Introduction: Micropipette
A micropipette is an instrument that is used commonly in the laboratory to transfer the liquid volumes accurately and precisely in the range of microliter. Various types of micropipettes are available for use in the laboratory. Different types of micropipettes offer different functions like single-channel micropipettes are commonly used for performing research for microbiology, cell culture, molecular biology, immunology, biochemistry, analytical chemistry and genetics. On the other hand, multichannel micropipettes are better used for diagnostic tests, kinetic studies, DNA amplification, and molecular screening.
What are the Components of a Micropipette?
Different sizes and designs can be found in micropipette. But, some components of a micropipette remain the same for all variations of this product. The common components include a digital display, the plunger, tip ejector, tip cone, and grippy. Some micropipettes are also available with a micropipette stand and a calibration tool.
Common Components:
1. The Plunger
The plunger has two functions, which are volume adjustment and liquid dispensing. For increasing or decreasing the volume adjustment, rotate the plunger anticlockwise or clockwise. While adjusting the plunger, a click sound can be heard to ensure the volume setting. For dispensing or aspirating the liquid, press and depress it.
2. Tip Ejector
To ensure that the liquid or sample doesn’t come in direct contact with the internal mechanism of this instrument, a disposable pipette tip is present to draw the sample and transfer it from the micropipette. Tip ejection ensures safe and effortless ejection from the tips. The tip ejection button can be pressed to safely remove the tip from the micropipette.
3. Volume Display
The function of this component is simple, that is to display the volume of the sample of the liquid that is to be dispensed or aspirated.
4. Tip Cone
A micropipette having the tip cone is preferred, as it provides fitment of the tip. Also, enhances the compatibility while using it with a micropipette.
What Are The Types Of Micropipette?
As said earlier, micropipettes come in various designs and sizes. The types of micropipette may be categorized on the following basis:
1. Based on the Working Principle:
a. Air Displacement Micropipette
As the name says, it functions on the principle of air displacement. In this type, the micropipette has a piston that dispenses/aspires the liquid depending on the movement of the air pocket. Here, the sample or liquid doesn’t come on directly with the internal mechanism of this instrument, as a disposable pipette tip is given preference for drawing the liquid.
b. Positive Displacement Micropipette
It works oppositely, as here, the pipette comes in direct contact with the sample liquid. The disposable tip in this type of micropipette is like a microsyringe. It is composed of various capillaries along with a piston to directly displace the liquid.
2. Based on Operating Mechanism:
a. Mechanical Micropipette
This type of micropipette is based on a spring mechanism including a piston shaft. Also, it is operated manually.
b. Electronic Micropipette
Unlike mechanical micropipettes, these are commonly automated. A one-touch button is used to perform the task of dispensing and aspirating the sample liquid. It helps the users to custom the device is suited to their diverse requirements.
3. Based on the Number of Channels:
a. Single Channel Micropipette:
This type of micropipette has a single channel to dispense or aspire the liquid.
b. Multi-Channel Micropipette:
This type of micropipette has multiple channels to dispense or aspire the liquid. Commonly the variant of this type includes eight channels, twelve channels, and sixteen channels. It helps in reducing the workload by providing multiple channels of dispensing the liquid.
4. Based on Volume/Capacity:
a. Fixed Volume Micropipette:
These types of micropipettes are used when the same volume of sample liquid needs to be dispensed or aspired multiple times, as the volume liquid, in this case, remains fixed.
b. Variable Volume Micropipette:
These micropipettes include a minimum and maximum volume range to adjust the volume of the sample that has to be dispensed.
What is the Technical Specifications/Size/Range?
