USER’S GUIDE

HIGH INTENSITY ULTRASONIC PROCESSOR

Microprocessor Controlled
250-Watt Model
500-Watt Model
TABLE OF CONTENTS

Important Safeguards
Low Surface Tension Liquids - Organic Solvents

SECTION 1 – INSTALLATION
Inspection
Electrical Requirements
Installing the Ultrasonic Processor

SECTION II – OPERATION
Principles of Ultrasonic Disruption
Functions of Controls, Indications, and Connectors
Preparations for Use
Tuning
Using the Ultrasonic Processor

SECTION III – SERVICE INFORMATION

Return of Equipment

SECTION IV – OPERATING SUGGESTIONS AND TECHNIQUES

The Ultrasonic Processor supplied with this instruction manual is constructed of the finest
material and the workmanship meets the highest standards. It has been thoroughly tested
and inspected before leaving the factory and when used in accordance with the
procedures outlined in this manual, will provide you with many years of safe and
dependable service.
Rev. 2003

1
 IMPORTANT SAFEGUARDS
READ BEFORE INSTALLING OR
USING THE EQUIPMENT
Your Ultrasonic Processor has been designed with safety in mind. However, no design
can completely protect against improper usage, which may result in bodily injury and/or
property damage. For your protection and equipment safeguard, observe the following
warnings at all times, read the operating instructions carefully before operating the
equipment, and retain this instruction manual for future reference. If the ultrasonic
Processor is used in a manner contrary to that specified in this instruction manual, the
protection features designed into the unit may be impaired.
When mounting the probe, always clamp the converter housing. Never clamp the
probe.
Make sure the Ultrasonic Processor is properly grounded via a 3-prong outlet.
High voltage is present in the power supply. Do not remove the cover. Refer all
servicing to qualified service personnel.
To avoid electric shock, disconnect the electrical power cord before removing the
cover prior to servicing.
Never operate the power supply unless it is connected to the converter.
Never secure anything to the probe, except at the nodal point (point of no activity).
Never touch a vibrating probe.
Never allow a microtip or extender to vibrate in air for more than 10 seconds.
When using a microtip, always keep the amplitude below 40%.
Never operate a probe with threaded end without a tip, extender or microtip.
It is recommended that ear protection be used when operating the Ultrasonic
Processor.
WARNING or CAUTION
Where you see this alert symbol and WARNING or
CAUTION heading, strictly follow the warning instructions
to avoid personal injury or equipment failure.

2
 CAUTION
LOW SURFACE TENSION LIQUIDS – ORGANIC SOLVENTS

The probes (solid or with a replaceable tip) are tuned elements that resonate at a specific
frequency. If the replaceable tip is removed or isolated from the rest of the probe, the
element will no longer resonate at that frequency, and the power supply will fail.
Unlike aqueous (water based) solutions, which rarely cause problems, solvents and low
surface tension liquids are problematic. These liquids penetrate the probe/replaceable tip
interface, and force the particulates into the threaded section isolating the tip from the
probe.
When processing low surface tension liquids ALWAYS use a solid probe

SECTION 1 – INSTALLATION
INSPECTION

Prior to installing the Ultrasonic Processor, perform a visual inspection to detect any
evidence of damage, which might have occurred during shipment. Before disposing of
any packaging material, check it carefully for small items.

The Ultrasonic Processor was carefully packed and thoroughly inspected before leaving
our factory. The carrier, upon acceptance of the shipment, assumed responsibility for its
safe delivery. Claims for loss or damage sustained in transit must be submitted to the
carrier.

If damage has occurred, contact your carrier within 48 hours of the delivery date. DO
NOT OPERATE DAMAGED EQUIPMENT. Retain all packing materials for future
shipment.

3
ELECTRICAL REQUIREMENTS

The Ultrasonic Processor requires a fused, single phrase 3-terminal grounding type
electrical outlet capable of supplying 50/60 Hz at 100 volts, 115 volts, 220 volts, or 240
volts, depending on the voltage option selected. For power requirements, check the label
on the back of the unit.

WARNING
For your personal safety, do not, under any
circumstances, defeat the grounding feature of the
power cord by removing the grounding prong.

