CA2140896A1 - Improvements in or relating to germicidal compositions - Google Patents
Improvements in or relating to germicidal compositionsInfo
- Publication number
- CA2140896A1 CA2140896A1 CA002140896A CA2140896A CA2140896A1 CA 2140896 A1 CA2140896 A1 CA 2140896A1 CA 002140896 A CA002140896 A CA 002140896A CA 2140896 A CA2140896 A CA 2140896A CA 2140896 A1 CA2140896 A1 CA 2140896A1
- Authority
- CA
- Canada
- Prior art keywords
- composition according
- organisms
- light
- dyestuff
- results
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/40—Dyes ; Pigments
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/43—Solvents
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/48—Medical, disinfecting agents, disinfecting, antibacterial, germicidal or antimicrobial compositions
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Detergent Compositions (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Apparatus For Disinfection Or Sterilisation (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
A surface germicidal composition comprises a dyestuff which is capable of photo-dynamic inactivation of micro-organisms. The dyestuff preferably generates singlet oxygen on exposure to light and is substantive to microorganisms, conveniently being selected from the group comprising Rose Bengal, Erythrosin B and phthalocyanin sulphonates. The composition may optionally include other ingredients such as one or more surfactants (for cleaning purposes) and/or one or more solvents.
Description
2 ~ 1 ~ 0 8 9 ~ PCI /GB93/01478 I, I . ..
.......
"` . .
. . ~ .
Title: Im~rovements in or Relatinq to Germicidal~
,: , Compositions ;
, . ~
Field of the Invention .
This invention relates to germicidal compositions, ,~
particularly` for use on surfaces, ie compositions capable `~
of destroying or inactivating micro-organisms, particularly surface-bound micro-organisms.
Summary of the Invention -`~
. .
In its broadest aspect the present invention provides a `
surface germicidal composition comprising a dyestuff which is capable of photo-dynamic inactivation of micro- 9'''~,``.' organisms. ~
!:
It is preferred to use a dyestuff that generates singlet oxygen on exposure to light.~ On absorption of light ~ ~-energy a dye molecule is converted to a more energetic or ~;~
exited state ~S1 ) from its electronic ground state (SO). , -(Electron spins are paired and SQ these are singlet states ! ` `
in the language of spectroscopy, having a single energy level in a magnetic field.) ,L :`. .
;`."'.
The excited state is short lived and can lose energy and return to the ground state in a number of ways: by emission of a quantum of light as fluoresecence; by internal conversion as the energy is degraded ~o heat; by collision with a molecule of a different substance , !; `
if. . :`
. .
`. :.`.
W094/02022 PCT/GB93/014781 ~
Q,~?,g6 ~
(fluorescence quenching). `
. ~
The shor~ lived singlet s~ate may also undergo a pr~cess called intersys~em crossing to a longer-lived excited state, the triplet state. (The state is termed "tripletn because the electron in the higher energy level is no longer spin-paired with the electro~uin the lower level ~`
and the excited state has three energy levels in a magnetic field.) ;~
`~
Interaction of the excited triplet state with ground state molecular oxygen (which exists in the tripLet state normally) regenerates the dye ground state as energy is transferred to the oxygen which is promoted to the .
electronically excited singlet state. This means that, ~`
under idea~l circumstances, a single photosensitiser molecule can generate many times~its own concentration of -~
singlet oxygen.
Singlet~oxyg~en is highly reactive~and~photosensitised oxidation~proceedlng via this route is known as Type II
photo-o~xida~t~ion. ~Type II photo-o~xidation is independent of the~photosensit~iser used ~o gene~rate the s~ingIet 1~
oxygen.; ~An~important féature of the sensitiser is that it ~^
shoul~d have~a h~igh~quantum~yie}d~of triplet formation (that is~ ideally;,~a triplet state~should be produced for ~`
ea~ch~photon absorbed~ Intersystém crossing to the ~-~triplet~state~; i8~ ~facil~itated~by the presence of heavy atoms in the molecule. `~
.
Photo-oxidation of any vital component of an organism may . ``
result in ce}l de~ath (protein, polypeptide, amino-acids, lipids with allylic hydrogens, tocopherols, sugars and cellulose).
, :~
~: , .
2 i ~0896 Currently preferred dys~uffs include Rose Bengal (Ac;d Red 94, Colour Index No. 4S440), Ery~hrosin B (Acid Red 51, Colour Index No. 45430), and ph~halocyanin sulphonates such as aluminium phthalocyanin sulphonate (APS) and zinc pht~. locyanin sulphonate (ZPS). Rose ~engal and Erythrosin B are known food colourants (Rose Bengal is Food Colour Red No 105 and Ery~hrosin B is Food Colour Red -`
No 14), and Erythrosin B is on an EEC list of colouring agents allowed for use in cosmetic products, so these two ~
dyes are well suited for use in compositions intended for `;
domestic use. Mixtures of dyes can be used, and in some cases it may be desirable ~o include in a mixture a dye ~
that will remain visible a~ the end of the photodynamic ~^;
process~
The concentration of dyestuff in the composition is not critical and will typically be up to lOOppm, with good results having been obtained with concentrations in the i~
~range lOppm to 20ppm. Lower concentrations, down to lppm should~ also give reasonable results. ~"
Singlet oxygen has a short lifetime and therefore a short pathlength for diffusion, 50 to be effective a ir;
photosensitising dye generating singlet oxygen must be };~
close to the target substrate. Preferred dyestuffs are ~-~
therefore substantive to (ie capable of binding to) micro-. .
organisms, typically by binding to cellular protein on the organism surface or other cellu}ar components~ eg cellular fats. `-'...
The preferred dyes men~ioned above generate singlet oxygen on exposure to light and are substantive tO protein and so capable of binding to micro-organisms via cellular pro~ein. In this way, target~ed killing of organisms and '. .:
,`'`' `:
W094/02022 PCT/GB93/01478~ ~
21~0896 hence germicidal action is possible.
It is also preferred to use a dyestuff that is bleached by ~
exposure to light. By use of a photo-bleaching dyestuff ;
that is substantive to miCro-Qrganisms itican be Qossible for a visible indication of the pres~nce of micro-organisms to be provided. As the dyestuff bleaches, the i photo-dynamic action proceeds causing the death of, or `~
otherwise inactivating, micro-organisms. In the presence of low levels of light, both bleaching and the photo- ~
dynamic activity are believed to proceed more slowly, ;
whereas at higher light intensities both processes occur more quickly. Thus, depending on the relative rates o~
bleaching and photo-dynamic activity, the presence of -visible dyestuff indicates to the user that the photo- -dynamic inactivation of any micro-organisms present is -incomplete. i ~ ~ .
The photo-dynamic inactivation of micro-organisms in suspension by dyes such as Rose Bengal is known. See, for example, Journal of Applied Bacteriology 1985, ~58] pages 391-400/ Photochemistry and Photobiology 1988, 148] pages 607-612 (Neckers et al), and Shokuhin Eiseigaku Zasshi `~
1962, 10(5), pages 344-347. However, it has now been ;~-found that suitable dyestuffs are capable of photo-dynamic inactivation of micro-organisms on surfaces, and this is `~
the basis of the present invention. It is well-known that micro-organisms are much more susceptible to biocides in their planktonic or suspended state: they are much more difficult to inactivate when attached to surf`aces, which is their usual or preferred state. Micro-organisms will normally be on surfaces in the form of "biofilms", that is, embedded in a matrix of extracellular material. This extracellular material may sometimes be referred to as -~
W094/02022 21 ~ 0 8 9 ~ PCT/GB93/01478 ~
"adhesin" in the literature. It is therefore no~ obvious that a process which acts on micro-organisms in their planktonic state would act on surface-bound organisms without modification being required. Surface-bound mlcro-organisms represent an important and substantial source of contamination in domestic, institutional and industrial ~
environments, and the present invention can enable `~-targetted germicidal action on such micro-organisms.
Compositions according to the present invention are particularly suitable for use on hard domestic and industrial surfaces such as glass, plastics, ceramic and metal surfaces. In particularly, the compositions are ~'~
effective for use on surfaces which may harbour soils having the potential for bacteriological contamination in surface imperfections, joints and other relatively confined regions.
The composition is preferably acidic, eg having a pH in the range of 3 to 5, eg a pH of about 4, as acidic s compositions are found to have substantially enhanced ;
effectiveness against Gram-negative (G-) micro-organisms `~
as~compared with neutral compositions. The effectiveness i`
against Gram-positive (G+) micro-organisms seems not to be significantly affected by pH. The compositions are conveniently made acidic by use of relative}y mild organic acid, such as àcetic acid. ~ ;~
, ~ .
Neckers et al (above) review conflicting evidence that penetration of the dye Rose Bengal itself through the cell wall is essential for inactivation. They suggest that their own results support the dye penetration hypothesis i-primarily on the basis of differential inactivation of G
and G- species. The envelope of G- bacteria has an W094/02022 PCT/GBg3/01478~ ~
2~l~o896 additional outer membrane composed substantially of lipopolysaccharide which Necker et al regard as serving as a barrier to potentially toxic substances. An alternative interpretation could be that the ~moun~ of protein exposed in the cell walls is very different ;`
between G+ and G- species, being g~eater for G+ than G-, `r'~' with a binding affinity for the dye which varies with pH.
The concentration of dye bound to the cell wall would therefore be a function of pH. This interpretation would account for our observations of the variation in kill rate i;
with pH (but not the apparent resistance of the G+ species B. subtilis and B. megaterium). -. .
There is no difference between the endospore-forming -species B. s~lbtilis and the nonspore-forming S. aureus in their susceptibilities to photodynamia action by Rose 3engal in the absence of endospores. The apparent differences shown in our studies are presumably due to the ~presence of endospores both for B. subtilis and B. `~
mégaterium~which are clearly more resistant to sing}et ``
oxyge~n than the bacteria themselves. The spores survive exposure to Rose Bengal and light and subsequently l`~
germinate to give countable colonies. Spores are known to be difficult to stain and presumably have no affinity for ~
Rose 8engal under any of the conditions used. There `
appears to be little documentation on the toxicity of --~
singlet oxygen to spores in the literature. From studies ;~
on the possibly less resistant conidia of Neurospora `~
~crassa (Photochem. Photobiol., 33, 349 (1981)),singlet ~:
oxygen does have potential in this respect.
The composition msy optionally include other ingredients such as one or more surfactants (for cleaning purposes) and/or one or more solvents.
- W094/02022 214 089 6 PCT/GB93/01478 ;~
.:
- 7 ~
:.
The surfactant is preferably alkoxylated, more preferably `
ethoxylated, eg being in the form of ethoxyla~ed al~cohols.
The alcohol preferably has between 4 and 15 carbon a~oms, 1.. `,.3~"
is of straight or branched chain configuration, and has an HLB value (hydrophilic lipophilic balance) in the range 10 to 14, eg 12.
A wide range of suitable surfactants are commercially available, one such material being the surfactant available under the trade name Imbentin 91-~5, from Kolb, which is a nonionic C9-11 alcohol ethoxylate, having an average of 5 moles of ethylene oxide per mole of alcohol.
,. . ..
Primary ethoxy sulphates may also be used. `Y
:
Mixtures of surfactants may be used if desired.
The surfactant is preferably non-ionic or anionic, or a mixture of both types. `
, ;. ~
Preferred anionic surfactants for this purpose include ~;`;
primary~alkyl sulphates (PAS), preferably sodium dodecyl ~
sulphate (SDS). Commercial mixtures containing a ~-substantial proportion of dodecyl sulphate (eg Empicol LX) `
are espcially preferred. Dodecyl sulphate is a known -~
protein denaturant, is good for cleaning protein off ``
surfaces, and is biocidal.
~he composition is preferably substantially free of `~
cationic surfactant, but may include a minor amount of cationic germicide.
- ,, Surfactant preferably constitutes an amount in the range `~
. - ..
W094/02022 PCT/GB93/01478~
2140896 , '`'i' 0.05 to 2.5% by weight of the total weigh~ of the composition, typically 0.5% to 1.5% by weight, eg 0.7% by weight nonionic surfactant with an optional amount of up ~-to 0.2~ by weight of anionic surfactant. ~
.~ ~ .
The solvent is preferably polar an~is preferably a ~--straight or branched chain C2 to C~5 alcohol such as ethanol, butanol, isopropanol (propan-2-ol) (IPA), N-butoxy propan-2-ol (propylene qlycol n-butyl ether), 2-butoxy ethanol (ethylene glycol monobutyl ether). IPA is the currently preferred solvent.
Dihydric alcohol such as ethylene glycol, and water miscible ethers such as dimethoxyethane may also be used. -~
`: `
Mixtures of solvents can be used if appropriate, eg mi~xtures of ethanol and N-butoxy propan-2-ol.
Solvent is preferably present in an amount in the range 2 ~-~
to 20t by weight of the total weight of the composition.
: .
At least some of these solvents, eg ethanol, weaken the cell walls of micro-organisms,~making them more permeable and so more susceptible to penetration by singlet oxygen.
This has the effect of enhancing the micro-organism-~illing effect of the dyestuff.
