Micropipettes Components, Types, Applications, Techniques

What is a micropipette?

A micropipette is a common yet indispensable laboratory device used to transfer microliter-range liquid quantities accurately and precisely. Micropipettes are available in single-channel and multi-channel varieties. Multichannel micropipettes are recommended for ELISA (diagnostic test), molecular screening, kinetic studies, and DNA amplification. Single-channel micropipettes are utilised in laboratories that conduct research related to molecular biology, microbiology, immunology, cell culture, analytical chemistry, biochemistry, and genetics.

What is the purpose of pipetting?

Pipetting is the process of measuring and dispensing a specific volume of liquid using a laboratory tool called a pipette. The primary purpose of pipetting is to accurately and precisely measure and dispense small volumes of liquid, which is important in many laboratory applications such as molecular biology, biochemistry, genetics, and cell culture.

In a laboratory setting, pipetting is used for a variety of tasks, including:

• Preparing solutions and reagents: Pipettes are often used to measure and dispense specific volumes of liquid to make solutions or prepare reagents for experiments.
• Diluting samples: Pipettes are used to accurately measure and dispense a specific volume of a sample and then add an appropriate volume of solvent to create a dilute solution.
• Mixing samples: Pipettes are used to transfer small volumes of liquid from one container to another, which is useful for mixing samples before analysis.
• Transferring samples: Pipettes are used to transfer small volumes of liquid from one container to another, which is useful for preparing samples for analysis or storage.
• Measuring and dispensing small volumes: Pipettes are often used to measure and dispense small volumes of liquid, particularly in applications such as PCR, ELISA, and other types of assay.

Overall, pipetting is a critical tool in many laboratory applications as it allows scientists to handle small volume of liquid precisely and accurately, as well as creating specific solutions/reagents and measuring samples with high precision in order to achieve accurate and reliable results in their experiments.

How to Use a Micropipette?

Here are some general steps for using a micropipette:

1. First, make sure the micropipette is clean and properly calibrated. Most micropipettes have a small knob or button that can be adjusted to set the volume.
2. Next, select the appropriate micropipette tip size and attach it to the micropipette. Tips come in different sizes, so make sure to choose one that is appropriate for the volume of liquid you will be measuring.
3. Turn on the micropipette, if it is electronic one.
4. Hold the micropipette in a comfortable and stable position, with your thumb on the thumb wheel or electronic button, and your index finger on the top of the micropipette.
5. To draw up a specific volume of liquid, immerse the tip of the micropipette into the liquid, and then use the thumb wheel or electronic controls to slowly draw the liquid into the micropipette. Be sure to keep the tip of the micropipette submerged in the liquid at all times.
6. To release the liquid, press the button or thumb wheel to release the liquid from the micropipette tip. Make sure to direct the micropipette tip to the desired container or location of the release.
7. Repeat the steps as necessary, and after you finish using the micropipette, make sure to release all liquid and discard the used tip. Clean the micropipette with appropriate solution according to the manufacturer’s instruction, if necessary, and be ready for the next use.
8. Most importantly, use the micropipette in a safe and responsible manner, take care of the cleanliness of the micropipette and use it with caution in order to avoid contamination or damage.

Keep in mind that different types of micropipettes may have slightly different instructions for use, so be sure to consult the manufacturer’s instructions for specific details. If you are new to using micropipettes, it’s a good idea to get some hands-on training from a more experienced researcher or lab technician.

Components of a micropipette

Micropipettes are available in many designs and sizes. Nevertheless, there are a few components that are fundamental and shared by all micropipettes. These components include the plunger, the digital display, the tip cone, the tip ejector, and the grip. As an accessory, certain micropipettes include a calibration tool and a micropipette stand.

1. Plunger

The plunger is able to perform the following two roles:

• Adjusting the volume: Turn the plunger clockwise or anticlockwise to decrease or increase the volume level. A distinct click sound after each volume adjustment ensures a an exact volume setting, and also prevents any unintentional volume changes.
• Liquid aspiration/ dispensing: The process involves pressing and depressing the plunger in order to aspirate or disperse liquid.

2. Tip Ejector

The internal mechanism inside the micropipette is not in direct contact with the liquid/sample. Instead the tip of a disposable pipette is utilized for drawing the liquid into and disperse through the micropipette. In order to facilitate the safe, easy and rapid expulsion of tips micropipettes are equipped with a tip ejection mechanism. Tips can be easily taken off the micropipette pressing the button for tip ejection.

