Crystal BBL.pdf

BBL CRYSTAL™ IDENTIFICATION SYSTEMS Anaerobe ID KitCLIA COMPLEXITY: HIGH CDC IDENTIFIER CODES ANALYTE: 0412 TEST SYSTEM: 07561 INTENDED USE The BBL Crystal™ Anaerobe (ANR) Identification (ID) System is a miniaturized identification method employing modified conventional, fluorogenic and chromogenic substrates. It is intended for the identification of frequently isolated anaerobic bacteria.1,2,6,9,10,11,16,20,24 SUMMARY AND EXPLANATION Micromethods for the biochemical identification of microorganisms were reported as early as 1918.3 Several publications reported on the use of the reagent-impregnated paper discs and micro-tube methods for differentiating enteric bacteria.3,4,8,21,23 The interest in miniaturized identification systems led to the introduction of several commercial systems in the late 1960s, and they provided advantages in requiring little storage space, extended shelf life, standardized quality control and ease of use. In general, many of the tests used in the BBL CRYSTAL ID Systems are modifications of classical methods. These include tests for fermentation, oxidation, degradation and hydrolysis of various substrates. In addition, there are chromogen and fluorogen linked substrates, as in the BBL CRYSTAL ANR ID panel, to detect enzymes that microbes use to metabolize various substrates.5,8,12,13,14,15,17,18,19 The BBL CRYSTAL ANR ID kit is comprised of (i) BBL CRYSTAL ANR ID panel lids, (ii) BBL CRYSTAL bases and (iii) BBL CRYSTAL™ ANR, GP, RGP, N/H ID Inoculum Fluid (IF) tubes. The lid contains 29 dehydrated substrates and a fluorescence control on tips of plastic prongs. The base has 30 reaction wells. Test inoculum is prepared with the inoculum fluid and is used to fill all 30 wells in the base. When the lid is aligned with the base and snapped in place, the test inoculum rehydrates the dried substrates and initiates test reactions. Following an incubation period, the wells are examined for color changes or presence of fluorescence that result from metabolic activities of the microorganisms. The resulting pattern of the 29 reactions is converted into a ten-digit profile number that is used as the basis for identification.22 Biochemical and enzymatic reaction patterns for the 29 BBL CRYSTAL ANR ID substrates for a wide variety of microorganisms are stored in the BBL CRYSTAL ANR ID database. Identification is derived from a comparative analysis of the reaction pattern of the test isolate to those held in the database. A complete list of taxa that comprises the current database is provided in Table 1. 2 .15 with a UV light source.15. Visually inspect the package for holes or cracks in the foil package. Refer to Table 3 for a list of active ingredients. Reactions employed by various substrates and a brief explanation of the principles employed in the system are described in Table 2.12. Bases: Bases are packaged in two sets of ten. STORAGE AND HANDLING/SHELF LIFE Lids: Lids are individually packaged and must be stored unopened in a refrigerator at 2 − 8ºC. The tests used in the system are based on microbial utilization and degradation of specific substrates detected by various indicator systems. until ready to use. leaks.18 Chromogenic substrates upon hydrolysis produce color changes that can be detected visually. will retain expected reactivity until the date of expiration. reduce or otherwise utilize a substrate in the BBL CRYSTAL ID Systems. Enzymatic hydrolysis of fluorogenic substrates containing coumarin derivatives of 4-methylumbelliferone (4MU) or 7-amino-4-methylcoumarin (7-AMC). results in increased fluorescence that is easily detected visually 5.e. inoculum tubes. REAGENTS The BBL CRYSTAL ANR ID panel contains 29 enzymatic and biochemical substrates. in plastic bag. Do not use if the packaging appears to be damaged. and the Food and Drug Administration (FDA) under CLIA’88.S. In addition. The CDC Analyte Identifier Code is 0412. Do not use if there appears to be a leak. A bacterial suspension in the inoculum fluid is used for rehydration of the substrates. N/H ID Inoculum Fluid (IF) is packaged in two sets of ten tubes. Panel location in referred tables indicates the row and column where the well is located (example: 1J refers to Row 1 in column J).17. turbidity).. haziness.13. Empty trays should be used to incubate panels. in BBL CRYSTAL incubation trays. filter