A centrifuge is a laboratory device that uses rotation to apply a sustained centrifugal force, which substitutes for gravity. This force separates components within a mixture based on their density. By spinning samples at high speeds, a centrifuge forces denser particles to the bottom of a tube while lighter components remain toward the top, enabling the separation of substances like cells from plasma or proteins from a solution. Understanding proper procedures is essential for achieving accurate experimental results and maintaining a safe laboratory environment.
Essential Safety and Pre-Run Checks
Before engaging the equipment, mandatory safety and inspection protocols must be completed to prevent mechanical failure or injury. Always wear appropriate personal protective equipment (PPE), including safety eyewear, a lab coat, and gloves, to protect against potential splashes or aerosols. Ensure all clothing offers adequate coverage, such as long pants and closed-toe shoes.
A thorough inspection of the centrifuge is necessary before every use. Visually check the rotor and all accessories, like buckets or tubes, for cracks, corrosion, or deformities that could compromise integrity during high-speed operation. Confirm the rotor is correctly and securely seated on the drive shaft, and that the bowl and spindle are clean and dry. Never use tubes that are cracked or not rated for the intended speed and temperature.
Sample Preparation and Rotor Balancing
Proper preparation of samples and precise balancing of the rotor are essential for safe and effective centrifugation, as an imbalance can lead to catastrophic damage to the machine. Begin by using identical tubes of the correct material and volume capacity for all samples to ensure uniform weight distribution. Fill the tubes according to the manufacturer’s specified limits, avoiding both underfilling and overfilling, which can lead to tube collapse or leakage.
The fundamental principle of balancing is to ensure that the weight of opposing loads across the rotor’s center of rotation is equal. This is achieved by matching the total weight of the tube, its cap, and the sample, not just the volume of the liquid inside. Use a precision scale to weigh all tubes and adjust the liquid levels until opposing tubes fall within the manufacturer’s specified tolerance, often less than one gram.
When loading the tubes into the rotor, place them symmetrically across the center point, such as directly opposite one another in a fixed-angle rotor. For an odd number of samples, create a balance tube by filling an empty, identical tube with water or a similar liquid until its total mass matches the single sample tube it opposes. This symmetrical arrangement ensures the center of gravity remains centered during the spin, preventing excessive vibration and strain on the motor.
Running the Centrifuge
Once the rotor is correctly loaded and balanced, the next step is setting the operational parameters. Speed is set using either Revolutions Per Minute (RPM) or Relative Centrifugal Force (RCF), but RCF is the preferred measure for reproducibility. RPM indicates the number of rotations per minute, while RCF, also known as g-force, measures the actual force applied to the sample, factoring in the rotor’s radius.
Most protocols specify RCF because it guarantees the same separation force is applied regardless of the centrifuge model or rotor size. You can convert RPM to RCF using a formula or an automatic calculator often built into modern centrifuges. After setting the RCF and the run time, securely close the lid, ensure the latch mechanism is engaged, and then initiate the run.
Stay nearby until the centrifuge reaches its full speed and appears to be running smoothly. If unusual noises, such as grinding, or excessive shaking or vibration occur, stop the machine immediately and re-check the balance of the tubes. Do not attempt to open the lid while the rotor is in motion, even if the speed is low, as it presents a serious safety hazard.
Post-Spin Procedures and Basic Care
After the programmed run time is complete, wait for the rotor to come to a complete and unassisted stop before opening the chamber lid. For high-speed or biohazardous runs, wait an additional ten minutes after the stop to allow any generated aerosols to settle before opening. Retrieve the separated samples gently to avoid disturbing the newly formed pellet or gradient.
Immediately after the run, especially if tube breakage or spillage is observed, the centrifuge and rotor must be decontaminated. Wipe the rotor and chamber with a mild, pH-neutral detergent and warm water, taking care not to pour liquid directly into the chamber. Any spills, particularly biohazardous ones, require immediate decontamination using an approved disinfectant, such as a diluted bleach solution, followed by a rinse and thorough drying.
Regular cleaning prevents corrosion and prolongs the lifespan of the equipment; the centrifuge should be wiped down after every use. Remove the rotor and accessories periodically for a detailed inspection and cleaning, especially of the bores and seals. Only plug the centrifuge back into the power supply after ensuring all components, including the chamber and rotor, are completely dry.