Laboratory Equipment Maintenance Manual PDF

M AIN T E NAN CE Manual for Laboratory Equipment 2nd E d itio n WHO Library Cataloguing-in-Publication Data Maintenance manual for laboratory equipment, 2nd ed. 1.Laboratory equipment. 2.Maintenance. 3.Manuals. I.World Health Organization. II.Pan American Health Organizati...

M AIN T E NAN CE Manual for Laboratory Equipment 2nd E d itio n WHO Library Cataloguing-in-Publication Data Maintenance manual for laboratory equipment, 2nd ed. 1.Laboratory equipment. 2.Maintenance. 3.Manuals. I.World Health Organization. II.Pan American Health Organization. ISBN 978 92 4 159635 0 (NLM classification: WX 147) © World Health Organization 2008 All rights reserved. Publications of the World Health Organization can be obtained from WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail: [email protected]). Requests for permission to reproduce or translate WHO publications – whether for sale or for noncommercial distribution – should be addressed to WHO Press, at the above address (fax: +41 22 791 4806; e-mail: [email protected]). The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement. The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. All reasonable precautions have been taken by the World Health Organization to verify the information contained in this publication. However, the published material is being distributed without warranty of any kind, either expressed or implied. The responsibility for the interpretation and use of the material lies with the reader. In no event shall the World Health Organization be liable for damages arising from its use. Design and Layout: L’IV Com Sàrl, Morges Switzerland Printed in Spain Contact: Dr G. Vercauteren, Coordinator, Diagnostics and Laboratory Technology, Department of Essential Health Technologies, World Health Organization, 20 Avenue Appia, 1211 Geneva 2, Switzerland This document is available at www.who.int/diagnostics_laboratory  M A I N T E N A N C E M A N U A L F O R L A B O R ATO R Y E Q U I P M E N T Table of Contents TABLE OF FIGURES viii ACKNOWLEDGEMENTS x INTRODUCTION xi CHAPTER 1 MICROPLATE READER 1 Photograph of microplate reader 1 Purpose of the microplate reader 1 Operation principles 1 Installation requirements 3 Routine maintenance 3 Troubleshooting table 4 Basic definitions 5 CHAPTER 2 MICROPLATE WASHER 7 Photograph of microplate washer 7 Purpose of the microplate washer 7 Operation principles 7 Installation requirements 9 Routine maintenance 9 Troubleshooting table 11 Basic definitions 12 CHAPTER 3 pH METER 13 Purpose of the equipment 13 Photograph and components of the pH meter 13 Operation principles 13 pH meter components 14 Typical circuit 15 Installation requirements 16 General calibration procedure 16 General maintenance of the pH meter 17 Basic maintenance of the electrode 18 Troubleshooting table 18 Basic definitions 19 Annex: The pH theory 20 iii TABLE OF CONT E NTS CHAPTER 4 BALANCES 21 Photographs of balances 21 Purpose of the balance 22 Operation principles 22 Installation requirements 26 Routine maintenance 27 Troubleshooting table 28 Basic definitions 29 CHAPTER 5 WATER BATH 31 Diagram of a water bath 31 Operation principles 31 Water bath controls 32 Water bath operation 32 Troubleshooting table 34 Basic definitions 34 CHAPTER 6 BIOLOGICAL SAFETY CABINET 35 Illustration of a biological safety cabinet 35 Purposes of the equipment 35 Operation principles 35 Biological safety 39 Installation requirements 39 Using the safety cabinet 39 Routine maintenance 40 Functional evaluation (alternative) 41 Table of functional evaluation of biological safety cabinets 42 Troubleshooting table 43 Basic definitions 44 CHAPTER 7 CENTRIFUGE 45 Photograph of centrifuge 45 Purpose of the centrifuge 45 Operation principles 45 Components of the centrifuge 46 Installation requirements 48 Routine maintenance 48 Appropriate management and storage recommendations 48 Troubleshooting table 50 Basic definitions 52 CHAPTER 8 WATER DISTILLER 53 Diagram of a water distiller 53 Purpose of the water distiller 53 Operation principles 54 Installation requirements 54 Routine maintenance 55 Troubleshooting table 56 Basic definitions 57 iv  M A I N T E N A N C E M A N U A L F O R L A B O R ATO R Y E Q U I P M E N T CHAPTER 9 DILUTOR 59 Diagram of a dilutor 59 Purpose of the dilutor 59 Operation principles 60 Installation requirements 61 Routine maintenance 61 Troubleshooting table 63 Basic definitions 64 CHAPTER 10 DISPENSER 65 Photograph and diagram of the dispenser 65 Purpose of the dispenser 65 Requirements for operation 67 Routine maintenance 67 Troubleshooting table 68 Basic definitions 68 CHAPTER 11 SPECTROPHOTOMETER 69 Photograph of spectrophotometer 69 Purpose of the equipment 69 Operation principles 69 Spectrophotometer components 72 Installation requirements 73 Spectrophotometer maintenance 73 Good practices when using the spectrophotometer 75 Troubleshooting table 77 Basic definitions 79 CHAPTER 12 AUTOCLAVE 81 Photograph of the autoclave 81 Purpose of the autoclave 81 Operation principles 82 Operation of the autoclave 84 Installation requirements 87 Routine maintenance 88 Maintenance of specialized components 90 Troubleshooting table 91 Basic definitions 92 CHAPTER 13 DRYING OVEN 93 Photograph of drying oven 93 Purpose of the oven 93 Operating principles 93 Installation requirements 94 Oven operation 94 Oven controls 95 Quality control 96 Routine maintenance 96 Troubleshooting table 97 Basic definitions 98 v TABLE OF CONT E NTS CHAPTER 14 INCUBATOR 99 Photograph of incubator 99 Operating principles 99 Incubator controls 101 Installation requirements 101 Routine maintenance and use of the incubator 101 Troubleshooting table 103 Basic definitions 104 CHAPTER 15 MICROSCOPE 105 Photographs of microscopes 105 Purpose of the equipment 106 Operation principles 106 Installation requirements 108 Description of potential problems with microscopes 109 General maintenance of the microscope 111 Troubleshooting table 115 Basic definitions 116 CHAPTER 16 PIPETTES 119 Photographs of pipettes 119 Purpose of the pipettes 120 Operation principles of the pipette 120 Requirements for use 120 Using the pipette 121 Routine maintenance 122 Troubleshooting table 125 Basic definitions 126 CHAPTER 17 STIRRING HEATING PLATE 127 Photograph of the stirring heating plate 127 Operation principles 127 Controls of the stirring heating plate 127 Installation requirements 128 Operation of the stirring heating plate 128 Routine maintenance 128 Troubleshooting table 129 Basic definitions 129 CHAPTER 18 REFRIGERATORS AND FREEZERS 131 Photograph of a refrigerated storage unit 131 Purpose of refrigerated storage units 132 Operation principles 132 Installation requirements 133 Refrigerator control circuit 134 Refrigerator operation 134 Refrigerator routine maintenance 135 Troubleshooting table 137 vi  M A I N T E N A N C E M A N U A L F O R L A B O R ATO R Y E Q U I P M E N T Operation of ultralow freezers 138 Turning the unit on 138 Routine maintenance 139 Troubleshooting table 140 Basic definitions 141 