Method of killing microorganisms in the inside of a container utilizing a plasma initiated by a focused laser beam and sustained by an electromagnetic field

Method of killing microorganisms in the inside of a container which comprises directing an electromagnetic field into such container, inducing a plasma therein by focusing a single-pulsed, high-power laser beam into said field and exposing the inside of such container to said plasma for from about 1.0 millisecond to about 1.0 second by sustaining said plasma with said electromagnetic field.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to a method of killing microorganisms in the inside 
of a container. More particularly, this invention relates to a method in 
which the inside of such container is exposed to a plasma induced by 
focusing a high-power laser beam in an electromagnetic field. 
2. Prior Art 
Killing of microorganisms, which when carried to totality constitutes 
sterilization, in containers into which are filled such substances as 
parenteral and other medications, foods, beverages, dairy products, and 
the like, has been practised for decades for the purpose of preventing the 
transmission of disease. Many methods have been devised to accomplish this 
purpose. Heat, both dry and wet, has been a popular method of killing 
microorganisms in the food, beverage and pharmaceutical arts for a long 
time. The use of chemicals such as formaldehyde, phenol, ethanol, ethylene 
oxide, and the like for killing microorganisms has found many useful 
applications. More recently irradiation, such as beta, gamma, and 
ultraviolet rays have been employed in specialized applications for 
killing microorganisms. 
In 1968, U.S. Pat. No. 3,383,163 described a method of sterilizing the 
surface of a material which does not conduct electricity comprising 
contacting such surface with a gaseous plasma at an extremely high 
temperature. In this method a corona discharge was utilized to generate a 
plasma inside a container. The corona discharge was achieved by 
introducing a grounded electrode into the container, surrounding the 
container with a coil and pulsing from about 5000 to about 7000 volts and 
above into the coil. Exposure of the surface to a plasma for a very brief 
period of time, normally not longer than one-tenth of a second, is 
described. 
In the interval since U.S. Pat. No. 3,383,163 was issued, many attempts 
have been made to develop the plasma sterilization process into an 
economically feasible method because of the inherent advantage of killing 
and the microorganisms in the inside of a container just prior to filling. 
However, the mechanical problems associated with introducing a grounded 
electrode into a container and simultaneously surrounding the container 
with a high voltage coil have been found to be of such a magnitude as to 
defeat exploitation of the invention. Moreover, the volume of plasma 
generated by the corona discharge is dependent on the style and shielding 
of the electrode tip, the winding of the high voltage coil and the 
potential difference between said electrode and said coil at the moment of 
the pulsed discharge, and such requirements have presented problems in the 
location of the electrode and coil so as to fill the container with 
plasma. Furthermore, the voltage required to initiate the corona discharge 
is substantial and requires specialized electrical circuitry. 
U.S. Pat. No. 3,955,921 describes a novel method of killing microorganisms 
inside of a container by repeatedly sparking an ultra-short-pulsed laser 
beam in the inside of the container. Each spark resulted in an 
ultra-short-lived plasma. This method has the advantage of inducing the 
microorganism killing plasmas within the container without the need for 
the mechanical introduction of an element into the containers. Moreover, 
microorganisms were killed in the inside of the container without 
contacting the inside surfaces of the container with the plasma. However, 
the need for a succession of independently generated plasmas extended the 
time which each container had to be in position for the laser beam to be 
focused thereinto. 
Accordingly, it is an object of this invention to provide a method of 
killing microorganisms in the inside of a container with a continuous 
plasma that is induced by a single pulse of a focused high-power laser 
beam and is expanded and sustained inside such container for whatever 
interval is required to effect a complete killing of all microorganisms 
therein. 
SUMMARY 
It has now been discovered that a single pulse of a focused high-power 
laser beam in an electromagnetic field generated in the inside of a 
container immediately before the laser beam is pulsed therein will result 
in a plasma that can be sustained at will by maintaining sufficient energy 
in the electromagnetic field. The plasma is maintained for as long as it 
is needed to effect the desired degree of microorganism kill and is then 
stopped by extinguishing the electromagnetic field, or by moving the 
container out of the electromagnetic field. The duration of the plasma 
required for sterilization is a function of the power delivered to the 
plasma. 
DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The novel method of this invention of killing microorganisms in the inside 
of a container comprises generating an electromagnetic field in the inside 
of such container and pulsing a focused, high-power laser beam in such 
field inducing a plasma that is expanded and sustained by the energy in 
the electromagnetic field. Said plasma is achieved by focusing said beam 
to a point of convergence (focal point) inside of said container, and at a 
sufficient distance from the inside surface of said container to avoid a 
contact of said surface by said plasma at the instant of the pulse. 
The electromagnetic field on the inside of the container is achieved by 
positioning the container in an appropriate location where the 
electromagnetic field is concentrated or to which such field is guided or 
directed. Illustrative of such a positioning is the use of cavity at the 
end of a wave-guide tunnel in which there is an electromagnetic 
radiation-generating means; said cavity being tuned to concentrate the 
energy in the electromagnetic field at the locus of the container situated 
therein. Another illustration embraces the positioning of the container 
between the source of the electromagnetic radiation and a parabolic dish 
designed to reflect and concentrate the electromagnetic energy at the 
locus of the container. The mechanics of the dimension of the delivery 
means utilized to concentrate and direct the electromagnetic radiation are 
a function of the frequency of the generating means. Those skilled in the 
art will recognize this relationship. 
Electromagnetic radiation generating means can theoretically be provided to 
yield oscillating frequencies from very low frequencies (VLF) up through 
radio frequencies (rf) to gamma rays, but for the purposes of this 
invention the frequencies generally fall within the range of from about 
10.sup.5 to about 10.sup.16 hertz. Even so, from a practical consideration 
there are only a relatively small number of specific frequency bands in 
the electromagnetic radiation spectrum that have been allocated by the 
Federal Communications Commission (FCC) for industrial, scientific and 
medical use. Representative of these bands are: 13.36-14.00 megahertz 
(MHz), 27.23-27.28 MHz, 40.66-40.70 MHz, 0.915 gigahertz (GHz), 2.45 GHz 
and 22-22.125 GHz. The expressed limitations hereinabove described are FCC 
regulated limitations and do not otherwise constitute limitations 
attendant to the invention. Moreover, additional practical considerations 
involve the availability of electronic systems that limit the generation 
of electromagnetic fields to bands that are specific and narrow. No useful 
economic purpose is served by generating the electromagnetic fields at an 
infinite number of specific frequencies. Other electromagnetic energy 
bands useful in their invention are: 28.2 terahertz (THz), 282 THz and 431 
THz. Should additional bands be allocated to such purposes as are 
consistent with this invention, such additional bands will be equally 
operative within the 10.sup.5 to 10.sup.16 spectrum and will only require 
the adaptation of the mechanics of the delivery means to the additional 
frequency or frequencies; a procedure which is known to those skilled in 
the art. 
The electromagnetic radiation field is supplied with sufficient energy to 
expand and sustain the plasma once it has been initiated by the focused 
laser beam. For example, it was found that about 1.2 KW of energy output 
from a magnetron tube was needed to expand and sustain a laser 
beam-induced plasma in air in an electromagnetic radiation field having a 
frequency of 2.45 GHz. 
The maintenance of a plasma in argon at atmospheric pressure by means of 
electromagnetic energy requires a minimum field intensity. It was found 
that these figures were approximately the following: 
______________________________________ 
Power Input 
Field to 
Frequency Intensity Generating Means 
______________________________________ 
2.45 GHz 100 watts/cm.sup.2 
0.8 KW 
27 MHz 275 watts/cm.sup.2 
2.2 KW 
5.2 MHz 560 watts/cm.sup.2 
4.5 KW 
______________________________________ 
Additionally, there is a relationship between the energy in the 
electromagnetic field and the sterilizing capacity of the plasma. The 
greater the energy in the field, the greater the sterilizing capacity of 
the plasma and the shorter the exposure time required to effect 
sterilization. For example, it was found that at a frequency of 2.45 GHz 
it required about 1.2 second to achieve a one-decade, or 90%, kill of B. 
