LABORATORY PROCEDURE
Contents
1. Phoenix Panels Phoenix ID Broth Phoenix AST Broth Phoenix AST Indicator Solution Phoenix Inoculation Station Phoenix Transport Caddy BBL CrystalSpec or BD PhoenixSpec Nephelometer and Standards 25 uL pipettor and sterile tips 50 uL pipettor and sterile tips 2 Pipette stands Materials Required But Not Provided Gram stain reagents Sterile cotton swabs Page 4 of 23 Non selective culture plated media e g Trypticase Soy Agar with 5 Sheep Blood Incubators Biohazard disposable container Markers etc PHOENIX TEST PROCEDURE Note The Phoenix instrument should always be powered on If it is not power on the instrument and allow 2 hours for the instrument to warm up before loading panels Prepare the Phoenix instrument to receive new panels as described in the BD Phoenix System User s Manual Operation Daily System Maintenance Care should be exercised when handling Phoenix panels You should handle panels by the sides only to avoid marking smudging or obscuring the front or back of the panel in any way Accession barcode labels affixed to a Phoenix panel should Not be of fluorescent material Not cover any Phoenix panel reaction wells Not cover the Phoenix panel sequence number barcode Broth and Panel Preparation 1 Confirm the Gram stain reaction of the isolate before proceeding with the inoculum preparation for use in the Phoenix instrument Once the Gram stain reaction is confirmed select the appropriat2. AST AST ID AST AST AST AST AST Page 15 of 23 Listeria welshimeri Macrococcus caseolyticus Micrococcus luteus Micrococcus lylae Oerskovia xanthineolytica Paenibacillus alvei Paenibacillus macerans Pediococcus acidilactici Pedicococcus damnosus Pediococcus dextrinicus Pediococcus parvulus Pediococcus pentosaceus Rhodococcus equi Rothia dentocariosa Rothia mucilaginosa Staphylococcus arlettae Staphylococcus aureus Staphylococcus auricularis Staphylococcus capitis Staphylococcus capitis ssp capitis Staphylococcus capitis ssp ureolyticus Staphylococcus caprae Staphylococcus carnosus Staphylococcus chromogenes Staphylococcus cohnii Staphylococcus cohnii ssp cohnii Staphylococcus cohnii ssp urealyticum Staphylococcus condimenti Staphylococcus delphini Staphylococcus epidermidis Staphylococcus equorum Staphylococcus felis Staphyloccus fleurettii Staphylococcus gallinarum Staphylococcus haemolyticus AST ID AST ID AST ID AST ID AST ID AST ID AST ID AST ID AST ID AST ID AST ID AST AST AST ID AST ID AST ID AST AST ID AST ID AST Page 16 of 23 Staphylococcus hominis Staphylococcus hyicus Staphylococcus intermedius Staphylococcus kloosii Staphylococcus lentus Staphylococcus lugdunensis Staphylococcus lutrae Staphylococcus muscae Staphylococcus pasteuri Staphylococcus piscifermentans Staphylococcus pulvereri Staphylococcus saccharolyticus Staphylococcus saprophyticus Staphylococcus s
3. adequately fill the panel f Broth may now be used to inoculate the Phoenix AST Broth and or the Phoenix Panel If you are performing identification only proceed to Step 15 and continue the procedure Label a Phoenix AST Broth tube 8 0 mL with the patient s specimen number Holding the AST Indicator Solution bottle vertically add one free falling drop of AST indicator solution to the AST broth tube Invert to mix DO NOT VORTEX Note Allow AST Indicator Solution to warm to room temperature before dispensing into AST broth The unused portion of the indicator should be returned to 2 8 C as soon as possible Do not store at room temperature for more than 2 hours Opened bottles should be discarded after 14 days from initial opening If volume other than one drop is added inadvertently discard the tube and use a fresh tube of AST broth After the addition of the Indicator to AST broth the mixed solution can be stored in the dark at room temperature for as long as 8 hours Tubes must be used within 2 hours after the addition of the indicator solution if exposed to light If an inoculum density of 0 50 0 60 was used transfer 25 uL of the bacterial suspension from the ID tube into the AST broth tube If an inoculum density of 0 20 0 30 was used transfer 50 uL use 2 shots if utilizing a 25 pL pipettor of the bacterial suspension from the ID tube into the AST broth tube Note Panels must be inoculated within 30 minutes of the time th
4. performed in the Phoenix instrument at a time using Phoenix ID AST combination panels A sealed and self inoculating molded polystyrene tray with 136 micro wells containing dried reagents serves as the Phoenix disposable The combination panel includes an ID side with dried substrates for bacterial identification an AST side with varying concentrations of antimicrobial agents and growth and fluorescent controls at appropriate well locations The Phoenix system utilizes an optimized colorimetric redox indicator for AST and a variety of colorimetric and Page 1 of 23 fluorometric indicators for ID The AST Broth is cation adjusted e g Ca and Mg to optimize susceptibility testing performance The Phoenix panel is comprised of a 51 well ID side and an 85 well AST side The ID side contains 45 wells with dried biochemical substrates and 2 fluorescent control wells The AST side contains 84 wells with dried antimicrobial agents and 1 growth control well Panels are available as ID only Phoenix NID Panels Phoenix PID Panels AST only Phoenix NMIC Panels Phoenix PMIC Panels or ID AST combination Phoenix NMIC ID Panels Phoenix PMIC ID Panels Unused wells are reserved for future use Phoenix panels are inoculated with a standardized inoculum Organism suspensions must be prepared only with the BBL CrystalSpec or BD PhoenixSpec Nephelometer Once inoculated panels are placed into the instrument and continuousl