CHAPTER 19 CHEMISTRY ANALYSERS 143 Photographs of chemistry analysers 143 Purpose of chemistry analysers 144 Operation principle 144 Components 144 Installation requirements 145 Operation of the dry chemistry analyser 145 Operation of the wet chemistry analyser 146 Routine maintenance of chemistry analysers 146 Non-routine maintenance and troubleshooting 147 Troubleshooting table 148 Basic definitions 148 CHAPTER 20 COLORIMETERS 149 Photograph of colorimeter 149 Purpose of the colorimeter 149 Operating principle 149 Components 150 Installation requirements 150 Operation of the colorimeter 150 Operation of the haemoglobinometer 151 Routine maintenance 151 Troubleshooting table 154 Basic definitions 155 BIBLIOGRAPHY 157 vii TABLE OF FIGURE S Table of Figures Figure 1 Equipment used for ELISA tests 2 Figure 2 Microplate washer 8 Figure 3 Well profiles 8 Figure 4 Diagram of a pH meter 14 Figure 5 Types of electrodes 15 Figure 6 Example of a typical pH meter control circuit 15 Figure 7 Spring balance 22 Figure 8 Sliding weight scale 22 Figure 9 Analytical balance 22 Figure 10 Upper plate balance 23 Figure 11 Substitution balance 23 Figure 12 Components of the electronic balance 24 Figure 13 Compensation force principle 24 Figure 14 Classification of balances by exactitude 25 Figure 15 Analytical balance control panel 26 Figure 16 Water bath 31 Figure 17 Immersion and external resistors 31 Figure 18 Water bath controls 32 Figure 19 Biological safety cabinet 35 Figure 20 Centrifugal force concept 46 Figure 21 Water distiller 53 Figure 22 Dilutor diagram 59 Figure 23 Dilutor controls 60 Figure 24 Syringe and dispenser 61 Figure 25 Dispenser 65 Figure 26 Dispenser and accessories 66 Figure 27 Interaction of light with matter 70 Figure 28 Absorbance phenomenon 71 Figure 29 Spectrophotometer components 72 Figure 30 Refraction of light 79 Figure 31 Diffraction grid 80 Figure 32 Vapour circuit of an autoclave 83 Figure 33 Space required for autoclave 87 Figure 34 Compressed air connection 87 Figure 35 Vapour connection 88 Figure 36 Vapour generator 89 Figure 37 Electronic control of the oven 95 Figure 38 Electrical circuit of the oven 95 Figure 39 Heat transfer systems used in incubators 100 viii  M A I N T E N A N C E M A N U A L F O R L A B O R ATO R Y E Q U I P M E N T Figure 40 Incubator controls 101 Figure 41 Positive (convergent) lens 106 Figure 42 Optics of the convergent lens 106 Figure 43 Diagram of a microscope 107 Figure 44 Cross-section of a microscope 108 Figure 45 Binocular head 109 Figure 46 Lighting system 109 Figure 47 Platform, plate or mechanical stage 110 Figure 48 Revolving, objective holder 110 Figure 49 Body of the microscope 111 Figure 50 Diagram of a pipette 119 Figure 51 Types of pipettes 120 Figure 52 Phases of pipette use 121 Figure 53 Disassembly of a pipette 123 Figure 54 Stirring heating plate controls 127 Figure 55 Induction motor 129 Figure 56 Refrigeration circuit 132 Figure 57 Control circuit of the refrigerator 134 Figure 58 Blood bank refrigerator controls 135 Figure 59 Ultralow temperature freezer control 138 Figure 60 Basic diagram of reflectance photometry on a test strip 144 Figure 61 Ulbricht’s sphere 145 Figure 62 Basic components of a photometer 145 Figure 63 Controls of a portable colorimeter 150 ix PREFACE Acknowledgements This manual is a revised edition of “Manual de mantenimiento para equipo de laboratorio” (PAHO, 2005) translated from Spanish into English. Revisions include additional chapters on laboratory equipment commonly used in some laboratories and updates allowing global use of the manual. The revised version has been prepared under the direction of Dr Gaby Vercauteren, World Health Organization, Geneva, Switzerland and in coordination with Dr Jean-Marc Gabastou, Pan-American Health Organization/World Health Organization, Washington, DC, USA; translated by Ms Christine Philips; reviewed by Ms Mercedes Pérez González and adapted, revised and edited by Mrs Isabelle Prud’homme. WHO kindly expresses thanks to those who have participated at all levels in the elaboration of this manual. WHO wishes to acknowledge the original contribution of Dr Jorge Enrique Villamil who wrote the first edition of this manual in 2005 (Manual para mantenimiento de equipo de laboratorio, ISBN 92 75 32590 1) and Dr Jean-Marc Gabastou and Mr Antonio Hernández, Reviewers at Essential Medicines Vaccines and Health Technologies at PAHO. WHO also thanks manufacturers who have granted permission to use their images in this publication. x  M A I N T E N A N C E M A N U A L F O R L A B O R ATO R Y E Q U I P M E N T Introduction This manual has been developed to support personnel employed in health laboratories. Its purpose is to give a better understanding of the technical requirements regarding installation, use and maintenance of various types of equipment which play an important role in performing diagnostic testing. The manual also aims to provide support to personnel responsible for technical management, implementation of quality management and maintenance. Due to the diversity of origins, brands and models, this manual offers general recommendations. Equipment-specific details are explained in depth in the maintenance and installation user manuals from manufacturers. These should be requested and ordered through the procurement processes of the individual agencies and professionals responsible for the acquisition of technology, or directly from the manufacturer. This manual was originally developed by the Pan-American Health Organization (PAHO) to support improved quality programmes which PAHO promotes in regional laboratories. The English version was produced by WHO to further expand support for quality programmes in other regions. The revised edition now includes 20 equipment groups selected to cover those most commonly used in low to medium technical complexity laboratories across the world. Given the differences in technical complexity, brands and existing models, each chapter has been developed with basic equipment in mind, including new technology where relevant. The following information is included in each chapter: Groups of equipment, organized by their generic names. Alternative names have also been included. Photographs or diagrams, or a combination of both to identify the type of equipment under consideration. A brief explanation on the main uses or applications of the equipment in the laboratory. A basic description of the principles by which the equipment operates with explanations of principles or physical and/or chemical laws which the interested reader can – or should study in depth. Installation requirements with emphasis on the electrical aspects and the requirements for safe installation and operation, including worldwide electrical standards. Basic routine maintenance, classified according to the required frequency (daily, weekly, monthly, quarterly, annually or sporadically). The procedures are numbered and presented in the actual sequence in which these should take place (model-specific procedures can be found in the manuals