subtilis spores in a conventional 10 ml. ampoule when the power absorbed 
by the plasma was about 0.2 KW. The same kill rate ws achieved in 0.3 
seconds when the power absorption was about 0.5 KW. Similar ampoules were 
sterilized in 0.9 seconds by a 0.5 KW plasma. It was calculated that with 
1000 and 2000 watts, respectively, in the plasma, sterilization would be 
accomplished in 0.1 and 0.02 seconds, respectively. However, there is a 
mechanical and economic limit to the application of high energy to the 
electromagnetic field. Too much energy in the field will result in damage 
to the material with which the container is made by direct absorption of 
electromagnetic energy. Moreover, there is no economic benefit from the 
use of such energy that results in a rate of sterilization that is 
inconsistent with the need for rapid but not excessively rapid 
sterilization. 
It is also to be noted that the lower the frequency the greater the energy 
needs to sustain the plasma. Illustratively, frequencies of 13.36-14.00 
et. seq. MHz, and 0.915 GHz would require a greater energy input to 
sustain and/or expand the laser beam-induced plasma, and the 22-22.125 GHz 
frequency needs less energy input to accomplish the same phenomenon than 
the 2.45 GHz frequency. 
In a preferred embodiment of this useful invention, a mechanical system 
comprising a wave-guide tunnel with an electromagnetic radiation 
generating means, such as a magnetron tube positioned therein and a cavity 
defined at one end thereof for the positioning therein of the container to 
be sterilized is constructed of an electrical conducting material, such as 
12 gauge steel plate which is opaque to electromagnetic radiation at these 
frequencies, confines the field to the dimensions of the wave-guide tunnel 
and prevents the radiation energy from escaping into space. The cavity is 
tuned to the frequency of the electromagnetic field by means well known to 
those skilled in the art. It was found that a bi-modal cavity, at the 
least, was required for the efficient concentration of the electromagnetic 
radiation energy at the locus of the container positioned therein. At 
least two modes are required to accomodate the change in resonance as the 
plasma is ignited and expanded. A n-modal cavity will provide even greater 
efficiency. 
The wave-guide tunnel is also tuned in volume to the frequency of the 
electromagnetic radiation. The lower the frequency the greater the 
cross-sectional area reguired. Such a guide is essential to concentrate 
and direct the electromagnetic radiation into the area in which the 
container to be sterilized is positioned. An internally located heat 
transfer means, such as a three part circulator with a water load, was 
required in the wave-guide tunnel to dissipate the heat of the unused 
reflected energy. 
Inasmuch as the novel method of this invention contemplates a usefulness 
over a wide range of container sizes, from small vials employed for 
parenteral medications up to large food containers such as gallon jars, it 
can be seen that a single set of dimensions for a wave-guide tunnel and 
cavity are not appropriate as the physical size of the container will 
limit the dimension of such a tunnel and cavity. While a small container 
can be positioned in a cavity having a large volume, such would constitute 
an uneconomic arrangement inasmuch as the lower frequency radiations 
associated with large cross-sectional tunnels require a higher energy 
input to sustain the plasma for a sterilizing period. Consequently, the 
total installation should be considered in relation to the highest 
radiation frequency, requiring the lowest energy input, that is 
appropriate for the size of the container to be sterilized. 
Many materials of which containers are made such as glass, plastics in 
general, ceramic ware and the like, are transparent to electromagnetic 
radiation; and for these it is only necessary that they be positioned in 
the electromagnetic field. The electromagnetic radiation will fill the 
container spontaneously as it is generated. Other container materials such 
as steel, aluminum, copper and other conducting metals, and the like, are 
opaque to electromagnetic radiation; and in such an event the cavity at 
the end of the wave-guide tunnel must be so designed that the container is 
actually a part of the mechanics of the cavity, and the container must 
have an opening therein which is transparent to electromagnetic radiation 
to permit such radiation to enter and fill the container. Those skilled in 
the art will understand such needs as being integral with the design of 
the cavity. 