5. A N VAALA PNP AD GLUCOSIDE P PAGLU Enzymatic hydrolysis of the colorless aryl substituted glycoside release yellow PNP PHOSPHATE P PHOL p nitrophenol BETA GENTIOBIOSE R_BGEN D SUCROSE R_DSUC MALTOTRIOSE R_MTT N ACETYL GLUCOSAMINE R_NGU D TREHALOSE R DTRE Utilization of carbohydrate results in lower pH T and change in indicator phenol red D TAGATOSE R_DTAG MALTOSE R_MAL DEXTROSE R_DEX UREA S_URE Hydrolysis of urea and the resulting ammonia change results in pH rise and change in fluorescent indicator ESCULIN T_ESC Hydrolysis of esculin results in a black precipitate in the presence of ferric ion NITROCEFIN L_NCF Enzymatic hydrolysis of the B lactam ring results in a color change Page 22 of 23 Table C Recommended Media and Approved Use Recommended Media Approved Use ID AST Trypticase Soy Agar with 5 Sheep Blood Yes Yes Bromthymol Blue BTB Lactose Agar Yes Yes BBL CHROMagar Orientation Yes Yes Chocolate Agar Yes Yes Columbia Agar with 5 Horse Blood Yes Yes Columbia Agar with 596 Sheep Blood Yes Yes Columbia CNA Agar with 5 Sheep Blood Yes No Cystine Lactose Electrolyte Deficient CLED Agar Yes Yes Phenylethyl Alcohol Agar Yes No Trypticase Soy Agar without Blood Yes No Trypticase Soy Agar with Lecithin and Tween Yes No 80 The use of CHROMagar Orientation may produce false susceptibility results when testing erythromycin with Gra
6. Broth cessit Configuration Panel 0 50 Blackened 0 25 0 50 25 uL Blackened If also running AST Page 7 of 23 USER QUALITY CONTROL In order to ensure appropriate set up procedure and acceptable performance of the system the following organisms are recommended to be tested The user is advised to review the individual AST panel formats to determine if all test strains need to be tested for routine laboratory Quality Control Refer to the Package Insert that accompanies the Phoenix panels for expected ID results and AST results for QC organisms For instructions for QC panel login and loading refer to the BD Phoenix System User s Manual Panel Login and Inserting Panels in the Instrument ID PMIC ID and PID panels Staphylococcus aureus ATCC 29213 Enterococcus faecalis ATCC 29212 AST PMIC ID PMIC panels Staphylococcus aureus ATCC 29213 Enterococcus faecalis ATCC 29212 Staphylococcus aureus ATCC 25923 PMIC ID panels only QC for Nitrocefin Enterococcus faecalis ATCC 51299 For the most reliable results it is recommended that the QC organisms be subcultured at least twice on two consecutive days onto TSA II with 5 Sheep Blood Agar before use in the Phoenix system Compare recorded results to those listed in the Package Insert If discrepant results are obtained review test procedures as well as confirm purity of the quality control strain used before contacting B
7. D Diagnostics Technical Services Department Unacceptable QC results are documented as Fail and acceptable QC results are documented as Pass on the QC Report RESULTS Organism identification will appear on the Phoenix Report Form with a probability percentage from the Phoenix database based on the substrate reaction profile Results from each substrate will appear as V or X for each reaction The MIC results and Interpretive Categorical Results SIR will be shown for the appropriate organism antimicrobial agent combinations Special messages will be shown when the BDXpert System detects results that are of particular clinical interest Further information concerning results obtained from the Phoenix system can be found in the BD Phoenix System User s Manual Obtaining Results Messages Error messages may appear if the system detects unexpected reactivity due to inappropriate procedure or instrument malfunction For a complete listing of error codes and their meaning refer to the BD Phoenix System User s Manual System Alerts Needs Attention and Troubleshooting Page 8 of 23 Special Notes In general the Phoenix System provides a MIC for all organisms at any of the concentrations defined on a specific panel For certain antimicrobic organism combinations a specific minimum or maximum MIC is reported even if there is a lower or higher concentration on the panel These MIC values are applied b
8. IDE M BDGLU L PROLINE AMC A LPROB L PYROGLUTAMIC A LPYR ACID AMC L PHENYLALANINE AMC A LPHET L TRYPTOPHAN AMC A LTRY 4MU PHOSPHATE M PHOS METHIONINE AMC A META Enzymatic hydrolysis of the amide or glycosidic 4MU AD GLUCOSIDE M ADGLU bond results in the release of a fluorescent ARGININE ARGININE AMC A ARARR coumarin or 4 methylumbelliferone derivative GLYCINE PROLINE AMC A GLPRB 4MU BD GLUCURONIDE M BDGLC L LEUCINE AMC A LLEUH 4MU N ACETYL BD GLUCOS M NAG AMINIDE L ARGININE AMC A LARGH 4MU PHOSPHATE with M PHOT Trehalose L HISTIDINE AMC A LHIST L ISOLEUCINE AMC A LISO 4MU BD GALACTOSIDE M BDGAL Page 21 of 23 Substrate Name Code Principle COLISTIN C CLST Resistance to the antimicrobial agents results in a reduction of the resazurin based indicator POLYMYXIN B C PXB D GLUCONIC ACID C DGUA 3 METHYL GLUTARIC ACID C 3MGA D FRUCTOSE C DFRU IMINODIACETIC ACID C IMN ALPHA KETOGLUTARIC C KGA Utilization of a carbon source results in a ACID reduction of the resazurin based indicator D MANNITOL C_DMNT 3 METHYLADIPIC ACID C_MAA THYMIDINE C_THY FLOURESCENT POSTIVE FLR_CTL CONTROL i i Control to standardize fluorescent substrate FLUORESCENT POSITIVE FLR CTL results CONTROL ALANINE ALANINE PNA N ALALH L PROLINE PNA N LPROT Enzymatic hydrolysis of the colorless amide substrate releases yellow p nitroaniline VALINE ALANINE PN