published by the manufacturers). Troubleshooting tables with the most frequent problems affecting the equipment with possible causes and actions that may resolve these problems. A list of basic definitions of some of the specialized terms used. For some equipment, additional themes related to calibration, quality control and design (with operational controls). This information, along with good use and care, helps to maintain laboratory equipment in optimal condition. xi  M A I N T E N A N C E M A N U A L F O R L A B O R ATO R Y E Q U I P M E N T Chapter 1 Microplate Reader GMDN Code 37036 ECRI Code 16-979 Denomination Photometric micro-plate reader The microplate reader also known as “Photometric OPERATION PRINCIPLES micro-plate reader or ELISA reader” is a specialized The microplate reader is a specialized spectrophotometer. spectrophotometer designed to read results of the ELISA Unlike the conventional spectrophotometer which facilitates test, a technique used to determine the presence of readings on a wide range of wavelengths, the microplate antibodies or specific antigens in samples. The technique reader has filters or diffraction gratings that limit the is based on the detection of an antigen or antibodies wavelength range to that used in ELISA, generally between captured on a solid surface using direct or secondary, 400 to 750 nm (nanometres). Some readers operate in the labelled antibodies, producing a reaction whose product ultraviolet range and carry out analyses between 340 to 700 can be read by the spectrophotometer. The word ELISA is nm. The optical system exploited by many manufacturers the acronym for “Enzyme-Linked Immunosorbent Assay”. uses optic fibres to supply light to the microplate wells This chapter covers the use of microplate readers for ELISA containing the samples. The light beam, passing through testing. For additional information on the instrument the sample has a diameter ranging between 1 to 3 mm. principles of operation and maintenance, consult Chapter A detection system detects the light coming from the 11 discussing the spectrophotometer. sample, amplifies the signal and determines the sample’s absorbance. A reading system converts it into data allowing the test result interpretation. Some microplate readers use PHOTOGRAPH OF MICROPLATE READER double beam light systems. Test samples are located in specially designed plates with a specific number of wells where the procedure or test is carried out. Plates of 8 columns by 12 rows with a total of 96 wells are common. There are also plates with a greater number of wells. For specialized applications, the current trend is to increase the number of wells (384-well plates) Photo courtesy of BioRad Laboratories to reduce the amount of reagents and samples used and a greater throughput. The location of the optical sensors of the microplate reader varies depending on the manufacturers: these can be located above the sample plate, or directly underneath the plate’s wells. Nowadays microplate readers have controls regulated by microprocessors; connection interfaces to information systems; quality and process control programs, which by PURPOSE OF THE MICROPLATE READER means of a computer, allow complete test automation. The microplate reader is used for reading the results of ELISA tests. This technique has a direct application in immunology and serology. Among other applications it confirms the presence of antibodies or antigens of an infectious agent in an organism, antibodies from a vaccine or auto-antibodies, for example in rheumatoid arthritis. 1 C HAP TER 1 M I C R O P L AT E R E A D E R Equipment required for ELISA testing Biochemical phases of the ELISA technique1 In order to perform the ELISA technique, the following The ELISA technique from a biochemical point of view: equipment is required: 1. The plate wells are coated with antibodies or antigens. 1. Microplate reader. 2. Samples, controls and standards are added to the wells 2. Microplate washer (Chapter 2). and incubated at temperatures ranging between room 3. Liquid dispensing system (multi-channel pipettes may temperature and 37 °C for a determined period of be used). time, according to the test’s characteristics. During the 4. Incubator to incubate the plates. incubation, the sample’s antigen binds to the antibody coated to the plate; or the antibody in the sample binds Figure 1 illustrates how this equipment is interrelated. to the antigen coated on the plate, according to their presence and quantity in the sample analyzed. Mechanical phases of the ELISA technique 3. After incubation, the unbound antigen or antibodies are Using the equipment washed and removed from the plate by the microplate When an ELISA test is conducted, it typically follows these washer using an appropriate washing buffer. steps: 4. Next, a secondary antibody, called the conjugate, is 1. A first washing of the plate may be done using the added. This harbours an enzyme which will react with microplate washer. a substrate to produce a change of colour at a later 2. Using a liquid dispenser or the multi-channel pipettes, step. wells are filled with the solution prepared to be used in 5. Then begins a second period of incubation during the test. which this conjugate will bind to the antigen-antibody 3. The plate is placed in the incubator where at a controlled complex in the wells. temperature, a series of reactions take place. 6. After the incubation, a new washing cycle is done to remove unbound conjugate from the wells. Stages 1, 2 and 3 can be repeated several times depending 7. A substrate is added. The enzyme reacts with the on the test, until the reagents added have completed their substrate and causes the solution to change in colour. reactions. This will indicate how much antigen-antibody complex is present at the end of the test. Finally, when all the incubation steps have been completed, 8. Once the incubation time is completed, a reagent the plate is transferred to the microplate reader. The reading is added to stop the enzyme-substrate reaction and of the plate is done and a diagnosis can be deduced. to prevent further changes in colour. This reagent is generally a diluted acid. 9. Finally, the plate in is read by the microplate. The resulting values are used to determine the specific amounts or the presence of antigens or antibodies in the sample. Note: Some of the wells are used for Figure 1. Equipment used in ELISA tests standards and controls. Standards allow the cut-off points to be defined. The standards and controls are of known quantities and are used for measuring the success of the test, evaluating data against known concentrations for each ELISA Plate control. The process described above Washer is common, although there are many ELISA tests with test-specific variants. Incubator Dispensing System ELISA Reader Computer 1 More detailed explanations must be consulted in specialized literature. 