It is found that inclusion of surfactant can reduce the photo-dynamic effect of dyestuffs (possibly by solubilising ~he dyestuff and preventing adsorption on the cell wall), and inclusion of solvent can also reduce the photo-dynamic effect of dystuffs (possibly by competing for singlet oxygen). However, it is found that with three ` ;
component formulations, comprising dyestuff, surfactant ``;-.
'~,.
: . .
:` `
W094/02022 PCT/GB93/01478 ~
21ilO896 ~' i, ~ g~
and solvent, the reduction in photo-dynamic effect of the j;:
dyestuff is less than would be expected ~rom the combined effects of surfactant and solvent. The surfac~ant and 1`~
solvent together thus have a synergistic effect, the '~A
result of which is to lower the reduction in the photo-dynamic effect of the dyestuff.
In a preferred aspect, the present invention thus provides `i a surface cleaning and germicidal composition, comprising a dyestuff which is capable of photo-dynamic lnactivation of micro-organisms, a surfactant and a solvent.
The composition may include a number of optional ingredients including ~he following: ~
1. Detergent boosters, preferably metal chelating agents --such as ethylene diamine tetra acetic acid (EDTA). Metal chelating agents (including EDTA) have also been claimèd to permeabilise cell walls, thus making organisms more susceptible to biocides.
. ..
~;
2. Electrolyte such as a buffer or salt, eg Na2S04, which acts to assist binding of dye to protein by promoting ~`
movement of dye from the aqueous~ phase to the protein salt. -Electrolyte is commonly present in various dye :~
formulations as commercially available, although -~
~additional electrolyte~can be added if required. Total `~
electrolyte content of the composition would~ypically be in the range 0 to 1~ by weight, preferably about 0.1~.
3. Perfumes.
.......
"` . .
. . ~ .
Title: Im~rovements in or Relatinq to Germicidal~
,: , Compositions ;
, . ~
Field of the Invention .
This invention relates to germicidal compositions, ,~
particularly` for use on surfaces, ie compositions capable `~
of destroying or inactivating micro-organisms, particularly surface-bound micro-organisms.
Summary of the Invention -`~
. .
In its broadest aspect the present invention provides a `
surface germicidal composition comprising a dyestuff which is capable of photo-dynamic inactivation of micro- 9'''~,``.' organisms. ~
!:
It is preferred to use a dyestuff that generates singlet oxygen on exposure to light.~ On absorption of light ~ ~-energy a dye molecule is converted to a more energetic or ~;~
exited state ~S1 ) from its electronic ground state (SO). , -(Electron spins are paired and SQ these are singlet states ! ` `
in the language of spectroscopy, having a single energy level in a magnetic field.) ,L :`. .
;`."'.
The excited state is short lived and can lose energy and return to the ground state in a number of ways: by emission of a quantum of light as fluoresecence; by internal conversion as the energy is degraded ~o heat; by collision with a molecule of a different substance , !; `
if. . :`
. .
`. :.`.
W094/02022 PCT/GB93/014781 ~
Q,~?,g6 ~
(fluorescence quenching). `
. ~
The shor~ lived singlet s~ate may also undergo a pr~cess called intersys~em crossing to a longer-lived excited state, the triplet state. (The state is termed "tripletn because the electron in the higher energy level is no longer spin-paired with the electro~uin the lower level ~`
and the excited state has three energy levels in a magnetic field.) ;~
`~
Interaction of the excited triplet state with ground state molecular oxygen (which exists in the tripLet state normally) regenerates the dye ground state as energy is transferred to the oxygen which is promoted to the .
electronically excited singlet state. This means that, ~`
under idea~l circumstances, a single photosensitiser molecule can generate many times~its own concentration of -~
singlet oxygen.
Singlet~oxyg~en is highly reactive~and~photosensitised oxidation~proceedlng via this route is known as Type II
photo-o~xida~t~ion. ~Type II photo-o~xidation is independent of the~photosensit~iser used ~o gene~rate the s~ingIet 1~
oxygen.; ~An~important féature of the sensitiser is that it ~^
shoul~d have~a h~igh~quantum~yie}d~of triplet formation (that is~ ideally;,~a triplet state~should be produced for ~`
ea~ch~photon absorbed~ Intersystém crossing to the ~-~triplet~state~; i8~ ~facil~itated~by the presence of heavy atoms in the molecule. `~
.
Photo-oxidation of any vital component of an organism may . ``
result in ce}l de~ath (protein, polypeptide, amino-acids, lipids with allylic hydrogens, tocopherols, sugars and cellulose).
, :~
~: , .
2 i ~0896 Currently preferred dys~uffs include Rose Bengal (Ac;d Red 94, Colour Index No. 4S440), Ery~hrosin B (Acid Red 51, Colour Index No. 45430), and ph~halocyanin sulphonates such as aluminium phthalocyanin sulphonate (APS) and zinc pht~. locyanin sulphonate (ZPS). Rose ~engal and Erythrosin B are known food colourants (Rose Bengal is Food Colour Red No 105 and Ery~hrosin B is Food Colour Red -`
No 14), and Erythrosin B is on an EEC list of colouring agents allowed for use in cosmetic products, so these two ~
dyes are well suited for use in compositions intended for `;
domestic use. Mixtures of dyes can be used, and in some cases it may be desirable ~o include in a mixture a dye ~
that will remain visible a~ the end of the photodynamic ~^;
process~
The concentration of dyestuff in the composition is not critical and will typically be up to lOOppm, with good results having been obtained with concentrations in the i~
~range lOppm to 20ppm. Lower concentrations, down to lppm should~ also give reasonable results. ~"
Singlet oxygen has a short lifetime and therefore a short pathlength for diffusion, 50 to be effective a ir;
photosensitising dye generating singlet oxygen must be };~
close to the target substrate. Preferred dyestuffs are ~-~
therefore substantive to (ie capable of binding to) micro-. .
organisms, typically by binding to cellular protein on the organism surface or other cellu}ar components~ eg cellular fats. `-'...
The preferred dyes men~ioned above generate singlet oxygen on exposure to light and are substantive tO protein and so capable of binding to micro-organisms via cellular pro~ein. In this way, target~ed killing of organisms and '. .:
,`'`' `:
W094/02022 PCT/GB93/01478~ ~
21~0896 hence germicidal action is possible.
It is also preferred to use a dyestuff that is bleached by ~
exposure to light. By use of a photo-bleaching dyestuff ;
that is substantive to miCro-Qrganisms itican be Qossible for a visible indication of the pres~nce of micro-organisms to be provided. As the dyestuff bleaches, the i photo-dynamic action proceeds causing the death of, or `~
otherwise inactivating, micro-organisms. In the presence of low levels of light, both bleaching and the photo- ~
dynamic activity are believed to proceed more slowly, ;
whereas at higher light intensities both processes occur more quickly. Thus, depending on the relative rates o~
bleaching and photo-dynamic activity, the presence of -visible dyestuff indicates to the user that the photo- -dynamic inactivation of any micro-organisms present is -incomplete. i ~ ~ .
The photo-dynamic inactivation of micro-organisms in suspension by dyes such as Rose Bengal is known. See, for example, Journal of Applied Bacteriology 1985, ~58] pages 391-400/ Photochemistry and Photobiology 1988, 148] pages 607-612 (Neckers et al), and Shokuhin Eiseigaku Zasshi `~
1962, 10(5), pages 344-347. However, it has now been ;~-found that suitable dyestuffs are capable of photo-dynamic inactivation of micro-organisms on surfaces, and this is `~
the basis of the present invention. It is well-known that micro-organisms are much more susceptible to biocides in their planktonic or suspended state: they are much more difficult to inactivate when attached to surf`aces, which is their usual or preferred state. Micro-organisms will normally be on surfaces in the form of "biofilms", that is, embedded in a matrix of extracellular material. This extracellular material may sometimes be referred to as -~
W094/02022 21 ~ 0 8 9 ~ PCT/GB93/01478 ~
"adhesin" in the literature. It is therefore no~ obvious that a process which acts on micro-organisms in their planktonic state would act on surface-bound organisms without modification being required. Surface-bound mlcro-organisms represent an important and substantial source of contamination in domestic, institutional and industrial ~
environments, and the present invention can enable `~-targetted germicidal action on such micro-organisms.
Compositions according to the present invention are particularly suitable for use on hard domestic and industrial surfaces such as glass, plastics, ceramic and metal surfaces. In particularly, the compositions are ~'~
effective for use on surfaces which may harbour soils having the potential for bacteriological contamination in surface imperfections, joints and other relatively confined regions.
The composition is preferably acidic, eg having a pH in the range of 3 to 5, eg a pH of about 4, as acidic s compositions are found to have substantially enhanced ;
effectiveness against Gram-negative (G-) micro-organisms `~
as~compared with neutral compositions. The effectiveness i`
against Gram-positive (G+) micro-organisms seems not to be significantly affected by pH. The compositions are conveniently made acidic by use of relative}y mild organic acid, such as àcetic acid. ~ ;~
, ~ .
Neckers et al (above) review conflicting evidence that penetration of the dye Rose Bengal itself through the cell wall is essential for inactivation. They suggest that their own results support the dye penetration hypothesis i-primarily on the basis of differential inactivation of G
and G- species. The envelope of G- bacteria has an W094/02022 PCT/GBg3/01478~ ~
2~l~o896 additional outer membrane composed substantially of lipopolysaccharide which Necker et al regard as serving as a barrier to potentially toxic substances. An alternative interpretation could be that the ~moun~ of protein exposed in the cell walls is very different ;`
between G+ and G- species, being g~eater for G+ than G-, `r'~' with a binding affinity for the dye which varies with pH.
The concentration of dye bound to the cell wall would therefore be a function of pH. This interpretation would account for our observations of the variation in kill rate i;
with pH (but not the apparent resistance of the G+ species B. subtilis and B. megaterium). -. .
There is no difference between the endospore-forming -species B. s~lbtilis and the nonspore-forming S. aureus in their susceptibilities to photodynamia action by Rose 3engal in the absence of endospores. The apparent differences shown in our studies are presumably due to the ~presence of endospores both for B. subtilis and B. `~
mégaterium~which are clearly more resistant to sing}et ``
oxyge~n than the bacteria themselves. The spores survive exposure to Rose Bengal and light and subsequently l`~
germinate to give countable colonies. Spores are known to be difficult to stain and presumably have no affinity for ~
Rose 8engal under any of the conditions used. There `
appears to be little documentation on the toxicity of --~
singlet oxygen to spores in the literature. From studies ;~
on the possibly less resistant conidia of Neurospora `~
~crassa (Photochem. Photobiol., 33, 349 (1981)),singlet ~:
oxygen does have potential in this respect.
The composition msy optionally include other ingredients such as one or more surfactants (for cleaning purposes) and/or one or more solvents.
- W094/02022 214 089 6 PCT/GB93/01478 ;~
.:
- 7 ~
:.
The surfactant is preferably alkoxylated, more preferably `
ethoxylated, eg being in the form of ethoxyla~ed al~cohols.
The alcohol preferably has between 4 and 15 carbon a~oms, 1.. `,.3~"
is of straight or branched chain configuration, and has an HLB value (hydrophilic lipophilic balance) in the range 10 to 14, eg 12.
A wide range of suitable surfactants are commercially available, one such material being the surfactant available under the trade name Imbentin 91-~5, from Kolb, which is a nonionic C9-11 alcohol ethoxylate, having an average of 5 moles of ethylene oxide per mole of alcohol.
,. . ..
Primary ethoxy sulphates may also be used. `Y
:
Mixtures of surfactants may be used if desired.
The surfactant is preferably non-ionic or anionic, or a mixture of both types. `
, ;. ~
Preferred anionic surfactants for this purpose include ~;`;
primary~alkyl sulphates (PAS), preferably sodium dodecyl ~
sulphate (SDS). Commercial mixtures containing a ~-substantial proportion of dodecyl sulphate (eg Empicol LX) `
are espcially preferred. Dodecyl sulphate is a known -~
protein denaturant, is good for cleaning protein off ``
surfaces, and is biocidal.
~he composition is preferably substantially free of `~
cationic surfactant, but may include a minor amount of cationic germicide.
- ,, Surfactant preferably constitutes an amount in the range `~
. - ..
W094/02022 PCT/GB93/01478~
2140896 , '`'i' 0.05 to 2.5% by weight of the total weigh~ of the composition, typically 0.5% to 1.5% by weight, eg 0.7% by weight nonionic surfactant with an optional amount of up ~-to 0.2~ by weight of anionic surfactant. ~
.~ ~ .
The solvent is preferably polar an~is preferably a ~--straight or branched chain C2 to C~5 alcohol such as ethanol, butanol, isopropanol (propan-2-ol) (IPA), N-butoxy propan-2-ol (propylene qlycol n-butyl ether), 2-butoxy ethanol (ethylene glycol monobutyl ether). IPA is the currently preferred solvent.
Dihydric alcohol such as ethylene glycol, and water miscible ethers such as dimethoxyethane may also be used. -~
`: `
Mixtures of solvents can be used if appropriate, eg mi~xtures of ethanol and N-butoxy propan-2-ol.
Solvent is preferably present in an amount in the range 2 ~-~
to 20t by weight of the total weight of the composition.
: .