9. LABORATORY PROCEDURE BD Phoenix PMIC ID Panels BD Phoenix PMIC Panels BD Phoenix PID Panels INTENDED USE The BD Phoenix Automated Microbiology System is intended for the in vitro rapid identification ID of Gram Positive bacteria from pure culture belonging to the genera Staphylococcus Enterococcus and other Gram Positive cocci and Gram Positive bacilli The BD Phoenix Automated Microbiology System is also intended for the quantitative determination of antimicrobial susceptibility by minimal inhibitory concentration MIC of most Gram Positive bacteria from pure culture belonging to the genera Staphylococcus and Enterococcus SUMMARY AND EXPLANATION OF THE TEST Micromethods for the biochemical identification of microorganisms were reported as early as 1918 Several publications reported on the use of the reagent impregnated paper discs and micro tube methods for differentiating enteric bacteria 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 Many of the tests used in the Phoenix ID panels are modifications of the classical methods These include tests for fermentation oxidation degradation and hydrolysis of various substrates In addition to these the Phoenix system utilizes chromogenic and fluorogenic substrates as
10. McFarland must be met Only the BD PhoenixSpec Nephelometer can be used to measure inoculum density for this range Phoenix panels can be read only by the Phoenix instrument Visual interpretation of the Phoenix panels is not possible Any attempt to manually interpret results from the panel may lead to misidentification and or inaccurate AST interpretations Identification The unique panel environment combined with the shortened incubation time may result in Phoenix panel reactions varying from those obtained using conventional biochemical media Page 9 of 23 Antimicrobial Susceptibility Testing After the addition of the Phoenix AST Indicator Solution to the AST broth tubes mix by inversion DO NOT VORTEX Vortexing may cause air bubbles to form in the AST broth which can result in inappropriate filling of the Phoenix panel during inoculation Because of the low probability of occurrence or special growth requirements some organisms included in the ID taxa are not included in the AST database These organisms will display the message Organism not included in the AST database Perform alternate method For some organism antimicrobial combinations the absence of resistant strains precludes defining any result categories other than susceptible For strains yielding results suggestive of a nonsusceptible category organism identification and antimicrobial susceptibility test results should be confirmed Subsequently the isolates s
11. Penicillin Proc Soc Biol and Med 51 386 389 13 Marymont J H and Wentz R M 1966 Serial Dilution Antibiotic Sensitivity Testing with the Microtitrator System Am J Clin Pathol 45 548 551 14 Gavan T L and Town M A 1970 A Microdilution Method for Antibiotic Susceptibility Testing An Evaluation Am J Clin Pathol 53 880 885 15 Lancaster M V and Fields R D 1996 Antibiotic and Cytotoxic Drug Susceptibility Assays Using Resazurin and Poising Agents U S Patent 5 501 959 16 CLSI M100 S15 Performance Standards for Antimicrobial Susceptibility Testing Fifteenth Informational Supplement January 2005 17 Murray Patrick R et al ed Manual of Clinical Microbiology 8 Edition ASM Press Washington D C 2003 18 CLSI M7 A6 Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically Approved Standard Sixth Edition January 2003 Manufactured by Becton Dickinson and Company 7 Loveton Circle Sparks MD 21152 USA 800 638 8663 Made in USA Page 12 of 23 TECHNICAL INFORMATION Approved by Date Effective Supervisor Date Director Date Reviewed 9 2006 Phoenix BDXpert BBL CrystalSpec PhoenixSpec Trypticase and BD are trademarks of Becton Dickinson and Company ATCC is a trademark of American Type Culture Collection Sharpie is a trademark of Sanford CHROMagar is a trademark of Dr A Rambach 2005 BD Table A Taxa for ID AST Deter
12. amycin Daptomycin Nitrofurantoin Linezolid Chloramphenicol Quinupristin Dalfopristin Tetracycline time of comparative clinical testing MXF NOR OFX GM GMS STS NN RA AMC AM SAM OX MEM CZ FOX CF SXT VA CC DAP FM LZD C SYN TE 0 125 8 0 25 16 0 25 8 0 25 16 500 1000 0 5 16 0 25 32 0 25 0 12 32 16 0 06 32 2 1 32 16 0 06 4 0 06 32 0 5 16 2 32 1 32 0 5 64 0 5 9 5 16 304 0 5 32 0 06 8 0 12 8 0 125 32 16 512 0 25 32 1 32 0 25 4 0 5 16 1777 1252 1184 1223 953 1261 871 475 1240 1231 1256 620 597 1164 904 634 1981 1395 1242 1568 1757 1454 1447 2019 2040 96 0 96 9 98 7 91 9 93 5 98 3 94 1 93 3 97 2 95 4 93 6 98 4 99 5 96 3 96 2 96 4 97 7 95 0 98 2 97 4 99 8 91 1 93 4 94 5 96 9 Page 11 of 23 1777 1252 1184 1223 763 756 797 1261 871 475 1240 1231 1256 1198 597 1164 904 634 1981 1395 1242 1568 1757 1454 1447 1500 2040 90 1 97 4 98 2 95 2 98 6 97 8 98 5 98 2 96 7 98 5 97 3 96 6 97 5 96 6 99 7 90 1 98 0 97 9 99 4 94 6 98 7 98 8 99 8 95 3 93 4 95 5 96 5 REFERENCES 1 Bronfenbrenner J and Schlesigner M J 1918 A Rapid Method for the Identification of Bacteria Fermenting Carbohydrates Am J Public Health 8 922 923 2 Arnold W M Jr and Weaver R H 1948 Quick Microtechniques for Identification of Cultu