papers used for indole tests and panels must be autoclaved prior to disposal or incinerated. Lids in the original packaging. cotton swabs. there are tests that detect the ability of an organism to hydrolyze.PRINCIPLES OF THE PROCEDURE The BBL CRYSTAL ANR ID panels contain 29 dried biochemical and enzymatic substrates. Centers for Disease Control and Prevention (CDC). DO NOT FREEZE. Precautions: in vitro Diagnostic After review by the U. the CDC Test System Identifier Code is 07561. Store in a dust free environment at 2 − 25ºC. Inoculum Fluid: BBL CRYSTAL ANR.14. After use. degrade. if stored as recommended. this product has been identified as high complexity. all infectious materials including plates. Visually inspect the tubes for cracks. tube or cap damage or visual evidence of contamination (i. etc. Store unused bases in the tray. RGP. The bases are stacked facing down to minimize air contamination. GP. only the lids need to be stored at 2 − 8ºC. 20 BBL CRYSTAL Bases.5 g. RGP. store the BBL Crystal ANR kit at 2 − 8ºC. The plastic cover should remain on the lid until used.5 g. If the kit or any of the components are stored refrigerated. 2 incubation trays. It is strongly recommended that laboratories employ methods discussed in the Manual of Clinical Microbiology for specimen collection. purified water to 1000 ml.895 g.) Once lids are removed from the sealed pouches. CaCl2 0. The recommended humidity level is 40 − 60%. no more than 24 − 48 h old for most genera. On receipt. Only BBL CRYSTAL ANR.Store tubes at 2 − 25ºC. or Schaedler Blood Agar. transport and placement on primary isolation media.1 Other recommended readings relevant to anaerobic specimen handling include Wadsworth Anaerobic Bacteriology Manual24 and Principles and Practice of Clinical Anaerobic Bacteriology. Brucella Blood Agar. The usefulness of BBL CRYSTAL ID Systems or any other diagnostic procedure performed on clinical specimens is directly influenced by the quality of the specimens themselves. 1 BBL CRYSTAL ANR ID Report Pad. N/H ID Inoculum Fluid Tubes. The test isolate must be a pure culture.3 ± 0. 3 . (See “Limitations of The Procedure”. Use isolates from a nonselective blood agar medium such as CDC Anaerobe Blood Agar. Each tube has approximately 2. they must be used within 1 h to ensure adequate performance. Expiration dating is shown on the tube label. Only cotton-tipped applicator swabs should be used to prepare inoculum as some polyester swabs may cause problems with inoculation of the panels. The incubator used should be humidified to prevent evaporation of fluid from the wells during incubation. Tricine N-[2-Hydroxy-1. SPECIMEN COLLECTION AND PROCESSING BBL CRYSTAL ID Systems are not for use directly with clinical specimens. 20 BBL CRYSTAL ANR. Columbia Blood Agar. N/H ID inoculum fluid should be used with BBL CRYSTAL ANR panels. The remaining components of the kit may be stored to 2 − 25ºC. Once opened.6 TEST PROCEDURE Materials Provided: BBL CRYSTAL ANR ID Kit − 20 BBL CRYSTAL Anaerobe ID Panel Lids. GP. RGP. each should be brought to room temperature prior to use. 1-bis (hydroxymethyl)methyl] glycine 0.15 ml of Inoculum Fluid containing: KCl 7. GP. for some slow growing cocci (up to 72 h) and Actinomyces species (72 − 96 h) older cultures may be acceptable. covered lids should be used within 1 h.4 standard. 4 and No.15 ml). Test Procedure: BBL CRYSTAL ANR ID System requires Gram stain. If additional colonies are unavailable for preparation of a new inoculum.0% Tween 80 added is recommended.3 ± 0. Perform indole test per instructions provided in the package insert. For catalase test a 15. Do not use panel if there is no desiccant in the pouch.7. storage and handling of clinical specimens. and mark the patient's specimen number on the side wall. Suspend colonies in a tube of BBL CRYSTAL ANR. nonselective culture plate and catalase reagent.85% sterile saline to bring down the turbidity equivalent to a McFarland No. RGP. Prior to panel set-up. b. 3. so that the final volume of inoculum is approximately equivalent to that of the original volume in tube (2. 5 standards. 5. N/H ID Inoculum Fluid. dilute the inoculum by adding the minimum required volume (not to exceed 1. Roll back any excess fluid to the target area and place the base on a bench top. McFarland No. BBL™ DMACA Indole Reagent Droppers. 