Customer Notification
Customer Notification PN 9101002 Rev C.1 KCJr software includes a Calibration Test Plate Program that may be used with all Bio-Tek Automated Readers. If you are using a Bio-Tek Universal Calibration Test Plate, Bio-Tek (PN 9000547), please read this information carefully. The Calibration Test Plate Program may incorrectly report a Pass for the Turnaround and Repeatability test results. Other test features which will alert the user to potential inconsistencies include the Optics Test, the Run Time Self Test, and the Calibration Test Plate Program. This error is limited to the following versions of KCJr: ♦ Version 1.12 (1994) and Version 1.14 (1995) This software error is being corrected. An updated version of KCJr will be supplied to the Users of Record for this program. To expedite this process, please record the information below and fax to: Bio-Tek Instruments: 802-655-7941 Attention : Laboratory Product Manager Thank you for your cooperation. If you have any questions or comments, please contact the Laboratory Product Manager at: Phone: Fax: E-mail: 800-451-5172 802-655-7941 [email protected] Model and Serial Number of Autoreader: Serial Number of KCJr Software (from KCJr Operator’s Manual): Universal Calibration Test Plate Serial Number: Shipping Address: Contact Person: ________________________________ Name:_________________________________ ________________________________ Phone:_________________________________ ________________________________ Fax/E-mail:______________________________ ________________________________ 6FDQQLQJ$XWR5HDGHU 0LFURSODWH:RUNVWDWLRQ 2SHUDWRU V0DQXDO )RULQYLWUR'LDJQRVWLF8VH 0$18$/ 3$57 0$18$/ 5(9,6,21 & ,167580(17 5(9,6,21 * .&-U 9(56,21 &23<5,*+7 %,27(. ,167580(176 ,1& ii Notices ______________________________________________________ Copyright © Copyright 1995, Bio-Tek Instruments, Incorporated. No part of this publication may be reproduced, transmitted, transcribed, stored in a retrieval system, or translated into any language without the written permission of Bio-Tek Instruments, Incorporated. Trademarks Bio-Tek is a registered trademark, and CERES 900C is a trademark of Bio-Tek Instruments, Inc. Restrictions and Liabilities Information in this document is subject to change, and does not represent a commitment by BIO-TEK INSTRUMENTS, Inc. Changes made to the information in this document will be incorporated in new editions of the publication. No responsibility is assumed by BIO-TEK for the use or reliability of software or equipment that is not supplied by BIO-TEK, or its affiliated dealers. Revision Appendix Revision Date A 06/94 B 12/94 Page First Issue iv xiv x 1-1 4-1 C-1 C Change 2/95 x 1-1 Printed in the United States of America Bio-Tek Printed Material Part No. 9101002 iii Warnings Changed T.O.C. Changed Intended Use Statement Introduction Added Warnings Appendix C Cover Sheet Changed Intended Use Statement Introduction Warnings and Precautions When operated in a safe environment, according to the instructions in this manual, there are no known hazards associated with the CERES 900C. However, the operator should be aware of certain situations which could result in serious injury. Hazard Warnings Warning! Power Rating. the CERES 900C must be connected to a power receptacle that provides voltage and current within the specified rating for the system. Use of an incompatible power receptacle may produce electrical shock and fire hazards. Warning! Electrical Grounding. Never use a two-prong plug adapter to connect primary power to the CERES 900C. Use of a two-prong adapter disconnects the utility ground, creating a severe shock hazard. Always connect the system power cord directly to a three-prong receptacle with a functional ground. Warning! Internal Voltage. Always turn off the power switch and unplug the power cord before cleaning the outer surfaces of the instrument. Warning! Liquids. Avoid spilling liquids on the instrument; fluid seepage into internal components creates a potential shock hazard. Wipe up all spills immediately. Do not operate the instrument if internal components have been exposed to fluid. Warning! Software. The user of the CERES 900C, utilizing the KCJr software, must follow the specific manufacturer’s assay package insert when modifying KCJr software parameters and establishing result calculation methods. Warning! Data Reduction Protocol. KCJr. is a basic data reduction software program that does not flag out of range results nor prevent these results from being sent to any connected information system. It is the user’s responsibility to verify that all quality control materials are in their expected ranges prior to utilizing the assay’s results. iv Precautions The following precautions are provided to help you avoid damaging the system: Caution: Service. The system should be serviced by Bio-Tek-authorized service personnel. Only qualified technical personnel should perform troubleshooting and service procedures on internal components. Caution: Replacement Fuses. Use replacement fuseswith the required current rating and specification. Improper fuses or short-circuiting the fuse holders may causefire or damage to the instrument. Caution: Environmental Conditions. Do not expose the system to temperature extremes. System performance may be adversely affected. Caution: Sodium Hypochlorite. Do not expose any part of the instrument to Sodium Hypochlorite solution (bleach) for more than 30 minutes. Prolonged contract may damage the instrument surfaces. Be certain to rinse and thoroughly wipe all surfaces. Caution: Power Supply. Only use the specified power supply to ensure proper operation of the unit. v Electromagnetic Interference and Susceptibility USA FCC CLASS A Warning: Changes or modifications to this unit not expressly approved by BioTek Instruments could void the user's authority to operate the equipment. This equipment has been tested and found to comply with the limits for a Class A digital device, pursuant to Part 15 of the FCC Rules. These limits are designed to provide reasonable protection against harmful interference when the equipment is operated in a commercial environment. Like all similar equipment, this equipment generates, uses, and can radiate radio frequency energy and, if not installed and used in accordance with the instruction manual, may cause harmful interference to radio communications. Operation of this equipment in a residential area is likely to cause harmful interference in which case the user will be required to correct the interference at his own expense. Canadian Department of Communications Class A This digital apparatus does not exceed Class A limits for radio emissions from digital apparatus set out in the Radio Interference Regulations of the Canadian Department of Communications. Le present appareil numerique n'met pas du bruits radioelectriques depassant les limites applicables aux appareils numerique de la Class A prescrites dans le Reglement sur le brouillage radioelectrique edicte par le ministere des Communications du Canada. vi EC EN 55011 GROUP 1, CLASS A - EC DIRECTIVE 89/336/EEC The system has been type tested by an independent testing laboratory and found to meet the requirements of EC Directive 89/336/EEC and also VDE 0871/CISPR 11:1990 for Radiated Emissions and Line Conducted Emissions. Verification was to the limits and methods of EN 55011 and VDE 0876/0877. The device is classified as EN 55011 Group 1, Class A. EC EN 50082-1 The system was also tested and found to meet requirements for Electrostatic Discharge Susceptibility, Radiated Susceptibility, and Conducted Susceptibility. Verification of compliance was conducted to the limits and methods of EN 50082-1:1992; IEC 801-2:1984; IEC-801-3:1984; and 801-4:1988. Safety This device has been type tested by an independent laboratory and found to meet the requirements of: ■ Underwriters Laboratories Standard (UL) UL 1262 Laboratory Equipment; ■ Canadian Standards Association C22.2 No. 151-M1986 Laboratory Equipment; and ■ IEC 1010-1 (1990) "Safety requirement for electrical equipment for measurement, control and laboratory use. Part 1 General requirements." ■ Japanese Industrial Standard JIS t 1001 “ General Requirements for Safety of Medical Electrical Equipment” vii Safety Symbols The following warning and informational symbols may be found in various locations on the CERES 900C. The warnings are given here in English, French, German, Spanish and Italian. Only qualified personnel who recognize shock hazards and are familiar with the safety precautions should use this instrument. read the manual carefully before operating this instrument. Alternating current Courant alternatif Dreiphasen-Wechselstrom Corriente Atterna Corrente alternata Earth ground terminal Borne de terre Erde (Bettriebserde) Borne de Tierra Terra (di funzionamento) Protective conductor terminal Borne de terre de protection Schutzleiteranscluss Borne de Tierra de Protección Terra di protezione On (Supply) Marche (alimentation) Ein (Verbindung mit dem Netz) Connectado Chiuso viii Off (Supply) Arrest (alimentation) Aus (Trennung vom Netz) Desconectado Aperto (sconnessione dalla rete di alimentazione) Caution (refer to accompanying documents) Attention (voir documents d'accompanement) Achtung siehe Begleitpapiere Atención (vease los documentos incluidos Attenzione, consultare la doc annessa Caution, risk of electric shock Attention, risque de choc electrique Gefährliche elektrische Spannung Atención, riesgo de sacudida eléctrica Alta tensione (in questo documento Alta tensione non significa “tensione pericolosa” come definito in IEC 417) ix Intended Use Statement The CERES 900C is an automated, benchtop, general purpose, Enzyme Immunoassay Analyzer which performs diagnostic analyses of a variety of samples. The Performance Characteristics of the data reduction software have not been established with any laboratory (in vitro)diagnostic assay. The user must evaluate this software in conjunction with the specific laboratory(in vitro) diagnostic assay. This re-evaluation must include the establishment of new performance characteristics for the specific assay. This system is designed for use with a variety of enzyme immunoassays. Assay protocol variations are addressed by the developer of the ELISA test kit, in accordance with the test kit's procedure. KCJr, Bio-Tek’s PC based curve fitting and statistical software program is included preloaded on every CERES 900C reader. An unlimited number of assay protocols and blanking patterns can be defined, stored in memory, and instantly accessed. Plate templates and formulas are automatically combined with the protocol assay setup. Data results may be displayed on the monitor, printed out, sent to a mainframe, or merged with other software packages such as Lotus 1-2-3 . Basic DOS utilities are included for convenience. KCJr provides: ■ An easy-to-use menu-driven interface; ■ Kinetic and endpoint curvilinear regressional and statistical calculations; ■ Curve Fitting, with 4-parameter, cubic, quadratic, and linear methods; ■ Formula calculations for more complex mathematical operations; ■ Limited calculations for positive or negative cutoffs. Specimen Preparation Samples should be obtained, treated and stored following instructions and recommendations contained in the package kit. x Table of Contents ___________________________________ Notices ............................................................................................. iii Warnings and Precautions ............................................................. iv Intended Use Statement .................................................................. x Chapter 1 Introduction..................................................................1-1 Introducing the CERES 900C .............................................................. 1-1 Hardware Features ............................................................................. 1-2 Software Features............................................................................... 1-2 Package Contents............................................................................... 1-3 About This Manual .......................................................................................... 1-4 Registration Card ................................................................................ 1-4 Help .................................................................................................................. 1-5 Technical Assistance BBS .................................................................. 1-5 Writing to Bio-Tek ............................................................................... 1-5 Phone Support .................................................................................... 1-5 Chapter 2: Installation The Operating Environment ........................................................................... 2-1 Unpacking the Instrument .............................................................................. 2-1 Installing the Filter Wheel............................................................................... 2-3 Replacing and Installing Filters ..................................................................... 2-4 Installing a Printer ........................................................................................... 2-6 Serial Connections ......................................................................................... 2-6 Electrical Connections.................................................................................... 2-8 Fuses................................................................................................................ 2-8 xi Chapter 3: Introducing KC Jr. Software An Overview of KC Jr...................................................................................... 3-1 Supported Pointing Devices........................................................................... 3-3 Supported Output Devices ............................................................................. 3-4 KC Jr.'s Main Menu.......................................................................................... 3-5 Menu Overview ................................................................................... 3-6 Using the Main menu .......................................................................... 3-7 KC Jr.'s Function Keys ........................................................................ 3-8 KC Jr.'s AutoRun Feature ................................................................... 3-9 Before you Begin: The CERES 900C Setup ................................................ 3-11 g System Utilities/Setup ........................................................................3-12 Filter Table ........................................................................................ 3-13 Standard Filter Configuration ............................................................ 3-13 Filter Configuration For CERES 900C UV......................................... 3-14 Serial Setup....................................................................................... 3-15 A Look at Optics................................................................................ 3-15 Report Device (Printer) ..................................................................... 3-16 d Protocol - Create/Edit .........................................................................3-17 The Protocol Definition Screen ......................................................... 3-17 Editing the Protocol Parameters ....................................................... 3-19 Saving/Aborting Changes ................................................................. 3-20 Protocol Name .................................................................................. 3-20 Protocol Description .......................................................................... 3-21 Reader Control File ........................................................................... 3-21 Assay Type ....................................................................................... 3-22 Number of Reads .............................................................................. 3-22 Reading Interval ................................................................................ 3-23 Prompt for Plate ID............................................................................ 3-23 xii Fitting method ................................................................................... 3-23 Data/Axis Type.................................................................................. 3-23 Average Sample Replicates.............................................................. 3-24 Units of Measure............................................................................... 3-24 Wells Slopes/R^2 .............................................................................. 3-25 Kinetic / Scanning Y scale ................................................................ 3-25 Standards Results............................................................................. 3-25 Sample Results................................................................................. 3-26 Protocol Definition............................................................................. 3-27 Template Description ........................................................................ 3-27 Absorbance Data .............................................................................. 3-28 Reports of Means.............................................................................. 3-28 Transform Raw Data By.................................................................... 3-28 Report Transformed Data ................................................................. 3-29 Apply Formula................................................................................... 3-29 Report of Limits................................................................................. 3-30 eTemplate (create/edit)..................................................................3-33 Creating a New Template ................................................................. 3-34 Selecting an Existing Template......................................................... 3-34 Modifying a Template........................................................................ 3-35 Entering Standard Concentrations.................................................... 3-36 Replicating Standards....................................................................... 3-38 Entering Sample Unknowns.............................................................. 3-38 Naming a Sample Unknown.............................................................. 3-40 Erasing a Sample Set or Standard Concentration ............................ 3-41 Replicating a Sample Set.................................................................. 3-41 Entering Dilutions.............................................................................. 3-43 Serial Dilutions .................................................................................. 3-44 xiii Manual Dilutions................................................................................ 3-46 Entering a Description....................................................................... 3-47 Saving the Template ......................................................................... 3-47 Defining Two Templates for Standards per Assays .......................... 3-47 h Data Defaults............................................................................. 3-49 D- Data Drive/Dir............................................................................... 3-50 L- Lotus Drive/Dir .............................................................................. 3-51 P- Default Protocol ............................................................................ 3-51 T- Default Template .......................................................................... 3-52 R- Report Device............................................................................... 3-52 C- Printer Control Code..................................................................... 3-53 f Formula (create/edit) .................................................................. 3-55 Selecting / Creating a Formula File................................................... 3-57 Entering a New Formula File............................................................. 3-58 Modify an Exisiting Formula .............................................................. 3-60 Using the Formula in Calculations .................................................... 3-61 Create/Modify a Data Transformation ............................................... 3-61 Using Transformations in Calculations.............................................. 3-64 Chapter 4: Running KCJr. ........................................................... 4-1 c Calculating Results ............................................................................... 4-1 Starting the Calculations Process ....................................................... 4-3 Reading a Plate and Saving Data ....................................................... 4-4 Retriving Data from a Disk File ........................................................... 4-7 The Kinetics Assay ....................................................................................... 4-11 The 96-Well Graphics Screen ........................................................... 4-11 Printing the Screen............................................................................ 4-12 Saving the screen to disk file ............................................................ 4-12 Adjusting y-scale axis ....................................................................... 4-13 xiv Selecting time interval....................................................................... 4-13 Mean slope and R2 value ................................................................. 4-14 Zoom-a-well ...................................................................................... 4-15 Edit Graph Screen ............................................................................ 4-16 Change the data/axis type or curve-fit method ................................. 4-17 Return to 96-well screen ................................................................... 4-17 Display standard results.................................................................... 4-17 Fitted Curve Screen .......................................................................... 4-19 Return to Edit Graph Screen............................................................. 4-20 Printing the fitted curve ..................................................................... 4-20 Save as disk file ................................................................................ 4-20 Sample Evaluation ............................................................................ 4-21 Endpoint Assay ............................................................................................. 4-24 Statistical Assay............................................................................................ 4-25 Scanning Assay............................................................................................. 4-26 i Incubate Plate............................................................................4-28 Chapter 5: KC Jr's Utilities ...........................................................5-1 g System Utilities/Setup ........................................................... 5-1 D- DOS File Operations ...................................................................... 5-2 C- Copy a File (or Plate) ..................................................................... 5-3 D- Directory of Files ............................................................................ 5-5 E- Erase a Data File (or Plate)............................................................ 5-7 N-reName a Data File ......................................................................... 5-9 P- Erase a Protocol........................................................................... 5-11 T- Erase a Template ......................................................................... 5-11 Y-copY a Template ........................................................................... 5-12 F- Erase a Formula File .................................................................... 5-13 xv L- Lotus & Other Transforms ......................................................... 5-14 Lotus transformation ...................................................................... 5-14 VisiCalc transformation.................................................................. 5-15 Importing the data to Lotus ............................................................ 5-17 S-Scattergram ................................................................................... 5-19 Printing the Scattergram ................................................................... 5-21 O- Optics Test ................................................................................... 