Sigma Chemical Co., 3050 Spruce St., St. Louis, Missouri 63103. Phone (314) 771-5765, is ideally suited for
that purpose.

Glass Bead can be acquired from Catatphote, Inc. P.O. Box 2369, Jackson, Mississippi 39225-2369 USA.
Phone (601) 939-4631 or (800) 221-2574.

Always use an extender or a solid probe (without replaceable tip) when working with solvents or low surface ten-
sion liquids such as methylene chloride.

SECTION III — MAINTENANCE

GENERAL
It is suggested that an Ultrasonic Processor in needs 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 units 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 method of shipment indicated.

BY RETURNING ANY MATERIAL, THE CUSTOMER OR THE CUSTOMER’S AGENT THUS CERTIFIES THAT
ANY AND ALL MATERIALS SO RETURNED ARE, OR HAVE BEEN RENDERED, FREE OF ANY HAZARDOUS
OR NOXIOUS MATTER OR RADIOACTIVE CONTAMINATION AND ARE SAFE FOR HANDLING UNDER
NORMAL REPAIR SHOP CONDITIONS. Do not return any material for which such certification can not be
made.


N

CAUTION

When using a microtip or extender, never allow the tip to vibrate in air for more than 15 seconds
and do not set the OUTPUT CONTROL above ‘'5”. Ignoring these instructions will cause the
microtip or extender to fracture.

Set POWER SWITCH to ON. The switch will illuminate.

Momentarily depress TUNE SWITCH and rotate the tuner clockwise or counterclockwise until
a minimum (not maximum) reading (usually less than 20) is obtained on the POWER MONITOR. If a
minimum reading cannot be obtained, the probe, cup horn, tip, microtip, extender, 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.

NOTE
The probe is tuned to vibrate at a specific frequency. If the resonant frequency of the
probe has changed, due to cavitation erosion or fracturing, minimum reading will not
be obtained. If minimum reading cannot be obtained, check the unit without the probe.
If proper tuning is obtained without the probe, then the probe should be changed.

Set OUTPUT CONTROL to “4”.
Set POWER SWITCH to OFF.

With a dual output 600 watt Ultrasonic Processor, if two converters are going to be used simultaneously, con-
nect at this time the second converter cable to the top connector.

12


UTILITIES REQUIRED

ELECTRICAL POWER — The Ultrasonic Processor requires a fused, single phase 3-terminal grounding type
electrical outlet capable of supplying 50/60 Hz at 100 volts, 117 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, the electrical line cord is equipped with a 3-prong grounding plug. Do
not, under any circumstances, defeat the grounding feature of the power cord by removing the
grounding prong. The plug must be plugged into a mating 3-prong grounding type wall receptacle.

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.

The power supply is air cooled. Sufficient space should be allocated around the unit so that the air flow through
the cabinet is unrestricted. If air circulation is impaired, the ambient temperature within the power supply may
become excessive, causing a safety thermal cut-out device to switch the power off.Although the power will be
automatically reinstated when the temperature reaches an acceptable level, it is advisable that when this
happens, the cause be investigated.

Periodically clean the air intake grille.


If the probe has to come in contact with a solid sample, use a standard %4” (20 mm) diameter stainless steel
centrifuge tube cut to 3” (70 mm) length. Do not use a glass tube.

When processing difficult cells, the usage of enzymes might be considered. Glusulase can be used with yeast,
lysostaphin can be used with staphylococcus, collagenase can be used with skin and cartilage, and trypsin
hyaluronidase can be used with liver and kidney.

Glass beads in the 50 micron size range can also be added to the liquid to expedite processing. When using
glass beads, a good ratio is 1/3 beads, 2/3 liquid. Glass beads concentrate the energy released by the implosion
and facilitate the crushing of cell walls. After processing, the glass will sink to the bottom of the vessel or can be
centrifuged or filtered out.

Whenever possible, the tissues should be diced very small to permit movement within the liquid. Freezing
followed by powdering could also be resorted to, if this procedure is not detrimental to the experiment.

Large samples should first be liquified in a high speed mechanical blender for about 10 seconds. If sub-cellular
particles are desired intact, the amplitude control should be set low and the processing time increased.

Ultrasonic outgassing or deaeration of liquids is best done at low pressure or under vacuum.