INSTALLING THE ULTRASONIC PROCESSOR

The Ultrasonic Processor should be installed in an area that is free from excessive dust,
dirt, explosive and corrosive fumes, and extremes of temperature and humidity.

4
 SECTION II – OPERATION
PRINCIPLES OF ULTRASONIC DISRUPTION

The ultrasonic power supply converts 50/60 Hz line voltage to high frequency electrical
energy. This high frequency electrical energy is transmitted to the piezoelectric
transducer within the converter, where it is changed to mechanical vibrations. The
vibrations from the converter are intensified by the probe, creating pressure waves in the
liquid. This action forms millions of microscopic bubbles (cavities) which expand during
the negative pressure excursion, and implode violently during the positive excursion.
This phenomenon, referred to as cavitation, creates millions of shock waves in the liquid,
as well as elevated pressures and temperatures at the implosion sites. Although the
cavitational collapse last but a few microseconds, and the amount of energy released by
each individual bubble is minute, the cumulative effect causes extremely high levels of
energy to be released into the liquid. The larger the probe tip, the larger the volume that
can be processed but at a lesser intensity. For information regarding the processing
capability of each probe, consult the tables below.

TAPERED MICROTIPS STEPPED MICROTIP

TIP DIAMETER 1/8" (3 mm) 3/16" (5 mm) 1/4" (6.5 mm) 1/8" (3 mm)
INTENSITY ultra high very high high very high
VOLUME (batch) 1-10 ml 3-15 ml 5-25 ml 250ul-10 ml

STANDARD PROBES
TIP DIAMETER 1/2" (13 mm) 3/4" (19 mm) 1" (25 mm)
INTENSITY high medium low
VOLUME (batch) 10-250 ml 25-500 ml 50-1000 ml

5
 FUNCTIONS OF KEYS, CONTROLS, INDICATIONS, AND CONNECTORS
1 On / Off / Tune Switch ON position - energizes the power supply.
OFF position - de-energizes the power supply.
TUNE position - energizes the power supply
momentarily for tuning purposes.

2 Pilot Light Illuminates when the power supply is energized.

3 Continuous / Pulsed * Sets the duty cycle to continuous or pulsed mode.

4 % Duty Cycle Selector * Sets the pulse rate when CONTINUOUS / PULSED
Switch is set to pulsed. Intermittent operation inhibits
heat build-up in the solution and provides more efficient
processing by allowing the material to settle back under
the probe tip after each burst.
5 Timer Sets the duration of ultrasonic application (up to 15
minutes). In the HOLD position, the timing mode is
uninhibited and the ultrasonic energy is applied
indefinitely.
6 Output Control Controls the amplitude of vibration at the probe tip.
CAUTION
When using a microtip, never allow the Output control
to exceed MICROTIP LIMIT “5”.
7 Power Monitor Indicates the percentage of ultrasonic power delivered to
the probe.

8 3 Pin Connector Connects to the converter cable.

9 3 Pin Connector ** Connects to the converter cable (available only with 500
watt dual output units).
10 Tuning Control Optimizes performance by matching the frequency of
the power supply to that of the converter / probe
assembly
11 Fuse Protects against electrical overload.
12 Electrical Line Cord Connects the power supply to the electrical outlet.
* Available only with Ultrasonic Processors equipped with pulsers.
** Available only with dual output 500 watt Ultrasonic Processors.

6
PREPARATION FOR USE

CAUTION
Do not operate an Ultrasonic Processor that has been in a very cold or hot
environment for a prolonged period of time. Wait until it has reached room
temperature

1. Ensure that the AMPLITUDE dial is set fully counter-clockwise.

2. Plug the electrical line cord into the electrical outlet.
3. If the converter / probe assembly is not already assembled, check for cleanliness the
mating surface of the converter and probe or stepped microtip (consisting of coupler
and stepped tip), and using the wrenc hes provided, screw them securely to the
converter.
4. To attach a tapered microtip or extender to a probe, remove the replaceable tip from
the ½” (13mm) probe, and using the wrenches provided, screw them securely to the
probe.

