Guide to Pipetting Techniques | Pipette.com

06 May.,2024

 

Guide to Pipetting Techniques | Pipette.com

Guide to Pipetting


Understanding Pipettes


Pipette terms:

Adjustment - altering the pipette so that the dispensed volume is within the specifications.

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Air Displacement Pipettes - are meant for general use with aqueous solutions. In air displacement pipettes, a certain volume of air remains between the piston and the liquid.

Aspirate - to draw up the sample.

Blow-out - to empty the tip completely.

Calibration check - checking the difference between the dispensed volume and the selected volume.

Dispense - to deliver the sample.

Positive Displacement Pipettes - are used for high viscosity and volatile liquids. In positive displacement pipettes, the piston is in direct contact with the liquid.

Air Displacement Pipettes

Air Displacement Pipette, used for standard pipetting applications, is highly accurate.

However, conditions such as temperature, atmospheric pressure as well as the specific gravity and viscosity of the solution may have an effect on the performance of air displacement pipettes.

How does an Air Displacement Pipette work?

  • The piston moves to the appropriate position when the volume is set.
  • When the operating button is pressed to the first stop, the piston expels the same volume of air as indicated on the volume setting.
  • After immersing the tip into the liquid, the operating button is released. This creates a partial vacuum and the specified volume of liquid is aspirated into the tip.
  • When the operating button is pressed to the first stop again, the air dispenses the liquid. To empty the tip completely the operating button is pressed to the second stop (blow out).

Factors affecting the accuracy of Air Displacement Pipettes:

Temperature
The most important factor in pipetting accuracy is the liquid temperature. The figure below shows the change in volume when the liquid has a different temperature than the pipette and air. If the temperature of the liquid, pipette and air is the same, the accuracy is not significantly affected.

Density
Density is the mass/volume ratio of the liquid. The density varies according to the temperature and air pressure. Typically, the density of water is 0.996 kg/dm3, for ethanol 0.79 kg/dm3 and for sulfuric acid (H2SO4) 1.85 kg/dm3.

Altitude
The geographic altitude affects the accuracy through the air pressure. The air pressure decreases at higher altitudes and the conversion factor Z decreases as well. The boiling point of some liquids can also change to quite close to room temperature, increasing the evaporation loss dramatically.

Positive Displacement Pipette

Positive Displacement Pipette, is used for applications like PCR and other DNA amplification techniques. The micro-syringe tips used in positive displacement pipettes are disposable. This helps to avoid sample-to-sample cross-contamination (also known as sample carry-over), and contamination due to the aerosol effect.

How does a positive displacement pipette work?

  • The piston moves to the appropriate position when the volume is set.
  • When the operating button is pressed to the stop, the piston descends to the tip opening.
  • After the tip is immersed into the liquid, the operating button is released. The plunger is then raised and a partial vacuum is created. This causes the liquid to enter the tip.
  • When the operating button is pressed again, the piston descends, expelling the liquid from the tip.

General guidelines and Pipetting techniques

General guidelines

Check your pipette at the beginning of your working day for dust and dirt on the outside. If needed, wipe with 70% ethanol.

Set the volume within the range specified for the pipette.

Hold the pipette so the "grippy finger rest" rests on your index finger.

To maximize accuracy, the pipette, tip and liquid should be at the same temperature.

Check that you are using tips recommended for this pipette. To ensure accuracy, use only high-quality tips made from contamination-free polypropylene.

Tips are designed for single use. They should not be cleaned for reuse as their metrological characteristics will no longer be reliable.

*Pre-rinsing (1-3 times) the tip with the liquid to be pipetted may improves accuracy, especially when using positive displacement tips.

Avoid turning the pipette on its side when there is liquid in the tip. Liquid might go to the interior of the pipette and contaminate the pipette.

Avoid contamination to or from fingers by using the tip ejector and gloves.

Store pipettes in an upright position when not in use. Pipette stands are ideal for this purpose.

Check calibration regularly, depending on the frequency of use and on the application, but at least once a year. If used daily, a three-month interval is recommended. Follow the instructions for recalibration in the manufacturer�s instruction manual.

Forward Pipetting technique

  • Press the operating button to the first stop.
  • Dip the tip into the solution to a depth of 1 cm, and slowly release the operating button. Wait 1-2 seconds and withdraw the tip from the liquid, touching it against the edge of the reservoir to remove excess liquid.
  • Dispense the liquid into the receiving vessel by gently pressing the operating button to the first stop and then press the operating button to the second stop. This action will empty the tip. Remove the tip from the vessel, sliding it up the wall of the vessel.
  • Release the operating button to the ready position.

Reverse Pipetting technique

The reverse technique is used for pipetting solutions with a high viscosity or a tendency to foam. Reverse pipetting is only possible with air displacement pipettes

  • Press the operating button to the second stop.
  • Dip the tip into the solution to a depth of 1 cm, and slowly release the operating button. This action will fill the tip with a volume that is larger than the set volume. Wait 1-2 seconds and withdraw the tip from the liquid, touching it against the edge of the reservoir to remove excess liquid.
  • Dispense the liquid into the receiving vessel by pressing the operating button gently and steadily to the first stop. This volume is equal to the set volume. Hold the button in this position. Some liquid will remain in the tip, and this should not be dispensed.
  • The liquid remaining in the tip can be pipetted back into the original solution or disposed together with the tip.
  • Release the operating button to the ready position.

Repetitive Pipetting technique

This technique is intended for repeated pipetting of the same volume. Can only be used with electronic and repeator pipettes.

  • Press the operating button to the second stop.
  • Dip the tip into the solution to a depth of 1 cm, and slowly release the operating button. Withdraw the tip from the liquid, touching it against the edge of the reservoir to remove excess liquid.
  • Dispense the liquid into the receiving vessel by gently pressing the operating button to the first stop. Hold the button in this position. Some liquid will remain in the tip, and this should not be dispensed.
  • Continue pipetting by repeating steps 2 and 3.

