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Assignment: Staphylococcus Unknown Dichotomous Key (flow diagram) Objective: Using selected physiological traits and a list of coagulase negative Staphylococcus species, students will produce a dichotomous key separating all possible Staphylococcus species. Overview: This assignment is the first of two assignments relevant to the identification of an unknown bacterium in the genus Staphylococcus. In this handout, students will have a list of the species of genus Staphylococcus that are coagulase negative. The coagulase test is used to separate the genus Staphylococcus into two groups, either coagulase positive or coagulase negative. It is not necessary at this time to perform and interpret the coagulase test, however we will do so in lab 13. Subsequent pages of this handout include 1) A list of the possible unknown Staphylococcus species, and 2) A list of tests and traits that cannot be performed or used to determine the identity of the Staphylococcus unknown. 3) Following an introduction to Bergey's Manual of Determinative Bacteriology and an introduction to dichotomous keys, students will produce and turn in a dichotomous key that separates the possible Staphylococcus unknown species. This dichotomous key will be used to complete a later assignment called Staphylococcus Unknown ID. Students will be expected to use Table 17.15 in Bergey's Manual of Determinative Bacteriology to produce their dichotomous key. This book is located in the lab, and two copies are available at the Reserve Desk in the library at the Rancho Campus. 1. List of Possible Staphylococcus Unknowns The list of Staphylococcus species shown below are all coagulase negative Staphylococcus species that can cause infections in humans. In contrast, Staphylococcus aureus is coagulase positive and is not included in the list. S. capitis subsp. capitis S. capitis subsp. ureolyticus S. cohnii subsp. cohnii S. cohnii subsp. urealyticus 2. S. epidermidis S. lentus S. lugdunensis S.saccharolyticus S. saprophyticus S. schleiferi subsp. schleiferi S. sciuri S. simulans Use Table 17.15 from Bergey's Manual of Determinative Bacteriology to create a dichotomous key that will initiate the process of the identification of the Staphylococcus unknown. The dichotomous key will show you the steps to take to identify your Staphylococcus unknown. 1, SP 16
3. Tests to Omit: The following tests are listed in the Bergey's Table 17.15 but are tests we cannot perform and so cannot be used in your dichotomous key. 1. Colony diameter >5 mm 2. Lactic acid production L(+) isomer or D (-)-isomer 3. FDP-aldolase 4. Acid (anerobically) from: a. Cellobiose b. Fucose c. Raffinose d. Salicin e. Melezitose f. Turanose & Xylitol . 5. Hyaluronidase 6. Growth on (NH4)2804 7. Alkaline phosphatase 8. Arginine dihydrolase 9. Coagulase Recommendations: • Begin with the table from Bergey's and mark out the species not on our list (columns). . Consult the list of tests we cannot perform and mark them out as well (rows). Use the remaining information to make a dichotomous key (two-branch diagram) that separates all the possible species from each other. 10. Clumping factor 11. Fibrinolysin 12. Deoxyribonuclease Agar 13. Heat Stable Nuclease 14. B-Glucosidase 15. B-Glucouronidase 16. Novobiocin resistance Table 17.15 uses symbols to indicate what the expected test outcome will be. The interpretation for these symbols should be read carefully at the end of the table. I will require that you treat specific symbols accordingly. Failure to follow the directions below will result in a loss of points: 1. + indicates that the expected result is positive and the species name will be listed on the positive side only. 2. -indicates that the expected result is negative and the species name will be listed on the negative side. 3. D, d, ND, ds indicates that between 11-89% of the strains test positive or that not enough information is known. Treat this as a 50/50 outcome, and write the notation (D/d/ND/ds) next to the species name and include the species name on both the positive and negative sides. Write the letter "d" for the expected test result next to the species name on the dichotomous key. 4. Qindicates a delayed result (test will run longer than typical). Include the parentheses next to the species name on the dichotomous key. 5. w/+w indicates that the result might produce a weak positive result, that MIGHT possibly be interpreted as not clearly positive. Include the species on the positive side only, Write the notation "w" or "+w+ for the expected test result next to the species name on the dichotomous key. 2, SP 16 6. - indicates the organism may appear negative OR may appear with a weak positive. Treat this as a 50/50 outcome and write the "-w" next to the species name and include the species name on both the positive and negative sides.
