AN MicroPlate TM Instructions for Use

Transcription

1 AN MicroPlate TM Instructions for Use (For In Vitro Diagnostic Use Only) Cabot Blvd. Hayward, CA FAX ORDERS Copyright, Biolog, Inc. Part# 00P 003, Rev B, SEP 2004

2 AN MicroPlate A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 Water N-Acetyl-D- N-Acetyl-D- N-Acetyl-β-D- Adonitol Amygdalin D-Arabitol Arbutin D-Cellobiose α-cyclodextrin β-cyclodextrin Dextrin Galactosamine Glucosamine Mannosamine B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 Dulcitol i-erythritol D-Fructose L-Fucose D-Galactose D-Galacturonic Gentibiose D-Gluconic D-Glucosaminic α-d-glucose Glucose- Glucose- Acid Acid Acid 1-Phosphate 6-Phosphate C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 Glycerol D,L-α-Glycerol m-inositol α-d-lactose Lactulose Maltose Maltotriose D-Mannitol D-Mannose D-Melezitose D-Melibiose 3-Methyl-D- Phosphate Glucose D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12 α-methyl-d- β-methyl-d- α-methyl-d- β-methyl-d- Palatinose D-Raffinose L-Rhamnose Salicin D-Sorbitol Stachyose Sucrose D-Trehalose Galactoside Galactoside Glucoside Glucoside E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 E11 E12 α- β- Turanose Acetic Formic Fumaric Glyoxylic Hydroxybutyric Hydroxybutyric Itaconic α-ketobutyric α-ketovaleric D,L-Lactic L-Lactic Acid Acid Acid Acid Acid Acid Acid Acid Acid Acid Acid F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 Succinic Acid D-Lactic Acid D-Malic L-Malic Propionic Pyruvic Pyruvic Acid D-Saccharic Succinamic Succinic Mono-Methyl m-tartaric Urocanic Methyl Ester Acid Acid Acid Acid Methyl Ester Acid Acid Acid Ester Acid Acid G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12 L-Alaninamide L-Alanine L-Alanyl-L- L-Alanyl-L L-Alanyl-L- L-Asparagine L-Glutamic L-Glutamine Glycyl-L- Glycyl-L- Glycyl-L Glycyl-L- Glutamine Histidine Threonine Acid Aspartic Glutamine Methionine Proline Acid H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 L-Methionine L-Phenylalanine L-Serine L-Threonine L-Valine L-Valine plus 2 -Deoxy Inosine Thymidine Uridine Thymidine-5 - Uridine-5 - L-Aspartic Adenosine Monophosphate Monophosphate Acid

3 If the identification does not match, review the test procedures and check the purity of your culture. Repeat the test. Call Biolog Technical Service if you have further problems or questions. Technical Assistance For help or to report problems with this product contact Biolog Technical Service either by phone ( ) by fax ( ) or by during business hours (7:30 A.M. to 5 P.M. Pacific Standard Time), or contact your local Biolog Distribution Partner. General information or MSDS sheets may be found at For Certificates of Analysis contact csorders@biolog.com or contact your local distribution partner. References 1 Bochner, BR Sleuthing out Bacterial Identities. Nature 339: Bochner, BR Breathprints at the Microbial Level. ASM News 55: Biolog, Inc., US Patent # 5,627,045. INSTRUCTIONS FOR USE OF THE BIOLOG AN MICROPLATE Intended Use The AN MicroPlate test panel provides a standardized micromethod using 95 biochemical tests to identify and/or characterize a broad range of anaerobic and microaerophilic bacteria. Biolog s MicroLog 1, MicroLog 2, or MicroLog 3 software is used to identify the bacterium from its metabolic pattern in the AN MicroPlate. Description Biolog MicroPlates test the ability of a microorganism to utilize or oxidize compounds from a preselected panel of different carbon sources. The test yields a characteristic pattern of purple wells, which constitutes a Metabolic Fingerprint. 1,2 All necessary nutrients and biochemicals are prefilled and dried into the 96 wells of the plate. Tetrazolium violet is used as a redox dye to colorimetrically indicate the utilization of the carbon sources. Testing is performed very simply. The isolate to be identified is grown on agar medium and then suspended in a special gelling inoculating fluid 3 (AN-IF) at the recommended cell density. Then the cell suspension is inoculated into the AN MicroPlate, 100µl per well. All of the wells start out colorless when inoculated. During incubation there is a burst of respiration in the wells that contain chemicals that can be oxidized and the cells reduce the tetrazolium dye forming a purple color. Negative wells remain colorless, as does the reference well (A-1) with no