Note: Descriptions are shown in the official language in which they were submitted.
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SYNERGISTIC ANTIMICROBIAL COMPOSITION OF PEROXYACETIC ACID
AND A PHOSPHORUS COMPOUND
FIELD OF THE INVENTION
The present invention relates to synergistic antimicrobial compositions which
are
generally useful for inhibiting microbial growth wherever such microbial
growth is found,
for example in aqueous systems related to a wide variety of industrial
applications. More
particularly, the present invention relates to synergistic admixtures of
peroxyacetic acid
and a phosphorus compound. Methods for using the same are also disclosed.
gACKrROUND OF THE INVENTION
Both peroxyacetic acid (sometimes referred to as peracetic acid) and certain
phosphorus compounds such as tetrakis (hydroxy methyl) phosphonium sulfate,
referred
to herein as PAA and THPS respectively, are known individually as
antimicrobial agents.
The unexpected finding of the present invention is that they are synergistic
when used in
combination. As used herein, the terms "synergy" and "synergistic" refer to
instances in
which the effectiveness of a composition comprising two or more biocides, such
as PAA
and THPS, exceeds the sum of the efficacies of the individual components taken
alone.
Thus, using a synergistic biocidal combination may allow for use of a lower
overall
concentration of biocide or the realization of an enhanced antimicrobial
effect at a
comparable dosage.
Peroxyacetic acid, is known for its antimicrobial properties and its use as
an antimicrobial agent is disclosed in U.S. Patent Nos. 5,368,749, 5,494,588,
5,624,575,
5,658,467, and 5,670,055. Disclosed is also synergistic mixtures of
peroxyacetic acid and
certain biocides as well as methods of using the same.
Likewise, the use of certain phosphorus compounds as antimicrobial agents,
including tetrakis (hydroxy methyl) phosphonium sulfate (THPS) and related
compounds
is known and is disclosed in U.S. Patent Nos. 4,673,509 arid 5,670,055.
However, the
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synergistic combination of PAA and THPS related compounds is not disclosed or
suggested in the art.
As used herein, the phrases "antimicrobial", "biocide", and "inhibiting
microbial
growth" refer to the killing of, the inhibition of, or the control of the
growth of bacteria,
yeast, mold and/or algae. A number of important industries have experienced
serious
adverse effects from the activity of such biological growth on the raw
materials which
they employ, in their process waters, on various components of their
manufacturing
processes, and in the finished products which they produce. Such industries
include the
paint, wood, textile, cosmetic, leather, tobacco, fur, rope, paper, pulp,
plastics, fuel, oil,
7 0 rubber, and machine industries.
Systems which utilize circulating water or aqueous media become infected with
microorganisms and experience substantial impairment of their efficiency when
deposits
of the microorganisms build up in the system. The deposits coat the walls of
tanks and
other vessels and any machinery or processing equipment which is employed and
create
blockages in pipes and valves. The deposits also create discolorations and
other
imperfections in the products being produced, forcing costly shutdowns.
Control of
microorganisms is particularly important in aqueous media in which there are
dispersed
particles or fines in the aqueous media, for example, dispersed cellulosic
fibers and
dispersed fillers and pigments in papermaking, and dispersed pigments in paint
manufacture.
Slime control in papermaking processes is of particular importance. The
control
of bacteria and fungi in pulp and paper mill water systems which contain
aqueous
dispersions of papermaking fibers in various consistencies is especially
critical. The
uncontrolled buildup of slime produced by the accumulation of bacteria and
fungi may
cause off grade production, decreased production due to down-time and greater
cleanup
frequency, increased raw material usage, and increased maintenance costs. The
problem
of slime deposits is especially critical in light of the widespread use of
closed white water
systems in the paper industry.
Another important area that requires the use of good antimicrobial
compositions to
control bacterial and fungal growth is in clay and pigment slurries. These
slurries
comprise various clays (e.g., kaolin) and pigments (e.g., calcium carbonate
and titanium
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dioxide) and usually are manufactured at a location separate from the end use
application
This means that they are generally transported and stored for later use at the
application
site. Because of high quality standards for the paper and paint products in
which such
slurries are used, it is essential that these clay or pigment slurries have a
very low
microorganism count per gram of sample.
