BIOTIX INC. THE BIOTIX GUIDE TO PIPETTING understand and get the most out of your pipettes to get better results in your experiments GET A BETTER HANDLE ON LIQUID HANDLING Biotix Inc. Technological breakthroughs found only in the most advanced liquid handling tools available. http://biotix.com/ [email protected] +1 858 875 7696 01 | THE HISTORY OF PIPETTE DESIGN 0 02 | TYPES OF PIPETTES 2 03 | FINDING THE RIGHT PIPETTE TIP 4 04 | CARING FOR AND MAINTAINING YOUR PIPETTE 8 05 | ERGONOMICS AND PIPETTING IN THE LAB 10 06 | SUSTAINABILITY 12 07 | FURTHER READING 14 TABLE OF TABLE CONTENT CHAPTER 1 THE HISTORY OF PIPETTE DESIGN 4 THE HISTORY OF PIPETTE DESIGN In the 19th century, Louis Pasteur needed a way to Contrary to popular belief, the earliest pipettes 3 transfer small amounts of liquid without contamination. 1 were created not by Louis Pasteur in the 19th He would take a glass tube, heat the center over a burner, century but in the 18th century by two French chemists, draw the tube out to make it very thin in the center, and Francois Descroizilles and Joseph-Louis Gay-Lussac. then snap it in half to yield two fine-tipped pipettes. The Descroizilles in particular is considered the patriarch of fine-tipped end was placed in liquid and a balloon or rubber the volumetric analysis field. He created the precursor bulb was placed on the larger diameter end. When the bulb to today’s buret and pipettes called the berthollimetre was squeezed, it creates a vacuum that draws liquid into and alcalimetre, respectively. In an 1824 paper, Gay- the pipette. Similar pipettes are still used today in this form Lussac made some modifications to the alcalimetre and or as an all-in-one plastic version, which is referred to as a coined the resulting instrument a “pipette.” transfer pipette. Typically, a transfer pipette does not have measurement markings and are not meant for transferring accurate amounts of liquid. Descroizilles’s and Gay-Lussac’s pipettes are most 2 similar to the modern day serological pipette. Today’s serological pipettes are either made of disposable plastic or reusable glass. Like Descroizille’s design, these pipettes have a gradation scale on the side to accurately measure and transfer between a fraction of a milliliter and 50 mL of material. In use, serological pipettes are inserted in to a pipette plunger apparatus. When depressed, the plunger creates a partial vacuum that allows the liquid to be drawn up in to the serological pipette. The user can easily control the amount of liquid drawn in to the pipette. Francois Descroizilles and Joseph-Louis Gay- Lussac created the first pipettes in the 18th century. Like most modern day pipettes, they transfer between a fraction of a milliliter and 50 mL of material. In the 19th century, Louis Pasteur needed a way to transfer small amounts of liquid without contamination. THE BIOTIX GUIDE TO PIPETTING 5 THE HISTORY OF PIPETTE DESIGN Heinrich Schnitger, a postdoctoral student at the The Carlsberg pipettes took Pasteur pipettes one 5 University of Marburg, Germany was frustrated with 4 step further. In the 1930s, Kaj Ulrik Linderstrøm– trying to accurately draw small volumes of materials using Lang in the Carlsberg laboratory in Denmark wanted to the Carlsberg pipette. To overcome this issue, he created study single cell metabolism. He passed the problem a piston-pump driven glass pipette starting from a syringe. of scaling down the typical laboratory techniques to a His design created a more accurate and durable pipette that research fellow named Milton Levy. For his research, didn’t require mouth pipetting. He was awarded a patent for Levy created the first reported pipette for small his design in 1961. Schnitger’s pipette design was adopted measurements. Similar to Pasteur, he made these by the scientific device industry and this basic design is still pipettes by drawing a glass tube over a lit Bunsen used today. burner and pulling one end to create a thin, capillary tube. However, each pipette was then calibrated with mercury or dye to measure a specified volume of liquid. Also, the liquid was drawn into the pipette by For his research, Levy created the first reported mouth pipetting—a technique in which the scientist pipette for small measurements. uses his/her mouth to suck up the liquid into the pipette. Schnitger’s design created a more accurate Carlsberg pipettes had a number of limitations—they and durable pipette that didn’t require mouth pipetting. were not consistently manufactured (though some were mass-produced), broke easily, did not ensure accurate volumes despite calibration, required extensive experience, and could cause the user to ingest whatever was being drawn up in to the pipette. THE BIOTIX GUIDE TO PIPETTING CHAPTER 2 TYPES OF PIPETTES 7 TYPES OF PIPETTES While micropipettes have been developed in all sizes to address specific laboratory