Pipetting whole blood

Use forward technique steps 1 and 2 to fill the tip with blood (do not pre-rinse the tip). Wipe the tip carefully with a dry clean cloth.

  • Dip the tip into the blood and press the operating button to the first stop. Make sure the tip is sufficiently below the surface.
  • Release the operating button slowly to the ready position. This action will fill the tip with blood. Do not remove the tip from the solution.
  • Press the operating button to the first stop and release slowly. Repeat this process until the interior wall of the tip is clear.
  • Press the operating button to the second stop and completely empty the tip. Remove the tip by sliding it along the wall of the vessel.
  • Release the operating button to the ready position.

Recommendations for Pipetting different Solutions / Compounds

Solution/Compound Examples Pipette Tip Technique Comments Aqueous solution Buffers, diluted salt solution Air Displacement Standard Forward   Viscous solution Protein and nucleic solutions, glycerol, Tween 20/40/60/80 Air Displacement Pos. Displacement Standard wide orifice Pos. Displacement Reverse Pipette slowly to avoid bubble formation. Volatile compounds Methanol, Hexane Air Displacement Pos. Displacement Filter Pos. Displacement Forward Pipette rapidly to avoid evaporation. Carbon filter tips prevent vapors from going into the Pipette. Nucleotide solutions Genomic DNA, PCR Products Air Displacement Pos. Displacement

Filter or wide orifice

Pos. Displacement Forward For genomic DNA wide orifice should be used to avoid mechanical shearing. Radioactive compounds Carbonate, H-thymidine Air Displacement Pos. Displacement Filter Pos. Displacement Forward   Acid / Alkalis H2SO4, HCI, NaOH Air Displacement Filter Forward   Toxic samples   Pos. Displacement Pos. Displacement Forward  

Pipetting guidelines for selected compounds

Body Fluids

Whole Blood

  • Pipette + tip combination: choose an air displacement pipette and a standard or wide orifice tip.
  • Technique: use the whole blood pipetting technique. Reverse pipetting should be used if high accuracy is needed.
  • Notice: some blood can remain in the tip and on the outer surface. Wipe the tip against the edge of the vessel to remove excess liquid outside the tip before dispensing.

Serum

  • Pipette + tip combination: choose an air displacement pipette and a standard or wide orifice tip.
  • Technique: use the whole blood pipetting technique. Reverse pipetting should be used if high accuracy is needed.
  • Notice: Residual serum can sometimes be found on the outer surface of the tip. Wipe the tip against the edge of the vessel to remove excess liquid outside the tip before dispensing.

Oily Fluids

Glycerol

  • Pipette + tip combination: choose an air displacement pipette and a standard or wide orifice tip.
  • Technique: For high accuracy of performance, use the reverse pipetting technique.
  • Notice: Oily fluids are difficult to pipette because of formation of air bubbles. Filling must be done very slowly to prevent air bubbles. Wipe the tip against the edge of the vessel to remove excess liquid outside the tip before dispensing. The use of a positive displacement pipette and tip is also useful for pipetting glycerol.

Tween 20, 10% solution

  • Pipette + tip combination: choose an air displacement pipette and a standard or wide orifice tip.
  • Technique: use the reverse pipetting technique.
  • Notice: Tween has a very high viscosity; to make pipetting easier, it should be diluted to a 10% solution. In any case pipetting will not be accurate; some liquid will stay inside the tip. Aspiration and dispensing should be done slowly. The use of a positive displacement pipette and tip is also advisable for pipetting Tween 20.

Bronidox L, 10% (preservative)

  • Pipette + tip combination: choose an air displacement pipette and a standard or wide orifice tip
  • Technique: use the reverse pipetting technique.
  • Notice: Bronidox L is very viscose; the aspiration and dispensing should be done slowly or a positive displacement pipette and tip should be used.

Salt solutions

10 x PBS, 0.1M NaCl, 3M

  • Pipette + tip combination: choose an air displacement pipette and a standard tip.
  • Technique: use the forward pipetting technique. Pre-wetting of the tip before aspiration increases accuracy.

Concentrated acids and bases

H2SO4

  • Pipette + tip combination: choose an air displacement pipette and a filter tip.
  • Technique: use the forward pipetting technique.

NaOH

  • Pipette + tip combination: choose an air displacement pipette and a filter tip.
  • Technique: use the forward pipetting technique.
  • Notice: some acids or bases vaporize easily (e.g. trifluoroacetic acid). Do the pipetting quite rapidly to minimize vapor formation.

Nucleic acids

For genomic DNA wide orifice should be used to avoid mechanical shearing.

  • Pipette + tip combination: choose an air displacement pipette and a filter tip or a positive displacement pipette and tip.
  • Technique: use the forward pipetting technique.
  • Notice: for genomic DNA wide orifice tips can be used to eliminate mechanical shearing.

Volatile compounds

  • Pipette + tip combination: choose an air displacement pipette and filter tip or positive displacement pipette and tip.
  • Technique: use the forward pipetting technique.
  • Notice: 1.) To get accurate results, calibrate the pipette with the volatile compound you want to pipette. If you use air displacement pipettes, aspirate and dispense the liquid a few times keeping the tip in the liquid. By doing so, the air inside the pipette will be saturated with vapor of the volatile compound. 2.) Pipette rapidly to avoid evaporation when using air displacement pipettes. 3.) It is recommended to use positive displacement pipettes for highly volatile compounds, since the built-in piston tip is in direct contact with the liquid.

Preventing cross-contamination

Pipette-to-sample

A contaminated pipette or contaminated tips can cause contamination of samples.
Prevention:

  • Use sterilized tips or sterilized filter tips and if possible autoclave the pipette.
  • Change the tip after pipetting of each sample.

Sample-to-pipette

Samples or aerosols from samples can enter the cone of the pipette.
Prevention:

  • Keep the pipette vertical when pipetting in order to prevent liquid from running into the pipette body.
  • Release the push-button slowly.
  • To avoid aerosol contamination, use filter tips or use a positive displacement pipette and tips.
  • Store the pipette vertically.

