Multichannel Pipettes: Parts and Calibration

An ELISA technician in a hospital laboratory is running a batch of 80 hepatitis B surface antigen tests. Using a single-channel micropipette, she pipettes 100 µL of patient serum into each well of the microtiter plate — 80 individual pipetting steps, each requiring tip change, aspiration, dispensing, and ejection. By the 60th sample, her pipetting angle has drifted slightly. Fatigue has made her plunger depression less consistent. The last 20 samples receive marginally different volumes. When the plate is read, the optical density values in the final rows show higher background noise than the first rows. Three borderline results in those rows cannot be confidently called positive or negative and must be repeated.

A multichannel pipette would have delivered all 80 samples in 10 pipetting steps — consistent volume, consistent angle, consistent pressure across every channel. The clinical difference between a single-channel and multichannel pipette in high-throughput work is not convenience. It is reproducibility.

Pipetting are ideal for contamination-free liquid handling. Some procedures require transferring the same fluid in multiple wells or tubes. So, switching to a multichannel pipette is a good idea.

Multichannel pipettes are the pipettes having various channels or shafts (8 to 384 channels available) for pipetting samples into many wells at the same time. These significantly increase productivity and output with minimal production cost and testing time in the laboratory.

The multichannel pipette is similar to the single channel micropipette or regular pipette used in the laboratory in its function, parts, and utility. But a multichannel pipette is better than a single-channel pipette in laboratories repeatedly handling the same liquids (reagents/samples).

Figure: Electronic and Manual Multichannel pipette

Why Multichannel Pipettes Matter in Clinical Microbiology

The microtiter plate — a plastic tray with 96 or 384 individual wells — is the format for several of the most important tests in clinical microbiology:

ELISA (Enzyme-Linked Immunosorbent Assay): Detecting antibodies or antigens in patient samples across 96 wells simultaneously. Each well must receive an identical volume of sample and reagent for the optical density readings to be comparable across the plate.

Broth Microdilution MIC Testing: Determining the minimum inhibitory concentration (MIC) of an antimicrobial agent against a bacterial isolate. Serial dilutions of antibiotic are prepared across a row of wells; each well must contain an exactly defined concentration. A volume error in one well shifts the concentration and produces a false MIC result.

Serological Titrations: Quantitative antibody tests (anti-streptolysin O, anti-HBs titers) that use serial dilutions across a plate format.

In all of these, the 96-well plate demands that identical volumes reach identical wells simultaneously. The multichannel pipette is not just faster than single-channel pipetting for this work — it is more reproducible, because all channels fire from a single plunger depression under the same conditions.

Parts of Multichannel Pipette

The parts of the manual multichannel pipette are the same as a manual single-channel micropipette which are as follows:

• Plunger: It has two stops, first for aspirating liquid and second for dispensing the aspirated fluid.
• Tip ejector button: Pressing down this button helps in the ejection or removal of pipette tips.
• Volume adjustment knob: Like micropipettes, multichannel pipettes are also available with adjustment volume. So, a knob near the plunger helps set the desired volume.
• Digital volume display window: It displays the volume of the pipette.
• Plastic shaft: It is a tube-like structure used to displace air in an air displacement pipette and plays the collar role for ejecting tips.
• Tip cone: It holds the pipette tips. The only difference in this part between the multi and single-channel pipette is the number. The single channel has only one tip cone, but multichannel comes in the range of 8 to 18 tip cones.
• Pipette tip: These are attachments made of disposable or autoclavable polypropylene. It attaches to the tip cone and comes in the direct compound of the fluid to be aspirated.

The electronic multichannel pipette has almost the same parts as the manual. However, instead of the volume adjustment knob and plunger, the electronic multichannel pipette has buttons to change the volumes and start the pipetting procedure.

How Does a Multichannel Pipette Work?

The correct pipetting method is essential for obtaining precision in liquid handling. The following things to consider before using a multichannel pipette:

1. Use gloves to ensure personal safety.
2. Adjust to the correct or desired volume in a calibrated and well-maintained pipette.
3. Use a clean pipette and ensure there is no blockage in the tip cone area.
4. Once the pipette is ready to use, attach the pipette tips, keep the pipette in an upright position and use the pipette tips in a box.
5. Ensure the pipette tip is firmly attached to the pipette.

Methods for Dispensing Liquids

Two of the commonly used methods are forward and backward pipetting techniques.

Forward pipetting

It is best for liquids that do not foam easily or are in a limited amount. It is also known as exact pipetting.

1. Depress the plunger into stop one before dipping the dip into the liquid.
2. While dipping the tips into the container, ensure it is as low as 1 cm, and all the tips are in equal depth.
3. Release the plunger to fill the tips.
4. Withdraw the tips from the container slowly from the edge to remove any extra liquid.
5. Repeat the steps 1-2 times for prewetting or priming the tips for aspirating and dispensing equal volumes in all the channels.
6. Check the tips for air bubbles, improper filling, or leakage before dispensing in the target container.
7. If there are air bubbles or the tips are filled improperly, dispense the liquid and repeat aspiration and if there is leakage, discard the tips and reattach the new ones.
8. For dispensing liquid in the target wells, place the tips as close to the bottom of the wells.
9. Depress the plunger into the first stop, pause, and then depress to the last stop. This action dispenses the liquid. Remove the pipette slowly while the plunger is down by keeping the tips inside the well.
10. Release the plunger and press the ejector button to discard the tips.

Backward Pipetting

It is also known as overfilling technique.

• The difference between this method and forward pipetting is depressing the plunger up to the second stop before dipping into the liquid, which overfills the liquid in the tips.
• Also, while dispensing the liquid, press the plunger to the first stop and discard the remaining liquid.