Various sizes and ranges are available for micropipettes. However, the most commonly used micropipette is the single-channel variable. Here is a list of error limits according to the ISO 8655-2 standard.
| Volume Range (ul) | Increment (ul) | Accuracy
(±ul) |
Accuracy
(± %) |
CV
( ± ul) |
CV
(± %) |
| 0.2-2 | 0.002 | 0.04 | 2 | 0.024 | 1.2 |
| 0.5-10 | 0.02 | 0.1 | 1 | 0.05 | 0.5 |
| 20-Feb | 0.02 | 0.16 | 0.8 | 0.08 | 0.4 |
| May-50 | 0.1 | 0.4 | 0.8 | 0.2 | 0.4 |
| 10-100 | 0.2 | 0.6 | 0.6 | 0.2 | 0.2 |
| 20-200 | 0.2 | 1.2 | 0.6 | 0.4 | 0.2 |
| 100-1000 | 1 | 6 | 0.6 | 2 | 0.2 |
| 500-5000 | 10 | 30 | 0.6 | 10 | 0.2 |
| 1000-10000 | 20 | 60 | 0.6 | 20 | 0.2 |
Working of a Micropipette
Using an air displacement micropipette is easier, as it is piston-driven. On pressing downwards, the air inside the space of the micropipette gets expelled out, removing out the liquid present inside it.
On the other hand, when the piston is moved upwards, it creates a vacuum in the vacant space of the piston. Due to this, the air from the tip rises and fills the vacant space. It then gets replaced by the sample liquid that is drawn up inside the tip.
Positive displacement micropipettes are also operated by piston displacement. In this case, the piston is in direct contact with the liquid. On pressing the piston downwards, the liquid inside the micropipette also moves downwards and gets removed from the tip. The same goes on pressing the piston upwards; the liquid also gets drawn in an upward direction.
How to Use an Air Displacement Micropipette?
To ensure the precise measurement of the sample liquid, it is essential to use the micropipette correctly. To use the air displacement micropipette, the common air displacement principle is applied. At first, the plunger is depressed, and then released. Meanwhile, the liquid is drawn inside the disposable tip. Pressing the plunger again dispenses the liquid. However, there are small steps that must be used correctly in between the major steps.
| Position | Direction | Usage | ||
| 1 | Rest position of the micropipette | Here, it is essential to press fit the tip, and not directly touching the tip. | ||
| 2 | Depressing the plunger till the first stop
Releasing the plunger |
Here, the plunger is depressed till the first stop to aspire the liquid inside the tip. Hold it vertically.
While the tip is immersed, release the plunger slowly, and aspire the liquid into the tip as per the volume required. |
||
| 3 | Depressing the plunger | In the inner wall of the vessel, place the tip of the pipette at steep angle to dispense the liquid. First, press the plunger slowly to the first stop. Next, press the plunger till its second stop to empty the tip. | ||
While some micropipette allows the fixed volume of the sample liquid, most of them are available through adjustable variable volume setting. Also, the variable volume micropipettes are present in versatile ranges, including upper and lower limits. However, using a variable volume micropipette may lead to varied error percentile. Dispensing more than the upper range will fill the tip leading the liquid to enter the body of the pipette. Likewise, dispensing less than the lower range will make the measurement inaccurate.
Correct Ways of Storage, Cleaning, and Maintenance a Micropipette
It is essential to take good care and maintenance of the micropipettes in laboratories. It helps in reducing the cost of purchasing a new expensive micropipette. It takes time and focuses to clean the micropipette else it may get damaged. It is vital to properly handle this instrument with delicate care. The following is a guide for cleaning and maintaining a micropipette.
a. External Cleaning: Typical laboratory cleaning agents may be used to clean the micropipette externally, such as soap or alcohol. Let to cleaning solution sit for 10-15 minutes to ensure full sterilization of the instrument.
b. Internal Cleaning: It requires more time and energy to clean this instrument internally, as full disassembling is required in this case. Depending on the sample liquid, every internal part of this instrument has to be cleaned properly.
c. Contamination Cleaning: If the micropipette has been contaminated with an external and unknown substance, a specific cleaning procedure must be followed depending on the substance type. Proper contamination cleaning must be present to eliminate any foreign substance from the instrument.
When the pipette is cleaned, it helps in reducing the chances of any miss-happening and ensures proper lab safety. Moreover, it specifically helps the pipette become more reliable, accurate, and long-lasting, reducing the recurring maintenance cost required.