Sample-to-sample (carry-over)

The remains of sample A can mix with next sample B inside the tip and may cause a false test result. Prevention:


  • Change the tip after each sample.
  • If you suspect that your pipette is contaminated, autoclave or clean your pipette.

Tip Selection

One of the most important factors in accurate Pipette measurement is the Tip. The Pipette and the Tip form a System. The best Pipetting results are naturally achieved by those manufactured together to ensure complete compatibility between Pipettors and Pipettor Tip.

Many tips on the market look fine, but when studying them more carefully, it appears that the price really reflects quality. Many of the cheap tips may have flashes, protrusions, and scratches, air bubbles, be bent or contain impurities. All these influence Pipetting results. For example, if the tip is not straight this alone may result in a 10% error in Pipetting accuracy (ISO 8655-2). Moreover, if the tip orifice is not accurately centered, the dispensing of the liquid might be affected significantly. If premium grade pure polypropylene is not used or the mold and processing are not up to the highest standards, part of the liquid stays inside the tip as a droplet, which causes an error in the Pipetting result.

GUIDELINES FOR SELECTING TIPS

All tips should be:

  • Made to match the Pipettor in use: It is recommended to use tips specified by the manufacturer as well as high quality Unifit tips.
  • Made of high quality pure virgin polypropylene
  • Free from dust or particles
  • Uniform in size and shape
  • Accurately centered for tip orifice
  • Free from metals, like cadmium
  • Securely sealed: There should be good tip cone match for a perfect seal and tip eject
  • Free from blemishes, flashing or any protrusions
  • Highly resistant to chemicals
  • Highly stable thermally
  • Autoclavable or presterilised for use in sterile applications
  • Traceable: Lot numbers and mold cavity identification markings in each tip

Calibrating your Pipettes

Calibration of Pipettes

Calibration of Pipettes, officially means determining the difference between the dispensed volume and the selected volume. Adjustment means altering the pipette so that the dispensed volume is within certain specifications. During factory calibration, performance is checked with different weighings at both the maximum volumes of the range and at the minimum or 10% of the maximum volume, whichever is higher. When you purchase a Pipette, you should choose one that is designed to permit recalibration and adjustment for different temperatures and various viscous liquids.

Calibration of Pipettes in a Quality System

The main objective of pipette calibration in a quality system is to ensure that measurements are made with the intended accuracy. Very often error limits are taken from the manufacturer�s specifications, while far less accuracy is required to perform the work. If these limits are not easily obtained, or vary, another option is to set the limits according to accepted standards (DIN 12650 or ISO 8655). However, if the laboratory work requires the highest accuracy, the manufacturer�s limits should be used. Basically every user should define their own limits, according to the application used and the ambient conditions.

Device requirements and test conditions

Balance:

The scale graduation value of the balance should be chosen according to the selected pipette volume.

Volume range Readable graduation Under 50 vl 0.001 mg Above 50 vl 0.01 mg

Note: check the calibration of your balance regularly using known weights.

Test liquid:

Water, distilled or de-ionized, "Grade 3", conforming to ISO 3696. The test water is held in the calibration room for at least 2 hours before calibration to reach equilibrium with the test room conditions.

Test room:

Tests are performed in a draught-free room at a constant (+/- 0.5C) temperature of 20C to 25C. Relative humidity is recommended to be above 45%. Especially with volumes under 50 vl, the air humidity should be as high as possible to reduce the evaporation loss effect.

- The pipette, the water and the air in the test room should be at the same temperature.

- A new tip should be pre-wetted 1 to 3 times to improve the accuracy.

- Always pipette water from a reservoir, do not take it back from the balance.

- Check the calibration regularly, depending on the frequency of use and on the application, but at least once a year. If used daily, a three-month interval is recommended.

Procedures to check calibration:

General Pipette Calibration Procedure

Incoming Inspection procedure

  • Upon receipt, each Pipette carefully unpacked and inspected for any signs of visible damage.
    • Any visible damage found is reported back to customer immediately. Customer will be advised if the damage is Repairable and what additional cost will be charged. Customer will issue a new Work Order to cover the cost of Repair.
  • Afterwards each Pipette is scanned for radiation contamination.
    • Any radiation contamination discovered is reported back to customer immediately, and Pipette is Rejected.
  • All customer Pipettes are counted and compared to quantity reported on supplied Work Order.
    • Any quantity discrepancies are reported back to the customer immediately. Customer will have opportunity to locate any missing quantities, or Work Order will need to be revised and revision signed by Customer.
  • Each Work Order will be placed in a separate in-house storage box until scheduled for Calibration.
  • Separate Job ID numbers are assigned for every Work Order.
  • Customer information is checked for any discrepancies from that on file and will be corrected, if needed. If this is a New Customer, a Customer information file will be started.
  • All Pipettes are entered in Cal Lab database under assigned Job ID number with ID number assigned to each instrument.
  • Pre-calibration procedure

  • As Found� data obtained and recorded prior to Calibration and any Pipette adjustments are also recorded.
  • Each Pipette will be tested at lowest and highest range points as specified by manufacture. Ten tests performed at each point.
  • As part of Preventive Maintenance, each Pipette disassembled according to manufacturer�s diagrams and cleaned thoroughly with the appropriate chemical solutions.
  • All parts are carefully inspected, piston is polished in most cases, piston seal and o-ring are replaced as well as all defective and malfunctioning parts. [see 12-Point Inspection diagram]
  • Upon completion each Pipette is reassembled according to the manufacturer�s diagram and procedures. Pipette is again visually checked for proper functionality.
  • Calibration procedure
    At the start of Calibration, each Pipette pre-checked 3 times before any adjustments are done. If Pipette fails, appropriate Calibration adjustments are performed. After corrections, each instrument will be Calibrated at the lowest and highest range points with 10 tests at each test point.
  • Calibration Data will be recorded and reported on a Calibration Report.
  • Calibration reports are issued for each Pipette as well as Calibration Summary for the whole job. Appropriate technicians sign reports.
  • Calibration Label created and attached to each Pipette.
  • All Pipettes crosschecked back to customer supplied paperwork as well as in-lab created documentation.
  • Randomly selected Pipettes are checked for overall performance by QC Supervisor.
    • If any error is detected, the entire Job is sent back for recheck.
  • QC Supervisor approves Job, and Pipette is carefully packaged and delivered to customer with all Calibration Documentation enclosed.

