Pipetting Techniques and Types

Pipetting Definitions

• Adjustment involves altering the pipette to ensure the dispensed volume is within specified limits.
• Air displacement pipettes are suitable for general use with aqueous solutions.
• Aspirating is drawing up the sample into the pipette.
• Blow-out refers to completely emptying the tip of the pipette.
• Calibration check is verifying the difference between the dispensed volume and the selected volume.
• Dispensing is the act of delivering the sample.
• Positive displacement pipettes are used for liquids with high viscosity and volatile liquids.

Automatic Pipettes: Air Displacement

• Air displacement pipettes use a piston to create suction, drawing the sample into a disposable tip.
• The disposable tip must be changed after each use.
• The piston does not come into contact with the liquid.

Automatic Pipettes: Positive Displacement

• Positive displacement pipettes function by moving the piston in the pipette tip or barrel, similar to a hypodermic syringe.
• These pipettes do not require a different tip for each use.
• Rinsing and blotting between samples is needed because of carryover concerns.

Automatic Pipettes: Dispensing Pipettes

• Dispensing pipettes repeatedly obtain liquid from a common reservoir and dispense it.
• Dispensing pipettes come in bottle-top, motorized, or handheld forms, or are attached to a dilutor.
• Dilutors often combine sampling and dispensing functions.

General Pipetting Guidelines

• Inspect the pipette for dust and dirt at the beginning of each work day and clean with 70% ethanol if necessary.
• Ensure the volume is set within the pipette's specified range.
• Hold the pipette with the 'grippy finger rest' resting on the index finger.
• The pipette, tip, and liquid should be at the same temperature to maximize accuracy.
• Use only recommended, high-quality, contamination-free polypropylene tips.
• Tips are designed for single use; reuse compromises their metrological characteristics.
• Pre-rinsing the tip 1-3 times with the liquid to be pipetted improves accuracy, especially with positive displacement tips.
• Prevent liquid from entering the pipette by avoiding turning it on its side when there is liquid in the tip.
• Prevent contamination by using the tip ejector and gloves.
• Store pipettes in an upright position on pipette stands when not in use.
• Regularly check calibration based on use frequency, at least annually, or every three months if used daily.
• Adhere to the manufacturer's recalibration instructions.

Preventing Cross Contamination: Pipette to Sample

• Contaminated pipettes or tips can cause sample contamination.
• Use sterilized tips or sterilized filter tips and autoclave the pipette, if possible, for prevention.
• Change the tip after each sample pipetted.

Preventing Cross Contamination: Sample to Pipette

• Samples or aerosols can enter the pipette cone.
• Keep the pipette vertical to prevent liquid from running into the pipette body.
• Release the push-button slowly.
• Use filter tips or a positive displacement pipette with tips to prevent aerosol contamination.
• Store the pipette vertically to prevent contamination.

Preventing Cross Contamination: Sample to Sample (Carry-Over)

• Remains of sample A can mix with sample B inside the tip, leading to false results.
• Change the tip after each sample.
• Autoclave or clean the pipette if contamination is expected.

Common Sources of Error

• Volatile liquids evaporate, so use in saturated air.
• High-density liquids sink, therefore change technique.
• Viscous liquids stick to the tip, so change technique.
• Hydrophilic tips retain liquid, thus pre-wet the tip.
• Immersed tips carry over liquid; immerse 2-3mm.
• Surface tension reduces accuracy; change technique.
• Angled pipettes take up more liquid, keep vertical.
• Hot and cold liquids affect accuracy, so pipette liquids at room temperature.
• Human factors affect results, so practice and observe technique.

Centrifugation

• Centrifugation separates solid matter from a liquid suspension using centrifugal force.
• Centrifuge is the machine used, which consists of a head or rotor, carriers, or shields attached to a motor's vertical shaft, enclosed in a metal covering.

Centrifugal Force

• Centrifugal force is expressed in relative centrifugal force (RCF) or in gravities (g).
• It depends on mass, speed (rpm), and radius.
• RCF = 1.118 x 10^-5 x r x (rpm)^2.
• The constant is 1.118 x 10^-5, for angular velocity.
• Radius (r) is in centimeters, from the centrifuge axis center to the bottom of the test tube shield.

Types of Centrifuges

• Microcentrifuges are for microcentrifuge tubes.
• Benchtop centrifuges are low-speed and useful for separating bodily fluids.
• Ultracentrifuges spin at very high speeds and g-forces.
• Industrial centrifuges process large volumes of materials continuously.
• Low-speed centrifuges are typically used for general purposes.
• High-speed centrifuges are suitable for collecting larger cellular organelles and precipitated proteins.
• Refrigerated centrifuges are similar to benchtop models, but with temperature control.
• Continuous flow centrifuges can centrifuge large volumes of samples without impacting the rate of sedimentation.

Centrifuge Care and Maintenance

• Care involves daily cleaning of spills or debris like blood or glass.
• It should be properly balanced and free from excessive vibrations.
• Balancing a centrifuge requires equalizing the volume and weight distribution across the centrifuge head.
• The centrifuge cover should remain closed until it has come to a complete stop to avoid aerosol contamination.
• Check the timer, brushes (if present), and speed periodically.
• Consult the manufacturer's service manual for details on brush changes and lubrication requirements.
• The speed is easily checked using a tachometer or strobe light.