tasks, mechanistically there are two main types: Air displacement and positive displacement. In overall basic mechanism, both air and positive displacement pipettes are similar. Both types of micropipettes work similarly to Schnitger’s basic pipette design. Both pipettes require depression of a piston-driven plunger. The user sets the volume to be drawn up prior to pressing the plunger. Depressing and releasing the plunger creates a vacuum that draws the set amount of liquid up into the pipette. However, because of slight differences in design and how the sample is drawn up, they are used for very different purposes and materials in the lab. THE BIOTIX GUIDE TO PIPETTING 8 TYPES OF PIPETTES Figure 1. Comparison of Air Displacement and Positive Displacement Pipettes. AIR DISPLACEMENT Air displacement starts with dialining in your volume which moves the internal piston to the correct position to be able to draw the appropriate volume. Once that volume is set you depress the plunger which moves the piston down in the pipette. Once you emerge the tip into the liquid is when the vacuum is created and once you release the button the piston moves up and aspirates the liquid to whatever setting you put on the pipette. Once you have the sample in the pipette tip and you push the plunger again the piston will move back towards the position you drew the liquid from. Then the blowout stage which is fully pressing the top button pushes the piston past its position when you began the draw which creates pressure greater than the volume drawn making it expel all the remaining liquid. THE BIOTIX GUIDE TO PIPETTING 9 TYPES OF PIPETTES A key feature of air displacement pipettes is that a cushion of air is left between the pipette shaft and the sample, so the sample is never allowed to come into contact with the shaft. This, in combination with the use of disposable pipette tips makes air displacement pipettes very convenient for repeated, routine, accurate pipetting work. In biological labs for example, air displacement pipettes are the instrument of choice for measuring most aqueous solutions and buffers, including common biological materials like DNA, RNA and proteins. The downsides of the air displacement mechanism are that the accuracy of the pipettes is affected by temperature, atmospheric pressure, and density/viscosity of the sample. In addition, the air cushion can allow highly volatile samples to evaporate into the air space, which can lead to contamination of the pipette. Fixed volume Single channel Calibrated to dispense one volume One pipette tip per pipette Variable/adjustable volume Multi-channel Can change and set volume dispensed One pipette holds many tips that by pipette simultaneously measure and dispense the same volume of liquid (often used with multi-well plates Manual Manually press and depress plunger Repeat function (repeaters) A larger volume is taken up into a Electronic reservoir. The sample volume is taken up and dispensed with a push of a button Pipette operated by battery force THE BIOTIX GUIDE TO PIPETTING 10 TYPES OF PIPETTES POSITIVE DISPLACEMENT PIPETTES Positive displacement pipettes act more like In comparison to air displacement pipettes, syringes. A disposable barrel/tip is placed the types of positive displacement pipettes on to the pipette (see Figure 1). The desired are limited. They are generally only available pipetting volume is selected and the piston as repeat or manual, single channel variable moves down into the disposable barrel. The pipettes. piston comes into direct contact with the sample so no air cushion exists between the sample and the piston. When the plunger is released, a partial vacuum draws the liquid up into the barrel. To release the sample, the plunger is pressed again and the piston pushes the liquid out of the barrel. The entire barrel/tip is disposed of between samples. A positive displacement pipette applies constant aspiration force to the sample that is not affected by the physical characteristics of the sample. Therefore, they are useful for accurately pipetting viscous or dense solutions. They are also used to pipette corrosive or radioactive samples to prevent contamination of the pipette as well as cross- contamination between samples. Positive displacement pipettes are also used when working with volatile samples that would be susceptible to evaporation in the air cushion of air displacement pipettes. THE BIOTIX GUIDE TO PIPETTING 11 TYPES OF PIPETTES Figure 2. Exploded Diagram of an Air Displacement Pipette. THE BIOTIX GUIDE TO PIPETTING CHAPTER 3 FINDING THE RIGHT PIPETTE TIP 13 FINDING THE RIGHT PIPETTE TIP In the days of mouth-drawn Pasteur pipettes (and even earlier), one size of pipette tip had to fit all applications. But today’s technology has advanced to the point where there many types of tips are available to suit a myriad of applications.