Assignment: Staphylococcus Unknown Dichotomous Key (flow diagram) Objective: Using selected physiological traits and a list of coagulase negative Staphylococcus species, students will produce a dichotomous key separating all possible Staphylococcus species. Overview: This assignment is the first of two assignments relevant to the identification of an unknown bacterium in the genus Staphylococcus. In this handout, students will have a list of the species of genus Staphylococcus that are coagulase negative. The coagulase test is used to separate the genus Staphylococcus into two groups, either coagulase positive or coagulase negative. It is not necessary at this time to perform and interpret the coagulase test, however we will do so in lab 13. Subsequent pages of this handout include 1) A list of the possible unknown Staphylococcus species, and 2) A list of tests and traits that cannot be performed or used to determine the identity of the Staphylococcus unknown. 3) Following an introduction to Bergey's Manual of Determinative Bacteriology and an introduction to dichotomous keys, students will produce and turn in a dichotomous key that separates the possible Staphylococcus unknown species. This dichotomous key will be used to complete a later assignment called Staphylococcus Unknown ID. Students will be expected to use Table 17.15 in Bergey's Manual of Determinative Bacteriology to produce their dichotomous key. This book is located in the lab, and two copies are available at the Reserve Desk in the library at the Rancho Campus. 1. List of Possible Staphylococcus Unknowns The list of Staphylococcus species shown below are all coagulase negative Staphylococcus species that can cause infections in humans. In contrast, Staphylococcus aureus is coagulase positive and is not included in the list. S. capitis subsp. capitis S. capitis subsp. ureolyticus S. cohnii subsp. cohnii S. cohnii subsp. urealyticus 2. S. epidermidis S. lentus S. lugdunensis S.saccharolyticus S. saprophyticus S. schleiferi subsp. schleiferi S. sciuri S. simulans Use Table 17.15 from Bergey's Manual of Determinative Bacteriology to create a dichotomous key that will initiate the process of the identification of the Staphylococcus unknown. The dichotomous key will show you the steps to take to identify your Staphylococcus unknown. 1, SP 16
3. Tests to Omit: The following tests are listed in the Bergey's Table 17.15 but are tests we cannot perform and so cannot be used in your dichotomous key. 1. Colony diameter >5 mm 2. Lactic acid production L(+) isomer or D (-)-isomer 3. FDP-aldolase 4. Acid (anerobically) from: a. Cellobiose b. Fucose c. Raffinose d. Salicin e. Melezitose f. Turanose & Xylitol . 5. Hyaluronidase 6. Growth on (NH4)2804 7. Alkaline phosphatase 8. Arginine dihydrolase 9. Coagulase Recommendations: • Begin with the table from Bergey's and mark out the species not on our list (columns). . Consult the list of tests we cannot perform and mark them out as well (rows). Use the remaining information to make a dichotomous key (two-branch diagram) that separates all the possible species from each other. 10. Clumping factor 11. Fibrinolysin 12. Deoxyribonuclease Agar 13. Heat Stable Nuclease 14. B-Glucosidase 15. B-Glucouronidase 16. Novobiocin resistance Table 17.15 uses symbols to indicate what the expected test outcome will be. The interpretation for these symbols should be read carefully at the end of the table. I will require that you treat specific symbols accordingly. Failure to follow the directions below will result in a loss of points: 1. + indicates that the expected result is positive and the species name will be listed on the positive side only. 2. -indicates that the expected result is negative and the species name will be listed on the negative side. 3. D, d, ND, ds indicates that between 11-89% of the strains test positive or that not enough information is known. Treat this as a 50/50 outcome, and write the notation (D/d/ND/ds) next to the species name and include the species name on both the positive and negative sides. Write the letter "d" for the expected test result next to the species name on the dichotomous key. 4. Qindicates a delayed result (test will run longer than typical). Include the parentheses next to the species name on the dichotomous key. 5. w/+w indicates that the result might produce a weak positive result, that MIGHT possibly be interpreted as not clearly positive. Include the species on the positive side only, Write the notation "w" or "+w+ for the expected test result next to the species name on the dichotomous key. 2, SP 16 6. - indicates the organism may appear negative OR may appear with a weak positive. Treat this as a 50/50 outcome and write the "-w" next to the species name and include the species name on both the positive and negative sides.