2  M A I N T E N A N C E M A N U A L F O R L A B O R ATO R Y E Q U I P M E N T INSTALLATION REQUIREMENTS 5. Verify that the reader is calibrated, column by column. In order for the microplate reader to operate correctly, the Place a clean, empty plate and carry out a reading. If following points need to be respected: there is no difference between each of the average 1. A clean, dust free environment. reading of the first to the last column, it can be assumed 2. A stable work table away from equipment that vibrates that the reader is calibrated. (centrifuges, agitators). It should be of a suitable size so that there is working space at the side of the microplate reader. The required complementary equipment for ROUTINE MAINTENANCE conducting the technique described above is: washer, Maintenance described next focuses exclusively on the incubator, dispenser and computer with its peripheral microplate reader. The maintenance of the microplate attachments. washer is described in Chapter 2. 3. An electrical supply source, which complies with the country’s norms and standards. In the countries of the Basic maintenance Americas for example, 110 V and 60 Hertz frequencies Frequency: Daily are generally used, whereas other regions of the World 1. Review that optical sensors of each channel are clean. use 220-240V, 50/60HZ. If dirt is detected, clean the surface of the windows of the light emitters and the sensors with a small brush. Calibration of the microplate reader 2. Confirm that the lighting system is clean. The calibration of a microplate reader is a specialized process 3. Verify that the reader’s calibration is adequate. When which must be executed by a technician or trained engineer the daily operations begin, let the reader warm up for following the instructions provided by each manufacturer. 30 minutes. Next, do a blank reading and then read a In order to do the calibration, it is necessary to have a set full plate of substrate. The readings must be identical. of grey filters mounted on a plate of equal geometric size If not, invert the plate and repeat the reading in order to those used in the analyses. Manufacturers provide these to determine if the deviation originated in the plate or calibration plates for any wavelength the equipment uses. the reader. 4. Examine the automatic drawer sliding system. It must Calibration plates are equipped with at least three pre- be smooth and constant. established optic density values within the measurement ranges; low, medium, and high value. In order to perform Preventive maintenance the calibration, follow this process: Frequency: Quarterly 1. Place the calibration plate on the equipment. 1. Verify the stability of the lamp. Use the calibration plate, 2. Carry out a complete reading with the calibration plate. conducting readings with intervals of 30 minutes with Verify if there are differences in the readings obtained the same plate. Compare readings. There must be no from well to well. If this is the case, invert the plate (180°) differences. and repeat the reading to rule out that differences are 2. Clean the detectors’ optical systems and the lighting attributed to the plate itself. In general, it is accepted systems. that the instrument does not need further calibration if 3. Clean the plate drawer. the plate results are as expected at two wavelengths. 4. Verify the alignment of each well with the light emission 3. Verify if the reader requires calibration. If so, proceed and detection systems. with the calibration following the routine outlined by the manufacturer, verifying that the reading’s linearity is maintained as rigorously as possible. 4. If the instrument does not have a calibration plate, verify it by placing a coloured solution in the wells of a plate and immediately carry out a complete reading. Then invert the plate 180° and read the plate again. If both readings display identical, average values in each row, the reader is calibrated. 3 C HAP TER 1 M I C R O P L AT E R E A D E R TROUBLESHOOTING TABLE PROBLEM PROBABLE CAUSE SOLUTION The reader gives a reading that does not make sense. The illumination lamp is out of service. Replace the lamp with one with the same characteristics as the original. The reader’s readings vary from row to row. Dirty optical sensors. Clean the sensors. The illumination system’s lenses or parts are dirty. Clean the lighting system’s lenses. Lack of calibration in one or more channels. Verify the calibration of each one of the channels. The reader displays high absorbance values. Reagents expired and/or incorrectly prepared. Check to see if the TMB is colourless and the preparation adequate. Contamination with other samples. Repeat the test verifying the labelling, the washer and how the pipette was used. Incorrect wavelength filter. Verify the recommended wavelength for the test. Adjust if it is incorrect. Insufficient or inefficient washing. Verify the washing method used. Use an appropriate quality control test. Very long incubation time or very high temperature. Check incubation times and temperatures. Incorrect sample dilution. Check process for sample dilution. Some reagent was omitted. Verify that the test has been carried out according to the established procedure. The reader displays low absorbance values. Very short incubation time and very low Check temperatures and incubation times. temperature. The reagents were not at room temperature. Check that the reagents are stable at room temperature. Excessive washing of the plate. Adjust the washing process to what the test manufacturers indicate. Incorrect wavelength filter. Verify the wavelength selected. Use wavelength recommended for the test. Expired or incorrectly prepared reagents. Check the used reagents. Test the dilutions. A reagent was omitted. Verify that the test was done according to the established procedure. The plate displays scratches at the bottom of the Prepare a new plate and repeat the test. wells. Incorrectly selected or dirty plate. Verify the type of plate used. Prepare a new plate and repeat the test. The plate wells have dried up. Change the manner in which the plate is washed. The plate is incorrectly placed or is seated unevenly Check the placement of the plate. Repeat the in the reader. reading. Humidity or fingerprints on the outer part of the Verify that the plate under the bottom of the wells bottom of the plate. is clean. Residual quantities of washing buffer in the wells Confirm that the washing buffer is completely before adding the substrate. removed. The substrate tablets do not dissolve completely. Verify that the tablets dissolve correctly. The substrate tablet has been contaminated by Test the integrity and handling of substrate tablets. humidity or metal clips or is not complete. The position of the blank well could have been Verify that the plate set-up is correct. changed and an incorrect quantity has been subtracted at each reading. The reader displays unexpected variation in the The reader’s lamp is unstable. Replace the lamp with one that has similar optical density readings. characteristics as the original. The reader displays a gradual increase or decrease Inappropriate calibration of the plate’s advance Calibrate the advance so that at each step the wells from column to column. motor. remain exactly aligned with the lighting system. The optical density readings are very low compared The reading is being carried out with a different Verify the wavelength used when conducting to the operator’s optical evaluation criteria. wavelength than required for the test. the reading. If this is the problem, adjust the wavelength and repeat the reading. Verify that the recommended wavelength filter has been selected. 