Liquid phase disinfection/sterilization with microwave energy

Dental, medical and laboratory instruments may be disinfected and/or sterilized by exposure to a biocidal solution composed of hydrogen peroxide and a weak acid in the presence of microwave irradiation. The process typically occurs at low temperature, generally less than 65.degree. C. In a preferred embodiment, the contaminated instruments are placed in a microwave transparent vessel containing the biocidal solution. A lid is placed over the vessel and the vessel is then placed into a cavity of a microwave oven and irradiated for a very short period of time. At the end of irradiation, instruments are safely and aseptically removed from the cooled container.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to a process for fast high level 
disinfection/serialization of dental, medical or laboratory instruments in 
liquids through a synergistic combination of microwave irradiation with a 
sterilant/disinfectant containing hydrogen peroxide and weak organic acid. 
2. Description of the Prior Art 
In the past, several unsuccessful attempts have been made to decontaminate 
dental instruments within microwave ovens at a frequency of about 2450 
MHZ. Metal instruments were quickly overheated. Further, intense microwave 
fields created localized arcing which pit metal surfaces. Although this 
could be somewhat minimized by proper humidification, instruments having 
different dielectric characteristics reach widely different temperatures 
even when irradiated with the same exposure time. Therefore, it is 
impractical to process metal tools in the presence of other instruments 
containing plastics or rubber type components. 
In the early eighties, there was a revival of the use of microwave fields 
as a means to create gas plasma with biocidal characteristics. A gas 
plasma is a highly ionized body of gas which is created, for instance, in 
an enclosed chamber under vacuum using radio frequency (RF) or microwave 
energy. These types of plasma are classified as low energy plasma and are 
called non equilibrium or glow discharge gas plasmas. Vacuum pressure is 
also an important variable. The deeper the vacuum, the greater the energy 
and reactivity of the plasma. 
U.S. Pat. No. 3,753,651 discloses an experimental set up to gas plasma 
sterilize instruments. This patent describes a chamber transparent to 
radiation which may be inserted into the microwave oven cavity. The 
chamber further contains both the sterilant gas (or vapor) and the 
instruments to be decontaminated. Other approaches are mentioned in U.S. 
Pat. No. 3,948,601 and U.S. Pat. No. 3,968,248 which describe continuous 
or batch sterilization within a highly reactive plasma atmosphere. 
Industrial sterilization systems based upon the principles described in 
these patents have been successfully developed using hydrogen peroxide 
(STERRAD) or peracetic acid (PLAZLYTE) as the active vaporized agents in 
the gas plasma. Unfortunately these industrial processes are rather 
complicated because they require creation and maintenance of a uniform 
stable gas plasma through vacuum control while also accurately injecting 
various amounts of gas sterilants. 
A small reliable and inexpensive dental instrument sterilizer cannot be 
based upon the sophisticated designs developed for gas plasma industrial 
or hospital systems. In the late 1980s, attempts to build dental 
sterilizers based upon gas plasma technology wherein traces of biocidal 
agents were injected into the gas phase, were reported. See, for instance, 
"Science Watch", June 20, 1989, New York Times. However no prototype or 
equipment ever reached the market despite promising news releases. One 
obvious reason was that to achieve sterilization according to FDA 
requirements (6 logs kill of spores) the best large size hospital plasma 
units required a contact time of 4 hours (PLAZLYTE with peracetic acid) or 
90 minutes (STERRAD with hydrogen peroxide). 
Another reason for the failure to develop a gas plasma sterilizer for 
dental instruments has been cost. Gas plasma sterilization is an expensive 
and delicate technology due to the difficult stabilization of plasma 
gases. Attempts to scale down such systems have failed. Inexpensive 
systems are needed which are capable of achieving sterilization or high 
level disinfection within a very short contact time with a minimum risk of 
corrosion for dental tools. 