carbon source. The MicroPlates are incubated for hours to allow the pattern to form. The pattern of purple wells is then keyed into Biolog s MicroLog computer software, which automatically cross-references the pattern to an extensive library of species. If an adequate match is found, an identification of the isolate is made. Precautions To obtain accurate and reproducible results, the recommendations below must be followed. Pure cultures must be used to obtain identifications. The system is not designed to identify individual bacterial strains from within mixed cultures. Culture media and repeated subculturing prior to testing is very important. Many strains will produce different metabolic patterns depending upon how they are cultured prior to inoculation. Refer to the sections titled Specimen Preparation and Limitations for details. Sterile components and asceptic techniques must be used in set-up procedures. Contamination will affect results. Disposable glassware should be used to handle all cell suspensions and solutions. Glassware that has been washed may contain trace amounts of soap or detergent that will affect results. Prewarm the AN-IF and the MicroPlates to room temperature before use. Some species may be sensitive to cold shocks. Calibrate your turbidimeter carefully and always prepare your inoculum within the specified density range. Refer to the section titled Inoculum Preparation. Biolog s chemistry contains components that are sensitive to temperature and light. Dark brown wells in the MicroPlate indicate deterioration of the carbon source. Some wells may have an inherent yellow or pink hue, which is normal. 8 Biolog AN MicroPlate 1

4 Always keep in mind that you are testing the metabolic properties of live cells. Some species can lose their metabolic vigor when subjected to stresses (e.g. temperature, ph, and osmolarity) for even a few seconds. To get the best performance possible from these MicroPlates, be aware that the cells are alive and careful with how you handle them. Read the entire Instructions for Use prior to using the MicroPlate. On Receipt Inspect each foil pouch and MicroPlate for damage in shipping. AN MicroPlates are packaged with a special packet that absorbs the oxygen inside the foil pouch giving the appearance of a package that is vacuum packed. If the foil pouch loses its seal, it will take in air and give a pillowed appearance. Do not use MicroPlates if the packaging indicates that the foil seal has been broken. To preserve the full shelf life, the MicroPlates must be stored at 2-8 C inside their foil pouch. The expiration date is printed on each pouch. Do not use MicroPlates after the expiration date. MATERIALS Materials Provided 10 Biolog AN MicroPlates (Biolog Catalog #1007). Materials Not Provided BUA Agar: BUA (Biolog Universal Anaerobe) Agar dehydrated medium (Biolog Catalog #70007), or plated BUA Agar with 5% sheep blood (Biolog Catalog #71007). Be sure to use sheep blood with a hematocrit of at least 40%. Kanamycin disks: 1 mg. AN-IF: Screw capped test tubes (20 x 113 mm) containing ml of anaerobically prepared, sterile gelling inoculating fluid (0.40% NaCl, 0.03% Pluronic F-68, 0.02% Gellan Gum, 0.084% NaHCO3, % methylene green, % sodium thioglycolate, final ph of AN-IF should be , Biolog Catalog #72007). LongSwabs : Sterile 7-inch disposable cotton-tipped swabs (Biolog Catalog # ). Streakerz : Sterile 6-inch taperd wooden streaking sticks (Biolog Catalog # ). Transfer Pipets: Sterile disposable 9 inch transfer pipets (Biolog Catalog #3019). Reservoirs: Sterile disposable reservoirs for multichannel pipetters (Biolog Catalog #3102). Pipettor: 8-Channel Repeating Pipettor (Biolog Catalog #3501 or 3505). Pipettor Tips: Sterile disposable pipettor tips (Biolog Catalog #3001electronic, Catalog # 3101 manual pipettor). Turbidimeter: (Biolog Catalog #3531 or 3532). Turbidity Standards: AN (Biolog Catalog #3427). Anaerobic Chamber/Incubator: C. Jar/Box/Bag with Anaerobic Sachet: Oxygen impermeable container that can be made anaerobic. Use only new style sachets that create an anaerobic atmosphere without hydrogen. Material Preparation Prewarm MicroPlates and tubes of AN-IF to at least 25 C before use. If all wells are negative, make sure that: The MicroPlate packaging maintained anaerobic conditions. If it did not, then oxygen will be absorbed