4  M A I N T E N A N C E M A N U A L F O R L A B O R ATO R Y E Q U I P M E N T Low reproducibility. Sample homogeneity. Mix the reagents before use. Allow these to equilibrate to room temperature. Incorrect pipetting procedure. Ensure pipette’s tips are changed between samples and that excessive liquid inside is removed. Reader not calibrated. Check the calibration. Use an appropriate quality control set. Wait until the reader has warmed up to its operating instrument. temperature. Expired reagents. Verify the expiry dates of the reagents. when washed. The blank sample shows high absorbance. Contaminated substrate. Check that TMB is colourless and its preparation. The data are not transferred from the reader to the Verify selected codes. microprocessor. transmission plugs. manufacturer. Misaligned light beam. The reader was transferred or moved without using Call the specialized service technician. the necessary precautions. The light source – lamp – has been changed and Verify its assembly and alignment. the replacement has not been installed or aligned correctly. The plate was incorrectly loaded. reading carrying out the adjustments. the reader. reading carrying out the adjustments. Computer fails to indicate the error codes. The programme which controls the activation of Call the specialized service technician. alarms and warning messages is defective or is not validated by the manufacturer. The reader demonstrates failure in detecting errors. Various components of the system display failure, Call the specialized service technician. such as the liquid level detection system. BASIC DEFINITIONS Chemiluminescence. Emission of light or luminescence resulting directly from a chemical reaction at environmental temperatures. ELISA (Enzyme-Linked Immunosorbent Assay). Biochemical technique used mainly in Immunology to detect the presence of an antibody or an antigen in a sample. ELISA plate. Consumable standardized to carry out the ELISA technique. Generally, plates have 96 wells in a typical configuration of 8 rows by 12 columns. There are also ELISA plates with 384 wells or up to 1536 wells for specialized high throughput testing in centres with high demand. Microplate washer. Equipment used for washing plates during specific stages of an ELISA test with the aim of removing unbound components during reactions. Microplate washers use special buffers in the washing process. Enzyme. Protein that accelerates (catalyses) chemical reactions. Fluorophore. Molecules absorbing light at a determined wavelength and emitting it at a higher wavelength. Microplate reader. The name given to spectrophotometers with the capacity to read microplates. TMB. Tetramethylbenzidine, a substrate for the horseradish peroxidase (HRP) enzyme. 5  M A I N T E N A N C E M A N U A L F O R L A B O R ATO R Y E Q U I P M E N T Chapter 2 Microplate Washer GMDN Code 17489 ECRI Code 17-489 Denomination Micro-plate washer The microplate washer or“plate or ELISA washer”is designed to OPERATION PRINCIPLES perform washing operations required in the ELISA technique. The microplate washer has been designed to perform The microplate washer performs the washing of the ELISA washing operations in the ELISA technique. The equipment plate’s wells during the different stages of the technique. possesses at least, the following subsystems which vary depending on the manufacturer’s design. Control subsystem. Generally, the washer is controlled PHOTOGRAPH OF MICROPLATE WASHER by microprocessors allowing programming and controlling steps to be performed by the washer such as: number of washing cycles2 (1–5); expected times; supplying and extracting pressures; plate format (96–384 wells); suction function adjustment according to the type of well3 (flat bottom, V bottom or rounded bottom or strips used); volumes distributed or aspirated; the soaking and agitation cycles, etc. Supply subsystem. In general, this comprises a reservoir Photo courtesy of BioRad Laboratories for the washing solution; one or several pumps; usually a positive displacement type syringe and a dispenser head that supplies the washing solution to the different wells by means of needles. The head usually comes with eight pairs of needles for washing and aspirating simultaneously the wells of the same row (the supply and extraction sub-systems converge on the head). There are models with twelve pairs of needles and others that conduct the washing process simultaneously in all PURPOSE OF THE MICROPLATE WASHER the wells. Some washers offer the possibility of working The microplate washer has been designed to supply cleaning with different types of washing solutions, performing buffers required for the ELISA technique in a controlled the solution changes according to the program entered manner. In the same fashion, the equipment removes from by the operator. each well, substances in excess from the reaction. Depending on the test performed, the washer can intervene from one to four times, supplying the washing buffer, agitating and removing the unbound reagents1 until the programmed times and cycles are completed. The washer has of two reservoirs; one for the washing buffer, the other for the waste generated during the washing process. 1 See a brief explication of the ELISA technique in Chapter 1, Microplate Reader. 2 The exact number of washing operations required depends on the assay used. This is explained in each manufacturer’s test instruction manual. 3 If the bottom is flat, the suction needle is located very close to one of well’s faces; if it is rounded or V-shaped, the suction needle is centered. 