CAUTION
Never place a washer between the probe and the converter.
Never apply grease to the mating surfaces or threads of the converter, probe,
replaceable tip or microtip.

5. Mount the converter / probe assembly in a laboratory stand. Secure the clamp to the
2½” (63mm) diameter converter housing only. Do not secure the clamp to the probe.
6. Connect the converter cable to the power supply. With a dual output 500 watt
ultrasonic processor, if two converters are going to be used simultaneously, do not
connect the second converter at this time.

NOTE
Should it become necessary to remove a probe, use the wrenches supplied. If the
probe has been attached to the converter for a long period of time it might be
necessary to use a vise. Be sure the vise has soft jaws or other means to prevent
scratching. Secure the wide diameter portion of the probe in the jaws of the vise.
Never grip the converter in the vise. Using a wrench, twist the converter off the
probe. A tap of a hammer may be applied to the end of the wrench. Never attempt
to remove the probe by twisting the converter housing, as this may damage the
electrical connections within the housing.

7
8
 Order
No DESCRIPTION
Number
1 Converter Model CV17 CV00017
2 Coupler 630-0421
3 Stepped tip 1/8” (3mm 630-0422
4 Probe ½” (13mm) solid 630-0219
Probe ½” (13mm) with threaded end and replaceable tip 630-0220
Probe ¾” (19mm) solid 630-0208
Probe ¾” (19mm) with threaded end and replaceable tip 630-0207
Probe 1” (25mm) solid 630-0209
Probe 1” (25mm) with threaded end and replaceable tip 630-0210
5 Tapered microtip 1 /8 ” (3mm) 630-0418
Tapered microtip 3 /16 ” (5mm) 630-0419
Tapered microtip ¼” (6mm) 630-0420
6 Same as 4
7 Replaceable tip ½” (13mm) 630-0406
Replaceable tip ¾” (19mm) 630-0407
Replaceable tip 1” (25mm) 630-0408
8 Extender ½” (13mm) 630-0410
Extender ¾” (19mm) 630-0409
Extender 1” (25mm) 630-0444
9 Same as 7
10 High gain probe ¾” (19mm) – solid 630-0306
High gain probe 1” (25mm) – solid 630-0310

CAUTION: Do not use tapered microtip with coupler. Do not use stepped tip without a coupler. Do not use
probes with threaded end and replaceable tip, when working with low surface tension liquids.

9
 TUNING

Tuning optimizes performance and insures maximum transfer of energy by matching the
frequency of the power supply to that of converter/probe assembly. The power supply
should be tuned 1) every time a new probe or accessory is used, 2) on occasions to
compensate for the frequency variation caused by cavitation erosion 3) following 10
minutes of continuous operation and 4) when the sample temperature is significantly
higher or lower than room temperature.
The piezoelectric crystal within the converter is part of the circuitry, which controls the
frequency at 20kHz. Any changes in t the crystal’s capacitance resulting from a variation
in temperature, will cause the equipment to operate in an out-of-tune condition. For
reliable performance, and equipment protection, it is important that the unit be tuned after
the probe temperature has had a chance to stabilize. When relocating the ultrasonic
processor from a very cold or very hot environment, allow 30 minutes for the unit
temperature to stabilize before operating. Continuous operation causes temperature
elevation in the sample. This increase in temperature is transmitted through the probe to
the crystal assembly. Always tune the power supply after the probe has reached operating
temperature. When working with low or high temperature samples, immerse the probe in
the sample for a few minutes, withdraw the probe from the sample, then tune the power
supply.
IMPORTANT
Tuning must be performed in air with the probe out of the sample. While tuning,
do not allow the probe to contact anything.

1. Ensure that the probe or microtip is not immersed in the sample and that it does
not come in contact with anything. If a cup horn is used, make sure that the water
has been drained out of it. If a flow through cell is used, make sure that the
sample has been drained out of it.
2. Set TIMER to HOLD.
3. SET output control to “10” (to “4” when using a microtip of extender).

CAUTION
When using a microtip, never allow the tip to vibrate in air for more than 10 seconds and do not set the
OUTPUT CONTROL above “5”. Ignoring these instructions will cause the microtip to fracture.