5-22 C- Calibration Plate Test ................................................................... 5-22 P- Programming Information ............................................................. 5-22 j Exit to DOS Shell.................................................................................5-23 Chapter 6: Troubleshooting ......................................................... 6-1 Troubleshooting .........................................................................6-1 CMOS Utility Software for the CERES 900C ...................................... 6-4 Running the Utility Program ................................................................ 6-5 Removing Junk Files from the CERES 900C ...................................... 6-6 Deleting Unnecessary Files ................................................................ 6-6 Chapter 7: Maintenance................................................................ 7-1 Before you begin .........................................................................7-1 Safety Precautions .............................................................................. 7-1 Replacing the Light Bulb ..................................................................... 7-2 Replacing the Fuses ........................................................................... 7-3 Configuring the CERES 900C for Different Voltages .......................... 7-4 Changing the Line Voltage for Incubation ........................................... 7-5 Cleaning The Optics............................................................................ 7-5 Cleaning the Exterior........................................................................... 7-7 Storage and Shipping.......................................................................... 7-7 xvi Chapter 8: Performance Verification ...........................................8-1 Testing the System/ Calibration ......................................................... 8-1 Calibration Plate Test.......................................................................... 8-1 Running the Calibration Plate ............................................................. 8-2 Defining the Calibration Plate ............................................................. 8-3 Calibration Test Results...................................................................... 8-9 Test Failures ....................................................................................... 8-9 The Optics Test................................................................................. 8-10 Quality Control .................................................................................. 8-11 Optical Verification ............................................................................ 8-12 Verification of Alignment ................................................................... 8-14 Linearity ............................................................................................ 8-15 Appendix A: Error Messages ...................................................... A-1 Appendix B: Serial, Parallel Pinouts........................................... B-1 Appendix C: Show Scan Scanning Software .......................... C-1 Appendix D: Specifications ......................................................... D-1 Appendix E: Accessories ............................................................ E-1 Appendix F: Reader Control File..................................................F-1 xvii xviii Chapter 1: Introduction Instrument Revision G KCJr Version 1.12 This chapter introduces the CERES 900C, and describes its hardware and software features. Also included are instructions on how to contact Bio-Tek, if needed, and information about this manual. Introducing the CERES 900C The CERES 900C EIA Scanning Autoreader and Microplate Workstation is an integrated reader-assay management system for in-vitro diagnostic use, designed to automatically perform complete kinetic, endpoint and scanning analysis (up to 35 scan points across a microwell diameter). The CERES 900C can measure the optical density of solutions in 96-well microplates, and create files from the resulting data that can be printed in report form or stored as data files for a variety of ELISA-based applications. The CERES 900C features superior optical specifications, with an extended dynamic range of up to 4.300 absorbance units, and an extended wavelength range of 340nm to 900nm. Assay definitions (consisting of protocols, templates and formulas ) and the data they produce, are managed by an onboard processor, with a detachable VGA display and QWERTY keyboard. The CERES 900C is designed to serve either as a stand-alone system, or as part of a larger laboratory data network, sending, receiving and manipulating assay data and/or RCD files as needed. The CERES 900C provides direct interfacing to sample processors, LIS, Robotics or Systems components. Its Incubation Module is programmable to 50° C. The CERES 900C is backed by Bio-Tek's superior support staff. If the CERES 900C ever fails to work perfectly, please refer to the phone, fax, and BBS numbers at the end of this chapter to contact Bio-Tek's Technical Support Staff. Hardware Features _________________________________________________ The CERES 900C hardware features include: • An external 14-inch color VGA display • A 120 MB hard disk drive • A 3.5" high density (1.4 MB) floppy disk drive • QWERTY membrane keyboard with numerical keypad, function and direction keys • 96-well microplate carrier • Two factory-set serial COM ports (COM1 and COM2) • LPT1 port (Centronics female 25-pin parallel port for printer) • Four PC expansion slots for optional circuit boards ( full, 3 halfs) • External keyboard port (5-pin female) • Incubator voltage selector switch (120/240) Software Features_________________________________________________ KC Jr. , Bio-Tek's PC-based curve fitting and statistical software program is included preloaded on every CERES 900C reader. An unlimited number of assay protocols and blanking patterns can be defined, stored in memory, and instantly accessed. Plate templates and formulas are automatically combined with the protocol assay setup. Data results may be displayed on the monitor, printed out, sent to a mainframe, or merged with other software packages such as Lotus 1-2-3. Basic DOS utilities are included for convenience. KC Jr. provides: ■ An easy-to-use menu-driven interface; ■ Kinetic and endpoint curvilinear regressional and statistical calculations; 1-2 ■ Curve Fitting, with 4-parameter, cubic, quadric, and linear methods; ■ Formula calculations for more complex mathematical operations; ■ Limited calculations for positive or negative cutoffs. Introduction Package Contents _______________________________________________________________________________________________________________________________________________________ The contents of the CERES 900C package as shipped includes: • CERES 900C Microplate Reader • CRT Monitor (optional) • Power Cord(s) (2 if monitor is included) • Filter wheel with 4 standard filters- 405nm, 450 nm, 490nm, 620nm, (340 nm is included with CERES 900C UV) • CERES 900C User Guide & Registration Card including: ∗ KCJr Diskette ∗ Show Scan Diskette ∗ CMOS Diskette (Directions in Manual) CERES 900C User's Guide • DOS Manual and disks • Fuses • Light Bulb • Dust Cover 1-3 About This Manual ____________________________________________________________________________________________________________________________________________________ The intent of this user's guide is to quickly instruct the new user on how to set up and operate the CERES 900C EIA Reader. To this end, we've employed certain conventions to help you read and understand this manual. ;;; First, any important information will be indicated in this way, with the associated information icon as appears to the left of this paragraph. Next, anytime a key is discussed, the key is represented this way: Major topic headings start a new page (like About > [email protected] This Manual, above) to give you a visual and style clue that a new major subject is being broached. One or more subheadings will appear below each major heading. Registration Card Once you have got the CERES 900C up and running successfully, please take a moment to fill out the postage-paid Registration card and mail it to Bio-Tek. 1-4 Introduction Help _________________________________________________________________________________________________________________________________________________________________________________________ If your new CERES 900C system fails to start or operate successfully, please contact Bio-Tek immediately. Writing To Bio-Tek If you prefer, you can write a letter with your comments and send it to: Bio-Tek Instruments, Inc. Technical Assistance Center Highland Park, Box 998 Winooski, VT USA 05404-0998 Phone Support You can also telephone our Technical Assistance Center between 8:30 AM and 6:00 PM Eastern Standard Time, Monday through Friday, except Holidays. The number is (800) 242-4685 ; Outside of the USA (802) 655-4044; European (32) 3-829 0455. Emergency Assistance is available Monday through Friday, 6:00 PM to 9:00 PM. Whichever method of contact you choose, please provide the following information. • Product name ("CERES 900C") and serial number • Revision level of your software • The specific steps which reproduce your problem • A daytime phone number. CERES 900C User's Guide 1-5 1-6 Introduction Chapter 2: Installation This chapter includes instructions for unpacking and setting up the CERES 900C and instructions for connecting printers and/or serial devices. The Operating Environment The CERES 900C is designed to operate optimally when installed on a level surface in an area where ambient temperatures remain between 15°C (59°F) and 40°C (104°F). The reader is sensitive to extreme environmental conditions, and these conditions should be avoided: Excessive humidity. Excessive light (particularly bright sunlight) as it may affect the instrument's optics and readings. Dust. Optical density readings may be affected by extraneous particles (such as dust) in the microplate wells. A clean work area is necessary to ensure accurate readings. Always keep the reader covered with a dust cover when not in use. Unpacking the Instrument Use Figure 2-1 as a guide when unpacking and packing the instrument. ■ If the shipping box is damaged, inspect the instrument for visible dents and scratches as you unpack it. ■ If the instrument is damaged, notify the carrier and your Bio-Tek representative. Keep the shipping cartons and the packing material for the carrier's inspection. Bio-Tek will arrange for the repair or replacement of your instrument immediately, before the shippingrelated claim is settled. If the CERES 900C is shipped to Bio-Tek for repair or replacement, the instrument must be carefully packed (use Figure 2-1 as a guide), using the original packing materials. If the original packing materials have been damaged, contact Bio-Tek Instruments for replacement materials. Shipping with improper packaging materials may void your warranty. To unpack the instrument: ■ Carefully open the top of the box, and remove the accessories box. ■ Lift the CERES 900C out of the box and remove the foam end caps. Place the instrument on a level surface. Figure 2-1: Packing the CERES 900C Installing the Filter Wheel The filter wheel is packaged separately during shipment to protect it from damage. Carefully unwrap the filter wheel and install it inside the reader: 2-2 Installation ■ Loosen the two wingnuts holding the Optical Assembly Compartment cover in place and open the compartment. Power Module On/Off Switch Serial (COM) port 3-prong power cable connector Printer (LPT1) connector Fuse compartment Keyboard connector Video display connector RS232 (COM) port Optical Assembly Optical Asembly Access Panel wing nuts Figure 2-2: Optical Assembly Compartment ■ CERES 900C User's Guide Slide the Optical Assembly outward until it is fully exposed. 2-3 Attach the magnetic Filter Wheel Hub to the Mounting Hub (Figure 2-3) so that the Registration Key on the Filter Wheel Hub fits into the Registration Slot on the Mounting Hub. Registration slot Lamp Assembly Registration key Spring-loaded bracket for lamp Figure 2-3: Attaching the Filter Wheel to the Optical Assembly Once the Filter Wheel is in place, slide the Optical Assembly back into position within the instrument, close the Access Panel and re-tighten the two Access Panel Wingnuts. Replacing and Installing Filters The Filter Wheel comes factory-equipped with four filters. A total of 6 optical interference filters can be installed. Additional filters are available for the CERES 900C. For information regarding price and availability, contact your authorized Bio-Tek Representative, or call (800) 451-5172 or (802) 655-4040 Monday - Friday, 8 AM to 6 PM, Eastern 2-4 Installation Standard Time. The standard filters are placed in the following positions on the filter wheel: CERES 900C CERES 900C-UV Position #1 - 405nm Position #1 - 405nm Position #2 - 490nm Position #2 - 340nm Position #3 - Plug (Blank / no pass filter) Position #3 - Plug Position #4 - 450nm Position #4 - 490nm Position #5 - 620nm Position #5 - 620nm Position #6 - Plug (Blank / no pass filter) Position #6 - Plug To replace or install filters: Figure 2-4: Attaching a filter to the filter wheel ;;; Be sure to handle the filter wheel by the rim to avoid placing fingerprints on the filters. Filters may be cleaned using photographic lens cleaner and lens paper, or Q-tips and isopropyl alcohol. Remove the Filter Wheel by pulling it firmly out of the Mounting Hub, being careful not to touch the filter surfaces (Figure 2-3). Gently press on the Filter tabs until the Filter is released from its slot. Install new Filters by inserting them carefully into the desired slot until the tabs click into place. CERES 900C User's Guide 2-5 ■ The filter(s) that will be used for testing are software selectable using the CERES 900C operating program, KCJr. When the CERES 900C is powered on, KC Jr. automatically loads. Filters may be selected by choosing the System Utilities item on KCJr.'s Main Menu. Select S-CERES 900 setup, from the System Utilities submenu. Then select F-Filter Table. For a detailed discussion of the filter table, refer to Chapter 5, KCJr.'s Utilities. Installing a printer The CERES 900C features a parallel port (LPT1) to allow connection to a variety of printers. The port is shown in Figure 2-5 on the following page. The connector is a 25-pin D-sub state connector. The pinout is illustrated in Appendix B. Most printers come equipped with a parallel cable designed to connect the printer to a computer or other output device. Should the cable that came with the printer become lost or damaged, Bio-Tek offers a variety of printer cables for IBM-compatible printers. Contact your authorized Bio-Tek Dealer for information regarding cable prices and availability, and the correct cable for your particular printer. To attach a printer to your CERES 900C: ;;; Place the printer in a location within five or six feet of the CERES 900C. Attach one end of the cable to the printer's parallel port. Attach the other end of the cable to the CERES 900C Parallel Port LPT1. Make sure the securing screws on both ends of the cable are tightened. System instability may result if the printer is not connected to the CERES 900C prior to system powerup. Serial Connections The CERES 900 provides two Serial Ports. One has a 9-pin D sub connector, and the other, a 25-pin D sub connector. The Serial ports may be used to send data to an external PC or serial device. The pinouts for the connectors are illustrated in Appendix B. 2-6 Installation If serial communications are required between the CERES 900C and another device, make sure that the communication parameters (Baud Rate, Data Bit Configuration and Parity Status) are set to match those of the receiving device. Serial Port (COM1) Power module (power cord connector, fuses, on/off switch) Serial Port (COM2) LPT1 (Parallel) Port for printer Figure 2-5: CERES 900C Connectors for Printers (LPT1) Serial (COM) ports and Power Module The CERES 900C default settings are: ■ COM1 and 2: 9600 Baud, 8/2 Data Bits and Even Parity. These settings may be changed via the CERES 900C operating software, KCJr., which is automatically loaded when the CERES 900C is powered on. To change the settings, select System Utilities from KCJr.'s Main Menu. Changing the default COM port settings is discussed in Chapter 5, KCJr.'s Utilities. CERES 900C User's Guide 2-7 Electrical Connections Ensure that the CERES 900C’s Incubator Voltage Select Switch, located on the back panel of the instrument, is matched to the line voltage being used (100 - 120VAC, set to "115" position / 220 240VAC, set to "230" position). Attach the female end of the power cable provided to the 3-prong male socket located on the back panel as shown in Figure 2-5 on the previous page. ■ Plug the male end of the cable into an earth-grounded 120VAC electrical source. (see Chapter 7, Maintenance for instructions on how to configure the CERES 900C for 220 or 240 Volt operation). ;;; WARNING!! Avoid using extension cords or cheater plugs that do not have a protective (Ground) conductor. The protective earth terminal (chassis) must be connected to the protective earth connector of the power source, which normally happens when the main power cable is properly connected to the AC power source. Failure to properly ground this unit can result in a hazardous condition Fuses The CERES 900C may be fused for a variety of operating voltages (AC), including: 120, 220 and 240. Detailed instructions on changing fuses can be found in Chapter 7, Maintenance. 2-8 Installation Chapter 3: Introducing KC Jr. Software This chapter introduces KC Jr. software for the CERES 900C. An Overview of KC Jr. KC Jr. is an IBM PC or PC-compatible software program that controls the operation of the CERES 900C and is designed to provide data reduction for most ELISA applications using Bio-Tek's microplate readers. The "junior" version of Bio-Tek's full-featured KinetiCalc program, which is preloaded on the CERES 900C, provides data reduction for four basic assay types: ■ End point curvilinear regression ■ Kinetic curvilinear regression ■ Statistical nonregression-based calculations ■ Well scanning KC Jr. is designed to retrieve microplate data in an 8 x12 matrix from a Bio-Tek reader, store the data in multiple-plate files for future use, and analyze the data based on a user-specified set of protocol parameters. In addition to the 96 optical density values, the reading date and time of the original file is recorded as part of the disk file. KC Jr. does not require the user to attend the CERES 900C during each plate reading. If the program and the reader are configured for CONTINUOUS READ, the program will continue to accept data from the reader and store each 96-well matrix in a microplate file until the specified number of plates or readings are made, the disk is full, or the program is interrupted. KC Jr. allows the user to: ■ Retrieve data from the reader and store the data on a disk (either the reader's hard disk, or a floppy) under a user-specified file name. ■ Display absorbance data on screen, or print them in raw, 8 x 12 format or as a scattergram. ■ View a directory of data already stored on disk. ■ Copy, erase, or rename microplate files. ■ Create data files usable with other commercial spread sheet and statistics programs, such as Lotus 1-2-3 or VisiCalc. ■ Calculate mean, standard deviation, variance, and coefficient of variation for any user-specified set of standard or sample wells on a plate. ■ Define formulas to analyze optical density data from any user-specified set of wells. 3-2 KC Jr. Main Menu ■ Correct any group of wells by a constant value to effectively eliminate background absorbance and optionally save the corrected data to disk. ■ Display or print a report that shows the relationship of all wells to as many as 4 user-specified limit values. ■ Store, retrieve, and modify microplate templates that map any user-specified combination of standard and sample wells. ■ Store, retrieve, and modify default protocol files that contain parameters specifying the assay type, curve calculation, and sample evaluation. ■ Edit the data points used to calculate the kinetic or end-point models. ■ Calculate a linear or curvilinear regression model based on the default protocol parameters. ■ Use back-calculation or extrapolation to evaluate the concentration values of samples on a microplate. ■ Save most reports to disk files. Supported Pointing Device (optional) ■ CERES 900C User's Guide Microsoft-compatible or IBM PS/2 mouse 3-3 Supported Output Devices 3-4 ■ IBM graphics or Proprinter; Star NX1001 ■ Epson FX, JX-80, LQ1500, LX, and MX printers ■ Epson-compatible dot-matrix printers ■ Kodak Diconix 180 si ■ Okidata (all models) ■ HP Laserjet, Laserjet + or Laserjet II or compatible ■ Texas Instruments printers (or compatibles) KC Jr. Main Menu KC Jr.'s Main Menu KC Jr.'s Main Menu provides an easy, graphical way to customize the reader's operating environment, design various protocols, and specify how data will be calculated and reported. KCJr. software will automatically load when the CERES 900C is powered on. To access KC Jr.'s Main Menu from the DOS prompt, change to the drive and directory containing KC Jr. software, and start the program by typing: KCJR A menu much like the one shown below will appear: Figure 3-1: The KCJr Main Menu CERES 900C User's Guide 3-5 Menu Overview Main Menu options can be accessed in four ways: ■ Pressing the function key assigned to the option (e.g.: F7 to set Data Defaults) ■ Using the up-down cursor arrow keys and pressing Enter ■ Using an installed mouse to point and click on an option ■ Typing the first letter of the option and pressing Enter Select any option on the Main Menu, and a pop-up window prompts for information, or provides a list of choices. Pressing Esc aborts the current operation, and returns you to the previous level in the menu hierarchy, and ultimately back to the Main Menu. KC Jr.'s Main Menu also contains a number of options that allow file maintenance without leaving the software program. For example, the System Utilities Menu option (F6) allows you to perform common DOS file operations such as copying or erasing data files, Templates or Protocols. You may also perform an optics test, verify instrument calibration, or transform data for use in Lotus 1-2-3, VisiCalc or Scattergrams. And the "Exit to DOS shell" option allows you to exit to DOS, and return to KC Jr. without actually exiting the program. The Main Menu also allows you to set an incubation interval. The status line appears as the bottom line of the Main Menu. Here you'll find information on the protocol and template being used for calculations, and the current operation. The status line also displays prompts for action. The area at the far right of the status line indicates whether the keyboard's Num Lock and Cap Lock features are active. When the cursor control pad is set for numeric function, "NUM" is displayed. If the Caps Lock key is pressed on the keyboard, "CAPS" is displayed on KC Jr.'s Main Menu. Otherwise this space is empty. 3-6 KC Jr. Main Menu Using the Main Menu The first time you begin KC Jr., you'll want to execute the program functions in the following order: Create an operating Protocol (F3). The Protocol file contains information that tells KC Jr. how to calculate and report ELISA data. Create a Reader Control File. The RCF must be created before the CERES 900C can read a microplate. The Reader Control File is selectable on the (F3) Protocol definition screen. Create a Microplate Template (F4). A Template defines multiple sets of standards and samples, and defines concentrations, dilutions and replicates. Create a Formula (F5). A formula for the transformation of optical densities or kinetic slopes can be created, edited and saved for later use in data analysis. Set Data Defaults (F7). This option allows you to specify the drive/directory for storing O.D. data; and how the data will be reported (displayed on the screen, printed, or both). Calculate Results (F2). The calculation process allows you to retrieve and manipulate the data, and report the results. These options are discussed in detail in the sections that follow. CERES 900C User's Guide 3-7 KC Jr.'s Function Keys KC Jr. utilizes the keyboard Function Keys (F1-9) making it easy to access commonly-used program functions. The F10 key is used to "save" Protocol, Template and Formula definitions. Function key assignments are: 3-8 F1 Help F2 Calculate results F3 Protocol (create/edit) F4 Template (create/edit) F5 Formula (create/edit) F6 System Utilities F7 Data Defaults F8 Incubate Plate / Home Plate F9 Exit to DOS shell F10 Save KC Jr. Main Menu KC Jr.'s "AutoRun" Feature (Batch File Operations) Data analysis may also be run directly from the DOS prompt as long as the protocol and template names are the same. This feature applies both to existing data files and new plate data taken directly from the reader. The options are as follows: ■ P Option: Using this option, enter the following command at the DOS prompt: KCJR /P:ENDPOINT:ELISA.DAT where both the protocol and template names are "ENDPOINT", and the data file name is "ELISA.DAT". ;;; This feature allows the user to quickly calculate a standard curve using existing data before additional plate(s) of samples are read. ■ B Option: This option allows the user to run an analysis of data being read directly from a reader. The user has the choice of either assigning a file name to the data to be read as part of the initial command, or waiting until KC Jr. prompts the user to enter a name for the data. For example, entering KCJR /B:ENDPOINT:ELISA.DAT or KCJR / B:ENDPOINT:* will initiate a new plate read, the results of which will be a new data file that KC Jr. will entitle "ELISA.DAT." Using an asterick for a datafile name will result in KCJR making a filename based on the current system date: YYMMDD.