Accordingly, there.remains a very real and substantial need for antimicrobial
compositions capable of effectively controlling and/or inhibiting microbial
growth in
industrial aqueous systems and on articles of manufacture. Because of
increasing
environmental regulations, there is still a further need to provide biocidal
compositions
having enhanced antimicrobial effect which are effective in lower doses than
historically
used. Use of lower amounts of biocides has a favorable impact on the
environment, and
allows users to realize significant cost savings.
Additionally, there remains a substantial need for antimicrobial compositions
that
are, even at relatively high levels, safe for the environment.
SI~AI~Y OF THE INVENTION
The synergistic antimicrobial combination according to the present invention
comprises an effective amount of peroxyacetic acid (PAA) and an effective
amount of a
phosphorus compound selected from the group consisting of tetrakis (hydroxy
methyl)
phosphonium sulfate (THPS), tetrakis (hydroxy methyl) phosphonium phosphate
(THPP),
and tetraltis (hydroxy methyl) phosphonium chloride (THPC). This amount of PAA
and
phosphorus compound can be any amount that provides an antimicrobial effect.
Preferably this amount is an amount that, even if added in much higher amounts
to the
system, results in a synergy index (K value) of less than 1 after partial
biocide
degradation.
The synergistic antimicrobial combination according to the present invention
can
also comprise: a) PAA and b) a phosphorus compound selected from the group
consisting
of THPS, THPP, and THPC, wherein the weight ratio of a) to b), on an active
basis,
ranges between about 1000: l and 1:1000.
The synergistic antimicrobial combination according to the present invention
can
also be in an aqueous concentrate that comprises: a) about 0.0001 to about 0.1
weight
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PAA; b) about 0.0001 to about 0.1 weight % of a phosphorus compound selected
from the
group consisting of THPS, THPP, and THPC; and c) the remainder being water
(and other
raw materials of the system being treated); wherein the weight ratio of a) to
b), on an
active basis, ranges between about 1000:1 and 1:1000.
The present invention also provides a method for inhibiting microbial growth
in
aqueous systems and on articles of manufacture prone to such growth comprising
adding
to said systems or applying to said articles an effective amount the
synergistic
antimicrobial combinations above.
DETAILED DESCRIPTION OF THE INVENTIOjV
It has unexpectedly been discovered that the combination of PAA and the
phosphorus compound (THPS, THPP, and/or THPC) is a synergistic antimicrobial
combination, in that this combination generally meets the described
antimicrobial needs
for many applications. This synergistic combination is of even more value
because of the
desire in some industries to avoid the use of halogens to lessen the risk of
adverse impact
upon the environment.
As used herein, the term "effective amount" refers to that amount of a
composition
comprising PAA and the phosphorus compound necessary to achieve the desired
level of
inhibition or control of microbial growth in the aqueous system or on the
article being
treated.
The phosphorus compound used in the synergistic blend of the present invention
is disclosed in U.S. Patent No. 4,673,509, the disclosure of which is
incorporated herein
by reference in its entirety. The phosphorus compound is preferably selected
from the
group consisting of THPS, THPP, and THPC, with THPS being most preferred. THPS
is
most preferred due to proven efficacy and availability, along with the fact
that it is not a
halogenated biocide.
It is contemplated that the synergistic admixture of PAA and the phosphorus
compound, as disclosed herein, and the methods for using the same, will be
useful in
virtually any aqueous system or on any article or product of manufacture in
which
inhibition of microbial growth is desired, absent compatibility problems.