6. prepare a new inoculum equivalent to a McFarland No. Also required are the necessary equipment and labware used for preparation. Due to the high cell concentrations used in BBL CRYSTAL ANR ID panels. 4. Take a base. 7. BBL CRYSTAL™ ID System Electronic Codebook or BBL CRYSTAL ANR Manual Codebook. GP. With a fresh tube of inoculum fluid. Pour entire contents of inoculum fluid into target area of the base.0% solution of hydrogen peroxide with 1. If the inoculum concentration is in excess of the recommended McFarland standard. Using aseptic technique. 2. pick colonies of the same morphology from one of the recommended media (see section "Specimen Collection and Processing"). Remove the excess amount added to the tube with a sterile pipet.24 1. the inoculum should 4 .4. Once removed from the pouch.4 standard (not to exceed McFarland No. Failure to do this will result in spilling of the inoculum over the black portion of the base rendering the panel unusable. Remove lids from pouch. Hold base in both hands and roll inoculum gently along the tracks until all of the wells are filled. BBL CRYSTAL™ Panel Viewer (includes BBL CRYSTAL Color Reaction Charts). incubator (35 − 37°C) non-CO2 (40 − 60% humidity). The turbidity should be equivalent to a McFarland No. catalase and indole tests should be performed. using aseptic techniques. one of the following steps is recommended: a. Discard desiccant.0 ml) of 0.Materials Not Provided: Sterile cotton swabs (do not use polyester swabs). Recap tube and vortex for approximately 10 − 15 sec. 5 standard). Take an inoculum fluid tube and label with patient's specimen number. with the tip of a sterile cotton swab (do not use a polyester swab) or a wooden applicator stick or disposable plastic loop. catalase and indole test results. Trays should not be stacked more than two high during incubation. label facing down) using the BBL CRYSTAL Panel Viewer.be slowly rolled across the tracks to ensure a proper fill of all wells. Align the lid so that the labeled end of the lid is on top of the target area of the base. this is the profile number. Place thumb on edge of lid towards middle of panel on each side and push downwards simultaneously until the lid snaps into place (listen for two "clicks"). 9. corresponding to the row where the test is located. All panels should be read face down (larger windows up. which is used as a fluorescence negative control) scored positive is given a value of 4. 2. Read columns A thru F (fluorescent substrates) using the UV light source in the panel viewer. Read columns G thru J first. remove the panels from the incubator. The purity plate or slant may also be used for any supplementary tests or serology. A fluorescent substrate well is considered positive only if the intensity of the fluorescence observed in the well is greater than the Negative Control well (A4). Reading: After the recommended period of incubation. label facing down) in a non-CO2 incubator with 40 − 60% humidity. The incubation time for panels is 4 h at 35 − 37°C. Calculation of BBL CRYSTAL Profile Number: Each test result (except 4A. A B C * + + + + + 1 6 3 Profile *(4A) = fluorescent negative control Example: 4 2 1 D + 2 E + + 5 F + + 6 G + 4 H + + 3 I + + + 7 J 0 5 . using the regular (white) light source. Purity Plate: Using a sterile loop. Push down until a slight resistance is felt. Incubate the slant or plate for 24 − 48 h at 35 − 37°C under anaerobic conditions. NOTE: The incubator door should not be opened repeatedly during the incubation period (preferably less than 3 times). Ten panels can fit in one tray (5 rows of 2 panels). if required. a. 8. or 1. All panels should be incubated face down (larger windows facing up. recover a small drop from the inoculum tube either before or after inoculating the base and inoculate an agar slant or plate (any nonselective medium) for purity check. Discard inoculum tube and cap in a biohazard disposal container. b. A value of 0 (zero) is given to any negative result. Refer to the color reaction chart and/or Table 3 for an interpretation of the reactions. Make sure there is no excess fluid between the wells before the lid is aligned. Use the BBL CRYSTAL ANR Report pad to record reactions. Incubation: Place inoculated panels in incubation trays. A 10-digit number is generated. The numbers (values) resulting from each positive reaction in each column are then added together. then incubate panel for 4 h at 35 − 37°C. The type of primary plate used to prepare the inoculum will determine the appropriate database. If any of the wells. 