria 3. Explain how the decarboxylation of lysine can be detected in culture 4. Perform lysine and ornithine decarboxylase tests Suggested Reading in Textbook 1. Protein and Amino Acid Catabolism, section 9.9; see also figure 9.23. Pronunciation Guide Citrobacter freundii (SIT-ro-bac-ter FRUN-dee) Enterobacter aerogenes (en-ter-oh-BAK-ter a-RAH- jen-eez) Klebsiella pneumoniae (kleb-se-EL-lah nu-MO-ne-ah) Proteus vulgaris (PRO-te-us vul-GA-ris) Why Are the Above Bacteria Used in This Exercise? This exercise gives the student experience using the lysine and ornithine decarboxylase test to differentiate between bacteria. Two lysine decarboxylase-positive (Enterobacter aerogenes and Klebsiella pneumoniae) and two lysine de- carboxylase-negative (Proteus vulgaris and Citrobacter freundii) bacteria, and two ornithine decarboxylase-positive (E. aerogenes and Citrobacter freundii) and two ornithine decarboxylase-negative (K. pneumoniae and P. vulgaris) bacteria were chosen to demonstrate the lysine and or- nithine decarboxylase tests. 189 EXERCISE Proteins,Amino Acids, and Enzymes IX: Lysine and Ornithine Decarboxylase Test 32 SAFETY CONSIDERATIONS Be careful with the Bunsen burner flame. No mouth pipetting. Keep all culture tubes upright in a test-tube rack or in a can. [...]... SpotTest Nitrate Reagent B zinc powder or dust or Difco’s SpotTest Nitrate Reagent C test-tube rack incubator set at 35 C 5 sterile test tubes wax pencil disposable gloves Escherichia coli (esh-er-I-ke-a KOH-lee) Pseudomonas fluorescens (soo-do-MO-nas floor-esshens) Staphylococcus epidermidis (staf-il-oh-KOK-kus epee-DER-meh-diss) Why Are the Above Bacteria Used in This Exercise? In this exercise, the student... phenylalanine deamination 2 Describe how to perform the phenylalanine deamination test 3 Perform a phenylalanine test Suggested Reading in Textbook 1 Protein and Amino Acid Catabolism, section 9.9; see also figure 9.23 Pronunciation Guide Escherichia coli (esh-er-I-ke-a KOH-lee) Proteus vulgaris (PRO-tee-us vul-GA-ris) Phenylalanine deaminase catalyzes the removal of the amino group (NH3+) from phenylalanine... decarboxylase transforms lysine into a basic primary amine, cadaverine This amine causes a pH rise in the acid-buffered system and a change in the indicator from yellow to red Lysine 2.0 mg ODC Ornithine decarboxylase transforms ornithine into a basic primary amine, putrescine This amine causes a pH rise in the acid-buffered system and a change in the indicator from yellow to red Ornithine 2.0 mg CIT Citrate... nitrogen Biochemistry within tubes Sulfanilic acid + N,N-dimethyl-1-naphthylamine + nitrite ions (colorless) (colorless) water + sulfobenzene azo-N,N-dimethyl-1-naphthylamine (red color) (C) (1+) (3+) (5+ ) Proteins, Amino Acids, and Enzymes XI: Nitrate Reduction 203 Harley−Prescott: Laboratory Exercises in Microbiology, Fifth Edition IV Biochemical Activities of Bacteria Laboratory Report © The McGraw−Hill... each of the compartments are recorded and interpreted according to the manufacturer’s instructions This is done by determining a five-digit code from the results and consulting a coding manual, or by comparing the results obtained with those outlined in a differential chart provided by the manufacturer (see appendix G) This quick multitest 2 15 Harley−Prescott: Laboratory Exercises in Microbiology, Fifth... phenylalanine can be used by P vulgaris? 2 What is the purpose of the ferric chloride in the phenylalanine deamination test? 3 When would you use the phenylalanine deamination test? 4 Name some bacteria that can deaminate phenylalanine 5 Describe the process of deamination 6 Why must the phenylalanine test be determined within 5 minutes? 7 Describe the color of an uninoculated tube of phenylalanine agar... beta-galactosidase releases yellow orthonitrophenol from the colorless ONPG; ITPG (isopropylthiogalactopyranoside) is used as inducer ONPG ITPG 0.2 mg 8.0 Ȗg ADH Arginine dihydrolase transforms arginine into ornithine, ammonia, and carbon dioxide This causes a pH rise in the acid-buffered system and a change in the indicator from yellow to red Arginine 2.0 mg LDC Lysine decarboxylase transforms lysine... positive phenylalanine test must be interpreted immediately after the addition of the FeCl3 reagent because the green color fades quickly (2) Rolling the FeCl3 over the slant aids in obtaining a faster reaction with a more pronounced color All phenylalanine tests should be read within 5 minutes After 5 minutes, the green color disappears Proteins, Amino Acids, and Enzymes X: Phenylalanine Deamination 197... Enterobacteriaceae 5 ml of sterile 0. 85% saline in test tube with cap 50 -ml plastic squeeze bottle containing tap water 5- ml Pasteur pipettes with pipettor oxidase test reagent or disk/strip petri plate TSA plate Whatman No 2 filter paper 10% ferric chloride Barritt’s reagents A and B sterile mineral oil tube containing McFarland No 3 (BaSO4) reference standard platinum inoculating loop (Nichrome wire... obtained if the test is run in tubes that have no gas from fermentation No bubbles The following abbreviations are used: ONPG (ȋ-galactosidase), ADH (arginine dihydrolase), LDC (lysine decarboxylase), ODC (ornithine decarboxylase), CIT (citrate), H2S (hydrogen sulfide), URE (urease), TDA (tryptophan deaminase), IND (indole), VP (Voges-Proskauer), GEL (gelatin), GLU (glucose), MAN (mannitol), INO (inositol), . 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. 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. freundii (SIT-ro-bac-ter FRUN-dee) Enterobacter aerogenes (en-ter-oh-BAK-ter a-RAH- jen-eez) Klebsiella pneumoniae (kleb-se-EL-lah nu-MO-ne-ah) Proteus vulgaris (PRO-te-us vul-GA-ris) Why Are