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GUIDE to PIPETTING Third Edition HOW the PIPETTE STORY BEGAN

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GUIDE to PIPETTING Third Edition HOW the PIPETTE STORY BEGAN GUIDE TO PIPETTING Third Edition HOW THEHOW PIPETTE BEGAN... STORY HOW THE PIPETTE STORY BEGAN… Over a century ago, Louis Pasteur invented the glass Pasteur pipette to reduce contamination when transferring samples. The Pasteur pipette is still in use today. The next significant improvement on pipettes occurred in the late 1950s with the introduction of a handheld, piston-operated pipette as a safe alternative to potentially dangerous mouth pipetting. The first handheld pipettes had pre- established volume setting (fixed volume pipettes). Further improvement was provided with the more flexible stepper volume setting (variable volume pipettes). In 1972, Dr. Warren Gilson invented the first continuously adjustable pipette. As an original error-preventing feature, the selected volume was now clearly displayed on the Gilson pipette (direct digital readout). Today, Gilson precision pipettes are still the world standard for accuracy, precision, and reliability. Today, the company still maintains its continuous effort of creativity and offers innovative, robust, and reliable pipettes to help scientists in their daily work. The pipetting system is our core expertise, and we truly enjoy sharing this knowledge and experience with pipette users so that they may achieve their goals. This guide will provide you with keys to understand how pipettes should be used and to get the most out of them. Examples given are based on our own pipette range, but the techniques described are equally applicable to other brands of pipettes and tips. Gilson is one of the winners of the Red Dot Award: Product Design for MICROMAN E. GILSON GUIDE TO PIPETTING TABLE OF CONTENTS TABLE OF CONTENTS TABLE OF CONTENSTABLE CHAPTER 1 - SELECTING THE RIGHT PIPETTE | 8 Appendix | 45 Working Principle of Pipettes | 8 Appendix A: Pipetting Terms | 45 The Right Choice for your Application | 11 Appendix B: Example of a Performance Check | 47 SpecificP ipettes for SpecificV essels | 13 Appendix C: Z Factor | 48 High Throughput and Repetitive Pipetting | 14 Appendix D: Evaporation Loss | 49 Appendix E: Chemical Resistance of Plastics | 50 TABLE OF CONTENTS TABLE CHAPTER 2 - PIPETTING TECHNIQUES | 16 Adjust the Volume Display | 16 FAQ | 51 Forward or Reverse Mode Pipetting | 17 Tips for Mistake-Free Pipetting | 23 Pipetting Ergonomics | 23 CHAPTER 3 - SELECTING THE RIGHT TIP | 26 Fitting a Disposable Pipette Tip | 26 Choosing the Best Tip for Your Application | 28 Evaluating Tip Quality | 30 CHAPTER 4 - PREVENTING CONTAMINATION | 32 Types of Contamination and How to Prevent Them | 32 Decontaminating Your Pipette | 34 CHAPTER 5 - PIPETTE SERVICE & MAINTENANCE | 36 Pipette SpecificationsA ccording to ISO 8655 | 36 Repair in the Lab or Return for Service? | 36 Quick Pipette Diagnosis | 37 How to Calculate Volumetric Accuracy and Precision | 38 Pipette Calibration | 39 Calibration with the Gravimetric Method | 41 Performance Check Procedure | 43 3 TABLE OF CONTENT | GILSON GUIDE TO PIPETTING GILSON GUIDE TO PIPETTING | USER’S GUIDE 4 Air-Displacement Pipette Positive-Displacement Pipette POSITIVE-DISPLACEMENT PIPETTE AIR-DISPLACEMENT PIPETTE 5 GLOSSARY | GILSON GUIDE TO PIPETTING GILSON GUIDE TO PIPETTING | USER’S GUIDE 6 Chapter 1 SELECTING THE RIGHT PIPETTE SELECTING THE RIGHT PIPETTE SELECTING THE RIGHTPIPETTE Working Principle of Pipettes Air-Displacement Pipettes Positive-Displacement Pipettes • Recommended for aqueous samples • Recommended for problem samples and for general laboratory work. (viscous, dense, volatile, radioactive, • Always have a cushion of air (dead corrosive, contaminating, hot and cold). volume) between the pipette piston • Direct contact of the piston and the liquid sample. with the sample (no air cushion). • The piston is integrated into the • The disposable piston is part of the tip lower part of the pipette. (not integrated into the pipette). Piston Shaft Shaft Disposable piston Disposable capillary Air cushion Disposable tip Piston seal Sample Sample 7 SELECTING THE RIGHT PIPETTE | GILSON GUIDE TO PIPETTING GILSON GUIDE TO PIPETTING | USER’S GUIDE 8 Working Principle of Air-Displacement Pipettes Working Principle of Positive-Displacement Pipettes When the push button is pressed on an air-displacement pipette, the piston inside the Positive displacement pipettes, such as MICROMAN, work like a syringe. There is no air cushion instrument moves down to let air out. Air is displaced by the piston. The volume of air between the disposable piston and the sample. With no elastic air cushion to expand or contract, displaced is equivalent to the volume of liquid aspirated. the aspiration force remains constant, unaffected by the physical properties of the sample. SELECTING THE RIGHTPIPETTE The schematic drawings show how the piston determines the volume of air displaced and This allows the positive-displacement operator to pipette very viscous or high density samples, subsequently the volume of sample aspirated. such as glycerol and blood. 