2. Pipettes

Pipettes

  • Pipettes are an extremely important piece of laboratory equipment.

  • Can be made of glass or plastic.

  • Can be reusable or disposable.

  • Can be either graduated or volumetric.

Graduated Glassware

  • Glassware with calibrated markings.

  • Used for liquid measurements:

    • Measures approximate volumes.

  • Degree of accuracy is poor as compared to volumetric glassware.

Volumetric Glassware

  • Measures accurate volumes ensuring measurement accuracy.

  • Volume calibration of glassware is a lengthy and time-consuming process.

  • Calibration: Means by which glassware is checked to determine the exact units it will measure or deliver by relating them to a known volume.

  • Calibrated glassware is more expensive than non-calibrated glassware.

Requirements for Volumetric Glassware

  • Must be of high quality.

  • Must be transparent.

  • Must be free from irregularities/defects.

Reading the Meniscus

  • The meniscus is the curvature in the top surface of a liquid.

  • The liquid level is always read at the bottom of the meniscus for transparent liquids.

  • Significant Figures: When reading the meniscus, it's crucial to determine how many significant figures to include, especially for providing measurements.

    • There is always a margin for uncertainty.

  • Example Images:

    • First image: Measurement is exactly 6.60 mL.

    • Second image: The meniscus is between 6.60 and 6.70 mL, indicative of uncertainty in the measurement—6.64 mL is a reasonable estimate.

Pipetting in the Laboratory

  • Pipettes are used extensively in the lab.

  • Accuracy of pipetting is one of the determining factors in the accuracy of laboratory testing.

  • Pipettes are calibrated to deliver or to contain specific volumes.

Manual and Automated Pipettes

  • Types of Pipettes:

    • Manual pipettes have at least one calibration/graduation mark.

    • Automated pipettes available for rapid, repetitive measurements of predetermined volumes.

  • Two main types of manual pipettes:

    • Volumetric (or transfer).

    • Graduated (measuring).

  • Classification of pipettes based on calibration:

    • To Contain (TC): Calibrated to contain a specific amount.

    • To Deliver (TD): Calibrated to deliver the specified volume.

To Contain (TC) Pipettes

  • Calibrated to hold a specific amount of liquid.

  • A small amount of liquid clings to the inside wall; should be rinsed with a diluting fluid to ensure complete contents are used.

  • Example of TC pipette: Serological pipette.

To Deliver (TD) Pipettes

  • Calibrated for exact delivery of designated volume.

  • Gravity causes liquid to flow out when held vertically; small amount remains in the tip.

  • Example of TD pipette: Volumetric pipette.

  • Instruction: Always hold the pipette vertically.

Blow Out Pipettes

  • To accurately deliver the liquid, any remaining fluid in the tip must be expelled manually using a bulb.

  • Indicated by an etched ring or double rings on the upper end.

  • Serological pipettes are also blow-out pipettes.

Volumetric Pipettes

  • Calibrated to deliver a fixed volume through drainage.

  • High degree of accuracy, used for specific volumes between 1 and 1000 mL.

  • Engraved ring above the bulb indicates calibration area, must be rinsed with the solution before use.

  • Specifications: Indicate calibration temperature, volume transferred at calibration mark.

  • Liquid is allowed to drain out without forcing it out; do not blow out remaining liquid.

How to Use a Volumetric Pipette

  1. Use a bulb to draw liquid up past the mark.

  2. Remove the bulb, place index finger over the opening.

  3. Wipe the outside tip to remove excess.

  4. Release pressure to adjust meniscus to the mark, ensure eye level alignment.

  5. Transfer liquid to the receiving container by touching the pipette tip to the side and allowing free flow.

  6. Do not blow out remaining liquid.

Ostwald Folin Pipette

  • Designed to deliver a fixed volume.

  • Similar to volumetric pipette but with bulb closer to the bottom, pear-shaped.

  • Primarily used for viscous fluids like blood or serum.

Volumetric Glassware Techniques

  • Correct technique is critical as it can affect volume measurements significantly:

    • Blowing out the last drops increases the delivered volume.

    • Using the top of the meniscus decreases the measured volume.

  • Pipettes must be clean to avoid reacting with reagents or changing volume.

  • Broken tips necessitate the disposal of pipettes to ensure safety and control.

Graduated Pipettes

  • Deliver specific liquid amounts between two calibration marks.

  • Also known as measuring pipettes.

  • Types of graduated pipettes:

    • Serological: Graduated to the tip, less accurate.

    • Mohr: Calibration between two marks on the stem.

Serological Pipettes

  • Larger orifice and drains rapidly.

  • Graduated to the end of the delivery tip, calibrated from 0 to the tip.

  • Etched ring indicates the last drop must be blown out.

  • Used for relative value testing.

How to Use a Serological Pipette

  1. Use a bulb to draw liquid past the 0 mark.

  2. Remove the bulb and place index finger over the top.

  3. Adjust the meniscus to the 0 mark.

  4. Drain fluid freely to the desired mark, blowing out the last drop if necessary.

Mohr Pipette

  • Space between last calibration mark and the tip is not accounted for measurement.

  • Various volumes can be delivered, calibrated between 0 and a mark on the stem.

  • Instruction: Draw liquid up to desired mark and release without blowing out the last drop.

Manufacturer Information

  • Brand: Represents the highest quality of volumetric pipettes.

  • Nominal Volume: The specified volume for a pipette with listed error limits.

  • ISO Association Mark: Indicates compliance with international standards for glassware.

  • Reference Temperature: Typically set at 20°C, indicating temperature at calibration.

  • Class A: Indicates the highest quality grade for pipettes.

Cleaning Non-disposable Pipettes

  • Soaked in a 10% hypochlorite solution tip-side up for 30 minutes.

  • Rinsed with tap water and distilled water.

  • Air-dried or dried using a warm oven.

  • Caution against drying in an oven or with air jets to prevent contamination or glass breakage.

Disposable Transfer Pipettes

  • Commonly used for liquid transfer.

  • One-piece plastic pipettes, sterile or non-sterile options available.

  • Primarily utilized in transfusion medicine (blood bank).

Automatic (Mechanical) Pipettes

  • Allows for rapid measurements of predetermined volumes.

  • Common type: Automatic or semi-automatic MicroPette.

  • Piston-operated device enabling accurate and repeatable delivery in small amounts.

How to Use a Mechanical Pipettor

  1. Attach appropriate tip and set delivery volume.

  2. Depress piston to stop position, then place the tip in liquid and allow the piston to return.

  3. Drain liquid into receiving vessel by depressing the piston against the wall to allow proper drainage.

  4. Dispose of the tip properly; some pipettors have automatic eject functions.

Parts of the Automatic Pipettor

  • Components include:

    • Plunger Button

    • Tip Ejector Button

    • Tip Ejector Arm

    • Volume Adjustment Knob

    • Digital Volume Indicator

    • Plastic Shaft

    • Disposable Tips

Conclusion

  • The precise understanding and execution of pipetting techniques are critical for ensuring accuracy and reliability in laboratory results.

  • Adequate cleaning and proper use of different types of pipettes directly affect laboratory outputs, making adherence to recommended guidelines essential.