5. For use with Brucella or Columbia Blood Agar media. Prior to incubation. confirm purity of quality control strain before contacting Becton Dickinson Microbiology Systems Technical Services. DO NOT USE PANELS from this lot. are positive per color reaction chart (after 1 − 2 min). The resulting profile number and off-line test results (Gram stain. If discrepant results are obtained.Select the appropriate BBL CRYSTAL Anaerobe database from the offered menu. Expected test results for additional quality control test strains are listed in Table 5. catalase and indole) should be entered on a PC in which the BBL CRYSTAL ID System Electronic Codebook has been installed. Taxa other than those listed in Table 1 are not intended for use in this system. 3. let panel remain at room temperature for 1 min (not more than 2 min). 8. Contact Becton Dickinson Microbiology Systems Technical Services. except 1F. Compare recorded reactions with those listed in Table 4. If all wells are negative. 6. to obtain the identification. If a PC is not available contact Becton Dickinson Microbiology Systems Technical Services for assistance with the identification. Read panel with the panel viewer and color reaction chart. (NOTE: Well 1F [Escosyl] should be positive upon rehydration). 4. LIMITATIONS OF THE PROCEDURE The BBL CRYSTAL ANR ID System is designed for the taxa provided. select alternate blood-agar database from the menu. record reactions using the Report Pad. Read and record reactions with the aid of the panel viewer and color reaction chart. 7. 6 . QUALITY CONTROL User Quality Control: follows: Quality control testing is recommended for each lot of panels as 1. 2. The incubator door should not be opened repeatedly during the incubation period (preferably less than 3 times). Inoculate a panel with Bacteroides fragilis ATCC® 25285 per recommended procedure (refer to “Test Procedure”). BBL CRYSTAL Identification Systems use a modified microenvironment. Use of panels and interpretation of results require a competent microbiologist. Only cotton-tipped applicator swabs. and Schaedler (see “Availability”). This may result in insufficient inoculum fluid to fill the wells. colonial characteristics on various media as well as metabolic end products as determined by gas-liquid chromatography. expected values for its individual tests may differ from information previously established with conventional test reactions. Once lids are removed from the sealed pouches. then it is likely that (i) the test isolate is producing atypical BBL CRYSTAL reactions (which may also be caused by procedural errors). Schaedler Agar with Vitamin K1 and 5% Sheep Blood. after inoculation. Conventional test methods are recommended when user error has been ruled out. The plastic cover should remain on the lid until used. Colonies should be taken from nonselective blood agar plates such as BBL™ CDC Anaerobe. Columbia. Reactivity of some substrates in rapid identification systems may be dependent upon the source media used in inoculum preparations. The incubator where panels are placed should be humidified to prevent evaporation of inoculum fluid from the wells during incubation. We recommend the use of the following BBL media for use with the BBL CRYSTAL ANR ID System: CDC Anaerobe Blood Agar. or wooden applicator sticks. cell morphology. Brucella. Columbia Agar with 5% Sheep Blood and Brucella Blood Agar with Hemin and Vitamin K1 (see "Availability”). label facing down) to maximize the effectiveness of substrates. should only be incubated face down (larger windows facing up. aerotolerance.All BBL CRYSTAL Anaerobe ID databases were developed with BBL™ media. The reproducibility of the individual substrate reactions ranged from 7 . therefore. The accuracy of the BBL CRYSTAL ANR ID System is based on statistical use of specially designed tests and an exclusive database. The panels. (ii) the test species is not part of the intended taxa or (iii) the system is unable to identify the test isolate with the required level of confidence. it should be recognized that minor variations may exist in strains within species. The final identification of the isolate should take into consideration the source