Calibration

  • The pipette is held in the calibration room for at least 2 hours before calibration to reach equilibrium with the test room conditions.
  • The pipette is checked at the maximum volume (nominal volume) and at the minimum volume or 10% of the maximum volume, whichever is higher.
  • A series of ten pipettings is performed with both volumes.
  • Calculate the accuracy and precision using the Formulas section below. If the calculated results are within the limits given in the Instructions-for-use booklet, the pipette calibration is correct. If not, the pipette has to be adjusted with the lower volume and checked again.

Sources of pipette error:

  • Volatile liquids evaporate. Use in saturated air.
  • High density liquids sink. Change technique.
  • Viscous liquids stick to tip. Change technique.
  • Hydrophilic tips will retain liquid. Pre-wet tip.
  • Immersed tips carry over liquid. Immerse 2-3mm.
  • Angled pipettes take up more liquid. Keep vertical.
  • Surface tension reduces accuracy. Change technique.
  • Hot and cold liquids. Pipette liquids at room temp.
  • The human factor. Practice and observe technique.

Procedures to adjust the pipette:

Manual pipettes

  • The adjustment is done at the lower volume.
  • Place the service tool that comes with the pipette into the openings of the calibration nut at the top of the handle.
  • Turn the service tool clockwise to increase the volume or counter clockwise to decrease the volume.
  • After the adjustment, check the calibration as described above.

Electronic pipettes

  • Please follow specific manufacturers� instructions due to substantial variations in calibration procedure.

Formulas for calculating results

Conversion of mass to volume

V = (w + e) x Z

V = volume (vl)

w = weight (mg)

e = evaporation loss (mg)

Z = conversion factor for mg/vl conversion

Note: evaporation loss can be significant with low volumes. To determine mass loss, dispense water into the weighing vessel, note the reading and begin timing with a stopwatch. Check how much the reading decreases during the 5 seconds. Compare this to the pipetting time. Typically, the pipetting time might be 5 seconds and the mass loss up to 2 mg. If an evaporation trap or lid on the vessel is used, an evaporation correction is unnecessary.

The conversion factor Z is for calculating the density of water suspended in air at the test temperature and pressure. See the conversion Table 1 on page 15.

Accuracy (systematic error)

Accuracy is the difference between the dispensed volume and the selected volume of a pipette.

Accuracy is expressed on the calibration certificate as a relative value:

Precision (random error)
Precision refers to the repeatability of the pipettings. It is expressed as standard deviation (s) or coefficient of variation (cv). In addition to the features of the pipette, laboratory practice and user experience are the main factors affecting precision.

Error according to DIN 12650 (F-value)

The DIN standard does not give individual limits for accuracy and precision, but uses a combined error limit: the F-value.

F = | A | + 2 x s

The relative F-value is calculated:

F% = | A% | + 2 x cv

Table 2 shows the error limit according to the DIN 12650 standard for single-channel air displacement pipettes. For multichannel pipettes, these limits are doubled. With variable volume pipettes, the nominal volume is the maximum volume. The absolute vl limit of the nominal volume applies to every selected volume throughout the volume range.

For example, for a 20 - 200 vl pipette, the error limit is 2.0 vl for every selected volume. If the nominal volume of the pipette is between those in the table, the relative error limit F% of the nearest volume is used. If the nominal volume is exactly between the two volumes in Table 2, the relative error limit F% of the lower volume is used.

Table:
DIN 12650 error limits for single channel air displacement pipettes

Best Practices

Ensuring optimum performance

Error-free pipetting requires both precision and accuracy. A number of factors can effect these specifications, which are the main quantitative parameters for evaluating pipette performance.

What are accuracy and precision?

For example when the set volume is 20 vl:

Accurate, but not precise: The mean volume is the correct (set) volume, but separate pipettings differ from the set volume.

Precise, but not accurate: There is no variation between the separate pipettings, but the mean volume differs from the set volume.

Accurate and precise: The mean volume is the set volume and there is no variation between different pipettings.

1. Pipette vertical, tip immersed about 1 cm into the liquid.

2. Pipette vertical, tip immersed about 3 cm into the liquid.

3. Pipette at a 30 � 40 degree angle; tip immersed about 3-4 cm into the liquid.

Autoclaving (Sterilization)

Please, follow these instructions carefully in order to avoid damage to tips and pipettes.

  • Autoclave tips at 121�C (248'F) for 20 minutes. Immediately after autoclaving, the tips are moist. Allow moisture to evaporate before using the tips, preferably overnight.
  • Some digital model Pipettes can be autoclaved in one piece at 121'C (248'F) for 20 minutes. The tip cone modules of the single-channel models can also be autoclaved.
  • After autoclaving, the pipette must be cooled at room temperature for at least two hours before use.
  • Check the calibration of the pipette after every autoclave treatment.

Note: Autoclaving has a limited spectrum of action and will not destroy RNA for example. It is also important to note that certain parts of a pipette - for instance the piston and the handle cannot be autoclaved without altering accuracy and precision

General guidelines for decontaminating Pipettes when Working with Different Liquids

General liquid handling questions

Question:
What is the difference between air displacement and positive displacement pipettes?

Answer: Both types of pipettes have a piston that moves in a cylinder, or capillary. In air displacement pipettes, a specified volume of air remains between the piston and the liquid. In positive displacement pipettes, the piston is in direct contact with the liquid. This keeps air from entering the tip, preventing contamination effectively. Air displacement pipettes are meant for general use with aqueous solutions. Positive displacement pipettes are used for high viscosity and volatile liquids.