7 C HAP TER 2 M I C R O P L AT E WA S H E R Extraction or suction system. This requires a vacuum The sub-systems previously described are shown in Figure mechanism and a storage system for gathering the fluids 2. Figure 3 shows the different types of wells most commonly and waste removed from the wells. The vacuum may be found in microplates. Each kind of well is suitable for a supplied by external and internal pumps. Extraction is particular type of test. done by a set of needles mounted on the washer/dryer’s head. The number of needles varies from one to three, Washing process according to the washer model used. The washing of the microplate is one of the stages of the If it uses only one needle, the washing and ELISA technique. Special solutions are used in the washing extraction operation is done with this single needle. steps. Among those most commonly used is phosphate If it uses two needles, one is used for supplying the buffer solution or PBS. The phosphate buffer solution has a washing solution and the other for extraction. If it uses stability of 2 months if kept at 4 °C. It is estimated that 1 to three needles, the first is used for supplying the washing 3 litres of solution is required for washing one microplate solution, the second for extraction and the third for and that 300 µl is used in each well per cycle. Washing can controlling (extracting) any excess volume in the well. be done manually, but it is preferable to use an automated Generally, the extraction needle is longer than the microplate washer for a better throughput and to minimize supply needle, which enables it to advance (vertically) handling of potentially contaminated substances. up to a height ranging between 0.3 and 0.5 mm from the bottom of the well. Among the washing processes used by microplate washers Advance sub-system. This is composed of a are featured: mechanism which moves the supply and extraction Aspiration from top to bottom. When the aspiration head horizontally to reach each well in the ELISA plate. phase is initiated, the needles move vertically and the When the horizontal movement to the following row aspiration is initiated immediately as these enter into occurs, there is a vertical movement towards the well the liquid. The process continues until the needles to dispense or extract the washing solution. There reach their lowest position very close to the bottom are washers which carry out these operations in a of the wells. At this point they are stopped in order to simultaneous manner. avoid suctioning the air that should flow against the interior lateral walls of the wells. This type of aspiration prevents air currents from drying the bound protein on the surface of the wells. Figure 2. Microplate washer Extraction Pump Positive Displacement Pumps Supply and Extraction Head Waste Receptacle Horizontal and Vertical Displacement Supply Pumps Washing Solution Wells ELISA Plate Figure 3. Well profiles Flat Round V-shaped Easy Bottom Bottom Bottom Wash 8  M A I N T E N A N C E M A N U A L F O R L A B O R ATO R Y E Q U I P M E N T Simultaneous distribution and aspiration. In certain Washing process verification types of washer, the washing and aspiration systems To verify that the washing process is done according to operate simultaneously, generating a controlled the specifications of ELISA techniques, manufacturers turbulence inside the well which removes the unbound of ELISA tests have developed procedures to be carried substances during the incubations. out regularly. One of the controls1 is based on using the Aspiration from the base of the wells. In this system, peroxidase reagent, which is dispensed using a pipette in the aspiration of the fluid contained in the wells is the plate wells to be read at 405, 450 and 492 nm. At once performed initially with the aspiration needles in a the wells are washed and a colourless substrate is added position very close to the bottom, immediately (TMB/H2O2–Tetramethylbenzidine/Hydrogen Peroxide). beginning a suctioning cycle, usually time-controlled. Whatever conjugate remains will hydrolyze the enzyme This system may aspirate air if there are differences in and the chromogen will change to blue. After stopping the levels of the tanks. the reaction with acid, the TMB will turn yellow again. The resulting colour intensity is directly related to the washing Washer calibration process efficiency. The microplate washer is critical for guaranteeing that the ELISA technique performs as expected. The alignment to be taken into account for the effective functioning of the INSTALLATION REQUIREMENTS equipment is presented next: For the microplate washer to operate correctly, the following Position of the needles (supply and aspiration head). is necessary: The horizontal and vertical position adjustment with 1. A clean, dust-free environment. respect to the wells must be verified carefully. If the 2. A stable work table located away from equipment plate has flat bottom wells, the supply needle must be that generates vibrations, (centrifuges, and agitators). checked to see that it is situated very close to the well’s It must be of a suitable size to locate the necessary wall. If the bottom is round or V-shaped, the suction complementary equipment: reader, incubator, needle should be located in the centre of the well: distributor and computer with its peripheral attachments upon the vertical movement, a needle-base distance at the side of the microplate washer. is maintained in the well, usually between 0.3 to 0.5 3. An electric outlet in good condition with a ground pole mm. The needles must never be allowed to touch the and, an electrical connection which complies with the bottom of the wells to avoid mechanical interferences country’s or the laboratory’s norms and standards. In the between the needle point and the well’s base during countries of the Americas, the 110 V and 60 Hz frequency the aspiration function. is generally used. In other parts of the World, the 220-240 Aspiration time. Appropriately adjust the aspiration V and 50/60 Hz frequency is generally used. time so that a solution film adhered to the well’s wall can flow towards the bottom. Avoid very long time lapses to prevent the coating on the wells from drying ROUTINE MAINTENANCE up. Check that the suction system’s needles are clean The routine maintenance described next focuses exclusively (free of obstructions). on the microplate washer. Maintenance of the microplate Distributed Volume. Check that the volume distributed reader is dealt with in the Chapter 1. is as close as possible to the maximum capacity of the well; confirm that all the wells are filled uniformly (at Basic maintenance the same level). Verify that the distributing needles are Frequency: Daily clean (free of obstructions). 1. Verify the volume distributed. Vacuum. The suctioning system must be calibrated 2. Test the filling uniformity. efficiently. If the vacuum is too strong, the test can 3. Verify the aspiration sub-system’s efficiency. be altered. In fact, it could dry out the wells and 4. Confirm the cleaning of the supply and extraction considerably weaken the enzyme activity in the wells needles. and completely alter the test result. The majority of 5. Clean the washer with distilled water after use, to remove washers function with a vacuum ranging between 60 every vestige of salt in the supply and extraction sub- and 70% of atmospheric pressure. In some washers, the systems’ channels. The needles may be kept submerged vacuum is made in an external pump which operates as in distilled water. an accessory of the washer. Its operation is controlled 6. Verify that the body of the washer has been cleaned. by the washer, which means that the vacuum pump If necessary, clean the exterior surfaces with a piece of operates only when required. cloth, moistened with a mild detergent. 1 Procedure developed by PANBIO, ELISA Check Plus, Cat. Nº E-ECP01T. 9 C HAP TER 2 M I C R O P L AT E WA S H E R Preventive maintenance 4. Confirm the integrity of the electrical connector and the Frequency: Quarterly inter-connection cable. 