of the specimen. they must be used within 1 h to ensure adequate performance. While BBL CRYSTAL ANR ID System aids in microbial differentiation. PERFORMANCE CHARACTERISTICS Reproducibility: In an external study involving four clinical laboratories (total of five evaluations). or disposable plastic loops should be used to prepare the inoculum suspension as some polyester swabs may cause the inoculum fluid to become viscous. when warranted. the reproducibility of BBL CRYSTAL ANR ID substrate (29) reactions was studied by replicate testing. The recommended humidity level is 40 − 60%. If the BBL CRYSTAL test profile yields a “No identification” result and culture purity has been confirmed. 1% . 245010 Description BBL CRYSTAL™ Anaerobe ID Kit. containing 20 each: BBL CRYSTAL Anaerobe ID Panel Lids. A total of five studies were conducted in four independent laboratories. BBL CRYSTAL™ Panel Viewer White Light Tube BBL CRYSTAL™ ID System Electronic Codebook. as well as to conventional reference identification methods based on VA Wadsworth Laboratory recommendations. Domestic model. BBL CRYSTAL ™ Panel Viewer. routine isolates arriving in the clinical laboratory. Out of 633 total isolates tested from the five studies. 110 V. 588 (93%) were correctly identified (including isolates that required supplemental testing) by the BBL CRYSTAL ANR Identification System. 100 V. BBL CRYSTAL™ Panel Viewer. 60 Hz. BBL CRYSTAL Bases and BBL CRYSTAL Anaerobe ID Inoculum Fluid. No. GP.25 AVAILABILITY Cat.96. Japanese model. 220 V. 50/60 Hz.2% to 100%. BBL™ CDC Anaerobe Blood Agar with 5% Sheep Blood. BBL CRYSTAL™ Panel Viewer Longwave UV Tube. 50 Hz. European model. RGP. N/H ID Inoculum Fluid. ctn of 100 plates. pkg of 20. The overall reproducibility of BBL CRYSTAL ANR panel was determined to be 99. A total of 36 (6%) isolates were incorrectly identified. ctn of 10.25 Accuracy of Identification: The performance of BBL CRYSTAL ANR ID System was compared to a currently available commercial system. BBL CRYSTAL™ Identification Systems Anaerobe Manual Codebook. as well as previously identified isolates of the clinical trial sites' choice were utilized to establish performance characteristics. BBL CRYSTAL™ ANR. 8 . using clinical isolates and stock cultures. pkg of 20 plates. and a message of "No Identification" was obtained for 9 (1%) isolates. BBL CRYSTAL™ Panel Viewer. BBL™ CDC Anaerobe Blood Agar with 5% Sheep Blood. Fresh. BBL™ Schaedler Agar with Vitamin K1 and 5% Sheep Blood. ctn of 100. Principles and practice of clinical anaerobic bacteriology. Manual of clinical microbiology.M. Stewart. J.T. Miniaturized microbiological methods. and V. 9 . Jr. pkg of 20. Manual for the identification of medical bacteria. Am. 8.. J.V. 6.J. American Society for Microbiology. Washington. ctn of 100.BBL™ Schaedler Agar with Vitamin K1 and 5% Sheep Blood. Clin. 5th ed. 8:922-923. P. Microbiol. 24:368-371. W. New York. 7.J. 1968. BBL™ Columbia Agar with 5% Sheep Blood.C.).). Jr. Public Health. A reliable test for differentiation and presumptive identification of certain clinically significant anaerobes. A. Shadomy (ed. Cowan. Baron. Steel.C. S. Cambridge.. Edberg. Academic Press. visit our website http://www. and C. Kontnick. St. Clin. 2. Herrmann. Mosby Company. BBL™ DMACA Indole Reagent Droppers. 1978.. S. K. Hartman.L. D. 1990. Bronfenbrenner. BBL™ Brucella Blood Agar with Hemin and Vitamin K1.. pkg 4 x 250 ml bottles. Am. BBL™ Brucella Blood Agar with Hemin and Vitamin K1. E.D. REFERENCES 1. Duben-Engelkirk.J.bd. and M... and B. Dowell. The C. J. Balows. A rapid method for the identification of bacteria fermenting carbohydrates.G. Belmont.J. S. Comparison of β-glucoronidase-based substrate systems for identification of Escherichia coli. 1986. (ed. Finegold. ctn of 50. 4. 2nd ed. 3. or contact the nearest Becton Dickinson Microbiology Systems office.L.M. Bailey and Scott's diagnostic microbiology. Louis. Slide catalase. 1991. J.. For specific catalog number information. BBL™ Columbia Agar with 5% Sheep Blood. 1 992. 1974. pkg of 20. J. Engelkirk. 5. Calif. 1918. and K. Hausler. PA. pkg of 100.J. Star Publishing Company.com/microbiology. BBL™ Gram Stain Kit. Schlesinger. Hansen.R. H. Microbiol. Cambridge University Press. Isenberg. 13:444-448.. and S. and H. 8th ed.J. E. Bennett. Douglas. P. Bascomb. Rapid presumptive identification of black-pigmented gram-negative anaerobic bacteria by using 4methylumbelliferone derivatives. Qadri. 