A Micropipette is a standard type of laboratory equipment that is used to measure the small volumes of liquids. Micropipettes are also used to transfer a small volume of liquids accurately from one vessel to another.
In 1957 Doctor Heinrich Schnitger at the University of Marburg in Germany invented and patented the first micropipette. He named the first invented pipette piston stroke micropipette. It had a removable plastic tip that can contain the liquid and a spring-loaded piston to load the liquid in the tip. It was a simple micropipette having all the features of modern days micropipettes incorporated in it.
In 1961 doctor Heinrich Netheler the founder of the Eppendorf inherited the rights of commercial production of micropipettes and started its commercial production. In 1972, Warren Gilson and Henry Lardy a professor of biochemistry at the University of Wisconsin-Madison developed adjustable micropipettes. The modern-day adjustable micropipettes are Wisconsin’s invention.
Introduction To Micropipettes:
Pipettes and micropipettes are used to measure and deliver an accurate volume of liquid. Pipettes are used to measure small volumes starting from 1 millimeter whereas micropipettes are used to measure the very small volume of liquid usually in microliters.
Micropipette works by displacing the air from the pipette shaft and allow the liquid to be drawn into the resulting vacuum. Pipettes and micropipettes are very essential tools used in microbiology labs, medical labs, and environmental sciences for various academics and research studies.
There are different types of micropipettes available in the market. Although these micropipettes don’t look the same but the basic parts of all the micropipettes are the same. It is important to identify the parts of a micropipette. Following are the basic parts of all micropipettes:
A plunger is the topmost part of the micropipette which is used to expel and take up the desired amount of liquid into micropipette tip.
The eject button is used to eject the tip from the micropipette without touching the micropipette tip. It is connected with the meter bar down which then pushes the micropipette-tip off.
At the top of the micropipette, a circle is present which is rotated to decrease and increase the set volume. The required volume intake is ensured by adjusting the volume with volume adjustment.
The adjusted volume is shown in the volume window. Different values of liquid can be set within the range of that pipette. Different micropipette allows a different range of measured volume.
Pipette barrel is a tube filled with air present with the pipette tip. Pushing the plunger forces out a volume of air and releasing the plunger lets that air come back in the barrel. There are two stops in the plunger which are used differentially for taking up liquid in forward and reverse pipetting.
Pipette tips are tips attached with micropipette for taking in liquid and then transferring it from one place to another. Different size tips are used for taking in different volumes of liquid.
Micropipette tips are usually available in 3 colors that are white, yellow, and blue. A specific micropipette tip is used for measuring a specific volume of liquid.
White Micropipette tips:
White micropipette tips are used for P2, P10, and P20. They micropipette tips are further divided into micro white tips and medium white micropipette tips. Micro white tips are used for P2 and P10 size micropipette used for taking volume from 0.2 µ to 10 µ. Medium white micropipette tips are used for P20 micropipette measuring a volume range of 2 µ to 20 µ
Yellow Micropipette tips:
Yellow micropipette tips are used for P100 and P200 micropipettes. These tips can be used to take a liquid volume from 20 µ to 100 µ.
Blue Micropipette tips:
Blue micropipette tips are used for P 1000 micropipettes. These tips are used to take a high liquid volume from 200 µ to 1000 µ. These are the micropipettes tips that can take in liquid volume even in milliliters.
Volume adjustment on Micropipette
For adjusting the proper volume rotate the volume adjustment dial present at the top of the micropipette. It can be rotated right or left to decrease or increase the volume. The volume that is going to be measured by micropipette is shown on the digital layout. As the volume adjustment dial is rotated the number on the digital layout is also changed. The volume you have set is shown on the digital readout.
On all sizes of micropipettes, the digital readout has three numbers. These numbers correspond to different volumes set on different size pipettes.
- In a P1000, the Bottom number refers to 10’s of µ (microliters)| Middle number refers to the 100’s of µ whereas the top number refers to 1000’s of µ.