4. Momentarily hold down On/OFF/TUNE switch to TUNE and rotate the tuning
control clockwise or counterclockwise until a minimum (not maximum) reading
(usually less than 20) is obtained on the power monitor. If minimum reading
(sometimes referred to as null) cannot be obtained, the probe, cup horn, tip,
microtip, or accessory is loose or out of resonance, or the power supply or
converter require servicing. A loose probe will usually generate a loud piercing
sound.

10
 NOTE
If minimum reading cannot be obtained, check unit without the probe to ascertain whether the
power supply or probe is at fault.

5. Set OUTPUT CONTROL to “4”.

6. Release ON/OFF/TUNE Switch.

7. With a dual output 500 watt Ultrasonic Processor, if two converters are going to
be used simultaneously, connect the second converter cable to connector.

CAUTION
On dual output 500 watt Ultrasonic Processors manufactured prior to June 1985, a
switch is located above the tuning control.
Check that the switch is in the down position.

11
USING THE ULTRASONIC PROCESSOR

The speed control on an automobile, can, to a certain extent, be compared to an

Ultrasonic Processor. The speed control is designed to maintain the vehicles rate of
travel constant. As the terrain changes, so do the power requirements. The speed
control senses these requirements, and automatically adjusts the amount of power
delivered by the engine in order to compensate for these ever changing conditions.
The greater the terrain rate of incline and greater the resistance to the movement of
the vehicle, the greater the amount of power that will be delivered by the engine to
overcome that resistance.

The Ultrasonic Processor is designed to deliver constant amplitude. As the

resistance to the movement of the probe increases, additional power will be delivered
by the power supply to ensure that the excursion at the probe tip remains constant.
Using a more powerful power supply will not deliver more power into the liquid.
Rather, it is the resistance to the movement of the probe that determines how much
power will be delivered into the sample.

The AMPLITUDE control allows the ultrasonic vibrations at the probe tip to be set
to any desired level. Although the degree of cavitation required to process the
sample can readily be determined by visual observation, the amount of power
required cannot be predetermined. A sensing network continuously monitors the
output requirements, and automatically adjusts the power to maintain the amplitude
at the preselected level. The greater the resistance to the movement of the probe due
to higher viscosity, deeper immersion of the probe into the sample, larger probe
diameter or higher pressure, the greater the amount of power that will be delivered to
the probe. Setting the AMPLITUDE control fully clockwise will not cause the
maximum power to be delivered to the sample. The maximum power that the
Ultrasonic Processor is capable of delivering will only be delivered when the
resistance to the movement of the probe is high enough to draw maximum wattage.

This phenomenon can be demonstrated as follows: depress the probe down against a
piece of wood. The greater the down pressure, and consequent greater resistance to
the movement of the probe, the greater the amount of power that will be delivered by
the power supply.

12
OPERATING INSTRUCTIONS

CAUTION
Never allow liquid to spill into the converter. Do not use the cup horn without a splash
shield
Do not allow a microtip or extender to vibrate in air for more than 10 seconds. When
working with a microtip never allow the OUTPUT CONTROL to be set above the
microtip limit “5”. Ignoring these instructions will cause the microtip to fracture.
Do not allow the vibrating microtip to contact anything but the sample.
When working with low surface tension liquids, do not use a probe with a replaceable
tip.
Never energize a threaded probe without the replaceable tip, extender, or microtip
attached.

1. Ensure that the power supply is properly tuned. Tuning optimizes performance
and insures maximum transfer of energy by matching the frequency of the power
supply to that of the converter / probe assembly
2. If a standard probe is used, immerse the probe approximately 2 inches (5 cm) into
the sample. If a microtip is used, immerse the microtip approximately ½” (1 cm)
into the sample.

NOTE
The probe should be immersed to a sufficient depth to preclude air from being injected into the
sample, and inhibit aerosoling and foaming.

3. Set ON/OFF/TUNE switch to ON.

4. Set CONTINUOUS/PULSED switch, % DUTY CYCLE SELECTOR, TIMER,

and OUTPUT CONTROL as desired.