Proper storage of micropipette is as essential as its cleaning and calibration. To ensure safe storage of this instrument, it is advised to store it in a cool, dry and clean place. The temperature of the storage place must be between the range of -20 °C to 50 °C (from -4 °F to 120 °F) along with the relative humidity ranging between 5% and 95%. Moreover, this instrument must always be stored in an upright position.
Different Pipette Techniques
The most commonly used pipette techniques are reverse pipetting and forward pipetting. Before moving to the techniques, here are some general instructions that must be understood.
• When working with sensitive and delicate solutions, the plunger must be pressed and released slowly to avoid snapping.
• The tip must be attached firmly to the tip cone.
• Before beginning with the experiment, make sure to fill and empty the tip of the tip cone, 2-3 times using the solution that needs to be pipetting.
• While aspirating the micropipette must be held in an upright position, resting the grippy on your index figure.
• Also, the temperature of all the micropipettes, the tips and the solution/reagent must be the same.
The Technique of Forwarding Pipetting
The steps involved in the forward pipetting technique are given below:
1. First, press the plunger till its first stop to aspire the liquid in the tip. The pipette tips must be immersed vertically in the liquid.
2. Next, release the plunger slowly, keeping the tip immersed in the liquid to aspire the liquid in the pipette tip.
3. Make sure to place the tip over the inner wall of the vessel to dispense the liquid.
4. Now, press the plunger slowly till its first stop and dispense off the liquid.
5. For emptying the tip, press the plunger till its second stop and wipe the tip on the vessel’s inner wall.
The Technique of Reverse Pipetting
The steps in the reverse pipetting technique are given below:
1. For aspirating the liquid inside the tip, the plunger must be pressed till its second stop, immersing the tip vertically inside the liquid.
2. Next, release the plunger slowly, keeping the tip immersed in the liquid to aspire the liquid in the pipette tip.
3. Make sure to place the tip over the inner wall of the vessel to dispense the liquid.
4. Keep pressing the plunger slowly to its first stop, and wipe the Make sure to place the tip over the inner wall of the vessel to dispense the liquid.
Choosing the Right Micropipette
People use micropipettes for versatile purposes, the dilemma may put people in the confusion of choosing the right micropipette for their specific task.
To determine the right micropipette for individuals, simple steps may be followed. Here, is a simple guide to choose the right micropipette for specific purposes. It includes the types, the purpose, the volume, the number of samples, and other conditions. Here is a step-wise comprehensive list of how to decide the right micropipette.
1. Selecting the type:
As mentioned earlier, there are two types of micropipettes- air displacement and positive displacement micropipettes. To determine the right one, one should match his application of the experiment with the specifications of the particular type.
2. Criteria for Volumes:
Once the type is determined by the person, the next factor to consider must be based on the volume of the samples. Various ways are available to handle the volume of the solution. To ensure the complete accuracy of the experiment, the following minute details are essential to look at:
a. Adjustable or Fixed Micropipettes
The decision between fixed and adjustable micropipette is essential to look for. The requirement of the experiment must be considered before selecting between the fixed and adjustable micropipettes. If the volume for the experiment remains the same regularly, fixed micropipettes may be the best choice. However, if the experiment includes various ranges of volume, adjustable or variable micropipette may be the ideal choice in this case.
b. Handling the Volume
It is a fundamental rule, that the smallest pipette must be used, as it is capable of meeting the smallest volume requirements. It helps in bringing accuracy and preciseness in the reading degree, as the volume is set closest to the minimum capacity.
c. Deciding Micropipette Tips
Once the micropipette is selected, the next decision that must be made is about the micropipette. There are various types of micropipette present to choose from. They can be universal, or specific, graduates or not graduated, filter and without filter, and sterile or non-sterile. On deciding the micropipette tips, the conditions and requirements of the experiment must be considered, and make the final decision. Moreover, the capacity of the micropipette must be matched with the capacity of the tips before use.
d. Consider Accuracy
The micropipette must provide the correct and same accuracy that is required for the sampling. As micropipettes are created to operate with the accuracies allowing a few % of the required value, choosing the right and compatible micropipette is essential.