into the plastic and will be toxic to the anaerobes. Your cells are freshly grown and you have used the recommended BUA+B agar medium. The inoculating fluid was anaerobic (i.e. the methylene green indicator was colorless) before the tube was opened. The inoculating fluid was prewarmed, prepared correctly, has the correct ph and salinity, and does not contain preservatives. You are handling the cells with all disposable hardware (soap residues are toxic). Your inoculum density is sufficient check the calibration of your turbidimeter. You did not leave the cells exposed to aerobic conditions for more than 20 minutes during the inoculation process. You incubated the MicroPlates under anaerobic conditions. Your jar /box /bag produced and maintained an anaerobic atmosphere. Your incubation temperature is correct for the organism that is being tested. The A-1 well is not over-filled. It is used as a reference well by the MicroStation. See the MicroLog User Guide for further assistance in interpreting identification results. Performance Characteristics The AN MicroPlate performance characteristics have been determined by establishing a database from a large collection of well characterized organisms including species type strains. The database is designed to give identifications of all species in the database in accordance with current taxonomic nomenclature. To obtain accurate and reproducible results, all procedures and recommendations in these Instructions for Use must be followed precisely. Limitations The AN MicroPlate is designed to identify pure cultures of anaerobic and certain microaerophilic bacteria. The panel will only recognize members of the species in the current database. Other species will usually be reported out with the message No identification. Atypical strains may also yield a similarity index that is less than 0.5 at hours and therefore will be reported out as No identification. Quality Control Biolog MicroPlates are tested and meet internal quality control stardards before being released for sale. However, some laboratories may desire or may be required to perform independent validations on each manufacturing lot. To test the performance of the AN MicroPlate use the 4 strains specified below. These are available from Biolog as a set (Biolog Catalog #8047). 1. Bifidobacterium breve ATCC Clostridium sordellii ATCC Lactobacillus casei ATCC Micromonas micros ATCC Inoculate each bacterium following the TEST PROCEDURES as specified. When lyophilized or frozen cultures are used, they should be subcultured at least twice before being tested. Read the MicroPlates at hours of incubation. The resulting identification should correctly correspond to the identity of the quality control strain. 2 Instructions for Use Biolog AN MicroPlate 7

5 RESULTS Interpretation of Results Read MicroPlates using the MicroLog 1, MicroLog 2, or MicroLog 3 software. Refer to the User Guide for detailed instructions. False positive color is defined as purple color forming in the control well (A-1) and in other negative wells. This problem is caused by the bacterial suspension reducing the buffer and subsequently reducing the tetrazolium dye and is typically seen with strains of certain genera such as Actinomyces, Bacteroides, and Propionibacterium. In most cases the false positive color is faint, and it is not a problem so long as the true positive reactions are discernible. If the strain produces a high background color making it hard to determine which wells are positive, repeat the setup and inoculation of the MicroPlate after exposure of the MicroPlate to air for 20 minutes (refer to step 4 and table on page 5). Most species give dark, clearly discernible positive reactions. However, it is normal for the positive reactions of some strains of certain genera such as Clostridium, Eubacterium, Fusobacterium, Prevotella, and Porphyromonas to be light or faint purple. For MicroPlates read at hours of incubation, the similarity index must be at least 0.50 to be considered acceptable. Trouble Shooting If you experience a problem in using the AN MicroPlate, start by rereading these Instructions for Use and review whether you have deviated from the recommended procedures. Then refer to the list below. If all wells are positive, make sure that: You are incubating the bacteria in an anaerobic atmosphere