Question:
How can I pipette viscous liquids?

Answer:
You can do so using an air displacement pipette with standard or wide orifice tip (reverse pipetting, slowly). An alternative to this is to use a positive displacement system.

Question:
How can I prevent liquid dropping out of the tip when pipetting volatile compounds?

Answer:
If you use air displacement pipettes, aspirate and dispense the liquid a few times keeping the tip in the liquid. By doing so, the air inside the pipette will be saturated with vapor of the volatile compound. It is recommend using positive displacement pipettes for highly volatile compounds, since the built-in piston tip is in direct contact with the liquid.

Question:
How accurately can I pipette warm or cold liquids.

Answer:
The pipettes are calibrated by weighing distilled or de-ionized water of 20 - 25C. With warm liquids, you will get a smaller mass with a certain volume, with cold liquids, you will get a higher mass.

Micropipette Guide: How to Use & Functions | Microlit USA

What is a micropipette?

A micro pipette is a common yet essential laboratory instrument used to accurately and precisely transfer volumes of liquid in the microliter range. Micropipettes are available in single-channel and multi-channel variants. While the single-channel micropipettes are used in labs that perform research related to molecular biology, microbiology, immunology, cell culture, analytical chemistry, biochemistry and genetics, the multichannel micropipettes are recommended for ELISA (diagnostic test), molecular screening, kinetic studies and DNA amplification.

Components of a micropipette:

Micropipettes are available in different designs and sizes. However, there are certain components that are basic and common to all micropipettes. These include the plunger, digital display, tip cone, tip ejector and grippy. Certain micropipettes are provided with a calibration tool and a micropipette stand as an accessory, Micropipette Diagram:

Plunger:

The plunger performs the following two functions- 

  1. Volume adjustment:

    Rotate the plunger clockwise/ anticlockwise to decrease/ increase the volume setting. A distinct click sound at every volume change ensures perfect volume setting and prevents any accidental volume change.

  2. Liquid aspiration/ dispensing:

    Press and depress the plunger to aspirate or dispense liquid.

Tip Ejector:

The internal mechanism of the micro pipette does not come in direct contact of the sample/liquid. Instead, a disposable micropipette tip is used to draw the liquid into and dispense from the micropipette. So in order to allow the safe, effortless and quick ejection of tips, micropipettes are provided with a tip ejection system. The tips can be easily removed from the micropipette by pressing the tip ejector button. 

Volume Display:

This shows the volume of the liquid to be aspirated or dispensed.

Tip Cone:

The tip cone provides fitment to the tips. A pipette with a universal tip cone is preferred as it enhances the compatibility of the instrument with most of the standard tips.

Micropipette Uses in Laboratory

Micropipettes are vital in labs for precise liquid handling. Their uses in microbiology and molecular biology are extensive. From measuring tiny liquid volumes to conducting ELISA tests, micropipettes are indispensable. They come in various types, like single-channel or multi-channel, each serving specific lab needs. Their accuracy makes them essential for experiments in genetics, cell culture, and biochemistry.

In laboratories, micropipettes are used for accurate liquid transfer. They are crucial in tasks like DNA amplification and kinetic studies. Different pipette types, including automatic and manual, cater to diverse uses. In microbiology, micropipettes ensure precision, which is key for successful experiments. Their versatility extends to various fields, making them a fundamental tool in any lab setting.

Types of micropipette:

Micropipettes can be classified depending upon:

  • Working Principle:

The plunger performs the following two functions- 

  1. Air Displacement Micropipette:

    This type of micropipette works on the air displacement principle. It consists of a piston that aspirates and dispenses liquid samples as the air pocket moves up and down, respectively. The internal mechanism of the pipette does not come in direct contact of the sample/liquid. Instead, a disposable micropipette tip is used to draw the liquid into and dispense from the pipette.

  2. Positive Displacement Micropipette:

    In these pipettes, the piston comes in direct contact of the sample. The disposable tip in a positive displacement micropipette is a microsyringe composed of a capillary and a piston (movable inner part) which directly displaces the liquid.

  • Operating Mechanism:

  1. Mechanical Micro pipette:

    These pipettes are operated manually based on a piston-shaft spring mechanism.

  2. Electronic Micropipette:

    An electronic micropipette is mostly automated. The aspirating and dispensing of liquid is performed by the one-touch buttons instead of manual plunger pressing and depressing. Electronic pipettes also often enable the user to create custom programs on the device allowing the pipettes to suit diverse application needs.

  • Number of Channels:

  1. Single Channel Micropipette:

    A single-channel micropipette is one that has a single channel to aspirate or dispense the liquid.

  2. Multi-Channel Micropipette:

    A multi-channel micropipette has multiple channels to aspirate or dispense the liquid. The commonly available multi-channel micropipette variants are the 8-channel, 12-channel and 16-channel. Multichannel micropipettes reduce the workload of a single-channel micropipette when working with large volumes of samples.

  • Volume/Capacity:

  1. Fixed Volume Micropipette:

    In a Fixed Volume Micropipette, the volume of liquid to be aspirated or dispensed remains fixed. These micropipettes are used when the same volume of liquid is to be dispensed multiple times.

  2. Variable Volume Micropipette:

    This micropipette comes with a specific minimum and maximum volume range. The volume of the liquid to be aspirated or dispensed can be adjusted (within the instrument’s volume range) depending on the requirement of the user.

Size and Range / Technical Specifications:

Micropipettes are available in different volumes ranging from 0.1 µl to 10,0000 µl. The commonly used variants of single-channel variable volume micropipettes are listed below along with their permissible error limits as specified in the ISO 8655-2 standard.

They are sometimes referred to as P10, P20, P1000, P5000 pipettes based on the maximum volume that can be aspirated/dispensed using the pipette. For instance, a 0.5-10ul Micro pipette may be commonly referred to as a P10 pipette.