1 2 1 2 1 1 0 0 0 0 Set Volume Prepare for Set volume Prepare for Aspiration aspiration The required The required volume is set. The push button volume is set. The push button SELECTING THE RIGHT PIPETTE The piston is pressed prior to The piston is pressed prior moves to the sample aspiration. moves down to to sample appropriate The piston the appropriate aspiration. position. descends and start position. The piston expels a volume descends down of air equal to the to the end of the selected volume of capillary. liquid. 3 4 3 4 Aspirate sample Dispense Aspirate sample Dispense sample sample As the push button The orifice is then is released, a The push button is immersed below The push button partial vacuum is pressed again. Air the liquid surface. is pressed again. created inside the pressure increases As the push button The piston tip. The ambient inside the shaft is released, the moves down atmospheric and the tip. The piston moves up and expels the pressure forces the compressed air and the ambient liquid out of the desired volume of pushes the liquid pressure forces the capillary. liquid through the out of the tip. desired volume orifice into the tip. of liquid through the orifice into the capillary. 9 SELECTING THE RIGHT PIPETTE | GILSON GUIDE TO PIPETTING GILSON GUIDE TO PIPETTING | USER’S GUIDE 10 The Right Choice for Your Application Accuracy and Precision While Pipetting Problem Liquids The type of analysis to perform, the physical properties of the liquid, and the volume to be Positive-displacement pipettes like MICROMAN are the right solution for complete and rapid handled will determine which pipette to use. It is recommended to select a pipette with a pipetting of viscous and dense liquids such as oil, syrup, cosmetic cream, liquefied food, paint, nominal (maximum) volume as close as possible to the desired volume to transfer. glycerol, or buffers. SELECTING THE RIGHTPIPETTE Positive-displacement pipettes are the unique solution to avoid leakage when pipetting high vapor pressure liquids such as acetone, chloroform, alcohol, or other solvents. Recommendations for Pipetting Different Volumes 0 µL 10 µL 50 µL 100 µL 200 µL 1000 µL 5000 µL 10 mL Volume of sample % Systematic error 5 Regular air-displacement pipette Molecular biology 4.5 4 Microbiology 3.5 Clinical assays Cell culture 3 Immunoassays Analytical chemistry 2.5 2 1.5 Air-displacement SELECTING THE RIGHT PIPETTE P2, P10, P100, P200, P1000 P5000, P10mL P20 pipettes 1 Positive-displacement 0.5 M10, M25, M50 M100, M250, M1000 pipettes 0 MICROMAN® E positive-displacement pipette Consider the Physical Properties of Your Sample 0 40 60 80 85 90 95 100 % Glycerol For volumes higher than 10 mL, it is suggested to work with a pipette filler like the Viscosity/Density MACROMAN with plastic or serological pipettes. Regardless of the volume you require, the nature of the sample directly impacts precision and accuracy. Air-displacement pipettes will be better for aqueous liquids whereas positive- Figure 3 displacement pipettes should be used for problem liquids. MICROMAN® E, Positive-displacement pipette, Systematic error SAMPLE TYPES EXAMPLES RECOMMENDED PIPETTES % Random error 3.5 Aqueous Water, sucrose, Tris, buffers with a pH of 7 Air-displacement. Regular air-displacement pipette 3 Biological DNA, RNA, proteins Air-displacement with filter tips Viscous Glycerol, surfactants, oil 2.5 Volatile Ethanol, hexane, formaldehyde 2 Radioactive isotopes, blood, infectious 1.5 Hazardous bacteria or viruses 1 Acids such as hydrochloric acid or sulfuric acid, bases such as ammonium hydroxide, 0.5 Corrosive salts such as sodium chloride Positive-displacement 0 MICROMAN® E positive-displacement pipette 0 40 70 80 85 90 95 100 % Glycerol Viscosity/Density Figure 4 MICROMAN® E, Positive-displacement pipette, Random error 11 SELECTING THE RIGHT PIPETTE | GILSON GUIDE TO PIPETTING GILSON GUIDE TO PIPETTING | USER’S GUIDE 12 SpecificP ipettes for SpecificV essels High Throughput and Repetitive Pipetting When pipetting in a high throughput setting it is important to have reliable results and to be as efficient as possible. Reliable results means not only having reproducible results with one technician's samples, but also among all technicians SELECTING THE RIGHTPIPETTE in the lab. There are a variety of ways to improve reliability and efficiency, some of Test tubes and centrifuge tubes are which include using motorized pipettes and/or repetitive pipettes. used with all single channel pipettes for sample preparations, such as qPCR templates. User-to-User Variability Motorized
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