13. at the AST inoculum is prepared Cap the AST tube and invert several times to mix Do not vortex Wait a few seconds for air bubbles to surface Tap the tube gently to aid in eliminating bubbles Pour the ID tube inoculum into the fill port on the ID side of the panel 51 well side Allow the fluid to traverse down the tracks before moving the panel If using an AST only panel DO NOT inoculate the ID side of the panel Retain the ID tube for a purity check Page 6 of 23 16 Pour the AST tube inoculum into the fill port on the AST side of the panel 85 well side Allow the fluid to traverse down the tracks before moving the panel 17 Before placing panel closure check for residual droplets of inoculum on the edge of the fill ports If a droplet is present remove the droplet with absorbent material The used absorbent material must be discarded along with your biohazard waste 18 Snap on the panel closure Make sure that the closure is fully seated Visually inspect panels to be sure each of the wells is full Look at both sides of the panel Make certain that the wells are not overfilled If any of the wells are unfilled or overfilled inoculate a new panel Note Panels must be loaded into the instrument within 30 minutes of inoculation Panels must be kept in the inoculation station after inoculation until the excess fluid has been completely absorbed by the pad Panels should stay vertical in the transport caddy until loaded into
14. ated with universal precautions Please refer to CDC manual Bio safety in Microbiological and Biomedical Laboratories 4 Edition 1999 as well as other recommended literature Prior to discarding sterilize specimen containers and other contaminated materials by autoclaving Panels once inoculated should be handled carefully until placed in the instrument STORAGE AND HANDLING Phoenix Panels Panels are individually packaged and must be stored unopened at room temperature 15 25 C Do not refrigerate or freeze Visually inspect the package for holes or cracks in the foil package Do not use if the panel or packaging appears to be damaged If stored as recommended the panels will retain expected reactivity until the date of expiration Phoenix ID Broth Tubes are packaged as 100 tube packs Visually inspect the tubes for cracks leaks etc Do not use if there appears to be a leak tube or cap damage or visual evidence of contamination i e haziness turbidity Store Phoenix ID Broth tubes at 2 25 C Expiration dating is shown on the tube label Phoenix AST Broth Tubes are packaged as 100 tube packs Visually inspect the tubes for cracks leaks etc Do not use if there appears to be a leak tube or cap damage or visual evidence of contamination i e haziness turbidity Store Phoenix AST Broth tubes at 2 25 C Expiration dating is shown on the tube label Phoenix AST Indicator Solution The indicator solution is individually pou
15. categorical interpretative criteria susceptible intermediate resistant The table below summarizes the data from these studies Additionally testing performed at multiple clinical sites demonstrated at least 95 reproducibility or greater within 1 doubling dilution for all antimicrobial agents listed in the table below DRUG CLASS DRUG NAME DRUG DRUG EA EA CA CODE RANGE N N ug mL 5 Fluoroquinolone Gatifloxacin GAT 0 25 8 1180 98 6 1180 5 Fluoroquinolone Levofloxacin LVX 0 25 8 1878 96 8 1878 Page 10 of 23 CA 90 1 95 1 5 Fluoroquinolone 5 Fluoroquinolone 5 Fluoroquinolone Aminoglycoside Aminoglycoside Aminoglycoside Aminoglycoside Ansamycin B Lac B Lac Inh B Lactam Pen B Lactam Pen Inh B Lactam Pen B Lactam Pen Carbapenem Cephem Cephem Cephem Folate Antagonist Glycopeptide Macrolide Lincosamide Lipopeptide Nitrofurantoin Oxazolidinone Phenicol Streptogramin Tetracycline The ability of the system to detect resistance for this antimicrobic with Enterococcus species and Staphylococcus species is unknown because resistant organisms were not available at the Moxifloxacin Norfloxacin Ofloxacin Gentamicin Gentamicin Synergy Streptomycin Synergy Tobramycin Rifampin Ampicillin Clavulanate Ampicillin Ampicillin sulbactam Oxacillin Penicillin Meropenem Cefazolin Cefoxitin Cephalothin Trimethoprim Sulfamethoxazole Vancomycin Erythromycin Clind
16. ccus viridans Alloiococcus otitidis Dermacoccus nishinomiyaensis Enterococcus avium Enterococcus casseliflavus Enterococcus durans Enterococcus faecalis Enterococcus faecium Enterococcus gallinarum Enterococcus hirae Enterococcus raffinosus Gemella haemolysans ID AST ID AST ID AST ID AST IID AST ID AST ID AST ID AST ID ID AST ID AST Page 18 of 23 Gemella morbillorum Globicatella sanguinis Helcococcus kunzii Kocuria kristinae Kocuria rosea Kocuria varians Kytococcus sendentarius Lactococcus lactis ssp cremoris Lactococcus lactis ssp hordniae Lactococcus plantarum Leuconostoc citreum Leuconostoc lactis Leuconostoc mesenteroides ssp mesenteroides Listeria innocua Listeria monocytogenes Macrococcus caseolyticus Micrococcus luteus Micrococcus lylae Pediococcus acidilactici Pedicococcus damnosus Pediococcus dextrinicus Pediococcus parvulus Pediococcus pentosaceus Rothia mucilaginosa Staphylococcus aureus Staphylococcus auricularis Staphylococcus capitis Staphylococcus caprae Staphylococcus carnosus Staphylococcus chromogenes Staphylococcus cohnii ssp cohnii Staphylococcus cohnii ssp urealyticum Staphylococcus epidermidis Staphylococcus equorum Staphylococcus felis ID AST ID AST ID AST ID AST ID AST ID AST ID AST ID AST ID AST ID AST ID AST Page 19 of 23 Staphylococcus gallinarum Staphylococcus haemolyticus Staphylococcus hominis Staphylococcus hyicus Staphylococcus intermediu