Bergey's Manual of Determinative Bacteriology-9 Table 17.15 Characteristics differentiating the species and subspecies of the genus Staphylococcus S. aureus S. capitis Characteristics Colony diameter > 5 mm/ Colony pigment (carotenoid) Aerobic growth Anaerobic growth (thioglycolate) Growth on NaCl agar: 10% (w/v) 15% (W/V) Growth at: 15°C 45°C Cytochrome (oxidase test) Lactic acid production: L(+)-Isomer o(-)-Isomer Acetoin production FDP-aldolase: Class I Class II Acid (aerobically) from: D-Xylose L-Arabinose D-Cellobiose D-Fucose Ratinion Salicin Sucrose Maltose D-Mannitol D-Mannose D-Trehalose a-Lactose o-Galactose B-D-Fructose D-Melezitose o-Turanose D-Ribote Xylitol Hyaluronidaso Growth on (NH₂)₂50 (nitrogen source) Nitrate reduction Alkaline phosphatase Footnotes are at end of table arlettae ND ND ND ND 33 물을 1+1+ ND ND ND ND ND subsp. anaeroblus 221 22 ND ND ND subsp. aureus +W +3+1+1 11 auricularis 11110112261 NO ND (0) subsp capitis (+) $1.1 IC+121 subsp. ureolyticus (+) 22 22 22 22 22 222 222 2 ND ND ND ND ND ND d ND ND d ND ND NO d ND ND
Table 17.15 (continued) Characteristics Arginine dihydrolase Urease Coagulase (rabbit plasma) Clumping factor Fibrinolysin Hemolysis Deoxyribonuclease (DNase agar) Heat-stable nuclease -Glucosidase p-Glucuronidase Galactosidase CNPC Novobiocin resistance (MIC z 1.6 µg/ml) Table 17.15 (continued) Characteristics Colony diameter>5 mm/ Colony pigment (carotenoid) Aerobic growth Anaerobic growth (thioglycolate) Growth on NaCl agar: 10% (W/V) 15% (w/v) arlettae Growth at 15°C 45 C Cytochrome c (oxidase test) Lactic acid production: L(+)-Isomer of-Isomer Acetoin production FOP aldolase: Class I Class II Footnotes are at end of table * ND ND ND ND ND S. aureus ND subsp. anaerobius 222* ND ND ND subsp. aureus +W ND ND auricularis ND ND ND S. caprae camnosus caseolyticus chromogenes FISI (d) subsp. capitis ND ND 012 BILI ND S. capitis subsp. ureolyticus d 2 2521 ND ND (d) ND S. cohnil subsp. subsp. cohnil urealyticus (+) ND ND
Table 17.15 (continued) Characteristics Acid (aerobically) from: D-Xylose L-Arabinose D-Cellobiose D-Fucose Ratinose Salicin Sucrose Maltose D-Mannitol D-Mannose D-Trehalose a-Lactose o-Galactose B-D-Fructose b-Melezitose o-Turanose D-Ribose Xylitol Hyaluronidase Growth on (NH₂)₂SO4 (nitrogen source) Nitrate reduction Alkaline phosphatase Arginine dihydrolase Ureaso Coagulase (rabbit plasma) Clumping factor Fibrinolysin Hemolysis Deoxyribonuclease (DNase agar) Heat-stable nuclease B-Glucosidase B-Glucuronidase B-Galactosidase Novobiocin resistance (MIC > 1.6 g/ml) 3. S. caprae carnosus III ND ++++1 3+1 (+) TEXTFI aaa++ 2221121 ND ND ND ND 30+1 S. S. caseolyticus chromogenes 2222210+++21+122 +2222 222221 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND LILLET ND +++01111330111 subsp. cohnil FILIF60 +11+1190 196. (d) (d) (d) NO ND (d) S. cohnil subsp. urealyticus TILTFIF 0+0+691331196311.. (+) (d) ND d +W -W ND (d)
