Laboratory Cell Disrupters
A review covering apparatus and techniques of cell disruption
Practical aspects of mechanical cell disruption are discussed. Sources
and approximate prices of equipment are given. A more extensive review
of the subject by the author, Tim Hopkins, is available in Purification
and Analysis of Recombinant Proteins, Seetharam and Sharma, editors, published
by Marcel Dekker, Inc (New York)1991.
BEAD MILL HOMOGENIZERS
In bead milling, a large number of minute glass beads are vigorously
agitated by shaking or stirring. Disruption occurs by the crushing action
of the glass beads as they collide with the cells. The method has been
used for years to disrupt microorganisms and works successfully with tough-to-disrupt
cells like cyanobacteria, yeast, spores and microalgae where other techniques
have failed. It is considered the method of choice for disruption for
yeast and fungi. More recently, bead mill homogenization has been applied
to plant and animal tissue. The size of the glass beads is important.
Optimal size for bacteria and spores is 0.1 mm , 0.5 mm for yeast, mycelia,
microalgae, and unicellular animal cells such as leucocytes or trypsinized
tissue culture cells and 1.0 or 2.5 mm for tissues such as brain, muscle,
leaves and skin. The speed of disruption is increased about fifty percent
by using like-sized ceramic beads made of zirconia-silica rather than
glass (Hopkins, unpublished observations), presumably because of their
greater density. The loading of the beads should be at least 50% of the
total liquid-biomass volume but can be up to 90%, provided adequate agitation
of the bead slurry is still possible. Generally, the higher the volume
ratio of beads to cell suspension, the faster the rate of cell disruption.
After treatment, the beads settle in seconds by gravity and the cell extract
is easily removed.
Microorganisms can be disrupted manually by this technique. A cell
suspension is added to the beads and the mixture is agitated at top speed
on a vortex mixer for several minutes. The hand-held method is slow and
tedious. Therefore, electromechanical bead mill agitators have been developed.
Bead agitation by these devices is either by shaking or stirring.
Shaking-type Bead Mills. Most shaking-type bead mills are restricted
to sample sizes of 3.0 mL or less. Heat generation is a concern in larger
shaking bead mills and effective cooling is difficult to achieve. For
example, bead milling of a 250 mL sample without external cooling increases
the temperature l0 degrees per minute of homogenization. The German made
Braun MSK (B. Braun Biotech, Bethlehem, PA) disrupts samples sizes up
to 40 mL but relies on somewhat unreliable cooling with liquid CO2 to
keep the sample at acceptable temperatures. Smaller volume shaking bead
mills have smaller breakage chambers with surface area to volume ratios
high enough to permit adequate heat dissipation without external cooling.
Four such commercial devices are the Mini-BeadBeater (BioSpec Products,
Bartlesville, OK), the Micro-Dismembrator II (B. Braun Biotech, Bethlehem,
PA), Retsch Mixer (Brinkmann, Westbury, NY) and the FastPrep (Bio 101,
Vista, CA). Disruption of microorganisms takes about 1-5 minutes. BioSpec
Products manufactures two machines, one which holds a single 2 mL screw-cap
microvial and the other capable of disrupting eight sample vials at a
time. BioSpec Products has a machine capable of holding 96-well assay
blocks under development. The price of shaking bead mills range from $600
Rotor-type Bead Mills. Larger capacity laboratory bead mill cell disrupters
agitate the beads with a rotor rather than by shaking. Equipped with efficient
cooling jackets, larger sample volumes can be processed without overheating.
The most widely used is the Bead-Beater (BioSpec Products, Bartlesville,
OK). This unit will disrupt about 250 mL or, with smaller chamber attachments,
50 or 15 mL batches of cell suspension in 3-5 minutes. Cell concentrations
as high as forty percent (wet wt) can be used. VirTis Company (Gardiner,
NY) offers an attachment for its line of high speed rotary homogenizers
which efficiently agitate glass beads in a special test-tube or fluted
flask. Complete homogenizer units cost about $500 and $800, respectively.