3. Volume Display

This indicates the volume of the liquid that will be dispersed or aspirated.

4. Tip Cone

The tip cone offers fitting for the tips. A micropipette equipped with tip cone that is universal is highly recommended as it improves the ability of the instrument to work with all the tip types.

Types of micropipette

A. Classification of micropipette based on Working Principle

Based on the Working Principle Micropipettes can be classified into two categories:

1. Air Displacement Micropipette:

The micropipette type works by using the air displacement principle. It is comprised of a piston that is able to aspirate and disperse liquid samples while an air pocket is moved upwards and downwards and back. The internal mechanism inside the pipette doesn’t come directly into contact with liquid or sample. Instead an unreplaceable pipette tip is employed to pull the liquid into and out of the pipette.

2. Positive Displacement Micropipette:

In these micropipettes, the piston is in direct contact with the sample. The disposable tip of a positive displacement micropipette an incredibly small syringe that is comprised of a capillary, and the piston (movable inside) that is able to directly move the liquid.

B. Classification of micropipette based on Operating Mechanism

Based on the Operating Mechanism, micropipettes can be classified in two types;

1. Mechanical Micropipette

The micropipettes are operated by hand using a spring piston mechanism.

2. Electronic Micropipette

The electronic micropipette is usually automated. The aspiration and dispensing of liquids is done with single-touch buttons, not manually pressing and depressing the plunger. Electronic pipettes often allow users to develop customized programs for the device, allowing the pipettes to be adapted to various requirements of the application.

C. Classification of micropipette based on Number of Channels

Based on the number of Channels micropipettes fall in two categories:

1. Single Channel Micropipette:

Micropipette with a single channel will be the one with one channel that can be used to aspirate or disperse the liquid.

2. Multi Channel Micropipette:

A multi-channel micropipette is equipped with multiple channels for aspiration or to disperse the liquid. The most commonly used micropipette models that are multichannel include the 12 channel, 8 channel channels and the 16 channel. Multichannel micropipettes can reduce the work of one channel micropipette dealing with large quantities of samples.

D. Types of micropipette based on Volume/Capacity:

Based on the volume/capacity of micropipettes, they are divided into two categories.

1. Fixed Volume Micropipette

In a fixed-volume micropipette, the amount of liquid that is to be dissipated or aspirated is fixed. They are utilized to dispense the same volume of liquid needs to be dispersed multiple times.

2. Variable Volume Micropipette

The micropipette has the specified limit and maximum range of volume. The quantity of liquid to be aspirated and dispensing can be altered (within the range of volume) according to the needs for the individual.

Size and Range / Technical Specifications of Micropipette

Micropipettes are offered in a variety of sizes ranging from 0.1 up to 10,0000 ul. The most commonly used versions of micropipettes that have a single channel of variable volume are listed below with their error tolerances as defined in the ISO-8655-2 standards.

They may be referred to as P10 or P20, P1000 or P5000 pipettes based upon the largest volume of liquid that is aspirated and disseminated by the pipette. For example the 0.5-10ul micropipette is often called P10 pipette.

Volume Range (ul)	Classification	Increment (ul)	Accuracy (± %)	Accuracy (±ul)	CV (± %)	CV ( ± ul)
0.2-2	P2	0.002	2	0.04	1.2	0.024
0.5-10	P10	0.02	1	0.1	0.5	0.05
2-20	P20	0.02	0.8	0.16	0.4	0.08
5-50	P50	0.1	0.8	0.4	0.4	0.2
10-100	P100	0.2	0.6	0.6	0.2	0.2
20-200	P200	0.2	0.6	1.2	0.2	0.4
100-1000	P1000	1.0	0.6	6	0.2	2
500-5000	P5000	10.0	0.6	30	0.2	10
1000-10000	P10000	20.0	0.6	60	0.2	20

The measuring ranges for the micropipettes most frequently used are:

• P10 – 1.0-10.0µL – White tips (slightly translucent)
• P20 – 2.0-20.0 µL – Yellow tips
• P100 -10.0-100.0 µL – Yellow tips
• P200 – 20.0-200.0 µL – Yellow tips
• P1000 – 11000.0 µL –Blue tips (should only be used for volumes 200 or greater)

Working Principle of micropipette

Air displacement micropipettes function via the piston driven air displacement. As the piston pulled down, air contained within the sleeve of the micropipette is released because of the force with which the liquid in the micropipettes ‘ tip is also eliminated.