The electromagnetic field utilized in the preferred embodiment of the 
useful method of the instant invention can be either a pulsed or a CW 
field. When a 2.45 GHz magnetron tube was employed in the wave-guide 
tunnel the field was pulsed 120 times a second. Such a field is suitable 
for the propagation of a plasma and the sustaining thereof when sufficient 
energy is fed thereinto. Inasmuch as the pulse of the laser beam which 
initiates the plasma is measured in nanoseconds, which is described 
hereinafter, it is essential that the laser pulse shall be fired before 
the pulse of the electromagnetic field moves much past peak field 
strength; preferably the laser pulse occurs just before the 
electromagnetic field pulse reaches peak field strength. If the laser 
pulse is fired too early in the ascendancy of the energy in each 
individual pulse of the electromagnetic field, there will not be efficient 
coupling of the energy in the laser individual plasma and the 
electromagnetic field; and, consequently, the plasma will not be 
sustained. A similar phenomenon occurs when the laser is pulsed too far 
after the electromagnetic pulse has reached peak field strength. It is 
also to be noted that as the energy in each electromagnetic pulse is 
increased, the interval during which the laser can be pulsed to effect a 
coupling of the energies to sustain the plasma is increased. 
When a CW electromagnetic field is used in the novel method of this 
invention the electrical circuitry provides for the ascendancy of properly 
phased following pulses to intersect with the descendency of a preceding 
pulse creating a continuous wave of energy, although such a CW field 
exhibits an undulating energy level. When the CW field is fed sufficient 
energy that at the point of intersection between following and preceding 
pulses a plasma sustaining intensity of energy is present, it is of no 
consequence when the laser is pulsed. 
The focused, high-power laser beam, employed in the novel method of this 
invention to initiate the plasma, was achieved by Q-switching or 
mode-locking a laser beam, each pulse thereof having a duration of from 
about one-tenth to about 300 nanoseconds. The mechanics and methods of 
Q-switching and mode-locking laser beams to produce pulsed beams of short 
duration are old and well known to those skilled in the art, and 
constitute no part of the instant invention. In general, a high-power 
laser beam is one that contains megawatts of power. 
The mechanics and methods of generating a plasma by focusing a high-power 
laser beam at a focal point where the cone of said beam converges are also 
well known to those skilled in the art. Optics appropriate to the wave 
length are employed in focusing a high-power laser beam. The focal point 
of the cone of the high-power laser beam must be sufficiently short to 
assure the generation of a spark on each pulse. Such focal point is a 
function of the energy in said beam; there being a direct relationship 
between the beam energy and the maximum focal point distance which 
unfailingly achieves a plasma with each pulse. The maximum focal length 
can be increased by increasing the energy in the beam. 
The instant invention does not depend on the energy in the beam, but rather 
the generation of a plasma at the focal point. Any high-power laser beam 
which will form plasma at its focal point when such focal point is located 
within the inside of a container will effectively initiate a plasma in the 
inside of said container when said container is filled with an 
electromagnetic field. It will be understood that it is a function of the 
mechanics of an installation in which the inside of containers are 
sterilized to coordinate the positioning of the optics which are employed 
to focus the high-power laser beam with the beam energy which will produce 
a plasma at the focal point of said beam located inside of said 
containers. 
A laser beam can be focused through a material that is optically clear and 
which does not appreciably distort the converging cone of the beam. So it 
is possible to accomplish the killing of microorganisms on the inside of a 
container in which there is no opening, or one in which the opening is 
insufficient for focusing a laser beam therethrough by focusing a laser 
beam through the material of which the container is made, if such material 
is also transparent to an electromagnetic field so that such a field is 
present in the inside of the container when the laser beam is focused 
therein. However, the greater number of the containers in which 
microorganisms will be killed by the useful method of this invention will 
be made of materials which do not meet the criteria noted above. 
Consequently, it is preferable that the containers, in which 
microorganisms will be killed by the novel process of the present 
invention, have an opening therein, and that the high-power laser beam be 
focused through such opening. 