###, where ### can be 001, 002, 003 etc. CERES 900C User's Guide 3-9 If, however, only KCJR /B:ENDPOINT is entered, KC Jr. will prompt the user for a file name during the reading. After the analysis is completed, KC Jr. will return to the DOS prompt. ■ L Option: This option automatically converts any data that is read during the batch run to a Lotus ".PRN" file. ■ G Option: This option automatically prints the standard curve generated during the batch run. KCJR /B:ENDPOINT:ELISA.DAT /L ;;; 3-10 /G These options are only available when the "B" option is used. KC Jr. Main Menu Before You Begin: The CERES 900C Setup The CERES 900C is set up at the factory for certain default operating parameters. You'll want to check that these parameters are right for your operating environment. These parameters include: ■ The Filter Table: The filter table may be accessed through the KC Jr. Main Menu by selecting F6 System Utilities, and then CERES 900 Setup. ■ Communications Parameters: Also accessed through F6 System Utilities/ CERES 900 Setup, communications parameters allow the CERES 900C to communicate with another device, like a computer. ■ Reporting Device Parameters: Specifying a Reporting Device (printer) can be accomplished by selecting Data Defaults on the KC Jr. Main Menu. Data Defaults are discussed in detail in this chapter. CERES 900C User's Guide 3-11 C System Utilities / Setup CERES 900C Setup is accessed from the KC Jr. Main Menu. To check the instrument's setup: ■ From KC Jr.'s Main Menu, select F6 System Utilities. ■ From the Options submenu, select S - Ceres 900 setup. A screen similar to the following will appear on the display: 3-12 KC Jr. Main Menu The Filter Table This window allows the user to notify the CERES 900C software program when changes are made to the selection or position of the filters installed on the Filter Wheel. The factory standard filter configuration is shown in the table below: CERES 900C Standard Filter Configuration FILTER TABLE CERES 900C User's Guide Position 1 405nm Position 2 490nm Position 3 plug/blank Position 4 450nm Position 5 620nm Position 6 blank 3-13 Filter Configuration for CERES 900C UV: FILTER TABLE YYY Position 1 405nm Position 2 340nm Position 3 blank/plug Position 4 490nm Position 5 620nm Position 6 blank/plug The Part Number for the blank / no pass filter (plug) is 3122037. To install a 660 nm filter in the empty third position, for example, the user would: ■ Remove the filter wheel from the CERES 900C, and install the new filter into position 3. Installing new filters is discussed in Chapter 2. ■ Select F6- System Utilities from the KC Jr. Main Menu, and then select S - CERES 900 setup; ■ Select F- Filter table to access the filter options, and the up/down cursor keys to highlight the fifth filter position on the Filter Table; ■ Using the number keypad on the CERES 900C keyboard, enter 660 next to POS 5; ■ Press the k key to save the new setting. Once the filters have been saved, a warning message appears on the display: WARNING: The filter table has changed. Check your protocols for correct filters. 3-14 KC Jr. Main Menu ;;; Position #1 must contain a filter. The CERES 900C requires that a 405nm filter reside in Position #1 for calibration purposes. Serial Setup This parameter allows the user to set up communication parameters with another serial device (example: another computer or a modem). ■ To change parameters: Select Serial on the System Utilities option submenu; ■ Use the cursor up/down arrow keys to highlight the parameter you want to change. Use the spacebar to scroll through the available options; ■ Press the kkey to save the changes. A Look at the Options Baud Rate: The baud rate, or rate of data flow may be changed using the spacebar. The factory set default is 9600 bits/sec. Options include 300, 1200, 2400, 9600 and 19200 bit/sec. Data Bits: The Data Bits setting defines two parameters: Character string bits (each character string is composed of either 7 or 8 bits), and Stop Bits (either 1 or 2 bits). The default setting is 8 and 2. CERES 900C User's Guide 3-15 Parity: The parity setting is used to prevent serial communications errors between devices. The options are odd or even parity. The default is Even Parity. ;;; All three parameters -- Baud Rate, Data Bits and Parity should be matched in the communications software on the device receiving the data. Report Device (Printer) If you've attached a printer to the CERES 900C, you'll need to ensure that the reader recognizes the print device, and and that any required Printer Control Codes have been entered. Selecting the Reporting Device and specifying Printer Control Codes is accomplished by selecting F7 Data Defaults on KC Jr.'s Main Menu. Data Default options are described elsewhere in this chapter. @Protocol - Create/Edit The Protocol Definition Screen A Protocol file contains information that tells KC Jr. how to calculate and report ELISA data. Once a protocol is created, the parameters it contains are saved on the 3-16 KC Jr. Main Menu default program disk. The Protocol can then be recalled and used for calculating curves and evaluating sample unknowns by entering its name as a Data Defaults parameter or by selecting its name whenever a Protocol is requested within KC Jr. The name of the active protocol file always appears on the Main Menu status line. The first time you create a protocol (F3 from the Main Menu), the screen displays a blank protocol definition screen with a prompt window requesting the new protocol filename. Figure 3-2: Protocol Definition screen with filename prompt Enter a file name up to 8 characters, and press Enter, (A file extension is unnecessary, as KC Jr. automatically adds a .PRT extension when it saves the protocol to disk.) The new filename appears at the top of the display with a default set of parameters that may be edited. CERES 900C User's Guide 3-17 Figure 3-3: Protocol Definition Screen with default parameters If protocol files have already been created and saved, a pop-up window appears, listing available protocol files. To edit an existing protocol file, select the file name from the window by using a mouse, or up/down cursor control keys and pressing Enter, as shown in the illustration that follows: Figure 3-4: Selecting a Protocol from a list of saved filenames 3-18 KC Jr. Main Menu Editing the Protocol Parameters If you want to create a new protocol file at this point, press Esc to clear the screen, then enter a new protocol filename. ;;; If you edit an existing Protocol file, when you save the file, KC Jr. will overwrite the original file with the new parameters. To edit the Protocol parameters: ■ Keyboard method: Use the up/down cursor control keys to move to a parameter field. Key in parameter information, or use the space bar to cycle through setting options. ■ Mouse method: Point at the parameter field, and click to key in information or view setting choices. The setting displayed on the screen when you move to the next parameter will be the setting used in the protocol file when it is saved. Saving/Aborting Changes When editing is completed: ■ Press F10 to save parameters in the Protocol file, or ■ Press Esc to return to the Main Menu without saving. Protocol Name Any name, up to 8 characters, may be used. KC Jr. automatically adds a .PRT extension to the file name you specify. CERES 900C User's Guide 3-19 While the protocol name is highlighted, you can press the space bar to bring up a window listing the protocol file names stored in the default program directory, in the event the protocol displayed on the definition screen is not the one you wish to modify. You may then select another protocol to create or edit. Figure 3-5 Protocol name highlighted Protocol Description This line, initially blank, allows you to enter a protocol file description up to 40 characters long. Reader Control File The Reader Control File must be created before the CERES 900C reads any plate data. This option is selectable as part of the Protocol definition screen. ■ To create/edit a Reader Control File, highlight the Reader Control File field on the Protocol definition screen, and press the space bar. The Reader Control File definition screen will appear (Figure 3-6 ). For a more detailed explanation of the Reader Control File, refer to Appendix F, Reader Control File. 3-20 KC Jr. Main Menu ;;; The Reader Control File is automatically given the same name as the Protocol, with an .FIL extension. Figure 3-6: The Reader Control File Definition Screen ■ Use the up/down cursor keys, or a mouse to highlight parameter fields in the Reader Control File. If reading Method or Blanking Pattern is highlighted, pressing F1 will pop up a menu of choices. ■ Use the spacebar (or mouse click) to view options. ■ Move to the next parameter to select any given option. ■ Press F10 to save changes in the Reader Control File ■ Press Esc to abort all changes and return to the Protocol Definition screen. CERES 900C User's Guide 3-21 Assay Type The assay type definition can be: kinetic, end point, statistical or scanning. To change the setting, highlight this field and press the space bar to cycle through the four choices. Number of Reads If the Assay Type is kinetic, this parameter must be specified in the Reader Control File. If the Assay is end point or statistical, this parameter specifies the number of plates to read. The value you enter here must be in the range of 1 to 250. ;;; If the reader control file specifies kinetic as the reading method, the number of readings per plate is automatically taken from the reader control file. 3-22 KC Jr. Main Menu Reading Interval This setting specifies the time interval between repeated reads on the plate, and is applicable only if the Assay Type is kinetic. If the reader control file specifies kinetic as the reading method, this reading interval is automatically taken from the reader control file. Prompt for Plate ID ■ Highlight this parameter and use the spacebar to toggle between Yes and No. ■ If Yes is selected, the user will be asked to enter a plate-specific ID each time a plate is read. Fitting Method This parameter can only be changed if the Assay Type is kinetic or End Point. The options are linear, quadratic, cubic, 4-parameter, and logit/log. ■ To change this setting, highlight this parameter, and press the space bar to cycle through the 5 options. Data / Axis Type This setting can only be accessed if the Assay Type is kinetic or end point. The setting options are linear, linear/log, or log/log. ■ To change the setting, highlight the parameter and press the spacebar to cycle through the options. If the Fitting Method parameter is either 4-parameter or logit/log, this parameter only affects the display of the curve. For the other fitting methods, the data will be transformed by the selected data type. Linear/Log is linear Y -axis, log X-axis. CERES 900C User's Guide 3-23 Average Sample Replicates This setting can only be changed if the Assay Type is kinetic or end point. The options are before, after, or none. ■ To change this setting, highlight the parameter field and press the spacebar to cycle through the options. ■ If set to before, the program averages the replicate optical densities (or slopes) before evaluating for the unknown concentrations. ■ If set to after, the back-calculated concentrations of replicates are averaged. ■ If set to none, the optical densities are not averaged; each one is evaluated separately. ;;; Bio-Tek recommends setting this parameter to before to reduce the amount of error in calculations. Units of Measure This parameter allows you to specify the concentration label, up to a 10-character maximum, for the X-axis on the fitted curve graph. It can only be changed if the Assay Type is kinetic or end point. ;;; Spaces entered in this label will become underscored. ■ The Y-axis label is automatically set to Optical Density for an end-point assay or to Rate for a kinetic assay. 3-24 KC Jr. Main Menu Well Slopes/R^2 If the Assay Type parameter is set to kinetic, this parameter allows you the option of reporting the well slopes and R-squared values for each well on all plates in the run. The report either will be sent to the printer, displayed on the screen, or both, depending on what was selected as a Report device during setup of Data Defaults. ■ To change this setting, highlight the parameter field and press the space bar to cycle through the options. Kinetic/Scanning Y-Scale Enter the maximum Y-scale value in the highlighted field. If a default value exists, press the spacebar to clear the field for input. Standards Results If the Assay Type parameter is set to either kinetic or end point, this parameter allows you to report the results of evaluating and back-calculating the standards (Figure 37). Figure 3-7: Standards Results CERES 900C User's Guide 3-25 The report either will be sent to the printer, displayed on the screen, or both, depending on what was selected as a Report device during setup of Data Defaults. ■ To change this parameter, highlight this item, then press the space bar to toggle between Yes and No. Sample Results If the Assay Type parameter is set either to kinetic or endpoint, you may opt to report the results of evaluating and back calculating the sample unknowns (see Figure 3-8). The report either will be sent to the printer, displayed on the screen, or both, depending on what was selected as a Report device during setup of Data Defaults. Figure 3-8: Sample Evaluation Report Screen (End Point Assay) ■ To change this parameter, highlight the parameter field and press the space bar to toggle between Yes and No. 3-26 KC Jr. Main Menu Protocol Definition This parameter allows you to list all the parameters and their settings from the currently active protocol. ■ To change the parameter, highlight the Protocol Definition field and press the spacebar to toggle between Yes and No. The report either will be sent to the printer, displayed on the screen, or both, depending on what was selected as a Report device during setup of Data Defaults. Template Description This parameter allows you to map the active template in an 8 x 12 matrix, showing the location and concentration of standards, and the location and dilutions of sample unknowns (Figure 3-9). The report either will be sent to the printer, displayed on the screen, or both, depending on what was selected as a Report device during setup of Data Defaults. Figure 3-9: Template Map Screen CERES 900C User's Guide 3-27 ■ To change the parameter, highlight the Template Description field and press the spacebar to toggle between Yes and No. Absorbance Data This parameter allows you the option of reporting the optical density data of each plate reading. If the parameter is set to Yes, the program will automatically produce an 8 x 12 matrix report of the optical density values for each reading (if the assay type is set to kinetic), or each plate (if the assay type is set to endpoint or statistical). ■ To toggle between the Yes/No settings, highlight the parameter and press the space bar. The report will be sent to the printer, displayed on the screen, or both, depending on what was selected as a Report device during setup of Data Defaults. Report of Means This parameter allows you the option of producing a statistical report of the optical density data. If the parameter is set to Yes, the program will automatically produce a means report of optical density data for all sets defined on the template. ■ Highlight the parameter and press the space bar to toggle between Yes and No. The report will be printed, displayed on the screen, or both, depending on what was selected as the Report device during Data Defaults setup. Transform Raw Data By_____ This parameter allows you to specify the name of the formula file which specifies the transformation to be performed on optical density data, before any other calculations or reporting is done. ■ Highlight the parameter field and press the spacebar to select from a list of existing formulas. 3-28 KC Jr. Main Menu ■ If a formula file has not yet been created, return to the Main Menu and select F5 Formula (create/edit). Report Transformed Data This parameter allows you to produce a matrix report of absorbance or kinetic rate data after it has been transformed by the formula(s) in the "transform raw data by___" file. ■ To toggle between Yes and No, highlight the parameter field and press the space bar. Apply Formula This parameter allows you to specify the name of the formula file that contains the calculation to be performed on the optical density data. To select a formula file name: ■ Highlight the parameter field and press the space bar. ■ A pop-up window will list formula files saved in the default program directory (Figure 3-10). ■ Use the cursor up/down keys, or the mouse to highlight a formula, and press Enter or click the mouse button. CERES 900C User's Guide 3-29 Figure 3-10: Applying a Formula Report of Limits This parameter allows you the option of producing a limit report for each plate in the run (Figures 3-11 and 3-12). The options are no, matrix, column, formula-mat, and formula-col. Figure 3-11: Matrix Report of Limits 3-30 KC Jr. Main Menu Figure 3-12: Column Limit Report Screen ;;; A formula must be entered in the Protocol's Apply Formula field in order to access the formula-mat and formula-col options. ■ To change the parameter, highlight the parameter field and press the spacebar to cycle through the options. After calculation is done, the Limits report will be sent to the printer, displayed on the screen, or both, depending on what was selected as a Report device during setup of Data Defaults. ■ If you set this parameter to anything other than No, the program will calculate and print a minus sign (-) for all plate values below or equal to the first limit, a +1 for all values between the first and second limit, a +2 for all values between the second and third limit, a +3 for all values between the third and fourth limit, and a +4 for all values above the fourth limit. ■ To edit the limit settings, press the down arrow key to move down to the limit settings line. Then use the left/right cursor control keys to move among the five limit settings and enter up to 4 limit values for the the report. Make sure you enter these limit values in increasing order. CERES 900C User's Guide 3-31 If set to formula-mat or formula-col, the limit values will be automatically calculated by the formula file specified by the Apply Formula parameter. ;;; If you want to specify your own limits and you have chosen to apply a formula to the data, make sure the selected formula does not contain limit calculations. Likewise, if you set this parameter to formula-mat or formula-col, make sure the selected formula contains limit definitions. Otherwise the results may be erroneous. ■ When you are finished editing the protocol, press F10 to save the new protocol and return to the Main Menu. 3-32 KC Jr. Main Menu ATemplate (create/edit) The Template defines multiple sets or groups of standards and samples and specifies their concentrations, dilutions, and replicates. Once a Template is created, the parameters it contains can be saved on the default program disk. The Template can then be recalled and used in calculations by entering its name as a Data Defaults parameter or by selecting its name whenever a Template is required within KC Jr. The status line at the bottom of KC Jr.'s Main Menu lists the active template file. (Figure 3-13 shows Main Menu with the Template file window.). Figure 3-13: Main Menu With Active Template Line ;;; Throughout this section, a sample template will be used to illustrate how to create a new template, enter and replicate sample sets, and enter dilutions. If you wish to create a template that duplicates the one used in this example, follow the steps marked with the pointer. ☛ CERES 900C User's Guide 3-33 Creating a New Template ☛ Press F4, or highlight Template (create/edit) on the Main Menu and press Enter. ☛ At the prompt window, enter a file name (up to 8 characters) and press Enter (or click the mouse button). A blank template appears on the screen with the well marker in well A1. Figure 3-14: Blank template map ■ Begin entering data, or press the Esc key to return to the Main Menu. Selecting an Existing Template ■ Select F4 - Template (create/edit) from the Main Menu. A window appears listing the template filenames stored in the default directory. 3-34 KC Jr. Main Menu ■ To select a Template to edit, use the up/down cursor keys (or enter the first letter of the Template name to highlight the next file beginning with that letter) and press Enter. KC Jr. will retrieve the template from disk and display it on the screen. Wells with a number prefixed by an S are standard concentration values. Wells holding only numbers are sample locations. Modifying a Template From the template screen, you can enter or modify standards, samples, dilutions, and replicates. ■ Use the up/down, left/right cursor arrow keys to move to a well location. If a previously created template is being edited, changes to the standards, concentrations, and dilutions may be made in any order you wish. ■ Press Esc to abandon the template and return to the Main Menu. Or, press F10 to save the template in the default program directory and return to the Main Menu. If a new template is being created, entries are usually made in the following order: ■ Enter the standard concentrations. ■ Replicate standard concentrations. ■ Enter the sample unknowns. ■ Name the sample unknowns. ■ Replicate the sample unknowns. ■ Enter sample dilutions. ■ Enter a template description. CERES 900C User's Guide 3-35 ■ Save the template to disk. Entering Standard Concentrations ☛ To enter standard concentration values: ■ Move the marker to the well location designated for the Standard concentration. ■ Press the S key. On the status line you will be prompted to enter the concentration number. ■ Key in the number and press Enter or an arrow key. The well displays the value entered, preceded by an S to indicate a standard concentration. Up to 96 unique standards or replicated standards may be entered. Bio-Tek recommends no more than 36 unique standards be entered, to insure readability if a standards report is requested. ;;; For the regression calculations to work properly, the total number of standards must be at least one greater than the degree of fit requested in the active protocol. Thus, for a linear (1st-order) fit, there must be at least 2 standards; for a Quadratic (2nd order) fit there must be at least 3 standards; for a cubic (3rd-order)fit, there must be at least 4. For either a 4-parameter fit or a logit/log fit, there must be at least 4. If these requirements are not met, the error message "Unable to calculate curve with this data!" will appear when the program begins its calculations. If you want to correct a standard you've already entered: ■ Move the well marker to the appropriate well, then re-enter the correct value. Press Enter or an arrow key. If you enter a standard concentration value that is too wide to fit in the well space (five or more characters), an S replaces the value in the well. When the highlighted 3-36 KC Jr. Main Menu marker is moved to this well, its standard concentration appears in the top left of the screen. ☛ Example: If you enter standard concentrations of 1000, 750, 500, 250, 125, 62.5, 31.25, and 15.75 in wells A1 through H1, your screen should show the template as in Figure 3-15: Figure 3-15: New template with standard concentrations Note on the sample template that wells G1 and H1 contain an S because their concentration values are too wide to fit in the space. The well marker is on well H1, and its concentration value of 15.75 is displayed in the upper left corner. CERES 900C User's Guide 3-37 Replicating Standards ☛ Move the well marker to the standard you want to replicate and press Ctrl-R. Then move the well marker to the last standard of the set. (As you expand the area covered by the standard you are replicating, the status line will display the expanded range of wells.) ☛ When the entire standard area is specified, press Enter. The standard number will appear in each of the wells that belong to the set. Entering Sample Unknowns When the plate data is analyzed, calculations are performed individually and by sample sets within the plate. Thus, if a plate has four sample sets and the Report of Means parameter is set to Yes in the active protocol, the program will calculate a mean, standard deviation, variance, and coefficient of variation for each of the sample sets. Similarly, if the Sample Results parameter is set to Yes in the active protocol, the program will report the slope, dilution, and concentration for each individual well. ☛ To specify a sample set on the template, use the arrow keys to move the highlighted well marker to the well where that sample set begins. Then enter the set number between 1 and 96 and press Enter, or a cursor arrow key. If the remaining wells in the set are contiguous to this well, you can either replicate the first sample (see description below), or move the well marker to the appropriate wells and manually enter the sample number. ;;; Sample sets do not have to be located in contiguous wells. For example, set 1 may be wells A1 through A12 and H1 through H12. Or A1, A12, H1, and H12 may be a sample set. In this case, move the well marker to the desired wells and enter the sample set number. 