As the piston is moved upwards there is a vacuum created in the empty space left due to the movement of the piston. The air at on the tips to expand to fill in the empty space. The tip air is replaced by the liquid that is drawn upwards to the tip.

Positive displacement micropipettes work via piston-driven displacement. The piston inside the positive displacement micropipette is directly in touch with liquid. If the piston gets pulled down, liquid that is inside the sleeve of the micropipette is also moved downwards and is ejected from the tips. As the piston moves upwards it draws the liquid with it in an upward direction.

Using an Air Displacement Micropipette

Accurate measurement of liquids is dependent on the proper micropipette use. Air displacement micropipettes operate using the principle of air displacement. The plunger is compressed by the thumb, and when it releases the liquid is drawn towards the tip of a disposable. If the plunger is then pressed again and the liquid is dispersed. Between these stages, there are many smaller steps that assist in making the process of liquid dispensing more precise.

Position 1

• In this, the micropipette is at rest position: You can press-fit the tip of the micropipette, but not touching directly to the edge.

Position 2

• In this, the plunger is depressed till the first stop: To aspirate the liquid at the tip push the plunger until its first stop. Place the pipette tip horizontally within the liquid.
• Release the plunger: Release the plunger slowly while the tip is submerged. The liquid will aspirate into the tip of the pipette. The liquid is then poured into the tip according to the micropipette’s volume that is set.

Position 3

• Depress the plunger: place the tip on the interior wall of the vessel at an angle of steep. The plunger should be pressed slowly to the first stop in order to disperse the liquid. To completely empty the tip push the plunger until next stop. Clean the tip with the wall before taking the tip from the vessel.

Some micropipettes are able to deliver a set amount of liquid. But, the majority of them can be adjusted with their variable volume settings. Variable volume micropipette is available with different ranges of measurement and upper and lower limit of measurement. In these situations, the error percentages can differ as according to the liquid being measured. Dispensing lesser than the lowest limit of the range could result in inaccurate measurements of liquid while attempting to use the upper limit will fill the tip and allow liquid to flow into the pipette’s body.

Cleaning, maintenance and storage of a micropipette

Maintaining and caring for the micropipette is a crucial routine in labs. Following a well-planned maintenance plan will reduce the expense of purchasing the expensive equipment. Cleaning the micropipette is a process that requires time, effort and focus as otherwise, the micropipette could be damaged. It is therefore essential to be careful when cleaning it. This is a complete guide to cleaning micropipettes.

External Cleaning

The majority of the pipette is able to be cleaned using the typical lab cleaning chemicals such as soaps, alcohol or soaps. To ensure complete sterilization you should allow the cleaning solution to sit on the micropipette 10 to 15 minutes before taking it off with a cloth.

Internal Cleaning

Cleaning the inside of the micropipette may be time-consuming as it requires complete disassembly. Additionally, every component of the micropipette must be cleaned in a proper manner based on the fluid used in the sampling.

• Check the instruction manual for precise instructions for micropipette
• Make use of a cotton swab soaked in the cleaning solution and distil water.
• Make sure to lightly grease your pistons using the lubricant supplied upon purchase.
• Assemble all the components and test to make sure the micropipette is operating efficiently.

Contamination Cleaning

If the micropipette is found to be contaminated by a known ingredient, then there’s certain cleaning procedures that need to be followed based on the kind of substance. Cleaning the micropipette with the above procedure won’t suffice if the micropipette is cross-contaminated.

Solution Types	Process to clean
For aqueous solutions, organic solvents and proteins	Rinse the contaminated parts with distilled water or 70 percent ethanol and air dry at approx 60°F temperature.
For infectious liquids	Autoclave the lower section at a temperature of 120°C for 15-20 minutes then allow it to return at room temperature before reassembling.
For radioactive substances	Place the pipette in a solution like Decon and then rinse and air dry.
For nucleic acids	Boil lower parts of micropipette in glycine/ HCI buffer (pH2) for 10 minutes, rinse with distilled water, and air dry.