Suggested
applications of the synergistic antimicrobial combinations of the present
invention
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include, for example: inhibiting the growth of bacteria and fungi in aqueous
paints,
adhesives, latex emulsions, inks, joint cements and caulking compounds;
preserving
wood; preserving cutting oils and metal working fluids; controlling slime-
producing
bacteria and fungi, including yeast and mold, in pulp and ;paper mills and
cooling towers;
as a spray or dip treatment for textiles and leather to prevent mold growth;
as a
component of anti-fouling.paints to prevent adherence of fouling organisms;
protecting
paint films, especially exterior paints, from attack by fungi which occurs
during
weathering of the paint film; protecting processing equipment from slime
deposits during
manufacture of cane and beet sugar, food, foodstuffs and food additives;
preventing
microorganism buildup and deposits in air washer or scrubber systems and in
industrial
fresh water supply systems; controlling microorganism contamination in closed
loop and
recirculating water cooling systems; controlling microorganism contamination
and
deposits in oil field drilling fluids and muds, and in secondary petroleum
recovery
processes; preventing bacterial and fungal growth in paper coating processes
which might
adversely affect the quality of the paper coating; controlling bacterial and
fungal growth
and deposits during the manufacture of various specialty boards, e.g.,
cardboard, particle
board and food grade board; preventing sap stain discoloration on freshly cut
wood of
various kinds; controlling bacterial and fungal growth in clay and pigment
slurries of
various types which are manufactured for later use in paper coating and paint
manufacturing and which are susceptible to degradation by microorganisms
during
storage and transport; as a hard surface disinfectant to prevent growth of
bacteria and
fungi on walls, floors, etc.; in swimming pools to prevent algal growth,
including green
algae and cyanobacteria (blue green algae); and to control bacterial and
fungal growth in
various cosmetic products. It is further contemplated that the synergistic
admixture of the
present invention will be useful in various types of non-aqueous systems as
well.
The synergistic antimicrobial composition disclosed in the present invention
is
particularly applicable to microbial slime control in papermaking processes.
The need to
control bacteria and fungi in pulp and paper mill water systems which contain
aqueous
dispersions of papermaking fibers in various consistencies is disclosed above.
Another important area in which the antimicrobial compositions of the present
invention are particularly useful is in the control of bacterial and fungal
growth in clay
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and pigment slurries, as disclosed above. It is essential that these clay or
pigment slurries
have a very low microorganism count per gram sample.
In addition, the synergistic combination of the present invention and methods
of
using the same have been found especially useful in controlling the harmful
effects of
microorganisms in water or aqueous media. Particular examples of aqueous
systems that
have a need for the use of the present invention to control microorganisms
include cooling
waters used in the chemical industry and in power generation, wastewater
treatment from
municipal and industrial plants, as well as pasteurization systems in the food
and beverage
industry.
As disclosed above, any system which utilizes circulating water or aqueous
media
becomes infected with microorganisms and experience substantial impairment of
their
efficiency when deposits of the microorganisms build up in the system and can
be treated
according to the present invention. Control of microorganisms by the present
invention in
aqueous media is particularly important where there are dispersed particles or
fines in the
aqueous media, for example, dispersed cellulosic fibers and dispersed fillers
and pigments
in papenmaking, and dispersed pigments in paint manufacture.
The present invention is directed to a synergistic antimicrobial composition
comprising: a) PAA; and b) a phosphorus compound selected from the group
consisting
of THPS, THPP, and THPC, wherein the weight ratio of a) to b), on an active
basis,
preferably ranges from about 1000:1 to 1:1000. The present invention is
further directed
to a method for inhibiting microbial growth in an aqueous system or on an
article of
manufacture prone to such growth, which method comprises treating said system
or said
article with an effective amount of an antimicrobial composition comprising:
a) PAA; and
b) a phosphorus compound selected from the group consisting of THPS, THPP, and
THPC, wherein the weight ratio of a) to b), on an active basis, ranges from
about 1000:1
to 1:1000.
In accordance with the present invention, the weight ratios of the two
components
of the synergistic combination are dictated by the dosage levels of each
component which
demonstrate synergism, based on 100% active ingredient, relative to each end
use
application. Typically, the weight ratio of component a), PAA, and component
b), the
phosphorus compound, ranges from about 1000: I to 1:1000 on an active basis,
preferably
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from about 100:1 to 1:100, more preferably from about 10:1 to 1:10. As will be
understood by one skilled in the art, however, the synergistic weight ratio of
the two
components generally varies to some extent depending on the application and
the
organism being controlled. For example, a higher ratio of PAA to the
phosphorus
compound might be more effective in one application, while a higher ratio of
the
phosphorus compound to PAA might be more effective in another application. The
PAA/phosphorus compound composition has been found particularly effective
against
bacteria when used in a weight ratio of between about S: l to 1:5.