IMPORTANT
Although the Ultrasonic Processor is capable of delivering maximum
power to the probe, the actual power delivered, as read on the power
monitor, will only be that required by the application
Full meter deflection will only take place when the Ultrasonic
Processor is called upon to deliver maximum output.

13
 SECTION III – SERVICE INFORMATION

Your Ultrasonic Processor was designed to provide you with years of safe and
dependable service. Nevertheless, because of component failure or improper usage, the
possibility does exist that it might not perform as it should, shut down due to an overload
condition or that it will stop working all together. The most probable causes for
malfunction are listed below and should be investigated.
The unit was plugged into an electrical outlet that provides a different voltage from that
required. See Electrical Requirements.
The probe and/or microtip is not secured properly.
If the probe has a replaceable tip, the tip is not secured properly, or the probe has been
used with low surface tension liquids.
A fuse(s) has failed. If a fuse(s) has failed, proceed as follows:
1. Ensure that the power switch is set to OFF.
2. Replace the fuse(s).
3. Set the OUTPUT CONTROL to “5” and the power switch to ON. With the probe
in air (out of sample), the wattmeter should read below 20 watts. If the reading
exceeds 20 watts, set the power switch to OFF, and disconnect the probe from the
converter.
4. Set the power switch back to ON. If the wattmeter reads below 20 watts, the
probe has failed or is out of tune due to excessive erosion, and should be replayed,
if the wattmeter reads above 20 watts, either the converter or power supply has
failed and the complete Ultrasonic Processor should be returned for repair.

14
RETURN OF EQUIPMENT

It is suggested that an Ultrasonic Processor in need of repair be sent back to the factory.

In order to receive prompt service; always contact the factory before returning any
instrument. Include date of purchase, model number and serial number. For instruments
not covered by the warranty, a purchase order should be forwarded to avoid unnecessary
delay. Care should be exercised to provide adequate packing to insure against possible
damage in shipment. The Ultrasonic Processor should be sent to the “Service
Department” with all transportation charges prepaid and return of shipment indicated.

Please obtain a Return Authorization Number prior to returning the instrument.

IMPORTANT
I CERTIFY THAT THE ULTRASONIC PROCESSOR AND / OR ACCESSORIES RETURNED
FOR REPAIR ARE FREE OF ANY BIOHAZARDOUS OR RADIOACTIVE MATERIAL AND ARE
SAFE FOR HANDLING.
DO NOT RETURN ANY EQUIPMENT UNLESS SUCH CERTICATION CAN BE MADE.

15
 SECTION IV - OPERATING SUGGESTIONS AND TECHNIQUES

DISRUPTING CELLS

The disruption of cells is an important stage in the isolation and preparation of

intracellular products. From research levels through to production, many areas of
biotechnology, particularly recombinant technology, necessitate the use of ultrasonics for
cell disruption. Although some biological products are secreted from the cell or released
during autolysis, many others require sonication to release intracellular material. Cell
disruption focuses on obtaining the desired product from within the cell, and it is the cell
wall that must be disrupted to allow cell contents extraction.

Single-cell organisms (micro-organisms) consis t of a semipermeable, tough, rigid outer

cell wall surrounding the protoplasmic membrane and cytoplasm. The cytoplasm is made
up of nucleic acid, protein, carbohydrates, lipids, enzymes, inorganic ions, vitamins,
pigments, inclusion bodies, and about 80% water. In order to isolate and extract any of
these substances from inside the cell, it is necessary to break the cell wall and
protoplasmic membrane. In some cases the cell may excrete the desired substance
without assistance, but in most cases, the cells must be lysed and sonicated in order for
these substances to be released. Breaking cell membranes and releasing the contents
present significant challenges. The process must be fast and thorough to maximize the
protein yield. Because the energy applied must be great enough to break the cell
membranes or walls, yet gentle enough to avoid physically or chemically damaging cell
contents, the Vibra-Cell with its variable intensity capability is ideally suited for this
application.

The level of intensity that should be used is application dependent. For example high
intensity might be recommended for the break up of cells, but should never be used when
the release of intracellular components might be objectionable e.g. Organelle isolation.