While the above small volume cell disrupters are used mostly for the disruption
of microorganisms, they can also be used to homogenize and extract plant
and animal tissue. This newer application is suitable for both soft tissue
and tough or fibrous samples such as skin, tendon or leaves. Extraction
yields of biomolecules such as nucleic acids, viruses, and receptor complexes
are often superior to that of other methods. For nucleic acid isolation,
consider disrupting the cells directly in the nucleic extraction solution
(phenol, guanidinium SCN, etc). Nuclease concerns will be eliminated and
yields enhanced. And, if PCR techniques are being used, shaking-type bead
mills which use disposable micro-vials totally eliminate cross contamination
between samples. Selective homogenization is sometimes possible using
different bead sizes or speeds of bead agitation. For example, it is possible
to selectively disrupt only the epidermal layer of whole leaves or to
obtain intact subcellular organelles by using smaller charges of beads
and/or shorter disruption times.
ROTOR-STATOR HOMOGENIZERS (also called colloid mills or Willems homogenizers)
are well suited for plant and animal tissue and generally outperform cutting-blade
type Benders. Compared to a blender, foaming, swirling and aeration are
minimized and smaller sample volumes are accommodated. Most tissues are
quickly and thoroughly homogenized with the apparatus. The cellular material
is drawn into the apparatus by a rotor sited within a static tube or stator.
The material is then centrifugally thrown outward to exit through slots
or holes on the tip of the stator. Because the rotor is turning at very
high speed, the tissue is rapidly reduced in size by a combination of
turbulence and scissor-like mechanical shearing occurring within the gap
of the rotor and stator. Since most rotor-stator homogenizers have an
open configuration, the product is repeatedly recycled. The process is
quite fast and, depending upon the toughness of the tissue sample, desired
results are usually be obtained in 10-60 seconds. For the recovery of
intracellular organelles or receptor site complexes, shorter times and/or
reduced rotor speeds are used. The sample size prior to processing with
the homogenizer must be small enough to be drawn inside the hole at the
tip of the stator. Therefore, samples often must be pre-chopped or - fragmented
with a scissors, single-edge razor blade or cryopulverizer (a device that
quickly powders tissue at liquid nitrogen temperatures - see below). Unlike
many other types of cell disrupters, rotor-stators homogenizers generate
negligible heat during operation.
Most laboratory rotor-stator homogenizers are top driven with a compact,
high speed electric motor which turns at 8,000 to 60,000 rpm. The size
of the rotor-stator probe (also called the generator) can vary from the
diameter of a drinking straw for 0.5-50 mL sample volumes to much larger
units handling 10 liters or more. There is an important relationship between
rotor speed and stator diameter. In principle, the top rotor speed of
the homogenizer should double for each halving of the rotor diameter.
It is not rpm per se but the tip velocity of the rotor that is the important
operating parameter. Ten to twenty meters per second (2000 to 4000 fpm)
are acceptable tip speeds for tissue disruption. Unfortunately, some of
the smaller-sized commercial rotor-stator homogenizers do not meet this
standard. Other factors such as rotor-stator design (there are many),
materials used in its construction and ease of cleaning are also important
factors to consider in selecting a rotor-stator homogenizer. Some manufactures
are BioSpec Products (Bartlesville, OK), Brinkmann Instruments (Westbury,
NY), Charles Ross & Son Company (Hauppauge, NY), Craven Laboratories
(Austin, TX), IKA Works (Cincinnati, OH), Omni International (Gainsville,
VA), Pro Scientific (Monroe, CT), Silverson Machines (Bay Village, OH),
and VirTis Company (Gardiner, NY). The cost of complete units (motor plus
rotor-stator head or generator) range from $600 to $5000.
Most laboratory sized homogenizers function properly only with liquid
samples in the low to medium viscosity range (<10,000 cps). The speed
and efficiency of homogenization is greatly degraded by using too small
a unit, and the volume range over which a given homogenizer rotor-stator
size will function efficiently is only about ten fold. Foaming and aerosols
can be a problem with rotor-stator homogenizers. Keeping the tip of the
homogenizer well submerged in the media and the use of properly sized
vessels helps with the first problem. Square shaped homogenization vessels
give better results than round vessels and it is also beneficial to hold
the immersed tip off center. Aerosols can be minimized by using properly
covered vessels (VirTis, Brinkmann and Omni). Even though a number of
the laboratory rotor-stator homogenizers use fully enclosed motors, none
of them are truly explosion-proof. Therefor, due caution should be followed
when using flammable organic solvents by conducting the homogenization
in a well ventilated hood.