Proper care of the probe is essential for dependable service. The intense cavitation will, after a prolonged period,
cause the tip to erode, and the power output to decrease without showing up on the power monitor. The
smoother and shinier the tip, the more power will be transmitted into the liquid. Any erosion of the probe tip will
increase the rate of future erosion. For that reason, it is recommended that after every 5 or 6 hours of use the tip
be examined, and if necessary, polished with a fine emery cloth. Since the probe and tip are tuned to vibrate ata
specific frequency, it is most important that only the contaminated surface be removed. If tip wear is excessive,
replace the tip.

High viscosity and concentration are problematical. 8,000 cP and 15% concentration by weight are maximum
limits. If the liquid is so thick that it will not pour or circulate easily, it is too thick.

16


NOTE

Should it become necessary to remove a probe, use the spanner wrenches supplied. If
the probe has been on the converter for along 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 spanner wrench, twist the converter off the probe. A tap of ahammer may
be applied to the end of the spanner wrench. Never attempt to twist the converter
housing as this may damage the crystal or the electrical connections within the
housing.

8. Connect the converter cable to the ae
bottom connector. With a dual Je ip
output 600 watt Ultrasonic Proces- {
sor, if two converters are going to
be used simultaneously, do not
connect the second converter at
this time.

bly(ies) in a laboratory stand. Secure

the clamp(s) to the 22” (63.5 mm) —

diameter converter housing only. Do ee
not secure the clamp(s) to any other
portion of the converter/probe assem-

bly(ies). Never allow the probe to touch

the workbench or other surfaces.

9. Mount the converter/probe assem- |



FUNCTIONS OF CONTROLS, INDICATORS, AND CONNECTORS

FRONT PANEL

POWER MONITOR (METER)

Indicates the percentage of maximum available ultrasonic power delivered
to the probe.

300 watt unit: 100% = 300 watts, 50% = 150 watts.

375 watt unit: 100% = 375 watts, 50% = 187.5 watts.

600 watt unit*: 100% = 600 watts, 50% = 300 watts.
*When using two probes with the dual output unit, the power delivered
to each probe is half that displayed on the power monitor.

"

POWER SWITCH

ON position — energizes the power supply.
OFF position — de-energizes the power supply.
\lluminates when the power supply is energized.

TUNE SWITCH

When depressed, allows the power supply to be tuned.

TIMER

Sets the duration of ultrasonic application from .1 second to 999 hours.
The right hand push button selects the timing option as follows:
-S=tenth of seconds, S=seconds, .M=tenth of minutes, M=minutes
.H=tenth of hours, H=hours.

The other three push buttons select the timing increments.

START BUTTON

When depressed, energizes the ultrasonics.

RESET BUTTON

When depressed, de-energizes the ultrasonics and resets the timer.



SECTION II — OPERATION

PRINCIPLES OF ULTRASONIC DISRUPTION

The ultrasonic power supply (generator) converts 50/60 Hz line voltage to high frequency 20 kHz (20,000 cycles
per second) 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. It is this phenomenon, referred to as cavitation, which produces the powerful shearing action
at the horn tip, and causes the molecules in the liquid to become intensely agitated.

The larger the tip of the probe, the larger the volume that can be processed but at lesser intensity. For information
regarding 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 Very High Very High High Ultra High
VOLUME (batch) 1-10 ml 3415 ml 5:25 ml 1/4-10 ml

STANDARD HORNS. HIGH GAIN “Q” HORNS
TIP DIAMETER | 3/8" (9.5mm) | 1/2” (13mm) | 3/4" (19mm) | 1” (25.4mm) TIP DIAMETER |3/4" (19mm) | 1” (25.4 mm)
INTENSITY Very High High Medium Low INTENSITY High Medium
VOLUME (batch)} 5-100 ml 10-250 ml 25-500 ml 50-1000 ml VOLUME (batch) | 25-500 ml 50-1000 mi



During cavitation free radicals are formed which, if they are allowed to accumulate, can greatly affect the
biological integrity of the sample. Although during short periods of processing their formation is not normally
considered a problem; for prolonged processing, the addition of free radical scavengers such as cysteine or
glutahione might be considered. Saturating the liquid with hydrogen or carbon dioxide will often eliminate free
radical formation. A small pellet of dry ice dropped in the liquid will often resolve the problem.

If aerosoling presents difficulties, use the Sealed Atmosphere Treatment Chamber.

The problem of oxidation is a serious one particularly where the study of sulfhydril enzymes is concerned. This
may be partially controlled by the use of free radical traps such as cysteine, reduced gluthathione orcomparable
substances or by insonating in the presence of an inert atmosphere. 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 has essentially the same efficiency for cellular disruption. A Sealed Atmosphere Treatment Chamber
permits extraction to be made in presence of helium or nitrogen. Forcing inert gas through the liquid will also
reduce aerobic oxidation.