The ability to control the amplitude at the probe tip is a prerequisite for process
optimization. And because each application requires its own set of processing parameters,
due to variation in volume and composition, the optimum amplitude can only be
determined empirically. When processing a new sample, it is recommended that the
amplitude be set first at 50% (30% with a microtip) and then increased of decreased as
required.

Yeast, gram-positive bacteria, and to a lesser extent, gram-negative bacteria have

considerably harder cell walls in comparison to animal cells, and require relatively high
power for cell disruption.

Gram negative bacteria typically require 10 to 15 minutes of processing, while

staphylococcus requires 20 to 30 minutes.

16
Microorganisms differ greatly in their sensitivity to ultrasonic disintegration. For
example, the most readily disintegrated are the rod- like forms (bacilli), while the
spherical organisms (cocci) are much more resistant. The group Mycobacteria, to which
the tuberculosis organism belongs, is particularly difficult to disrupt. Generally, animal
cells are more easily disintegrated that plant cells, and red blood cells are more readily
disintegrated than muscle cells because they lack a protective cell wall.

Cellular disruption is the first step in RNA isolation and one of the most critical steps
affecting yield and quality of the isolated RNA. Typically, cell disruption needs to be fast
and thorough. Slow disruption, for example placing cells or tissue in guanidinium
isothiocyanate (GITC) lysis solution for a long time prior to sonication, may result in
RNA degradation by endogenous RNases released internally. This is especially a concern
when working with tissues high in endogenous RNase such as spleen and pancreas.

Disrupting frozen tissue is more time consuming and cumbersome that processing fresh
tissue, but freezing samples is sometimes necessary. Samples are usually frozen when, 1)
they are collected over a period of time and thus, cannot be processed simultaneously; 2)
there are many samples, 3) samples are collected in the field, or 4) mechanical processing
of fresh samples is insufficient for thorough disruption. A mortar and pestle or bag and
hammer are typically used when the starting material is frozen. RNA will remain intact in
tissues for a day at 37ºC, a week at 25ºC a month at 4ºC and indefinitely at subzero
temperatures.

Ultrasonic processing will typically cause the temperature of the sample to increase
especially with small volumes. Since high temperatures inhibit cavitation, the sample
temperature should be kept as low as possible - preferably just above its freezing point.
This can be accomplished by immersing the sample vessel in an ice-salt-water bath.
Temperature elevation can also be minimized by using the pulser.

Increasing hydrostatic pressure (typically 15-60 psi) and viscosity can enhance cell
disruption. For microorganisms, the addition of glass beads in the 0.5 to 1mm size range
promotes cell disruption. Beads are almost a prerequisite when working with spores and
yeast. A good ratio is one volume of beads to two volumes of liquid. Glass beads are
available from Cataphote, Inc. P.O. Box 2369, Jackson, Mississippi 39225-2369 USA,
phone (800) 221-2574 or (601) 939-4612, FAX (601) 932-5339, Jayco Inc. 675 Rahway
Ave., Union NJ 07083 USA, phone (908) 688-3600, FAX (908) 688-6060 or Sigmund
Lindner GmbH. P.O. Box 29. D-95483 Warmensteinach, Germany. Phone (49) 0 92 77 9
94 10, FAX (49) 0 92 77 9 94 99.

When processing difficult cells such as yeast, pretreatment with an enzyme is beneficial.
Lysozyme, byaluronidase, glycosidase, glucalase, lyticase, zymolase and lysostaphin
digestion are among the enzymatic methods frequently used with yeast and Lysozyme
with bacteria. Enzymatic treatment is usually followed by sonication in a GITC lysis
buffer. Collogenase may be used with collogen, lysostaphin with staphylococcus, and
trypsin hyaluronidase with liver and kidney.

17
If enzymes cannot be used, the following procedures should be considered: Freezing the
sample at -70?C overnight, then thawing it in water immediately prior to ultrasonic
processing.

Most animal tissues can be processed fresh (unfrozen). It is important to keep fresh tissue
cold and to process it quickly (within 30 minutes) after dissection. When working with
fresh tissue, the cells must be sonicated immediately at the time the GITC lysis solution is
added. This can be done by dispensing the lysing solution in the tube, adding the tissue
and immediately sonicating. Samples should never be left sitting in lysis solution,
undisrupted. Large samples of hard tissues should be first treated in a blender or a
mechanical homogenizer.