At least some of these solvents, eg ethanol, weaken the cell walls of micro-organisms,~making them more permeable and so more susceptible to penetration by singlet oxygen.
This has the effect of enhancing the micro-organism-~illing effect of the dyestuff.
It is found that inclusion of surfactant can reduce the photo-dynamic effect of dyestuffs (possibly by solubilising ~he dyestuff and preventing adsorption on the cell wall), and inclusion of solvent can also reduce the photo-dynamic effect of dystuffs (possibly by competing for singlet oxygen). However, it is found that with three ` ;
component formulations, comprising dyestuff, surfactant ``;-.
'~,.
: . .
:` `
W094/02022 PCT/GB93/01478 ~
21ilO896 ~' i, ~ g~
and solvent, the reduction in photo-dynamic effect of the j;:
dyestuff is less than would be expected ~rom the combined effects of surfactant and solvent. The surfac~ant and 1`~
solvent together thus have a synergistic effect, the '~A
result of which is to lower the reduction in the photo-dynamic effect of the dyestuff.
In a preferred aspect, the present invention thus provides `i a surface cleaning and germicidal composition, comprising a dyestuff which is capable of photo-dynamic lnactivation of micro-organisms, a surfactant and a solvent.
The composition may include a number of optional ingredients including ~he following: ~
1. Detergent boosters, preferably metal chelating agents --such as ethylene diamine tetra acetic acid (EDTA). Metal chelating agents (including EDTA) have also been claimèd to permeabilise cell walls, thus making organisms more susceptible to biocides.
. ..
~;
2. Electrolyte such as a buffer or salt, eg Na2S04, which acts to assist binding of dye to protein by promoting ~`
movement of dye from the aqueous~ phase to the protein salt. -Electrolyte is commonly present in various dye :~
formulations as commercially available, although -~
~additional electrolyte~can be added if required. Total `~
electrolyte content of the composition would~ypically be in the range 0 to 1~ by weight, preferably about 0.1~.
3. Perfumes.
4. Thickeners. ``
- .
.
- ., .
..
W094/02022 PCT/GB93/01478~
2~40896 1 ~ _ ;
The composition is in the form of an isotropic, single phase composition and is of particular use as a germicide (possibly also with a cleaning effec~) on hard surfaces, `
finding application in a wide range of contexts, including domestic applications, eg kitchen and-bathroom surfaces -including toilet bow~s, in institu~tions such as schools, hospitals etc, and in commercial premises such as factories, offices, hotels etc.
:..
For domestic use at least, the composition is preferably formulated as a product intended for application by spraying and is conveniently packaged in a suitable container, eg having a hand operated trigger spray or an ;
aerosol propellant dispenser. The container is preferably `
light-opaque. ,~-, . ~.
In use, the composition is applied to a surface to be treated in any convenient manner, eg by spraying from a 1;~
suitable dispenser, wipinq on with a carrier such as a cloth or sponge, or pouring from a container etc. This might in some cases, particularly in industrial cleaning, be followed by exposure to a light source, eg a white ~;
light source such as a quartz halogen lamp or fluorescent ;~
"daylight~ source. (The process would be an alternative to using danqerous germicidal radiation, for example from a low pressure mercury discharge lamp emitting resonance ~--radiation at 254 nm. Such radiation is harmful to the unprotected eye.) This would genérally be followed by a `
rinsing step, if required, eg by wiping with a carrier, application of a stream of running water etc.
In a further aspect, the invention thus provides a method of killing bacteria on a surface, comprising applying to the surface a composition in accordance with the ~ ,:
W094/02022 PCT/GB93/01478 s:
21~0896 ~
invention.
The invention will be further described, by way of illus~ration, in the following Examples and by reference tO the accompanying Figures in which:`~
` ,s`
Figure 1 shows two bar charts of log (reduction) values`-i illustrating the lethal effect of Rose Bengal and light on ;-~
various micro-organisms in suspension at pH4 and pH7, with Figure la showing results for Gram-positive micro- .-organisms, and Figure lb showing results for Gram-negative `
organisms and yeasts;
.. .
Figure 2 is a pair of graphs of log (reduction) versus pH
showing the biocidal effect of Rose Bengal and light on S. `
aureus and E. coli as a function of pH, with Figure 2a.~.
showing results after 20 minutes expo~ure to light and Figure 2b showing results after 60 minutes exposure; -Figure 3 is a pair of graphs similar to Figure 2 obtained using Erythrosin B in place of Rose Bengal; :~
j~
Figure 4 shows two bar charts of ?9 ( reduction) r ~,' illustrating the photohygiene effect of various combinations of Rose Bengal (RB), Imbentin C91-35 (AE),.
isopropranol (IPA) and Empicol LX (PAS), with Figure 4a showing results obtained without exposure to light andL`
Figure 4b showing results obtained with èxposure to light; ```:
. :~
Figure 5 is a graph of adsorption of Rose Bengal by E..
coli, with amount adsorbed (nanomoles) versus equilibrium ::
concentration (micromoles/l), with results at pH 4 shown ~.
by squares and results at pH 7 shown by crosses; ':.
:
W094/02022 PCT/GB93/01478~ `
21~0896 ' ,.".,.
FiguLe 6 is a pair of graphs similar to Figure 5 showing ;.~
the effect of electrolyte, with Figure 6a showing results ;
at pH 4 and Figure 6b showing results at pH 7, with results without additive shown by squares, results with sodium sulphate (1%) shown by crosses.and results with :
sodium sulphate (5%) shown by double~;.crosses; ~.:
Figure 7 is a pair of graphs similar to Figure 5 showing the effect of surfactant, with Figure 7a showing results at pH 4 and Figure 7b showing results at pH 7, with .
results without additive shown by squares, results with ~-non-ionic surfactant (0.7%) (NI) shown by crosses and -~;
results with PAS (0.7%) shown by double crosses; .~:
Figure 8 is a graph similar to Figure 5 showing the effect of solvent at pH 4, with results without additive shown by sma}l squares, those for 10% IPA shown by crosses, those ~.
for 0.7~ Imbentin shown by double crosses and those for .`
10% IPA and 0.7% Imbentin shown by large squares;
Figure~9 is~ a qraph of log (reduction) versus exposure :time~(minutes) showing the effectof pH on thie rate of kill ~.
of E. coli by Rose Beng:al, with results at pH 4 shown by . -squares and results at pH 7 shown by crosses; and .-: Figure 10 is a graph s:imilar to Figure 9 showing the .`
: effect of electrolyte at pH 7 on the rate of kill of .-:
E.coli by Rose Bengal,:wi~th r.esults without electrolyte:i`
shown by squares and r~esults with Na sulphate shown by `.
crosses. ; .
',`':~''''.
. Examples .~..
: . , ~ ... "
.
~ , .... ... .
~ ~, ...
''.`,.'.
; .
. .
W094/02022 21~ 0 8 9 6 PCT/GB93/01478 l;~
,:
- 13 - :
EXPERIMENTAL PROCEDURES -~
~ ` .
Preparation of Inocula ~ .~
... .
~.
The following micro-organisms (generally either fro... he !'''"
National Collection of Type Cultures (NCTC) or the American Type Culture Collection (ATCC~) were used in the experiments described:
.,'`-''-Bacteria: ~:
,~."~,.
Staphylococcus aureus NCTC 6538 (Gram positive) Escherichia coli NCTC 8196 (Gram negative) "
~;
Pseudomonas aeruginosa NCTC 5940 (Gram negative) ~.
Enterobacter sp. NCTC 3281 (Gram negative) rr~.
Klebsiella sp. ATCC 11677 (Gram negative) Bacillus subtilis .NCTC 6432 (Gram positive) Bacillus megaterium NCTC 7581 (Gram positive) ;~.
Yeast:
Candida albicans j ., ~ .
Organisms were grown up by overnight incubation in . .
: nutrient broth at 37-C for bacteria (28-C for Ps. .-. .
aeruginosa) or SABS broth (SABS is Sabourand Dextrose Agar, with liquid medium in the case of SABS broth, from Oxoid ~td) at 28-C for yeast. Cultures were isolated by vacuum filtration using a 0.45um Millipore fi}ter and washed with quarter-strength Ringers solution before resuspension in Ringers solutio~n (lOml). The organisms in `~
suspension were enumerated by serial ailution and plating with nutrient aqar (bacteria) or SABS agar (yeast) and the .`
total viable count (TVC) expressed as the decadic !,~,~
- W094/02022 4089 6 PCT/GB93/01478l logarithm of the number of colony-forming units (cfu) per -~
ml.
..
Experiments were carried out at pH 7 unless otherwise specified. -~`
1 ) AGAR DIFFUSION DISC METHOD `~
Aqueous solutions containing 100 ppm of dye were prepared.
Aliquots (1Oml) of each dye solution were ste;rilized in glass universal screw cap vials. Antibiotic assay discs (13mm from BDH) were also sterilized. All organisms were `
grown overnight in nutrient or SABS broth (10ml).
For each micro-organism, two nutrient agar plates ( SABS "
agar for the yeast) were seeded with the overnight culture `
t10ul) to give confluent growth over the whole plate. -~Using aseptic techniques, an antibiotic disc was dipped `~
into the first dye solution and placed on the surface of a seeded agar plate. This was repeated with two other dye solutions to give three discs on duplicate plates. -One of each pair of duplicate plates was immediately placed in an incubator at the appropriate temperature with m~inimal exposure to light. The remaining plates were placed on top of a light box for 3 hours. Illumination `;
from the light box was diffused white light of mean intensity 4000 lux from fluorescent "daylight" tubes (2 x 15 watt, Exal X-ray Accessories Ltd, Hemel Hempstead). -~
Light intensities were measured at the surface of the ``
diffuser using a Megatron DA10 light meter. After ~`
exposure the plates were incubated overnight and then examined for zones of inhibition around the disc.
,-~
- W O 94/0202~ 2 ~ 4 08g 6 P ~ /GB93/01478 ~,',.
U~ing this approach, the following results weL-e obtained:
:
Example 1 (Disc Method) , ~.
Results on agar for aqueous dye solutions of Rose Bengal, Erythrosin B and aluminium phthalocyanin sulphonate (APS) -(100 ppm) at pH 7 after exposure to light for 180 minutes ,::
as described above are summarised in Table 1. In the Table, the results are expressed as the difference (in .
millimetres) between the radius of ~he clear zone of inhibition (the area of no bacterial growth on the spread agar plate) and the radius of the disc. Thus, the higher the value, the greater the baterial kill. .
The agar diffusion disc method ranked Rose Bengal as more ,:`
efective than the structurally similar Erythrosin B. It is tempting to ascribe this ranking to a difference in the quantum yield for singlet oxygen formation. In methanol, the quantum yield for singlet oxygen formation is 0.76 for ~
Rose Bengal compared to 0.6 for Erythrosin B. However, a .number of other factors might also be expected to ~-contribute to the observed differences such as the rate of `ii dye diffusion or differences in dye binding to the agar gel or disc material. ~`' . .
A particular feature of the results from the disc method .
is the clear distinction in the sensitivity of Gram~
positive (G+) and Gram-negative (G-) organisms to photodynamic action, at least at pH 7. Possible reasons -~for the relative resistance of G- organisms are discussed ~.`
elsewhere. ~`
F ~;
. 2) SURFACE TEST ~ `
~`'`.`.
~-~
"'~
':
.,.-`,.. ~. ., , , ,. . , . ,, :
W094/02022 PCT/GB93/01478i ~
2~0~96 Test Solutions Rose sengal (20ppm) in pH 4 b,ùffer and: `
.. `~ ' ~;' 1. No further addition ~
2. Ethanol (10 ~ v/v) and Imbentin C91-35 (0.7%). `
3. Propan-2-ol (10% v/v) and Imbentin C91-35 (0.7%). ~-Sodium hypochlorite solution (0.125 %) was used as a positive control.
Estimation of Number of Organisms Adherent to Base of -~
Petri Dish ,., Micro-organism, eg S. aureus, suspension (0.5ml) was added to aliquots (100 ml) of quarter-strength Ringers solution and the average cfu per ml determined (TVC). Aliquots ~`
(20ml) of these solutions were pipetted into sterile petri `
dishes and left at room temperature for 5 hours. The inoculum was then removed by pipette into a sterile bottle and the average cfu per ml remaining in suspension es~imated. The number of organisms (as cfu) per square cm adherent to the Petri dis~h was calculated from the differencè in the solution concentrations.
.
Test Method ~.
Bacterial suspensions in quarter-strength Ringers ~20mls) ~;
were pipetted into sterile plastic Petri dishes which were ;~`~
left at ambient temperature for 5 hours. The inoculum was then removed, the dishes washed once with Ringers solution ~
with gentle swirling by hand, and the solution poured off. ~;`-Pairs of bacterially contaminated plates were treated with ~`
W094/02022 21 ~ n 8 9 6 PCT/GB93/01478 the test solutions. An aliquot (20ml) of ~est solution was poured into one dish and ~he solution decanted after 30 seconds. The dish was rinsed with pH 4 buffer and placed on the light box for 20 mins. In a separate /~
experiment, the solution was exposed in the second dish on -~
the light box fol- 20 minutes before being decanted and the dish rinsed with pH 4 buffer. Both experiments weLe `
duplicated. -,.. ...