that is hydrogen-free. Many anaerobes have a strong hydrogenase activity and will reduce the tetrazolium dye and form a purple color in all wells due to the presence of hydrogen gas. You have accurately prepared your AN-IF. Excessive levels of thioglycolate can cause artifactual positive reactions. You are not carrying over any nutrients from the agar growth medium into the inoculating fluid. Your inoculum density is not excessive check the calibration of your turbidimeter. You have allowed the MicroPlate to be exposed to aerobic conditions for minutes following inoculation. Your inoculum is free of all clumps. The A-1 well is not under-filled. It is used as a reference well by the MicroStation TM. You have checked for kanamycin resistance if your organism is a rapidly growing GNR. TEST PROCEDURES Step 1. Culture Isolation on Biolog Recommended Media Isolate a pure culture on agar media. Use Biolog recommended media (BUA Agar with 5% sheep blood) and incubate anaerobically at 26, 30, or C. All species that can be identified with the AN MicroPlate will grow under these conditions. For best results, always streak for isolation. Subculture anaerobic becteria twice on BUA, and prepare a suspension from the second plate only. Step 2. Specimen Preparation and Characterization Perform a Gram stain on your isolate and observe the cell morphology in the Gram stain: coccus or rod. This will allow you to categorize the bacterium into one of the following groups: gram-negative coccus (GNC), gram-negative rod (GNR), gram-positive coccus (GPC), or gram-positive rod (GPR). Categorization will aid the identification procedure by directing the MicroLog database search to the appropriate class. Grow the bacterium using the recommended conditions. The choice of the agar medium is very important since it must support growth and promote retention of full metabolic activity to accurately match the metabolic patterns in the AN database. All species that can be identified should be grown anerobically on BUA with 5% sheep blood (BUA+B) and in most cases should be incubated at C. Only a few species require 30 C (Lactobacillus malefermentans, Pectinatus cerevisiiphilus and frisingensis, Tetragenococcus halophilus, Zymomonas mobilis, Zymophilus raffinosivorans and paucivorans) or 26 C (Lactobacillus collinoides and hilgardii, Leuconostoc gelidum). For gram-negative rapidly-growing rods, a 1 mg kanamycin disk should also be included in the first quadrant. All species are cultured under strictly anaerobic conditions. The cells must be freshly grown since many strains lose viability and metabolic vigor in stationary phase. The recommended incubation period for most organisms is hours. If insufficient growth is obtained to inoculate the panel, continue to incubate the plate to obtain more growth, or restreak heavily (as a lawn) onto one or more agar plates. Incubate for hours. This should give sufficient growth to inoculate the panel. Step 3. Inoculum Preparation Examine your AN-IF to make certain that it is anaerobic. The inoculating fluid contains methylene green as an indicator. If it is anaerobic, it will be colorless. If oxidized, the indicator will turn a faint blue-green color and the AN-IF should not be used. Establish the acceptable turbidity range on your turbidimeter. First, set the transmittance adjustment to 100% transmittance using an uninoculated tube. Then, determine the desired turbidity with the AN Turbidity Standard described in the section titled Materials. Using the Biolog turbidimeter and 20 mm diameter tubes, this should give a transmittance level of about 65%. These readings may vary slightly on different Biolog turbidimeters. With other instruments or with other tubes, the transmittance readings may vary substantially. Blank the turbidimeter (transmittance = 100%) with a tube containing uninoculated AN-IF. Because the tubes used are not optically uniform, they should be blanked individually and not rotated in the light path of the turbidimeter. 6 Instructions for Use Biolog AN MicroPlate 3