Animal tissues that have been frozen after collection should be disrupted by grinding in
liquid nitrogen with a mortar and pestle. During this process, it is important that the
equipment and tissue remain at cryogenic temperatures. The tissue should be dry and
powdery after grinding. Grinding should be followed by thorough sonication in a GITC
lysis buffer. Processing frozen tissue in this way is cumbersome and time consuming, but
effective.

Cultured cells are normally easy to disrupt. Cells grown in suspension are collected by
centrifugation, rinsed with PBS to remove culture medium, and then lysed by sonicating
in a GITC lysis buffer. Placement of the vessel on ice while washing and lysing the cells
will further protect the RNA from endogenous RNases released during the disruption
process.

Soft, fresh plant tissue can often be disrupted by sonicating in a lysis buffer. Other plant
tissues, like pine needles, need to be ground dry, without liquid nitrogen. Some hard,
woody plant materials require freezing and grinding in liquid nitrogen prior to being
ultrasonically processed. Plant cell suspension cultures and calluses can typically be
sonicated in a lysis buffer within 2 minutes. The diversity of plants and plant tissue make
it impossible to give a single recommendation for all. However, most plant tissues
typically contain polysaccharides and polyphenols that can coprecipitate with RNA and
inhibit downstream assays. Treating a plant tissue lysate with polyvinylpyrrolidone
(PVP) will precipitate such problematic components from the lysate before the actual
RNA isolation is carried out.

Whenever possible, the tissues should be diced very small to permit movement within the
liquid. Tough tissues such as skin and muscle should be macerated first in a blender or
the like for about 10 seconds, and confined to a small vessel during ultrasonic treatment.
If sub-cellular particles are desired intact, the amplitude should be kept low, and the
processing time increased.

Yeast can be extremely difficult to disrupt because their cell walls may form capsules or
nearly indestructible spores. To process yeast, sonicate in a tube containing the sample,
guanidinium-based lysis buffer and small glass beads (0.5 – 1 mm). Pretreatment with

18
zymolase, glucalase and / or lyticase to produce spheroplasts that are readily lysed may
also be useful.

To disrupt filamentous fungi, scrape the mycelial mat into a cold mortar, add liquid
nitrogen and grind to a fine powder with a pestle. The powder can then be thoroughly
sonicated in lysis buffer to solubilize completely. As fungi may also be rich in
polysaccharides, pretreatment with polyvinylpyrrolidone (PVP) may be beneficial.

Bacteria, like plants, are extremely diverse; therefore, it is difficult to make one
recommendation for all bacteria. Ultrasonic processing will lyse most Gram positive and
Gram negative bacteria, including mycobacteria. Although it is recommended that glass
beads and lysis solution be used; it is possible to lyse some Gram negative bacteria by
sonicating in lysis solution without beads. Bacteria cell walls can be digested with
lysozyme to form spheroplasts. Gram positive bacteria usually require more rigorous
digestion and longer processing time. The spheroplasts are then lysed with sonication in
GITC lysis buffer.

Disruption of cells found in soil and sediments is accomplished one of two ways. One
technique isolates the bacterial cells from the material prior to the RNA isolation
procedure. This is accomplished by homogenization of wet soil in a mechanical blender
followed by a slow speed centrifugation to remove fungal biomass and soil debris. The
supernatant is centrifuged again at a higher speed to pellet the bacteria cells. Cells can
then be lysed as described above for bacteria. Other techniques describe RNA isolation
from the soil or sediment directly. For example, one method requires soil to be added to a
diatomaceous earth and lysis buffer, and then sonicated. The sample is then centrifuged
to remove solid debris.

Always immerse the probe deep enough below the surface of the sample to inhibit
aerosoling or foaming, foaming substantially reduces cavitation. Processing at a lower
power setting without foam is much more effective than processing at a higher power
setting with foam. Decreasing the power, increasing processing time and lowering the
temperature of the sample will usually prevent aerosoling and foaming. Do not use any
antifoaming agents or surfactants.