3-38 KC Jr. Main Menu ■ Set designations also can be changed. If a well initially is part of set #1, and you wish to change it to set #2, move the highlighted well marker to the well, type 2, and press Enter. ■ To remove a well from a group, move the well marker to the appropriate well and press the spacebar. Press End to move the highlighted well marker to well H12 (the last well on the plate), and press Home to move the well marker back to well A1 (the first well on the plate). ☛ Example: In the sample template we are creating, sample 1 will occupy wells A5 through D5. Figure 3-16: New template with sample sets ■ Move the well marker to each of these wells and enter 1. Likewise, create sample 2 in wells E5 through H5, sample 3 in wells A9 through D9, and sample 4 in wells E9 through H9. The template should look like Figure 3-16. ;;; Any well not defined as part of a set will not be included in the analysis. Be sure to define your sets carefully and accurately. CERES 900C User's Guide 3-39 Naming a Sample Unknown Once a set is designated on the template, you can give the set a name. Figure 3-17: Sample Template, Naming a Sample Set ☛ Place the well marker on any well in the sample set, then press Ctrl-N. ☛ When prompted in the window, enter a name up to 12 characters, and press Enter. Now when you move the well marker around the template, the name of the set where the well marker rests is displayed above the template. The set names also appear when the calculations are displayed or printed. Figure 3-17 shows the prompt for a set name. Set 2 has been given the name, sample #2. The well marker rests in cell E5, so the name of set #2 appears at the top left corner of the template. 3-40 KC Jr. Main Menu Erasing a Sample Set or Standard Concentration ■ To erase a sample set or standard concentration from the template, move the well marker to any well within the set or standard and press Ctrl-E. ■ At the status line, KC Jr. will ask for confirmation before the program erases the set or standard. If the response is N(o), KC Jr. will return to the template screen. If the response is Y(es), the sample set will be removed from the template in memory. To make the erasure permanent, save the template (F-10) before returning to the Main Menu. Replicating a Sample Set Replication of wells produces exact copies of sample unknowns into contiguous wells on the template currently being edited. This procedure is performed from the main template screen, and is normally done after the sample sets have been entered but before dilutions are entered. ☛ Move the well marker to one corner of the sample set and press Ctrl-R. Then move the well marker to the opposite corner of the sample set. (As you expand the area covered by the sample set you are replicating, the status line will display the range of wells.) When the entire sample area is specified, press Enter. The sample set number will appear in each of the wells that belong to the current sample set. ☛ If you want to use all 96 wells of the plate in a single set, move the well marker to well A1, and key in 1. Press Ctrl-R to replicate, move the well marker to the last well, then press Enter. The screen will display 1 in each well, and the program will perform the analyses using the optical density values in all 96 wells as a single sample set. ☛ Example: Using the sample template, replicate sample 1 in wells A5 through D7. CERES 900C User's Guide 3-41 ■ To do this, move the well marker to well A5, and press Ctrl-R. Then move tthe well marker to well D7 an press Enter. The sample number 1 should appear in each of the 12 wells in the replicated range. ■ Replicate sample 2 in wells E5 through H7; sample 3 in wells A9 through D11, and sample 4 in wells E9 through H11. The template should look like Figure 3-18: Figure 3-18: Template With Replicated Sample Sets ;;; Don't try to replicate more than one sample set at a time, or the sample number where the replication procedure was initiated will be replicated over the entire range. If you replicate after dilutions have been entered, press Ctrl-D to display the dilutions screen, then check the dilution values. You may have to re-enter them. 3-42 KC Jr. Main Menu Entering Dilutions You cannot enter dilutions unless you have first entered standard concentrations or sample locations. ☛ Press Ctrl-D to enter dilutions. The program will redraw the screen to show the underlying dilution values on the template. On the newly created template, all the standard and sample wells will show a value of 1, indicating a 1:1 dilution ratio (Figure 3-19). Figure 3-19: Template Showing 1:1 Dilution The dilution factor shown is a ratio of 1 to the value shown (i.e., if a well on an existing template shows 2.5, KC Jr. will accept that as a 2.5 dilution). ■ From the dilutions screen, in addition to entering dilutions, you can press F10 to save the template and return to the Main menu or Esc to return to the main template screen. Once the dilution template is displayed, you can let the program calculate serial dilutions automatically, or you can enter the dilutions manually. CERES 900C User's Guide 3-43 Serial Dilutions: ☛ Press Ctrl-X to begin serial dilutions. The program will prompt for the dilution ratio. Enter the dilution ratio 2.000 The dilution ratio shown is either the program default value (2) or the last dilution ratio entered during this session. The ratio value you enter here will be the dilution ratio between successive wells within the sample. That is, it is the multiplying factor to be used in creating successive dilutions. Example: If the first dilution is 1:10 and the dilution ratio is 2, the dilutions would be 1:10, 1:20, 1:40, 1:80, etc., each dilution being multiplied by the dilution ratio to create the next dilution. Next, the program will prompt you for the starting dilution: ■ Enter the starting dilution 1. The starting dilution shown is either the program default value (1) or the last starting dilution ratio you entered during this session. ■ To accept the displayed value, press Enter. Or, enter a new value and press Enter. Before calculating the dilutions, the program needs to know the direction to dilute the samples: ■ Dilute DOWN or dilute RIGHT Your response tells KC Jr. whether to calculate the serial dilutions down the column or right across the row from the starting point you specify. ■ Enter D if your dilutions go down, or R if your dilutions go right from the starting well. ■ Finally, before KC Jr. can calculate and display the dilutions, you must specify the starting well for the first dilution. The windows are removed from the screen 3-44 KC Jr. Main Menu to redisplay the current template, and you are prompted for the starting well for the serial dilution. ■ Move the well marker to the beginning well for the sample dilution and press Enter to lock in the first well for the dilution set. Then move to the opposite corner of the diluted sample set and press Enter again. The screen will be redrawn with the dilution values calculated and displayed. ☛ Example: On the sample template we will dilute sample #1 with a dilution ratio of 2.00 and a starting dilution of 2.00. ■ Press Ctrl-X and enter these values. ■ Specify Dilute Down when prompted. ■ Next, move the cursor to well A5 and press Enter. ■ Then, move the cursor to well D7 and press Enter. Using the same procedure (Ctrl X, 2, 2, Dilute Down) for diluting samples 2, 3, and 4, the dilution screen should appear as in Figure 3-20: Figure 3-20: Dilution screen with Serial Dilutions CERES 900C User's Guide 3-45 Manual Dilutions ☛ To enter dilutions manually, ■ Press Ctrl-D from the main template screen to display the dilutions screen. ■ Move the marker to the wells where you want to add dilution values, and enter the dilutions manually. ☛ Example: To enter the same dilutions manually that you previously entered in the sample template, move the well marker to A5 and enter dilutions of 2, 4, 8, and 16 in wells A5 through D7. As you enter the dilution values, they are immediately displayed. Continue until all dilutions are entered. If you try to enter dilutions in wells where samples have not been entered from the main template screen, you will hear a beep, and KC Jr. will wait for valid key input. Entering a Description ☛ To enter a description for the template, press Ctrl-I. ■ At the prompt, type a description of the current template (up to 40 characters). This description will be printed/displayed with the template. Saving the Template ☛ When finished editing the template, press F10 from either the main template screen or the dilutions screen to save the template on the default program disk and return to the Main Menu. To use this template for calculations, enter the template's name during Data defaults setup, or select it during the "Calculate Results" process. 3-46 KC Jr. Main Menu Using a Previous Standards Curve (Defining two templates for standards) To run standards-per-assay (as opposed to standards-per-plate), 2 templates must be defined. Using standards per assay allows you to have standards (with or without samples) on the first microplate. Subsequent microplates would have samples only. For example, Template #1 would have standards (and samples if desired) defined on it. Only samples would be defined on Template #2. To run a standards-per-assay using the two templates: ■ Make template #1 the default template. (Data Defaults) ■ "Calculate" the standard curve and any samples using the appropriate data file. ■ Return to the Main Menu. ■ Change the default template to template #2 (samples only). ■ "Calculate" just the samples this time, using the appropriate data file(s). A message appears confirming that there are no standards on template #2: No standards! Use the previous standard curve? Yes. If the user defines template #2 as the first template, the following warning message will appear: No standards! Use a template w/standards to calculate a curve! ■ CERES 900C User's Guide Press Enter to continue. 3-47 The user must then return to the Main Menu and change the default template to template #1 (the template with standards defined on it). 3-48 KC Jr. Main Menu D Data Defaults The Data defaults option allows you to specify how and where data is saved and reported within KC Jr. Figure 3-21: Selecting Data Defaults from the Main Menu ■ Select Data Defaults from the Main Menu by pressing F7 or D. You may also use the curser up/down arrow keys to highlight Data Defaults and press Enter. If another KC Jr. menu is on the screen, press Esc to clear the screen, then select Data Defaults. The changes you make here will be saved in the KIN.SYS file on your default program disk and used by KC Jr. as a reference whenever the program is executed. CERES 900C User's Guide 3-49 ■ To change a Data defaults parameter, enter the initial letter (i.e. D for Data drive\dir) or highlight the parameter using the up/down cursor keys and press Enter, or point and click with the mouse. ■ Enter the appropriate data at the pop-up window prompt, or select from the choices provided. D - Data Drive/Dir ■ When D is selected from the Data Defaults menu, a window prompts you to enter the destination drive and directory for absorbance data files: Enter drive & directory for the O.D. data Data will be stored in an \EIA subdirectory (created by KC Jr.) of whatever drive and directory you enter here. ■ For example, if you want to store your data on drive B, then enter B:, and the platefiles will be stored in an EIA subdirectory on drive B. Or, if your KC Jr. program is in the KCJ subdirectory on your hard disk drive C:, then you should enter C:\KCJ for this parameter. KC Jr. will create the EIA subdirectory for you if the subdirectory doesn't already exist. ;;; Caution: Unless absolutely necessary, do not store data on the working program disk. Use an alternative drive and data disk. ■ After you enter the destination drive and directory and press Enter, KC Jr. writes the new parameter to the Default file in the program directory and returns the Data defaults menu to the screen. 3-50 KC Jr. Main Menu L - Lotus Drive/Dir ■ When L is selected from the Data Defaults menu, a window prompts you to enter the destination drive and directory for data files transformed into a .PRN format for use by Lotus or VisiCalc applications. Enter drive & directory for the Lotus data ■ For example, if your KC Jr. program is in a subdirectory on your hard disk named KCJ, then you should enter C:\KCJ\LOTUS for this parameter. If the subdirectory doesn't already exist, KC Jr. will create it for you. ■ After you press Enter, KC Jr. writes the new parameter to the Default file in the program directory and returns the Data defaults menu to the screen. P - Default Protocol ■ When P is selected from the Data Defaults Menu, a window listing the protocol file names stored in the default directory appears (Figure 3-42). ■ Highlight the protocol name that will become the default and press Enter or click the mouse button. Or, enter the first letter of the protocol file name to highlight the next file beginning with that letter. Press Enter to select the file. ■ If you don't want to make a change, continue to the next data defaults parameter, or press Esc to return to the Main Menu. When you change the default protocol, KinetiCalc saves the new parameters to the Default file in the program directory and displays the new protocol name on the left corner of the status line. CERES 900C User's Guide 3-51 T - Default Template ■ When T is selected from the Data Defaults menu, a window listing the template file names stored in the default program directory appears (Figure 3-43). ■ Highlight the template name that will become the default and press Enter or click the mouse button. Or, enter the first letter of the template file name to highlight the next file beginning with that letter. Press Enter to select the file. ■ If you don't wish to make a change, continue to the next parameter, or press Esc to return to the Main Menu. When a template is selected, KC Jr saves the new parameter in the program's Default file and displays the template name on the status line. R - Report Device ■ When R is selected from the Data Defaults menu, the program prompts for an output device for calculations and reports: P - Printer only D - Display only B - Printer & Display ■ If you press Esc while this window is displayed, the program returns to the Main Menu and no change is made. If this parameter is set to D, reports are displayed on your screen. If set to P, reports are printed. If set to B, the reports are both displayed and printed, with a pause after each report so you can examine the results. 3-52 KC Jr. Main Menu ■ After you enter P, D, or B, KC Jr. writes the new parameter to the Default file in the program directory and returns to the Data Defaults menu. C - Printer Control Code ■ When C is selected from the Data Defalts menu, a window prompts for a special escape code sequence to send to the printer before any reports are printed. Entering an escape code sequence is optional. The escape code sequence is used by some printers to specify print instructions (condensed print, bold, letterquality print etc.). Refer to the operator's manual that accompanied your printer for more information on printer control codes Enter the printer control escape sequence (27)(15) ■ The program displays the current sequence and waits for input. If you don't want to change the displayed sequence, press Enter and the program will return to the Data Defaults menu. ■ To change the control code, enter the decimal value for each code in parentheses. You can enter a maximum of 15 control code characters, including the parentheses. For example, if your printer requires an ESC G sequence to select double-strike printing, you would enter that sequence here as (27)(71). Or if you want to set condensed print, you might enter the escape sequence here as (27)(15). ■ After entering a new code and pressing Enter, KC Jr. writes the new parameters to the Default file in the program directory and returns to the data defaults menu. CERES 900C User's Guide 3-53 When you have finished editing Data default parameters, press Esc to return to the Main Menu. 3-54 KC Jr. Main Menu BFormula (create/edit) ■ Option F5 Formula (create/edit) from the Operations menu allows you to define plate and transformation formula(s) for the transformation of optical densities or kinetic slopes. ;;; Important! Transformations take place before other reports; formulas take place after other reports. The formula, which can be named and stored on disk for later recall and modification, may consist of one equation or Multiple Individually Calculated Equations (MICE). Once created, the formula may be used in data analysis by entering its name in a Protocol's Apply Formula parameter. Keep in mind that the formula or transformations must conform to the following rules in order for the calculations to be performed properly: Form. Trans. Chars. Description ✔ ✔ + Represents addition ✔ ✔ - Represents subtraction ✔ ✔ * Represents multiplication ✔ ✔ / Represents division @ Represents averaging () Represents an inclusive unit ✔ ✔ ✔ for calculation ✔ & Separates MICE (used only in the plate formula section) ✔ CERES 900C User's Guide S Represents set definition 3-55 Form. Trans. Chars. Description ✔ L Represents limit definition ✔ ' Separates elements for averaging ✔ ✔ N Represents all sets [] Represents wells to be referenced by absolute location(s), no change when replicated (used only in the transformation section) ✔ ✔ !! Represents a comment in the transformation section, and a label that will be printed in the plate formula report. Table 3-1: Allowed letters and symbols in a formula Aside from well designators A through H, the only other letters and symbols that may be used to describe arithmetic or mathematical relationships in formulas and transformations are listed in Table 3-1. The plate formula may be composed of up to 140 text characters, including single equations or MICE, separated by ampersands. ■ The transformation formula(s) can each contain up to 15 characters. ■ The only arithmetic operations allowed are addition, subtraction, multiplication, and division. 3-56 KC Jr. Main Menu ;;; All formulas are calculated from left to right, and not by mathematical order of precedence (i.e. multiplication and division functions first, followed by addition and subtraction functions). If a different order of precedence is required, use parentheses. Selecting/Creating a Formula File ■ Select Formula (create/edit) from KC Jr.'s Main Menu. If formulas have previously been created and stored in program's default directory, a window appears listing the file names. Figure 3-22: Saved formula files available for selection ■ Select a formula from the list, or press Esc to clear the window and enter a name for a new formula at the prompt. ■ If no formulas have been created, a prompt windows asks for the name of the new formula to be created. Enter a name up to 8 characters, and press Enter. An extension is unnecessary. KC Jr. automatically adds a .FRM extension to the CERES 900C User's Guide 3-57 filename. If you press the Enter key in response to the file name prompt, KC Jr. will return to the Main Menu. Figure 3-23: System prompts for a new formula name Entering a New Formula When a new file name is entered, the program displays an empty formula/transformation entry screen. ■ Use the down arrow key to position the cursor after Plate formula(s). Use the numeric keypad, the left and right cursor control keys, the delete key, the backspace key, and the appropriate symbols to enter your formula. You can enter up to 41 characters for a single formula or single MICE, or up to 140 characters comprising several individual equations (MICE). The maximum (MICE) length is 41 characters. ;;; Make sure you separate the MICE with ampersands (&) so the program will recognize and calculate them properly. Note that there is no space between characters in a formula line. 3-58 KC Jr. Main Menu ☛ Example: If the template contains one sample set in each row, you may wish to define a formula that: ■ subtracts the average of set 2 (wells B1 through B12) from the average of set 1 (wells A1 through A12) and ■ subtracts the average of set 4 (wells D1 through D12) from the average of set 3 (wells C1 through C12), etc. Using S to represent the set number, the formula to accomplish this would be entered as follows: @[email protected]&@[email protected]&@[email protected]&@[email protected] Alternatively, you may wish to have paired wells, such as negative and positive controls, and may wish to compensate each positive based on the associated negative. The formula might look like this: A1-B1&A2-B2&A3-B3&A4-B4& ... or, more succinctly: S1-S2&S3-S4&S5-S6&S7-S8 A more complex example might be demonstrated by the use of both paired variates and blanking. Suppose you have paired positive and negative values in rows A and B, C and D, E and F, and G and H. You want to blank the plate based on the average of the corrected pairs A1-B1 and A12-B12. The formula might look like this: @(A1-B1,A12-B12)- S1-S2&@(A1-B1,A12-B12)-S3-S4& @(A1-B1,A12-B12)-S5-S6& ... CERES 900C User's Guide 3-59 If you precede individual equations, up to four MICE, with L, the program will calculate the equation(s) and set the results as the values for the limits parameter when this formula is entered for a Protocol's Apply Formula parameter. ■ When you have finished entering a formula, press Enter, and the program will display the formula editing screen: Figure 3-24: The Formula Editing Screen with example formula Modify an Existing Formula When you retrieve a formula file from the default data disk, KC Jr. displays the formula- editing screen, with the cursor located at the beginning of the formula. ■ Use the cursor arrow keys to position the cursor after Plate formula(s), and the cursor directional keys to move through the formula and make any necessary changes. Any characters you type will be inserted to the left of the cursor position. Use the Del and Backspace keys to delete any unwanted characters. 