In the aqueous system being treated with the synergistic composition of PAA
and
the phosphorus compound, the amount of each biocide present can range from
about 0.05
parts per million (ppm) to about 200 ppm. Preferably, between about I ppm and
about
100 ppm of each biocide based on the weight of water in the system being
treated, are
added, more preferably, between about 2 ppm and 60 ppm. However, an effective
amount of the synergistic combination of the present invention must be added
to the
aqueous system being treated even if there is only a very small amount of one
biocide and
a larger amount of the other. This amount of the synergistic composition of
the present
invention should at least be about 0.1 ppm based on the weight of water in the
system
being treated, preferably at least 1 ppm, more preferably at least 10 ppm.
The upper limits of each component and the upper limits of the total
synergistic
combination of the present invention depends upon economics and environmental
concerns. The total amount of the inventive synergistic biocide combination
present in an
aqueous system should be less that about 300 ppm, preferably less that about
200 ppm,
more preferably no more than about 150 ppm, with an amount no more than about
100
ppm being most preferred. However, it is well within the ordinary skill of one
practicing
in the art to determine the effective amount of biocide for a given system
based on various
system parameters including but not limited to the size of the system, pH of
the system,
the types of organisms present and the amount of control desired.
Additionally, in some applications a higher overall percent biocide is
required to
control or kill the microbes. In these applications, higher amounts of each
biocide can be
added while maintaining a measurable synergistic effect. Thus, the upper limit
of the
synergy of the combination varies, depending on the application. In
preservation
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applications for example, each biocide in the blend could be present in a
concentration
ranging from about 0.05 ppm up to as high as 1000 ppm.
Much higher amounts of one or both biocides can be added to the system to
provide protection from microbial growth since the concentration throughout
the entire
system will not necessarily be the same and the localized ratios can very
widely. Also,
initial higher amounts can be added with the anticipation that one or both
biocides will
degrade or decompose over time to then be within the ranges stated above. Even
though
higher amounts than stated above are added to the system to be treated, what
is important
is that the localized amounts or time degraded amounts fall within the stated
range at
some point in time, thereby benefiting from the synergistic combination as the
microbes
in the system are controlled.
Additionally, when one of the two biocides in the present biocide blend is
added at
a relatively high concentration (e.g. 200 ppm or more) there still is a
synergistic effect at
the addition of the second biocide but that effect is masked by the overall
high
concentration of the total biocide present in the system. For example, if the
phosphorus
compound THPS is added in and amount greater than 200 ppm to a system
containing
E.coli at the same time an amount of PAA is added, the kill rate would be
faster than with
THPS alone. However, the final kill or inhibition effect would not be
significantly
different because all of the E.coli bacteria are eventually killed with 200
ppm of THPS all
by itself.
An effective amount of a synergistic combination of PAA and the phosphorus
compound can also be applied to the article of manufacture being treated.
Generally, a
solution of the synergistic antimicrobial combination described above having a
concentration of at least 0.1 ppm is incorporated into, sprayed or poured
onto, used to dip,
or otherwise applied, for example by dipping or submersing, to the substrate
being treated
in order to prevent growth of bacteria, mold, yeast and algae. Again, it is
well within the
ordinary skill of one practicing in the art to determine the effective amount
of biocide to
apply to a given article of manufacture being treated and to determine
suitable modes of
application.
The active ingredients of the synergistic antimicrobial compositions of the
present
invention may also be used in diverse formulations: solid, including finely
divided
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powders and granular materials; as well as liquid, such as solutions,
emulsions,
suspensions, concentrates, emulsifiable concentrates, slurries and the like,
depending
upon the application intended, and the formulation media desired. Further,
when the
synergistic antimicrobial combinations are liquid, they may be employed neat
or may be
incorporated into various formulations, both solid and liquid, as an adsorbate
on suitable
inert carriers such as talc, clays, diatomaceous earth and the like, or water
and various
organic liquids such as lower alkanols, kerosene, benzene, toluene, and other
petroleum
distillate fractions or mixtures thereof.
