Proper Pipette Usage

by Jack Silver

Introduction

Although micropipettes have been used in labs for many years, there always seem to be new users. These lab workers range from new employees to temporary workers who may not have worked with pipettes in the past. Since most micropipettes are manual, there exists the possibility of variation due to differences in user technique.

While these pipettes are simple to use, a user needs to use proper technique to obtain repeatable results, and to obtain results comparable to other members of a lab.

The easiest way to test your technique is by using a pipette with an analytical balance. Pipette deionized water into a tared container on the balance to check your accuracy and reproducibility. One-thousand microliters of water weighs about 1,000 milligrams, which can be easily measured on an analytical balance.

Types of pipettes

All popular pipettes work on the same principles a piston moves up and down to aspirate or dispense a liquid. In positive displacement pipettes, the piston is in direct contact with the liquid. These pipettes are useful for viscous liquids such as oils, fats or glycerin.

The most popular pipettes are air displacement pipettes. The piston in these units moves to aspirate the liquid as described for positive displacement pipettes, but the piston is separated from the liquid by an air space. The air space allows the user to work with different samples by changing the tip; cleaning the pipette is not necessary. These pipettes can have only a single channel, or multiple channels that allow dispensing multiple samples into microplates. Air displacement pipettes can be either manual or electronic.

Electronic pipettes reduce the stress of repetitive pipetting. Being under electronic control, they also reduce variances due to users aspirating or dispensing at different rates.

Manual pipettes with selectable volume come in two forms. Most common is the micrometric screw mechanism, used in the Wheaton/Socorex Acura line. This mechanism is easy to use, but it is time consuming to change volumes, especially if the user is working at the ends of the pipettes' volume range. The user needs to "wind" the volume setting to reach the ends of the pipette operating range. Friction can progressively cause wear of the micrometer screw, and there can be mechanical backlash, both leading to inaccurate transferred volumes. The twin cam system, used in the Wheaton/Socorex Calibra line, is more accurate since it relies on a series of solid calibrated steps as plunger stops. This system allows instant setting of the key volume and fine adjustment no winding is needed. This increased accuracy comes with the minor cost of locking the plunger before changing volumes (much like using the clutch in a manual transmission car).

Types of tips

There are many different types of tips available for pipetters. Only tips supplied or approved by the manufacturer should be used, since other tips may not seal on the pipette cone properly. An improper tip seal will cause inaccuracies in the amount of liquid transferred. Improper tips may also damage the nose cone of the pipette. Wide bore tips are useful for reducing DNA or other large molecule shearing. They are also useful for viscous liquids or transferring cells, but are more susceptible to changes in barometric pressure leading to calibration problems. Smaller bore tips are better able to reach inside small tubes or vials and sequence gel wells.

Pipette working position

When aspirating the sample, the pipette must be held vertically, or else too much liquid will be drawn in. Tilting the pipette by 30 degrees can cause nearly 5% more liquid to be drawn!When dispensing the sample, the tip should be held at an angle against the container to draw out the liquid in the tip. Under normal pipetting operations, analytical chemists will recognize the pipette as a "to contain" pipette.

The tip should generally be immersed to 2-3 mm. Placing the tip deeper into the sample allows pressure from the liquid to help push more sample into the tip, reducing accuracy.

Working conditions

Under ideal conditions, the sample should be at the same temperature used to calibrate the pipette (generally room temperature). Cold liquids are denser than warm liquids. If the pipette was calibrated at room temperature, but used in a cold-room and at the cold-room temperature, smaller samples than expected would be dispensed.

Figure 2. Course and fine setting of Calibar pipette line. Because the course and fine settings are seperate, it is not necessary to "wind" the pipette as would be done on a screw-type adjustment. Figure 3. The cams in Calibar pipettes provide great reproducibility and the accuracy of a fixed volume pipette, but with the ability to easily adjust the volume setting.

Normal pipetting

1. Fit the tip, set the volume (variable pipettes only), and press down the pipetting plunger to the first stop (metering stroke) with the thumb. Immerse the tip 2-3 mm in the sample while holding the pipette vertically.

2. Slowly retract the pipetting plunger while watching the liquid fill the tip. You should not observe bubbles or turbulence, which indicate gasses being pulled from the liquid. These gasses reduce the vacuum that draws the liquid, reducing the amount of liquid aspirated into the pipette.

3. When the pipetting plunger has been retracted to its upper stop, remove the thumb since the absence of pressure increases the precision of the pipette. Slowly withdraw the pipette from the liquid. Wipe any drops on the outside of the tip on the wall of the vessel that you are drawing liquid from.

4. To dispense liquid, hold the tip against the side of the receiving container at an angle (30-45 degrees). Use the thumb to push down the pipetting plunger to the first stop, and hold it for one second. Next push the button to the second stop sliding the pipette slightly against the container wall. Pushing to the second stop blows out any liquid left in the tip.

1. For work with volatile solvents, such as methanol, this procedure may be performed on a "dummy" sample to saturate the system with vapor. This may improve accuracy, as pre-saturating the air will reduce the tendency to "blow out" the liquid before you are ready to dispense the sample. Tips should be saturated in this fashion every time they are changed. Remember that solvents other than water will have a different density, and they will pipette at differing volumes than indicated on the pipetter.

Reverse pipetting

Reverse pipetting is used to aspirate an additional volume of liquid. This technique is useful when working with thick, viscous liquids. This is also useful for volatile solvents.

1. Press the pipetting plunger with the thumb to the second stop. This is different from the procedure listed above for regular operation.

2. Holding the pipette vertically, slowly retract the plunger to its upper stop. Wait for the liquid to properly fill the tip. With viscous liquids, this will take longer than when pipetting water. A larger amount of liquid will be aspirated than with normal operation since the plunger was pushed down to the second stop. Wipe any drops on the outside of the tip on the wall of the vessel from which liquid is being withdrawn.

3. When dispensing, push the plunger only as far as the first stop. Wipe any liquid hanging on the tip on the side of the receiving container. Any remaining liquid will be discarded with the tip.

Storage

Pipettes should be stored in an appropriate rack or stand. This reduces the risk of scratching or damaging the nose cone. Damage to the nose cone could result in a poor seal to the pipette tip, which will reduce accuracy.

Testing and calibration

During regular use, the operator should inspect the pipette for scratches or other damage to the nose cone. Also, users should visually inspect the pipette for loose parts or connections.

You should periodically check the operation of your pipette by checking its calibration. Wheaton/Socorex air-displacement pipettes are warranted for calibration for 2 years. Under modern quality management such as GLP/GMP, ISO-9000, or regulatory requirements, pipettes calibration should be tested. If a pipette is dropped, or you suspect any type of damage, again, calibration should be checked.

Testing is generally performed gravimetrically, using an analytical balance. Calibration is usually done at room temperature, away from drafts or direct sunlight. The ISO-8655 standard states that the calibration be carried out between 15 and 30 C, with a relative humidity greater than 50% to reduce the effects of evaporation.

Pipettes should be tested for leaks. Aspirate the maximum listed volume for your pipette of degassed distilled water (your pipette and tip should be at the same temperature as the water to prevent expansion effects) and put the pipette on a vibration-free stand. There should be no droplets seen on the tip before a period of one minute.

The actual calibration details vary between pipettes, and are listed in the pipette instructions.

About the author

Jack Silver is Product Manager for Life Sciences with Wheaton Science Products.

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