1990. Microbiol. M. A. B. Churchill Livingstone Inc. 20. Rodloff. Cato and W. Rabe. Moore. R.P. Virginia Polytechnic Institute and State University. Jr. 1 5. Holdeman. 5:36-40. Supplement to the 4th edition. 4th edition.L. M. and T. Clin. Maddocks. 1977. 1976. Rapid diagnosis of Enterobacteriaceae 1: detection of bacterial glycosidases.C. Principles and practice of infectious diseases. Rapid identification of Bacteroides fragilis group organisms with the use of 4-methylumbelliferone derivative substrates. Rauhoff. New York. Virginia Polytechnic Institute and State University.L. Cox. 1 3. L. Rapid method for identifying bacterial enzymes.... 84:245-251. D. Rapid test for esculin hydrolysis by anaerobic bacteria.C. Hiller. Sanders. E. J.C. 18. Mangels. Virginia Polytechnic Institute and State University. 1981. 3rd ed.E.E. 1993.E. Infect. Practical anaerobic bacteriology. P. J. Cato and W. 11. Pathol.J.V.9.M. 21. Antonie van Leeuwenhoek 47:371-379. Supplement to the 4th edition.C. and W. 1991..J.C. 1987.K. 1975.C. Blacksburg. L. 10.C. Bulow. LV. Johnson. Holdeman. and M. Microbiol. 1957. 19. Anaerobe laboratory manual update. A. Glycosidase profiles of members of the family Enterobacteriaceae.. A. EP. Scand. J. 10 .. Clin. 29:2877-2879. 1991. Coordinating ed. Greenan. Moncla. Blacksburg. Rodloff. Kilian. Rev. G. 1993. Clin. 29:1955-1958.. Blacksburg. and S. Dott. J.E. Cook. and J. O. Edvalson. and R. S. 1991. Mandell. Holdeman. 16. Washington. 14.. and S. Dis. Fluorogenic and chromogenic substrates used in bacterial diagnostics. Clin..M. 1991. Kampfer. Microbiol.E.P.G.. Braham. 12. Anaerobe laboratory manual update.. Sect. Microbiol. 28:686-687. W.. Moore. Appelbaum. American Society for Microbiology. Acta Pathol. Anaerobe laboratory manual. P. L.. and S. A rapid method for the characterization of enteric pathogen using paper discs. B. Faber. Appl.. Kneifel. 55:335-348. 16(54):5319-5321. J. and P. and M.L. Microbiol. I. Cumitech 5A. J.V. 17.. Manafi. Cato and W.C. Moore. Zabransky. A. The application of computers to taxonomy. 1/99 (PI 7/99) © 1999 Becton Dickinson and Company BBL. Sneath.M.H. H. Belmont. Wadsworth anaerobic bacteriology manual. Trop. 1957. P.:96-100. Public Health. 25. selection 2.M. Rev.M. 5th ed. and BBL CRYSTAL are trademarks of Becton Dickinson and Company.22. Puerto Rican J. Fermentation reactions with dried paper discs containing carbohydrate and indicator.A. Microbiol. telephone Technical Services. Strong.J. ATCC is a trademark of the American Type Culture Collection. 1949. D. and S. Approved by: Date effective: Supervisor: _________________________________ Date: _____________ Director: ___________________________________ Reviewed by: Date: _____________ 11 . Data on file at Becton Dickinson Microbiology Systems. C. TECHNICAL INFORMATION: In the United States. Citron.. 24. Wexler. P. Summanen. J. toll free (800) 638-8663. Barron. Finegold. 17:201-221.B. E. Star Publishing Company. Med. 1993. Calif. O. Gen. Soto. 23. pyogenes A. jensenii L. minutum Bifidobacterium B. sporogenes C. denticola P. morbillorum Peptostreptococcus P. mulieris M. odontolyticus A. wadsworthia Desulfomonas D. asaccharolyticus P. granulosum4 P. micros P. limosum C. veroralis4 Non Pigmented. BBL™ Schaedler and BBLCRYSTAL alternate Blood Agar databases only. catenaforme L. clostridioforme C. BBL™ Schaedler database only. merdae. nucleatum F. sphenoides C. propionicum Lactobacillus L. praeacuta Bile Tolerant Non Pigmented Bilophila B. russii F. viscosus Atopobium A. species2. pigra Desulfovibrio species Campylobacter C. aerofaciens E. varium Leptotrichia L. caccae B.Table 1 Taxa in BBL CRYSTAL™ ANR ID System Gram. curtisii M. indolicus P. granulosum4 P. bifermentans C. difficile C. stercoris B. ureolyticus Campylobacter C. 4 = These taxa have < 10 unique BBL CRYSTAL profiles in the current database. necrophorum F. ovatus B. naeslundii A. oralis P. adolescentis B.. fragilis B. disiens P. buccalis P. productus4 Staphylococcus S. constellatus S. bovis A.4 F. innocuum C. prevotii P. cadaveris C. acnes P. sordellii C. Non Pitting Bacteroides B. endodontalis P. meyeri A. 2= Taxon in BBL CRYSTAL.Negative Bacilli Bile Tolerant Bacteroides fragilis group B. buccalis Clostridia Clostridium C. melaninogenica Porphyromonas P. botulinum