The amount of the synergistic combination of PAA and the phosphorus compound
can be added to the aqueous system being treated as solid single components or
as a dry
blend. However, the synergistic combination is preferably added as a solution
that is an
aqueous concentration to be diluted in situ to the above amounts. This
synergistic
combination is preferably added as two separate single component solutions to
be blended
and diluted in situ. This separate addition is preferred due to the
instability of blend
solution over time due to degradation. PAA is an oxidizing biocide and the
phosphorus
compound is a non-oxidizing biocide. Thus, over time PAA degrades in the
solution
blend as it degrades and oxidizes the phosphorus compound (THPS, THPP, and/or
THPC).
The aqueous concentrate of each of the single component solutions used in the
synergistic antimicrobial combination would contain much higher amounts of
each of the
biocides than would be present in the final treated aqueous system and is well
within the
ordinary skill of one practicing in the art . This concentrate can vary widely
depending
upon the weight savings desired with reduced amounts of water while
maintaining ease of
application with minimal mixing in situ. What is important is the ratio and
final
concentration of each of the biocides in the treated aqueous system. These
should be
within the limits stated above. Each concentrate can contain other standard
components
and be added in combination with other standard additives used in the
particular aqueous
system.
PAA is commercially available in liquid form from English China Clays
Inc./Calgon Corporation (ECC/Calgon), Pittsburgh, PA as Metasol~ PAA, which is
12%
active PAA, CH3COOOH. This product also contains about 18% hydrogen peroxide,
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about 20% acetic acid, and about 50% water. THPS is also commercially
available from
ECC/Calgon, Pittsburgh, PA and is in liquid form as Metasol~ LT, which is 35%
active
THPS in a water solution.
To prepare the two solutions of the synergistic composition under this
invention,
an effective amount of each active ingredient should be combined in a suitable
Garner
such as water, organic solvents and the like. The preparation of such a
composition is
within the ordinary skill of one practicing in the art.
It will also be understood by one skilled in the art that the synergistic
antimicrobial combination disclosed herein may be used in combination with
other
antimicrobial materials. For example, the combination can be combined with
other
fungicides and bactericides in appropriate concentrations and in appropriate
instances so
as to combine the action of each to obtain particularly useful results. Such
combinations
might find particular application in the preparation of germicidal soaps, in
the production
of cosmetics and aqueous coatings and in combating paper riiill microbial
slime
accumulations. It is clear also that the synergistic antimicrobial combination
of the
present invention can be combined with other algicidal agents as well.
In accordance with the present invention there is still further provided a
method of
inhibiting the growth of at least one of the following: bacteria, yeast, mold
and algae.
According to the methods of the present invention, this growth is inhibited in
aqueous
systems or on articles or products of manufacture prone to such growth. These
methods
comprise adding to the aqueous system or treating the article or product
containing said
bacteria, yeast, mold and/or algae with an effective amount of a synergistic
combination
of PAA and the phosphorus compound (e.g. THPS). This addition can be
accomplished
either by simple addition of components together as a single admixture, or by
the
preferred addition of the two components separately. Such separate
administration can
either be at the same time or at different times. The net effect will be the
same--the
system, article or product being treated will ultimately have incorporated
therein or have
applied thereto the desired dosage concentration of each component.
Further, the compositions of the present invention are believed to be
effective
irrespective of the method of application (unless the blend is stored for a
period of time
prior to addition). For example, the antimicrobial compositions described
herein can be
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added to a system being treated as two separate or one stream via a low level,
continuous
feed practice, a semi-continuous feed practice or through slug feeding. All of
these
feeding practices will be familiar to one having ordinary skill in the art.
Slug feeding is
particularly effective and therefore is a preferred manner of employing the
methods of the
present invention. This type of feed allows the user to monitor the
microorganism
concentration in the system and feed product only when microorganism
concentrations
increase. The user realizes a cost savings by feeding an effective amount of
PAA and the
phosphorus compound only when needed.