The probe tip may be autoclaved, or sterilized either by immersing in boiling water or in a detergent
bactericide such as Staphene and a disinfectant

Use the Sealed Atmosphere Treatment Chamber for processing pathogenic and biohazardous materials.

Use the Continuous Flow Cell for processing large volumes. If it is necessary to keep the flow cell cold, immerse
the coiled copper tubing carrying the water to the water jacket in a salted ice bath.

Use the Cup Horn for processing pathogenic, radioactive and biohazardous materials in complete isolation.
Because plastic tubes have a tendency to absorb vibrations, it is advisable to use stainless steel tubes or glass
tubes when working with a cup horn. To expedite processing, add glass beads to the liquid.

Ifconcerned with sample loss in test tube due to sticking, siliconize the test tube inaccordance with the following
procedure. Wash and dry the tube thoroughly, coat with silicone, then air-dry. “Sigmacote” manufactured by

17


PREPARATION FOR USE

CAUTION

If the Ultrasonic Processor has been left in a very cold or hot environment for a prolonged
period of time, do not operate until it has reached room temperature.

To safeguard the fuse against failure, always switch the power supply off before connecting
or disconnecting the footswitch plug.

Ensure that the POWER SWITCH is set to OFF.

Plug the electrical line cord into the electrical outlet.

If a footswitch is used, plug into the FOOTSWITCH CONNECTOR. Make sure that the plug is inserted
forcefully all the way in.

4. If the converter, and accessories are not already assembled, observe steps 5, 6, and 7.

5. Clean the mating surfaces of the converter and probe or stepped microtip, as well as the threaded stud and
hole. Check that the stud is tight.

N=

CAUTION

Never assemble or disassemble a probe by holding the converter in a vise.

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.

6. Hand assemble the probe or stepped microtip assembly (consisting of coupler and stepped tip) to the converter,
and using wrenches provided, tighten securely. See Page 9.

7. Tosecure a replaceable tip, extender, or tapered microtip to a probe, use a spanner wrench and an open wrench.
See page 9.

7


High Intensity Ultrasonic Processor

300-Watt Model
375-Watt Model
600-Watt Model

600-Watt Model Dual Output

INSTRUCTION MANUAL



No. DESCRIPTION Order Number
1 Converter Model CV17 Cvo0017
2 Four element coupler 630-0425
3. | Stepped tip(s) 1%.” (3 mm) 630-0422
4 Booster BHNVCGD
5 Standard horn "/2" (13 mm) solid 630-0201

Standard horn '/2" (13 mm) with threaded end and replaceable tip 630-0200
Standard horn 3/4" (19 mm) solid 630-0208
Standard horn 3/4” (19 mm) with threaded end and replaceable tip 630-0207
Standard horn 1” (25 mm) solid 630-0209
Standard horn 1” (25 mm) with threaded end and replaceable tip 630-0210
6 Replaceable tip 1/2" (13 mm) 630-0406
Replaceable tip 2/4" (19 mm) 630-0407
Replaceable tip 1” (25 mm) 630-0408
7 | Coupler 630-0421
8 Stepped tip '/s” (3 mm) 630-0422
9 | Standard horn 1/2” (13 mm) with threaded end and replaceable tip 630-0200
10 | Tapered microtip "/s” (3 mm) 630-0418
Tapered microtip 3/16” (5 mm) 630-0419
Tapered microtip 1/4” (6 mm) 630-0420
11 Standard horn - solid or with threaded end and replaceable tip - same as 5
12 Replaceable tip - same as 6
13 Extender "/" (13 mm) 630-0410
Extender 3/4” (19 mm) 630-0409
Extender 1” (25 mm) 630-0444
14 | High gain Q horn 2/," (19 mm) solid 630-0306
High gain Q horn */," (19 mm) with threaded end and replaceable tip 630-0305
High gain Q horn 1” (25 mm) solid 630-0310
High gain Q horn 1” (25 mm) with threaded end and replaceable tip 630-0311
15 | Replaceable tip 3/4" (19 mm) or 1” (25 mm) - same as 6
46 | Extender 3/4” (19 mm) or 1” (25 mm) - same as 13
CAUTION

Do not use tapered microtip with coupler.

Do not use stepped tip without coupler.

10



HORN REMOVAL HORN TIGHTENING

lg ; vl

TIP REMOVAL TIP TIGHTENING


FUNCTIONS OF CONTROLS, INDICATORS, AND CONNECTORS (CONT.)