Bottom-driven laboratory rotor-stator homogenizers are a new entry to
the laboratory. The rotor-stator assembly is usually placed within a sealed
chamber or container, fits blender motor bases and have working volumes
of 100-1000 mL. They costs about $250 - $400 and are available from BioSpec
Products (Bartlesville, OK) and Eberbach Corporation (Ann Arbor, MI).
Closely related homogenizers, called dispersers, are used for preparing
large volumes of crude plant and animal aqueous extract. Operating like
a household garbage disposal unit, the rotor-stator mechanism quickly
homogenizes and liquefies kilogram quantities of biomass. The sample is
suspended in one or more liters of media, loaded into a top reservoir
and homogenized either in a continuous or batch mode. Costing $600 to
$7000, two manufacturers are BioSpec Products and IKA Works.
BLADE HOMOGENIZERS. Although less efficient than rotor-stator homogenizers
and aeration and foaming can be a problem, blade homogenizers (commonly
called blenders) have been used for many years to produce fine brie and
extracts from plant and animal tissue. Blenders are not suitable for disruption
of microorganisms. In this class of homogenizer a set of stainless steel
cutting blades rotate at speeds of 6,000-50,000 rpm inside a glass, plastic
or stainless steel container. The blades are either bottom- or top-driven.
Some of the higher speed homogenizers can reduce tissue samples to a consistent
particulate size with distributions as small as 4 microns, as determined
by flow cytometric analysis. After blending, some plant tissue homogenates
undergo enzymatic browning - a oxidation and cross-linking process which
can complicate subsequent separation procedures. Enzymatic browning is
minimized by carrying out the extraction in the absence of oxygen or in
the presence of oxygen scavenging thiol compounds such as mercaptoethanol.
Sometimes, addition of polyethylene imine, metal chelators, or certain
detergents such as Triton X-100 or Tween 80 also help.
When using a blender, use caution when blending with flammable solvents
such as alcohol or acetone or when homogenizing diseased tissues. Blenders
use brush motors to achieve their high speeds and, therefor, spark during
operation. Also aerosols are readily formed while blending. Use a sealed
blender container and operate it in a well ventilated hood. Blade homogenizers
can process liquid sample sizes from 2 mL to one gallon. Accessories for
blenders include cooling jackets for temperature control, closed containers
to minimize aerosol formation and entrapment of air, special vessels made
of stainless steel, semi-micro containers and even insulated vessels for
use with cryogenic solvents (see Freeze fracturing). Manufactures of a
scientific line of blenders include British Medical Enterprises (London,
England), ESGE (Basel, Switzerland), Hamilton Beach Commercial (Washington,
NC), Omni International (Waterbury, CT), Professional Diagnostic (Edmonton,
Alberta Canada), The VirTis Company (Gardiner, NY) and Waring Products
Division (New Hartford, CT). Accessory vessels for Hamilton-Beach brand
blenders are manufactured by BioSpec Products (Bartlesville, OK) and for
Waring brand blenders by Eberbach Corporation (Ann Arbor, MI). Prices
for blade homogenizers range from about $100 to $2000.
FREEZE-FRACTURING. Both microbial pastes and plant and animal tissue can
be frozen in liquid nitrogen and then ground with a common mortar and
pestle at the same low temperature. Presumably the hard frozen cells are
fractured under the mortar because of their brittle nature. Also, ice
crystals at these low temperatures may act as an abrasive.
