What is the basic mechanism of how a centrifuge separates mixtures?

A centrifuge rapidly spins a rotor containing samples, generating centrifugal force directed away from the axis of rotation. This force causes particles with different densities to separate, with heavier particles moving toward the bottom of the tube. The separation speed depends on particle size and density, making centrifugation much faster than gravity-based sedimentation.

How should centrifuge tubes be prepared before use?

Select tubes compatible with your centrifuge's maximum RPM and verify limits in the documentation. Inspect tubes for cracks or damage and ensure they are intact. Fill each tube to no more than three-quarters full to prevent spills, then balance tubes and cap them tightly before placing them in the rotor.

What precautions are necessary when centrifuging hazardous materials?

Wear appropriate personal protective equipment and prepare samples in a fume hood or laminar flow cabinet. Use secondary containment such as glass or plastic inserts for hazardous chemicals or biological samples. Sealed safety tubes provide an additional contained environment. Always ensure proper use of personal protective equipment when handling potentially dangerous materials.

What rotor inspection steps should be completed before centrifugation?

Verify the rotor is appropriate for your centrifuge and check that it and its cover are clean, dry, and free of cracks or deformities. Inspect O-rings for wear and examine the centrifuge spindle for bending or damage. Ensure the rotor is correctly seated on the spindle and that sample tubes fit snugly without gaps.

What should you do if unusual sounds or vibrations occur during centrifugation?

Immediately turn off the power button to stop the centrifuge. Evacuate the immediate area and allow the rotor to stop completely before approaching. Inspect samples for spillage and the rotor for abnormalities or imbalances. Do not attempt to restart until the cause is identified and resolved.

How should a centrifuge be decontaminated after use with biological samples?

Decontaminate the rotor with 10% bleach regardless of whether a spill occurred. Allow the bleach to stand for 30 minutes, then rinse with 70% ethanol and air dry completely. After decontamination, remove and inspect the rotor for damage and document your use in the logbook.

Working with Centrifuges

0 views07:07 min

1. General Protocol for Safe Centrifuge Usage

The laboratory supervisor or laboratory safety officer should ensure all users are properly trained in the selection of the rotor, sample tube, and proper operation of the centrifuge.
Any new user should be closely supervised by a trained user until he/she is confident on how to properly use the instrument.
Prior to using the instrument, each user should carefully read and understand the safety instructions for the specific centrifuge.
Depending on sample, compatible rotors should be used.
The rotor, tube, and spindle should be clean, dry, and free from cracks or deformities.
The O-rings should be checked for any wear or cracks.
It is important to make sure that the tubes used for centrifugation are compatible with the sample and the rotation speed (low, high, or ultra).
The centrifuge tubes should not be filled more than three-fourths of their maximum fill level.
The tubes should be capped tightly.
It is extremely important to balance the tubes opposite to each other. Failure to do so may lead to incomplete separation and, in extreme cases, can cause dispersion of the tube contents in the housing of the centrifuge.
Before turning on the centrifuge, the rotor should be checked to ensure it is correctly seated on the drive and the lid is properly closed.
The lid should be kept closed at all times when the centrifuge is in operation, and should never be open until the rotor completely stops.
The operator should remain in the vicinity of the centrifuge until it has safely reached the target operation speed. In the case of unusual sounds or vibrations, the instrument should be immediately turned off. The speed limit of the instrument should never be exceeded. Upon turning off, allow sufficient time for the rotor to slow down to a stopped position. Modern centrifuges have indicators that will signify that it is safe to open the centrifuge.
Once the centrifugation is over, samples should be removed and the instrument should be checked for any spills or leaks. If necessary, the rotor should be cleaned thoroughly.

2. Additional Precautions While Handling Hazardous Chemicals or Infectious Materials

When using hazardous chemicals or biological samples that might be infectious, it is important to label the instrument while in usage to alert other workers to the danger.
Appropriate gloves¹should be worn to handle hazardous samples.
Sealed safety tubes should be used. It is preferable to use secondary confinement, if available, for the centrifuge tubes. These are usually in the form of plastic or glass inserts that are placed directly in the sample buckets of the centrifuge.
Once the centrifugation is over, the tubes should be removed from the rotor and opened only in the fume hood or biological safety cabinet, as applicable.
Cleaning, if required, should be done with extreme care, so as to not expose the operator to hazardous chemicals or infectious materials. In case of spills of such samples, see step 3, Emergency Situations, below.
For specific types of hazardous samples, it may be preferable to maintain standard procedures and cleaning protocols in laboratory. These should be reviewed and updated accordingly, as necessary.

3. Emergency Situations and Their Safety Measures

An emergency situation may arise if the rotor malfunctions or if there is a spill in the centrifuge due to tube breakage or mishandling. In such situations, the following steps should be taken.

The centrifuge should be turned off immediately and the lid should be kept closed until others are notified and evacuate the location. The power should be terminated at the power button on the instrument. The button will be located either on the front, side, or back of the instrument. The user should familiarize themselves with its location. If the power button is malfunctioning, then the user should remove the main power plug from the wall socket.
To reduce the risk of aerosols, it is sometimes better to leave the centrifuge untouched for at least 0.5 h. Consult with the centrifuge technical document.
For cleaning up spills, the operator should be well acquainted with the sample being used and should wear proper attire including lab-coat, gloves, facial shield, and safety glasses.
1. Once cleaned, the spilled material should be properly disposed of and the contaminated protective clothing should be disposed of or cleaned properly. Hands and any exposed skin should be washed thoroughly.
If overtly exposed to highly hazardous materials, 911 should be called or immediate medical attention should be administered to the afflicted person.