TUNER Optimizes power supply performance by tuning the power supply to
the converter/probe assembly.

FOOTSWITCH CONNECTOR Connects to footswitch cable.

PULSER Applies the ultrasonic energy on a pulsed mode.

In the OFF position the ultrasonics is continuous.

In the ON position the ultrasonics is intermittent.

The % DUTY CYCLE SELECTOR sets the pulse rate.

Intermittent operation inhibits heat build-up in the liquid and provides
more efficient processing by allowing the material to settle back under
the probe tip after each burst.

OUTPUT CONTROL Controls the amplitude of vibrations at the probe tip.

CAUTION: Whenusing a microtip, never allow the output control!
setting to exceed MICROTIP LIMIT “5”.

REAR PANEL
3 PIN CONNECTOR (S) Connect to the converter cable(s).
FUSE(S) Protects against electrical overload.
ELECTRICAL LINE CORD Connects the power supply to the electrical outlet.

6


SECTION I - 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.

It was carefully packed and thoroughly inspected before leaving our factory. Responsibility for its safe delivery
was assumed by the carrier upon acceptance of the shipment. Claims for loss or damage sustained in transit
must, therefore, be made upon the carrier.

Visually inspect all external controls, indicators and surfaces to detect any damage in transit. If damage has
occurred contact your carrier within 48 hours of delivery date. DO NOT OPERATE DAMAGED EQUIPMENT. Retain
all packing materials for future shipment.


USING THE ULTRASONIC PROCESSOR

CAUTION

* Do not operate the power supply unless it is connected to the converter(s).

° High voltage is present in the power supply — Do not operate with the cover off.

° 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 15 seconds.

° When using a microtip, never exceed MICROTIP LIMIT “5” on OUTPUT CONTROL.
Do not allow the vibrating microtip to come in contact with anything but the liquid.

* Do not operate the converter in an explosive, humid, or caustic atmosphere.

1. Ensure that the power supply is properly tuned (see Pages 11 and 12).

2. If astandard probe is used, immerse the probe 1 to 1% inches (3 cm) into the liquid. If a microtip is used,
immerse the microtip % inch (1 cm) into the liquid.

3. Set TIMER as required. If the footswitch is used, set TIMER to 999 HOURS. Depressing the footswitch will
not energize the power supply, unless the TIMER is activated.

Set PULSER as required. If the pulsing mode is not required, set PULSER to OFF.
Set POWER SWITCH to ON. The switch will illuminate.

Activate the timer by depressing the START button.

If the footswitch is used, depress footswitch.

OM @ or oe

Using OUTPUT CONTROL increase or decrease intensity as required.
13


belongs, is particularly difficult to disrupt. Generally, animal cells are more easily disintegrated than plant cells, and
red blood cells are more readily disintegrated than muscle cells because they lack a protective cell wall.

Before each experiment, place the probe tip in water or alcohol and energize the power supply for a few seconds
to remove any residue.

The probe must never be allowed to come in contact with anything but the liquid. When using a microtip, the
stress resulting at the point of contact with the vessel could cause the microtip to fracture. Standard probes will
not fracture if they contact the treatment vessel, but they can damage the vessel.

Insert the probe deep enough below the surface of the liquid to inhibit aerosoling or foaming. Foaming
substantially reduces cavitation. Processing at a lower power setting without foam is more efficient than
processing at a higher power setting with foam. Decreasing the power, increasing processing time, using a
narrower vessel, and lowering the temperature of the liquid will usually prevent aerosoling and foaming. Do not
add anti-foaming agents or any materials that might lower surface tension. Reducing surface tension, such as by
adding a surfacant, will lower the power required but will decrease intensity. Use a conical shaped tube such asa
reaction vial whenever possible.

Allowing the probe to come in contact with the vessel will decrease the power output, and cause tiny glass
particles to migrate into the liquid. Although these glass particles will not adversely affect the chemical
composition, they will form a thin grey layer on centrifuging.

In ultrasonic processing the molecular motion in the liquid causes temperature elevation — especially with small
volumes. Since high temperature reduces cavitation, the liquid should be kept as cold as possible. This can be
accomplished by immersing the sample vessel in an ice-salt-water-alcohol bath, or by using a water-jacketed
processing vessel through which very cold water or alcohol is circulated.