C. bivia P. histolyticum C. uniformis B. distasonis and B. asaccharolytica P. lentum E. thetaiotaomicron B. casei L. Pitting Bacteroides B. baratii C. buccae P. subterminale C. eggerthii B. oris P. beijerinckii C. Bile Sensitive Non Pigmented Prevotella P. putrificum C. butyricum C. loescheii P. intermedius Gram-Negative Cocci Veillonella species 12 . tetradius Ruminococcus R. corporis P. species Eubacterium E. perfringens C. israelii A. fermentum L. intermedia P. gonidiaformans1. hastiforme C. septicum C. 3 = Includes B. acidophilus L. gracilis Fusobacterium F. splanchnicus Porphyromonas levii4 Bile Sensitive Pigmented Capnocytophaga species Prevotella P. distasonis group3 B. saccharolyticus Streptococcus S. limosum Mobiluncus M. anaerobius P. avidum P. glycolicum C. capillosus Tissierella T. curvus/rectus Non-Pigmented. gingivalis Key: 1= Taxon in BBL CRYSTAL. novyi A C.4 Propionibacterium P. dentium B. magnus P. tertium C. ramosum C. paraputrificum4 C. mortiferum F. johnsonii L. rhamnosus Gram-Positive Cocci Gemella G. vulgatus Other: B. tetani4 1 Non-Spore Forming GramPositiveBacilli Actinomyces A. 5.5.14.5.Table 2 Principles of Tests Employed in the BBL CRYSTAL ANR ID Sysytem Panel Location 4A 2A 1A 4B 2B 1B 4C 2C 1C 4D 2D 1D 4E 2E 1E 4F 2F 1F 4G 2G 1G 4H 2H 1H 4I 2I 1I 4J 2J 1J Test Feature Fluorescent negative control L-arginine-AMC L-histidine-AMC 4MU-α-D-mannoside L-serine-AMC L-isoleucine-AMC 4MU-β-D-mannoside Glycine-AMC L-alanine-AMC 4MU-N-acetyl-β-D-galactosaminide L-pyroglutamic acid-AMC L-lysine-AMC L-methionine-AMC 4MU-β-D-cellobiopyranoside 4MU-β-D-xyloside L-phenylalanine-AMC L-leucine-AMC Escosyl Disaccharide Furanose Pyranose p-nitrophenyl-α-D-galactoside p-nitrophenyl-β-D-galactoside p-nitrophenyl-phosphate p-nitrophenyl-α-D-glucoside p-nitrophenyl-N-acetyl-glucosaminide L-proline-p-nitroanilide p-nitrophenyl-α-L-fucoside p-nitrophenyl-β-D-glucoside L-alanyl-L-alanine-p-nitroanilide Code FCT FAR FHI FAM FSE FIS FBM FGL FAL FGA FPY FLY FME FCE FXY FPH FLE FSC DIS FUR PYO AGA NPG PHO AGL NAG PRO AFU BGL ALA Enzymatic hydrolysis of the colorless amide substrate releases yellow p-nitroaniline.2.4.12.1.13.12.15.17.14.8 Hydrolysis of the glycosidic bond results in the release of nonfluorescent esculetin.14.13.15 Enzymatic hydrolysis of the colorless aryl substituted glycoside releases yellow p-nitrophenol.14.15 Enzymatic hydrolysis of the colorless aryl substituted glycoside releases yellow p-nitrophenol.19 Enzymatic hydrolysis of the amide or glycosidic bond results in the release of a fluorescent coumarin derivative.12.15 Enzymatic hydrolysis of the colorless amide substrate releases yellow p-nitroaniline.13.15 Utilization of carbohydrate results in lower pH and change in indicator (Phenol Red).12.13.18 Principle (Reference) Control to standardize fluorescent substrate results 13 .5. Neg. FCT FAR FHI FAM FSE FIS FBM FGL FAL FGA FPY FLY FME FCE FXY FPH FLE FSC n/a blue fluorescence >FCT well blue fluorescence >FCT well blue fluorescence >FCT well blue fluorescence >FCT well blue fluorescence >FCT well blue fluorescence >FCT well blue fluorescence >FCT well blue fluorescence >FCT well blue fluorescence >FCT well blue fluorescence >FCT well blue fluorescence >FCT well blue fluorescence >FCT well blue fluorescence >FCT well blue fluorescence >FCT well blue fluorescence >FCT well blue fluorescence >FCT well blue/green fluorescence >FCT well Gold/Yellow Gold/Yellow Gold/Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow n/a blue fluorescence ≤FCT well blue fluorescence ≤FCT well blue fluorescence ≤FCT well blue fluorescence ≤FCT well blue fluorescence ≤FCT well blue fluorescence ≤FCT well blue fluorescence ≤FCT well blue fluorescence ≤FCT well blue fluorescence ≤FCT well blue fluorescence ≤FCT well blue fluorescence ≤FCT well blue fluorescence ≤FCT well blue fluorescence ≤FCT well blue fluorescence ≤FCT well blue fluorescence ≤FCT well blue fluorescence ≤FCT well blue/green fluorescence ≤FCT well Orange/Red Orange/Red Orange/Red Colorless Colorless Colorless Colorless Colorless Colorless Colorless Colorless Colorless Active Ingredients Fluorescent coumarin derivative L-arginine-AMC Approx. Fluorescence will decrease when the enzyme is present. Amt (g/L) ≤1 ≤1 ≤1 ≤1 ≤1 ≤1 ≤1 ≤1 ≤1 ≤1 ≤1 ≤1 ≤1 ≤1 ≤1 ≤1 ≤1 ≤1 ≤300 ≤300 ≤300 ≤7 ≤7 ≤7 ≤7 ≤7 ≤7 ≤7 ≤7 ≤7 L-histidine-AMC 4MU-α-D-mannoside L-serine-AMC