3-60 KC Jr. Main Menu ■ Pressing Esc while you are entering or editing a formula returns you to the Main Menu without saving the formula. Pressing F10 saves the formula on your default program disk and returns you to the Main Menu. You must save the new or modified formula before it can be used for calculations. Using the Formula in Calculations ■ To use this formula in calculations, select Protocol (create/edit) from the Main Menu. ■ At the Protocol description screen, move to Apply Formula parameter field and press the spacebar. ■ Select the formula name from the window listing saved formulas. Create/Modify a Data Transformation When Formula (create/edit) is selected from the Main Menu, the program displays a window listing the file names of formulas stored in the default directory. On the status line, the program prompts you to select the formula file to be edited. ■ Select one of the formula listed, or press Esc to bring up a prompt for the name of a new formula. When a new file name is entered, the program displays an empty formula entry screen and waits for you to begin a new formula. The blank Formula Input Screen features an 8 X 12 matrix of 40-character spaces for defining transformation formulas. CERES 900C User's Guide 3-61 Figure 3-25: Delta OD formula applied to readings ☛ Example: A Delta O.D. formula of A1-A2 (Figure 3-25) is applied to a set of microplate well readings. The calculations are carried out and the results shown in Figure 3-26. Figure 3-26: Results of well transformations 3-62 KC Jr. Main Menu ;;; Because only 5 character spaces are available in each well location , the formulas shown in column 9 of Figure 3-26 display "A9-A1", rather than "A9-A10" which requires an additional character space to show the zero. As each individual well is referenced, however, lines one and two at the top of the screen will display the current well referenced and the complete formula for that well. Options for simplifying transformation entry include: Ctrl -D ■ eDit an existing transformation formula: Move the cursor to the well to be edited. Press Ctrl - D. The cursor moves to line #2 on the screen, allowing editing (insertion, deletion, etc.) of the formula. Ctrl - R ■ Replicate transformation formula to other well(s): Press Ctrl - R. Move the cursor to the well to be replicated. Notice the Prompt on the status line. ■ Press Enter ■ Move the cursor to the first well in which the formula is to be replicated. Press Enter. ■ Move the cursor to the last well in which the formula is to be replicated. Press Enter. The formula will then be replicated to the selected wells. ;;; Any well intended as a formula constant (i.e. any or all well(s) - A2 should be in brackets so that the formula reads: "A1 - [A2]". If no bracket is used, the formula will reference the wells in relative increments (i.e. A1 - A2, B1 - B2, rather than A1 - A2, B1 - A2). CERES 900C User's Guide 3-63 Ctrl - E ■ Ctrl- E will erase a well, a column of wells, a row of wells, or an entire plate of transformation formulas. Alternately, pressing the spacebar on an individual well will erase that well only. ■ Press Esc while you are entering or editing a formula, to return to the Main Menu without saving the formula / transformation(s). Press F-10 to save the formula(s) and return you to the Main Menu. ;;; You must save the new or modified formula before it can be used for calculations. Using Transformations in Calculations To use these transformations in the calculations: ■ Select Protocol (create/edit) from the Main Menu. ■ At the Protocol definition screen, move to the parameter field Transform Raw Data by_____, and press the spacebar. ■ Select the transformation formula from the list of saved formulas in the pop-up window. ■ Move to the next parameter field, Report Transformed Data. Use the spacebar to toggle the setting to Yes. 3-64 KC Jr. Main Menu Chapter 4: Running KC Jr. This chapter explains how to calculate results, read a plate and save data, retrieve data from a disk file, the Kinetics Assay, the 96-well graphics screen, endpoint, statistical and scanning assays and incubating a plate using KC Jr. software for the CERES 900C. ?Calculating Results ;;; Warning! Software. The user of the CERES 900C utilizing KCJr Software must follow the specific manufacturer’s assay package insert when modifying KCJr software parameters and establishing result calculation methods. ;;; Warning! Data Reduction Protocol. KCJr is a basic data reduction software program that does not flag out of range results nor prevent these results from being sent to any connected information system. It is the user’s responsibility to verify that all quality control materials are in their expected ranges prior to utilizing the assay’s results. Use the checklist that follows to ensure you've completed some preliminary tasks before you begin your calculations: ■ Define the protocol for the current plate run, and make sure its name appears on the KC Jr. status line. See Chapter 3, Protocol (create/edit) for instructions on creating a protocol file, and Data Defaults for information on installing the protocol as the default. ■ Create a Reader Control File from within the Protocol. Information on creating a Reader Control File as part of a Protocol is included in Protocol (create/edit) in Chapter 3. ■ Design the template for the current plate run, and make sure its name appears on the status line. For instructions on creating a template see Chapter 3, Template (create/edit), and Data Defaults for information on installing the template as the default. ■ Set Data Defaults parameters. Otherwise, the data may not be saved where you want it. Refer to Chapter 3, Data Defaults for details. 4-2 Using KC Jr. Starting the Calculations Process Selecting F2 Calculate results from KC Jr.'s Main Menu begins the calculation process that retrieves data either from disk or directly from the reader, performs all necessary calculations, and reports the results based on the predefined protocol currently in use. Figure 4-1: Selecting Calculate from the Main Menu Since KC Jr.'s data files can contain multiple plates, the program uses the Assay Type parameter in the active Protocol file to determine the data configuration. If the Assay Type is either end point or statistical, KC Jr. assumes that any multipleplate data file contains independent plates of data and performs all calculations separately on each plate of the file. But if the Assay Type is kinetic, then the program assumes the multiple plates in the file are consecutive readings of the same plate and should be treated as kinetic data. CERES 900C User's Guide 4-3 ■ When Calculate results is selected from the Main Menu, the program prompts for the source of the data: R - get plate(s) from Reader D - get plate(s) from Disk ■ Select R (Reader) to retrieve, display, report, and save the optical density values from one or more plates read by the CERES 900C to a computer through the RS232 port. ■ Select D (Disk) to retrieve platefiles of data previously read and stored or entered manually and saved in the EIA subdirectory on the default data disk. ■ To select the source of the data, highlight the option and press Enter or click the mouse button. Or, key in either R or D to choose the corresponding option. ■ Press Esc to return to the Main Menu without retrieving any data. Reading a Plate and Saving Data When you indicate you wish to read a plate, (Select R) KC Jr.: 4-4 ■ Asks you to confirm Protocol and Template defaults ■ Requests the Number of Readings ■ Prompts for Plate ID (option selected in Protocol) Using KC Jr. Figure 4-3: KC Jr.'s plate read prompts ■ Prompts for a file name to be used for saving data ■ Enter a name that will uniquely identify this file on the disk. The name can be alphanumeric as long as it does not exceed 8 characters, with a three-character extension. After you enter the file name, the program checks the EIA sub-directory on the default data disk for a file with that name. (The EIA subdirectory is created when you specify where you want to save the absorbance data files, during Data Defaults setup). If a file of plates already has the name, KC Jr. gives you the option of overwriting, or appending data to, or entering a new name for the specified file. CERES 900C User's Guide 4-5 Figure 4-4: Selecting Plate File Options: Overwrite, Append or New KC Jr. uses the setting from the Number of Readings parameter in the active Protocol file to determine the number of readings to make. ■ If the Assay Type is either End Point or Statistical, KC Jr. uses the Number of Readings setting as the number of plates to read. When a correct file name has been entered, the program displays the message: Waiting for plate #1 Plate reading and data transmission begins automatically. ■ If you press Esc to interrupt the reading process, the program will close the file, close the RS232 port, and begin the specified calculations and reports based on the data already read. When the appropriate key is pressed, the CERES 900C will read the plate and send the data to the computer. As each plate is received by the computer, it will be saved in the EIA subdirectory on the default data drive 4-6 Using KC Jr. specified during Data Defaults setup. The transmission speed depends on the reading options selected in the active protocol, as well as the baud rate selected on the CERES 900C and specified during KC Jr. Setup. ■ If you want to read another plate, press the appropriate key on the reader, and it will perform another plate read. ■ If you don't want to read more plates for this file, press Esc, and the program begins calculations. Retrieving Data from a Disk File ■ Selecting D - "get plate(s) from disk," allows you to retrieve and make calculations based on any plate or plates of optical density data stored on disk. If you've previously retrieved one or more platefiles, either from disk or from the reader when D is selected from the submenu, KC Jr. gives you the option of making more calculations on the same plates: Figure 4-5: KC Jr. prompts for data source CERES 900C User's Guide 4-7 ■ If you select N, KC Jr. reads a microplate and calculates using that data; if you select P, the program bases its calculations on previously retrieved plates. The program displays a window listing the platefile names in the EIA subdirectory, then prompts you to accept the existing Protocol, Template and Formula defaults or enter new ones. When the defaults have been changed and/or accepted, a second window will prompt you for the platefile: Figure 4-6: KC Jr. prompts for the platefile ■ Highlight the name of the file you wish to retrieve, and press Enter, or enter the first letter of the platefile name to move to the next file beginning with that letter. ■ For information about any stored platefile, highlight the name of that file, and press Shift-F1. Another window will appear, showing platefile name, the number of plates it contains, the date the file was created, and the descriptive text for the first plate in the file. To proceed, press Esc to remove the help window from the screen. 4-8 Using KC Jr. ■ You can also use DOS wildcard characters to retrieve multiple platefiles. For example, to retrieve all platefiles with an extension of .TST, press Esc until the file window disappears from the screen. Then enter *.TST in response to the prompt as follows: Figure 4-7: Retrieving multiple plate files using DOS wildcard ■ The program will then retrieve the selected file names under the prompt. If the selected file contains more than one plate, you will be asked which plates you want to retrieve: A - get All plates in file S - get Specific plates ■ If you want to retrieve only some of the plates in the file, select S. The program then prompts for the plates to use: Which of the X plates to use? where X is the total number of plates in the file. CERES 900C User's Guide 4-9 ■ Enter the range of plate numbers or the individual plate numbers, separated by commas, that you want to retrieve; then press Enter. Example: If a platefile contains 9 plates of data and you want to retrieve plate numbers 4 through 7, and 9, enter 4-7, 9 in response to the prompt. Or if you want to retrieve plate numbers 3, 6, and 8, enter 3, 6, 8 when prompted. ■ Enter E for all even-numbered or O for all odd- numbered plates in the file. ■ If you enter 0 (zero) or press Enter in response to this prompt, no data will be retrieved and you will be asked if you wish to retrieve more files or to proceed with calculations. After you have entered the plate numbers you wish to retrieve, a cumulative total of plates to be read is displayed in the upper right corner of the screen. ■ If you want to retrieve all the plates in the selected file, select A and press Enter or click the mouse button. ■ After you specify which plates to retrieve, the program will give you the opportunity to retrieve more files: R - Retrieve more files/plates P - Proceed with calculations ■ You can choose to retrieve up to 100 plates by selecting the desired file, then specifying which plates from the file you wish to analyze. If you select P to proceed, the program reads the plates into memory, and begins the calculations. 4-10 Using KC Jr. Kinetics Assay The 96-Well Graphics Screen Once you choose to proceed with the calculations for a kinetic assay, KC Jr. plots the absorbance data points for all 96 wells on a graphics screen as each reading is made. Figure 4-8: 96-Well Graphics Screen As the plates are read, a counter in the upper right corner of the screen displays the number of the current reading. Each well is represented as a box, which contains one mark for each reading. The optical density scale range at the top of the screen is based on input data, and changes as the readings are made. When the run is completed, KC Jr. calculates the slopes for each well, then displays a scaled time axis at the bottom of the screen with one tic mark for each reading. A pointer (+) in the center of the screen can be used to select a well to zoom in on, or to specify the time interval to include in the curve calculations. CERES 900C User's Guide 4-11 Depending on how you want to treat the data, you have several options while the 96well graphics screen is displayed: ■ Print the 96-well graphics screen ■ Save the 96-well graphics screen to disk file. ■ Adjust the y-axis scale ■ Select the time interval for the calculation ■ Display the mean slope and R-squared value table ■ Zoom in on a specific well ■ Edit the standard concentrations Printing the screen ■ To print the 96-well graphics screen, press Ctrl-P. Saving the screen to a disk file ■ To save the 96-well graphics screen to disk file, press Ctrl-D. The file "KINETIC.PCX" will be created. 4-12 Using KC Jr. Adjusting y-scale axis ■ To adjust the maximum O.D. for the y-scale: Type in the new maximum O.D. value and press Enter. Selecting time interval ■ To select time interval: By selecting the readings to use for the calculation of rate (slope), this option allows you to select only the linear portion of the kinetic reaction for the ELISA. Selecting the interval is a 4-step procedure: 1) Move the pointer to the time axis at the bottom of the screen and press Enter. A small box or arrow appears at the right end of the time axis. 2) Use the cursor control keys or the mouse pointer to move the small box or arrow to the tic mark that represents the last reading you wish included in the calculations and press Enter. The screen will be redrawn to include only the data points within the selected time interval. 3) Move the pointer to the left end of the time axis and press Enter. 4) Again, use the cursor control keys to move the box or arrow to the the tic mark that represents the first reading you wish to include in the calculations, and press Enter. The 96-well graphics screen is then redrawn so that each well shows only those data points within the time interval just selected. ■ To restore a reading to the calculation: move the pointer to the appropriate end of the time axis and press Enter. Then use the cursor control keys to move the box or arrow to the tic mark you wish to restore and again press Enter. CERES 900C User's Guide 4-13 You can continue to change the time interval until it includes the readings you want. Each time you change the interval, the screen will be redrawn and the slopes will be recalculated. Mean slope and R2 value To display slope/R-squared table: From the 96-well graphics screen, press CtrlPgDn to display the mean slopes and R-squared values for each well. Figure 4-9: Sample slope / R2 Table The top value in each well in the matrix is the mean slope, and the bottom is the R squared value for all the readings. From this screen, you can press Ctrl-PgUp to return to the 96-well graphics screen or PgDn to continue to the edit graph screen. 4-14 Using KC Jr. Figure 4-10: Edit Graph Screen Figure 4-11: Sample Zoomed Well Screen Zoom-a-well ■ To zoom in on a specific well: move the screen pointer to the desired well and press Enter or click the mouse button. CERES 900C User's Guide 4-15 When the zoomed well Screen appears, it will show the selected data points, the slope line, the standard deviation, and the R-squared value for the selected well (Figure 411) . The scaled time-axis becomes the X-axis for the zoomed well, and the optical density scale is the Y-axis along the left edge of the screen. ■ To print the zoomed well, press Ctrl-P. To save the zoomed well to a disk file "ZOOM_ _ _.PCX", press Ctrl-D. The "_ _ _" will be the location of the particular zoomed well. Press Esc to return to the 96-well graphics screen. Edit Graph Screen ;;; If the assay type is kinetic, the Edit Graph screen plots slope (rate) values on the Yaxis against the standard concentration values on the X-axis. If the assay type is end point, the screen shows optical density values on the Y-axis and standard concentration values on the X-axis. The edit graph screen deletion of standard values from the equation calculation. An asterisk represents each standard slope/optical density value on the retrieved plate. If only one asterisk appears for a standard concentration with replicates, the optical densities for the replicates of that concentration are too close to plot separately. On this editing screen, you can now mark for deletion any values you want excluded from the curve calculation. ■ Use the right, left, up, and down cursor keys to move the blinking cursor to the value you wish to exclude, and press D to delete that value. A lower-case o will replace the * asterisk, indicating a deleted value. If you change your mind and want to restore a standard concentration value, move the cursor to the deleted value and press R. This action will replace the o with the original * asterisk and the value will be included in the calculations. 4-16 Using KC Jr. ■ If you decide you want to abort the calculation procedure, press Esc to return to KC Jr.'s Main Menu. From the edit graph screen, you have the following options: 1) Press Shift-PrtSc to get a printed copy 2) Change the data type or curve-fit method for the plate run. 3) Return to the 96-well graphics screen (kinetic analysis only) 4) Display standards results report 5) Calculate and display the fitted curve and equation Change the data/axis type or curve-fit method ■ Press F9 to change the curve fit method. Press F10 to change the data/axis type. Return to 96-well screen ■ Press PgUp from the edit graph screen to review the 96-well graphics screen (kinetic analysis only - see Figure 4-8). For details, refer to the section entitled 96-Well Graphics Screen. Display standards results To display the standards results report, press Ctrl-PgDn. CERES 900C User's Guide 4-17 Figure 4-12: Sample Standards Results Report Figure 4-12 shows the standards results for a kinetic analysis. If the assay type were end point, the second column of data entitled Mean Slope would be replaced by a column entitled Average Absorbance. Estimated concentrations whose values are outside the range of the standard concentrations are shown in parentheses. Figure 4-13: Fitted Curve (Kinetic) 4-18 Using KC Jr. If the report contains more data then can be displayed on the screen at one time, the program pauses at the end of the first screen of data and waits for you to press PgDn to continue. When all the data has been displayed, press PgDn to display the fitted curve (Figure 4-13), or cubic curve (Figure 4-14); or Ctrl-PgUp to return to the previous page of the report or to the Edit Graph screen (as shown in Figure 4-8). Fitted Curve Screen ■ To calculate the curve model and display the fitted curve, press PgDn from either the Edit Graph screen (Figure 4-10) or the standards results table (Figure 4-13). Figure 4-14: Fitted Cubic Curve (End Point) The fitted curve screen plots standard concentration values on the X-axis against rate (slope) on the Y-axis if the Assay Type is kinetic (Figure 4-14), or against optical density values on the Y-axis if the Assay Type is end point. The line represents the least-squares best fit for the data, given the calculating parameters set on the active protocol and in Data Defaults Setup. Each point is a plot of an individual value. The CERES 900C User's Guide 4-19 range of values on the X- and Y-axes is automatically scaled from the data on the plate and the plate map. From the fitted curve, you have the following options: 1) Return to the Edit Graph screen 2) Print the Fitted Curve screen. 3) Evaluate the sample unknowns Return to the Edit Graph Screen ■ From the Fitted Curve screen (Figure 4-13 or Figure 4-14), press PgUp to return to the Edit Graph screen (Figure 4-10). You can then delete or restore standard concentration values from the equation and change the curve fit or data type, if desired. ;;; If you press PgUp to return to the edit graph screen, any previously deleted points will be restored to the screen and therefore to any further calculations unless you delete them again. Printing the fitted curve ■ To print the fitted curve, press Ctrl-P to send a copy of the fitted curve to the installed printer. Save as disk file ■ To save the fitted curve to a disk file, press Ctrl-D. The file, STDCRV.PCX will be created. 4-20 Using KC Jr. Sample Evaluation ■ From the fitted curve, press PgDn to evaluate the sample unknowns. KC Jr. feeds the sample optical density values through the formula calculated from the curve and produces the calculated concentrations of unknowns for linear (Figure 4-15) and cubic (Figure 4-16). Figure 4-15: Calculated concentrations of unknowns (Linear) CERES 900C User's Guide 4-21 Figure 4-16: Calculated concentrations of unknowns (Cubic) KC Jr. then produces the reports called for in the active protocol. If the Report device parameter in Data Defaults is set to printer only, the results will be sent to the printer. Otherwise, they will be displayed on the screen, one screenful at a time. ■ When the screen display pauses, press PgDn to continue to the next screen or report. KC Jr. prints the series of reports specified by the active protocol for each reading. For example, if the active protocol specifies 7 readings, 7 sets of absorbance data reports would be printed or displayed. The following is a list of available report screens that may be printed: 4-22 ■ Kinetic Slopes/^R2 ■ Kinetic/Endpoint Standard Report ■ Kinetic Sample Evaluation Results ■ End Point Sample Evaluation Using KC Jr. ■ Protocol Definition ■ Template Description ■ Absorbance Data ■ Report of Transformed Data ■ Formula Translation ■ Report of Means ■ Report of Limits When all the reports have been displayed or printed, the program returns to the Main Menu. CERES 900C User's Guide 4-23 End Point Assay If the currently active protocol specifies an End Point Assay, the program displays the absorbance values on the screen as each plate is read. If the plate was logged from the reader, all values have already been corrected by the blanking pattern selected on the reader. Blank wells have either not been read or have no value. Figure 4-17: End Point Assay Screen A minus sign (-) precedes any value that was measured or entered as a negative optical density; otherwise all values are positive. Any well showing asterisks (*****) has a value outside the range of readability. As each plate is received from the reader, it is also saved in the EIA subdirectory on the default data drive as specified in Data Defaults Setup. After the last plate is read or the reading is interrupted, the program proceeds to the Edit Graph screen (Figure 4-10). From there the program follows the same sequence as it would with a kinetic assay. Except for the fitted curve and the sample evaluation results, the screens and reports are the same as for a kinetic assay. 4-24 Using KC Jr. With an end point assay, the sample results report shows average absorbance instead of average slope. Additionally, the fitted curve for an end point assay plots optical density, (not rate) against standard concentration values. Statistical Assay If the currently active protocol specifies a statistical assay type, the plates are read and the absorbance data values displayed. If the data is being retrieved from the reader, it is saved to disk. KC Jr. bypasses all the slope calculations and immediately prints the reports specified in the active protocol, then returns to the Main Menu. CERES 900C User's Guide 4-25 Scanning Assay If the currently active protocol specifies a scanning assay type, the reader performs a user-defined number of multiple reads of each microplate well, based on dividing each well into a pre-selected number of discrete scan areas. To perform a scanning assay: ■ Set the operating Protocol's Assay Type parameter to Scanning ■ In the Reader Control File, set the Read Method parameter to Scanning ■ In the Reader Control File enter the appropriate number of scans to the corresponding well width in the Number of Scans and Well Diameter parameter fields, using the table below. Number of Scans Well Width 1-17 4.0 - 4.4 1-19 4.5 - 4.9 1-22 5.5 - 5.9 1-28 6.0 - 6.4 1-30 7.0 - 7.4 Table 4-1: Valid scans for specific well diameters When the microplate is read, the absorbance data points are plotted on the 8 x 12 kinetic matrix graph. ■ To zoom-a-well, position the pointer on the well in the matrix graph, and press Enter. ■ To print the graph, press Ctrl-P while the matrix graph or any of the zoomed well graphs are displayed. 