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Introduction & History
The modern pipette has had a colorful history as a standard tool in the scientist’s arsenal. What began as the usage of straw, one’s mouth, and the scientific principles of suction is now one of the most technologically-evolved yet extremely straightforward devices in the modern laboratory.
Commonly used in genetic research, chemistry, microbiology, and pharmacological testing; pipettes and micropipettes are glass or plastic tubes used to measure, transfer, and deliver substances of accurate volumes. Most pipettes function by creating a vacuum above the space that the liquid shall fill and then control the uptake of the liquid by releasing this vacuum, suctioning the liquid upwards. Simple enough as this may sound, pipettes have come a long way–let’s look at its evolution through an exhaustive list of products that are widely used today.
The First Pipettes
The Pasteur Pipette
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Aside from his more popular invention of the process of pasteurization, we also have Louis Pasteur to thank for the first pipette ever invented. As the founder of medical microbiology and the proponent of Germ Theory, it was then important for Pasteur and his experiments that things be kept clean and germ-free: hence, the invention of the first pipette. The Pasteur pipette, which are the familiar eye droppers and chemical droppers that we still encounter in everyday use, was then merely designed to prevent contamination in transferring small amounts of liquids. The addition of rubber teats at the end of long, thin, glass tubes made aspirating and dispensing of liquids a quick and easy affair. Today, these pipettes are used for the transfer of rough, uncalibrated volumes of liquids of up to 2.5 milliliters.
How is it used? Pinch the rubber teat of the Pasteur pipette with thumb and forefinger, and immerse the tip of the pipette on the surface of the liquid. Then, slowly release pressure on the rubber teat and wait for the liquid to go up. Once pressure is released, and the proper amount of liquid is aspirated, bring the full Pasteur pipette to the receiving container and pinch the rubber teat again to dispense the liquid on the side of the container’s wall.
The Transfer Pipette
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Pasteur’s glass pipettes were a 19th-century hit, but problems arose from the fragility of its material. The invention of plastics in the 1940s, therefore, paved the way for a modification to the original pipette design–plastic Pasteur pipettes, also called transfer pipettes, were single pieces of molded plastic manufactured using the blow-molding process on low-density polyethylene (LDPE). Both the stem and bulb are part of the single piece of plastic, in place of the rubber attachment in glass pipettes. Today, these disposable transfer pipettes are made in a variety of sizes and shapes, and some include graduation marks for approximate volume calibrations. Note, however, that though transfer pipettes may be perfect for use with aqueous solutions, organic solvents such as acetone may dissolve the plastic material.
How is it used? Transfer pipettes use a similar procedure as with the usage of the Pasteur pipette, but instead of a rubber teat, the plastic bulb is pinched.
Volumetric and Measuring Pipettes
The high-end and complex pipette varieties that offer more accuracy and precision than Pasteur and transfer pipettes emphasize on the other aspect of pipetting, aside from the transfer and delivery of liquids–measurement. And, though considered a reliable high-precision instrument, the pipette does not achieve accuracy and precision on its own. A tool is only as good as its user, and, in the laboratory context, the wielder of the pipette must be well-prepared and knowledgeable with common pipetting techniques to make full use of its features. More importantly, scientists are expected to make informed decisions when selecting which pipette to use. Which pipette is the right pipette? How are they used? Let’s look at our list again.
The Volumetric Pipette
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Volumetric pipettes resemble thin rolling pins with large bellies, blunt on one end and tapered on the tip. Typically, these pipettes are used to deliver single, specific volumes between 1 and 100 milliliters, at which they are calibrated. These glass pipettes offer more accuracy than Pasteur and transfer pipettes, emphasizing on the other aspect of pipetting, aside from the transfer and delivery of liquids–measurement. By having the middle bulge where the bulk of the liquid is stored, and the single volume marker at the thin portion, errors in accuracy are extremely lessened. The narrow diameter of the thin portion makes errors more obvious and easier to point out.
With these types of pipettes, procedures require the use of separate pipette bulbs, devices that provide suction. The common rubber bulb is fairly simple and easy to use–shaped like a fist-sized balloon, the rubber bulb is squeezed and touched to the mouth of the pipette, while the pipette tip is placed in liquid. The liquid draws up as pressure on the bulb is slowly released. Other similar tools, such as safety pipette fillers and pipetting aids, also serve the same purpose.
How is it used? First, hold the pipette at the upper portion using your thumb, pinky, ring and middle finger. Free your index finger. With your other hand, squeeze the rubber bulb and touch its opening to the top mouth of the pipette. Insert no more than a half-centimeter of the pipette into the rubber bulb. Afterwards, immerse the tip of the pipette into the liquid and slowly release your hand’s pressure on the rubber bulb. The liquid will then be drawn up into the pipette. Once the curved surface of the liquid, called the meniscus, reaches the target measurement, remove the bulb and quickly slip your free index finger over the tip of the pipette’s mouth. With your finger still firmly closed on the pipette, lift the pipette out of the liquid and into the receiving container.
It is a safe option always to collect more than, but closest to, the graduation mark so that adjustments can be easily made. Observing the pipette at eye-level, carefully raise your index finger to lessen the liquid and reach the desired amount. Depending on the physical properties of the liquid, the meniscus may either be concave or convex–for both cases, the center of the meniscus is used as the basis for measurement.
The Mohr & Serological Pipette
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As opposed to volumetric pipettes, measuring pipettes are thin glass tubes that are calibrated into small divisions of volume range so that different amounts of liquid may be measured and transferred. The two types of measuring pipettes–Mohr and serological pipettes–differ according to the placement of graduations. In Mohr pipettes, graduations always end before the tapered tip, while in serological pipettes, the graduations continue to the tip.