SUMMARY OF THE INVENTION 
Disinfection and/or sterilization proceeds by liquid chemical sterilization 
with microwave energy versus gas plasma. Liquid chemical sterilization 
with microwaves does not deal with reactions in gas but instead in liquid 
phase irradiated by microwaves. The disinfection and/or sterilization of 
medical, dental or other scientific instruments in accordance with the 
invention may proceed by exposure of the instrument to a biocidal solution 
comprising hydrogen peroxide and a weak acid. The instruments are further 
exposed to the biocidal solution in the presence of microwave irradiation 
at a temperature less than or equal to 65.degree. C. 
The contaminated instruments are placed in a vessel containing the biocidal 
solution. The vessel may then be placed into a microwave oven and 
irradiated for a short period of time. 
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The method of invention provides a quick and efficacious means of combining 
the action of microwave irradiation with two inexpensive chemical agents. 
As defined by the EPA as well as the FDA, high level disinfection consists 
of substantial destruction of bacterial endospores while achieving 
complete kill of the most resistant vegetative organisms, such as 
Mycobacterium tuberculosis var. bovis. 
The method of the invention is of particular significance in dental and 
doctor offices which are typically small areas. In such offices, it is 
typically necessary for the disinfection/sterilization units to be capable 
of handling quick turnover of instruments. In addition, biocidal solutions 
employed in such areas need to be odorless. 
In accordance with the invention, medical, dental or laboratory 
instruments, generally metallic in nature, are submerged in the biocidal 
solution. Alternatively, the biocidal solution may be added to the vessel 
containing the contaminated instrument. The vessel containing the biocidal 
solution and instrument(s) is then placed within a microwave energy 
source, such as a microwave oven. Thus, there is no "arcing" between the 
walls of the microwave energy source and the instruments. Arcing only 
occurs when metals are directly exposed to the microwave energy source. 
The instrument may be one used in a hospital, veterinary establishment, 
dental office, or medical doctor's office, coroner's office and/or 
laboratory or other establishment which requires the use of sterilized or 
bacteria-free instruments. 
The biocidal solution employed in the invention is composed of hydrogen 
peroxide and a weak organic acid. Suitable weak organic acids are those 
having a pKa greater than or equal to 4. Such acids include acetic acid, 
citric acid and lactic acid. Typically, the hydrogen peroxide employed in 
the invention is between 8 to about 11, most typically 10, weight percent. 
The peroxide solution is diluted down to about 6 or about 7.5 weight 
percent with an aqueous solution of the weak organic acid. 
While the weak organic acids or their salts are not the active ingredients 
in the biocidal composition of the invention, they capture some oxygen 
from the hydrogen peroxide molecule. The captured oxygen enters into a 
--OOH group which is either very reactive per se or later releases a 
highly reactive oxygen atom. Thus, the improved biocidal efficacy observed 
in the biocidal composition of the invention is the result of synergistic 
biocidal mechanisms created by combining microwave energy, heat and 
oxidizing chemical reactions. 
The composition of the invention may further contain a non ionic 
surfactant. Suitable non ionic surfactants are ethoxylates of isomeric 
linear alcohols such as TERGITOL from Union Carbide, TRITON from Rohm and 
Haas Co., PLURONIC from BASF Wyandotte Corporation and alkali metal 
alkylaryl sulfates and sulfonates. 
The biocidal composition may further contain a stabilizer such as 
methylparaben or acetominophen. Generally less than 1 weight percent of 
stabilizer is employed. 
In addition, the pH of the biocidal solution is between about 1 to about 7. 
Suitable buffering agents include alkali metal carbonates, bicarbonates, 
phosphoric acid, phosphates and borates, carboxylic acids, piperazine, 
sulfonic acid, organic carboxylate salts, and mixtures thereof. Preferred 
are monobasic potassium or sodium phosphate with anhydrous dibasic sodium 
phosphate, phosphoric acid, mono or disodium phosphate, maleic acid, 
trisodium citric acid, citrate-phosphate buffer, succinic acid, cacodylate 
buffer, n-(2-acetamido) iminodiacetic acid, piperazine-n, n.sup.1 -bis 
(2-ethane sulfonic acid), and 2 (n-morpholino) ethane sulfonic acid. 
Particularly preferred are monobasic potassium, sodium phosphate and 
anhydrous dibasic sodium phosphate and mixtures thereof. 