17. ched and packaged as a package of 10 dropper bottles Visually inspect the bottle for cracks leaks etc Do not use if there appears to be a leak bottle or cap damage or any change from a dark blue color Store Phoenix AST Indicator Solution at 2 8 C Each bottle contains enough solution to test up to 100 panels Expiration dating is shown on the box pouch and bottle label and is for unopened bottles An opened bottle is stable for up to 14 days if stored at 2 8 C Be sure the bottle is held vertically when dispensing the AST Indicator Solution SPECIMEN COLLECTION AND PROCESSING The Phoenix system is not for use directly with clinical specimens Only pure culture isolates of Gram Positive organisms are acceptable for testing The test isolate must be a pure culture It is recommended that cultures be no more than 24 hours old unless additional incubation is required to achieve sufficient growth Page 3 of 23 Isolates must be tested with a Gram stain test to assure the appropriate selection of Phoenix panel type Once the Gram stain reaction is confirmed select the appropriate Phoenix panel for inoculation e g PMIC ID panel for use with Gram Positive organisms Selection of the incorrect panel type could lead to incorrect results For AST testing in the Phoenix system isolates recovered from non selective media are recommended It is recommended that media containing antibiotics not be used for organisms to be tested in the Phoenix syst
18. chleiferi Staphylococcus schleiferi ssp coagulans Staphylococcus schleiferi ssp schleiferi Staphylococcus sciuri Staphylococcus simulans Staphylococcus succinus ssp casei Staphylococcus succinus ssp succinus Staphylococcus vitulinus Staphylococcus warneri Staphylococcus xylosus Streptococcus acidominimus Streptococcus agalactiae Strep group B Streptococcus anginosus Streptococcus bovis Strep group D Streptococcus bovis II Strep group D Streptococcus canis Streptococcus constellatus Streptococcus cristatus Streptococcus dysgalactiae ssp dysgalactiae Streptococcus dysgalactiae ssp equisimilis Streptococcus equi Streptococcus equi ssp equi ID AST ID AST ID AST ID AST ID AST ID AST AST AST ID AST AST AST AST ID AST ID AST ID AST ID AST ID AST ID AST AST AST ID AST ID AST ID AST Page 17 of 23 Streptococcus equi ssp zooepidemicus Streptococcus equines Streptococcus gordonii Streptococcus intermedius Streptococcus mitis Streptococcus mitis pneumoniae Streptococcus mutans Streptococcus oralis Streptococcus parasanguinis Streptococcus pneumoniae Streptococcus porcinus Streptococcus pyogenes Strep group A Streptococcus salivarius Streptococcus sanguinis Streptococcus sobrinus Streptococcus uberis Streptococcus vestibularis 1 Not all species encountered during clinical performance evaluations Gram Positive 0 25 McFarland Gram Positive Taxa Aerococcus urinae Aeroco
19. e Phoenix panel for inoculation Selection of the incorrect panel type could lead to incorrect results 2 Examine the pouch and do not use the panel if the pouch is punctured or opened Remove the panel from the pouch Discard the desiccant Do not use the panel if there is no desiccant or if the desiccant pouch is torn Note Panels must be used within 2 hours of being removed from the pouch 3 Place the panel on the Inoculation Station with ports at the top and pad on the bottom 4 Label a Phoenix ID Broth tube with the patient s specimen number Using aseptic technique pick colonies of the same morphology with the tip of a sterile cotton swab do not use a polyester swab or a wooden applicator stick from one of the recommended media See Table C 5 Suspend the colonies in the Phoenix ID Broth 4 5 mL 6 Capthe tube and vortex for 5 seconds 7 Allow approximately ten seconds for air bubbles to surface Tap the tube gently to aid in eliminating bubbles 8 Confirm default settings for inoculum density before inoculating panels Insert the tube into the BBL CrystalSpec or BD Phoenix Spec Nephelometer Make sure the tube is inserted as far as it will go Note Only the BD PhoenixSpec Nephelometer can be used to make inoculum densities of 0 25 McFarland Refer to the BBL CrystalSpec Nephelometer or BD PhoenixSpec product insert for correct usage instructions and calibration verification 9 Ifthe inoculum density is set to 0 5 for
20. em Selective media may inhibit some strains of bacteria therefore caution must be used when selecting isolated colonies from these media For ID and AST testing refer to Table C for recommended media For ID only testing of Gram Positive organisms isolates from one of the following media may be used Trypticase Soy Agar without blood Columbia Colistin Nalidixic Acid CNA Agar with 5 sheep blood and Phenylethanol Agar PEA When swabs are used only cotton tipped applicators should be used to prepare the inoculum suspensions Some polyester swabs may cause problems with inoculation of the panels The usefulness of the Phoenix system or any other diagnostic procedure performed on clinical specimens is directly influenced by the quality of the specimens themselves It is strongly recommended that laboratories employ methods discussed in the Manual of Clinical Microbiology for specimen collection transport and placement on primary isolation media Inoculum for use on the Phoenix system is prepared by the CLSI recommended direct colony suspension method Due to variations in inoculum concentrations prepared with McFarland standards use of the BBL CrystalSpec or BD PhoenixSpec nephelometer is required for adjusting the test inoculum prior to use in the Phoenix system It is highly recommended that the purity of the inoculum be checked by preparing a purity plate See Purity Check below MATERIALS REQUIRED Materials Provided