How are they used? The procedure for the transfer of liquids in measuring pipettes is similar to that of volumetric pipettes, necessitating the usage of rubber bulbs or pipette aids. With measuring pipettes, it is also recommended that the volume of liquid to be collected is more than, but closest to, the target measurement so that adjustments can be easily made.
The invention of the micropipette by Heinrich Schnitger in 1957 paved the way for measurement of smaller volumes in units of microliters through an automatic, push-button and piston technology. With the push of a retractable plunger or push-button, aspiration, dispensing, and purging of liquids are all made simpler. The automatic micropipette is also highly specialized–as opposed to manually measuring the liquid to line up to a graduation mark, as is the tradition in ordinary pipettes, automatic micropipettes allow the scientist to set the volume of liquid that needs to be transferred beforehand through a digital volumeter.
All micropipettes operate like an automatic syringe, but the two primary types–the air-displacement pipette and the positive-displacement pipette–differ based on the presence of an air cushion between the pipette piston and the liquid sample.
The Air-Displacement Pipette
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Air-displacement pipettes always have a cushion of air between the pipette piston and the liquid. The simpler of the two instrument types, air-displacement pipettes are easily influenced by factors such as temperature, atmospheric pressure, specific gravity, and the liquid’s viscosity. Keeping the liquid away from the piston or barrel is an advantage, but also provides many limitations in terms of maintaining accuracy. For instance, working with liquids that may easily evaporate would require the use of the other type of pipette–positive-displacement pipettes.
How is it used? Air-displacement and positive-displacement micropipettes involve a quicker and more efficient procedure for transferring liquids of specific volumes. The first step to using these automatic micropipettes is the push-button volume adjustment. Hold the body of the micropipette in one hand and use the other hand to rotate the push-button. Do not attempt to force the volume setting beyond the limits of your micropipette, at the risk of internal damage. Also, make sure that the numbers of your volume setting are in proper alignment. Afterwards, a disposable pipette tip is attached.
After setting the volume and attaching the disposable tip, the next step is the process of pre-rinsing. Pre-rinsing, also called pre-wetting, is a highly-advised procedure for achieving greater uniformity and precision, as it provides identical contact surfaces for the liquid to be aspirated. To pre-rinse, simply aspirate with the tip and then dispense back into the original reservoir or to a waste receptacle. Pre-rinsing should be performed each time you change a pipette tip, and when you increase the volume setting.
The operation of automatic micropipettes involves two points of resistance — the first stop, the point at which the piston reaches the calibrated volume on the digital volumeter, and the second stop, at which the remaining liquid is fully expelled or purged. When aspirating, two factors can influence the accuracy of your measurement — the aspiration angle and immersion depth. Prepare the micropipette by holding it in a nearly vertical position and depressing the plunger or push-button to the first stop position using your thumb. Once the push-button is in the first stop, immerse the pipette tip just below the surface of the liquid, to avoid droplets sticking on the outside of the pipette tip and slowly and smoothly release the plunger or push-button back to the rest position as you wait for the liquid to move up and into the tip. Do not let the plunger snap back up as this will result in air bubbles.
Once the liquid is collected, place the pipette tip at an angle of 10 to 45 degrees against the inside wall of the receiving vessel, and depress the plunger smoothly back to the first stop position to dispense the liquid. Wait one second, and then depress the plunger once more into the second stop position to “purge” or “blow-out” the tip of any residual liquid. Finally, remove the pipette tip by sliding it off the sidewall. The procedure is the same for positive-displacement micropipettes, except both the disposable pipette tip and piston are ejected and replaced after each transfer.
The Positive-Displacement Micropipette
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The mechanism of positive-displacement pipettes involves a direct contact of the piston with the sample. This set-up makes positive-displacement pipettes more accurate for pipetting volatile solutions because there is no dead space for the liquid to evaporate. The absence of the air cushion also lessens the risk of contamination with corrosives and other bio-hazardous materials. However, these pipettes are more expensive as they replace both piston and tip after each use.
Pipette Tips & Multichannel Pipettes
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A crucial step in using automatic micropipettes is choosing the right pipette tip. Pipette tips, which hold the aspirated liquid provide efficiency in laboratory procedures by not requiring thorough washing or cleaning, through quick replacement of the reusable or disposable tips after every use. Multichannel pipettes, meanwhile, provide maximum efficiency by allowing the aspiration of multiple pipette tips at the same time.
Taking Care of Pipettes and Micropipettes
Finally, like any working device, micropipettes have many moving parts that are susceptible to wear and tear or breakage. Therefore, any responsible owner of pipettes and micropipettes must exhibit regular care and maintenance, even when these tools are not in use. Preventive pipette maintenance includes the process of calibration in preserving high-quality performance, accuracy, and precision. It is important to have your pipettes serviced at least twice a year by experienced pipette handlers and pipette clinics. Pipette owners must also clean pipettes after every session, and check pipettes daily for damage before use. In storing pipettes, pipette holders are recommended to ensure that the instruments are kept vertical and upright.
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Klingenberg, M. (2006). The Original Micropipette. Retrieved from The Scientist, https://www.the-scientist.com/?articles.view/articleNo/19720/title/The-Original-Micropipette/
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Helmenstive, A. M. (2017). How to Read a Meniscus in Chemistry. ThoughtCo. Retrieved from https://www.thoughtco.com/how-to-read-a-meniscus-606055