How Is A Micropipette Calibrated?
Micropipette calibration may be a vital part of the routine of any laboratory for precise and correct pipetting results. It’s vital to measure the pipette calibration each month or during a few months’ gaps to make sure accuracy of sampling. Conducting a correct micropipette activity in step with ISO 8655 commonplace needs special close conditions. Factors like atmospheric pressure, humidity, temperature and even altitude affect the pipetting results. The below text explains the specified material or instrumentality and complete steps that must simply be compelled to perform and check the activity of a micropipette.
To calibrate the micropipette, the following items are essentially required:
• Micropipette and the tips
• Thermometer
• Clean Beaker
• Distilled Water
• Density Chart of Distilled Water with Temperature
• Calculator
• Semi-Micro Balance having readability upto 0.0001 g
• Notebook for writing the results
Use Balance to Calibrate the Micropipette
Following are the steps of calibrating a micropipette by using the balance:
1. To make sure of the consistent temperature, distilled water must be left out for 15-20 minutes.
2. Use the thermometer and measure the distilled water’s temperature.
3. Now, place the beaker that must be empty and clean on the balance.
4. Weigh the beaker.
5. Make sure the micropipette is in proper condition to function.
6. While filling the micropipette for calibration, make sure there is no formation of the bubbles in the flask and the micropipette.
7. The distilled water needs to be dispensed in the beaker by using the micropipette.
8. Measure the weight of the distilled water using the balance.
9. Note the measurement by repeating the process 5-10 times.
10. Now, using the formula V = W * Z the volume dispensed needs to be calculated by the pipette.
I. W is for the distilled water’s weight.
II. Z is the factor of conversion upon the density of distilled water.
III. V is the dispensed water’s volume that is calculated.
Make sure to keep the water temperature consistent as the density of the water is determined by the temperature of the water.
11. Next, the results need to be average to accurately calibrating them.
12. Use the formula and solve the equation manually by taking the W as distilled water’s average weight in the equation.
13. Now, the accuracy of the pipette has to be determined either by the software or manually using the formula A = 100 x Vavg/V0
I. A is the Accuracy.
II. Vavg is the average of calculated volume.
III. V0 is the assigned value of the pipette to dispense.
It is recommended that the assigned value should be between 99 and 101%.
14. Record the results. If the result is consistent and calibrated properly, it can be used for the experiment. On the other hand, if it is not giving consistent results, it needs to be fixed or changed before using again.
When Should a Micropipette Be Calibrated?
Calibration is essential to make the micropipette in good condition and maintain the accuracy of the sampling results. The interval of calibrating micropipette depends on various factors, such as:
• Frequency of using the pipette
• Type of liquid used for dispensing
• Taking care of the pipette
• Application of pipette when superior accuracy is required
Regulations and standards printed by organizations like the ASTM and FDA International provide minimum necessities to confirm the standard of laboratory testing results. Rules guide that each laboratory instrument employed in productions & sampling, as well as micropipettes, should be habitually graduated at appropriate intervals.
Similarly, the Clinical and laboratory standards institute (CSLI) also provides the rules for multi and single-channel micropipettes. They specify that these micropipettes should be calibrated each three to six months. Also, a minimum of 2 volumes should be tested with ten replicas at each nominal and lowest setting.
Steps to Sterilize the Micropipette
• To sterilize the micropipette, it is recommended to autoclave them at 121°C (252°F), and the duration must be 15-20 minutes.
• Also, it doesn’t require any special preparation. However, steam sterilizing bags may be used if needed.
• After finishing the autoclaving, the micropipette has to be cooled down to room temperature for at least 2 hours. Also, make sure the instrument is dry before pipetting.
• To achieve the best accuracy and precision, the calibration must be checked after every sterilisation.
What is a Micropipette Starter Kit?