As noted above, the present invention is based upon the discovery that use of
PAA
in conjunction with certain types of phosphorus compounds produces synergistic
results
and is effective in controlling the growth of bacteria, yeast, mold and algae
in a variety of
industrial and other applications. The utility of the synergistic
antimicrobial combination
disclosed herein derives from its versatility both in the numerous industries
in which it
can be applied, as well as the numerous microorganisms against which it is
effective. In
particular, the large economic losses in papermaking operations caused by the
accumulation of bacterial and fungal slimes in various parts of the system can
be
eliminated to a significant extent by use of the synergistic combination
described herein.
The superior antimicrobial activity of the synergistic antimicrobial
combination of
PAA and the phosphorus compound has been confirmed using standard laboratory
techniques. The antimicrobial combination has been found effective, for
example, in
inhibiting bacterial growth including but not limited to Klebsiella pneumoniae
and
Escherichia coli and has been found to be particularly effective against
Pseudomonas
aeruginosa. The combination is also believed to be effective against other
aerobic
bacteria, such as Bacillus, Staphylococcus, Flavobacterium, Enterobacter, and
Xanthomonas, anaerobic bacteria, freshwater organisms such as filamentous
bacteria,
fungi including but not limited to various species of Candida and
Saccharomyces, white
and pink yeasts, molds, and various species of green algae and blue green
algae.
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The following examples are set forth to illustrate the present invention and
should
not be construed as limiting the invention in any way.
The biocidal efficacy in microtiter tests of the antimicrobial composition of
the
present invention is demonstrated below. Three different bacterial strains,
Klebsiella
pneumoniae, Escherichia coli, Pseudomonas aeruginosa, as well as a mixture of
all three
of the strains, were used.
Each of the three bacteria were separately grown on Standard Methods Agar
(STM) plates and incubated at 37°C for a period of between about 24-48
hours. Each
species of bacteria was inoculated into a small tissue flask containing
approximately 25
ml of Allen's Media using a sterile swab. The cultures were then diluted by
adding 10 ml
of bacterial suspension to 90 ml of fresh Allen's Media. To prepare a mixed
culture of all
three of the organisms, an equal amount of approximately 20 ml, of each of the
diluted
cultures was mixed together in a large tissue culture flask. Samples from the
mixture and
the individual bacterial suspensions were then used in the microtiter test.
An 8X stock solution of PAA to use in combination with THPS was prepared by
dissolving about 6.6 grams (g) of 12% active PAA in about 100 ml of deionized
water. A
4X stock solution of THPS to use in combination with PAA was prepared in the
same
manner only using 0.106 g of THPS (35% active THPS). A 4X stock solution of
THPS
to use alone was prepared in the same manner only using 0.53 g of THPS. A PAA
8X
stock solution to use in combination with THPS was prepared by dissolving
about 6.6 g
of 12% active PAA in 100 ml with deionized water. A 4X stock solution of THPS
to use
in combination with PAA was prepared in the same manner only using 0.05 g
THPS. The
4x stock solution of PAA to use alone was prepared in the same manner only
using 3.3 g
PAA. Subsequently, two other plates were made up using 6.6 grams of PAA and
0.16 g
and 0.26 g THPS respectively. Eight microtiter plates were used in the example
(not
counting the plates for PAA and THPS alone), each microtiter plate having 8
rows, A-H,
and 12 columns, 1-12. The amount of each biocide in each well of the eight
plates is
depicted below.