After light exposure, one of ~he duplica~e plates was overlaid with Tryptone Soya agar containing 1% glucose and 0.015~ Neutral Red cooled to abou~ 50C. The other ```~
duplicate plate was stained with 0.01% Acridine Orange for ~;
30 seconds, rinsed and examined microscopically (Nikon "Optiphot" microscope equipped with a 100x apochromat oil- ~;
immersion objective, 10x eyepiece and epifluorescence ;;~-`
attachment with a B2-A combined filter/dichroic mirror / ~`~
block and super high pressure mercury lamp). The overlaid ~-agar plates were incubated at 37C for 48 hours, by which time colonies had grown out of the adherent bacteria which had not been killed. The colonies took up the ~eutral Red and could be seen and counted under the agar on the surface of the dish. (A M R MacKenzie and R L Rivera-Calderon, Agar Overlay Method to Measure Adherence of Staphylococcus epidermidis to Four Plastic Surfaces, `~
App}ied and Environmental Microbiology, 50, 1322 (198S).) Plates stained with Acridine orange were examined and photographed. The number of stained bac~eria were counted in a field of view (clumps counted as one) which had previously been estimated by photographing a micrometer ~;
scale.
Example 2 (Surface Test) ``
W094/02022 PCT/G~93/01478f ~40S96 ' ~;
- 18 ~
Control experiment surfac~ tests using both direct epifluorescence microscopy as described above and suspension depletion gave similar values for the number of bacteria (S. aureus) that could,~e attached to the surface of a plastic dish (of the ord~r,~of one million per square '' centimetre). '' , ''' ., ~
Contracting the surface with the positive control (sodium ,~-,, hypochlorite solution, 30 seconds) reduced the number of '~, viable bacteria to zero. The results for photo~
dynamically inactivated bacteria are summarised in Table 2 '~
as the decadic logarithm of the reduction (log (reduction)) of viable bacteria before and after exposure, ie log (start count) - log (final count).
; ,~, . ` ',`
Example 3 . ., ~
Formulation: Rose Bengal (20 ppm), nonionic surfactant (Imbentin C91-3S, 0.7%), propan-2-ol (10 %) (pH 4). ,i"
. . .
Experimental procedure was as previously described éxcept i~
that surface attachment of the~bacteria required them to be in the exponential growth phase. This was achieved by incubation in nutrient broth (for bacteria) or SABS broth (for yeast) in the plastic Petri-dish for 3 hours. In ' Example 2, a suspension of non-growing bacteria in Ringers' solution was simply allowed to stand for 5 hours. '~
This worked well for S. aureus but not other bacteria. `'`
, The results are shown in Table 3. '~`"
'Previous experience using direct epifluorescence '~`
microscopy suggests that where confluent growth was `,'' obtained, this would be equivalent to an initial surface '`'' .
"~
, ~.. W094/0202~ PCT/GB93/01478 ~
21~0896 `~`
- 19 - '~
density of viable microbes of the order of 1 million/sq.
cm. The sur~ace area of the Petri-dishes used was abou~
57 sq.cm. so tha~ the treatmen~ in all cases has reduced the level of surface contamination by a~ leas~ 5 orders of magnitude (log 5). ~;
, ~ ,, Surface tests are more difficult to perform than ;
suspension tests, and for this reason mos~ experimental .-~
work has been performed in suspen~ion to demonstra~e the effect on varying conditions.
.~,;
3) SUSPENSION TEST
Preparation of Solutions Stock solutions of the following were prepared by weighing and sterilized (except those containing solvent): :
Rose Bengal (0.2 percent) in propan-2-ol (95 percent) Nonionic surfactant (Imbentin:C91-35, 14 percent) (sometimes referred to by ~the abbreviation AE, for alcohol ethoxylate) Anionic surfactant (Empicol LX, 14 percent) (sometimes referred to as PAS) pH 4 buffer (citric acid (0.1 M,307 ml) + dibasic sodium phosphate (0.2 M, 193 ml) :.
pH 7 bùffer (sodium dihydrogen orthophosphate (0.4M,468 ~:
ml) ~ disodium hydrogen orthophosphate dodecahydrate, (0.4M,732 ml) Buffers pH 5,6,8,9 were prepared as indicated in the CRC
Handbook of Chemistry and Physics, 8-36, 73rd Ed., CRC .
Press (1992-1993) ' Final concentrations in the test solutions were ' ~, W094/02022 PCT/GB93/01478~
i -:
2~0~96 ~.
:
typically~
.. .
Rose Bengal - 20ppm Ethanol - 10.0% (v/v) ~ ~-Surfactant - 0.7% (w/v) ~
. ' ',~,.
In some examples, different concentrations were used as specified. ``
", . ..
Test Method ~`
Test solutions were made up in sterile plastic petri dishes to a depth of 5mm (30mls). A suspensian of micro-organism tO.3ml) was added to each solution and gently mixed in. If Rose Bengal was to be included in the test solution it was added last to minimise light exposure.
Solutions were either exposed on a light box, placed in the dark (conditions of reduced light exposure) or left on the bench. The average intensity at the surface of the light~box diffuser was 4000 lux measured with~a Megatron DA 10 Iight meter tfrom Megatron Ltd). After specified `~
exposure times, surviving bacteria were enumerated as colony-forming units (cfu/ml) following incubation after ~
serial dilution and plating onto agar. The decadic ;.~, logarithm of the number of bacteria remaining (as colony- ;
forming units per ml) was determined and compared to the -~
number before exposure as log (start count) - log (final `~
count). The higher the value, the greater the bacterial `-kill~ -Suspension tests have been carried out against a variety ;
of organisms under a variety of conditions to optimise photo-dynamic action against a range of micro-organisms, including Gram-negative organisms and yeast. Salient '~
. `
`:
.
W094/02022 2 14 0 8 9 6 PCT/GB93/01478 i~;
~ .
results are summarised in the associated Tables. In these Tables results are expressed as the decadic lagarithm of the ratio of the initial number of colony-forming u~its per ml to the number remaining after exposure, the log (reduction~. Using this notàtion a value of zero means no change in the number of organisms following exposure to the conditions. The notation ~+" preceding a log reduction figure indicates that no micro-organism growth ~5 could be observed (ie total kill).
Exam~le 4 Suspension tests were carried out as described above to show the lethal effect of Rose Bengal and light on micro-organisms in suspension; particularly the synergy of low pH and ethanol solvent. Tests were carried using Rose Bengal (20ppm) alone or with ethanol (1~ (v/v), with light exposure of 20 minutes (light box). The results expressed as log (reduction) values are qiven~in Table 4. ~-Longer exposure times (of 60 and 100 minutes) improve `
performance for Gram negative organisms, part~icularly at pH 7.
It will be seen that performance for Gram negative ~`
organisms is much improved at pH 4 as compared with pH 7.
~;
Example S ~
. .
Suspension tests were carried out as described above at pH
4 and pH 7 to show the lethal ef..~ct of Rose Bengal (20ppm) and light (20 minutes exposure on a light box) of various Gram-positive and Gram-negative micro-organisms `
and yeast in suspension, and the results expressed as log :
W094/02022 PCT/GB93/01478 ' ~ ~
2~4~96 (reduction) are shown graphically in Figures la and 1b, ;
with Figure 1a giving results for Gram-positive micro- -~
organisms and Figure lb givin~ esults for Gram-negative A,.
micro-organisms and yeast. ~
... ...
... ..
Example 6 ,-. .
Suspension tests were carried out as described above at a s;`
range Oe different pHs to show the biocidal efSect of Rose Bengal ~20ppm) and light on S. aureus (G~) and E. coli (G-) as a function of pH, and the results expressed as log (reduction) are shown graphically in Figures 2a (exposure time 20 minutes) and 2b (exposure time 60 minutes). In the Figures, crosses show results for control (G-), double crosses show results for E. coli, inverted triangles show results for~control (G+) and triangles show results for S.
aureus~. X
Similar suspension tests were carr~ied out to show the `~
biocidal effect of Erythrosin B and light on S. aureus and `' E. coli as~a function of pH, and the results are shown graphically in Figures 3a and 3b, which are otherwise identical to~Figures 2a and 2b. These show that Erythr~osin~B performs similarly ~o Rose Behgal in terms of ``
its photobiocidal profile with p~
:
, .
Example 7 Further suspension tests were carried out as described ~
above at pH t using the following solutions: -Nonionic surfactant Imbentin C91-35 (0.7%) `
bentin C91-35 ~0.7%) with Rose Bengal (100 ppm) Anionic surfactant Empicol LX (0.7~
Empicol LX with Rose Bengal (100 ppm) .~:
., Imbenlin C91-35, Empicol LX (bo~h 0.7~) and Rose Bengal ;~
(lO0 ppm).
The resul~s are set OUt below in Table 5. In ~he Ta~le ~he term "dark" indica~es conditions of reduced light light exposure rather ~han total darkness because of the practical difficul~ies of avoiding some light exposure.
In the Table the term '~light~ indicates results that are the average of several experiments carried OUt: over a range of times (20 mins, 1 hour, 3 hours) in a statistically designed experiment.
Results obtained in similar manner for E. coli at pH 7 are shown graphically in the bar charts of Figure 4. In this '`~''i-~!'`'', Figure PAS is used as an abbreviation for Empicol LX, IPA
is used as an abbreviation for isopropanol and AE as an abbreviation for Imbentin C91-3S. This figure represents averaged data for AE 0.7%, PAS 0.7%, IPA lO~.
`:
Example 8 ~
"`',','`
Further suspension tests were performed at pH 4 for E.
coli using one or more of the following: Rose Bengal 40ppm, surfactant 0.7% (Imbentin C91-35 or Empicol LX), IPA (isopropanoI) 10~. Results are given in Table 6.
The following examples concern suspension tests carried out generally as described above, but at pH 4. Rose Bengal when used was present at a concentration of 20ppm, although in some cases control solutions without Rose Bengal were exposed to light and the results for these are given in the column headed UNo Rose Bengal".
. ,~..
Examp es 9, 10, 11 and 12 used the Gram positive organism ~`
S. aureus, and Examples 13 and 14 the Gram negative organism E. coli. Other reagents used are indicated in `
W094t02022 PCT/GB93/01478 o~96 `~
the examples. In all these Examples, samples were exposed for 20 minutes on a light box. The average intensity at ~he surface of the diffuser was~4000 lux measured w~lth a Megatron DA10 light meter (~om Megatron Ltd).
.~ .. , ,. ~ .
Example 9 i ,~,.
Suspension tests were carried out using Rose Bengal, ethanol and Imbentin C91-35, with S. aureus. The decadic ~
logarithm of the starting concentration, log (start), of -`;
S. aureus was 6.8. Results are given in Table 7. ;
:
Example 10 ,~.
Suspension tests were carried out using Rose Bengal, Dowanol PnB and Imbentin C91-35, with S. aureus. The log (start) was 6.9. Results are given in Table 8. ;-~ . .
This example shows that Dowanol PnB has certain biocidal ~:
properties. ~-Example 11 Suspension tests were carried out using Rose Bengal, ethylene glycol and Imbentin C91-35, with S. aureus. The ~-~
log (start) was 6.8. Results are given in Table 9. `~
.
Example 12 -Suspension tests were carried out using Rose Bengal, IPA
and Lialet 111, with S. aureus. Lialet 111 is the trade ~;
name of an ether sulphate formulation commercially ¦~
available from Enichem, having an average chain length 11 '~
W O 94/02022 2 1~ 0 8 9 6 P(~r/GB93/01478 '~
!. ~, with an average degree of ethoxylation of 3. The log (start) was 6.7. Results are given in Table 10.
Example 13 Suspension tests were carried out using Rose Bengal, propan-2-ol and Imbentin C91-35, with E. coli. The log ;~
(start) was 6.8. Results are given in Table 11.
-~.
Example 14 Suspension tests were carried out using Rose Bengal, ethanol and Imbentin C91-35, with E. coli. The log (start) was 7.1. Results are given in Table 12.
Example 15 Rose Bengal adsorption was detérmined from the depletion in solution concentration. Concentrations were obtained spectroscopically from absorbances measured at the wavelength of maximum absorbance (ca. 549 nm) using a WPA
Linton S110 spectrophotometer on supernatant liquors freed from microbes by centifugation. -~
';~
The results ar shown in Figures 5 to 8. ~;
The results show that the photobiocidal effect is ~-dependent on dye adsorption. Dye adsoprtion is:
a) increased by low pH (Figure 4); ~`~
b) increased by netural electrolyte (which also increases the photobiocidal effect at neutral pH ~-~
on E. coli) (Figure 6); -~
:, .
c) decreased by surfactant (Figure 7); ~
W094/OZ02~ 96 PCT/GB93/01478 26 ,.. ! :;
d) increased by propan-2-ol (Figure 8).
: .
Calculations show the amou~t`;adsorbed onto E. coli ig of ~he right order of magnitude for monolayer coverage, given `~
that dyes are known to aggregate and the calculated 1?'`' surface are of E. coli must be an underestimate (no ~`~
. .
account taken of fimbriae/pili). ~rief details of the ~-calculations are given below. The molecular dimensions of `-Rose Bengal were taken from a scale model (Catalin Ltd., ~`
~ondon). -', Surface area of E. Coli lE7 sq.nm Area of Rose Bengal 2 sq.nm (flat) ~`
0.5 sq.nm (side) ~.