A clean and well-maintained pipette will protect the lab from potentially harmful substances. This makes the pipette more efficient, durable, and reliable and also reduces the expense of sample collection.

The proper storage of micropipettes is just as crucial as cleaning and calibrating the devices. The micropipette as well as accessories must be kept in a cool, clean and dry area. The storage space should have temperatures ranging between -20°C and 50° degC (from -4 degF up to 120°F) with the relative humidity ranging between 5 and 95 percent. Another important thing to keep in mind is the fact that your instrument needs to be kept in an upright place. To put pipettes away, Microlit recommends the use of its carousel stand Microlit Faveo.

Different pipetting techniques

The most frequently employed pipetting techniques comprise forward pipetting and reverse pipetting. Before we learn about these techniques in greater detail the basic guidelines for pipetting that are listed below will be worth mentioning.

1. The plunger should be released and pressed gradually, always especially when working with viscosity reagents or solutions. Check that the plunger doesn’t snap.
2. Make sure that the tip is secured to the cone.
3. Before beginning your experiment, you should fill then empty your tip three times with the reagent , or solution you’ll be pipetting.
4. Make sure to hold the micropipette upright in a position while you aspirate. The Grippy should rest on your index finger.
5. Check your tips and the pipette as well as the solution/reagent are all at the same temperature.

Forward Pipetting Technique

1. For aspiration of the liquid from the tip push the plunger until at least the initial stop. Place the pipette tip horizontally into the fluid.
2. Release the plunger slowly while the tip is submerged. The liquid will aspirate into the tip of the pipette.
3. To disperse the liquid put the tip against the inside of the vessel receiving the liquid at an acute angle
4. The plunger should be pressed slowly until the first stop in order to disperse the liquid.
5. To fully empty the tip push the plunger up to the second stop.
6. Cleanse the tip with the inside of the wall, while taking the tip from the vessel.

Reverse Pipetting Technique

The reverse method is ideal for dispensing solutions or reagents with an extremely high viscosity or tendency to form foams easily. It is also recommended to dispensing small amounts.

1. To expel the liquid from the tip, push the plunger to the next stop, then immerse the tip of the pipette in the liquid.
2. The plunger should be released slowly as the tip is submerged. The liquid will aspirate into the tip of the pipette.
3. To disperse the liquid, put the tip of the tube on the wall in the tube. It should be at an upward angle.
4. The plunger should be pressed slowly until the first stop.
5. Wipe the tip clean on the inside of the wall, while taking the tip from the vessel.

Note: Any residual liquid stays in the end. It is not part of the volume of dispensing.

Choose the right micropipette

Since micropipettes can be used for many different applications The issue is how to select the appropriate micropipette for the specific job.

To do this, follow our easy, step-by-step guide. The first step is writing down the specifics of the experiment you’re conducting using the micropipette. It should include details about the amount of sample to be taken, the volume of the sample to be transferred as well as the number of duplicates and whether you require sterile conditions and other details. In this manner can aid in deciding the best micropipette for you.

Selecting the Type of Micropipette

Micropipettes are generally classified into two kinds namely air Displacement Micropipettes as well as Positive Displacement Micropipettes. It is possible to choose the best type by matching the application to the research you want to conduct.

Next Steps

When you’ve decided on the best type, let’s examine the various sub types that are that are available, based on factors such as the amount of material of work to be done, the quality of tips, the manual or electronic pipettes as well as other factors. This is where the specific details about the procedure will be useful.

1. An adjustable micropipette or a fixed micropipette

You could either choose the micropipette with a volume that is already fixed, or one that is able to be altered as needed. If you typically employ a single volume like 100 ul to conduct your experiments, opt for an instrument with a fixed volume, such as MICROLIT RBO Fixed Volume (Single Channel). If your project requires you working with several volumes, go for the flexible or variable pipette such as MICROLIT RBO Variable Volume (Single Channel or Multichannel).

2. The volume to be handled

As a general rule it is recommended to select the smallest pipette that can handle the required quantity because if the quantity is near the capacity limit of the micropipette accuracy and precision of readings diminish.