Further, the composition may contain an anticorrosive agent such as 
benzotriazole, tolytriazole, sodium molybdate or benzoate. 
In a preferred embodiment, the hydrogen peroxide solution is poured into 
the container containing the contaminated instruments. The weak organic 
acid is then added to the hydrogen peroxide solution. This biocidal 
solution renders unnecessary the use of low concentration glutaraldehyde 
solutions which often display satisfactory sporicidal activity under 
microwave excitation but release toxic glutaraldehyde vapor at the end of 
the processing period once the lid of the vessel is opened and the 
instruments are removed. Contrary to glutaraldehyde based sterilizing 
solutions, hydrogen peroxide formulations provide fast cidal action 
without the release of toxic vapors at the end of processing (when 
removing the disinfected instruments from the irradiated solutions). 
With microwave irradiation, the biocidal composition of the invention 
releases only small amounts of non toxic organic acid, hydrogen peroxide, 
water and oxygen - none of which are dangerous from the environmental view 
point. 
The container for use in the invention may be any container which is safe 
and transparent to microwave energy including those containers of 
appropriate shape made of glass ceramic (CORNINGWARE tin, PYROCERAM, etc), 
heat resistant glass (PYREX), microwave safe plastics (typically those 
used for an autoclave) as well as pottery, stoneware, porcelain or even 
paper. Preferred are FLASHPAK.RTM. and the Rubbermaid SERVIN SAVER plastic 
container since if the container is overheated the lid rim is designed to 
quickly release excess steam due to sudden increases in vapor pressure. 
(This is not an issue if disinfection is conducted at an end temperature 
of the biocidal composition lower than 65.degree. C.) 
In a preferred embodiment, the instruments to be disinfected/sterilized may 
be placed on a tray which is, in turn, inserted into the vessel. The tray 
most desirably has openings--such as horizontal slits of approximately 2 
to 5 mm in diameter--to increase the flow of biocidal solution to the 
contaminated instruments. Alternatively, the tray may be meshed having 
openings of about 2 to about 5 mm. 
The method of the invention further provides an ultrafast means of 
disinfection unlike conventional means of heating (conduction, convection, 
etc.) which create a lot of drawbacks from the corrosion view point. 
The contaminated instrument(s) is exposed to microwave irradiation and the 
biocidal composition for a time and at a temperature sufficient to 
disinfect or sterilize the instrument depending upon the objective of the 
one performing the operation. 
Conventional heat does not have the same effect as microwave heat on 
microorganisms and their surroundings on account of fundamental 
differences between conventional thermal energy and microwave energy. 
Microwave energy is coherent electromagnetic energy. In other words, it is 
ordered. As such, its characteristics may readily be identified and 
controlled with precision. Thermal energy, on the other hand, has random 
disordered characteristics which are not so easily controlled. 
Although the term microwave, in general, may cover a rather wide range of 
frequencies (from 100 MHZ up to several hundred thousand MHZ), in a 
preferred embodiment of the invention the frequency is between about 100 
to about 23,000 MHZ. Microwave oven outputs are typically rated according 
to the International Electrotechnical Commission (IEG) 705 test procedure 
in compliance with the standards set by the FCC. Most commercial ovens are 
rated with outputs between about 500 and about 900 watts at a frequency of 
2450 MHZ. The minimum average power density should be at least 0.01 
W/cm.sup.3. 
In an alternative embodiment, the microwave irradiation may be a continuous 
or pulsed wave emission having a repetition rate of the order of between 
about one per nanosecond to about one per minute. 
The mechanism through which the microwave heating occurs at such 
frequencies is based upon the dipole moment or "polarization" of the 
molecules of the irradiated substance. When the polar molecules (absorbed 
water in cellular organisms for instance) are subjected to a strong 
alternating field, their rapid reorientations within the field create 
internal friction resulting in heat. Microwave transfer of energy takes 
place directly without the necessity of an intermediate medium such as a 
hot surface or a high temperature air stream. Energy transfer occurs 
wherever the field penetrates. No contact with the substance itself is 
required. Microwave heating eliminates the inherent inefficiency of 
transferring heat from an external source to the processed loan. Since 
microwave energy can be switched on to full power levels and off again by 
simply flipping a switch, the time lags associated with thermal processes 
is eliminated. This, indeed, is extremely important for instance to a 
dentist because it shows that he does not need to maintain a permanent 
heating system which continuously will create toxic vapors (case of 
glutaraldehyde) when introducing equipment or removing it from the 
sterilant container. 