1. Disassemble and clean the channels and connectors. 5. Clean the washer with distilled water after using it in Verify their integrity. If leaks or any vestiges of corrosion order to remove every vestige of salt in the supply and are detected, adjust and/or replace. extraction subsystems’ channels. 2. Verify the integrity of the mechanical components. 6. Verify the integrity of the fuse, and that its contact Lubricate according to the manufacturer ’s points are clean. instructions. 3. Test the adjustment of each one of the sub- Note: Trained technical personnel must carry out systems. Calibrate according to the manufacturer’s maintenance of the control system. If necessary, call the recommendations. manufacturer or representative. 10  M A I N T E N A N C E M A N U A L F O R L A B O R ATO R Y E Q U I P M E N T TROUBLESHOOTING TABLE PROBLEM PROBABLE CAUSE SOLUTION Upon completion of washing, residual solution The washer extraction system demonstrates failure. Verify if the vacuum system is functioning at the remains in the wells. appropriate pressure. The conducts/pipes of the vacuum system are of a Check that the diameter of the channels corresponds different diameter than that recommended. to the recommendation by the manufacturer. The suction line shows obstructions. Verify that the vacuum lines are clean. The container for storing the waste is full. Confirm the waste recipient’s level. The line filter is damp or blocked. Verify the state and integrity of the suctioning system’s filter. The needles’ points are not placed correctly and do Examine the placement of the needles’ points. not reach the bottom of the wells. A different microplate is used in the test. Verify the type of plate required for the test. The washer has not been purged sufficiently. Check the purging process. The operator has not followed the manufacturer’s Examine the process recommended by the instructions correctly. manufacturer. Carry out the required adjustments. The plate placed in the washer is incorrectly aligned. Check the placement of the plate in the washer. The washing cycle is performing inadequately. The washing solution reserve is exhausted. Examine the cleaning solution storage receptacle. Replace the volume missing. The washer was not purged sufficiently at the Clean adequately in order to homogenize the beginning of the work cycle. humidity in each one of its components and to eliminate air bubbles. The volume of washing solution distributed has been Verify the required volume for each type of test and programmed erroneously. for each plate. The plate was placed incorrectly in the washer. Check the correct installation of the plate in the washer. The cycle setting was incorrectly selected. Review the cycle setting recommended for each type of plate. The plates used are different from those Verify that the plates used are completely recommended by the manufacturer. compatible with the washer. The fluid level in the wells is inadequate. The washing solution supply tube is not of the Check the manufacturer’s specifications. If necessary, diameter or thickness specified by the manufacturer. correct. The pressure is insufficient for delivering the Check the supply system and supply channels, there adequate amount of washing solution. might be an obstruction in the filling line. The washing container shows fungal and bacterial The system is not used frequently. Check the procedures used for preventing fungal and growths. bacterial growth. An adequate control procedure (disinfection) is not Check the procedures used for preventing fungal and used. bacterial growth. The tubes and connectors are not changed with the Verify the change frequency suggested by the required frequency. manufacturer and or the technical department. The washing solution has been contaminated. Confirm the procedures used in the preparation and management of the washing solution with the aim of determining the cause of contamination and eliminate it. Maintenance has not been carried out according to Check the dates planned for carrying out its schedule. maintenance. Inform those responsible. 11 C HAP TER 2 M I C R O P L AT E WA S H E R BASIC DEFINITIONS Buffer. A solution containing either a weak acid and its salt or, a weak base and its salt, which makes it resistant to changes in pH at a given temperature. PBS. One of the solutions used to perform washing operations in ELISA tests. PBS is the acronym for Phosphate Buffer Solution. This is made of the following substances: NaCl, KCl, NaHPO42H2O and KH2SO4. The manufacturers supply technical bulletins which indicate the proportions and instructions for preparing PBS. In general, one part of concentrated PBS is mixed with 19 parts of deionised water. Plate (ELISA). Consumable with standard dimensions, designed to hold samples and reactions for the ELISA technique. In general, these have 96, 384 or 1536 wells and are made of plastics such as polystyrene and polypropylene. There are plates specially treated to facilitate the performance of the tests. Positive displacement pump. A pump adjusted by a plunger moving along a cylinder. The mechanism is similar to that of a syringe. It is equipped with a set of valves for controlling the flow to and from the pump. TMB/H2O2. (Tetramethylbenzidine/hydrogen peroxide). A set of reagents used for verifying the quality of washing done on the wells used in the ELISA technique. 12  M A I N T E N A N C E M A N U A L F O R L A B O R ATO R Y E Q U I P M E N T Chapter 3 pH Meter GMDN Code 15164 ECRI Code 15-164 Denomination pH Meter The pH meter is used for determining the concentration of PHOTOGRAPH AND COMPONENTS OF THE hydrogen ions [H+] in a solution. This equipment, provided pH METER it is carefully used and calibrated, measures the acidity of an aqueous solution. pH meters are sometimes called pH analysers, pH monitors or potentiometers. 2 1 PURPOSE OF THE EQUIPMENT The pH meter is commonly used in any field of science related to aqueous solutions. It is used in areas such as agriculture, water treatment and purification, in industrial processes such as petrochemicals, paper manufacture, foods, pharmaceuticals, research and development, metal mechanics, etc. In the health laboratory, its applications are related to the control of culture mediums and to the measurement of the alkalinity or acidity of broths and buffers. In specialized laboratories, diagnostic equipment microelectrodes are used to measure the pH of liquid blood components. The plasma pH allows the patient’s health to be evaluated. It normally measures between 7.35 3 and 7.45. This value relates to the patient’s metabolism Photo courtesy of Consort in which a multitude of reactions occurs where acids and 1 Electrode carrying arm and electrode bases are normally kept in balance. Acids constantly liberate 2 Digital display 3 Control panel with temperature adjustment control, mode hydrogen ions [H+] and the organism neutralizes or balances selection (Standby/mV/pH) and calibration controls