A freeze-fracturing device called the Bessman tissue pulverizer is useful
for fragmenting 10 mg to 10 g quantities of fibrous tissue such as skin
or cartilage to the size of grains of salt. This material is then easily
homogenized by other methods. Looking somewhat like a tablet press, the
pulverizer consists of a hole machined into a stainless steel base into
which fits a piston. The base and piston are pre-cooled to liquid nitrogen
temperatures. Ten mg to ten grams of hard frozen animal or plant tissue
is placed in the hole. The piston is placed in the hole and given a sharp
blow with a hammer. The resulting frozen, powder-like material can be
further processed by Pestle and Tube, Bead Mill or Rotor-stator homogenizers.
Manufactured by BioSpec Products (Bartlesville, OK) and by Spectrum Medical
Industries (Carson, CA), the pulverizers come in several sizes and cost
$200 to $400. In larger capacity models, BioSpec Products (Bartlesville,
OK) has incorporated a built-in, spring loaded hammer looking much like
a staple gun. This same company also makes a small screw press designed
to pulverize 50-500 mg of hard frozen tissue. This pulverization tool
works slower than the Bessman device (perhaps a minute rather than a few
seconds per sample), but it is well suited for fresh bone and other hard
GRINDERS. Grinding biological material in a mortar or tube with fine sand,
alumina or glass powder is roughly the equivalent of bead-milling (see
Part 1). The method works reasonably well with all types of biomass but
is strictly small scale and is labor intensive. Cell pastes or solid mass
with a minimum volume of buffer are mixed with 0.5-1 volume of grinding
media and ground with a mortar and pestle. Disruption efficiency is poor
if lower cell densities or smaller charges of grinding media are used.
Glass powder, having a high surface area, may adsorb significant amounts
of charged biomolecules such as nucleic acids and proteins.
PESTLE AND TUBE HOMOGENIZERS (also called tissue grinders) are used to
disrupt animal tissue. While variations of the pestle and tube homogenizer
have names like Potter, Potter-Elvehjem, Dounce, and Ten Broeck, as a
group they consist of test-tubes made of glass, inert plastic or stainless
steel into which is inserted a tight-fitting pestle (clearance about 0.1-0.2
mm) made of like material. The walls of the test-tube and pestle can be
smooth or have a ground finish. Most tissues must be cut or chopped into
small pieces (1-5 mm) with scissors or a single-edge razor blade before
being suspended in a 4-10 fold volume excess of medium in the test-tube.
The pestle is manually worked to the bottom of the tube, thus tearing
and fragmenting tissue as it is forced to pass between the sides of the
pestle and the wall of the tube. The grinding action occurs again as the
pestle is withdrawn. Five to thirty repetitions of this low shear method
homogenizes the tissue. Rotation of the pestle at about 500-1000 rpm with
an electric motor while the test-tube is manually raised and lowered speeds
up the process. While pestle and tube homogenization is simple and the
equipment used is usually inexpensive, it is both labor intensive and,
in the case of fragile glass homogenizers, potentially dangerous. Even
so, this homogenizer will continue to be popular because of its extremely
gentle action. Often it is the method of choice for the preparation of
small quantities of subcellular organelles from soft animal tissues such
as brain or liver. Microorganisms cannot be disrupted with pestle homogenizers.
Commercially available glass or plastic pestle homogenizers with batch
capacities of 0.1-50 mL generally cost $15-$100 and are available from
many manufacturers including Ace Glass (Vineland, NJ), Bellco Glass (Vineland,
NJ), BioSpec Products (Bartlesville, OK), Kontes (Vineland, NJ), Thomas
Scientific (Swedesboro, NJ), Tri-R Instruments (Rockville Center, NY),
Sage Products (Crystal Lake, IL) and Wheaton Industries (Milville, NJ).
Disposable, plastic pestles which fit into microcentrifuge tubes are available
from Kontes. They also offer a small, hand-held motor unit to drive the
pestle. While precision stainless steel tissue grinders are more expensive
($200 - $250, BioSpec Products and Wheaton), they are efficiently cooled
and tolerate vigorous homogenization without risk of breakage. A 'Rolls-Royce'
homogenizer costing about $3000 has a variable speed motor, cooling jacket,
and hand- operated lever to rise and lower the pestle (B. Braun Biotech
Bethlehem, PA). A continuous pestle homogenizer is available from Yamato
USA (Northbrook, IL). Grooves machined on the upper one-third of the pestle
catch and guide tissue through the close tolerance region of the lower
two-thirds of the cylinder pestle. The resultant homogenate exits from
the bottom of the cylinder. Recycling is usually necessary. The machine
comes in two sizes and costs $2000-$3000.