While thermal death of bacterial cells and spores is generally logarithmic, 
sigmoidal curves is not uncommon. The theory called "The Distribution of 
Resistance" points out the existence of non-uniform heat resistant spores. 
When applying heat to a microorganisms population (spores, for instance) 
deviation from the logarithmic nature of the survival curve is generally 
attributed to two basic factors: (1) The presence of a hump or "lag" in 
the initial portion of the survival curve due to heat "activation;" and 
(2) the presence of a tailing of the final portion due to the presence of 
the more resistant variants in the population. 
An energy of "activation" is generally necessary to initiate a chemical or 
biological process. In the case of spores, it is the energy necessary to 
release spores from their dormant state to begin their germination 
process. There is also an activation energy requirement to inactivate 
(lethal effect) microorganisms. Heat activation and inactivation both obey 
first order kinetics in combination and in that order. The moment a spore 
becomes activated it is subjected to the inactivation law. The effect of 
heat on microorganisms is the result of enzyme inactivation, proteins 
denaturation or both. This is, in other words, the integer of several 
complex phenomena. The type of heat flux (dry or moist), the manner into 
which the heat is generated or penetrates through the microorganisms 
(convection, conduction, radiation, etc) are, therefore, extremely 
important. This, indeed, could explain the faster killing rates observed 
with microwave energy which acts at once at molecular level and creates a 
coherent state of molecular turbulence throughout the entire irradiated 
mass. 
Two other phenomena may also contribute to an explanation of the faster 
microwave cidal action on microorganisms present on the surface of 
contaminated instruments or in the liquid sterilant. The first phenomenon 
is the interaction of intense microwaves with the molecular structure of 
the liquid disinfectants. Several overlapping phenomena have been observed 
such as: high speed molecular oscillations which produce chemical bonds 
breaking, accelerated diffusion of ions through membranes, electrical 
charges modification at interfaces or even pH variations. The second 
phenomenon recognizes that microwaves may affect a metabolic system 
distinct from that of thermal energy. 
Studies in soil sterilization indicated that a large soil fungus 
(Rhizoctonia solani) was killed at a temperature about 10.degree. C. below 
that of its normal thermal death point. Another fungus (Verticillium 
albo-atrum) with extremely small spores was killed at about 3.degree. C. 
below its lethal temperature. Non-spore forming bacteria are, in general, 
killed by microwave energy at points as much as 10.degree. C. below 
thermal death point. 
Regarding chemical bond cleavage, a few seconds irradiation at 2450 MHZ of 
0.1 N solutions of NaOH produces hydrogen peroxide at a rate of about 
0.01% every five seconds. The temperature at the end of three 10 second 
(i.e. 30 seconds) exposures is about 100.degree. C. When similar samples 
are treated in a water bath to the same temperature, no hydrogen peroxide 
is detected with the UV absorption technique. This, among other things, 
demonstrates that the result of chemical bond breakage leads to the 
production of new chemicals or radicals with sporicidal or bactericidal 
characteristics. 
Another important advantage of the present invention is the simplicity of 
the process which enables the dentist, doctor or technician to safely load 
contaminated instruments into the vessel. The vessel is typically 
transparent to microwave irradiation. The vessel is covered with a lid. 
The loaded vessel with its lid is then placed into a microwave oven cavity 
and it is irradiated during a few minutes. In a preferred embodiment, a 
switch is used to activate the irradiation for the time chosen by the 
experimenter for high level disinfection. Following the conclusion of 
irradiation, the vessel is cooled down a few minutes before the lid is 
open for safe aseptic removal of the instruments. Where a tray is used, 
the tray may be slightly shaken to rid it of the liquids which may have 
remained on the surfaces of the instruments prior to removal. The whole 
operation takes only a few minutes and there is no release of toxic vapors 
into the atmosphere. The biocidal solution in the vessel may be reused as 
long as the peroxide content is in excess of about 6 weight percent. 
