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Respect the Ultra

If a random sampling of my hallway is any indication, then half of you don’t think too much of using a ultracentrifuge, while the other half of you are scared to death of it. I think the best approach is right in between these two perspectives: have a healthy respect for the ultra. Here are some hints and tips for using a preparative ultracentrifuge, disaster free.

Ultracentrifuges generally come in two flavors: analytical and preparative. Analytical ultras are a specialized piece of equipment, and you can read more about them here. Preparative ultras are the simpler, more widespread machines that you are more likely to use in the course of purifying viruses, DNA, or proteins, and handle larger volumes that the analytical ultras.

These beasts of the lab spin samples fast enough to create centripetal forces as high as 1,000,000 times gravity. That is a serious amount of force, which deserves a serious amount of respect. Here’s what you need to know:

Balance your tubes
I know it’s obvious, but it has to be said. You CANNOT ‘balance’ your ultracentrifuge tubes by volume (ie – pipetting the same volume into two centrifuge tubes and assuming they’re balanced), or by sight (ie they look like they have the same volume in them). You need to put them on the scale and, as a rule of thumb, balance them to within 0.1 grams. If you’re using a tube with a screw-on top, then include the tops while weighing the tube just in case the tube/cap assemblies you are using aren’t perfectly matched.

Know Your Rotor
You might not think of centrifuge rotors as perishable, but they are. During the lifetime of a rotor, the metal can start to fatigue and form microscopic cracks. If you have a clumsy labmate that drops or bangs the rotors together, then these microscopic cracks develop even faster. Low speed rotors usually have an expiration date based on the manufacturer’s estimate of how often they are used. For precise estimates of a ultracentrifuge rotor’s lifetime, typically the rotor identification is recorded in a log book along with the speed and time spent running. (Newer ultracentrifuges keep track of this information in their on-board computer.) Based on this, the rotors are derated (the maximum rpm for that rotor is reduced) over time. So rather than knowing what the maximum speed for a rotor was when it was new, it is more important to know what it is now. Failure to do this could result in the overused rotor breaking during a run, undergoing what is called catastrophic failure. To be fair, however, almost any failure over 100,000g is catastrophic

Know Your Tubes
The maximum speed of the rotor is only part of the equation. You also have to know the maximum speed for the tubes you are using given the volume of solution you will be placing inside it.  To put this a different way, reuseable ultracentrifuge tubes have a maximum volume and a minimum volume, and the maximum speed of the tube depends on the volume in the tube. A friend of mine unintentionally tested this idea several years ago, when they had to scale back a protocol, but tried to use the same centrifuge tubes they normally used in the protocol.  The result was a pair of collapsed tubes wedged into the rotor in a truly impressive manner.

In case you were wondering how many PhDs it would take to unscrew a collapsed ultra tube from a rotor, the answer is 6.5 – 6 PostDocs and a Grad Student.  (But 4.5 of the PhDs were ‘supervising’.)

The reason for this is that the fluids that we work with are, by and large, non-compressible; however, air is quite compressible.  So when the tube contains more fluid, it is more structurally sound.  So if your lab uses reusable tubes like the one above (it used to be reusable, anyway), make sure everybody using them knows the minimum and maximum volumes that can be spun in the tubes, the maximum speeds the tubes can be spun at those volumes, and the acceptable and unacceptable solvents and pH’s the tubes can be exposed to. There are some conditions that may weaken the tube without causing a noticeable effect on the tube.

If you are using thin-walled sealed tubes, the tube must be completely full or it will collapse.  Only open-top tubes in swing-bucket rotors can have air in them, but even these are usually quite full to achieve the maximum resolution of the gradients they often contain.