4-26 Using KC Jr. ■ To save the graph to a disk file, press Ctrl-D while the matrix graph or any of the zoomed well graphs are displayed. ;;; Curve fit line, and associated statistics reports are not available. Figure 4-18 A Twelve- Scan Read CERES 900C User's Guide 4-27 EIncubate Plate Selecting F8 / Incubate plate from KC Jr.'s Main Menu allows you to set an incubation time interval for the microplate. When you press F8, a prompt appears requesting a time interval (Figure 4-18). Figure 4-18: Incubate plate time prompt ■ Enter the time interval (hh:mm:ss) and press Enter. The microplate carrier is retracted and incubation begins. If no time is entered, the incubation interval is set for an indefinite time (user determined). ■ Press Enter at the time prompt to initiate an indefinite incubation period. The Incubate Plate line on KC Jr.'s Main Menu, changes to Home Plate. ■ Press F8 again to retrieve the microplate and halt the incubation process. A window will appear confirming that incubation was interrupted, and post the incubation time. 4-28 Using KC Jr. Chapter 5: KCJr's Utilities This chapter discusses the system utilities available with KC Jr. software on the CERES 900C. These utilities include DOS file operations, Lotus and other Transformations, and exiting through a DOS shell. gSystem Utilities Selecting F6 from KC Jr.'s Main menu allows you to perform a number of "housekeeping" tasks without leaving the KC Jr. environment. These tasks include copying, deleting and renaming data files, protocols, templates and formulas; transforming data into a format readable by Lotus 1-2-3 or VisiCalc, and receiving information on the version of KC Jr. you are running. Systems Utilities also includes the Calibration and Optics Test options. We'll discuss using the Calibration Plate with KC Jr.'s Calibration Verification program and the Optics Test in Chapter 8, Performance Verification. Figure 5-1: System Utilities option on Main Menu D - DOS File Operations Selecting D - DOS File Operations pops up a menu of options that allows you to display a directory of files in any user-specified subdirectory on the default data disk, and to copy, erase, and rename microplate data files stored in the EIA subdirectory. ■ Move the cursor to highlight the desired function and press Enter or click the mouse button. Or, type the letters to select the corresponding option. ■ Press Esc while this window is displayed to abandon the DOS operations and return to the Main Menu. ■ If you wish to perform DOS file operations in a directory other than EIA, press Esc to return to the Main Menu, and select F9-Exit to DOS shell. When you are ready to return to KC Jr., type Exit at the DOS prompt. 5-2 KCJr Utilities Figure 5-2: File Utilities Menu C - Copy a File (or Plate) The first item on the File Utilities Menu allows you to copy a file or plate within the EIA subdirectory, and give that copy a different name. When you select C Copy a data file (or plate), the program lists the files in the EIA subdirectory. (If there are more than 40 files, press Esc to view additional files.) ■ Highlight the file you wish to copy and press Enter or click the mouse button. You can also type the first letter of the file name to highlight the next file beginning with that letter, and press Enter. ■ If you want information about a stored file, highlight the name of the file and press Shift-F1. Another window will pop up, showing you the file name, the number of plates it contains, the date and time of the first plate, and any other descriptive information for the first plate in the file. Press Esc to remove the help window from the screen. When you select a file to copy, KC Jr. prompts for a new name for that file. CERES 900C User's Guide 5-3 Figure 5-3: Copy a data file (or plate) Copy FILENAME.EXT To: where FILENAME.EXT is the file you chose to copy. ■ ;;; Type the new name for the file copy, and press Enter. If you enter the name of a file that already exists, KC Jr. will allow you to cancel the copy command before the original file is overwritten. If you press Enter without entering a file name in response to this prompt, the program returns to the File Utilities Menu without copying the file. You will be asked which plates from that file you want to copy: A - copy All plates in FILENAME.EXT S - copy Specific plates where FILENAME.EXT is the file you chose to copy. ■ 5-4 To copy all the plates in the file, enter A. Otherwise, enter S. KCJr Utilities ■ You will be asked which plates you want to copy. Key in the range of plate numbers or the individual plate numbers (separated by commas) that you want to copy, and press Enter. ;;; For example, if a platefile contains 9 plates of data and you want to copy plate numbers 4 through 7 and 9, enter 4-7, 9 in response to the prompt. Or if you want to copy plate numbers 3, 6, and 8, enter 3,6, 8 in response to the prompt: Which of the 9 plates to copy? 3, 6, 8 After copying the plates to the new file, KC Jr. will return you to the File Utilities Menu. ■ If you change your mind, or don't want to copy any plates from the specified file, press Enter in response to the prompt. The program returns the File Utilities Menu to the screen. D - Directory of Files Option D from the file utilities window allows you to display a list of the files stored on a given drive and directory, and checks for the amount of free space on the disk. ■ When D is selected, you are prompted for the drive/ directory name you wish to display: Directory of: CERES 900C User's Guide 5-5 ■ Enter the directory or subdirectory name and press Enter. You can also use wildcard characters (? and *) to display selected files. For example: If you wish to see all the platefiles with the extension of .TST stored in the EIA subdirectory, the prompt and your response would appear as follows: Directory of: EIA\*.TST The names of the files with the extension of .TST currently stored in the specified subdirectory on the disk will be displayed. 5-6 ■ Press Shift-Prt Sc keys to obtain a printout of the displayed directory. ■ Press any key to return to the File Utilities Menu. KCJr Utilities E - Erase a Data File (or Plate) Option E from the file utilities menu allows you to erase a plate within a file or to erase a complete file within the EIA subdirectory. When you select E from the file utilities window, a pop-up windows lists the files in the EIA subdirectory. ■ Highlight the file you wish to erase and press Enter or click the mouse button. You can also enter the first letter of the file name to highlight the next file beginning with that letter, and press Enter. CERES 900C User's Guide 5-7 ■ If you want information on a stored platefile, highlight the name of the file and press Shift-F1. A pop-up window will list the file name, the number of plates it contains, the date and time of the first plate, and any descriptive information for the first plate in the file. Press Esc to remove the help window from the screen. ■ KC Jr. will prompt for information on the plates to be erased: A - erase All plates from FILENAME.EXT S - erase Specific plates where FILENAME.EXT is the name of the file you chose to erase. ;;; If there is only one plate in the file, the file is erased immediately upon selection. ■ If you want to erase all the plates in the file, enter A. Otherwise, enter S to indicate specific plates to erase. 5-8 KCJr Utilities ■ If you plan to erase specific plates (S) you will be asked to enter the range of plate numbers or the individual plate numbers, separated by commas, that are to be deleted. Example: If a platefile contains 9 plates of data and you want to erase plate numbers 4 through 7 and 9, enter 4-7, 9 in response to the prompt. Or if you want to erase plate numbers 3, 6, and 8, enter 3,6, 8 in response to the prompt: Which of the 9 files to erase? 3,6,8 Only the plates you specify will be erased from the file. After performing this function, KC Jr. returns to the File Utilities Menu. ■ If you decide not to erase any plates from the file, press Enter in response to the prompt. The program returns the File Utilities Menu. N - reName a Data File Option N from the File Utilities Menu allows you to change the name of a plate or file in the EIA subdirectory. When you select N from the file utilities window, the program lists the files in the EIA subdirectory. CERES 900C User's Guide 5-9 Figure 5-4: Renaming a data file ■ Highlight the name of the file you wish to rename, and press Enter, or click the mouse button. You can also enter the first letter of the file name to highlight the next file beginning with that letter, and press Enter. ■ For information on any stored platefile, highlight the name of the file and press Shift- F1. After you select a file, you will be prompted for the file's new name. Rename to: ■ Key in the new file name (up to 8 characters), and press Enter. The program will rename the file and return you to the File Utilities Menu. ;;; If you specify the name of a file that already exists, or if you press Enter without entering a file name in response to this prompt, KC Jr. immediately returns to the File Utilities Menu without renaming the file. 5-10 KCJr Utilities P - erase a Protocol Option P from the File Utilities Menu allows you to erase one or more protocol files within the KC Jr. directory. When P is selected, a window listing protocol files appears. ■ Highlight the protocol file you wish to erase and press Enter or click the mouse button. You can also enter the first letter of the file name to highlight the next file beginning with that letter, and press Enter. T - erase a Template Option T from the File Utilities Menu allows you to erase one or more template files in the KC Jr. directory. When T is selected, a list of the template files appears. CERES 900C User's Guide 5-11 ■ Highlight the template file you wish to erase and press Enter or click the mouse button. You can also enter the first letter of the file name to highlight the next file beginning with that letter, and press Enter. Y - copY a Template Option Y from the file utilities window allows you to copy template files in the KC Jr. directory. When Y is selected, a list of the template files appears. 5-12 KCJr Utilities ■ Highlight the template file you wish to copy and press Enter, or click the mouse button. ■ You will be prompted to enter a new name for the copied file. Key in the new name and press Enter. After the file is copied, you will be returned to the File Utilities Menu. F - erase a Formula file Option F from the file utilities window allows you to erase one or more formula files in the KC Jr. directory. When F is selected, a list of the formula files appears. ■ Highlight the formula file you wish to erase and press Enter, or click the mouse button. You can also enter the first letter of the file name to highlight the next file beginning with that letter, then press Enter. CERES 900C User's Guide 5-13 L- Lotus & Other Transforms Selecting option L from the System Utilities Menu allows you to transform microplate file data into a format readable by software applications such as Lotus 1-2-3 or VisiCalc, or create a scattergram. The original file data remains on disk in its logged form, and a new file or display is created. When Lotus & other transforms is selected, a pop-up menu appears. Figure 5-4: Selecting Lotus Transformation ■ Highlight the desired option, and press Enter or click the mouse button. You can also key in either L, V, or S to select the corresponding option. Lotus transformation Option L arranges the plate data in a 8 x 12 matrix and saves the matrix in an ASCII text file that is readable by Lotus. Each well on the plate will be 9 characters wide to conform to the Lotus default cell width. The new file will have the same 8-character file name as the logged data but is given a .PRN extension. 5-14 KCJr Utilities VisiCalc transformation Option V creates the 8 x 12 matrix in a new disk file that contains the standard DIF (Data Interchange Format) tuple and vector markers recognized by all programs that work with DIF files. Selecting the File When either L or V is selected, the screen will display a window containing the microplate files in the EIA subdirectory. ■ Move the highlighted cursor to the name of the file that contains the plate(s) you wish to transform and press Enter or click the mouse button. You can also enter the first letter of the plate filename to highlight the next file beginning with that letter. Press Enter to select the file. As files are selected for transformation, their names are copied on the screen below the window. ■ If you want information about any stored file, highlight the name of that file, and press Shift- F1. Press Esc to remove the help window from the screen. You can also use DOS wildcard characters to retrieve multiple platefiles in a set. For example: ■ To retrieve all platefiles with an extension of .TST, press Esc until the file window disappears from the screen. Then enter *.TST in response to the prompt as follows: Enter file(s) to retrieve: *.TST <Enter> KC Jr. will copy the selected file names to the screen beneath the file window. A cumulative total of the number of plates to be transformed is displayed in the upper right corner of the screen. CERES 900C User's Guide 5-15 ■ If any selected file contains more than one plate, you will be asked which plates from that file you want to transform: A - get All plates in the file S - get Specific plates ■ If you want to transform all the plates in the file, enter A. Otherwise, enter S to indicate specific plates. ■ If you select S, you will be asked to indicate which of the plates you want to analyze. Enter the range of plate numbers or the individual plate numbers, separated by commas, that you want to transform, then press Enter. For example, if a platefile contains 9 plates of data and you want to transform plate numbers 4 through 7 and 9, enter 4-7, 9 in response to the prompt. If you want to transform plate numbers 3, 6, and 8, enter 3, 6, 8 in response to the prompt: Which of the 9 plates to use 3, 6, 8 ■ If you change your mind, or don't want to transform any plates from the specified file, press Enter. Once you've specified the plates to retrieve, the program displays another window: R - Retrieve more files/plates P - Proceed with calculations 5-16 KCJr Utilities ■ The program will retrieve more platefile names if you enter R in response to this prompt. When you've finished selecting platefiles, press P(roceed) to continue. The program then transforms the data and saves it to disk in the Lotus drive/dir subdirectory you specified during Data Defaults setup. If you try to save a transformed file with a name that already exists in the spreadsheet directory, the message center will display the following message: FILENAME exists. Do you want to overwrite it? (y/n) If you answer Yes, the transformed data will overwrite the existing file. If you answer No, the program will return to the Main Menu without creating any transformed data or overwriting the existing file. Importing the data to Lotus To import a PRN file of transformed data into a Lotus spreadsheet: ■ Place the cell cursor in the upper left corner of the 8-row by 12-column area where you wish the data to be. Then choose the /FIN (File Import Numbers) option from the menu. ■ When prompted for the file name to copy, enter the name of the plate transformed by KC Jr. ( See your Lotus manual for details on importing an ASCII file into the spreadsheet.) You may also wish to explore the /FCAE and /FCSE options to add or subtract values in one plate from those in another. CERES 900C User's Guide 5-17 5-18 KCJr Utilities S - Scattergram Option S from the transformation submenu allows you to display/print a scattergram of plate data. KC Jr. scales the Y-axis to the range of optical densities for the selected plate. When S - Scattergram is selected, if a platefile is still held in memory after having been retrieved by some other option, the program will give you the opportunity to use the plates already retrieved: ■ N - get New plate(s) P - use Previous plates If you want to work with previously retrieved files, answer P to this prompt, and the program will display a scattergram of the current file(s). Figure 5-5: Scattergram CERES 900C User's Guide 5-19 ■ If there are no files currently in memory, or if you answer N, the screen will display a window containing the files in the EIA subdirectory and prompt you for the name of the file to be displayed as a scattergram. ■ Highlight the name of the file that contains the plate(s) you wish to retrieve. Press Enter or click the mouse button. You can also key in the first letter of the platefile name to highlight the next file beginning with that letter, and press Enter. As files are selected, their names are copied on the screen below the window. ■ If you wish information about any stored platefile, highlight the name of the file, and press Shift- F1. Press Esc to remove the help window from the screen. You can also use DOS wildcard characters to retrieve multiple platefiles in a set. For example, to retrieve all platefiles with an extension of .TST, press Esc until the file window disappears from the screen. Then enter *.TST in response to the prompt. If a selected file contains more than one plate, you will be asked which plate(s) from that file you want to retrieve: A - get All plates in the file S - get Specific plates ■ If you want all the plates in the file, enter A. Otherwise, enter S to indicate specific plates. ■ If you select S, you will be asked which of the plates you want to analyze. Key in the range of plate numbers or the individual plate numbers, separated by commas, that you want to use, and press Enter. 5-20 KCJr Utilities ■ If you change your mind, or don't want to retrieve any plates from the specified file, press Enter in response to the above prompt. Once you've specified the plates to retrieve, the program again prompts: R - Retrieve more files/plates P - Proceed with calculations ■ The program will retrieve more plate filenames if you enter R in response to this prompt. When you've finished selecting platefiles, press P to continue. After a brief pause while the program is working, the scattergram will appear on the screen if the Report device parameter in Data Defaults is set to either Display only or Printer & Display. Printing the Scattergram If the Data Defaults parameter is set to either Printer only or Printer & Display the scattergram will be printed. The program automatically scales the range of optical density values on the Yaxis to the low-high values for the plate being drawn. The X-axis represents columns 1 through 12 on the plate. For each column, row letters mark the relative measure of the optical density of a well compared to the Y-axis scale. Up to five (5) well row labels can appear at one scale location for a given column. If more than five wells in a column cluster at a scaling level, then an asterisk (*) will be displayed. Over-range and blank (nonread) wells appear at the top of the scale. CERES 900C User's Guide 5-21 If the platefile contains more than one plate, or you specified additional files, press any key to display the scattergram of the next plate. You can also press Esc to return to the Main Menu. O - Optics Test Selecting O causes an optics test to be run on the microplate reader. The Optics Test is discussed in detail in Chapter 8, Performance Verification. C- Calibration Plate Test Selecting C allows you to run a Calibration plate with the CERES 900C to verify the instrument's calibration. Running the calibration plate with KC Jr.'s Calibration Plate Test is discussed in Chapter 8, Performance Verification. P - Program Information Selecting P from the System Utilities submenu brings up a window that displays the version of KC Jr. being run. You'll need to have this information if you call Bio-Tek's Technical Service representative with a question. 5-22 KCJr Utilities jExit to DOS Shell Selecting F9 from KC Jr.'s Main Menu allows you to temporarily return to the DOS prompt to issue a command such as formatting a disk or erasing a file you no longer need. ■ When you are finished with DOS and want to return to KC Jr., type exit at the DOS prompt, and the program will return you to KC Jr.'s Main Menu screen. CERES 900C User's Guide 5-23 5-24 KCJr Utilities Chapter 6: Troubleshooting This section is designed to provide solutions to common problems that may occur when operating the CERES 900C EIA Workstation. Troubleshooting If a problem occurs that cannot be solved using this guide or the DOS Manual, contact Bio-Tek's Technical Assistance Center. Problem Cause Solution No display Instrument not plugged in Check power cable connection Faulty outlet Move instrument to working outlet Monitor not plugged in, or turned on, or VGA cable not attached Check power , VGA cable connections. monitor on/off switch Faulty display connection Replace monitor cable Fuse blown Inspect the fuses Replace if necessary Faulty bulb connection Remove and reinstall bulb Bulb burned out Remove and replace bulb Reader bulb does not light Reported optical density values are near zero, but solution in well is obviously colored Incorrect interference filter installed or filter table not correct Check the wavelength on the side of filter and replace if necessary. Check filter table and correct if necessary. Repeated optical density readings of same well do not agree to within +0.005 absorbance units Defective lamp Visually inspect microplate and replace if necessary. Replace bulb Using U-bottom PVC microplates with single wavelength setting Use dual wavelength setting Contaminated sample solids in well Replace samples Optical Density above 2.500 abs Repeatability specified to 2.500 abs Pipetting error, or nonuniform washing procedure Check against former method of reading microplate Optical density readings of replicate wells do not agree to within +1% plus 0.010 OD Non uniform binding Reported phenomena that peripheral wells differ in binding capacity (edge effect) or temperature from other wells on the microplate Optical density readings vary considerably from earlier readings of the same microplate 6-2 Scratches or fingerprints on well bottom or foreign particles in well Use new plate or dual wavelength feature Carrier out of alignment Run CAL Plate Test Direct sunlight or extreme ambient light reaching photo detectors Relocate reader Not enough stop solution in wells Add more stop solution after reviewing assay kit instructions Defective lamp Replace bulb Troubleshooting Read error occurs while a microplate is being read Printer not activated when printout is requested Printer head moves but does not print Paper jams in printer Ceres 900C User's Guide Bulb burned out Replace bulb No filter wheel Install filter wheel No filter in position #1 or in test wavelength position Re-install filters and check filter table setup Instrument or printer not plugged in properly Turn on printer first, check when connection, then turn on the Ceres 900C CERES 900C System print defaults not properly set Review the print setup section in this manual No power at wall outlet Plug another device, or a lamp into the outlet to ensure the outlet has power Printer not online Depress ONLINE button on the printer Printer cable damaged or improperly installed Refer to printer manual for installation instructions. Replace cable if necessary Ribbon jammed or worn out Replace ribbon Paper jammed Try advancing paper by hand, using thumb wheel on printer. If paper is jammed, refer to printer manual for instructions on paper feeder release. Refeed paper Improper paper feed Remove jammed paper and reinsert paper according to printer manual instructions 6-3 CMOS Utility Software for the Ceres 900C The CERES 900C contains a battery which powers the CMOS RAM (Random Access Memory) on the CERES 900C’s PC motherboard. This CMOS RAM holds the system’s configuration information. When the battery loses power, the configuration, or system setup, data is lost from the system’s memory (CMOS RAM). This data must be restored before the computer can communicate with its hard disk, and thus operate the CERES 900C. ;;; The normal battery life is two years. Battery power can be lost if the CERES 900C is left powered off for an extended period of time. The CERES 900C distribution disks contain a utility program, CMOS.EXE, which provides a means to read, restore, compare and view the contents of the CERES 900C CMOS memory. You’ll find the CMOS Utility disk packaged with the CERES 900C program disks. The utility is first run at the factory to read the system’s CMOS RAM data when the system is initially assembled. This instrument-specific information is then save in a file called cmosdata.sav, and stored on the CMOS Utility disk that is shipped with the instrument. Should the CERES 900C system lose its configuration information, this floppy disk can be used to restore the configuration to original factory settings, after the cause of the problem has been determined or a new battery inserted. After restoring the data, the user would then set the system’s date and time, using the DOS date, and time commands. Remember: The CMOS data file, cmosdata.sav is specific to your CERES 900C instrument. This file cannot be used to restore CMOS information on other CERES 900C instruments. 