Laboratory Exercise: Pipetting

Objectives

  • At the end of this laboratory activity, students should be able to:

    1. Identify the appropriate use of different types of pipettes.

    2. Demonstrate the proper technique of delivering an aliquot of a solution.

    3. Practice the proper technique for aspirating and dispensing liquids.

    4. Analyze how to calibrate a pipette to ensure its accuracy by comparing the volume dispensed to the known standard volume and adjusting as necessary.

Definition of Pipette

  • Pipette: A pipette is a glass or plastic laboratory apparatus used for the transfer of a volume of liquid from one container to another.

    • Applications of pipettes include:

    • Reconstituting controls and calibrators.

    • Preparing serum or plasma dilutions.

    • Aliquoting specimens.

Classification of Pipettes

I. Calibration Marks/Design

  1. To Deliver (TD)

    • Definition: Delivers the exact amount it holds into a container.

  2. To Contain (TC)

    • Definition: Holds a particular volume but does not dispense the exact amount.

II. Drainage Characteristics

  1. Blowout

    • Definition: Pipette has continuous etched rings on top; the exact volume is obtained when the last drop is blown out.

  2. Self-draining

    • Definition: No etched rings; liquid drains by gravity.

III. Types of Pipettes

  1. Transfer Pipets

    • Volumetric Pipet

      • Use: For non-viscous liquids.

      • Characteristics: Self-draining, smaller amount left in the tip should not be blown out.

    • Ostwald Folin Pipet

      • Use: For viscous fluids.

      • Characteristics: Equipped with an etched ring.

    • Pasteur Pipet

      • Use: Transfers fluids without consideration of a specific volume.

    • Automatic Pipettes

      • Types: Macro or micropipettes.

  2. Graduated or Measuring Pipets

    • Serological Pipet

      • Characteristics: Graduated to the tip; designed for blowout.

    • Mohr Pipet

      • Characteristics: Lacks graduation to the tip; calibrated between two marks and is self-draining.

    • Bacteriologic Pipet

    • Ball, Kolmer, and Kahn Pipets

    • Micropipettes.

Details Regarding Types of Pipettes

Type

Graduations

Purpose

Method

Orifice

Serological

Up to the tip

Non-viscous

Blow-out

Smaller

Mohr

Between two marks

Viscous

Self-draining

Larger

Correct and Incorrect Pipette Positions

  • Correct Positions:

    • For Serologic/Mohr and Volumetric/Ostwald-Folin pipets, maintain proper vertical alignment while working.

Manual Pipetting Technique

Filling the Pipet:

  • Instructions:

    1. Hold the pipet vertically at eye level and insert the tip into the liquid.

    2. Use a rubber bulb for filling the pipet.

    • Caution: Do not insert the pipet too far into the bulb or force the bulb onto the pipet.

    1. Place the rubber bulb at the top of the pipette and squeeze it to expel air.

    2. With the tip submerged in the solution, relax the bulb gently to draw the solution into the pipet.

    • Note: Always keep the tip below the liquid surface to prevent air intake.

    1. When the solution is approximately 20 mm above the calibration mark, remove the bulb and place your finger on top to prevent loss of liquid.

    2. Remove the pipet from the liquid and gently wipe the outside with soft tissue.

    3. Release the pressure of your finger to allow the liquid to fall until it reaches the calibration mark.

Dispensing from a Pipet:

  • Instructions:

    1. Position the pipet into the receiving container and release the solution.

    2. Avoid splashing into the container.

    3. Avoid quick release of the liquid.

    4. Do not blow any remaining liquid out from the lower tip.

Reading a Meniscus

  • Meniscus: The curve seen at the top of a liquid in response to the walls of its container.

    • Types of Meniscus:

    • Concave: Observed in most liquids including water.

    • Convex: Less common.

  • Instructions for Reading:

    • Align your eye level with the meniscus.

    • For clear liquids like water, read the bottom of the meniscus.

    • For colored liquids, read the top of the meniscus.

Classification of Mechanical or Automatic Pipets

  1. Air Displacement Pipet

    • Function: Uses a piston for suction to draw the sample into a disposable tip.

    • Note: Tips must be changed after each use.

  2. Positive Displacement Pipet

    • Function: Operates by moving the piston in the pipet tip or barrel, similar to a hypodermic syringe.

    • Note: Does not require a different tip for each use.

  3. Dispenser/Dilutor Pipet

    • Function: Retrieves liquid from a reservoir and dispenses it repeatedly.

    • Options: May be bottle-top, motorized, handheld, or attached to a dilutor, often combining sampling and dispensing functions.

Points of Resistance in Pipetting Mechanism

  1. First Stop

    • Definition: The point where the piston reaches the calibrated volume displayed on the dial.

  2. Second Stop

    • Definition: Engaged to fully expel the aspirated volume into the receiving vessel, equivalent to blowing out a glass pipette.

Calibration of Pipettes

  • Calibration Standards:

    • Class A pipets do not require recalibration.

    • Calibration Mediums:

    • TD pipets use distilled water.

    • TC pipets use mercury.

    • A 0.1% phenol red solution in distilled water is used for reproducibility comparison of different brands of pipet tips.

    • Cleaning Solution:

    • Acid dichromate (H₂SO₄ + K₂Cr₂O₇) used for cleaning glassware.

References

  • Bishop, M. L., Fody, E. P., Siclen, C. V., Mistler, J. M., & Moy, M. (2010). Clinical chemistry: principles, techniques, and correlations (6th ed.). Lippincott Williams & Wilkins, a Wolters Kluwer.

  • Rodriguez, M.T. (2014). Clinical Chemistry: Review Handbook for Medical Technologists.

Acknowledgment

  • Prepared by Kathleen May Y. Balda-Lopez, RMT at St. Alexius College, Inc., Medical Technology Department, City of Koronadal, South Cotabato.