Laboratory centrifuges are common,
everyday instruments. Yet selecting
the proper centrifuge and rotor
system for a specific application can
sometimes be a challenge. Understanding
the various types of rotors
available and the applications they
are best suited for requires a solid
understanding of centrifugation
requirements. Furthermore, since
rotors are a significant investment
for the laboratory, it is also essential
to provide the proper level of rotor
care and maintenance.
This article examines the different
types of centrifuge rotors available and
the applications they are designed to
support. Proper rotor maintenance
procedures are outlined to ensure not
only the maximum performance and
longevity of the rotors, but also the
safety of the laboratory personnel handling
these systems.
Three types of centrifuge rotor
Centrifuge rotors fall into three categories:
swinging-bucket rotors, fixed-angle
rotors, and vertical rotors. Each
category is designed to address three
key factors: 1) type of centrifugation
(differential, rate-zonal, or isopycnic),
2) speed, and 3) volume range.
Of these categories, fixed-angle and
swinging-bucket rotors are the most
common styles for benchtop, low-speed,
and high-speed floor-model centrifuge
applications. Vertical rotors are
used primarily in ultracentrifugation.
Rotors can be fabricated from a range
of materials including carbon fiber,
aluminum, and titanium. Each material
has different characteristics that
lend themselves to particular applications.
Selecting and caring for the
different types of rotor materials are
addressed later in this article.
Swinging-bucket rotors
Swinging-bucket rotors are ideal
for separating large-volume samples
(up to 12 L) at low speeds. A
swinging-bucket rotor system consists
of three parts: 1) the rotor body
attaches to the centrifuge drive and
has four or six arms to support the
buckets, 2) the buckets are placed
onto the arms of the rotor body,
and 3) trunnion pins are used to
hold the buckets in place.
Figure 1 - Sorvall® STEPSAVER™ rotor system (Thermo Fisher Scientific, Asheville, NC).
The above components form
the basic swinging-bucket rotor
framework; additional accessories
can be added as needed to tailor
the rotor for a specific application
or sample format. For example,
large-volume rotors frequently
offer a wide variety of adapters (plastic
inserts) that can be placed into
the buckets to hold the desired tube
size (see Figure 1). Certain buckets
offer sealing lids, which provide biocontainment
for potentially hazardous
samples.
Swinging-bucket rotor
applications
The most common benchtop and
superspeed centrifuge applications
requiring a swinging-bucket rotor
include: 1) high-throughput protocols
such as batch harvesting of
whole cells from growth media, 2)
high-capacity processing of blood
collection tubes, and 3) large-volume
tissue culture processing. For these
applications, the user can start with
volumes as large as 1000 mL and scale
down to smaller volumes of 1.5-mL
conical tubes.
For ultracentrifuge applications, a
swinging-bucket rotor typically supports
sample volumes ranging from
36 mL to 2.2 mL (adapters are not
normally used). An ultracentrifuge
equipped with a swinging-bucket rotor can support two types of separations:
rate-zonal (i.e., based on mass
or size) or isopycnic (i.e., based on
density). For rate-zonal separations,
a swinging-bucket rotor is advantageous
because the pathlength of the
gradient (i.e., distance from Rmin
[inside top of meniscus] to Rmax
[outside bottom of tube]) is long
enough for separation to occur. Also,
since the buckets are positioned at
90° during the run and returned to
a vertical position at the end of the
run, the sample retains its orientation,
which minimizes any pellet or
band disturbance.
Fixed-angle rotors
Fixed-angle rotors are the most ubiquitous
rotors used in centrifugation.
The majority are used for basic pelleting
applications (differential separations),
either to pellet particles
from a suspension and discard the
excess debris, or to collect the pellet.
The cavities in these rotors range
in volume from 0.2 mL to 1 L, with
speeds ranging from single digits to
1,000,000 × g (relative centrifugal
force, RCF).
Two factors determine the type of
fixed-angle rotor required: desired
g-force (RCF) and desired volume.
Generally speaking, the size of the
rotor is inversely proportional to
its maximum speed capability (i.e.,
the larger the rotor, the lower
the maximum speed). An important
specification when selecting
a fixed-angle rotor is the K factor,
which indicates the pelleting efficiency
of the rotor at top speed,
taking into account the maximum
and minimum radius (pathlength)
of the rotor cavity. A low K factor
indicates a higher pelleting efficiency;
therefore, the K factor can
be a useful metric for comparing the
speed at which particles will pellet
across a range of rotors.
Vertical rotors
Vertical rotors are fairly
specialized—their most common
use is during ultracentrifugation
for isopycnic
separations, specifically
for the banding of DNA in
cesium chloride. In this type of
separation, the density range of
the solution contains the same
density as the particle of interest;
thus the particles will orient
within this portion of the
gradient. Isopycnic separations
are not dependent on the pathlength
of the gradient but rather
on run time, which must be sufficient
for the particles to orient
at the proper position within
the gradient. Vertical rotors have
very low K factors (typically in the
range of 5–25), indicating that the
particle must only travel a short distance
to pellet (or in this case form
a band); therefore run time is minimized.