The process of the invention is further normally conducted at temperatures 
not to exceed 65.degree. C., typically between about 54 to about 
65.degree. C. Such temperatures do not create problems with high vapor 
pressure in the irradiated vessel. 
The invention has particular applicability in the dental field since 10% 
hydrogen peroxide is available at corner drugstores since it is typically 
used in hair bleaching, etc. The hydrogen peroxide may be poured into a 
container transparent to microwaves. A mark may then be made on the 
plastic to indicate a the upper level of the solution of peroxide to be 
poured into the container after positioning the instruments to be 
decontaminated. A second level mark may be made to indicate the amount of 
a weak acid, such as 5% commercial vinegar) to be added to the container. 
The lid is placed into the container and the container is then placed into 
the microwave. The microwave is operated at approximately 2450 MHZ. 
The process of the invention proceeds at a faster rate than autoclaving. In 
the process of the invention, the container is radiated slightly more than 
0.01 watt/cm.sup.3 in the loaded cavity. Disinfection of the instruments 
occurs in about 3 to about 5 minutes at a temperature less than 65.degree. 
C. No additional time is needed for the removal of sporicidal activity. 
This is in contrast to an average 20 minute contact time with steam 
sterilization in an autoclave and a ten hour contact time for liquid 
sterilants, for instance CIDEX. The loaded container may then be removed 
from the cavity, cooled and opened. Decontaminated residues, with toxic 
residuals removed, may then be aseptically removed. 
In light of the predictability in the requisite time for decontaminating 
the instrument, the microwave energy source may be set on a timer, such as 
by the sound of an automatic acoustic signal on a microwave oven. 
The invention may further be employed to sterilize medical and dental 
instruments. Sterilization can be achieved in about 8 to about 12 minutes, 
typically around 10 minutes. This is in sharp contrast to the 10 hours 
needed presently with glutaraldehyde containing solutions.

EXAMPLES 
Example 1 
A biocidal solution was prepared by adding a solution of hydrogen peroxide 
into a container. A weak organic acid, such as acetic acid, was then added 
to the container containing the hydrogen peroxide. The temperature in the 
liquid phase was permitted to increase until approximately 55 to about 
60.degree. C. The exposure time was determined by the volume of liquid 
being irradiated. Table 1, for instance, illustrates that 8 minutes of 
irradiation time is needed for a 2000 cc container, 3 minutes for a 750 cc 
container and a little less than one minute for a 250 cc container. Adding 
metal instruments into the irradiated solution slightly decreased the 
exposure time. 
The operating parameters were as follows: 
Microwave unit: Dualwave 2-Microsystem General Electric 
Power Rating: 700 watts frequency 2450 MHZ Q25 MHZ) 
Fresh tap water: Starting temperature 26.7.degree. C. 
Volume of irradiated liquid: 250 cc (1 quart vessel), 750 cc (2 quarts 
vessel) and 2000 cc (1 gallon vessel) 
Results are illustrated in Table 1: 
TABLE 1 
______________________________________ 
Irradiation 
End-Temperature in 3 vessels containing: 
in min. 
250 cc 750 cc 2000 cc 
______________________________________ 
0.5 42.2 (108.degree. F.) 
1.0 58.9 (138.degree. F.) 
35 (95.degree. F.) 
1.5 70.0 (15.degree. F.) 
2.0 84.4 (184.degree. F.) 
45 (13.degree. F.) 
2.5 95.0 (208.degree. F.) 
3.0 55.6 (132.degree. F.) 
4.0 63.9 (147.degree. F.) 
5.0 78.3 (173.degree. F.) 
54.4 (130.degree. F.) 
6.0 85.0 (185.degree. F.) 
8.0 65.6 (150.degree. F.) 
11 77.2 (171.degree. F.) 
14 83.3 (182.degree. F.) 