6 Prepare a uniform suspension while maintaining anaerobic conditions. If several MicroPlates are to be inoculated, it is best to prepare the suspensions inside an anaerobic chamber. If only a few MicroPlates are to be inoculated, the suspensions can be prepared and inoculated quickly under aerobic conditions. Preparation of all suspensions should be completed within a 5 minute period. For this reason, best results will be obtained if the number of suspensions prepared at any one time is limited to six. To prepare the suspensions, remove cells from the anaerobic agar plate with a sterile cotton swab so as not to carry over any nutrients from the agar medium into the suspension. Twirl and press the swab against the inside surface of the tube on the dry glass above the fluid line to break up clumps and release cells. Move the swab up and down the wall of the tube until the organism mixes with the fluid and becomes a homogenous, clump-free suspension. Use the swab with a vertical stirring motion to generate a uniform suspension all the way to the bottom of the tube. A sterile transfer pipet may also be used to mix the suspension without creating an aerosol. If clumps are still present, let the tube stand for several minutes and allow them to settle to the bottom. Adjust the inoculum density. Watch as the meter needle goes toward the acceptable turbidity range. The acceptable range is defined by the turbidity standard plus or minus 2% transmittance. This must be done with precision since it establishes the oxygen concentration for the cells and for the redox chemistry. The density can be lowered by adding more inoculating fluid or raised by adding more cells. Inoculate the cell suspension into the MicroPlate promptly. Some strains lose metabolic activity if held too long (no more than 20 minutes) in inoculating fluid without nutrients. Step 4. Inoculation of the MicroPlate The following steps must be performed under aerobic conditions, but should be performed quickly. For Kanamycin resistant gran-negative rods that have grown well enough to prepare a suspension overnight, the pouches should be opened and the AN MicroPlates exposed to air 20 minutes before inoculation. For all other bacteria, open the pouch and remove the AN MicroPlate only after all suspensions have been prepared. Inoculate the AN MicroPlate promptly (within 5 minutes). Label the MicroPlate with the organism name/number. Pour the cell suspension into the multichannel pipet reservoir. Fasten 8 sterile tips securely onto the 8-Channel Repeating Pipettor. Refer to manufacturer s instructions. Fill the tips and check to see that all tips are filling equally. If not, refasten any loose tips. Prime the tips if you are using a manual pipetter by dispensing the first delivery back into the reservoir. The electronic pipettor performs priming automatically. Fill all wells with precisely 100 µl. Be careful not to carry over chemicals or splash from one well into another. The length of time from starting to inoculate until all MicroPlates are inoculated should be no more than 5 minutes. The inoculating fluid will form a soft gel shortly after inoculation. Cover the MicroPlate with its lid. Allow the MicroPlate to sit in aerobic conditions for minutes after inoculation, preceding incubation. Step 5. Incubation Incubate the MicroPlate under anaerobic conditions in an oxygen impermeable jar or box, using an appropriately sized anaerobic sachet that generates a hydrogen-free anaerobic environment. Use indicator strips that use resazurin (Oxoid ) rather than methylene blue. Do not incubate the MicroPlate in a standard anaerobic chamber. Many anaerobic bacteria have strong hydrogenase activity and will reduce the tetrazolium dye forming a purple color in all wells due to the presence of hydrogen gas in the atmosphere. Incubate MicroPlates for hours. The following table summarizes the testing procedures Exceptions Use 30 C or 26 C as listed in Step 2. Anaerobe BUA+B C Anaerobic (must use a hydrogen-free anaerobic atmosphere for incubating the MicroPlates) AN-IF 65% T Less than 5 minutes exposure to air before inoculation min. exposure to air after inoculation. Microbe type Culture medium Temperature Atmosphere 20 minutes exposure to air before inoculation for (a) Gram-negative rods that are kanamycin resistant and that produce enough growth to prepare a suspension after overnight incubation, (b) strains that produce a high false positive purple color in the MicroPlate. Inoculating fluid Inoculum turbidity Exposure of AN MicroPlate to air 100 µl hours Inoculum per well Incubation time 4 Instructions for Use Biolog AN MicroPlate 5