Once it is determined that a
vertical rotor is appropriate for the
end-user application, volume and
speed become the deciding factors
for which rotor to use.
Rotor care
Proper rotor care is essential for ensuring
safety and longevity. In addition
to reducing the risk of accidents, regular
rotor maintenance can save time
and money and greatly extend rotor life
span. A simple and effective set of rotor
maintenance steps is outlined below.
Rinse
Whether using a carbon fiber, aluminum,
or titanium rotor, rinsing with
water after each use to remove any
residual sample or dirt, followed by a
thorough drying, yields dramatic results
in rotor longevity. If debris is firmly
lodged to the rotor, a mild detergent
and soft cloth or brush can be used to
remove it. Metal tools should never
be used to remove lodged debris. It is
important that rotors are completely
dried following any rinse or wash.
Disinfect
When working with potentially infectious
agents, a disinfection step is
recommended after each use. There
are many disinfection products available through major laboratory suppliers.
Because different rotor materials
require different disinfection products,
it is important to select the appropriate
cleaning agent for the rotor
material. For example, bleach can be
used on carbon fiber rotors but not
on aluminum. All rotor manufacturers
provide guidelines regarding the
acceptable active ingredients in a disinfecting
agent; therefore it is valuable
to review the literature provided.
Sterilize
In some cases, rotors must be sterilized
using autoclaving or UV exposure
to kill highly infectious agents.
All rotors, whether made from aluminum,
titanium, or carbon fiber, are
autoclavable, making this the most common and easiest method of sterilization.
In cases in which autoclaving
is not an option, users should consult
the manufacturer’s guidelines to identify
the active disinfecting ingredients
that are safe to use on the rotor.
Additional maintenance—rotor parts
In addition to the body of the rotor, most
rotors contain other parts that also require
regular attention, including O-rings, lid
threads, and locking mechanisms.
O-rings
O-rings provide the main source of
protection against sample leakage,
but they are often completely
neglected. O-rings must be lubricated
prior to installation on a
new rotor. More importantly,
since most detergents and many
disinfecting agents remove the
lubricant, the O-rings need to
be dried and relubricated after
washing or disinfecting. O-rings
degrade after repeated cleaning
and autoclaving; therefore
it is important to replace them
when they begin to show signs
of cracking or stretching.
Lid threads
The threads on bucket or rotor
lids must be cleaned regularly with
a soft, lint-free cloth to remove
built-up debris. Applying a light coat
of manufacturer-approved grease on
the rotor lid threads ensures easy
opening and closing; it also helps to
prevent inadvertent cross-threading
and general corrosion.
Locking mechanisms
Locking mechanisms, which hold the
lid to the rotor or bucket, wear with
time and can also become damaged
when attaching and removing the lids.
If the locking mechanism is threaded,
it should be checked regularly for
damage to the threads (either nicks
or wear on the edges of the threads).
The locking mechanisms can usually
be replaced by a field service technician.
By regularly checking and cleaning
the threads with a soft cloth to remove debris, the life of the mechanism
can be extended substantially.
Lubrication for these parts is not typically
required.
Manufacturer support
Whenever a factory-certified technician
is on site, it is worth asking
for a quick inspection of all rotors.
Most manufacturers will also conduct
rotor-inspection clinics upon
request. Over time, rotors will
begin to lose their finish or show
telltale signs of damage to the
surface, such as small pits in the
surface of the rotor, rust, or metal
corrosion. These are all strong
indicators that a rotor needs careful
inspection. If left unchecked,
microcracks can form at the bottom
of the pits, and corrosion can
spread until the rotor fails. Carbon
fiber rotors do not corrode; however,
the finish can wear off over
time and after repeated exposure to
chemicals. This type of rotor can
be refinished by the manufacturer
before the exterior surface becomes
a handling hazard.
Low-speed and superspeed centrifuge
rotors that have been in use
for 10 years or longer should be
closely inspected and considered
for replacement, especially if they
show any of the symptoms described
above. Ultracentrifuge rotors all
have recommended life spans based
on the number of hours in use or
cycles completed.
Summary
As in most endeavors, gathering the
right information is key. When selecting
a rotor, it is important to know the
type of separation required—differential,
isopycnic, or rate-zonal—as well
as the volume and RCF requirements.
This will help identify which of the
three rotor types to choose.
Once the rotors are in the laboratory,
it is important to establish a
regular rotor maintenance program.
This will increase laboratory safety,
extend rotor life span, and reduce
centrifugation and contamination
problems. Users should follow the
manufacturer’s recommended schedule
to have rotors inspected, and
retire them when they show wear.
Being preemptive will ensure that
centrifuges and rotors perform the
vital sample preparation function
they are designed to provide.
Ms. Goodman is Sample Preparation and Separations
Applications Product Manager, Thermo
Fisher Scientific Inc., 275 Aiken Rd., Asheville,
NC 28804, U.S.A.; tel.: 828-365-1348;
e-mail: [email protected].