- In a P200, the bottom number refers to 1’s of µ | Middle number refers to the 10’s of µ whereas the Top number refers to 100’s of µ.
- In a P20, the Bottom number refers to 0.1 of µ | Middle number refers to the 1’s of µ whereas the Top number refers to 10’s of µ.
SOP About How To Use A Micropipette:
Here is how to use a micropipette. For transferring a measured amount of liquid micropipette tips need to be attached with the shaft of the micropipette. Liquids are never drawn directly to the shaft. Different colored tips are available. Their color specifies the volume limits they can measure. White tips are used for P2, P10, and P20. Yellow-colored tips are used for P100 and P200. Blue colored micropipette tips are used for P1000. The tips are present usually in a sterilized form in a plastic box with a cover. The cover should be closed to prevent any chance of airborne contamination.
Attaching Micropipette Tip
Micropipette tips are attached with micropipette shaft according to the following procedure or SOP:
- First of all, select the proper and correct sized tips.
- Open the box having micropipette tips without touching the micropipette tips.
- Micropipette shaft is inserted into the tip and then is pressed down firmly so that the tip will attach to the shaft properly.
- Take the micropipette out of the box having tip attached with the shaft.
- Close the cover of the box without touching the tips with your hands.
Measuring the volume of a liquid
Before measuring the liquid and transferring it make sure that you have selected the appropriately sized micropipette, adjusted the correct volume, and the tip is inserted properly on the shaft.
For measuring the volume of a liquid follow following steps:
- After carrying the micropipette properly in your working hand press the upper knob to the first stop.
- Immerse the micropipette tip into the solution approximately 3 mm deep into the sample liquid.
- Slowly release the pressure from the knob and let it come to the initial position.
- The liquid will start coming into the tip. Watch the solution that is drawn up into the tip. Don’t release the plunger too quickly as it can cause bubble formation in the solution and can also cause splashing of the solution on the non-sterile shaft.
- Withdraw the tip from the solution. The liquid will now be in the micropipette tip.
Note: The most serious and common error is depressing the plunger to the second stop before filling the micropipette. Avoid this error.
Transferring volume of a liquid
For dispensing the liquid follow these steps:
- Take the volume of liquid in the tip then place the tip against the sidewall of the container or tube in which the liquid needs to be shifted. Surface tension will also help to dispense the liquid. Don’t dispense the liquid into the air.
- Smoothly depress the plunger to first stop then take a pause and after that, further depress the plunger to the second stop. Most of the content is released at the first stop the second stop ensures that the last drop of the liquid is also being released.
- Take the tip out of the container and return the plunger to the initial position.
- Remove and discard the tip into the waste container by using tip ejector.
Types of Pipetting Techniques
There are two pipetting techniques known as forward pipetting and reverse pipetting.
- Adjust the required volume with the help of volume adjustment knob.
- Attach the suitable tip to the pipette.
- Genteelly press the knob to the first stop.
- Dip the tip into the solution to a certain depth.
- After this slowly release the pipette knob till the starting position.
- The liquid will come in tip. Withdraw the tip from the liquid.
- Move the pipette to the receiving vessel.
- Dispense the liquid from the tip by pressing the knob to the second stop. This will remove the last drop of liquid present in the tip.
- After transferring liquid eject or release the tip with the help of tip ejector.
Reverse pipetting is usually done for volatile liquids. It is done as follows:
- Adjust the required volume by volume adjustment knob.
- Attach the suitable tip to the pipette.
- Press the knob to the second stop by passing the first stop.
- Dip the tip to the solution to a certain depth.
- After this slowly release the pipette knob till the starting position.
- Withdraw the tip from the liquid.
- Move the pipette to the receiving vessel and press the knob to the first stop to dispense the liquid from the tip. Be careful while pressing the knob. Knob should be pressed to the first stop not to the second stop.