21. escribed in Table B Antimicrobial Susceptibility Testing The Phoenix AST method is a broth based microdilution test The Phoenix system utilizes a redox indicator for the detection of organism growth in the presence of an antimicrobial agent Continuous measurements of changes to the indicator as well as bacterial turbidity are used in the determination of bacterial growth Each AST panel configuration contains several antimicrobial agents with a range of two fold doubling dilution concentrations Organism identification is used in the interpretation of the MIC values of each antimicrobial agent producing Susceptible Intermediate or Resistant SIR result classifications A complete list of taxa for which the Phoenix system can provide AST results is provided in Table A The list of antimicrobial agents and concentrations available for susceptibility testing in the Phoenix system is provided under Performance Characteristics There are antimicrobial agents for use with the Phoenix System that are not proven to be effective for treating infections for all organisms listed in the taxa For interpreting and reporting results of antimicrobial agents that have been shown to be active against organism groups both in vitro and in clinical infections refer to the individual pharmaceutical antimicrobial agent labeling Alternatively refer to the most recent CLSI M100 Performance Standard Table 1 Suggested Groupings of US FDA Approved Antimicrobial Agent
22. hould be saved and submitted to a reference laboratory that will confirm the result using the CLSI reference dilution method PERFORMANCE CHARACTERISTICS Gram Positive Identification In two internal studies the performance of the Phoenix Gram Positive identification was evaluated The 0 5 inoculum density configuration and the 0 25 inoculum density configuration were tested with 696 strains 0 5 and 755 strains 0 25 respectively Results were evaluated against commercial and non commercial methods The Phoenix Gram Positive identification performance is outlined below McFarland Agreement No Agreement No ID Species Level 0 5 95 496 3 996 0 796 0 25 98 0 1 6 0 4 An internal study was performed to simulate inter site reproducibility The identification results obtained using the Phoenix system were compared with expected results This performance testing demonstrated intra site and inter site reproducibility of at least 95 or greater Gram Positive Susceptibility Clinical stock and challenge isolates were tested across multiple clinical sites to determine Essential Agreement EA and Category Agreement CA of the Phoenix system to the CLSI broth microdilution reference method Essential Agreement occurs when the MIC of the Phoenix system and the reference method agree exactly or is within 1 dilution of each other Category Agreement occurs when the Phoenix system results agree with the reference method with respect to the CLSI
23. m positive organisms Antimicrobial susceptibility test results should be confirmed using Trypticase Soy Agar with 5 Sheep Blood The use of Bromthymol Blue lactose Agar for Gram Positive organism identification should be restricted to Staphylococci for both the 0 5 and 0 25 GP systems The use of Cystine Lactose Electrolyte Deficient Agar for Gram positive organism identification should be restricted to Staphylococci for the 0 25 GP system Page 23 of 23
24. mination There are antimicrobial agents for use with the Phoenix system that are not proven to be effective for treating infections for all organisms listed in this section For interpreting and reporting results of antimicrobial agents that have shown to be active against organism groups both in vitro and in clinical infections refer to the individual pharmaceutical antimicrobial agent labeling Alternatively refer to the most recent CLSI M100 Performance Standard Table 1 Suggested Groupings of US FDA Approved Antimicrobial Agents That Should be Considered for Routine Testing and Reporting on Organisms by Clinical Microbiological Laboratories Gram Positive 0 5 McFarland Gram Positive Taxa ID AST ID AST Aerococcus urinae ID Aerococcus viridans ID Alloiococcus otitidis ID Arcanobacterium haemolyticum ID Arcanobacterium pyogenes ID Bacillus cereus ID Bacillus circulans ID Bacillus coagulans ID Bacillus licheniformis ID Bacillus megaterium ID Bacillus pumilus ID Bacillus sphaericus ID Bacillus subtilis ID Bacillus thuringiensis ID Page 13 of 23 Brevibacillus brevis Brevibacterium species Cellulomonas turbata Corynebacterium amycolatum Corynebacterium bovis Corynebacterium diphtheriae Corynebacterium jeikeium Corynebacterium kutscheri Corynebacterium matruchotii Corynebacterium minutissimum Corynebacterium propinquum Corynebacterium pseudodiphtheriticum Corynebacterium pseudotuberculosis Corynebacterium renale Corynebac
25. res I Indole production J Lab Clin Med 33 1334 1337 3 Bachmann B and Weaver R H 1951 Rapid Microtechnics for Identification of Cultures V Reduction of Nitrates to Nitrites Am J Clin Pathol 21 195 196 4 Hannan J and Weaver R H 1948 Quick Microtechniques for the Identification of Cultures Il Fermentations J Lab Clin Med 33 1338 1341 5 Hartman P A 1968 Paper strip and disc methods p 123 132 Miniaturized microbiological methods Academic Press New York 6 Sanders A C Faber J E and Cook T M 1957 A Rapid Method for the Characterization of Enteric Pathogen Using Paper Discs Appl Microbiol 5 36 40 7 Synder M L 1954 Paper Discs Containing Entire Culture Medium for the Differentiation of Bacteria Pathol Bacteriol 67 217 226 8 Soto O B 1949 Fermentation Reactions with Dried Paper Discs Containing Carbohydrate and Indicator Puerto Rican J Publ Hlth Trop Med 25 96 100 9 Weaver R H 1954 Quicker Bacteriological Results Am J Med Technol 20 14 26 10 Kampfer P Rauhoff O and Dott W 1991 Glycosidase Profiles of Members of the Family Enterobacteriaceae J Clin Microbiol 29 2877 2879 11 Manafi M Kneifel W and Bascomb S 1991 Fluorogenic and Chromogenic Substrates Used in Bacterial Diagnostics Microbiol Rev 55 335 348 12 Rammelkamp C H and Maxon T 1942 Resistance of Staphylococcus aureus to the Action of