A micropipette starter kit is that the most convenient and price effective assortment of pipetting necessities. It includes a collection of micropipettes with totally different volumes vary micropipette tips that may be utilized in given micropipettes, and a few different accessories for an economical sampling method.
What are Applications/Fields to Use Micropipettes?
Commonly micropipettes are used in chemistry, microbiology, and labs for medical testing to attain the accurate and precise transfer of liquids. Different types of micropipettes offer different functions like single-channel micropipettes are commonly used for performing research for microbiology, cell culture, molecular biology, immunology, biochemistry, analytical chemistry and genetics. On the other hand, multichannel micropipettes are better used for diagnostic tests, kinetic studies, DNA amplification, and molecular screening.
Micropipettes are generally used in the following fields:
• Health Care
• Environmental Monitoring
• Diagnostic Kits
• Life Sciences
• Food & Beverage
• Pharmaceuticals
• Academic & Research Institutes
What are the Benefits of Using Micropipettes?
Micropipettes are reliable lab instruments to precisely transfer liquid quantities. It helps in increasing test effectiveness and efficiency. Some of the common benefits are mentioned below:
• It helps in saving time.
• It is designed ergonomically.
• It is easy to use.
• It provides precision and accuracy.
How to Get the Best Micropipette for the Lab?
Every laboratory wants to use an instrument that fulfils its experimental requirements by ensuring efficiency in the process. Micropipettes are used for presenting precise and accurate measurement values. Therefore, it is essential to select the right micropipette for use in the labs. The correct micropipette must meet your specific requirements for dispensing the sample liquid. The following points must be considered to avoid purchasing the wrong micropipette:
• It should be able to handle the pipetting for the required range of liquid.
• It should come with universal tip cones to support all kinds of tips.
• It must be able to autoclave at standard temperature.
• It should offer comfortable and smooth handling.
• It should be calibrated for providing accurate and precise dispensing.
• It should offer the reading per the ISO standards.
Conclusion
It was all about the types, uses, techniques, and application of micropipettes. It was an ultimate guide, including every aspect of micropipettes. Now, here are some common FAQs to help in solving any query.
FAQs/Suggestions:
1. Which pipette tip is suitable for micropipette?
The pipette tip depends on the micropipette that is being used. Having a micropipette with a universal tip cone makes it easier to use with any pipette tip according to the range of micropipette volume.
2. How to sterilize the pipette?
The micropipette must be autoclavable to sterilize it. The sterilization process is required when the micropipette is used for hazardous and sensitive liquid sampling. The steps required for sterilizing the micropipette are mentioned above in this blog. Those steps can be referred to sterilize any micropipette.
3. Why should micropipette tips not be touched?
When the micropipette tip is touched, it can force the liquid outside the tip. Moreover, the heat from the hand can affect the volume delivery. It can also increase the chances of cross-contamination.
4. How to check the calibration of the micropipette?
It is essential to check the micropipette to reading correctness, whenever it is used in the lab. To check the correctness, the micropipette must be set at the nominal value. Next, the water should be distilled five times, and measure the volume of dispensed liquid on the balance. After, check the readings; if they are within the standards of the ISO, it is calibrated. Otherwise, the micropipette must be calibrated and check again before use.
5. How frequently should the micropipette be calibrated?
Calibrating frequency generally depends on the type of sampling used. However, it should be calibrated every three or six months. According to the ISO standards, it may be calibrated annually.
6. What about the largest source of problems of pipetting?
The largest source of problems of pipetting is human error. It is mainly caused due to hand touching and improper handling.
7. How to set the volume of the micropipette?
The micropipette includes a small display of a three-digit volume range. In the case of the fixed volume micropipette, the range is fixed. However, the adjustment is required in a variable volume micropipette. The range can be adjusted based on the volume range of the sampling. The rotational dial is used to change the volume range, which is present over the plunger of the micropipette.
8. What happens on releasing the micropipette plunger too fast?
It may lead to a lower draw up of the liquid and less dispensing of the liquid. Therefore, the micropipette plunger must not be released too fast.