- Discard or eject the tip with the help of tip ejector.
Types of Micropipettes
Following are some of the types of micropipettes:
Air displacement Micropipettes
Air displacement micropipettes are precise and very accurate micropipettes used to measure a liquid volume range from 0.1 – 5000 µ. These micropipettes are available in different sizes from P 2 to P 5000 as mentioned above. These pipettes are used to measure and transfer the volume of liquid with the help of disposable micropipette tips. Micropipettes tips are available in different colors and sizes and are compatible with specific micropipettes.
An internally built piston displaces a specific volume of air as set by volume adjustment knob and shown on the reading layout.
When the plunger is depressed the piston moves upward creating a vacuum. The liquid moves inside the tip when the user releases that pressure. This liquid can be released from the micropipette tip by pressing the knob.
Types of Air displacement Pipettes:
The types of air displacement pipettes include:
- Volume handled micropipettes
- Adjustable of fixed micropipettes
- Single-channel, multi-channel or repeater micropipettes
- Manual or electronic micropipettes
- Cylindrical or conical tips micropipettes
- Locking or standard micropipettes
While performing repetitive pipetting there is always a risk of musculoskeletal disorders to minimize this disorder electronic micropipettes are used. Electronic micropipettes are available both as single-channel and multi-channel micropipettes. Volume adjustment can be done electronically and the adjusted volume is shown digitally on the digital readout.
Positive displacement Micropipettes
Positive displacement micropipettes work on the same principle as air displacement micropipettes do but these micropipettes are less commonly used in the laboratory. These micropipettes are used to avoid contamination of volatile or viscous substances at very small volumes such as DNA contamination.
The major difference between air displacement micropipettes and direct displacement micropipette is that direct displacement micropipettes have a disposable microsyringe in place of micropipette tip. These micro syringes are made up of plastic and consist of a piston and a capillary and it directly displace the liquid sample.
These are micropipettes are a recent introduction to micropipette fields invented by Alar Ainla in the biophysical technology lab of the Chalmers University of Technology Sweden. Polydimethylsiloxane (PDMS) is used to make these micropipettes. Multiple solutions can be loaded and switched in demand in microfluidic pipettes. A localized flow zone is created at the tip of the pipette to allow constant control of nano liter environment. This environment is directly operated in front of the pipette.
Despite the precision and accuracy of micropipettes, there are chances of human error which can affect the results. To limit this error robots are used for pipetting purposes. These robots are capable of operating micropipettes as humans can operate minimizing the chances of human error.
Micropipettes are used to measure and transfer the volume of liquid with great accuracy and precision in a laboratory. Accuracy in micropipette measurement is necessary as any discrepancy in volume dispensed can affect the result of the experiment. To ensure accuracy, it is necessary to check micropipette calibration after every week. The calibration of micropipette is done as follows:
- Prepare the micropipette by adjusting the volume with the help of volume adjustment knob and then attach an appropriate size tip with the shaft of the micropipette.
- Take a Petri plate and note its weight by placing the dry Petri plate on weighing balance.
- Tare the weight of the Petri plate; the weighing balance will show zero reading now.
- Aspirate the adjusted volume from micropipette.
- Dispense the volume of liquid taken on Petri plate.
- Note the reading shown by the Petri plate.
- Take at least six readings of the same volume.
Following calculations need to be done after taking the weighing balance readings:
Dispense volume = Final weight – weight of Petri plate
The dispense volume is in the gravimetric unit so convert this into volume unit. For converting the gravimetric unit into a volumetric unit multiple the calculated gravimetric value with a correction factor Z which is the density of water depending upon the atmospheric pressure and temperature.
Vi = mi x z
Where Vi is the volume value, mi is the gravimetric value, and Z is the correction factor.
Calculating the arithmetic average value of volume:
For calculating the average values from the volume, divide the sum of volume values by the number of measurements.
number of measurements
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