Table 17.15 (continued) Characteristics Colony diameter > 5 mm/ Colony pigment (carotenoid) Aerobic growth Anaerobic growth (thioglycolate) Growth on NaCl agar: 10% (w/v) 15% (w/v) Growth at: 15°C 45°C Cytochrome c (oxidase test) Lactic acid production: L(+)-Isomer D(-)-Isomer Acetoin production FDP-aldolase: Class I Class II Acid (aerobically) from: D-Xylose L-Arabinose D-Cellobiose D-Fucose Ratinose Salicin Sucrose Maltose D-Mannitol D-Mannose o-Trehalose a-Lactose D-Galactose B-D-Fructose D-Melezitose D-Turanose D-Ribose Xylitol Hyaluronidase S. S. delphini epidermidis 弓弓弓弓 +弓 +++弓弓弓弓弓弓弓弓弓弓 弓 ND ND ND ND ND aaagaa++++ 1€ 5. SP 16 S. equorum 물을 11 33 물을 +1. ND ND ND ND ND ++++++0+1 S. felis 1. 弓弓弓弓 ND S. S. gallinarum haemolyticus NO ND ND 1 10019 S. hominis
Table 17.15 (continued) Characteristics Urease Coagulase (rabbit plasma) Clumping factor Fibrinolysin Hemolysis Deoxyribonuclease (DNase agar) Heat-stable nuclease B-Glucosidase B-Glucuronidaso p-Galactosidase Novobiocin resistance (MIC 2 1.6 μg/ml) Table 17.15 (continued) Characteristics Colony diameter > 5 mm/ Colony pigment (carotenoid) Aerobic growth Anaerobic growth (thioglycolate) Growth on NaCl agar: 10% (w/v) 15% (w/v) Growth at: 15°C 45 C Cytochrome c (oxidase test) Lactic acid production: (+)-Isomer o(-)-Isomer Acetoin production FDP-aldolase: Class I Class II Acid (aerobically) from: D-Xylose L-Arabinose o-Celloblose D-Fucose Rulinone Salicin Sucrose S. S. S. delphini epidermidis equorum +11+ ND NO ND +10 11 +++ 11 -W (d) S. S. hylous intermedius (+) +0+1 +++ TIITTI ------ ND ND ND +0+1 22 221 S. S. kloosil lentus ND ND ND ND **222 W ND ND ND d S. S. fells gallinarum haemolyticus -W ND ND ND -W -W +1 d d • d + +11132. ND S. lugdunensis +2+1 ND 3++ 29212+ ND ND ND 0561938 1001 I ND ND (+) ds 05322 +1 312 +222 ND ND S. saccharolyticus -W ND S. hominis ND ND ND +27 TICIE ND
Table 17.15 (continued) Characteristics Maltose D-Mannitol -Mannose D-Trehalose a-Lactose o-Galactose Po-Fructose D-Melezitose -Turanose D-Ribose Xylitol Hyaluronidase Growth on (NH)80₂ (nitrogen source) Nitrate reduction Alkaline phosphatase Arginine dihydrolase Urease Coagulase (rabbit plasma) Clumping factor Fibrinolysin Hemolysis Deoxyribonuclease (Nase agar) Heat-stable nuclease Glucosidase 3-Glucuronidase -Galactosidase Novobiocin resistance (MIC 1.6 µg/ml) Table 17.15 (continued) Characteristics Colony diameter 5 mm Colony pigment (carotenoid) Aerobic growth Anaerobic growth (thioglycolate) Growth on NaCl agar: 10% (w/v) 15% (w/v) S. hylcus ND d S Intermedius kloosil lentus lugdunensis (W) S. mrophytou subsp. congutian NO 5. schleifer NO ND ND ND ND NO ND ND ND ND ND ND seluri simulans S. saccharolyticus 93999 99-9-9999999 ND ND NO ND xylosus
Table 17.15 (continued) Characteristice Growth at: 15/0 40/0 Cytochrome (oxidase Tw Lactic acid production: L-Isomer of-)-somer Acetoin production FOP-aidoilase Class Class Acid (erobically) from -Xylose L-Arabinoss e-Celobiose o-Fucose Raftinose Salon Sucrose Maloo Mannitol -Marnos D-Trehalose a-Lactose Galactose Bo-Fructose Melezitose Turanose Abon Xylo Hyaluronidase Growth on (NH),50, (nitrogen source) Nitrate reduction Alkaline phosphatase Arginine dihydrolase Urease Coagulase (rabbit plasma) Cumping factor Fibrinolysin Hemolysis Table 17.15 (continued) Characteristice Deoxyribonuclease (DNase agar) Heat-stable nuclease -Glucosidase -Glucuronidas B-Galactosidase Novobiocin resistance (MIC > 1.6 μg/ml) subsp. seprophyticus coagulans NO saprophyticus NO ND ND & schleifer ND 2010. 