• Single channel or multichannel micropipettes: The choice of this type is according to the quantity of replicates or samples you’re working with. Single channel micropipettes such as those of the MICROLIT RBO Single channel (Fixed volume and variable) are utilized when the quantity of samples is lower, while many sample or even well plate could be handled with ease with multichannel micropipettes such as those of the MICROLIT RBO Multichannel (8-channel and 12-channel).

3. Micropipette Tips

Once you’ve picked the micropipette you want to use then this is the next critical decision you have to make. Micropipette Tips are graduated or not either universal or pipette specific and with filter or no filter, sterile or not, and so on. If the procedure requires aseptic, sterile conditions, you should make use of sterile and filter-filtered tips. Universal tips can be utilized for a range of pipettes. It is crucial to make sure that how much capacity the tip has to that of the micropipette prior making use of.

4. Accuracy with specific tolerance

Micropipettes are made to work with accuracy just a few percent (generally less than 5 percent) of the value you want of the sample. Be sure that your micropipette has the same level of accuracy that you require for the sampling.

Micropipette calibration

Micropipette calibration is a crucial element in every laboratory’s procedure to guarantee precise and precise results from pipetting. To ensure that the sample is accurate it is essential to verify the calibration of the pipette each month or after between a few months. Making a correct micropipette calibration as per ISO 8655 standard requires special conditions in the ambient. Things like air pressure or humidity, temperature, or even altitude influence the outcomes of the pipetting. The following text will explain the material or equipment needed and the various steps you should follow and verify the calibration of a micropipette.

Here’s a list things you’ll need to test a micropipette

• Micropipette that must be calibrated and tips
• Distilled Water
• Clean Beaker
• Thermometer
• Distilled Water Density Chart and Temperature
• Semi-Micro Balance having an accuracy of at minimum 0.0001 grams
• Notebook to write down the results
• Calculator

How to calibrate a pipette using a balance

• Let the distilled water sit in the refrigerator for 15 to 20 minutes to guarantee the same temperature.
• Make use of the thermometer to gauge your temperature for the distillated water.
• Empty and clean the beaker on top of the balance.
• Consider the weight of the beaker.
• Make sure the micropipette is in good condition and functioning properly.
• While filling up the micropipette to the size that can be calibrated (1ml for instance) using the distilled water ensure that there aren’t any air bubbles in the flask , and also in the micropipette.
• Dispense the water distilled into the beaker by using the micropipette.
• Utilize the balance to determine the weight of the water that has been distilled.
• Repeat the procedure 5-10 times and take note of every measure.
• Utilize this formula: V = Z * W to determine the amount that is dispensing through the pipette.
• W is the amount of distillate water.
• Z stands for “conversion factor based on the density of the water” and
• V is the amount of water that is dispensed.
• The temperature of water is the determining factor for its density, this is why it’s crucial to keep the water’s temperature as stable as it is. If you are using software, automatically handle that.
• Take the test results and average them. If you completed 10 tests, then divide the total number of tests by 10.
• The average weight of water that is distilled can be represented as the number W. It is possible to do the calculation manually or employ software.
• Next, you must to determine the accuracy of the pipette, either by hand or using software. If you must do it manually, follow the formula A = 100x Vavg/V0
• A is a reference to Accuracy.
• Vavg is the standard calculated volume.
• V0 is the number that is assigned to the pipette in order to disperse.
• It is suggested that the number be between 99 and 101%..
• Note the results. If the micropipette has been properly calibrated, it is safe to use it. If you find that the results are inconsistent don’t make use of the pipettes, or correct them before you use it again.

Sterilization Process of a micropipette

• The micropipettes are sterilized using autoclaving at temperatures of 121°C (252degF) or 15 PSI for a period of between 15 and 20 minutes.
• There is no special preparation required.
• You can utilize steam sterilization bags when you require. When the autoclave is done, the pipette needs to be chilled to ambient temperature for at minimum two hours. Before you pipetting, ensure you have the pipette clean.
• It is suggested to test the calibration at the end of each sterilization process to get the greatest precision and accuracy.

When is micropipette calibration required?

To maintain the high quality of the results of the sampling The micropipettes need to be in good working order and calibrated properly. Normally, the period that a micropipette has to be calibrated is determined by several factors:

• Frequency of pipette use
• The type of liquid that is dispensable via pipettes
• Care and handling of the pipette
• Pipette-related applications that require high accuracy also require more frequent calibration

Regulations and standards issued by organizations like The FDA as well as ASTM International provides minimum requirements to ensure the accuracy of laboratory test results. Regulations stipulate that all laboratory equipment used for sampling and production, including micropipettes should be regularly checked at regular intervals.