During cavitation, free radicals are formed which, if they are allowed to accumulate, can
greatly affect the biological integrity of the sample by reacting with proteins,
polysaccharides, or nucleic acids. Although during short periods of processing their
formation is not normally considered a problem; for longer durations, the addition of free
radical scavengers such as, carbon dioxide, N2 O, cysteine, reduced glutahione,
dithiothreitol or other SH compounds, might be beneficial. Saturating the sample with a
protective atmosphere of helium or nitrogen gas, or dropping a small pellet of dry ice in
the sample, will also inhibit free radical formation. Whereas it is true that gas is required
for effective cellular disruption, it is not necessary that the vapor phase be oxygen or air
since any gas except carbon dioxide will work just as well.

19
Various methods can be used to measure the efficiency of the disruption. For example, a
visual count can be made using a microscope.
For greater accuracy, a protein assay could be used. This procedure is widely recognized
as a good method for measuring cell disruption by taking into account the amount of
protein released after disruption. The disrupted cells are then tested and checked against
this number for percentage breakage.
There are several types of protein assays. One commonly used is the Folin Reaction
(Lowry Assay) method, as it is comparatively simple and provides consistent results.
This colorimetric method has a sensitivity to protein of around 8 µg / mL in the assay
solution.
The assay turns blue in the presence of proteins due to the reaction of copper ions in the
alkaline solution with protein and the reduction of phosphomolybdate- phosphotungstic
acid in the Folin reagent by aromatic amino acids in the treated protein.
Fractional protein release, Rp, is calculated using the following equation and multiplying
the result by 100:
Rp = Cf – Cb
Ct – Cb
Cf = Free protein
Ct = total protein
Cb = Background protein
This gives the actual disruption percentage, taking into account the background levels of
protein before disruption.

Since the greatest concentration of energy is beneath the probe, it is imperative that the
sample be kept as close to the tip as possible, liquids are easily processed because the free
moving cells circulate repeatedly below the probe. Solid materials however have a
tendency to be repelled by the ultrasonic, and should be processed in a vessel large
enough to accommodate the probe, yet small enough to restrict sample movement. For
small samples, conical shaped test tubes are recommended.

Allowing the probe to contact the vessel will decrease the power output, and cause
minute grey glass particles to migrate into the sample. Although these glass particles will
not adversely affect the chemical composition of the sample, they will form a thin grey
layer on centrifuging. If the probe has to come in contact with a solid sample, use a
standard 20mm (3/4”) diameter stainless steel centrifuge tube cut to 70mm (3”) length.
Do not use a glass tube. Microtips must never allowed to come in contact with anything
but the liquid, because the stress resulting at the point of contact with a hard surface will
cause the microtip to fracture. Although larger probes will not fracture if they come in
contact with a glass vessel, they may cause the vessel to fracture.

Before each application, place the tip in water or alcohol and energize the power supply
for a few seconds to remove any residual substances. If concerned about contamination
from previous use, clean the probe with a 20% Virkon solution and rinse with distilled
water. For critical application, probes may be autoclaved.

20
To inhibit sample loss in test tube due to sticking, siliconize the test tube as follows:
Wash and dry the test tube thoroughly, coat with silicone, then air dry. “Sigmacote”
manufactured by Sigma Chemical Co., 3050 Spruce Street, St. Louis, Missouri 63103,
USA, phone (314) 771-5765, is ideally suited for that purpose.

High viscosity and concentration are problematic. 2,000 cps and 15% concentration by
weight are maximum limits. Because with ultrasonics the sound waves are propagated
through the sample, if the sample is so thick that it will not pour or circulate easily, it is
too thick for ultrasonic processing.

Use the Cup Horn for processing pathogenic, radioactive, and biohazardous materials in
complete isolation without probe intrusion. Because plastic tubes have a tendency to
absorb vibrations, it is preferable, whenever possible, to contain the sample in a stainless
steel tubes or glass tubes when working with a cup horn. To expedite processing, add
glass beads to the sample. If desired, crushed ice can also be added to the water inside the
cup horn, in order to optimize cooling. Processing samples in a Cup Horn will usually
take 4 times longer than processing with the direct probe intrusion method.

21