26. s Staphylococcus kloosii Staphylococcus lentus Staphylococcus lugdunensis Staphylococcus pasteuri Staphylococcus saprophyticus Staphylococcus schleiferi ssp coagulans Staphylococcus schleiferi ssp schleiferi Staphylococcus sciuri Staphylococcus simulans Staphylococcus vitulinus Staphylococcus warneri Staphylococcus xylosus Streptococcus agalactiae Strep group B Streptococcus anginosus Streptococcus bovis Strep group D Streptococcus bovis Strep group D Streptococcus bovis II Strep group D Streptococcus constellatus Streptococcus cristatus Streptococcus equi Streptococcus gordonii Streptococcus group C G large colony Streptococcus intermedius Streptococcus mitis Streptococcus mutans Streptococcus oralis Streptococcus parasanguinis Streptococcus pneumoniae Streptococcus porcinus Streptococcus pyogenes Strep group A ID AST ID AST ID AST ID AST ID AST ID AST ID AST ID AST ID AST ID AST ID AST ID AST ID AST ID AST ID AST ID AST ID AST Page 20 of 23 Streptococcus salivarius ID Streptococcus sanguinis ID Streptococcus sobrinus ID Streptococcus uberis ID Streptococcus vestibularis ID 1 Not all species encountered during clinical performance evaluations Table B List of Reagents and Principles Employed in the Phoenix System Substrate Name Code Principle 4MU BD CELLOBIOSIDE M BDCEL L ALANINE AMC A LALT 4MU BD GLUCOS
27. s That Should Be Considered for Routine Testing and Reporting on Organisms by Clinical Microbiological Laboratories The components required for testing using the Phoenix system include 1 Phoenix panels with panel closures 2 Phoenix ID Broth 3 Phoenix AST Broth 4 Phoenix AST Indicator solution 5 Phoenix Inoculation Station 6 Phoenix Transport Caddy 7 BBL CrystalSpec or BD PhoenixSpec Nephelometer 8 25 uL pipettor and sterile tips and 9 Miscellaneous lab supplies listed under Materials Required But Not Provided Page 2 of 23 Prior to inoculation the Phoenix panel is placed on the Inoculation Station with the inoculation ports at the top for filling Separate inocula are added manually to the ID and AST ports The inocula flow down the panel in serpentine fashion filling the panel wells as the liquid front progresses toward the pad The pad absorbs excess inoculum Closures are manually inserted in the fill ports An air admittance port is located in the divider area of the panel lid to ensure adequate oxygen tension in the panel for the duration of the test INGREDIENTS For a listing of biochemical substrates used in the Phoenix panel refer to Table B The package insert enclosed in the panel box provides a listing of the specific antimicrobial agents and concentrations found in the panel PRECAUTIONS For in vitro Diagnostic Use All patient specimens and microbial cultures are potentially infectious and should be tre
28. terium striatum Corynebacterium ulcerans Corynebacterium urealyticum Corynebacterium xerosis Dermabacter hominis Dermacoccus nishinomiyaensis Enterococcus asini Enterococcus avium Enterococcus casseliflavus Enterocooccus cecorum Enterococcus columbae Enterococcus dispar Enterococcus durans Enterococcus faecalis Enterococcus faecium Enterococcus flavescens Enterococcus gallinarum Enterococcus gilvus Enterococcus haemoperoxidus Enterococcus hirae Enterococcus malodoratus AST ID AST ID AST AST AST AST ID AST ID AST ID AST AST ID AST AST AST ID AST AST Page 14 of 23 Enterococcus moraviensis Enterococcus mundtii Enterococcus pallens Enterococcus pseudoavium Enterococcus raffinosus Enterococcus ratti Enterococcus saccharolyticus Enterococcus solitarius Enterococcus sulfureus Erysipelothrix rhusiopathiae Gardnerella vaginalis Gemella haemolysans Gemella morbillorum Globicatella sanguinis Helcococcus kunzii Kocuria kristinae Kocuria rosea Kocuria varians Kytococcus sendentarius Lactococcus garvieae Lactococcus lactis ssp cremoris Lactococcus lactis ssp hordniae Lactococcus lactis ssp lactis Lactococcus plantarum Lactococcus raffinolactis Leifsonia aquatica Leuconostoc citreum Leuconostoc lactis Leuconostoc mesenteroides ssp cremoris Leuconostoc mesenteroides ssp mesenteroides Leuconostoc pseudomesenteroides Listeria grayi Listeria innocua Listeria ivanovii Listeria monocytogenes AST AST
29. the instrument Inoculated panels should be handled with care Avoid knocking or jarring the panel Purity Check 1 Using a sterile loop recover a small drop from the inoculum fluid either before or after inoculation of the panel 2 Inoculate an agar plate any appropriate medium for a purity check 3 Discard inoculum fluid tube and cap in a biohazard disposal container 4 Incubate the plate for 24 48 hours at 35 C under appropriate conditions ID Inoculum Density Flexibility You may run the ID portion of a panel in the opposite mode from what is configured by darkening well A17 on the back of a panel before placing the panel in the instrument This allows you to run a panel at an inoculum density of 0 20 0 30 even if you are configured for a density of 0 5 for that particular panel type Likewise you can run the panel at an inoculum density of 0 50 0 60 if you are configured for a density of 0 25 There is no way to alter the density setting during Panel Login To use a panel in the opposite density mode using a black Sharpie permanent marker blacken the A17 well entirely See the BD Phoenix System User s Manual Operation ID Inoculum Density Flexibility for position of well A17 For instructions for panel login and loading refer to the BD Phoenix System User s Manual Panel Login and Inserting Panels in the Instrument eaan Ines npe cee Amount of ID Inoculum to EET UDINE Desired for Test Add to AST
