WEEK 2: PIPETTING TECHNIQUES AND LABORATORY MATHEMATICS IN MOLECULAR BIOLOGY LABORATORY

PIPETTE TYPES AND TECHNIQUES

PIPETTE CLASSIFICATION BASED ON DESIGN

• To Contain (TC)

  • Description/Uses:

  • Holds or dispenses a particular volume.

  • Does not dispense that exact volume; liquid may adhere to the lumen.

  • Rinsing is necessary for accurate solution transfer.

• To Deliver (TD)

  • Description/Uses:

  • Holds a particular volume that will flow by gravity to deliver the exact volume.

  • Further classified into:

    • To Blowout

    • Requires the remaining liquid after free delivery to be blown out to complete the volume transferred.

    • To Drain (Self-Draining)

    • Allows fluid to drain freely by gravity.

PIPETTE CLASSIFICATION BASED ON FUNCTION

• A. Measuring Pipets

  • Graduated Pipets (Serological Pipets)

  • Capable of dispensing different liquid volumes with graduation marks up to the tip.

  • Mohr Pipets

  • Does not have graduation marks up to the tip, classified as self-draining.

  • Macropipets

  • Dispenses volume greater than 1 mL.

  • Micropipets

  • Calibrated to contain a volume of less than 1 mL.

• B. Transfer Pipets

  • Volumetric Pipets

  • Long narrow tubes with an elongated or rounded bulb designed for maximum accuracy and precision for delivering exact fluid volumes.

  • Ostwald-Folin Pipet

    • Specialized for transferring biologic fluids with greater viscosity than water.

• Automatic Pipet

  • Most common type, permitting rapid, repetitive measurement and delivery of volumes.

  • Mechanism for liquid draw and dispense is integral to the pipet.

PIPETTING TECHNIQUES

MARKINGS ON VOLUMETRIC GLASSWARE

• Graduation Marks

  • All lines in volumetric glassware provide reference for measurement.

• Calibration Marks

  • Essential for accurate liquid measurements.

• Reading the Meniscus

  • Clear or Colorless Solutions: Read the lower meniscus.

  • Colored/Turbid Solutions: Read the upper meniscus.

  • Readings must be made with the observer’s eye level to avoid parallax error.

PARTS OF AN AUTOMATIC PIPETTE

• Serial Number

BASIC PROCEDURES WHEN USING PIPETTES (AIR DISPLACEMENT)

1. Set the Volume:

   • The required volume is set, and the piston moves to the appropriate position.

2. Prepare for Aspiration:

   • Press the push-button to expel air equal to the predetermined volume.

3. Aspirate the Sample:

   • Release the push-button to create a partial vacuum that draws the liquid into the tip.

4. Dispense the Sample:

   • Press the push-button again to increase air pressure that pushes the liquid out of the tip.

BASIC PROCEDURES WHEN USING PIPETTES (POSITIVE DISPLACEMENT)

1. Set the Volume:

   • Ensure correct volume is set; the piston moves to the starting position.

2. Prepare for Aspiration:

   • Press the button to move piston down, immersing the orifice below the liquid surface.

3. Aspirate the Sample:

   • Release the plunger to draw the liquid into the capillary.

4. Dispense the Sample:

   • Press down to expel the liquid out of the capillary.

TYPES OF AUTOMATIC PIPETTES

AIR DISPLACEMENT

• When the push-button is pressed, the piston moves down to let air escape.

• Air displaced equals the volume of liquid aspirated.

POSITIVE DISPLACEMENT

• Works like a syringe, with a constant aspiration force, unaffected by sample properties.

• Recommended for viscous, dense, volatile, radioactive, or corrosive samples.

• Direct contact with the sample for optimal results; disposable pistons are utilized.

MODES OF PIPETTING

FORWARD PIPETTING

• Preferred for dispensing aqueous solutions, low detergent, or protein concentrations.

• Steps:

  1. Hold the pipette vertically and depress the plunger to the first stop.

  2. Immerse the tip in the liquid and allow time for the sample to fill.

  3. Place the tip at an angle against the receiving vessel and press to the first stop.

  4. Wait and then press the plunger to the second stop for complete delivery.

REVERSE PIPETTING

• Used for viscous liquids or foaming samples.

• Steps:

  1. Depress plunger to the second stop initially.

  2. Immerse the tip to aspirate.

  3. Perform distribution as before, ensuring to wait each time.

  4. For purging, allow for waste to clear.

RECOMMENDATIONS FOR PIPETTING DIFFERENT SOLUTIONS

• Whole Blood:

  • Utilize forward pipetting to aspirate, dispel, and ensure clearing of the tip by repeating as necessary.

• Pre-rinsing or Pre-waiting:

  • Aspirate the liquid to be dispensed before timing for greater uniformity. This step should be performed multiple times upon changing tips or increasing volume settings.