In the same sequence, the Clinical and Laboratory Standards institute (CSLI) has provided the guidelines for multi-channel and single channel micropipettes. The guidelines state that these micropipettes should be calibrated every 3 to six months. In addition, a minimal of 2 volumes should be tested using 10 copies in both nominal and low settings.

Setting a suitable frequency of calibration minimizes the risk of a wrong liquid delivery in the laboratory . It also ensures transparency, accountability, and confidence in the result.

Uses of Micropipette

Micropipettes are appropriate for use in laboratories that handle minute sample volumes (0.1 to 1000 µl). These are some areas where micropipettes are utilised:

• Clinical and microbiological laboratory: The clinical and microbiological laboratory is utilised for a variety of diagnostic testing. It is used to transfer reagents/samples in serological testing (antibody/antigen detection utilising quick test kits, ELISA tests, etc.), molecular tests (sequencing, PCR, blotting techniques, electrophoresis, etc.), and virus growth in cell lines.
• Chemical laboratory: These laboratories utilise micropipettes to manipulate viscous and volatile liquids for a variety of studies.
• Forensic laboratory: These laboratories analyse blood, tissues, and fibres with the aid of micropipettes. Micropipettes are also useful for determining the genetic makeup of victims and analysing DNA and fingerprints obtained at a crime scene.
• Pharmaceutical laboratory: Pharmaceutical laboratories utilise micropipettes for the manufacturing of medicines and drugs. These also use micropipettes for assessing the quality control (QC) of the manufactured medications.
• Food and beverage industry: In the food and beverage sectors, micropipettes serve primarily as liquid handling instruments, with quality control tests constituting another application area.

Advantages of micropipette

The micropipette is a trusted device for dispensing precise amounts of liquid. It enhances the effectiveness of testing with efficiency. Other benefits that a micropipette provides are described below:

• Time Saving
• Ergonomics
• Accuracy & Precision
• Ease of Use

What you shouldn’t do with micropipettes?

• You should not drop your pipette. If you drop your pipette, it could cause it to not be in the calibration. Be delicate and cautious.
• Aspirate the contents into the pipette. It happens, we’ve seen it. If you’re working with chemicals that are harsh and it gets stuck in the pipette could cause to the build-up of substances and cause inaccurate results.
• Move the dial up or below the volume limit. This could cause damage to the volume indicator as well as other parts of the pipette.
• Jam pipette tip into pipette. This could not only harm the pipette’s tip but also more importantly, the pipette shaft. What this means is that you will most likely require a more fit pipette tip.

Make sure you treat your micropipette properly. It’s an extremely precise instrument, and, as such, careful care of its performance has to be observed. If you find that it’s out of performance or isn’t performing as it used to, it’s time to have it checked or calibrated. If you need further help regarding how to use a micropipette, we’ve created an extensive Guide to Proper Pipetting resources to better help you.

Precautions To Be Taken While Using Micropipettes

Micropipettes are pricey! To maintain their functionality, it is essential that they be handled with care. Please follow these rules to maintain their functionality:

• Never alter the volume beyond the micropipette’s range. No micropipette should be set below zero microliters. Never adjust the P20 over 20  µ µL, the P200 above 200  µL, or the P1000 above 1 mL.
• Never force the volume control dial to turn. If the knob gets difficult to adjust, you are likely surpassing the pipette’s capacity or the pipette is destroyed. Communicate the issue to your instructor.
• Avoid dropping the micropipette.
• Always use the micropipette with a steady motion. This will aid in providing accurate measurements and prevent pipette damage. There should not be any crackling sounds.
• Always store pipettes vertically.
• Never place a pipette containing liquid on the bench.
• Always select the correct size pipette for the volume being measured.
• Always dispose of cigarette ends in the correct waste receptacle.

FAQs

References

• https://www.microlit.us/micropipette-product-guide/
• https://paramedicsworld.com/biochemistry-practicals/micropipettes-guidelines-of-micropipetting/medical-paramedical-studynotes
• https://solutions.pipette.com/micropipette/