Monolayer coverage SE6 (flat) or 20E6 (side) ,'~
Measured adsorption 2 - 8 E-16 Moles/bacteria Number of molecules 120 - 480 E6 per bacterium Example 16 i"~
.,~,.
Suspension tests were carried out as described above to determine the effect on the rate of kill of E. coli by `~
Rose Bengal (20 ppm) of pH and addition of electrolyte (Na sulphate, 5~) at pH 7, and the results are shown graphically in Figures 9 and 10, respectively. ~;
'~
~ .
` ~'`
- 27 ~
'' .
Table 1 (Example 1) .
Photobiocidal Effect of Disclosing A~ents Zone of Inhibition Around Dlsc Gram Organism Type Rose Bengal Erythrosin B APS
S. aureus .+ 4 1 2 B subtilis + 3 - 1 t B meqaterium + 3 1 1.5 :
E coii - 0 0 0 :K Pneumoniae - 1.5 0 3 :~
Ps aeruqinosa - 0 0 0 ~
Enterobacter sp. - 0 0 0 ~.
C. albicans 0 0 0 .
.
Table 2 (Example 2) ~.:
~ ., : The Lethal Effect of Rose Bengal and Light on Staphylococcus Aureus Attached to a~P astic Surface Solution Log ~Ratio) Rose Bengal 4.7 Rose Bengal + - ~
Imbentin C91-35 + 6.0 . ~-Ethanol ' W094/02022 . PCT/GB93/01478( .. -;
" ~ ~o~96 _ 28 -;
:, : ',.
. .
Table 3 (Example 3) Organism Experiment Results from Agar Overlay Technique ~i Number Control After Exposure S. aureus 1 Confluent growth No growth ~`
2 Confluent growth No growth E. coli 1 Much growth 45 cfu `
2 Confluent growth No growth .
R. pneumoniae 1 Confluent growth No growth ~.
2 Confluent growth No growth i~.
P. aeruginosa 1 Much growth 5 cfu ~-2 Conf~uent growth No growth C albicans 1 Confluent growth 5 cfu . A
2 Confluent growth 4 cfu ~-~
,; , . .
, ' :!.:
.'~.'i'~';
~ Table 4 ~Example 4) . ", ..~:
Organism Gram Type Conditions of Exposure .~i No Solvent With Ethanol ~- .
~ pH4 ~ pH4 !'~
S~ aureus + 7.0 7.1 7.1 6.9 B subtilis + 0.6 2.1 3.1 0.8 ~1 B megaterium + 1.3 0.3 1.1 0.3 -:
E. coli - 0.2 5.3 0.1 6.9 ~;~
K. pneumoniae - 0.9 5.6 0 7.0 ~:
Ps. aeruqinosa - 0 7.0 0 6.9 Enterobacter sp. - 1.1 . 7.3 0 7.4 `-~`
C. aIbicans 0 5.7 0 5.7 ::.
Controls (No Rose Bengall E. coli - 0.1 0.8 ~``
~ : .
:.,. : , .. . .
. ;`,`' '~ ~
.
Table 5 (Example 7) :
, ORGANISMIMBENTIN IMBENTIN/RB EMPICOL EMPICOL/RB ~:
light dark light dark light dark light dark St. aureus 4.5 4.0 8.0 3.5 4.5 4.5 5.5 5.0 ~-~
: ` `.
E. coli 9.0 9.5 10.5 10.0 5.5 5.0 6.5 6.0 .
Entero 0 0 0 0 0 0 0 0 Klebsiell2 7.0 6.0 6.0 7.0 6.0 6.0 6.0 6.0 Ps. aerug. 0 0 0 0 0 0 0 0 ~' ~"
C. albicans 0 0 1.0 1.0 2.5 1.5 3.0 3.0 ~:
,~
-~ ORGANISM IMBENTIN/EMPICOL IMBENTIN/EMPICOL/RB ROSE BENGAL
3 ~ lioht dark liaht dark : liqht dark ,:
St. aureus 3.0 4.0 2.0 0 4.0 0 ~`
E. coli 4.0 4.0 4.0 4-5 Entero. 0 0 0 : 0 0 0 ,, Klebsiella 3.5 3.5 4.0 2.5 0 0 . . .~
Ps. aerug. 0 0 0 0 ~C. albicans 0 0 0 0 0 0 ---~ .
`:
.~
W094/02022 PCT/GB93/01478~
~,~4~9 .~
- 30 - ~
, ~ ;, ~:, .. ~ . ~ .
Table 6 (Example 8) ; ,.
.
~ ,.
EXPOSURE TIME
1 HOUR 2:HOURS 3 HOURS
light dark light dark light dark Rose Bengal 3.8 0.5 7.2 0.4 7.2 0.7 .
RB/Imbentin C91-35 0.2 0.2 1.0 0.3 3.8 0.5 `.-RB/Empicol LX 1.7 0.4 3.9 0.7 7.2 0.7 ~ .
RB/IPA 5.4 3.6 4.7 5.0 7.2 7.2 '- ~ Imbentin C91-35~ ~ 0.1 0.3 0.9 Empicol LX : 0.1 0.6 1.0 IPA 0.3 1.7 4.4 .
: : ,~
, ,, . W094~02022 ~1 4 n 8 9 6 PCT/~B93/01478 - 31 - -:
Table_7 (Example 9) ,~
, ....
Log (reduction) ~:
Ethanol Imbentin After No % C91-35 ~ Light Exposure Rose Bengal 0.2 +6.8 150 0 66 +6 8 0.6 +6.8 - +6.8 0.1 -+6 r 8 ~ O ~ 4 - +6.8` 0.0 - 0.2 4.6 2.5 - 0.6 3.5 2.3 - ..
- - +6.8 2.3 - '.
,~, .
Table 8 (Example 10) - Log (reduction) :~: Dowanol Imbentin ~fter No ; % C91-35 % Light Exposure Rose 8engal :~ ~ 3: . :0.7 +6.9 3 - - :. +6.9 4.0 `~
- 0.7 5.2 3.4 `~
_ - +6.9 : ~.
~:
~.
WO94/02022 PCr/GB93/01478 ( 2~40~96 !~ .
-Table 9 (Example 11) ~ "~
; .
,-~ ".
.. Log keduction) ~;
Ethylene Imbentin . ~ After No Glycol % C91-35 %Light Ex-posure Rose Bengal 0-7 +6.8 ~10 - +6.9 -0.2 - ~ 0.7 . +6.8 3.4 +5.8 ~ble 1 0 (Example 12) Log ( reduction) ~.
~Propan-2-ol Lialet 111 ~After No % ~ ~ % Light Exposure Rose Bengal 5~ o 5 +6 7 0 S ~ 6 7 6 . 7 . c Table 1~1 ~Example 13~
: ~ Log ( reduct ion ) . ` `.
Propan-2-ol ~ ~ Imbentin : ~ ~ ~After: No j.;.;.:
9 6 ~C9~t-35 :~:~ Light~ Ex:posure Rose::~Bengal 5 : ~ 0 ~ *6~3 i O: ~ O ~ S + 6 . 8 o.7 \ ~6.a r~
4 ~ 8 ~ 5 ~ 5 0 ~ 3 jt. ``
1 0 ~ 3 ~ 0 1 ~ 9 0~ 2 1 ~3 0-5 1-0 ~1~2 ~
0 - 7 1 . 1 , 1 - 1 ;'~' . 1'`,`
,~ ~ c - W094/02022 PCT/GB93/01478 ~
21~08~G
- 33 - ~
::
Table 12 (Example 14) ~
. ., . . .
Log (reduction) .
EthanolImbentin After No : % C91-35 ~Light Exposure Rose Bengal 0.2 4.1 ~i.
0.6 +7.1 ,.
- 5.0 0.2 :~`
- +7.1 0.2 _ +7.1 2.2 -- 0.2 3.3 2.5 .:~
- 0.6 3.4 2.6 :~
_ - 3 9 :: .
'.''~
.~
. ~
';'~
:
- .
.
- ., .
..
W094/02022 PCT/GB93/01478~
2~40896 1 ~ _ ;
The composition is in the form of an isotropic, single phase composition and is of particular use as a germicide (possibly also with a cleaning effec~) on hard surfaces, `
finding application in a wide range of contexts, including domestic applications, eg kitchen and-bathroom surfaces -including toilet bow~s, in institu~tions such as schools, hospitals etc, and in commercial premises such as factories, offices, hotels etc.
:..
For domestic use at least, the composition is preferably formulated as a product intended for application by spraying and is conveniently packaged in a suitable container, eg having a hand operated trigger spray or an ;
aerosol propellant dispenser. The container is preferably `
light-opaque. ,~-, . ~.
In use, the composition is applied to a surface to be treated in any convenient manner, eg by spraying from a 1;~
suitable dispenser, wipinq on with a carrier such as a cloth or sponge, or pouring from a container etc. This might in some cases, particularly in industrial cleaning, be followed by exposure to a light source, eg a white ~;
light source such as a quartz halogen lamp or fluorescent ;~
"daylight~ source. (The process would be an alternative to using danqerous germicidal radiation, for example from a low pressure mercury discharge lamp emitting resonance ~--radiation at 254 nm. Such radiation is harmful to the unprotected eye.) This would genérally be followed by a `
rinsing step, if required, eg by wiping with a carrier, application of a stream of running water etc.
In a further aspect, the invention thus provides a method of killing bacteria on a surface, comprising applying to the surface a composition in accordance with the ~ ,:
W094/02022 PCT/GB93/01478 s:
21~0896 ~
invention.
The invention will be further described, by way of illus~ration, in the following Examples and by reference tO the accompanying Figures in which:`~
` ,s`
Figure 1 shows two bar charts of log (reduction) values`-i illustrating the lethal effect of Rose Bengal and light on ;-~
various micro-organisms in suspension at pH4 and pH7, with Figure la showing results for Gram-positive micro- .-organisms, and Figure lb showing results for Gram-negative `
organisms and yeasts;
.. .
Figure 2 is a pair of graphs of log (reduction) versus pH
showing the biocidal effect of Rose Bengal and light on S. `
aureus and E. coli as a function of pH, with Figure 2a.~.
showing results after 20 minutes expo~ure to light and Figure 2b showing results after 60 minutes exposure; -Figure 3 is a pair of graphs similar to Figure 2 obtained using Erythrosin B in place of Rose Bengal; :~
j~
Figure 4 shows two bar charts of ?9 ( reduction) r ~,' illustrating the photohygiene effect of various combinations of Rose Bengal (RB), Imbentin C91-35 (AE),.
isopropranol (IPA) and Empicol LX (PAS), with Figure 4a showing results obtained without exposure to light andL`
Figure 4b showing results obtained with èxposure to light; ```:
. :~
Figure 5 is a graph of adsorption of Rose Bengal by E..
coli, with amount adsorbed (nanomoles) versus equilibrium ::
concentration (micromoles/l), with results at pH 4 shown ~.
by squares and results at pH 7 shown by crosses; ':.
:
W094/02022 PCT/GB93/01478~ `
21~0896 ' ,.".,.
FiguLe 6 is a pair of graphs similar to Figure 5 showing ;.~
the effect of electrolyte, with Figure 6a showing results ;
at pH 4 and Figure 6b showing results at pH 7, with results without additive shown by squares, results with sodium sulphate (1%) shown by crosses.and results with :
sodium sulphate (5%) shown by double~;.crosses; ~.:
Figure 7 is a pair of graphs similar to Figure 5 showing the effect of surfactant, with Figure 7a showing results at pH 4 and Figure 7b showing results at pH 7, with .
results without additive shown by squares, results with ~-non-ionic surfactant (0.7%) (NI) shown by crosses and -~;
results with PAS (0.7%) shown by double crosses; .~:
Figure 8 is a graph similar to Figure 5 showing the effect of solvent at pH 4, with results without additive shown by sma}l squares, those for 10% IPA shown by crosses, those ~.
for 0.7~ Imbentin shown by double crosses and those for .`
10% IPA and 0.7% Imbentin shown by large squares;
Figure~9 is~ a qraph of log (reduction) versus exposure :time~(minutes) showing the effectof pH on thie rate of kill ~.
of E. coli by Rose Beng:al, with results at pH 4 shown by . -squares and results at pH 7 shown by crosses; and .-: Figure 10 is a graph s:imilar to Figure 9 showing the .`
: effect of electrolyte at pH 7 on the rate of kill of .-:
E.coli by Rose Bengal,:wi~th r.esults without electrolyte:i`
shown by squares and r~esults with Na sulphate shown by `.
crosses. ; .
',`':~''''.
. Examples .~..
: . , ~ ... "
.
~ , .... ... .
~ ~, ...
''.`,.'.
; .
. .
W094/02022 21~ 0 8 9 6 PCT/GB93/01478 l;~
,:
- 13 - :
EXPERIMENTAL PROCEDURES -~
~ ` .
Preparation of Inocula ~ .~
... .