Volume Range
(ul)
Classification
Increment
(ul)
Accuracy
(± %)
Accuracy
(±ul)
CV
(± %)
CV
( ± ul)

0.2-2

P2

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0.01

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

 

How does a micropipette work?

Air displacement micropipettes operate by piston-driven air displacement. When the piston is pressed downwards, the air within the sleeve of the micropipette gets expelled out due to the force of which the liquid present in the tip of the micropipette also gets removed.

When the piston moves upwards, a vacuum is created in the space left vacant by the piston. This causes the air from the tip to rise in order to fill the vacant space, and the tip air is then replaced by the liquid, which is drawn up into the tip.

Positive displacement micropipettes operate by piston-driven displacement. The piston in a positive displacement micropipette is in direct contact with the liquid. When the piston is pressed downwards, the liquid which is present in the sleeve of the micropipette also moves downwards and gets removed through the tip. When the piston is pulled upwards, it also draws the liquid along with it in the upward direction.

  1. Using an Air Displacement Micro pipette:

    Precise measurement of liquid depends on the correct micropipette usage. The air displacement micropipettes work on the common air displacement principle. A plunger is depressed by the thumb and as it is released, the liquid is drawn into a disposable tip. When the plunger is pressed again, the liquid is dispensed. In between these steps, there are several small steps that help in making the liquid dispensing process more precise.

    Position 1

    In this, the micropipette is at rest position.

    Press fit a tip to the micropipette without directly touching the tip.

    Position 2

    In this, the plunger is depressed till the first stop.

    To aspirate the liquid in the tip, press the plunger to the first stop. Immerse the pipette tip vertically in the liquid.

    Release the plunger

    Slowly release the plunger while the tip is immersed. The liquid will be aspirated into the pipette tip.
    The liquid is filled in the tip as per the preset micropipette volume.

    Position 3

    Depress the plunger

    To dispense the liquid, place the tip on the inner wall of the receiving vessel at a steep angle.
    Slowly press the plunger to the first stop to dispense the liquid.
    To empty the tip completely, press the plunger to the second stop.
    Wipe the tip on the inner wall while taking the tip out of the vessel.

     

    Some micropipettes deliver a fixed volume of liquid. However, the majority are adjustable with the variable volume setting. Variable volume micropipette comes with different ranges and upper & lower limits of measurement. In such cases, the error percentage may vary as per the measured liquid. Trying to dispense less than the lower value of the range will result in inaccurate liquid measurements whereas trying to dispense over the upper range will completely fill the tip and allow the liquid to enter into the pipette body.

  2. Cleaning, maintenance and storage of a micropipette

    The care and maintenance of a micropipette is an important routine in laboratories. Implementing a proper maintenance schedule can reduce the cost of a new purchase of this expensive equipment. Cleaning the micropipette takes time, focus and practice; otherwise, the micropipette can be damaged. So, it is important to handle the micropipette carefully while cleaning. The following is a comprehensive guide for proper cleaning of micropipettes.

    External Cleaning: Most of the pipette can be cleaned externally with typical laboratory cleaning agents, soaps or alcohol. To ensure full sterilization, let the cleaning solution sit on the micropipette for 10-15 min before wiping it off.

    Internal Cleaning: Cleaning the interior of the micropipette can be more time-consuming because it requires full disassembly. Also, every part of the micropipette will need to be cleaned properly depending on the liquid used in sampling.
    – Refer to the instructions manual for a specific direction of the micropipette
    – Use a cotton swab with a cleaning solution and distilled water
    – Lightly grease the pistons with the lubricant provided upon purchase
    – Reassemble all parts and check to ensure the micropipette operates smoothly

    Contamination Cleaning: If the micropipette becomes contaminated with a known substance, there are specific cleaning steps that must be taken depending on the type of substance. The above cleaning routine will not be sufficient 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 the lower parts of the micropipette in glycine/ HCI buffer (pH2) for 10 minutes, rinse with distilled water, and air dry.

     

    A well-maintained and clean pipette ensures lab safety from hazardous solutions. It makes the pipette more accurate, reliable, long-lasting and reduces the cost of sampling.

    Proper storage of micropipettes is as important as cleaning and calibrating them. The micropipette and its accessories should be stored in a clean, cool and dry place. The storage place should have a temperature ranging from -20 °C to 50 °C (from -4 °F to 120 °F) with relative humidity between 5% and 95%. Another point to be remembered is that the instrument should be stored in an upright position. To store pipettes, Microlit recommends the use of its carousel stand, Microlit Faveo.

What are the different pipetting techniques used?

The frequently used pipetting techniques include forward pipetting and reverse pipetting. Before we understand these techniques in detail, the general instructions and micropipette diagram of pipetting listed below would be noteworthy.

  1. Press and release the plunger slowly, at all times, particularly when working with high-viscosity reagents/solutions. Make sure that the plunger does not snap.
  2. Make sure the tip is firmly attached to the tip cone.
  3. Before starting your experiment, fill and empty the tip 2-3 times with the reagent or solution that you will be pipetting.
  4. Hold the micropipette in an upright position while aspirating. The Grippy must rest on your index finger.
  5. Make sure that the tips, the micropipette and the reagent/solution are at the same temperature.

Forward Pipetting Technique:

  1. To aspirate the liquid in the tip, press the plunger to the first stop. Immerse the pipette tip vertically in the liquid.
  2. Slowly release the plunger while the tip is immersed. The liquid will be aspirated into the pipette tip.
  3. To dispense the liquid, place the tip on the inner wall of the receiving vessel at a steep angle
  4. Slowly press the plunger to the first stop to dispense the liquid.
  5. To empty the tip completely, press the plunger to the second stop.
  6. Wipe the tip on the inner wall while taking the tip out of the vessel.

Reverse Pipetting Technique:

The reverse technique is suitable for dispensing reagents/solutions that have high viscosity or a tendency to foam easily. It is also recommended for dispensing very small volumes.