MEAT MINCER AND SOLIDS PRESS. The household meat grinder or mincer has
been used for many years for the preparation of animal tissue extracts.
Tissue is mechanically pressed through holes in a metal sieve plate while
rotating blades slowly sweep across the face of the plate cutting the
meat in 0.3-0.5 mm fragments. While it is not an effective way to disrupt
cells per se , it is useful as a preliminary step for complete homogenization
using other physical or chemical methods. Meat grinders cut flexible tissue
like muscle better if the tissue is processed slightly frozen.
For small tissue samples, BioSpec Products (Bartlesville, OK) manufacture
hand operated screw presses for the preparation of tissue extracts as
does EDCO Scientific (Chapel Hill, NC). Capable of considerable force,
sample sizes from 0.1 grams up to 50 grams of soft tissue are pushed through
sieve plates having 0.5 to 3 mm holes, much like the action of a kitchen
garlic press. Hard or fibrous tissues like tendon, skin, leaves and seeds
will not pass through the press. The units cost from $25 to $400. Fred
S. Carver (Wabash, IN) has a compact hydraulic laboratory tissue press
for the extraction of intracellular liquids and oils for about $1600.
Designed for plant leaves, Bioreba (Chapel Hill, NC) makes a hand-held
grinder consisting of an circular array of steel balls which crush a few
leaves inside a mylar plastic bag. The grinder and bags cost about $200.
ULTRASONIC DISINTEGRATORS are widely used to disrupt cells. These devises
generate intense sonic pressure waves in liquid media. Under the right
conditions, the pressure waves cause formation of microbubbles which grown
and collapse violently. Called cavitation, the implosion generates a shock
wave with enough energy to break cell membranes and even break covalent
Modern ultrasonic processors use piezoelectric generators made of lead
zirconate titanate crystals. The vibrations are transmitted down a titanium
metal horn or probe tuned to make the processor unit resonate at 15-25
kHz. The rated power output of ultrasonic processors vary from 10 to 375
Watts. What really counts is the power density at the probe tip. Higher
output power is required to sustain good performance in large sized probes.
For cell disruption, probe densities should be at least 100 W/cm2 and
the larger the better for tip amplitude (typical range: 30-250 microns).
Some manufacturers of ultrasonic disintegrators are Artek Systems (Farmington,
NY), BioSpec Products (Bartlesville, OK), Branson Sonic Power Company
(Danbury, CT), B. Braun Biotech (Bethlehem, PA), RIA Research Corp. (Hauppauge,
NY), Sonic Systems (Newton, PA) and VirTis Company (Gardiner, NY). A new
development is a cordless ultrasonic processor supplied by BioSpec Products
(Bartlesville, OK). With a tip diameter of 1/8 inch, it easily fits into
microtubes and 96 well titer plates.
Ultrasonic disintegrators generate considerable heat during processing.
For this reason the sample should be kept ice cold if possible. For microorganisms
the addition of 0.1 -0.5 mm diameter glass beads in a ratio of one volume
beads to two volumes liquid is recommended. Tough tissues like skin or
tendon should be macerated first in a tissue press or grinder or pulverized
in liquid nitrogen (see above). Use small vessels during ultrasonic treatment
and place the probe tip deep enough in the sample to avoid foaming. Finally,
one should be aware that free radicals can be generated during sonication
and that these radicals can react with most biomolecules. Damage by oxidative
free radicals can be minimized by including scavengers like cysteine,
dithiothreitol or other -SH compounds in the media.
Other cell disruption apparatus or techniques not covered in this review
but discussed in detail by the author in the original book are High-Pressure
Homogenizers, Autolysis, Enzymatic lysis, Dehydration, Chemical lysis,
Solvent lysis and Programmed self-distruction.