~.
The following micro-organisms (generally either fro... he !'''"
National Collection of Type Cultures (NCTC) or the American Type Culture Collection (ATCC~) were used in the experiments described:
.,'`-''-Bacteria: ~:
,~."~,.
Staphylococcus aureus NCTC 6538 (Gram positive) Escherichia coli NCTC 8196 (Gram negative) "
~;
Pseudomonas aeruginosa NCTC 5940 (Gram negative) ~.
Enterobacter sp. NCTC 3281 (Gram negative) rr~.
Klebsiella sp. ATCC 11677 (Gram negative) Bacillus subtilis .NCTC 6432 (Gram positive) Bacillus megaterium NCTC 7581 (Gram positive) ;~.
Yeast:
Candida albicans j ., ~ .
Organisms were grown up by overnight incubation in . .
: nutrient broth at 37-C for bacteria (28-C for Ps. .-. .
aeruginosa) or SABS broth (SABS is Sabourand Dextrose Agar, with liquid medium in the case of SABS broth, from Oxoid ~td) at 28-C for yeast. Cultures were isolated by vacuum filtration using a 0.45um Millipore fi}ter and washed with quarter-strength Ringers solution before resuspension in Ringers solutio~n (lOml). The organisms in `~
suspension were enumerated by serial ailution and plating with nutrient aqar (bacteria) or SABS agar (yeast) and the .`
total viable count (TVC) expressed as the decadic !,~,~
- W094/02022 4089 6 PCT/GB93/01478l logarithm of the number of colony-forming units (cfu) per -~
ml.
..
Experiments were carried out at pH 7 unless otherwise specified. -~`
1 ) AGAR DIFFUSION DISC METHOD `~
Aqueous solutions containing 100 ppm of dye were prepared.
Aliquots (1Oml) of each dye solution were ste;rilized in glass universal screw cap vials. Antibiotic assay discs (13mm from BDH) were also sterilized. All organisms were `
grown overnight in nutrient or SABS broth (10ml).
For each micro-organism, two nutrient agar plates ( SABS "
agar for the yeast) were seeded with the overnight culture `
t10ul) to give confluent growth over the whole plate. -~Using aseptic techniques, an antibiotic disc was dipped `~
into the first dye solution and placed on the surface of a seeded agar plate. This was repeated with two other dye solutions to give three discs on duplicate plates. -One of each pair of duplicate plates was immediately placed in an incubator at the appropriate temperature with m~inimal exposure to light. The remaining plates were placed on top of a light box for 3 hours. Illumination `;
from the light box was diffused white light of mean intensity 4000 lux from fluorescent "daylight" tubes (2 x 15 watt, Exal X-ray Accessories Ltd, Hemel Hempstead). -~
Light intensities were measured at the surface of the ``
diffuser using a Megatron DA10 light meter. After ~`
exposure the plates were incubated overnight and then examined for zones of inhibition around the disc.
,-~
- W O 94/0202~ 2 ~ 4 08g 6 P ~ /GB93/01478 ~,',.
U~ing this approach, the following results weL-e obtained:
:
Example 1 (Disc Method) , ~.
Results on agar for aqueous dye solutions of Rose Bengal, Erythrosin B and aluminium phthalocyanin sulphonate (APS) -(100 ppm) at pH 7 after exposure to light for 180 minutes ,::
as described above are summarised in Table 1. In the Table, the results are expressed as the difference (in .
millimetres) between the radius of ~he clear zone of inhibition (the area of no bacterial growth on the spread agar plate) and the radius of the disc. Thus, the higher the value, the greater the baterial kill. .
The agar diffusion disc method ranked Rose Bengal as more ,:`
efective than the structurally similar Erythrosin B. It is tempting to ascribe this ranking to a difference in the quantum yield for singlet oxygen formation. In methanol, the quantum yield for singlet oxygen formation is 0.76 for ~
Rose Bengal compared to 0.6 for Erythrosin B. However, a .number of other factors might also be expected to ~-contribute to the observed differences such as the rate of `ii dye diffusion or differences in dye binding to the agar gel or disc material. ~`' . .
A particular feature of the results from the disc method .
is the clear distinction in the sensitivity of Gram~
positive (G+) and Gram-negative (G-) organisms to photodynamic action, at least at pH 7. Possible reasons -~for the relative resistance of G- organisms are discussed ~.`
elsewhere. ~`
F ~;
. 2) SURFACE TEST ~ `
~`'`.`.
~-~
"'~
':
.,.-`,.. ~. ., , , ,. . , . ,, :
W094/02022 PCT/GB93/01478i ~
2~0~96 Test Solutions Rose sengal (20ppm) in pH 4 b,ùffer and: `
.. `~ ' ~;' 1. No further addition ~
2. Ethanol (10 ~ v/v) and Imbentin C91-35 (0.7%). `
3. Propan-2-ol (10% v/v) and Imbentin C91-35 (0.7%). ~-Sodium hypochlorite solution (0.125 %) was used as a positive control.
Estimation of Number of Organisms Adherent to Base of -~
Petri Dish ,., Micro-organism, eg S. aureus, suspension (0.5ml) was added to aliquots (100 ml) of quarter-strength Ringers solution and the average cfu per ml determined (TVC). Aliquots ~`
(20ml) of these solutions were pipetted into sterile petri `
dishes and left at room temperature for 5 hours. The inoculum was then removed by pipette into a sterile bottle and the average cfu per ml remaining in suspension es~imated. The number of organisms (as cfu) per square cm adherent to the Petri dis~h was calculated from the differencè in the solution concentrations.
.
Test Method ~.
Bacterial suspensions in quarter-strength Ringers ~20mls) ~;
were pipetted into sterile plastic Petri dishes which were ;~`~
left at ambient temperature for 5 hours. The inoculum was then removed, the dishes washed once with Ringers solution ~
with gentle swirling by hand, and the solution poured off. ~;`-Pairs of bacterially contaminated plates were treated with ~`
W094/02022 21 ~ n 8 9 6 PCT/GB93/01478 the test solutions. An aliquot (20ml) of ~est solution was poured into one dish and ~he solution decanted after 30 seconds. The dish was rinsed with pH 4 buffer and placed on the light box for 20 mins. In a separate /~
experiment, the solution was exposed in the second dish on -~
the light box fol- 20 minutes before being decanted and the dish rinsed with pH 4 buffer. Both experiments weLe `
duplicated. -,.. ...
After light exposure, one of ~he duplica~e plates was overlaid with Tryptone Soya agar containing 1% glucose and 0.015~ Neutral Red cooled to abou~ 50C. The other ```~
duplicate plate was stained with 0.01% Acridine Orange for ~;
30 seconds, rinsed and examined microscopically (Nikon "Optiphot" microscope equipped with a 100x apochromat oil- ~;
immersion objective, 10x eyepiece and epifluorescence ;;~-`
attachment with a B2-A combined filter/dichroic mirror / ~`~
block and super high pressure mercury lamp). The overlaid ~-agar plates were incubated at 37C for 48 hours, by which time colonies had grown out of the adherent bacteria which had not been killed. The colonies took up the ~eutral Red and could be seen and counted under the agar on the surface of the dish. (A M R MacKenzie and R L Rivera-Calderon, Agar Overlay Method to Measure Adherence of Staphylococcus epidermidis to Four Plastic Surfaces, `~
App}ied and Environmental Microbiology, 50, 1322 (198S).) Plates stained with Acridine orange were examined and photographed. The number of stained bac~eria were counted in a field of view (clumps counted as one) which had previously been estimated by photographing a micrometer ~;
scale.
Example 2 (Surface Test) ``
W094/02022 PCT/G~93/01478f ~40S96 ' ~;
- 18 ~
Control experiment surfac~ tests using both direct epifluorescence microscopy as described above and suspension depletion gave similar values for the number of bacteria (S. aureus) that could,~e attached to the surface of a plastic dish (of the ord~r,~of one million per square '' centimetre). '' , ''' ., ~
Contracting the surface with the positive control (sodium ,~-,, hypochlorite solution, 30 seconds) reduced the number of '~, viable bacteria to zero. The results for photo~
dynamically inactivated bacteria are summarised in Table 2 '~
as the decadic logarithm of the reduction (log (reduction)) of viable bacteria before and after exposure, ie log (start count) - log (final count).
; ,~, . ` ',`
Example 3 . ., ~
Formulation: Rose Bengal (20 ppm), nonionic surfactant (Imbentin C91-3S, 0.7%), propan-2-ol (10 %) (pH 4). ,i"
. . .
Experimental procedure was as previously described éxcept i~
that surface attachment of the~bacteria required them to be in the exponential growth phase. This was achieved by incubation in nutrient broth (for bacteria) or SABS broth (for yeast) in the plastic Petri-dish for 3 hours. In ' Example 2, a suspension of non-growing bacteria in Ringers' solution was simply allowed to stand for 5 hours. '~
This worked well for S. aureus but not other bacteria. `'`
, The results are shown in Table 3. '~`"
'Previous experience using direct epifluorescence '~`
microscopy suggests that where confluent growth was `,'' obtained, this would be equivalent to an initial surface '`'' .
"~
, ~.. W094/0202~ PCT/GB93/01478 ~
21~0896 `~`
- 19 - '~
density of viable microbes of the order of 1 million/sq.
cm. The sur~ace area of the Petri-dishes used was abou~
57 sq.cm. so tha~ the treatmen~ in all cases has reduced the level of surface contamination by a~ leas~ 5 orders of magnitude (log 5). ~;
, ~ ,, Surface tests are more difficult to perform than ;
suspension tests, and for this reason mos~ experimental .-~
work has been performed in suspen~ion to demonstra~e the effect on varying conditions.
.~,;
3) SUSPENSION TEST
Preparation of Solutions Stock solutions of the following were prepared by weighing and sterilized (except those containing solvent): :
Rose Bengal (0.2 percent) in propan-2-ol (95 percent) Nonionic surfactant (Imbentin:C91-35, 14 percent) (sometimes referred to by ~the abbreviation AE, for alcohol ethoxylate) Anionic surfactant (Empicol LX, 14 percent) (sometimes referred to as PAS) pH 4 buffer (citric acid (0.1 M,307 ml) + dibasic sodium phosphate (0.2 M, 193 ml) :.
pH 7 bùffer (sodium dihydrogen orthophosphate (0.4M,468 ~:
ml) ~ disodium hydrogen orthophosphate dodecahydrate, (0.4M,732 ml) Buffers pH 5,6,8,9 were prepared as indicated in the CRC
Handbook of Chemistry and Physics, 8-36, 73rd Ed., CRC .
Press (1992-1993) ' Final concentrations in the test solutions were ' ~, W094/02022 PCT/GB93/01478~
i -:
2~0~96 ~.
:
typically~
.. .
Rose Bengal - 20ppm Ethanol - 10.0% (v/v) ~ ~-Surfactant - 0.7% (w/v) ~
. ' ',~,.
In some examples, different concentrations were used as specified. ``
", . ..
Test Method ~`
Test solutions were made up in sterile plastic petri dishes to a depth of 5mm (30mls). A suspensian of micro-organism tO.3ml) was added to each solution and gently mixed in. If Rose Bengal was to be included in the test solution it was added last to minimise light exposure.
Solutions were either exposed on a light box, placed in the dark (conditions of reduced light exposure) or left on the bench. The average intensity at the surface of the light~box diffuser was 4000 lux measured with~a Megatron DA 10 Iight meter tfrom Megatron Ltd). After specified `~
exposure times, surviving bacteria were enumerated as colony-forming units (cfu/ml) following incubation after ~
serial dilution and plating onto agar. The decadic ;.~, logarithm of the number of bacteria remaining (as colony- ;
forming units per ml) was determined and compared to the -~
number before exposure as log (start count) - log (final `~
count). The higher the value, the greater the bacterial `-kill~ -Suspension tests have been carried out against a variety ;
of organisms under a variety of conditions to optimise photo-dynamic action against a range of micro-organisms, including Gram-negative organisms and yeast. Salient '~
. `
`:
.
W094/02022 2 14 0 8 9 6 PCT/GB93/01478 i~;
~ .
results are summarised in the associated Tables. In these Tables results are expressed as the decadic lagarithm of the ratio of the initial number of colony-forming u~its per ml to the number remaining after exposure, the log (reduction~. Using this notàtion a value of zero means no change in the number of organisms following exposure to the conditions. The notation ~+" preceding a log reduction figure indicates that no micro-organism growth ~5 could be observed (ie total kill).
Exam~le 4 Suspension tests were carried out as described above to show the lethal effect of Rose Bengal and light on micro-organisms in suspension; particularly the synergy of low pH and ethanol solvent. Tests were carried using Rose Bengal (20ppm) alone or with ethanol (1~ (v/v), with light exposure of 20 minutes (light box). The results expressed as log (reduction) values are qiven~in Table 4. ~-Longer exposure times (of 60 and 100 minutes) improve `
performance for Gram negative organisms, part~icularly at pH 7.
It will be seen that performance for Gram negative ~`
organisms is much improved at pH 4 as compared with pH 7.
~;
Example S ~
. .