  1. To aspirate the liquid in the tip, press the plunger to the second stop and immerse the pipette tip vertically in the liquid.
  2. Slowly release the plunger while the tip is immersed. The liquid will be aspirated into the pipette tip.
  3. To dispense the liquid, place the tip on the inner wall of the tube at a steep angle.
  4. Slowly press the plunger to the first stop.
  5. Wipe the tip on the inner wall while taking the tip out of the vessel.

Note: Residual liquid remains in the tip. This does not belong to the dispense volume.

How to choose the right micropipette?

As micropipettes can be used for a number of applications, the dilemma one faces is how to choose the right micropipette while handling a specific task.

For this, you can follow our simple, stepwise guide. The preliminary step includes noting down the details of the experiment you are going to perform with the micropipette. This should ideally include the number of samples, the volume to be transferred, the number of replicates, whether sterile conditions are required and so on. Making a comprehensive list in this manner will make the process of deciding which micropipette to use easier.

  • Selecting the Type of Micropipette:

Types of  Micropipettes – Air Displacement Micropipettes and Positive Displacement Micropipettes. You can choose the right one by matching their applications to the experiment you wish to perform.

  • Next Steps:

After choosing the correct type, let’s look at the subtypes available based on criteria like the volume to be handled, quality of tips, manual or electronic pipettes, etc. This is where the more minute details regarding the experiment will come in handy.

  1. An adjustable micropipette or a fixed micropipette:

    You can either use a micropipette whose volume is already fixed or one whose volume can be adjusted as per requirement. If you regularly use a single volume such as 100 µl for your experiments, go for a fixed volume pipette like MICROLIT RBO Fixed Volume (Single Channel). If your experiment entails you working with a range of volumes, choose the variable or adjustable pipette, like MICROLIT RBO Variable Volume (Single Channel or Multichannel).

  2. The volume to be handled:

    As a standard rule, it is better to choose the smallest pipette capable of handling the required volume because when the set volume is close to the minimum capacity of the micropipette, the accuracy and precision of the readings decrease.

    1. Single channel or multichannel micropipettes:

      This decision can be made based on the number of samples or replicates you are working with. Single channel micropipettes like the MICROLIT RBO Single Channel (Fixed Volume and Variable Volume) are used when the number of samples is less whereas a large number of samples or well plates can be easily handled by multichannel micropipettes like the MICROLIT RBO Multichannel (8-channel and 12-channel).

  3. Micropipette Tips:

    Once you’ve selected your micropipette, this is the next crucial decision to make. Micropipette Tips can be graduated or not graduated, universal or pipette specific, with a filter or without filter, sterile or non-sterile etc. If the experiment requires sterile, aseptic conditions, one should use filtered and sterile tips. Universal tips can be used for a wide range of pipettes. It is also important to match the capacity of the tip with the capacity of the micropipette before use.

  4. Accuracy with specific tolerance:

    Micropipettes are designed to operate with accuracies within a few percent (generally <5%) of the intended value for the volume. Make sure your micropipette provides the same accuracy as you need for your sampling.

How is a micropipette calibrated?

Micropipette calibration is a very important part of any laboratory’s routine for accurate and precise pipetting results. To ensure the accuracy of sampling, it is important to check the pipette calibration every month or in a few months’ gap. Executing a proper micropipette calibration according to ISO 8655 standards requires special ambient conditions. Factors such as air pressure, humidity, temperature and even altitude affect the pipetting results. The below text explains the required material or equipment and all the steps that you need to perform and check the calibration of a micropipette.

Here is a list of things that you will need in order to calibrate a micropipette:

  • Micropipette that needs to be calibrated & tips
  • Distilled Water
  • Clean Beaker
  • Thermometer
  • Distilled Water Density Chart with Temperature
  • Semi-Micro Balance with a readability of at least 0.0001 g
  • Notebook to write down the results
  • Calculator

How to calibrate a pipette using a balance:

  1. Leave the distilled water out for 15-20 minutes to ensure a consistent temperature.
  2. Use the thermometer to measure the temperature of the distilled water.
  3. Place empty and clean the beaker on the balance.
  4. Take the beaker’s weight.
  5. Ensure the micropipette is clean and ready to function.
  6. When filling the micropipette up to the volume that will be calibrated (1ml, for example) with the distilled water, make sure air bubbles are not created in the flask and in the micropipette.
  7. Dispense the distilled water in the beaker using the micropipette.
  8. Use the balance to measure the distilled water’s weight.
  9. Repeat the process 5-10 times at least and note each measurement.
  10. Use the formula V = W * Z to calculate the volume dispensed by the pipette.
    W is the weight of the distilled water
    Z is the “conversion factor based on the density of the water” and
    V is the calculated volume of dispensed water
    The temperature of the water determines its density, which is why it is important to keep the water temperature as constant as possible. Skip if you have the software that automatically does that.
  11. Average the results. If you did 10 tests, divide the sum of the 10 tests by 10.
  12. The average weight of the distilled water is the  W in the equation. You can now solve the equation manually, or use the software.
  13. The next step is determining the pipette’s accuracy, manually or via software. If you have to do it manually, use the formula A = 100 x Vavg/V0
    A stands for Accuracy.
    Vavg is the average calculated volume.
    V0 is the value assigned for the pipette to dispense.
    It is recommended that the value should be between 99 and 101%.
  14. Record the results. If the micropipette is properly calibrated, you can use it. If the results are consistently wrong, do not use the pipettes or fix them before using it again.

How to sterilize a micropipette?

  • The micropipettes can be sterilized by autoclaving them at 121°C (252°F) and 15 PSI for a duration of 15-20 minutes.
  • No special preparation is needed.
  • You may use steam sterilization bags if needed. After autoclaving, the micropipette must be cooled to room temperature for at least two hours. Before pipetting, make sure that the instrument is dry.
  • It is recommended to check the calibration after every sterilization cycle to achieve the best possible precision and accuracy.