Since the greatest concentration of energy is immediately below 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 ultrasonics. To
alleviate this problem, the vessel should be large enough to accommodate the probe, yet small enough to restrict
sample movement. 15


CAUTION

Never allow a microtip or an extender to vibrate in air for more than 15 seconds. When
using a microtip never exceed setting ‘‘5’’ on output control. Ignoring these cautions
will cause the microtip or extender to fracture.

METHYLENE CHLORIDE ¢ ORGANIC SOLVENTS
The probes (solid or with a replacable tip) are tuned elements that resonate at 20kHz
+ 50 Hz. If the replaceable tip is removed or isolated from the rest of the probe, the
unit will not resonate at 20kHz causing the power supply to fail.

Unlike aqueous (water based) solutions which rarely cause problems, solvents and
low surface tension liquids — such as Methylene Chloride, are problematical. These
liquids penetrate the probe/replaceable tip interface, and force the particulates into
the threaded section isolating the tip from the probe.

When working with these liquids ALWAYS use a solid probe or as an alternate an
extender.

The extender screws into the probe, and takes the place of the replaceable tip.
Being 5” (13cm) long, the threaded section is above the level of the liquid, inhibiting
the particulates from penetrating the threaded section.


WARNING
SAFETY PRECAUTIONS
READ BEFORE INSTALLING OR
USING THE EQUIPMENT

This Ultrasonic Processor has been designed to assure maximum operator safety.
However, no design can completely protect against improper usage. Improper usage
may result in bodily injury and/or property damage. For maximum safety and
equipment protection, observe the following warnings at all times, and read the
instruction manual carefully before you attempt to operate the equipment.

— High voltage is present in the power supply. Never remove power supply cover
unless qualified to do so.

— Disconnect electrical plug before removing cover for servicing.

— Make sure the Ultrasonic Processor is Properly grounded with a 3-prong plug.
Before plugging in, test electrical outlet for Proper earth grounding.

— Never touch a vibrating probe.

The Ultrasonic Processor supplied with this instruction manual is constructed of the
finest material and the workmanship meets the highest manufacturing 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.


TUNING

Tune the power supply in accordance with the following procedure each time a new converter, probe, cup horn,
tip, microtip, extender, or accessory is used. .

CAUTION

The power supply should be tuned after the probe has reached operating temperature.
When working with low or high temperature liquids, immerse the probe in the liquid fora
few minutes, withdraw the probe out of the liquid, then, tune the power supply.
When using the optional cup horn or continuous flow cell, remove all the water from the cup
horn, and liquid from the continuous flow cell, before tuning the power supply.

1. Ensure that the probe or microtip is not immersed in the liquid and that it does not come in contact with
anything. If a cup horn or flow through cell is used, make sure that it does not contain any liquid.

2. Set PULSER to OFF.

3. Set OUTPUT CONTROL to “10” (to “5” when using a microtip).

W


IMPORTANT

As the volume or viscosity increases, so does the need for higher power output. A sensing network con-
tinuously monitors the output requirements, and automatically adjusts the power output in order to satisfy the
needs of the application. The actual power delivered will only be that required by the application. The greater
the resistance to the movement of the probe, because of higher viscosity, larger volume or deeper immersion
of the probe into the liquid, the greater the deflection on the POWER MONITOR.

With the 300 watt unit, full meter deflection will only take place when the unit is called upon to deliver 300
watts. With the 375 watt unit, full meter deflection will only take place when the unit is called upon to deliver
375 watts. With the 600 watt unit, full meter deflection will only take place when the unit is called upon to
deliver 600 watts.

The VC 600-dual can energize one converter at 600 watts or two converters at 300 watts each. When pro-

cessing two identical samples with two identical probes, the power delivered to each probe will be half that in-
dicated on the power monitor.

To demonstrate the above statement, perform the following: With a 2" (13mm), %" (19mm) or 1" (25mm) probe,
set OUTPUT CONTROL to maximum (to halfway point with a microtip). Depress the probe down against a piece
of wood. The greater the down pressure, and consequent greater resistance, the greater the

deflection on the POWER MONITOR.

OPERATING SUGGESTIONS AND TECHNIQUES

Cavitation induced by ultrasound has many advantages for cell disruption as compared with other methods, the
main advantage being that resistant organisms such as bacteria and yeasts, which have tough and resilient cell
walls, are broken relatively quickly. It has been established that micro-organisms differ greatly in their sensitivity to
ultrasonic disintegration. The most readily disintegrated for example are the rod-like forms (bacilli) while the
spherical organisms (cocci) are much more resistant. The group Mycobacteria, to which the tuberculosis organism

14