Pipettes

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Pipettes

Introduction

Pipettes, also known as chemical droppers, are small glass or plastic tubes used to transfer a measurable amount of liquid in laboratory practices. Our sophisticated pipettes are available in several designs with differing levels of accuracy and precision. Modern pipettes have allowed dispensing amounts of liquid down to about 0.1 μL manually. Pipettes work by creating a partial vacuum above the liquid-holding chamber and then releasing this vacuum to draw the liquid from the solution and then dispense it in another container. Precise and reproducible liquid dispensing is essential for research and diagnostic purposes in medical and molecular biology laboratories.

 

Over a century ago, Pasteur invented the glass pipette to eliminate contamination during sample transfer. These pipettes are still in use. In the 1950s, a handheld, piston-operated pipette was introduced as an alternative to potentially dangerous mouth pipetting. Previously, the handheld pipettes had a pre-established volume setting. Then they were further improved to adjust the volume manually. In 1972, Dr. Gilson invented the first continuously adjustable pipette. Since then the pipettes are prominently been used in laboratories for research as well as diagnostic purposes because of their accuracy, precision, and reliability.

Principle

Air-cushion principle

In the air cushion principle, an air cushion is present at the interface of the liquid in the tip and the piston inside the pipette. The piston is pushed to move the air cushion, and the liquid is thus drawn into the pipette tip or dispensed. This air cushion works like an elastic spring, to which the liquid to be drawn sticks. 

 

Air-displacement pipettes are used for aqueous samples in general laboratories. In this type of pipette, a cushion of air (dead volume) is present between the piston and the liquid sample. The piston is present in a cylinder or capillary tube that moves with the volume adjustment. The liquid is dispensed by pressing the operating button to the first stop, and the piston expels the same volume of air. The operating button is released when the tip is immersed in the liquid for aspiration. When the button is pressed again to the first stop, the air dispenses the liquid. To empty the tip, the operating button is pressed to the second stop. 

 

Positive-displacement principle

The positive-displacement principle is used in the pipettes having syringe-like pipette tips. The piston is constructed in the tip. Positive-displacement systems are closed systems as they do not require tips from outside. In this type of pipette, an air cushion is not present between the piston and the liquid. Therefore, the aspiration force remains unaffected by the physical properties of the sample. This enables the pipette to be used for viscous or high-density samples, for example, glycerol or blood.

Pipetting Techniques

Most commonly used dispensing techniques include forward pipetting, reverse pipetting, dispensing, diluting, and sequential dispensing. The selection of the dispensing technique depends on the experimental needs. 

 

Forward dispensing

Forward pipetting is used for aqueous solutions that contain low concentrations of protein or detergents. In this technique, the tip is wetted in the liquid before drawing to improve the analysis. It is the most commonly used pipetting technique to dispense an exact volume of the liquid from the tip repeatedly. The careful technique is essential as the operation plug is depressed only to the first stop during drawing and dispensing. 

 

Reverse dispensing 

Reverse pipetting techniques are only applied to air displacement pipettes. With this technique, the liquid could be aspirated more than the selected volume, but only a specified amount is dispensed. This technique is known for its precision with viscous, volatile, and foaming samples.

Pipette Types

Beral pipette

The Beral pipette is also known as a disposable pipette. It is made of plastic and is used for the non-quantitative transfer of solutions. Sometimes graduations are present on the stem of these pipettes. 

 

Pasteur pipette

Pasteur pipette is a thin glass transfer pipette like the Beral pipette. It requires a disposable rubber bulb to draw the sample and then dispense it. The thin stem allows for higher precision and more accuracy as compared to the Beral pipette for small volume transfers.

 

Serological pipette

Serological pipettes are graduated and are used for quantitative transfers. It requires a vacuum source for liquid dispensing typically a pipettor or a pipette bulb attached to its blunt end. It is frequently used in the laboratory for transferring milliliter volumes, from less than 1 ml to up to 50 ml. Serological pipettes are useful for mixing solutions and cell suspensions and transferring liquids.

 

Volumetric pipette

Volumetric pipettes, also known as bulb pipettes, allow the user to precisely transfer or measure the sample. These pipettes are equipped with a large bulb with a single graduation mark as it is calibrated for a single volume. Usually, the markings are for 10, 25, and 50 mL. These types of pipettes are used to make laboratory solutions from a base stock and prepare solutions for titration.

Transfer Protocol

  1. Carefully fix the tip on the holder. 
  2. Comfortably hold the pipette in one hand with the thumb positioned on the plunger.
  3. Press the push button smoothly to the first stop. 

Note: Do not immerse the tip in the liquid at this step. 

 

  1. Immerse the tip in the liquid to the correct depth. Keep the pipette straight and smoothly release the plunger to aspirate the liquid.
  2. Make sure that the plunger fully extends to its upper stop position. 
  3. Push the plunger down to the second stop to force the air through the tip to expel the remaining liquid.
  4. Remove the tip from the solution and release the press button.

 

Air-displacement pipettes

  1. Calibrate the pipette by adjusting the piston on the required volume of the liquid.
  2. Press the push button before sample aspiration to expel a volume of air equal to the set volume.
  3. Release the push button to create a partial vacuum inside the tip. This atmospheric pressure would force the desired volume of the liquid through the tip.
  4. Press the push button again. This will increase the air pressure inside the shaft and the tip. The compressed air will then push the liquid out of the tip.

 

Positive-displacement pipettes

  1. Set the required volume. 
  2. Press the push button for sample aspiration. The piston is lowered down to the end of the capillary.
  3. Immerse the tip orifice below the liquid surface. 
  4. Release the push button to move the piston. The ambient pressure forces the required volume of the liquid through the orifice.
  5. Press the push button again. This will down the piston to expel the liquid out of the capillary.

Precautions

  • Wear proper gloves, coat, and masks in the laboratory.
  • Do not use the same tip for two solutions to avoid contamination.
  • Set the volume carefully before drawing up the liquid.
  • Wipe the work area on the laboratory bench with a disinfectant before and after the operation.
  • Discard the used tips properly.

Maintenance and Care of Pipettes

  • Properly clean the pipette before and after every use. 
  • Rinse the contaminated parts of the pipette with distilled water or 70% ethanol and air dry at 60°F to be used for aqueous solutions.
  • Autoclave the lower part at 120°C for 20 minutes for infectious solutions.
  • For proteins, rinse the pipette with a detergent and air dry. Do not use alcohol for protein solutions as it will set the proteins.
  • Periodically inspect the pipette for proper functioning and damages. 

Strengths and Limitations

  • Pipette is a powerful laboratory tool to transfer accurate volumes of liquid.
  • Pipettes are equipped with an easily adjustable digital volumeter for optimal calibration to draw specific volumes.
  • The pipettes are easily cleaned and autoclaved that makes them useful for infectious solutions too.
  • Pipettes have made research more comfortable and accurate by allowing safe sample transfer.
  • They need proper maintenance and expertise to be handled.

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