Consequently, the size of the opening in the container must be considered 
in designing a beam and the focusing thereof as a partial distortion of 
the converging sides of the cone of said focused beam by a contact with 
the material of which the container is made can corrupt said beam and 
interrupt the sparking thereof. 
In the instant invention "plasma" defines a highly or essentially 
completely ionized body of gas which is composed of positively charge 
nuclei and negatively charged electrons, and exists at an extremely high 
temperature, perhaps approaching that of the sun. The life of the plasma 
of a focused, high-power laser beam is of exceptionally short duration, 
being in the neighborhood of from about 5 nanoseconds to about 5 
microseconds longer than the laser pulse which initiates and sustains it. 
While the exact mechanism by which the plasma initiated by a focused, 
high-power laser beam and expanded and sustained by electromagnetic 
radiation energy accomplishes the killing of microorganisms in the inside 
of a container in which said plasma is induced is not known, it is known 
that it is not necessary that the inside surfaces of said container be 
contacted by said plasma. 
The plasma which is the result of the ionization of the gas inside said 
container by the focused, high-power laser beam can be formed from many 
ionizable gases. Air, comprised of nitrogen and oxygen, will form a 
plasma. Other ionizable diatomic gases, such as the halogens, will form 
plasmas, however, the preferred gases for plasma formation are monatomic 
gases such as argon, helium, xenon, neon, and the like. Irrespective of 
the gas utilized, the firing of a focused, high-power laser beam within 
the body of said gas induces a plasma which is expanded and/or sustained 
by the energy in the electromagnetic field. 
In a preferred embodiment of the instant invention, a monatomic gas is 
introduced into the container, in which microorganisms are to be killed, 
prior to the generation of a plasma in said container. The monatomic gases 
are easier to ionize then oxygen or nitrogen; consequently, less energy is 
required to generate a plasma. An especially preferred embodiment 
constitutes the introduction of argon into the container prior to the 
generation of a plasma therein because such gas is plentiful and economic, 
and the residue thereof is limited to neutral argon. 
Furthermore, plasmas can be formed from ionizable gases when the pressure 
within said container, wherein said focused, high-power laser plasma is 
generated, is other than atmospheric. The pressure can be either sub- or 
superatmospheric. And again, regardless of the pressure of the ionizable 
gas, the pulsing of a focused, high-power laser beam can be adjusted to 
initiate a plasma, which in turn is effective in killing microorganisms in 
the inside of a container, when such plasma is generated therein. 
Moreover, the electromagnetic field in the inside of said container is 
effective in expanding and sustaining the plasma regardless of the 
identity of the gas or the pressure in the container, with the energy 
levels varying with such condition. Such levels being well known to those 
skilled in the art. 
The key to the useful method of this invention lies in combining the 
pulsing of a focused, high-power laser beam in the inside of a container 
with the presence of an electromagnetic field within such container, said 
field having sufficient energy therein to sustain the plasma induced by 
the laser beam spark for a duration of from about 1.0 millisecond to about 
1.0 second to effect a complete killing of the microorganisms in said 
container. The total elapsed duration of the plasma required to achieve 
the killing of microorganisms in the inside of a container wherein said 
plasma is initiated by a focused, high-power laser beam and sustained by 
an electromagnetic field varies with the energy absorbed in the plasma. 
The electrical properties of the material of which the container is 
constructed are of importance only with respect to the mechanical design 
of the apparatus which directs and concentrates the electromagnetic 
radiation energy in the container, as the plasma is generated by forces 
entirely within the confines of the container. It is preferred that the 
total accumulated exposure to the plasma be held to the minimum consistent 
with the total killing of the microorganisms in the inside of the 
container. Moreover, inasmuch as some aspects of the laser spark can be 
likened to a sonic boom, it is imperative that the focal point of the 
high-power laser beam be located at a sufficient distance from any point 
or part of the inside surface of said container to avoid the contacting of 
said inside surface by the plasma initiated by the laser beam pulse. 