30. the panel type being run then a range of 0 50 0 60 is acceptable If the inoculum density is set to 0 25 for the panel type being run then a range Page 5 of 23 10 11 12 13 14 15 of 0 20 0 30 is acceptable If the density of organisms is low you can add colonies from the isolate Re vortex the sample and reread to confirm that the correct density has been achieved If the density of organisms exceeds 0 6 McFarland follow the steps below to dilute the broth It is very important to accurately indicate the level of the liquid in the tube since this volume is needed to adequately fill the wells in the panel Note The standardized bacterial suspension in ID broth must be used within 60 minutes of preparation a Using a marker mark the broth level in the over inoculated Phoenix ID Broth tube b Using a sterile pipette aseptically add fresh Phoenix ID Broth to the inoculum Only Phoenix ID Broth may be used to dilute the inoculum c Vortex the tube and allow to sit for 10 seconds d Place the tube in the nephelometer and remeasure the turbidity of the suspension e Ifthe reading is greater than 0 6 repeat Steps b d e Ifthe reading is 0 5 0 6 go to Step e e Using a sterile pipette aseptically remove excess broth to the original level indicated by the mark on the tube created in Step a Remove excess broth to avoid overfilling the panel Also do not remove too much broth as there may be insufficient broth to
31. well as single carbon source substrates in the identification of organisms The modern broth microdilution test used today has origins in the tube dilution test used in 1942 by Rammelkamp and Maxon to determine in vitro antimicrobial susceptibility testing of bacterial isolates from clinical specimens The broth dilution technique involves exposing bacteria to decreasing concentrations of antimicrobial agents in liquid media by serial two fold dilutions The lowest concentration of an antimicrobial agent in which no visible growth occurs is defined as the minimal inhibitory concentration MIC The introduction in 1956 of a microtitrator system using calibrated precision spiral wire loops and droppers for making accurate dilutions rapidly allowed Marymont and Wentz to develop a serial dilution antimicrobial susceptibility test AST The microtitrator system was accurate and allowed the reduction in volumes of antimicrobial agents The term microdilution appeared in 1970 to describe the MIC tests performed in volumes of 0 1 mL or less of antimicrobial solution The Phoenix AST test is a modified miniaturized version of the micro broth doubling dilution technique Susceptibility testing in the Phoenix system is performed through determination of bacterial growth in the presence of various concentrations of the antimicrobial agent tested PRINCIPLES OF THE PROCEDURE A maximum of 100 identification and antimicrobial susceptibility tests can be
32. y incubated at 35 C The instrument tests panels every 20 minutes on the hour at 20 minutes past the hour and again at 40 minutes past the hour up to 16 hours if necessary Phoenix panels are read only by the instrument Phoenix panels cannot be read manually Bacterial Identification The ID portion of the Phoenix panel utilizes a series of conventional chromogenic and fluorogenic biochemical tests to determine the identification of the organism Both growth based and enzymatic substrates are employed to cover the different types of reactivity in the range of taxa The tests are based on microbial utilization and degradation of specific substrates detected by various indicator systems Acid production is indicated by a change in the phenol red indicator when an isolate is able to utilize a carbohydrate substrate Chromogenic substrates produce a yellow color upon enzymatic hydrolysis of either p nitrophenyl or p nitroanilide compounds Enzymatic hydrolysis of fluorogenic substrates results in the release of a fluorescent coumarin derivative Organisms that utilize a specific carbon source reduce the resazurin based indicator In addition there are other tests that detect the ability of an organism to hydrolyze degrade reduce or otherwise utilize a substrate A complete list of taxa that comprises the Phoenix ID Database is provided in Table A Reactions employed by various substrates and the principles employed in the Phoenix ID reactions are d
33. y the software and are reported out as less than or equal to lt for the minimum MIC or greater than gt for the maximum MIC The table below provides the range for these special antimicrobic organism combinations Antimicrobial Agent Organism s Applied Range pug mL Oxacillin Coagulase negative 0 0625 1 0 staphylococci Penicillin Staphylococcus spp 0 0625 1 0 Enterococcus spp 1 0 32 Gentamicin Staphylococcus epidermidis lt 4 and gt 16 Moxifloxacin Enterococcus spp other 0 25 8 than E faecium MICs of 4 8 16 not reported LIMITATIONS OF THE PROCEDURE See the package insert shipped with the panel for specific organism antimicrobial limitations General A Gram stain test is required for the selection of the appropriate Phoenix panel types Accurate identification and or AST results may not be made without this test Use only well isolated bacterial colonies from one of the recommended primary isolation media See Table C Media containing esculin should not be used Use of mixed colonies could result in inaccurate identification and or AST interpretations If the instrument inoculum density is configured to 0 5 for the panel type being used an inoculum density of 0 50 0 60 must be met Only the BBL CrystalSpec or BD Phoenix Spec Nephelometer can be used to measure the inoculum density If the instrument inoculum density is configured to 0 25 for the panel type being used an inoculum density of 0 20 0 30
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