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AMOUNT OF EACH BIOCIDE IN WELLS OF Microtiter PLATES 1-R
COLUMN
CONCENTRATIONS
(pPm
Active)
PLATEBIOCIDE
t 2 3 4 5 6 7 8 9 10 ll 12
1,5 PAA 1000 500 2S0 12S 62 31 16 8 4 2 - +
(8X)
THPS 200 200 200 200 200 200 200 200 200200 - +
(4X)
2,6 PAA 1000 S00 250 125 62 31 16 8 4 2 - +
(8X)
THPS 100 100 100 100 100 100 100 100 l00100 - +
(4X)
3,7 PAA 1000 500 250 125 62 31 16 8 4 2 - +
(8X)
THPS 300 300 300 300 300 300 300 300 300300 - +
(4X)
4,8 PAA 1000 500 250 12S 62 31 16 8 4 2 - +
(8X)
THPS 500 500 500 500 500 500 S00 500 500S00 - +
(4X)
9 PAA 1000 500 2S0 125 62 31 16 8 4 2 - +
(8X)
THPS 0 0 0 0 0 0 0 0 0 0 - +
(4X)
PAA 0 0 0 0 0 0 0 0 0 0 - +
(8X)
THPS 1000 S00 250 125 62 31 16 8 4 2 - +
(4X)
5
As is illustrated in the table above, the amount of PAA in the wells of plates
1
through 9 were varied in a serial dilution series ranging from 1000 ppm active
to 2 ppm
active while the other component's (THPS) concentration was kept constant at
200, 100,
300, 500, and 0 ppm active. Plate 10 represented use of THPS alone in a serial
dilution
10 series. Plates 9 and 10 represented the use of each biocide alone and were
used to
determine the minimum amount of each biocide which, when used alone, would
inhibit
microbial growth. No biocide was added to the wells of column 12 in any of the
plates,
which represented an organism control, or positive control. This positive
control was run
to ensure that the organisms were capable of growing in the environment
provided. No
bacteria were added to the wells of column 11 in any of the plates, which
represented the
Allen's Media control, or a negative control. This was done to ensure that
there was no
contamination of the plates. In each of the 10 microtiter plates Pseudomonas
aeruginosa
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was added to rows A and B, Klebsiella pneumoniae to rows C and D, Escherichia
coli to
rows E and F, and the mix of all three bacteria to rows G and H.
Plates 1-4 were used to determine the minimum inhibitory concentration (MIC)
for each biocide combination against each bacteria strain. The MIC is the
least amount of
biocide needed to prevent growth in the well, with growth being defined as a
turbidity in
the medium or a "pellet" of cells which came out of the medium and settled at
the bottom
of the well.
Plates 5-8 were then subcultured from plates 1-4, respectively, at 24 hours
following biocide addition. Subculturing was done to determine the minimum
biocidal
concentration (MBC). The MBC is the lowest concentration of biocide that
results in no
growth after subculturing and subsequent incubation.
All of the microtiter plates including the MIC plates and the MBC plates were
incubated for 24 hours at 37°C. Following the 24 hour incubation
period, the presence or
absence of growth in each well of the plates was determined. Growth in the
microtiter
plates was determined by subculturing the plates onto a solid agar plate
dipicting each
microtiter well. The plates were then incubated at 37°C far 24 hours.
The presence or
absence of growth in each well, along with the concentration of biocide in
each well, was
then used to determine the synergistic properties of the biocide combinations.
The
synergistic properties were evaluated by determining the Kull value/K value;
the K value
was determined for each bacterium tested. The method for calculating K value
is well
known to those skilled in the art. In this example, the K value was determined
by the
following formula:
K = jPA~]' In Combination + (THPS] In Combination
[PAA] Alone [THPS] Alone
where "[PAA] In Combination" means the concentration of PAA which, when used
in
combination with THPS, resulted in inhibition of microbial growth;
"[THPS] In Combination" means the concentration of THPS which, when used in
combination with PAA, resulted in inhibition of microbial growth;
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"[PAA] Alone" means the concentration of the PAA which, when used alone,
resulted in
inhibition of microbial growth; and
"[THPS] Alone" means the concentration of THPS which, when used alone,
resulted in
inhibition of microbial growth.
A K value of less than 1 indicates synergy between the two biocides, a K value
of greater
than 1 indicates antagonism between the two biocides, and a K value equal to 1
indicates
an additive effect of the two biocides.
The K values determined for each of the organisms used in the example are'
recorded in Tables 2 through 9.