6-4 Troubleshooting Running the Utility Program If the CERES 900C has lost its CMOS data, it is probable that the system will not boot successfully, or be able to read a floppy disk. The CMOS disk provided with the CERES 900C can be used as a “boot” disk, that is, the system will start successfully if the disk is inserted into the disk drive before the instrument is powered on. Use the procedure that follows to view and/or restore the system’s CMOS configuration data: • Place the CERES 900C CMOS Utility disk in disk drive A: and power up the CERES 900C. • When the A: prompt appears on the display, type: CMOS followed by the appropriate “switch” which determines the action you wish to invoke. For example: To restore the contents of the CERES 900C’s cmosdata.sav file to system CMOS memory, type: CMOS /R Either all upper or upper and lower case letters may be used. The available switches are: /R Restores the CMOS memory (overwrites it) with the contents of the cmosdata.sav file in the current directory. /C Displays the contents of CMOS memory and compare CMOS memory contents to the backup file in the current directory. Mismatches are highlighted in reverse video. Mismatches that are problems will blink. The time, date and some status registers will not blink, as the system updates these. Ceres 900C User's Guide 6-5 /F Displays the file contents of the cmosdata.sav file in the current directory. /M Displays the CMOS memory contents on the CRT /S Saves the CMOS data to the file, cmosdata.sav in the current directory. • When you have restored the CMOS data to the CERES 900C, remove the floppy disk, and reboot the CERES 900C. Removing Junk Files from the CERES 900C When using software installed on the CERES 900C, it is possible to inadvertently create “junk” files on the instrument’s hard disk drive. These junk files are created when the user exits a software program improperly, or shuts off the system without first “quitting” the software program. Junk files can also be created when a user exits incorrectly from a DOS shell, i.e. forgets that a DOS shell is in operation, and restarts the software that is already running in the background. To avoid creating junk files: • Type EXIT to return to the reader’s software program from a DOS shell. (This reminder appears on the screen when the DOS shell function is chosen by the user.) • After using software on the reader, select QUIT to back out of the software until the DOS prompt is displayed. Then turn off power to the reader. Deleting Unnecessary Files If you suspect you are accumulating these unnecessary files on the CERES 900C: 6-6 Troubleshooting • Check all directories of the software program for 8-character file names that are alphanumeric and do not have an extension. Ensure these file names were not intentionally created by the user. • Delete these files using a simple DOS delete command. If you are unfamiliar with DOS commands, and need assistance with this procedure, please call Bio-Tek Instruments at 1-800-242-4685. Ceres 900C User's Guide 6-7 Chapter 7: Maintenance This chapter describes the Maintenance Procedures that can easily be performed by the user to keep the CERES 900C operating properly. Before you begin... Service procedures other than cleaning or replacing minor components such as fuses, bulbs and filters, should be left to qualified service personnel. If the instrument malfunctions, refer to Chapter 6, Troubleshooting, or Appendix A, Error Messages. If the problem cannot be resolved by following these procedures, contact Bio-Tek's Technical Assistance Center at 1-(800) 242-4685, or (802) 6554040, or your local Bio-Tek Representative. Calls made to our European office can be made to (32) 3829 0455. Safety Precautions For safety purposes (and to avoid damage to the instrument) the CERES 900C must be turned OFF, and its power cord disconnected during the maintenance procedures described in this chapter. If you begin maintenance after operation of the instrument, remember that the bulb and bulb holder are hot, and must be handled with caution. Allow at least 15 minutes before beginning maintenance to allow the instrument to cool off. During general operation, ensure that the main power cord is plugged into a power outlet that has a protective earth contact to connect the chassis of the CERES 900C to ground. Do not disable this feature by using extension cords or cheater plugs without earth ground, or a hazardous condition may result. Replacing the Light Bulb While the tungsten-halogen bulb supplied with the CERES 900C will provide many hours of use, eventually it will need to be replaced. In addition, a failed self-test may indicate the need for a new bulb, even if the bulb appears to be functioning. To check a suspect bulb, lower the hinged access cover on the rear of the reader. If the bulb needs to be replaced: ■ Turn the CERES 900C off, and allow the instrument to cool for 15 minutes. ■ Loosen the two wing nuts and open the hinged Optics Access Cover on the rear of the reader. ■ Slide the Optics Assembly out of the compartment until it is fully exposed. ■ Remove the Bulb and Socket Assembly from the support bracket and remove the bulb from the socket. While the bulb is out of the assembly, you might wish to clean the optics inside the instrument. Refer to the section, Cleaning the Optics, in this chapter. ;;; ■ Plug the new bulb into the socket and gently reattach it to the bracket. ■ Slide optics assembly back into the compartment. Caution: Use caution when working inside of the instrument when power is on, and when working around the Lamp Assembly and Optics. Avoid touching the lenses and bulb reflector. 7-2 Chapter 7 ;;; Warning: Use of bulbs other than those recommended by Bio-Tek Instruments may cause a fire hazard. (PN 3400508) • Gently close the Access Cover, tighten the screws, and reattach the power cord and printer cable. ■ Test the CERES 900C by following the instructions in the section, Testing the System. Replacing the Fuses The CERES 900C fuse bay is located on one end of the Power Supply Module at the rear of the instrument. ;;; Warning: Using fuse(s) other than those recommended can cause fire and/or shock. (US/ PN-46063 European/ PN-46056) To replace the fuses: ■ Power down the CERES 900C, and unplug the main power cord. ■ Using a small-blade screwdriver or similar tool, carefully pry open the access cover to the fuse compartment access cover. The fuse block assembly is in the back of this cover (Figure 7-1). Chapter 7: Maintenance ■ Carefully lift the old fuse(s) from the fuse slot. ■ Replace the fuse(s) with a new one. 7-3 U.S Single Fuse (Hot) European Dual Fuse (Both Hot & Neutral) Figure 7-1: Fuse Assembly Configuring the CERES 900C for Different Voltages If you purchased your CERES 900C within the continental United States, it has been factory-set for US Voltage (either 110 or 120 Volts). Should you decide to use the CERES 900C at a location with 220 or 240 line voltage, you will need to reconfigure the fusing and change the Incubation Voltage Switch setting (if the Incubation Option is present). Configured for US current, the fuse block will be mounted on the backside of the Access Cover with the jumper bar exposed, providing 1 slot for a single fuse (Figure 7-1, upper insert ). To reconfigure the fusing for European voltages: ■ 7-4 Turn off the power and disconnect the line cord. Chapter 7 ■ Remove the fuse block holder by prying it out of the housing. ■ Loosen the Phillips screw that attaches the Fuse Block to the Access Cover (2 turns). ■ Slide the Fuse Block away from the Cover. Turn the Fuse Block over so that the jumper bar lies flat against the cover and the 2 fuse slots face up (Figure 7-1, lower insert). ■ ;;; Slide the fuse block back onto the Phillips screw and pedestal, and tighten the screw. Caution: Overtightening the screw may cause stripping of the threads on the fuse holder. ■ Replace the cover to the fuse assembly, and plug in the CERES 900C. Changing the Line Voltage for Incubation If the CERES 900C is shipped from Bio-Tek with the Incubation option, a voltage selection switch is provided that allows the Incubator to accommodate either 110/120 or 220/240 line voltage. The voltage switch is clearly marked, and is located on the rear panel of the instrument. If the instrument is moved to a location with one of these voltages, the switch must be reset for the new voltage. Additionally, the fuses will require reconfiguring as explained in the section titled Configuring the CERES 900C for Different Voltages in this chapter. Cleaning the Optics The optical elements of the CERES 900C should be cleaned periodically to insure proper operation. The optics may be conveniently cleaned when changing filters or replacing a bulb. Chapter 7: Maintenance 7-5 To clean the optics: ■ ;;; Turn the CERES 900C OFF and unplug the main power cord. Warning: To avoid injury, always allow the unit to cool down at least 15 minutes prior to performing this procedure. ■ Loosen the 2 Wing nuts on the hinged Optics Access Cover and open the cover. ■ Slide the Optical Assembly out until fully exposed. ■ To remove the filter wheel, firmly grab the wheel hub (center) and pull away from the mounting hub. The wheel is held onto the motor shaft magnetically and will require a certain amount of force to separate them. Be careful not to bump the lamp assembly while removing the Filter Wheel. ■ Use photographic lens-cleaning fluid (isopropyl alcohol may be substituted) and lens-cleaning tissue to clean both sides of the filters. ;;; Caution: When the filter wheel is removed, the large end of the fiber optics bundle is visible. Do not attempt to clean this except to blow away dust. Any direct cleaning of the surface of the fiber optics could scratch the assembly and ruin it. ■ Blow away any dust on the large focusing lens on the lamp assembly with pressurized photographic air. Pressurized air, designed for cleaning optical equipment, can be purchased at a photographic supply store. 7-6 Chapter 7 ■ To clean the surface of the large lens, use photographic lens-cleaning fluid and lens-cleaning paper. If you need to clean the lamp side of the lens, remove the bulb from the spring-loaded bracket by sliding it up and out, and clean the lens with lens-cleaning fluid and lens-cleaning paper, then reinstall the bulb. ■ Slide the optics assembly back into its compartment. ■ Close the hinged cover; replace and tighten the 2 Wing nuts. Cleaning the Exterior Clean all CERES 900C surfaces with a soft towel or sponge moistened with a mild soap and water solution. Do not use solvents or abrasive cleaners. Storage and Shipping The unit may be safely stored or shipped in environments with the following limits: ■ Temperature -20° to +60° C Humidity up to 96% Altitude 7,620 meters maximum (26,000 feet) The CERES 900C should be protected from temperature extremes that can cause condensation within the instrument, and from corrosive fumes and vapors. ■ When the CERES 900C is shipped to Bio-Tek for service or repair, the unit must be shipped in the original packing; other forms of commercially available packing are not recommended and may void the warranty. If the original packing has been damaged or lost, contact Bio-Tek for replacement packing. Repack the CERES 900C for shipping as follows: Chapter 7: Maintenance 7-7 ⇒ Remove microplate from the carrier bed. ⇒ Remove the filter wheel, and pack it in its original padded container. ⇒ Replace the foam end caps above and beneath the instrument and place the reader in the original shipping carton. ■ Obtain a Return Authorization Number from Bio-Tek Service representatives by calling tollfree in the US 1-(800)242-4685, or (802)655-4044; telex: 94-0136 BIO TEK SHVT; or Service FAX: 802-655-3399. Outside the US contact your BIO-TEK Sales Representative. ■ Include in the packing box the following information: ⇒ What repairs or adjustments are required (i.e., calibration, cleaning, repair, etc.) ⇒ A tag specifying the return authorization number, the full model and serial number. ⇒ Name and telephone number of someone Bio-Tek can contact if additional information is necessary. 7-8 ■ Securely fasten the packing carton flaps with packing tape. ■ Insure the CERES 900C for its full value and return it to Bio-Tek for service. Chapter 7 Chapter 8: Performance Verification The CERES 900C has been tested at the factory to insure that its performance is within the specifications listed in this manual. In addition to the calibration plate and optical tests, this chapter details a "wet test" procedure that can be used to verify CERES 900C performance. Testing the System / Calibration _________________________________ It is considered good laboratory practice to periodically validate the calibration of the CERES 900C. KCJr. software provides tests of several system parameters to ensure the system is installed and functioning properly. Calibration validation should occur on a weekly basis by performing the tests in this section. The tests detailed in this section include: ■ Calibration Plate Test or ■ Linearity Test (wet test) ■ Optics Test Calibration Plate Test This test is used to determine the alignment, repeatability, accuracy and linearity of microplates, and the CERES 900C plate carrier, with the CERES 900C optics system, and is crucial for ensuring read accuracy. An alternative method used to determine accuracy, repeatability and linearity is the Linearity or Wet Test described later in this chapter. Requirements: ■ Bio-Tek Calibration Plate (PN 9000547) Rev. B or later. ■ Bio-Tek Calibration Plate (PN 9000543) Rev. B or later. ■ Bio-Tek Calibration Plate (PN 9000517) Rev. B or later (Alignment test invalid for CERES 900C) Running the Calibration Plate ■ From the KC Jr. Main Menu, select F6- System Utilities. ■ From the Systems Utilities submenu, select option C- Calibration plate test. An option screen will appear, providing a choice between D - Define a calibration plate, and R - Run a calibration plate. 8-2 Performance Verification ■ Select D - Define calibration plate. Defining the Calibration Plate ■ From the option submenu, select: 3 - Test filter Cal Plate (P/N 3110517) 4 - Test filter Cal Plate (P/N 9000543), or 6 - Test filter Cal Plate (P/N 90000547) CERES 900C User's Guide 8 -3 depending on the number of filters in the version of calibration plate that you are using. A screen similar to the one pictured below appears when 6 is selected. The CERES 900C is shipped with a calibration plate with filters in all six positions (number 6). ■ Using the Specifications sheet provided with the Calibration Plate, enter the calibration values, using the up/down arrow, and backspace keys. Press the kkey to save the values. You will be returned to KC Jr.'s Main Menu. Running the Calibration Plate 8-4 ■ From KCJr.'s Main Menu, select F6- System Utilities. ■ Select C- Calibration Plate. ■ From the Options submenu, select R - Run calibration plate test. Performance Verification ■ Enter the Calibration plate serial number when prompted. ■ From the displayed list of filters, select the filter to be tested, using the up/down arrow keys, and pressing the y key. CERES 900C User's Guide 8 -5 ;;; ■ An options submenu will prompt you to select a R- get plate(s) from Reader, or D - get plate(s) from disk. ■ Select R - get plate(s) from reader, unless you wish to use data from a previously run calibration plate, which has been saved to disk. If the desired filter is not displayed, refer to Chapter 3, Introducing KC Jr. for information on setting up the filter table to match filters installed on the CERES 900C filter wheel. ■ You will be prompted to enter a file name for the data. Enter the file name, and place the Calibration plate in the microplate carrier. ;;; If the file name duplicates another already saved to disk, you will be asked if you wish to overwrite the old file with the new data, or select a new filename. ■ 8-6 Press y to start the calibration test. Performance Verification ■ After the first calibration plate read, the carrier will exit the reading chamber, and a screen prompt will direct you to turn the plate 180° and again press y. ■ At the conclusion of the second read, a Calibration Test Report will be displayed and/or sent to the printer. The report will be displayed on two screens -- press any key to get the second half of the report. CERES 900C User's Guide 8 -7 8-8 Performance Verification Calibration Test Results The Calibration Report has four report parameters: ■ Alignment: This portion of the test measures the alignment of the microplate carrier with the optical path. A reading of > .015 represents and out-of-alignment condition. Wells A2, A11, H2, H11 are valid for the CERES 900C when using Cal plates that are part numbered 9000543 and 9000547. ■ Accuracy: Accuracy is a measure of the absorbance (optical density) of Calibration Plate wells C1, D4, E2, F5, G3 and H6 with known standard values contained in the Specification Sheet that accompanies each Calibration plate. ■ Repeatibility: This parameter is a measure of the instrument's ability to read the same well with minimum variation between two reads with the well in the same location. ■ Turnaround: The Calibration Plate's six filtered wells (C1, D4, E2 , F5, G3, and H6) are each read from two positions - once with the Calibration Plate in the normal position, and once with the plate turned 180°. This portion of the test meaures the diference between the optics at opposite sides of the read chamber. Test Failures If any of the test parameters report as "FAIL", contact your Bio-Tek representative, or call Bio-Tek's Technical Assistance center at 1-(800)-2424685. CERES 900C User's Guide 8 -9 The Optics Test The Optics Test is designed to compare the integrity of each of the 12 reading channels to a 13th reference channel. The test checks the light level and system gain, notifying the user if the measured valves don’t adhere to program limits. The Optics Test should be performed weekly to ensure consistent assay/plateread accuracy. The optics test is performed every time a plate is read (transparent to the user). If the valves don’t fit programmed limits, an error message will be displayed (“Run Optics Test”). When run, the optics test will then report specific errors. Running the Optics Test ■ From KC Jr.'s Main Menu, select F6- System Utilities. ■ From the Options submenu, select O- Optics Test, and press y. The carrier will be retracted and the Optics Test will begin. . 8-10 Performance Verification ■ At the conclusion of the test, the Optics Test Report will be displayed on the screen. To send the test results to a printer, it would be necessary to press Shift/ Print screen. The Optics Test Report The displayed results include a PASS/FAIL determination for each of the optics channels, the reference channel, and signal (gain) level. Should any of the test results show a FAIL, contact Bio-Tek's Technical Assistance Center, at 1-(800)-242-4685. Quality Control It is considered to be good laboratory practice to run laboratory samples according to instructions and specific recommendations included in the package insert for the test to be conducted. Failure to conduct Quality Control checks could result in erroneous test data. CERES 900C User's Guide 8 -11 Optical Verification Achieving Maximum Performance Experience with this and similar instruments indicates that inaccuracies introduced by the environment, or by laboratory technique are far greater than any inaccuracies inherent in the basic instrument. What follows is a list of recommendations for achieving maximum performance from the CERES 900C. • The microplate should be perfectly clean and free of dust. Use new microplates from sealed packages. Do not allow dust to settle on the surface of the solution; use microplate covers if necessary to protect the solution (when not reading the plate). • Although the CERES 900C works with all flat, U-, and V-bottom microplates, it performs best with optically clear, flat bottomed wells. • Non-uniformity in the optical density of the well bottoms can cause loss of accuracy, especially with U- and V-bottom polyvinyl microplates. Check for this by reading an empty microplate. Dual wavelength readings can eliminate this problem, or bring the variation in density readings to within acceptable limits for most measurements. • The best repeatability from channel to channel is obtained when the wavelength used corresponds to the peak absorbance for the particular solution being used. This is most true when the shape of the absorbance curve is rather narrow. • Inaccuracy in pipetting has a large effect on measurements if smaller volumes of liquid are used. For best results, use at least 100 microliters. With half-area wells, smaller volumes are acceptable. 8-12 Performance Verification • To achieve the highest possible accuracy at optical densities approaching 2.0 OD and higher, eliminate illumination of the shutter by sunlight or strong ambient light. If interference from ambient light is suspected, read a microplate of high-density solutions under the suspect conditions, and again with all the light to the shutter area blocked (with a black cloth, for example); then compare results. The blocked shutter results will appear as an increase in optical density readings. • The inclination of the meniscus can cause loss of accuracy in some solutions, especially with small volumes. Agitate the microplate before reading to help bring this problem within acceptable limits. Use Tween 20 if possible (or some other wetting agent) to normalize the meniscus. • Some solutions develop menisci over a period of several minutes. This effect varies with the brand of microplate. As the center of the meniscus drops and shortens the light path, the density readings change. The shape of the meniscus eventually stabilizes. • A 10-minute warm-up of the instrument is suggested prior to reading to achieve the best repeatability from microplate-to-microplate measurements. • Always use the reader with a filter in the light path, as continuous operation without a filter in place may damage the optics. • To prevent damage to the carrier mechanism, never move the carrier by hand. CERES 900C User's Guide 8 -13 Verification of Alignment ___________________________________________ Use a new microplate, free from dust and scratches which could affect the readings. Fill the microplate with the time-stable solution you want to measure, such as the solution suggested for the Linearity Test described below. Use a filter wavelength corresponding to the maximum absorbance of your solution. Use different solution volumes (not less than 100 microliters) and densities. For example, put 100 microliters in rows A through D, and 150 microliters in rows E through H. The factory repeatability and accuracy tests that follow are performed with Costar™ flat (#3590) and round bottom (#3797) microplates with Durkee™ yellow food coloring, using a 405 nm filter. Different solutions and microplates, may vary repeatability and accuracy. Repeatability 1. Read the microplate using a single wavelength, no blanking (or blank on air), and an 8 x 12 matrix format. 2. When the readings have printed out, read the microplate a second time. 3. Compare the readings. Each well in the first printout should be within 0.5% of the OD, and 0.005 counts of the same reading in the second printout. For example, A1 in the first printout should be within +.5% OD, +0.005 OD of A1 in the second printout. If A1 was initially 1.000 OD, then subsequent readings should be between .990 OD and 1.010 OD Accuracy 1. Turn the microplate described in the Repeatability section around and read it again. 8-14 Performance Verification 2. Compare the printout with the last printout from the repeatability test. Be sure to compare the readings from the same wells. Remember that well A1 is now in the H12 position. The readings should be within 1% of the OD., and 0.010 counts of each other. For example, if A1 was initially 1.000, then the "turn-around" test should show results between .980 OD and 1.020 OD. Linearity Test_____________________________________________________ The linearity of the reader can be tested by preparing a series of solutions of varying absorbances. The absorbance of a solution is a function of how much light is absorbed by that solution when it contains a colored compound; therefore, a solution that has twice as much color as another solution will have twice the optical density. (Any solution that gives a stable color will suffice. This includes substrates incubated with an enzyme preparation and then stopped with an acidic or basic solution.) Some enzyme/substrate combinations are given in Table 8-1. A more basic method is to use food coloring available at supermarkets. (Durkee™ food coloring is one such brand.) Equipment Required: • Distilled water • Pipet • Yellow food coloring (Example: Durkee™ yellow food coloring, .3 oz. bottle) • Flat-bottom microplates (Example: Costar™ #3590) • Tween™ 20 (polyoxyethylenesorbitan Monolaurate) • Beakers • Rack of 10 test tubes (10 ml minimum volume) CERES 900C User's Guide 8 -15 1. Create a 0.5% solution of Tween 20 and distilled water. As an example, add 1 ml of Tween 20 to 200 ml of distilled water. Shake well for several minutes. 