______________________________________ 
The biocidal composition of the invention fulfills high level disinfection 
in a few minutes (2 to 5 min) without the creation of noxious vapors in 
the atmosphere at the end of processing (when the lid is removed), 
Example 2 
Tests were conducted with a General Electric Dualwave 2-microsystem whose 
cavity had the following dimensions: height: 11 inch, width: 16 inch, 
depth: 131/2 inch. Magnetron was emitted at a nominal frequency of 2450 
MHZ (.+-.25 MHZ). The AC line voltage was single phase 120 V, 60 HZ. The 
AC power input to the Magnetron was 1186 Watts and the average power 
microwave output in the cavity (1.4 CF) was approximately 700 Watts. 
Inside the cavity was placed a Rubbermaid microwave container (11" L, 61/2" 
W and 21/2" H) rated as a 2 quarts unit (1.9 liter) and having a capacity 
of 750 cm.sup.3 for the biocidal composition. 
Biocidal composition A was made of two active chemicals: a hydrogen 
peroxide solution (10%) which was poured first into a container and 
diluted with vinegar of 5 weight percent acidity. The final content in 
hydrogen peroxide was between 6 and 7.5 weight percent. The following 
dental tools were then submerged into the liquid: one plastic filling WI 
by Henry Schein, a 5 DE Explorer by HU-Friedy, one SE CS mirror handle by 
HU-Friedy and a front surface mirror from the same company. Between the 
instruments and inside the liquid disinfectant were placed ten SPORDEX 
bacterial spore strips from American Sterilizer. The Bacillus subtilis 
population of each strip averaged about 100,000. These strips are 
currently being used for checking ETO sterilization in gas phase but they 
have also been accepted by the FDA for liquid chemical sterilization 
monitoring. Since each package of SPORDEX contained several strips, half 
of them were used for the tests and the other half kept as a control. 
The microwave surface sterilizer was turned on and kept running for about 3 
minutes to reach an end temperature close to 60.degree. C. The container 
lid was then opened. The dental tools and spore strips were removed under 
sterile conditions. The strips were individually placed in labeled test 
tubes, each containing 25 cm.sup.3 of sterile fluid thioglycollate medium 
(FTM). The control strips left unsterilized were also placed into test 
tubes containing the same medium. B. subtilis was incubated at 37.degree. 
C. for up to seven days. Cultures producing turbidity were recorded as 
positive for growth. Cultures not producing changes after seven days of 
incubation were recorded as negative for growth (ie, sterile). The results 
are reported in Table 2. (Operating parameters: 10 B. subtilis strips and 
10 control strips used in each experiment. Microwave frequency: 2450 MHZ; 
energy density: 0.018 watt/cm.sup.3.) 
TABLE 2 
__________________________________________________________________________ 
Exposure Time 
Experimental 
in min. with end. 
Metal Instruments 
Growth in B. 
Growth in control 
Vapor at the end 
Conditions 
temp. in Liquid 
subtilis strips 
test tubes 
of process 
__________________________________________________________________________ 
Microwave heat 
3 min, 65.degree. C. 
yes none yes no 
& chemical 
disinfectant 
Microwave heat 
3 min, 56.degree. C. 
no yes yes no 
(no chemical) 
Chemical 3 min., 56.degree. C. 
no yes yes no 
disinfectant (no 
microwave heat) 
Chemical 3 min, 25.degree. C. 
yes yes yes no 
disinfectant (no 
heat whatsoever) 
Microwave heat & 
4 min, 83.degree. C. 
yes none yes yes 
chemical 
disinfectant 
__________________________________________________________________________ 
From Table 2, it is apparent that microwave heat alone without the chemical 
disinfectant (replaced by tap water) cannot kill all B. subtilis on the 
strips. The chemical disinfectant alone (no microwave heat) at 56.degree. 
C. exhibited growth in some B. subtilis strips. The chemical disinfectant 
alone at room temperature did not kill B. subtilis on any strip. 
Thus, the data of Table 2 demonstrates that the biocidal composition of the 
invention coupled with microwave irradiation at an energy density level 
higher than 0.018 Watt/cm.sup.3 exhibited a biocidal synergistic effect. 
Table 2 data also shows that to avoid the emission of noxious vapor at the 
end of a decontamination batch, the end temperature should not be higher 
than 65.degree. C. 