What are micropipette starter kits? How are they beneficial during lab setup?

A micropipette starter kit is the most convenient and cost-effective collection of pipetting essentials. It includes a set of micropipettes with different volume ranges, micropipette tips that can be used in given micropipettes and some other accessories for an efficient sampling process.

A micropipette starter kit is beneficial as it saves the time of selecting the micropipette and the compatible tips which can be used with it. Microlit Micropipette Starter Kit combines 4 single channel pipettes to cover the complete pipetting range (0.5-10ul pipette, 10-100ul pipette, 100-1000ul pipette, 1-10ml pipette). The micropipette kit also includes a sleek carousel stand and boxes of compatible tips for each pipette so that one does not have to go looking for the right tips.

When is micropipette calibration required?

In order to maintain the quality of sampling results, the micropipettes must be in good condition and properly calibrated. Normally, the interval at which a micropipette needs to be calibrated depends on many factors:

  1. Frequency of pipette use
  2. Type of liquid dispensed by the pipettes
  3. Handling & care of pipette
  4. Pipette applications that require superior accuracy also demand more frequent calibration

Regulations and standards published by organizations such as the FDA and ASTM International provide minimum requirements to ensure the quality of laboratory testing results. Regulations specify that all laboratory instruments used in sampling & production including micropipettes must be routinely calibrated at suitable intervals.

In the same series, the Clinical and laboratory standards institute (CSLI) has provided the guidelines for single and multi-channel micropipettes that specify these micropipettes must be calibrated every 3 to 6 months. And a minimum of two volumes must be tested with 10 replicas at both nominal and lowest settings.

Establishing an appropriate calibration frequency minimizes the chances of incorrect liquid delivery in the laboratory and ensures traceability, accountability and confidence in the results.

Applications/fields where micropipettes can be used

Micropipettes are generally used in microbiology, chemistry, and medical testing laboratories for the accurate and precise transfer of samples. While the single-channel micropipettes are used in labs that perform research related to molecular biology, microbiology, immunology, cell culture, analytical chemistry, biochemistry and genetics, the multichannel micropipettes are recommended for ELISA (diagnostic test), molecular screening, kinetic studies and DNA amplification.

Some of the major fields where micropipette is widely used are:

  1. Pharmaceuticals
  2. Health Care
  3. Food & Beverage
  4. Environmental Monitoring
  5. Academic & Research Institutes
  6. Diagnostic Kits
  7. Life Sciences

Benefits of using a micropipette

A micropipette is a reliable device to dispense precise quantities of liquid. It increases the performance of the testing efficiently. Some other benefits of using a micropipette are listed below:

  1. Time-Saving
  2. Ergonomics
  3. Accuracy & Precision
  4. Ease of Use

Where do we get the best micropipette for the lab?

Every lab technician wants a micropipette that is ergonomically designed, aesthetically presentable and provides accurate and precise measurement values. Selecting the best micropipette can be a challenging task as the market is full of micropipettes that appear to be good but do not provide reliable test results. Before going with any micropipette brand, it is important to ensure some points that can save you from wrong pipette purchasing:

– Is the micropipette able to handle your pipetting range of liquid?
– Does your micropipette have a universal tip cone for handling all kinds of tips?
– Is your micropipette autoclavable at the required standard temperature?
– Does your micropipette have smooth and comfortable handling?
– Is your micropipette calibrated for precise dispensing?
– Is the accuracy and precision in readings offered by the micropipette in accordance with the ISO standards?

The above points ensure your right micropipette purchasing which will work for a long time and improve the liquid handling activities in your laboratory.

Microlit designs and develops high-precision liquid handling instruments for leading laboratories around the world. It offers an advanced and innovative micropipette range that facilitates remarkable user experience in practical laboratory environments.

FAQs/Troubleshooting:

Which pipette tips can I use with the micropipette?

It depends on your micropipette, if your micropipette has a universal tipcone, you can use it with all international tips as per your micropipette volume range.

How should I sterilize the pipette?

You can sterilize your micropipette if it is autoclavable. If you use your micropipette for sensitive and hazardous liquid sampling, the sterilization process is required to clean your micropipette. Check your micropipette manual to know the maximum temperature and time duration for the sterilization process. Refer to the ‘Contamination Cleaning’ section to know how to sterilize your micropipette easily as per dispensed liquid in the sampling.

How can I check whether the micropipette is calibrated?

It is advisable that every day when you start the pipetting in the lab, check your micropipette for its reading correctness. For this, set the micropipette at the nominal volume and dispense distilled water at least 5 times. Measure the dispensed liquid volume on the electronic balance. If the mean of your readings is within the ISO standard limit, the micropipette is calibrated. If not, calibrate your micropipette and check it again.

How often do I need to calibrate the micropipette?

Generally, it depends on how often your micropipette is used for sampling. For normal use of a micropipette, it should be checked every 3 or 6 months for calibration. As per ISO 8655 standard, it is recommended to get your micropipette calibrated annually.

How do you set the volume on a micropipette?

A small display with 3 number volume range is given on the body of every micropipette. On a fixed volume micropipette, it is a fixed volume range. On the variable volume micropipette, you can adjust it within the given volume range as per your required volume sampling. To change the volume range numbers, use the rotational dial given at the top of the micropipette plunger or refer to the manual to know how to change the volume of the micropipette.

Why should you avoid touching the micropipette tips?

Touching the micropipette tip can cause the liquid to stick to the outside of the tip. The heat transferred from your hands to the tips can affect the delivery of volumes. It also increases the risk of cross-contamination in the laboratory. So, it is recommended to handle the tips only with the micropipette.

What is the largest source of pipetting problems?

Human error is the largest source of pipetting problems which is caused by touching tips, handling micropipettes improperly, etc.

What happens if you release the micropipette plunger too fast?

If the micropipette plunger is released too fast, you may face the issue of less draw-up and dispensing of liquid.

For more information regarding Microlit Products, drop a mail to info-usa@microlit.com.

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