acidity by liberating bicarbonate ions [HCO3–]. The acid-base ratio in the organism is maintained by the kidneys, (organs in which any excesses present are eliminated). The plasma pH is one of the characteristics that vary with factors such as age or state of health of the patient. Table 1 shows typical OPERATION PRINCIPLES pH values of some bodily fluids. The pH meter measures the concentration of hydrogen ions [H+] using an ion-sensitive electrode. Under ideal conditions, pH values of some bodily fluids this electrode should respond in the presence of only one type of ion. In reality, there are always interactions or Fluid pH Value interferences with other types of ions present in the solution. Bile 7.8 – 8.6 A pH electrode is generally a combined electrode, in which Saliva 6.4 – 6.8 a reference electrode and an internal glass electrode are Urine 5.5 – 7.0 integrated into a combined probe. The lower part of the Gastric Juice 1.5 – 1.8 probe ends in a round bulb of thin glass where the tip of the internal electrode is found. The body of the probe Blood 7.35 – 7.45 13 C HAP TER 3 pH ME T E R contains saturated potassium chloride (KCl) and a solution pH METER COMPONENTS 0.1 M of hydrogen chloride (HCl). The tip of the reference A pH meter generally has the following components: electrode’s cathode is inside the body of the probe. On the 1. The body of the instrument containing the circuits, outside and end of the inner tube is the anodized end. The controls, connectors, display screens and measuring reference electrode is usually made of the same type of scales. The following are among some of its most material as the internal electrode. Both tubes, interior and important components: exterior, contain a reference solution. Only the outer tube a) An ON and OFF switch. Not all pH meters have an has contact with the measured solution through a porous on and off switch. Some simply have a cord with a cap which acts as a saline bridge. plug which allows it to be connected to a suitable electrical outlet. This device acts like a galvanized cell. The reference electrode b) Temperature control. This control allows is the internal tube of the pH meter probe, which cannot lose adjustments according to the temperature of the ions through interactions with the surrounding environment. solution measured. Therefore as a reference, it remains static (unchangeable) c) Calibration controls. Depending on the design, during the measuring process. The external tube of the pH meters possess one or two calibration buttons probe contains the medium which is allowed to mix with or dials. Normally these are identified by Cal 1 and the external environment. As a result, this tube must be Cal 2. If the pH meter is calibrated using only one filled periodically with a potassium chloride solution (KCI) solution, the Cal 1 button is used; making sure for restoring the capacity of the electrode which would that Cal 2 is set at a 100%. If the pH meter allows otherwise be inhibited by a loss of ions and evaporation. two point calibrations, two known pH solutions covering the range of pH to be measured are used. The glass bulb on the lower part of the pH electrode acts In this case, the two controls are used (Cal 1 and Cal as a measuring element and is covered with a layer of 2). In special cases, a three-point calibration must hydrated gel on its exterior and interior. Metallic sodium be done (using three known pH solutions). cations [Na+] are diffused in the hydrated gel outside of d) Mode selector. The functions generally included the glass and in the solution, while the hydrogen ions [H+] in this control are: are diffused in the gel. This gel makes the pH electrode I. Standby mode (0). In this position the electrodes ion-selective: Hydrogen ions [H+] cannot pass through the are protected from electrical currents. It is the glass membrane of the pH electrode. Sodium ions [Na+] pass position used for maintaining the equipment through and cause a change in free energy, which the pH while stored. meter measures. A brief explanation of the theory on how II. pH mode. In this position the equipment can electrodes function is included in the appendix at the end take pH measurements after performing the of the chapter. required calibration procedures. Figure 4. Diagram of a pH meter High Impedance Voltmeter Ag/AgCI Electrode Temperature Regulator Active Termimal Reference Terminal KCI KCI Special Glass Permeable to Ions Saline Mesh Bridge Solution Under Analysis 144  M A I N T E N A N C E M A N U A L F O R L A B O R ATO R Y E Q U I P M E N T III. Millivolt mode (mV). In this position the reference electrode (also known as Calomel electrode) equipment is capable of performing millivoltage and an internal electrode, integrated into the same body. readings. Its design varies depending on the manufacturer. IV. ATC mode. The automatic temperature control mode is used when the pH is measured in TYPICAL CIRCUIT solutions for which the temperature varies. This Figure 6 features a typical circuit adapted to the control function requires the use of a special probe. Not system of the pH meter. Each manufacturer has its own all pH meters have this control. designs and variations. 2. A combined electrode or probe. This device must be stored in distilled water and stay connected to the measuring instrument. A combination electrode has a Figure 5. Types of electrodes Combined Electrode Reference Electrode (Calomel) h Platinum Wire (Pi) Silver Wire (Ag) Mercury [Hg] Reference Electrode Mercury Chloride [Hg CI] Semi-Permeable Mesh Potassium Chloride Buffer Solution Porous Stopper Figure 6. Example of a typical pH meter control circuit 1N 4002 7812 110 VAC 3,300 0.1 mfd mfd 560K 9,09 K 10K Variable Transformer resistor 110 V AC/ 12 V DC pH mV 1,00 K 0.1 30K mfd 7 3,300 2 mfd 6 12V 3 TL081 1 7912 Lamp 5 Exit 4 10K Zero Entrance 10K mV Reference pH 15 C HAP TER 3 pH ME T E R Description of typical control circuit elements System Element Description Electric feeding and correction. 110 V/12 V AC transformer.* A device converting the voltage of the 110 V to 12 V AC network. 1N4002 rectifier diodes. Diode controlling the type of wave and guaranteeing that is positive. Electrolyte condensers 3300 microfarads (µfd) (2). Condensers absorbing the DC voltage to the diodes. Tri terminal regulators (7812, 7912). A device regulating the voltage resulting from the interaction between diodes and condensers. 0.1 microfarad (µfd) (2) electrolyte condensers. Devices used to achieve stability at high frequency. 12 V D C signal light. Light indicating if the equipment is ON. Measurement of pH and millivolts. TL081 non-inverted type dual amplifier. Millivolts circuits. (R1) 9.09 K Ω (ohm) resistors. (R2) 1 K Ω (ohm) resistors. (R3) 560 K Ω (ohm) resistors. pH circuits. (R4) 10 K Ω(ohm) variable resistors. (R5) 30 K Ω (ohm) resistors. Ground resistance. The circuit gain is governed by means of the following equation: Gain = 1+ (R3+PxR4)/R5+ (1–P) xR4. Outlet section. Low cost DC voltmeter. Permits readings in millivolts. The voltage read is 10