Suspension tests were carried out as described above at pH
4 and pH 7 to show the lethal ef..~ct of Rose Bengal (20ppm) and light (20 minutes exposure on a light box) of various Gram-positive and Gram-negative micro-organisms `
and yeast in suspension, and the results expressed as log :
W094/02022 PCT/GB93/01478 ' ~ ~
2~4~96 (reduction) are shown graphically in Figures la and 1b, ;
with Figure 1a giving results for Gram-positive micro- -~
organisms and Figure lb givin~ esults for Gram-negative A,.
micro-organisms and yeast. ~
... ...
... ..
Example 6 ,-. .
Suspension tests were carried out as described above at a s;`
range Oe different pHs to show the biocidal efSect of Rose Bengal ~20ppm) and light on S. aureus (G~) and E. coli (G-) as a function of pH, and the results expressed as log (reduction) are shown graphically in Figures 2a (exposure time 20 minutes) and 2b (exposure time 60 minutes). In the Figures, crosses show results for control (G-), double crosses show results for E. coli, inverted triangles show results for~control (G+) and triangles show results for S.
aureus~. X
Similar suspension tests were carr~ied out to show the `~
biocidal effect of Erythrosin B and light on S. aureus and `' E. coli as~a function of pH, and the results are shown graphically in Figures 3a and 3b, which are otherwise identical to~Figures 2a and 2b. These show that Erythr~osin~B performs similarly ~o Rose Behgal in terms of ``
its photobiocidal profile with p~
:
, .
Example 7 Further suspension tests were carried out as described ~
above at pH t using the following solutions: -Nonionic surfactant Imbentin C91-35 (0.7%) `
bentin C91-35 ~0.7%) with Rose Bengal (100 ppm) Anionic surfactant Empicol LX (0.7~
Empicol LX with Rose Bengal (100 ppm) .~:
., Imbenlin C91-35, Empicol LX (bo~h 0.7~) and Rose Bengal ;~
(lO0 ppm).
The resul~s are set OUt below in Table 5. In ~he Ta~le ~he term "dark" indica~es conditions of reduced light light exposure rather ~han total darkness because of the practical difficul~ies of avoiding some light exposure.
In the Table the term '~light~ indicates results that are the average of several experiments carried OUt: over a range of times (20 mins, 1 hour, 3 hours) in a statistically designed experiment.
Results obtained in similar manner for E. coli at pH 7 are shown graphically in the bar charts of Figure 4. In this '`~''i-~!'`'', Figure PAS is used as an abbreviation for Empicol LX, IPA
is used as an abbreviation for isopropanol and AE as an abbreviation for Imbentin C91-3S. This figure represents averaged data for AE 0.7%, PAS 0.7%, IPA lO~.
`:
Example 8 ~
"`',','`
Further suspension tests were performed at pH 4 for E.
coli using one or more of the following: Rose Bengal 40ppm, surfactant 0.7% (Imbentin C91-35 or Empicol LX), IPA (isopropanoI) 10~. Results are given in Table 6.
The following examples concern suspension tests carried out generally as described above, but at pH 4. Rose Bengal when used was present at a concentration of 20ppm, although in some cases control solutions without Rose Bengal were exposed to light and the results for these are given in the column headed UNo Rose Bengal".
. ,~..
Examp es 9, 10, 11 and 12 used the Gram positive organism ~`
S. aureus, and Examples 13 and 14 the Gram negative organism E. coli. Other reagents used are indicated in `
W094t02022 PCT/GB93/01478 o~96 `~
the examples. In all these Examples, samples were exposed for 20 minutes on a light box. The average intensity at ~he surface of the diffuser was~4000 lux measured w~lth a Megatron DA10 light meter (~om Megatron Ltd).
.~ .. , ,. ~ .
Example 9 i ,~,.
Suspension tests were carried out using Rose Bengal, ethanol and Imbentin C91-35, with S. aureus. The decadic ~
logarithm of the starting concentration, log (start), of -`;
S. aureus was 6.8. Results are given in Table 7. ;
:
Example 10 ,~.
Suspension tests were carried out using Rose Bengal, Dowanol PnB and Imbentin C91-35, with S. aureus. The log (start) was 6.9. Results are given in Table 8. ;-~ . .
This example shows that Dowanol PnB has certain biocidal ~:
properties. ~-Example 11 Suspension tests were carried out using Rose Bengal, ethylene glycol and Imbentin C91-35, with S. aureus. The ~-~
log (start) was 6.8. Results are given in Table 9. `~
.
Example 12 -Suspension tests were carried out using Rose Bengal, IPA
and Lialet 111, with S. aureus. Lialet 111 is the trade ~;
name of an ether sulphate formulation commercially ¦~
available from Enichem, having an average chain length 11 '~
W O 94/02022 2 1~ 0 8 9 6 P(~r/GB93/01478 '~
!. ~, with an average degree of ethoxylation of 3. The log (start) was 6.7. Results are given in Table 10.
Example 13 Suspension tests were carried out using Rose Bengal, propan-2-ol and Imbentin C91-35, with E. coli. The log ;~
(start) was 6.8. Results are given in Table 11.
-~.
Example 14 Suspension tests were carried out using Rose Bengal, ethanol and Imbentin C91-35, with E. coli. The log (start) was 7.1. Results are given in Table 12.
Example 15 Rose Bengal adsorption was detérmined from the depletion in solution concentration. Concentrations were obtained spectroscopically from absorbances measured at the wavelength of maximum absorbance (ca. 549 nm) using a WPA
Linton S110 spectrophotometer on supernatant liquors freed from microbes by centifugation. -~
';~
The results ar shown in Figures 5 to 8. ~;
The results show that the photobiocidal effect is ~-dependent on dye adsorption. Dye adsoprtion is:
a) increased by low pH (Figure 4); ~`~
b) increased by netural electrolyte (which also increases the photobiocidal effect at neutral pH ~-~
on E. coli) (Figure 6); -~
:, .
c) decreased by surfactant (Figure 7); ~
W094/OZ02~ 96 PCT/GB93/01478 26 ,.. ! :;
d) increased by propan-2-ol (Figure 8).
: .
Calculations show the amou~t`;adsorbed onto E. coli ig of ~he right order of magnitude for monolayer coverage, given `~
that dyes are known to aggregate and the calculated 1?'`' surface are of E. coli must be an underestimate (no ~`~
. .
account taken of fimbriae/pili). ~rief details of the ~-calculations are given below. The molecular dimensions of `-Rose Bengal were taken from a scale model (Catalin Ltd., ~`
~ondon). -', Surface area of E. Coli lE7 sq.nm Area of Rose Bengal 2 sq.nm (flat) ~`
0.5 sq.nm (side) ~.
Monolayer coverage SE6 (flat) or 20E6 (side) ,'~
Measured adsorption 2 - 8 E-16 Moles/bacteria Number of molecules 120 - 480 E6 per bacterium Example 16 i"~
.,~,.
Suspension tests were carried out as described above to determine the effect on the rate of kill of E. coli by `~
Rose Bengal (20 ppm) of pH and addition of electrolyte (Na sulphate, 5~) at pH 7, and the results are shown graphically in Figures 9 and 10, respectively. ~;
'~
~ .
` ~'`
- 27 ~
'' .
Table 1 (Example 1) .
Photobiocidal Effect of Disclosing A~ents Zone of Inhibition Around Dlsc Gram Organism Type Rose Bengal Erythrosin B APS
S. aureus .+ 4 1 2 B subtilis + 3 - 1 t B meqaterium + 3 1 1.5 :
E coii - 0 0 0 :K Pneumoniae - 1.5 0 3 :~
Ps aeruqinosa - 0 0 0 ~
Enterobacter sp. - 0 0 0 ~.
C. albicans 0 0 0 .
.
Table 2 (Example 2) ~.:
~ ., : The Lethal Effect of Rose Bengal and Light on Staphylococcus Aureus Attached to a~P astic Surface Solution Log ~Ratio) Rose Bengal 4.7 Rose Bengal + - ~
Imbentin C91-35 + 6.0 . ~-Ethanol ' W094/02022 . PCT/GB93/01478( .. -;
" ~ ~o~96 _ 28 -;
:, : ',.
. .
Table 3 (Example 3) Organism Experiment Results from Agar Overlay Technique ~i Number Control After Exposure S. aureus 1 Confluent growth No growth ~`
2 Confluent growth No growth E. coli 1 Much growth 45 cfu `
2 Confluent growth No growth .
R. pneumoniae 1 Confluent growth No growth ~.
2 Confluent growth No growth i~.
P. aeruginosa 1 Much growth 5 cfu ~-2 Conf~uent growth No growth C albicans 1 Confluent growth 5 cfu . A
2 Confluent growth 4 cfu ~-~
,; , . .
, ' :!.:
.'~.'i'~';
~ Table 4 ~Example 4) . ", ..~:
Organism Gram Type Conditions of Exposure .~i No Solvent With Ethanol ~- .
~ pH4 ~ pH4 !'~
S~ aureus + 7.0 7.1 7.1 6.9 B subtilis + 0.6 2.1 3.1 0.8 ~1 B megaterium + 1.3 0.3 1.1 0.3 -:
E. coli - 0.2 5.3 0.1 6.9 ~;~
K. pneumoniae - 0.9 5.6 0 7.0 ~:
Ps. aeruqinosa - 0 7.0 0 6.9 Enterobacter sp. - 1.1 . 7.3 0 7.4 `-~`
C. aIbicans 0 5.7 0 5.7 ::.
Controls (No Rose Bengall E. coli - 0.1 0.8 ~``
~ : .
:.,. : , .. . .
. ;`,`' '~ ~
.
Table 5 (Example 7) :
, ORGANISMIMBENTIN IMBENTIN/RB EMPICOL EMPICOL/RB ~:
light dark light dark light dark light dark St. aureus 4.5 4.0 8.0 3.5 4.5 4.5 5.5 5.0 ~-~
: ` `.
E. coli 9.0 9.5 10.5 10.0 5.5 5.0 6.5 6.0 .
Entero 0 0 0 0 0 0 0 0 Klebsiell2 7.0 6.0 6.0 7.0 6.0 6.0 6.0 6.0 Ps. aerug. 0 0 0 0 0 0 0 0 ~' ~"
C. albicans 0 0 1.0 1.0 2.5 1.5 3.0 3.0 ~:
,~
-~ ORGANISM IMBENTIN/EMPICOL IMBENTIN/EMPICOL/RB ROSE BENGAL
3 ~ lioht dark liaht dark : liqht dark ,:
St. aureus 3.0 4.0 2.0 0 4.0 0 ~`
E. coli 4.0 4.0 4.0 4-5 Entero. 0 0 0 : 0 0 0 ,, Klebsiella 3.5 3.5 4.0 2.5 0 0 . . .~
Ps. aerug. 0 0 0 0 ~C. albicans 0 0 0 0 0 0 ---~ .
`:
.~
W094/02022 PCT/GB93/01478~
~,~4~9 .~
- 30 - ~
, ~ ;, ~:, .. ~ . ~ .
Table 6 (Example 8) ; ,.
.
~ ,.
EXPOSURE TIME
1 HOUR 2:HOURS 3 HOURS
light dark light dark light dark Rose Bengal 3.8 0.5 7.2 0.4 7.2 0.7 .
RB/Imbentin C91-35 0.2 0.2 1.0 0.3 3.8 0.5 `.-RB/Empicol LX 1.7 0.4 3.9 0.7 7.2 0.7 ~ .
RB/IPA 5.4 3.6 4.7 5.0 7.2 7.2 '- ~ Imbentin C91-35~ ~ 0.1 0.3 0.9 Empicol LX : 0.1 0.6 1.0 IPA 0.3 1.7 4.4 .
: : ,~
, ,, . W094~02022 ~1 4 n 8 9 6 PCT/~B93/01478 - 31 - -:
Table_7 (Example 9) ,~
, ....
Log (reduction) ~:
Ethanol Imbentin After No % C91-35 ~ Light Exposure Rose Bengal 0.2 +6.8 150 0 66 +6 8 0.6 +6.8 - +6.8 0.1 -+6 r 8 ~ O ~ 4 - +6.8` 0.0 - 0.2 4.6 2.5 - 0.6 3.5 2.3 - ..
- - +6.8 2.3 - '.
,~, .
Table 8 (Example 10) - Log (reduction) :~: Dowanol Imbentin ~fter No ; % C91-35 % Light Exposure Rose 8engal :~ ~ 3: . :0.7 +6.9 3 - - :. +6.9 4.0 `~
- 0.7 5.2 3.4 `~
_ - +6.9 : ~.
~:
~.
WO94/02022 PCr/GB93/01478 ( 2~40~96 !~ .
-Table 9 (Example 11) ~ "~
; .
,-~ ".
.. Log keduction) ~;
Ethylene Imbentin . ~ After No Glycol % C91-35 %Light Ex-posure Rose Bengal 0-7 +6.8 ~10 - +6.9 -0.2 - ~ 0.7 . +6.8 3.4 +5.8 ~ble 1 0 (Example 12) Log ( reduction) ~.
~Propan-2-ol Lialet 111 ~After No % ~ ~ % Light Exposure Rose Bengal 5~ o 5 +6 7 0 S ~ 6 7 6 . 7 . c Table 1~1 ~Example 13~
: ~ Log ( reduct ion ) . ` `.