Calibration of Pipette (Multi and Single Channel)

Calibration is the process of assessing and refining any instrument’s precision and accuracy, especially measuring devices. Likewise, the pipette needs calibration from time to time.

You need to know the following for the correct way of calibrating a pipette manually:

Things to Consider for Calibration

• Temperature controlled environment
• Anti-vibration table
• Controlled pressure
• Know the proper pipetting techniques
• Clean environment
• The balance should be calibrated and stable.

Materials Required for Calibration

• Pipettes
• Semi-micro Balance
• Distilled Water
• Density chart for water at different temperatures
• Clean beakers
• Calculator or software for calculation
• Notebook for recording data

Procedure of Calibration

According to Texas Tech University, gravimetric analysis is ideal for calibrating a pipette. The step-by-step method of calibration is as follows:

1. Keep water at room temperature for 15-30 minutes to obtain a temperature-controlled environment. Then record the temperature of the water.
2. Place the clean, empty beaker in the analytical balance and reset the balance to display 0.
3. Then, take the pipette needed to be calibrated and ensure it is unclogged and ready for pipetting.
4. Aspirate the water and make sure no air bubbles are formed during aspiration.
5. Dispense the water into the beaker and measure the weight of the water. Then record the weight.
6. Repeat steps two to five, 5-10 times.
7. Calculate the volume of water dispensed using the formula V= w **✕**Z, where V is calculated volume, w is the average weight of the pipetted water, and Z is the conversion factor based on the density of water in the particular temperature. The software can be used in the calculation.
8. An average number of times the test is done is calculated.
9. Use software to determine the accuracy of the pipette. Calculating the accuracy manually is also possible using the formula A =100 **✕**Vavg/V0, where A stands for accuracy, Vavg stands for average volume, and V0 stands for the assigned value of the pipette. The ideal accuracy should be between 99-101%.
10. Record the result; the pipette is ready to use if the value is between 99% and 101%.

The calibration of the multichannel pipette is the same as the single channel pipette. A center plunger mechanism is used for activating all the channels at the same time in a consistent way. However, the process becomes hectic in the standard laboratory, so it is best to reach out to companies providing calibration services for the multichannel pipette.

Difference Between Single and Multichannel Pipette

• A single channel pipette is not ideal for increasing efficiency in the laboratory that handles larger assays like 96 well ELISA. Pipetting from a single channel requires repeating the same task again and again.
• The single channel pipette has a capacity of 0.1 to 10,000 μl, and the multichannel pipette has a volume capacity of 0.5 to 1000 μl.
• A few years back, a multichannel pipette had inaccuracy in aspirating liquid in different channels due to difficulty in use. However, the issue is handled well by the new generation pipette.
• Calibration of the multichannel pipette is more time-consuming and tedious than a single channel pipette.
• A multichannel pipette helps to achieve a significant decrease in human error due to less repetition of pipetting.
• Repairing some multichannel pipettes is impossible because if one channel of the pipette gets damaged, the entire system needs to be changed.

Common Errors with Multichannel Pipettes

How to Remember

Multichannel = plate work; single-channel = tube work. The simplest selection rule: if your target container is a microtiter plate (96-well or 384-well), use a multichannel pipette. If it is a tube, an Eppendorf, or a single container, use a single-channel micropipette. The format of the target container tells you which pipette to reach for.

Forward pipetting = standard; backward pipetting = foamy or viscous. Forward pipetting (press to stop 1, aspirate, press to stop 1 to dispense, stop 2 to blow out) is the default. Switch to backward pipetting (press to stop 2, aspirate overfill, press to stop 1 only to dispense) when the liquid foams easily (serum, detergent solutions) or is viscous. Backward pipetting leaves excess in the tip — this is correct, not a mistake.

8 channels = column; 12 channels = row. A standard 96-well plate has 8 rows (A–H) and 12 columns (1–12). An 8-channel pipette fills one column at a time; a 12-channel fills one row at a time. Knowing this tells you immediately how many pipetting steps are needed to fill a full plate: 12 steps with an 8-channel pipette, 8 steps with a 12-channel pipette.

Calibration formula anchor: V = w × Z V (calculated volume) = w (average weight of water dispensed) × Z (conversion factor from density table at that temperature). This is gravimetric calibration — the gold standard for pipette accuracy assessment.

Available Multichannel Pipette

Major multichannel pipette manufacturers include Eppendorf, Gilson (PIPETMAN series), Thermo Fisher Scientific (Finnpipette series), Sartorius (Picus series), Rainin (Mettler Toledo), and Integra Biosciences (Viaflo series). Channel configurations range from 4 to 384; volume ranges vary by model. For current specifications, refer to manufacturer websites or your laboratory supplier catalogue.

For a comparison of all pipette types used in the microbiology laboratory, see Types of Pipettes in the Microbiology Laboratory

Key exam facts in one table

References:

1. Clinical and Laboratory Standards Institute (CLSI). (2016). Clinical Microbiology Procedures Handbook (4th ed.). American Society of Microbiology. https://doi.org/10.1128/9781555818814
2. ISO 8655-1:2022. Piston-operated volumetric apparatus — Part 1: Terminology, general requirements and user recommendations. International Organization for Standardization.
3. Eppendorf AG. (2019). The Lab Pipetting Guide. Eppendorf. https://www.eppendorf.com/pipetting-guide
4. Mahon, C. R., Lehman, D. C., & Manuselis, G. (2018). Textbook of Diagnostic Microbiology (6th ed.). Elsevier.
5. Cheesbrough, M. (2006). District Laboratory Practice in Tropical Countries, Part 2 (2nd ed.). Cambridge University Press.