Typical containers in which the microorganisms present therein can be 
killed by plasmas initiated by pulsing a focused, high-power laser beam 
inside thereof, and sustaining such plasma by an electromagnetic field, 
are ampoules and vials used for parenteral and other medications, beverage 
bottles and cans such as those used for soft drinks, beer and ale, orange 
and lemon concentrates, and the like, milk bottles and cartons, baby food 
jars and cans and canned food containers, and the like.

This invention is further illustrated by the following example. 
EXAMPLE 1 
This experiment was run to determine the effect of a plasma generated on 
the inside of a container by pulsing a focused high-power laser beam in an 
electromagnetic radiation field on the bacterial count within said 
container. 
Nine 10 ml. sterile vials having a 0.5 inch neck opening were each 
inoculated with about 125 spores of Bacillus subtilis. The B. subtilis was 
suspended in water. After inoculation, each vial was swirled to distribute 
the B. subtilis suspension on the inside surface thereof, and the vials 
were freeze dried. The dried inoculated vials were then closed with 
sterile rubber stoppers. For uninoculated sterile vials were also 
stoppered and utilized as controls. 
A tuned delivery means comprising a cavity at the end of an appropriate 
wave-guide tunnel with a 2.45 GHz magnetron tube in position therein was 
equipped with a receptacle for the positioning of the vials and a focused 
high-power laser was located at an appropriate place so that a beam 
therefrom would be focused through the opening in the vial to converge at 
a point within the inside of the vial. Electrical circuitry was installed 
to fire the laser about 1.1 millisecond following the initiation of 120 Hz 
pulses from the nagnetron tube. A means for purging the vials with argon 
gas prior to the generation of the plasma was provided. 
In turn each of the nine B. subtilis contaminated vials and the four 
sterile vials were treated as follows: 
Two of the sterile vials were left unopened at the work area. 
Two of the sterile vials were unstoppered, positioned in the delivery 
means, purged with argon, restoppered and left at the work area. 
Two of the contaminated vials were left unopened at the work area. 
Two of the contaminated vials were unstoppered, placed in the delivery 
means, purged with argon, restoppered and left at the work area. 
Three of the contaminated vials were unstoppered, placed in the delivery 
means, purged with argon, exposed to the electromagnetic field and an 
uncoupled pulse from the laser beam which did not yield a sustained 
plasma, restoppered and left at the work area. 
Two of the contaminated vials were unstoppered, placed in the delivery 
means, purged with argon, exposed to a plasma initiated by a pulse from a 
focused high-power laser beam and sustained and expanded for about 1.0 
second by a 2.45 gHz electromagnetic field providing a power absorption of 
about 0.2 KW, restoppered and left at the work area. 
All 13 of the vials were submitted for microbiological testing with the 
results shown in Table 1 below. 
Table 1 
__________________________________________________________________________ 
Contaminated Ampoules 
Unstoppered, 
Argon Swept, 
Unstoppered, 
Exposed to 
Argon Swept, 
Sterile Controls Microwave & 
Exposed 
Unstoppered, Unstoppered, 
Laser Spark, 
to 
Ampoule Argon Swept, Argon Swept, 
But Not Plasma, 
Plasma, 
Treatment Unopened 
Restoppered 
Unopened 
Restoppered 
Restoppered 
Restoppered 
__________________________________________________________________________ 
Final Condition 
(spores/ampoule) 
Sterile 
Sterile 113 + 6 115 .+-. 50 
22 .+-. 5 Sterile 
Actual &lt;3 &lt;3 119 65 20 &lt;3 
Microbiology 
&lt;3 &lt;3 107 165 29 
Report 17 
__________________________________________________________________________ 
The microbiology report "&lt;3" is a reflection of the aliquoting process in 
which the vial is washed thoroughly, one-third of the wash is cultured, 
and the result is multiplied by 3. Thus, a completely sterile ampoule is 
reported "&lt;3." 
The data in Table 1 indicate the effectiveness of a plasma induced by a 
single pulsed high power laser beam and sustained by electromagnetic 
radiation energy in killing microorganisms in the inside of a container 
wherein such plasma is generated and sustained.