Example 3 
Potential corrosion on metals other than stainless steel was investigated 
under the 3 min/65.degree. C. experimental conditions. The dental tools 
were replaced by a dozen hollow anodized aluminum tubes (length 25 cm, 
int. diam. 2-5 mm). No apparent damaged was observed after several days of 
repeated exposures. 
Example 4 
In the original text of Content and Format of Premarket Notification (510 
k) Submissions for Liquid Chemical Germicides (Jan. 1982), several 
definitions are given by the regulatory agencies of a High level 
disinfectant. On page 5, for instance, the following definition is 
provided: 
A GERMICIDE THAT KILLS ALL MICROBIAL PATHOGENS EXCEPT LARGE NUMBERS OF 
BACTERIAL ENDOSPORES, WHEN USED ACCORDING TO LABELING. 
This definition was repeated in the April 26, 1995 revised edition printed 
by the FDA on pages 5 and 36. High level Disinfection claims require a 
showing that the contact time for high level disinfection is sufficient to 
achieve a 6 log reduction of Mycobacterium tuberculosis var. bovis under 
the worst case conditions of germicide compositions. Testing may be 
conducted with Mycobacterium in suspension or on carriers, but the number 
of organisms on the carriers must be quantitated. 
Tuberculocidal and virucidal efficacy was determined using different 
chemical formulations set forth in Tables 3 and 4 respectively: 
TABLE 3 
______________________________________ 
TUBERCULOCIDAL ACTIVITY WITH 3 DIFFERENT SAMPLES 
Condition in 
Exposure End-Temperature 
Percent Reduction of 
Reaction Vessel 
Time with Microwave 
M. bovis var. BCG** 
______________________________________ 
1.0 ml M. bovis 
0.5 min 30.degree. C. 
99.86% 
plus 49.0 ml of 
1.0 35.degree. 99.90 
Sample A. 1.5 43 99.980 
(H.sub.2 O.sub.2 plus 5% 
2.0 45 99.985 
acetic acid) 
3.0 56 &gt;99.9999% (no 
surviving colonies) 
1.0 ml M. bovis 
0.5 min 31.degree. C. 
99.865% 
plus 49.0 ml of 
1.0 35 99.90 
Sample B 2.0 45 99.998 
H.sub.2 O.sub.2 plus 5% 
3.0 60 &gt;99.9999% (no 
citric acid surviving colonies) 
1.0 ml M. bovis 
0.5 31.degree. C. 
99.86 
plus 49.0 ml of 
1 35 99.90 
Sample C 2 45 99.998 
(H.sub.2 O.sub.2 plus 5% 
3 66 &gt;99.999% (no 
lactic acid) surviving colonies) 
______________________________________ 
**Weight percent reduction of M. bovis var. BCG is given by the formula: 
[1.0 - S.sup.+ /S.sub.0 ] .times. 100 
wherein S.sub.0 is the original number of M. bovis var. BCG in the vessel 
at zero time. In this experiment, it was equal to 5.2 .times. 10.sup.6 
CFU. 
S.sup.+ is the number of surviving colonies of M. bovis var. BCG at 
various exposure times. 
TABLE 4 
______________________________________ 
VIRUS INACTIVATION OF SAMPLE B AFTER ONE DAY 
REUSE AT 60.degree. C. WITH 3 MIN EXPOSURE TIME 
Titer if 
virus exposed 
Titer to microwave 
untreated virus 
& chemical Inactivation 
______________________________________ 
Polio virus type 2 
10.sup.6 10.degree. 6 logs 
Herpes simplex-type 1 
10.sup.5.5 10.degree. 5.5 logs 
Influenza A virus (PR8) 
10.sup.5.5 10.degree. 5.5 logs 
______________________________________ 
As set forth in Table 3, the biocidal compositions were always a mixture of 
10 weight percent of concentrated hydrogen peroxide diluted down to about 
6 to about 7.5 weight percent with a weak organic acid, such as acetic 
acid, C.sub.2 H.sub.4 O.sub.2, citric acid, C.sub.6 H.sub.8 O.sub.7 or 
lactic acid, C.sub.3 H.sub.6 O.sub.3. 