L-isoleucine-AMC 4MU-β-D-mannoside glycine-AMC L-alanine-AMC 4MU-N-acetyl-β-D-galactosaminide L-pyroglutamic acid-AMC L-lysine-AMC L-methionine-AMC 4MU-β-D-cellobiopyranoside 4MU-β-D-xyloside L-phenylalanine-AMC L-leucine-AMC Escosyl 4G 2G 1G 4H 2H 1H 4I 2I 1I 4J 2J 1J Disaccharide Furanose Pyranose p-n-p-α-D-galactoside p-n-p-β-D-galactoside p-n-p-phosphate p-n-p-α-D-glucoside p-n-p-N-acetylglucosaminide L-proline-p-nitroanilide p-n-p-α-L-fucoside p-n-p-β-D-glucoside L-alanyl-L-alanine-pnitroanilide DIS FUR PYO AGA NPG PHO AGL NAG PRO AFU BGL ALA Disaccharide Furanose Pyranose p-n-p-α-D-galactoside p-n-p-β-D-galactoside p-n-p-phosphate p-n-p-α-D-glucoside p-n-p-N-acetyl-glucosaminide L-proline-p-nitroanilide p-n-p-α-L-fucoside p-n-p-β-D-glucoside L-alanyl-L-alanine-p-nitroanilide * The Ecosyl substrate is fluorescent unhydrolized.Panel Location 4A 2A 1A 4B 2B 1B 4C 2C 1C 4D 2D 1D 4E 2E 1E 4F 2F 1F Substrate Fluorescent negative control L-arginine-AMC L-histidine-AMC 4MU-α-D-mannoside L-serine-AMC L-isoleucine-AMC 4MU-β-D-mannoside Glycine-AMC L-alanine-AMC 4MU-N-acetyl-β-Dgalactosaminide L-pyroglutamic acid-AMC L-lysine-AMC L-methionine-AMC 4MU-β-Dcellobiopyranoside 4MU-β-D-xyloside L-phenylalanine-AMC L-leucine-AMC Escosyl* Table 3 Reagents used in the BBL CRYSTAL ANR ID System Code Pos. 14 . 15 .9 + + +1 + + + + + − + + + Panel Location 4A 2A 1A 4B 2B 1B 4C 2C 1C 4D 2D 1D 4E 2E 1E 4F 2F 1F 4G 2G 1G 4H 2H 1H 4I 2I 1I 4J 2J 1J Substrate Fluorescent negative control L-arginine-AMC L-histidine-AMC 4MU-α-D-mannoside L-serine-AMC L-isoleucine-AMC 4MU-β-D-mannoside L-serine-AMC L-alanine-AMC 4MU-N-acetyl-β-D-galactosaminide L-pyroglutamic acid-AMC L-lysine-AMC L-methionine-AMC 4MU-β-D-cellobiopyranoside 4MU-β-D-xyloside L-phenylalanine-AMC L-leucine-AMC Escosyl Disaccharide Furanose Pyranose p-n-p-α-D-galactoside p-n-p-β-D-galactoside p-n-p-phosphate p-n-p-α-D-glucoside p-n-p-N-acetyl-glucosaminide L-proline-p-nitroanilide p-n-p-α-L-fucoside p-n-p-β-D-glucoside L-alanyl-L-alanine-p-nitroanilide Code FCT FAR FHI FAM FSE FIS FBM FGL FAL FGA FPY FLY FME FCE FXY FPH FLE FSC DIS FUR PYO AGA NPG PHO AGL NAG PRO AFU BGL ALA 1 = Negative from BBL™ Schaedler 6 = Variable from BBL™ Brucella 2 = Positive from BBL™ Schaedler 7 = Negative from BBL™ Columbia 3 = Variable from BBL™ Schaedler 8 = Positive from BBL™ Columbia 4 = Negative from BBL™ Brucella 9 = Variable from BBL™ Columbia 5 = Positive from BBL™ Brucella * Results shown are expected when BBL™ CDC Anaerobic Agar with 5% sheep blood is used.Table 4 Quality Control Chart for BBL CRYSTAL ANR ID System* Bacteroides fragilis ATCC® 25285 − V − V1 − − + − V + V1.4 V V + V1 V + − 3.6. 5 − − − − − − − − − − − − Lactobacillus acidophilus ATCC® 314 − + +6 − +6 + − V2 + − V4.5.9 +3.8 V2.9 − V1 V5.6.8 + + V1.9 − − V +3 V2.6. 16 .Table 5 Additional Quality Control Strains for BBL CRYSTAL ANR ID System Bacteroides distasonis ATCC® 8503 − + V + − −9 +3 V1.8 + V8 +3 V8 + V + + + + + + + + − − + + Peptostreptococcus asaccharolyticus ATCC® 29743 − + + − − − − V1 V1 − − + +3.6.9 +3.7 + +3 +3.6.9 − − − − − − − − + − V − − − V V5 − V + − − − − − − − − − Panel Location 4A 2A 1A 4B 2B 1B 4C 2C 1C 4D 2D 1D 4E 2E 1E 4F 2F 1F 4G 2G 1G 4H 2H 1H 4I 2I 1I 4J 2J 1J Substrate Fluorescent negative control L-arginine-AMC L-histidine-AMC 4MU-α-D-mannoside L-serine-AMC L-isoleucine-AMC 4MU-β-D-mannoside Glycine-AMC L-alanine-AMC 4MU-N-acetyl-β-D-galactosaminide L-pyroglutamic acid-AMC L-lysine-AMC L-methionine-AMC 4MU-β-D-cellobiopyranoside 4MU-β-D-xyloside L-phenylalanine-AMC L-leucine-AMC Escosyl Disaccharide Furanose Pyranose p-n-p-α-D-galactoside p-n-p-β-D-galactoside p-n-p-phosphate p-n-p-α-D-glucoside p-n-p-N-acetyl-glucosaminide L-proline-p-nitroanilide p-n-p-α-L-fucoside p-n-p-β-D-glucoside L-alanyl-L-alanine-p-nitroanilide Code FCT FAR FHI FAM FSE FIS FBM FGL FAL FGA FPY FLY FME FCE FXY FPH FLE FSC DIS FUR PYO AGA NPG PHO AGL NAG PRO AFU BGL ALA 1 = Negative from BBL™ Schaedler 6 = Variable from BBL™ Brucella 2 = Positive from BBL™ Schaedler 7 = Negative from BBL™ Columbia 3 = Variable from BBL™ Schaedler 8 = Positive from BBL™ Columbia 4 = Negative from BBL™ Brucella 9 = Variable from BBL™ Columbia 5 = Positive from BBL™ Brucella * Results shown are expected when BBL™ CDC Anaerobic Agar with 5% sheep blood is used.7 + + + − + + − 3.8 V − + V Fusobacterium varium ATCC® 27725 − − 3.