Pay Attention to the Vacuum Seals
The seals on tubes aren’t just there to keep the liquids in, but also to keep the vacuum out. Ultracentrifuges maintain low temperatures while spinning at such high speeds by running under high vacuum, and thereby reducing heat generated by air-rotor friction. However, if you exposed your sample to this vacuum, then it would start de-gassing your sample, potentially causing some liquid to escape even if you are using a swing-bucket rotor. To make matters worse, if you are spinning the samples overnight or longer, then exposure to the vacuum will cause that sample to evaporate while its paired sample doesn’t, potentially causing an imbalance in the rotor. So pay attention to the seals on the tubes or on the buckets for a swing-bucket rotor, they may require a little vacuum grease on the threads to seal properly depending on the design. For fixed angle rotors, there may be an additional seal between the lid and the rotor – make sure this is clean and undamaged before your run.

No Buckets Left Behind
One of the most common sources for ultra-disasters is somebody running a swing bucket rotor without all the buckets in place. These rotors are designed to be used with all the buckets in place, even if some of them are empty. Run a six-bucket rotor with only four buckets, and the rotor will deform and turn the four that are there into projectiles.  In addition, it is usually a good idea to have a small mirror handy to check that all the buckets are properly seated into the rotor once the rotor is positioned in the ultra.

So before you start using your laboratory’s ultracentrifuge, know that it is a potentially dangerous piece of equipment, but one that can be used safely.  Read the manuals, learn about your reusable and disposable ultracentrifuge tubes, and, if nobody else in the lab has, talk to the service technician for your ultracentrifuge to determine the maximum rate speeds for your rotors.

Do you approach the ultra with fear, respect or nonchalance? Have you ever had or witnessed an ultra disaster? Or do you have any other safety tips that I missed, please let us know in the comments!

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Know Your Tubes

The maximum speed of the rotor

is only part of the equation. You also have to know the maximum speed for the tubes you are using given the volume of solution you will be placing inside it.  To put this a different way, reuseable ultracentrifuge tubes have a maximum volume and a minimum volume, and the maximum speed of the tube depends on the volume in the tube. A friend unintentionally tested this idea several years ago, when they had to scale back a protocol, but tried to use the same centrifuge tubes they normally used in the protocol.  The result was a pair of collapsed tubes wedged into the rotor in a truly impressive manner.

5 Comments

  1. Jan Terhag on July 15, 2010 at 1:32 pm

    @Jode: I think the accident happened with disposable nitrocellulose tubes… But -80 °C was just too cold.

  2. Jode Plank on July 14, 2010 at 4:27 pm

    Kate – So you never even found the tube?

    Nick – I just spent 15 minutes trying to find a picture of Adam & Jamie from ‘Mythbusters’ cowering behind a plexiglass shield (a scene that you see every other episode, I swear), but my Google skills have failed me. Their explosion shields seem the perfect solution to your conundrum.

    BioBlubb – That is a new one to me. Freezing a centrifuge tube is bad idea, even for low-speed bottles. I know a lot of people do it, but every freeze-thaw is shortening the lifetime of the bottle. Only disposable centrifuge tubes are allowed in our freezers.

  3. Jan Terhag on July 14, 2010 at 8:52 am

    Another thing to keep in mind are suitable temperature ranges for the tubes. When I was an undergrad, one of my supervisors wanted to do a sucrose gradient sedimentation. Her PI (!) told her a little trick for making the gradient – freeze 25 M sucrose at -80 °C and let it slowly thaw at 4 °C. That way, a nice gradient forms. Unfortunately, the tubes weren’t made for such low temperatures, causing the tube to collapse. Although that happened quite early during acceleration, the centrifuge was FUBAR.

  4. Nick Oswald on July 13, 2010 at 10:10 pm

    My dilemma in using these things was always — do you stay close by to make sure it is ok, or get the hell out of there in case it goes haywire! 🙂

  5. Kate Cooper on July 13, 2010 at 2:20 pm

    The old rotor thing is definitely true. As an extreme example, in undergrad we used an ultra rotor that was probably 5-10 years past the date in a lab experiment isolating protein from bovine heart or something like that. It was a swinging bucket type and after my lab partner and I started the machine, we were almost out of the room when we heard a bang. We didn’t hear any other noises after that, however, so shrugged and came back after the spin was over. One of the buckets had completely split in half from the bottom (all parts staying connected to the rotor) and the tube/sample had been sucked out and was non-existent.

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