2. Add 6 drops of yellow food coloring to the 200 ml of the 0.5% Tween solution. This should give an absorbance of 1.4 to 2.0 absorbance units when using 200 ml in the well. If you wish to attain an absorbance up to 3.0 OD, add 3 additional drops of food coloring (a total of 9 drops). Table 8-1 Typical Enzyme-Substrate Combinations and Stopping Solutions Enzyme Substrate Alkaline Phosphate o-nitrophenyl phosphate 3N sodium hydroxide beta-Galactosidase o-nitrophenyl -beta-D galactopyranoside 1M sodium carbonate Peroxidase ABTS 2, 2 Amino-di-(3 ethyl[benzthazolin sulfonate] citrate-phosphate buffer, pH 2.8 Peroxidase o-phenylenediamine 0.03N sulfuric acid 3. Stopping Solution Stir well to insure all the color is spread throughout the solution. This is entirely a trial-and-error process. If it does not give enough absorbance, add another drop of food coloring. The reader will read absorbance units up to 4.300 absorbance units; it is specified to be linear to 3.000 absorbance units. 4. Set up a rack containing 10 tubes, numbered consecutively. Table 8-2 Test Tube Dilutions Tube Number Volume of Original Solution (ml) Volume of 0.5% Tween Solution (ml) 8-16 1 20 2 18 3 16 4 14 5 12 6 10 7 8 8 6 9 4 10 2 0 2 4 6 8 10 12 14 16 18 Performance Verification Absorbance (Expected if original solution is 2.0 at 200 ul) 2.0 5. 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 Perform a percentage dilution, beginning with 100% of the original dense solution in the first tube, 90% of the original solution in the second tube; 80% in the third tube, all the way to 10% in the last tube. Dilute using amounts of the remaining 0.5% solution of distilled water and Tween 20, as shown in Table 8-2. 6. Remove a 200 ml aliquot from each tube, and dispense it into the appropriate microplate well. There should be enough solution to have several replicates, if desired. 7. Read the microplate at 405 nm to determine the absorbance, and then graph the results, plotting absorbance versus percentage. You should get a straight line passing through zero, if your pipetting is accurate. You may also use Kineti-Calc™ or KC-Jr.™ (CERES 900C software) to plot your curve. 8. The choice of dilutions is strictly arbitrary, as is the absorbance of the original solution. Use Table 8-2 as a model for calculating the expected absorbances of a series of dilutions, given a different absorbance of the original solution. CERES 900C User's Guide 8 -17 Appendix A Error Messages This appendix contains an alphabetical listing of error messages that may be encountered when using the CERES 900C EIA Workstation or KC Junior Software. Other Error Messages If you encounter an error message not listed in this section, check the DOS manual that accompanied the CERES 900C, or contact Bio-Tek's Technical Assistance Center. Problem Cause Solution Blank pattern changed, please edit blank map! User has attempted to save a new RCD Blank Map option without viewing/editing the new map before saving it View and/or edit the new Blank Map by hitting the spacebar Calibration Error. Read aborted. Run Optics test Read values outside proper parameters Filter defined is not present. Bulb is failing Constant blank value out of range: valid range is -4.00 4.00 An invalid value has been entered within the RCD "User Blank" option Choose a value from -4.00 to 4.00 Disk full! Unable to proceed Floppy or hard disk is too full to accept additional data Download or delete nonessential data ( “junk files”) Appendix A A-1 Problem Cause Solution Factor value out of range: 0.010 -> 1000 A factor value outside of acceptable parameters has been entered Enter a value no less than 0.010 and no greater than 1000 Incubation Set Point out of range: valid range is 20.0 50.0 An incubation set point value outside allowable parameters has been entered Enter a value between 20.00 C and 50.00 C Invalid Calibration Plate values entered A non-real number value (0 or a non-numeric value, for example) has been entered as a Calibration Plate standard value during a calibration test Enter the standard value provided by the specification sheet included with the calibration plate Invalid characters in filename G\*y! Use '_' or '_' for separators. Press enter to continue Invalid characters used as separators Use underscores or minus signs as separators Invalid drive specified in default path Drive/path default parameter within Setup System is incorrect Enter C: for hard drive, or A: for floppy drive Invalid Number of scans: Valid range is 1 to 35 An invalid value has been entered within an RCD Read Method parameter Enter a value => than 1 and =< 35 Invalid path specified or disk not ready An improper drive and/or directory entered within System Setup, or no floppy disk has been inserted into the disk bay of the CERES 900C Correct drive/directory value within System Setup Drive/Path default box, or insert a floppy disk in the disk bay of the CERES 900C Limit value out of range: -4.0 -> 4.0 A value outside the allowable parameters has been entered within the RCD screen Limit option Enter a value => -4.00 or =< 4.00 A-2 Error Codes Problem Cause Solution Monitor OD value out of range: valid range is; -4.0 ->4.0 A value outside the allowable parameters has been entered within the RCD screen Read Method option Enter a value => -4.00 or =< 4.00 NO MCB communications. File AS_MCB.BIN not present in the AS directory Copy AS_MCB.BIN file from the master disk top the AS directory on the hard drive Bad monitor controller board Call The Technical Assistance Center Number of readings out of range: valid range is 1->250 A invalid value has been entered within an RCD Kinetic Read method Enter a whole number between 1 and 250 Range value out of range: -4.0->4.0 An invalid number has been entered within an RCD Range Report parameter Enter a value >= -4.0 or <=4.0 The Bottom value must be < or = to the top value! An RCD Range Report bottom range value has been entered that is greater than the top value Enter a bottom number that is < or = to the top value The file selected cannot be opened, or no file selected. Either no file has been selected or a file other than a data file has been selected Select a valid data file The file selected is not a compatible file. Format not understood Either a non-data file, or no data file has been selected Select a valid data file. Convert non standard data files (spreadsheet format) to standard format. The range for hours is 0 to 336 An invalid value has been entered for the Total Read Time parameter of an RCD Kinetic or Monitored Kinetic read method Bad or nonexistent board Appendix A A non-standard data file has been selected Enter a time value between 0 and 336 A-3 Problem Cause Solution The range for hours is 0 to 12 An invalid value has been entered for the Read Interval parameter on an RCD Kinetic or Monitored Kinetic read method Enter a time value between 0 and 12 hours The range for minutes is 0 to 60 An invalid value has been entered for the RCD shaking parameter Enter a time value between 0 and 60 minutes The range for minutes is 0 to 59 An invalid value has been entered for the Delay in Read or Kinetic/Monitored Kinetic Read Interval parameters Enter a time value between 0 and 59 minutes The range for seconds is 0 to 59 An invalid seconds value has been entered for any time parameter Enter a time value between 0 and 50 seconds There are too many files present in one directory to handle Combined application and data files exceed 640 kilobyte DOS capacity Move some data files into a separate data file directory Unable to run batch file CURV_FIT.BAT File CURV_FIT.BAT missing from AS directory Copy CURV_FIT.BAT from CERES 900C master disk to AS directory Unable to exit to DOS Shell Insufficient floppy or hard disk space to access DOS shell function Move some data files from the floppy or hard disk to a separate location Warning! Incubator has not reached setpoint Plate read has been attempted before Incubator has had time to reach preset temperature Allow 10 minutes for the incubator to reach the setpoint temperature Well width out of range: valid range is 4.0 -> 8.0 mm An invalid value has been entered within an RCD Scanning read Well Width parameter Enter a well width value =>4.0 mm or =< 8.0 mm A-4 Error Codes Appendix B: Serial, Parallel Pinouts This Appendix illustrates the pinout configurations for the CERES 900C’s parallel (printer) and Serial (data communication) connectors.. Parallel Port (LPT1) The CERES 900C’s parallel port (LPT1) is 100 % PC-AT compatible, It provides the necessary control signals for use as a Centronics-type parallel interface, allowing the instrument to be connected to a variety of printers. The connection is done through a DB-25 connector, J7, located at the end of the board, and visible at the rear of the reader. The pinout is illustrated below. Signal Signal Flow Pin # Pin # Signal Flow Signal STB* 0 1 2 I/O P0 P1 I/O 3 4 I/O P2 P3 I/O 5 6 I/O P4 P5 I/O 7 8 I/O P6 P7 I/O 9 10 I ACK* BUSY I 11 12 I PE SLCT I 13 14 0 AFD* ERR* I 15 16 0 INIT* SLIN* 0 17 18 - GND GND - 19 20 - GND GND - 21 22 - GND GND - 23 GND - 25 24 - GND Serial Port Connections The CERES 900 provides two Serial Ports. One has a 9-pin D sub connection, and the other, a 25-pin D sub connection. The Serial ports may be used to send data to an external PC or serial device. The pinouts for the connectors are illustrated below. COM1 (9-pin Male D Sub connector) Signal Signal Flow Pin # Pin # Signal Flow Signal DCD I 1 2 I RX TX 0 3 4 0 DTR GND - 5 6 I DSR RTS 0 7 8 I CTS RI I 9 COM2 (25-pin Male D Sub connector) Signal Signal Flow Pin # Pin # Signal Flow Signal DCD I 8 3 I DSR RX I 2 7 0 RTS TX 0 7 6 I CTS DTR 0 4 5 I RI GND N.C. B-2 21 1,9,10,11,12,13,14,15,16,17,18,19,22,23,24,25 Serial, Parallel Connector Pinouts Appendix C: ShowScan™ Scanning Software This appendix explains how to use ShowScan™ Software for pattern recognition assays. About ShowScan™ Software ShowScan is a software package for demonstration of pattern scanning capabilities of the CERES 900C. It has not been cleared for any in-vitro diagnostic use. ShowScan allows the user to visually evaluate the reading of the percentage transmission patterns on 96-well microplates. It does not, however, support mathematical interpretation of the scanned patterns (such as formula determination of positive and negative calls), nor does it allow editing or entry of reagent files. ShowScan automatically reads 30 points across each microplate well and graphically displays the transmittance profile of each well in single or dual wavelength. Individual wells, rows, columns or the total microplate may be viewed simultaneously. Here is how to access and use ShowScan, the on-board scanning software for the CERES 900C. Starting ShowScan ShowScan software is preinstalled on the CERES 900C hard disk drive at the factory. To start ShowScan: • Key in at the DOS prompt: CD \SC ShowScan • Press the <Enter> key. If the optional Power Menu has been loaded onto your system, your DOS prompt will resemble the following: C:\Menu> • Access the Power Menu by keying in at the DOS prompt: MENU • Select the ShowScan item from the Power Menu list of options. Press <Enter> to start the program. ShowScan's Main Menu Before using ShowScan for the first time, select the SETUP option from the Main Menu to configure the software for the microplate reader that you are using. After the first configuration you should not have to perform this procedure again. • Use the left/right cursor keys scroll across the Main Menu bar. As you highlight a Main Menu item, a pop-down submenu simultaneously displays available options. Use the up/down cursor keys to select an option, and press <Enter>. C-2 ShowScan Scanning Software The SETUP Option Highlight SETUP on ShowScan's Main Menu bar to configure ShowScan software for the CERES 900C. From the pop-down options menu, select: • Reader Type. At the device submenu, use the cursor keys to highlight CERES 900Cand press <Enter>. • RS232. At the device submenu, use the cursor keys to select preferences for: • Baud Rate: 1200, 2400, 4800, 9600, 19200 • COM port: 1 or 2 • Data bits: 7 or 8 • Stop bits: 1 or 2 • Parity: ODD, EVEN, or NO • Filters. At the device submenu, enter the filter list for the reader. Upon completion of Setup, the actual locations are downloaded from the reader. Other Main Menu Options READ Highlight READ on the Main Menu bar. From the pop-down options menu, highlight, and press <Enter> to choose: • Read Plate. Select the filter at which you wish to read the plate. Place the microplate on the plate carrier, and press <Enter>. To return to the Main Menu bar, press <Esc>. Appendix C C-3 • Read Reference: Select the measurement and reference reading wavelengths, and proceed as for Read Plate. The program will calculate and display the difference between the two readings. The left part of the display shows the individual well readings (up to 8), and the right part of the display shows the calculated reference readings (up to 4). • Read Scanfile: Select the data file to be read. The data file can be a scanfile stored with the SHOWSCAN module, or a scanfile stored by the ScanMaster package. The scanfile must be located in the actual working directory of the ShowScan program, and must have an extension of .SCN. FILES Highlight FILES on the Main Menu bar. From the pop-down options menu, highlight, and press <Enter> to choose: • Save Scandata: Select the measurement wavelength to be stored from all available in memory. A maximum of 8 scans (entire microplate assays) can be stored on the diskette. Enter the filename (8 characters, without extension) for the raw scanfile.The file will be stored in the ShowScan working directory. DISPLAY Highlight DISPLAY on the Main Menu bar. From the pop-down options menu, highlight, and press <Enter> to choose: • C-4 Display Well: Enter the well number (A1 to H12) to display. ShowScan Scanning Software All available scan patterns of the selected well will be shown, including calculated reference patterns. • Display Row: Enter the row number (A to H) to be displayed, and select the measurement available in memory. • Display Column: Enter the column number (1 to 12) to be displayed, and select the measurement available in memory. • Display Total MTP: Select the measurement to be shown from memory. The screen will show all 96 scan patterns. Each well can be individually highlighted by moving the cursor right or left, up or down. • To ZOOM-A-WELL, highlight the selected well, and press <Enter>. • Press <Esc > to return to the total 96-scan view. Press the <F1> key to reveal the following keys: • <Shift><p> Print Screen • <Esc> Return to menu • <F2> Select another scan from memory • <F3> Select measurement from memory and show color-coded interpretation of left and right AVERAGED transmission values. Appendix C C-5 • <F4> Select measurement from memory and show color-coded interpretation of transmission values. • <F5> Toggle color-coding for <F3> and <F4> options. RESET Up to 8 raw measurement wavelengths and 4 calculated (dual) reference wavelength readings can be retained in memory. Use these options to delete either one type or all data: • Reset Measurement. Clear raw wavelength measurement data from memory, retaining reference readings. • Reset Reference. Clear calculated reference readings from memory, retaining raw measurements. • Reset All. Clear all calculated reference and raw measurement readings from memory. PRINT • Select Print and press <Enter> to print the graphic scan patterns shown on the screen. EXIT • Select Exit and press <Enter> to return to DOS or the Power Menu's opening screen. C-6 ShowScan Scanning Software Appendix D: Specifications This chapter provides the specifications for the CERES 900C EIA Reader. All specifications are subject to change without notice. Specifications Size The CERES 900C measures 25.5" wide x 8.00" high x 15.5" deep. Weight The instrument weighs 35 pounds without monitor (will vary depending on options); the monitor weighs a maximum of 30 pounds. Speed The CERES 900C automatically reads 96-well microplates using a single wave length in 5 seconds, dual wavelengths in 10 seconds, and prints an 8 x 12 array of optical density values in approximately 20 seconds, depending on the printer. Measurement Range 0.000 to 4.300. Absorbance Units over the range of wavelengths specified. Negative optical densities will occur when the well being read absorbs less light than an accompanying blank well. While negative readings as low as -4.300 are possible, minimum possible optical density (OD) is 0.000. Wavelength Range and Selection CERES 900C- 380 to 900 nanometers (nm) using narrow band interference filters. CERES UV900C- Filters from 340 to 900 nm are available. Filter Wheel Configuration The CERES 900C has a 6-position filter wheel. The user selects the filter(s) desired for a particular assay from those resident within the filter wheel using the CERES 900C software. Four standard filters are supplied with the instrument in the following positions: Position Filter 1 405 2 450* 3 none 4 490 5 630 6 none *340 nm with the UV option Additional filters are available from Bio-Tek Instruments, Inc. For more information, contact Bio-Tek Customer service at (800) 451-5172 or (802) 6554040. Light Source User-replaceable Tungsten-halogen bulb with an average life of 500 hours or 50000 reads. Replace with Bio-Tek part number 3400508. D-2 Appendix D Display 15" VGA color CRT (optional) Linearity +/- 1% from 0 to 2.500 OD at 405 nm, 400-750 nm +/- 1% from 0 to 2.000 OD at 405 nm, 340-399 nm Reading Accuracy and Repeatability- Factory Acceptance Criteria +/- 1% +/- 0.010 OD from 0-2.500 OD, 400-750 nm +/- 1% +/- 0.010 OD from 0-2.000 OD, 340-399 nm Reading Repeatability Successive reading of any well will produce readings that are repeatable within +/- .5% +/- 0.005 OD from 0-2.500 OD, 400-750 nm +/- 1% +/- 0.005 OD from 0-2.000 OD, 340-399 nm assuming measurements are made at a single wavelength and blanked on air. Resolution 0.001 OD Microplates Accommodated Uses 96-well (8x12) microplates; flat, U-and V-bottom; polystyrene and polyvinyl, and removable and strip wells. (Polyvinyl microplates require an adapter, and strip wells require frames.) Photometry Method Single or dual wavelength Appendix D: Specifications D-3 Photo detectors 12 measuring photo diodes, 1 Reference photo diode Printout External printer, dot matrix or thermal type Power Requirements 150 VA at 100, 120, 220 or 240 V AC 50/60 Hz Fuses 2.5A SloBlo for 100-120 VAC operation / 2.5A SloBlo for 220-240VAC operation D-4 Appendix D Appendix E: Accessories This chapter lists CERES 900C accessories and their part numbers, and provides ordering information. Accessories The following accessories are available for the CERES 900C. For more information, contact your Bio-Tek representative or call (800) 451-5172. Item Part Number Calibration Plate 9000547 Fuses 2.5A Slo-Blo (U.S. application) 46063 2.5A Slo-Blo (European application) 46056 (2) are required Tungsten Halogen Bulb 3400508 Barcode Wand 97113 Mouse 48210 External Keyboard 48204 Dust cover 9102004 Filters Many wavelengths are available. Contact Bio-Tek. VGA 120V 48258 VGA Universal Power 48315 E-2 Appendix E Appendix F: Reader Control File This Appendix descibes the parameters and their options used in a Reader Control File for the CERES 900C microplate reader. CERES 900C Reader Control File Editing a Reader Control File When the Reader Control file name is opened, the definition screen is displayed, waiting for your input. Reader Control File Name This is the file name you specified to create or edit. Note that KC Jr. has added a .FIL extension to the file name. Reading Method You can choose from five reading options: EndPoint, Monitored EndPoint, Kinetic, Monitored Kinetic, and Scanning. To change the setting, move the cursor to highlight this parameter, then press the spacebar to cycle through the five options. ❑ EndPoint If you select EndPoint, the reader carries the microplate into the reading chamber, reads the 96 optical density values, makes the specified calculations and reports, and returns the microplate to its full-out position. ❑ Monitored EndPoint If you select Monitored EndPoint, the reader monitors one or more specified wells until the average optical density of the monitored wells reaches a certain value. You must select the Well locations to monitor,( i.e., A1 and H10 ), and the Value to monitor for. If the Wavelength parameter is set to single, the reader reads the microplate every 5 seconds. ❑ Kinetic If you select Kinetic to do kinetic reading of the microplate wells, you must also specify the time Interval between reads and the total number of Reads. You can set the Interval parameter to 5 seconds or greater if the Wavelength parameter is set to single, or to 10 seconds or greater if the Wavelength is set to dual. The Reads parameter can be set to a value between 1-250. ❑ Monitored Kinetic During a Monitored Kinetic read, the CERES 900C monitors from 1 through 96 preselected wells, until a blanked, unfactored average of those wells reaches a specified optical density threshold (from -4.300 to 4.300). After the threshold is reached, the kinetic portion of the read is performed. ❑ Scanning If you select Scanning, you must also specify the number of scans and the well diameter. Valid numbers are 4.0 to 7.5 mm. Based on this input, the reader will perform multiple reads of each microplate well, using a preselected number of discrete scan areas ( i.e., a 12-scan read ). F-2 Reader Control File Figure F-1 A Twelve-Scan Read Wavelength The options are Single, Dual and Multi wavelengths. To change the setting, move the cursor to highlight this parameter, then press the spacebar to toggle between the two options. ❐ If you select Single, the reader measures the optical density of each well with a single filter. Set the W1 parameter to the wavelength value for that reading. ❐ If you select Dual, the reader reads each well twice, each time with a different filter. Set the W1 and W2 parameters to the appropriate wavelength values. The microplate is not removed from the reading chamber between the two readings. The reported optical density is the difference between the two readings. Dual wavelength readings significantly reduce optical interference caused by scratched or fingerprinted microplates (scratches or fingerprints reduce the amount of light on both wavelengths). Since the substrate is sensitive to only one wavelength, the difference between the two readings is attributed to the substrate. Appendix F F-3 ❐ If you select Multi, the reader measures the optical density of each well with multiple single filters. Selection can be all the wavelengths installed in your reader. Shaking Available shaking options are determined by the selected read method. The options are 1st Read, Every Read, and No. In each case, the user determines the rate of shake speed, and the duration of the shake. The duration is limited by the read method selected. The speed options are Very Low, Low, Medium, High and Variable. The shaking options are: ❐ EndPoint and Scanning Options: 1st Read/No. The user specifies a shake duration from 1 second to 1 hour, and one of the shake speed options. ❐ Kinetic Options: 1st Read/Every Read/No. Shake times between 1 second and 1 hour may be selected. If Every Read is selected, Continuous shake time may be specified. ❐ Monitored EndPoint Options: 1st Read/Every Read/ No, until a predetermined threshold is reached. ❐ Monitored Kinetic Options: 1st Read/Every Read/ No, until a predetermined threshold is reached. The shake duration must be specified for the 'monitor' as well as the 'kinetic' portion of the read. Continuous shake may be selected via the space bar. F-4 Reader Control File Blanking Pattern The options are: Full, Half, Quarter, Row, Column, Constant, Rep. Down 1st, Rep. Down 2nd, Rep Across 1st, Rep Across 2nd, and Blank on Air. ❐ If you select Full, Half, Quarter, Row or Column, you can move the cursor to the Edit Blank Map prompt, and press the spacebar. The program then displays the corresponding plate map so you can select the blanking wells for each sector. For example, if the Blanking Pattern parameter is set to Full, the screen would appear as in (Figure F-2). Figure F-2 Blank Map ❐ Use the cursor keys to move the highlighted cursor to the appropriate well(s). ✔ As wells are selected by hitting the “B” key, their positions are highlighted. Appendix F F-5 ✔ If you deselect a well, the highlighting is removed. The values from the selected blanking wells in each sector will be subtracted from all wells in that sector before the optical density values are reported. ❐ When you've finished setting up the blanking map, press F10 to save the map and return to the Reader Control File definition screen. Delay Initial Reading Time This parameter allows you to specify a delay time after the plate enters the reading chamber before the reading begins. ❐ Enter any value from 00:00:00 to 12:00:00 seconds for this parameter. Incubation Setpoint This option is available if an incubator is installed. ❐ Use the spacebar to toggle between Off and a Setpoint. The setpoint can be between 22.0° C and 50.0° C, but must be at least 1.5° C above ambient temperature. Saving the Reader Control File When you are finished entering or editing the Reader Control File parameters, press F10 to save them to disk and return to the Protocol Definition Menu. F-6 Reader Control File
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