Propan-2-ol ~ ~ Imbentin : ~ ~ ~After: No j.;.;.:
9 6 ~C9~t-35 :~:~ Light~ Ex:posure Rose::~Bengal 5 : ~ 0 ~ *6~3 i O: ~ O ~ S + 6 . 8 o.7 \ ~6.a r~
4 ~ 8 ~ 5 ~ 5 0 ~ 3 jt. ``
1 0 ~ 3 ~ 0 1 ~ 9 0~ 2 1 ~3 0-5 1-0 ~1~2 ~
0 - 7 1 . 1 , 1 - 1 ;'~' . 1'`,`
,~ ~ c - W094/02022 PCT/GB93/01478 ~
21~08~G
- 33 - ~
::
Table 12 (Example 14) ~
. ., . . .
Log (reduction) .
EthanolImbentin After No : % C91-35 ~Light Exposure Rose Bengal 0.2 4.1 ~i.
0.6 +7.1 ,.
- 5.0 0.2 :~`
- +7.1 0.2 _ +7.1 2.2 -- 0.2 3.3 2.5 .:~
- 0.6 3.4 2.6 :~
_ - 3 9 :: .
'.''~
.~
. ~
';'~
:
Claims (19)
1. A surface germicidal composition effective against surface-attached microorganisms, comprising a dyestuff which is capable of photo-dynamic inactivation of micro-organisms.
2. A composition according to claim 1, wherein the dyestuff generates singlet oxygen an exposure to light.
3. A composiiton according to claim 1 or 2, wherein the dyestuff f is substantive to micro-organisms.
4. A composition according to claim 1, 2 or 3, wherein the dyestuff is bleached by exposure to light.
5. A composition according to any one of the preceding claims, wherein the dyestuff is selected from the group comprising Rose Bengal, Erythrosin B and phthalocyanin sulphonates.
6. A composition according to any one of the preceding claims, wherein dyestuff is present in an amount in the range 1 to 100 ppm.
7. A composition according to any one of the preceding claims, further comprising one or more surfactants.
8. A composition according to claim 7, wherein the surfactant is alkoxylated.
9. A composition according to claim 8, wherein the surfactant is ethoxylated.
10. A composition according to claim 7, 8 or 9, wherein the surfactant is at least predominantly non-ionic and/or anionic.
11. A composition according to any one of claims 7 to 10, wherein surfactant is present in an amount in the range 0.05 to 2.5% by weight of the total weight of the composition.
12. A composition according to any one of the preceding claims, further comprising one or more solvents.
13. A composition according to claim 12, wherein the solvent is polar.
14. A composition according to claim 13, wherein the solvent is a straight or branched chain C2 to C5 alcohol.
15. A composition according to claim 12, 13 or 14, wherein solvent is present in an amount in the range 2 to 20% by weight of the total weight of the composition.
16. A composition according to any one of the preceding claims, having a pa in the range 3 to 5.
17. A composition according to claim 16, having a pH of about 4.
18. A surface cleaning and germicidal composition, effective against surface-attached microorganisms, comprising a dyestuff which is capable of photo-dynamic inactivation of micro-organisms, a surfactant and a solvent.
19. A method of killing surface-attached microorganisms, comprising applying to the surface a composition in accordance with any one of the preceding claims.
Applications Claiming Priority (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB929215555A GB9215555D0 (en) | 1992-07-22 | 1992-07-22 | Improvements relating to cleaning compositions |
| GB9215555.5 | 1992-07-22 | ||
| GB929222813A GB9222813D0 (en) | 1992-10-30 | 1992-10-30 | Cleaning compositions |
| GB9222813.9 | 1992-10-30 | ||
| GB939304732A GB9304732D0 (en) | 1993-03-09 | 1993-03-09 | Improvements in or relating to germicidal compositions |
| GB9304732.2 | 1993-03-09 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2140896A1 true CA2140896A1 (en) | 1994-01-23 |
Family
ID=27266295
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002140896A Abandoned CA2140896A1 (en) | 1992-07-22 | 1993-07-14 | Improvements in or relating to germicidal compositions |
Country Status (15)
| Country | Link |
|---|---|
| EP (1) | EP0652709B1 (en) |
| JP (1) | JP3133336B2 (en) |
| KR (1) | KR100252797B1 (en) |
| CN (1) | CN1086255A (en) |
| AU (1) | AU4577493A (en) |
| BR (1) | BR9306767A (en) |
| CA (1) | CA2140896A1 (en) |
| CZ (1) | CZ14595A3 (en) |
| DE (1) | DE69324015T2 (en) |
| ES (1) | ES2130276T3 (en) |
| HU (1) | HUT70688A (en) |
| PL (1) | PL173758B1 (en) |
| SK (1) | SK6495A3 (en) |
| TW (1) | TW272114B (en) |
| WO (1) | WO1994002022A1 (en) |
Families Citing this family (20)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5679661A (en) * | 1995-07-25 | 1997-10-21 | The Procter & Gamble Company | Low hue photodisinfectants |
| US8974363B2 (en) | 1997-12-11 | 2015-03-10 | Provectus Pharmatech, Inc. | Topical medicaments and methods for photodynamic treatment of disease |
| US8557298B2 (en) | 1998-08-06 | 2013-10-15 | Provectus Pharmatech, Inc. | Medicaments for chemotherapeutic treatment of disease |
| US6420455B1 (en) * | 1999-06-18 | 2002-07-16 | 3M Innovative Properties Company | Antimicrobial composition containing photosensitizers articles, and methods of use |
| US6905672B2 (en) * | 1999-12-08 | 2005-06-14 | The Procter & Gamble Company | Compositions and methods to inhibit tartar and microbes using denture adhesive compositions with colorants |
| FR2853239B1 (en) * | 2003-04-01 | 2010-01-29 | Oreal | USE OF COMPOSITIONS COMPRISING A FLUORESCENT COLORANT AND A PARTICULARLY AMPHOTERIC OR NON-IONIC SURFACTANT FOR COLORING WITH A LIGHTENING EFFECT OF HUMAN KERATINIC MATERIALS |
| US20050059731A1 (en) * | 2003-09-16 | 2005-03-17 | Ceramoptec Industries, Inc. | Erythrosin-based antimicrobial photodynamic therapy compound and its use |
| GB0525504D0 (en) | 2005-12-14 | 2006-01-25 | Bristol Myers Squibb Co | Antimicrobial composition |
| US8673836B2 (en) * | 2007-03-20 | 2014-03-18 | The Procter & Gamble Company | Laundry detergent composition with a reactive dye |
| US20100266716A1 (en) * | 2007-10-25 | 2010-10-21 | Olson Merle E | Natural Photodynamic Agents and their use |
| DE102008020755A1 (en) * | 2008-04-18 | 2009-10-22 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Use of photosensitizer for cleaning air, water or contaminated surfaces and in filters, where the photosensitizer is covalently linked and/or linked over physical interaction to surface and activated by visible light to form singlet oxygen |
| JP5457447B2 (en) | 2008-07-10 | 2014-04-02 | スリーエム イノベイティブ プロパティズ カンパニー | Viscoelastic light guide |
| JP2011530718A (en) | 2008-08-08 | 2011-12-22 | スリーエム イノベイティブ プロパティズ カンパニー | Light guide with viscoelastic layer for managing light |
| GB0823265D0 (en) | 2008-12-20 | 2009-01-28 | Convatec Technologies Inc | Antimicrobial Composition |
| GB0901434D0 (en) | 2009-01-29 | 2009-03-11 | Univ Strathclyde | Ballast water treatment system |
| BRPI1011746A2 (en) | 2009-06-25 | 2018-02-06 | 3M Innovative Properties Co | light-activated antimicrobial articles and methods of use |
| WO2011008441A1 (en) | 2009-06-30 | 2011-01-20 | 3M Innovative Properties Company | Light-activated antimicrobial article and method of use |
| GB201020236D0 (en) | 2010-11-30 | 2011-01-12 | Convatec Technologies Inc | A composition for detecting biofilms on viable tissues |
| MX2015007771A (en) | 2012-12-20 | 2015-09-04 | Convatec Technologies Inc | Processing of chemically modified cellulosic fibres. |
| CN111328952B (en) * | 2020-03-03 | 2023-04-25 | 四川大学 | Photodynamic sterilization method for acidic food |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2387658A1 (en) * | 1977-03-25 | 1978-11-17 | Ciba Geigy Ag | PROCEDURE FOR FIGHTING MICROORGANISMS |
| US4497741A (en) * | 1981-12-09 | 1985-02-05 | Ciba-Geigy Corporation | Water-soluble zinc and aluminium phthalocyanines |
| FR2613626B1 (en) * | 1987-04-07 | 1990-12-14 | Bbc Brown Boveri & Cie | METHOD AND DEVICE FOR DISINFECTING UTENSILS |
-
1993
- 1993-07-14 ES ES93916071T patent/ES2130276T3/en not_active Expired - Lifetime
- 1993-07-14 CA CA002140896A patent/CA2140896A1/en not_active Abandoned
- 1993-07-14 EP EP93916071A patent/EP0652709B1/en not_active Expired - Lifetime
- 1993-07-14 BR BR9306767A patent/BR9306767A/en not_active IP Right Cessation
- 1993-07-14 JP JP06504251A patent/JP3133336B2/en not_active Expired - Fee Related
- 1993-07-14 KR KR1019950700238A patent/KR100252797B1/en not_active IP Right Cessation
- 1993-07-14 HU HU9500176A patent/HUT70688A/en unknown
- 1993-07-14 CZ CZ95145A patent/CZ14595A3/en unknown
- 1993-07-14 DE DE69324015T patent/DE69324015T2/en not_active Expired - Fee Related
- 1993-07-14 SK SK64-95A patent/SK6495A3/en unknown
- 1993-07-14 AU AU45774/93A patent/AU4577493A/en not_active Abandoned
- 1993-07-14 PL PL93307168A patent/PL173758B1/en unknown
- 1993-07-14 WO PCT/GB1993/001478 patent/WO1994002022A1/en active IP Right Grant
- 1993-07-15 TW TW082105640A patent/TW272114B/zh active
- 1993-07-22 CN CN93116562A patent/CN1086255A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| PL307168A1 (en) | 1995-05-15 |
| JPH07509236A (en) | 1995-10-12 |
| DE69324015D1 (en) | 1999-04-22 |
| EP0652709A1 (en) | 1995-05-17 |
| EP0652709B1 (en) | 1999-03-17 |
| WO1994002022A1 (en) | 1994-02-03 |
| PL173758B1 (en) | 1998-04-30 |
| KR100252797B1 (en) | 2000-04-15 |
| DE69324015T2 (en) | 1999-08-05 |
| SK6495A3 (en) | 1995-07-11 |
| ES2130276T3 (en) | 1999-07-01 |
| HUT70688A (en) | 1995-10-30 |
| CN1086255A (en) | 1994-05-04 |
| CZ14595A3 (en) | 1995-10-18 |
| JP3133336B2 (en) | 2001-02-05 |
| AU4577493A (en) | 1994-02-14 |
| BR9306767A (en) | 1998-12-08 |
| KR950702386A (en) | 1995-07-29 |
| HU9500176D0 (en) | 1995-03-28 |
| TW272114B (en) | 1996-03-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0652709B1 (en) | Improvements in or relating to germicidal compositions | |
| EP0631610B1 (en) | Improvements in or relating to cleaning compositions | |
| US5863882A (en) | Cleaner and sanitizer formulation | |
| US5403587A (en) | Disinfectant and sanitizing compositions based on essential oils | |
| US20160168384A1 (en) | Porphyrinoid components, method and apparatus for water photodisinfection | |
| US20170275572A1 (en) | Compositions for photodynamic control of infection | |
| HU221137B1 (en) | Synergistic antimicrobial cleaning composition and method of treating surfaces using it | |
| CA2227763A1 (en) | Low hue photodisinfectants | |
| CN1155900A (en) | Stable liquid enzyme compositions and methods of use in contact lens cleaning and disinfecting systems | |
| Brovko et al. | Photodynamic treatment: a novel method for sanitation of food handling and food processing surfaces | |
| US6004510A (en) | Articles, compositions and process for cleaning surfaces by use of a catalyst at the surface | |
| CA2219279A1 (en) | Stable liquid enzyme compositions and methods of use | |
| EP1379286B1 (en) | Composition and method for reducing odor and disinfecting | |
| GB2331703A (en) | Disinfectant compositions | |
| JPH04231054A (en) | Two-component mixed type composition of quaternary ammonia compound polymer and enzyme for washing and sterilization of contact lens | |
| KR20010091645A (en) | Antibacterial and deordorant compositions for hygiene of bath room | |
| WO2021072255A1 (en) | Light activated nanoparticle compositions and uses thereof | |
| Pompermayer et al. | EFFECT OF HYPOCHLORITE ON MIXED CULTURES OF STAPHYLOCOCCUS AUREUS AND ESCHERICHIA COLI ADHERING TO POLYPROPYLENE SURFACES | |
| CS238946B1 (en) | Liquid disinfecting detergent | |
| JPH06145008A (en) | Cleansing sterilizer for industry |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Dead |