TABLE 2
"K" VALUES OF PLATE 1 (MIC1
Organism [PAA] [THPS (PAA] [THPS] K Weight
In In
Alone, Alone, Combination,Combination,Value Ratio
ppm
PPm PPm PPm PAA:
THPS
Pseudomonas 1000 125 3.9 200 1,6 0,0195:1
aeruginosa
Klebsiella 93.75 125 5.85 200 1.7 0.0293:1
pneumoniae
Escherichia 7.8 125 1.95 200 1.9 0.0098:1
coli
Mixture of 12S 125 5.85 200 I .6 0.0293:
above three I
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TABLE 3
"K" VA UES OF PLATR 2~'~;
Organism [PAA] [THPS] [PAA]
In [THPS] K Weight
Alone, Alone, Combination,In Value Ratio
ppm
PPm ppm Combination ppA;
PPm THPS
Psuedomonas 1000 125 5.8 100 0.81 O.OS8:1
aeruginosa
Klebsiellapneumoniae93.75 t25 3.9 100 0.84 0.04:1
Escherichia 7.8 125 I .9S 100 1.05 0.019:1
coli
Mixture of 125 125 23.4 100 0.99 0.234:1
above three
TABLE 4
"K" VALUES OF PLATE 3 fMICI
Or anism
g [PAA) [THPS] [PAA) [THPS) K Weight
In
Alone, Alone, Combination,In Value Ratio
ppm
PPm ppm Combination ppp:
PPm THPS
Pseudomonas 1000 125 15.6 300 2.42 0.052:1
aeruginosa
Klebsiello 93.75 125 31.2 300 2.73 0.104:1
pneumoniae
Escherichia 7.8 125 I .95 300 2.65 0.007:
coli T
Mixture of 125 125 15.6 300 2.52 0.052:1
above three
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TABLE 5
"K" VALUES OF PLATE 4lMIC1
Organism [PAA] [THPSJ (PAA] [THPSJ K Weight
In
Alone, Alone, Combination,tn Value Ratio
ppm
ppm ppm Combination ppp;
PPn~ THPS
Pseudomorras 1000 125 3.9 500 4.00 O.OOS:I
aeruginosa
Klebsiella 93.75 125 5.85 500 4.06 0.012:
pneumoniae I
Escherichia 7.8 125 1.95 500 4.25 0.004:1
coli
Mixture of 125 125 5.85 500 4.05 0.012:1
above three
TABLE 6
Organism [PAA] (THPS] [PAAJ [THPSJ K Weight
In
Alone, Alone, Combination,In Value Ratio
ppm
ppm ppm Combination PAA:
PPn~ THPS
Pseudomonas 62.5 125 3.9 200 1.66 0.0195:1
aeruginosa
Klebsiella 78. 125 5.85 200 I .67 0.029:1
pneumoniae l
Escherichia 15.6 125 1.95 200 1.73 0.009:
coli I
Mixture of 125 125 5.85 200 1.65 0.029:1
above three
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TABLE 7
"K" VALUES O~ PLATE 6 iMBC'.)
Organism [PAAj [THPSj [PAAJ [THPSJ K Weight
In
Alone, Alone, Combination,In Value Ratio
ppm
ppm ppm Combination PAA:
PPm THPS
Pseudomonas 62.5 125 2.9 100 0.85 0.029:1
aeruginosa
Klebsiella 78. 125 3.9 100 0.85 0.04:1
pneumoniae I
Escherichia 15.6 125 1.95 100 0.93 0.019:1
coli
Mixture of 125 125 3.9 100 0.83 0.04:1
above three
TABLE 8
Organism [PAAj [THPS] (PAA] (THPS] K Weight
In
Alone, Alone, Combination,In Value Ratio
ppm
ppm ppm Combination pAp;
PPm THPS
Pseudomonas 62.5 125 IS.G 300 2.65 0.052:1
aeruginosa
Klebsiella 78.1 125 3 I .9 300 2.80 0.1:1
pneumoniae
Escherichia 15.6 125 1.95 300 2.53 0.006:1
coli
Mixture of 125 125 15.6 300 2.52 0.052:1
above three
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TABLE 9
Organism [PAA] [THPS] [PAAJ In [THPS] K Weight
Alone,Alone, Combination,In Value Ratio
ppm
ppm ppm Combination PAA:
ppm THPS
Pseudomonas 62.5 125 2.9 500 4.05 0.006:1
aeruginosa
Klebsiella 78. 125 5.85 500 4.07 0.012:
pneumoniae I I
Escherichia 15.6 125 1.95 500 4.13 0.004:1
coli
Mixture of 125 125 7.8 500 4.06 0.015:1
above three
19