333-3 S. schleifer ND ND ND NO ND ND d ND ▼ ND NO ND ND scurt almulans warner xylosus NO NO subsp. subsp. coagulans schleifert selurt simulans warner xylosus ND NO ND (da) ND NO
Symbols: +, 90% or more strains are positive; -, 90% or more strains are negative; d, 11-89% strains are positive: (), delayed reaction; w, weak reaction; -w, negative to weak reaction; +w, positive to weak reaction; ds, test differentiates subspecies (not separated out above in heading); de, test differentiates ecotypes; ND, test not determined. Schleifer et al. (Int. J. Syst. Bacteriol. 35: 223-225, 1985; Syst. Appl. Microbiol. 5: 501-509, 1984). C d S. hyicus subsp. hyicus and S. hyicus subsp. chromogenes in Bergey's Manual of Systematic Bacteriology; raised to species status by Hájek et al. (Int. J. Syst. Bacteriol. 37: 179-180, 1987; effective publication: Syst. Appl. Microbiol. 8: 169-173, 1986). Bannerman and Kloos (Int. J. Syt. Bacteriol. 41: 144-147, 1991); the spelling of the second subspecific epithet is as in the original, urealyticus; however, the preferred form (Appendix 9 of the Bacteriological Code) is not clear. • Kloos and Wolfshohl (Int. J. Syst. Bacteriol. 41: 284-289, 1991) formally named the subspecies S. cohnii subsp. cohnii and S. cohnil subsp. urealyticus (originally spelled urealyticum through a typographic error), which in Bergey's Manual of Systematic Bacteriology were referred to as S. cohnii subsp. 1 and S. cohni subsp. 2, respectively. The subspecific epithet urealyticus is, however, illegitimate because of the prior subspecific epithet ureolyticus in S. capitis subsp. ureolyticus (Rules 12b and 13c of the Bacteriological Code), of which urealyticus is a later homonym as an orthographic variant. The epithet urealyticus in S. cohnil subsp. urealyticus will therefore have to be replaced. Varaldo et al. (Int. J. Syst. Bacteriol. 38: 436-439, 1988). Igimi et al. (Int. J. Syst. Bacteriol. 39:373-377, 1989). h Freney et al. (Int. J. Syst. Bacteriol. 38: 168-172, 1988). Igimi et al. (Int. J. Syst. Bacteriol. 40: 409-411, 1990). Colony diameter is determined after incubation on P agar (Kloos et al., Int. J. Syst. Bacteriol. 24: 79-101, 1974; Kloos and Schleifer, J. Clin. Microbiol. 1:82-88, 1975) at 34-35°C for 3 days and at room temperature ( 25°C) for an additional 2 days. N May grow on repeated subculture. Important Notes for Users of This Manual Unless otherwise indicated in footnotes to tables, the meanings of symbols are as follows: + 90% or more of strains are positive - 90% or more of strains are negative d 11-89% of strains are positive v strain instability (not equivalent to "d") D Different reactions in different taxa (species of a genus or genera of a family) All other symbols are defined in footnotes to tables.