4. Maintenance of the Centrifuge

NOTE: The body of the centrifuge is usually made from metals that can corrode in the presence of moisture, chemicals, or strong cleaning agents. For proper maintenance of the centrifuge, it is recommended to carefully follow the manufacturer's instructions for cleaning and maintenance. Other than that, a few general measures for maintenance to be kept in mind are as follows.

The centrifuge should always be kept clean and dry.
Any kind of spills should be immediately cleaned.
The rotor should be decontaminated after use with biological and radioactive materials. It is recommended to use 10% bleach for 30 min followed by 70% ethanol, letting the rotor air dry.
Abrasive brush wires should never be used to clean the rotor and associated parts.
The rotor should be inspected after each use by the operator and at least annually by manufacturer inspector.
A log book should be maintained for each centrifuge to keep a record of users, samples, and service history. Additionally, copies of any warranty-related materials can be included in this log book.

Centrifugation is one of the most powerful tools used in labs for the separation of heterogeneous mixtures, but it can be hazardous, and therefore safety precautions should be applied.

A centrifuge rapidly spins samples to separate heterogeneous materials, which induces extreme centrifugal forces on the samples and the instrument. Due to the high speed at which a centrifuge operates, it can cause serious injury to the user if mishandled. Therefore, special care must be taken when using a centrifuge to prevent spills and damage to the equipment.

This video will illustrate the mechanism of a centrifuge and how to safely operate and maintain one.

Before going into the details of the safety measures, let's quickly review the basics of centrifugation. Centrifuges contain a rotor housed on a spindle. Differently sized rotors can usually be fitted on a single spindle to accommodate different sample bottles, tubes, or bags. A motor spins the rotor at a specified RPM or revolutions per minute. This, in turn, imparts the centrifugal force on the heterogeneous mixture directed away from the axis of rotation, which separates particles with different densities.

Different types of centrifuges operate at different RPMs. Low-speed centrifuges run at less than 10,000 RPMs, high-speed up to 21,000, and ultracentrifuges to 30,000 and above. Each type has different rotors that are compatible with it. After the run, a braking system usually slows down the rotor. In any centrifuge, high speed deforms the rotor.

In normal use, it will return to its previous size. However, if the force due to spinning exceeds the maximum force that the rotor can handle, then it might get permanently deformed. This can lead to cracks, which may, in turn, lead to serious failure. Centrifuge failures, on the other hand, may lead to sample leakage, which can be dangerous if hazardous materials are involved and should be handled with care. Now, we will show you how to prepare samples and safely use them in the centrifuge.

First, select appropriate centrifuge tubes that are compatible with the rotating speeds you want to use. Check the documentation to find out the tube's limits. Tubes should be intact and free of any cracks. Fill each to no more than 3/4 full to prevent spills. Then make sure that the tubes are balanced and cap them tightly.

When centrifuging hazardous chemical or biological samples, take special precautions. Wear appropriate protective equipment, and prepare samples in an appropriate work environment, like a fume hood or laminar flow cabinet. For more information on these safety features, see JoVE's videos on Fume Hoods.

If necessary, use a secondary containment for hazardous materials, like a glass or plastic insert. Sealed safety tubes providing a contained environment can be used as well. Everyone must be trained before using any centrifuge or rotor.

Select a rotor appropriate to the centrifuge. Check that the rotor and its cover are clean and dry and do not have cracks or deformities. Make extra sure that the O-rings on the rotor or cover are not worn. Also, check the centrifuge spindle to see if it is bent or has any other deformities. Then place the rotor on the spindle making sure it is correctly seated. Insert samples into the rotor, being sure that the tubes fit snugly in the rotor.

Always put balanced tubes opposite each from each other. Cover the rotor, ensuring that the seal is tight. Close the centrifuge lid and start your program. Again, see JoVE's previous centrifuge video, for more information about running a centrifuge program.

Stay at the centrifuge until it has reached its maximum speed. Familiarize yourself with the location of the power button. Turn off the power button if there are any unusual sounds or vibrations to stop the centrifuge immediately. Then evacuate the immediate area until the rotor has stopped. Once it has stopped, inspect the samples for spillage and the rotor for any abnormalities or imbalances. If there were no problems, wait until the centrifuge has completed its program and completely stopped.

Wait an additional half-hour if aerosol-forming chemicals are present. Otherwise, you may open

the centrifuge immediately. Remove the samples and take them back to a specialized area for opening if necessary. In case of a spill, clean up the centrifuge immediately. Use appropriate PPE to any hazard present, and dispose of contaminated materials according to guidelines. Do not use abrasive brush wires to clean as they may damage the rotor. Alert emergency personnel, and do not attempt to clean the centrifuge if life-threatening hazards have spilled.

Decontaminate the rotor with 10% bleach for any biological samples used in the centrifuge regardless of spill or not. Let the bleach stand for 30 minutes, and then rinse 70% ethanol and air dry. Lastly, remove and inspect the rotor for any damage, and note down your use in the logbook.

You've just watched JoVE's Introduction Into Centrifuge Use. You should now understand how to prepare your samples as well as how to operate and maintain the instrument.

Thanks for watching.