The quantitative tuberculocidal efficacy test employed is an EPA approved 
method based on the modified work of Ascenzi et al (Appl. Environ. 
Microbiol, 93: 2189-2192, 1987). In the test, 1.0 ml of M. bovis var. BCG 
was added to 49.0 ml of the tested disinfectants in a 750 ml. reaction 
vessel previously described (Rubbermaid, microwave container). The 
reaction vessel holds more that 10 Colony Forming units (CFU) of M. bovis. 
var BCG. The reaction vessel with its content was microwave irradiated 
during 3 minutes and reached an end-temperature of 65.degree. C. At 
various exposure time intervals such as 0.5, 1.0, 1.5, 2.0, 3.0 and 5 
min., 1.0 ml is removed from the reaction vessel added to neutralizing 
medium (phosphate buffered saline plus catalase), and immediately further 
diluted and measured for surviving CFU of M. bovis. var. BCG. This 
mycobacterium was very slow to grow and colonies were counted only after 
28 days incubation period at 37 degrees C. The weight percent reduction of 
M. bovis var BCG are given in table 3 for various compositions and 
exposure times. M. tuberculosis var bovis is more difficult to kill than 
most other non-sporulated bacteria (such as P. aeruginosa, S. aureus and 
S. cholerasuis) whose kill is required to make a hospital germicide 
disinfectant claim. Likewise, Trichophyton mentagrophyte (ATCC 9533) is 
the requisite fungus for making an EPA claim according to the official 
method of the Association of Official Analytical Chemists. Dilution tests 
according to the AOAC procedure were conducted in the presence of 3 weight 
percent serum. All tests with the above referenced bacteria showed 
complete kill in 3 minutes at 60.degree. C. in the presence of microwave 
irradiation (energy density: 0.018 watt/cm.sup.3). The biocidal 
composition used for these experiments contained a 6 to 7.5 weight percent 
dilution of hydrogen peroxide mixed with a vinegar of 5 weight percent 
acidity. 
Example 5 
Virucidal capabilities of the process of the invention was further 
demonstrated by testing with one hydrophilic polio virus type 2 and two 
lipophilic viruses (herpes simplex type 1) and influenza A virus, (PR8). 
The viruses were dried on stainless steel penicylinders according to the 
AOAC method (paragraph 4.007 through 4.014) and treated for 3 minutes with 
microwave irradiation (60.degree. C. and the chemicals (hydrogen peroxide 
diluted with a 5 weight percent citric acid solution). The results given 
at the bottom of Table 4 show complete eradication of the viruses by a 
sample which had already been used five times in succession in one day. 
This indirectly confirmed that the biocidal Sample B contained still 
enough active ingredient at the end of one day reuse. 
In accordance with the above it must be well understood that according to 
the desired results the present invention can be applied with different 
sizes and shapes of containers with different microwave power units and 
with different exposure times as long as one can achieve a full 
destruction of microbial spores, microorganisms and viruses present on the 
surface of the instruments. Still without departing from the scope of the 
invention the structural details of the described apparatuses (shape of 
containers to accommodate various instruments, microwave cavity sizes and 
corresponding supporting shelves etc) may be modified and that certain 
members may be replaced by other equivalent means (Magnetrons replaced by 
Klystrons or Amplitron tubes). The present invention can be used to 
sterilize or disinfect any solid interface accessible to the liquid 
biocidal agent. Microwave radiation will not only penetrate the different 
materials supporting the microorganisms but will also act upon the 
molecular structure of the microorganisms themselves. In other words many 
intricate contaminated surfaces such as channels of fiber optic devices, 
small tubings in both rigid and flexible endoscopes, laparoscopes etc. 
could be decontaminated as well as simpler instruments such as front 
surface dental mirrors, explorers, scalpels, scissors etc. 
The invention may take form in various steps and arrangements of steps and 
in various parts and arrangements of parts. Various modifications may be 
made therefore in the nature, composition, operation and arrangement of 
the various elements, steps and procedures described herein without 
departing from the spirit and scope of the invention as defined in the 
following claims.