This invention relates to an antimicrobial indicator composition, particularly to a colourchange indicator composition, which indicates the presence and absence of live microbes and particularly to a visual indicator composition containing or for use with an antimicrobial agent. The invention also relates to a method of indicating the presence of microbes on a substrate and determining the absence of live microbes. The invention relates, in particular, to an antimicrobial indicator composition comprising an antimicrobial agent and a colour-change indicator. The indicator composition is useful in providing an indication of the presence of microbes, for example bacteria, on a surface or a substrate by changing colour upon contact with the microbes, cleaning or disinfecting the surface or substrate and indicating absence of live microbes.

The presence of microbes, for example bacteria, fungi, yeast and viruses, in a wide range of environments and circumstances presents a hazard to public health, particularly in an environment where food is present, in the home environment and in healthcare establishments such as hospitals and clinics and certain industrial settings especially where microbial, particularly bacterial, quality control is important. Antimicrobial compositions are also widely used in personal care applications and as hygiene products, for example as hand wash and hand sanitiser products. In some cases, drug resistance to pathogens presents a particular threat to public health, especially in hospitals. Common bacteria often found in the home, commercial or industrial environments, particularly in kitchens and bathrooms include Pseudomonas aeruginosa; Escherichia coli; Staphylococcus aureus; Enterococcus hirae; Salmonella typhimorium; Enterobacter cloacae; Lactobacillus brevis; Listeria monocytogenes; Campylobacter; Clostridium perfringens; Bacillus cereus, methicillin-resistant Staphylococcus aureus among other siderophore -producing bacteria.

The prevention of bacterial contamination in domestic environments requires antimicrobial products and/or disinfectant products, which typically provide a high level, for example at least 99.99%, of antimicrobial action or efficacy, that are effective against different microbial species, that are safe during use and may have residual bacterial action to protect surfaces. However, the user may apply an antimicrobial agent to a surface, for example in the home or a hospital or in a commercial and industrial setting, but not be sure as to the efficacy of the product and whether the antimicrobial agent has rendered inactive any microbes present. Viruses also present a significant hazard to public health, examples of such viruses including common cold viruses, influenza viruses, coronaviruses including sever acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and SARS-CoV-2. Viruses are not typically present in isolation but in combination with other microbes/bacteria.

Cleaning products, disinfectants and antimicrobial agents are used to clean and to kill pathogenic organisms and microbes on surfaces and substrates. Such products may be used as homecare products to clean or disinfect hard surfaces in a wide range of environments including in commercial, medical and domestic settings, for example in bathrooms, kitchens and areas requiring a high degree of cleanliness such as in food preparation, hospitals, clinics, sterile or “clean” rooms in a research or manufacturing context and the like. Cleaning products are formulated to have certain properties depending on the intended use. For example, bathroom products typically have a pH of 3 or lower and contain chelating agents for removal of limescale. Kitchen products may be strongly alkaline, with a pH above 10, to aid certain cleaning functions, for example to saponify fats. Other cleaning products may contain organic cleaning agents, for example anionic, amphoteric and non-ionic surfactants and may be slightly acidic, alkaline or of neutral pH. Examples of known antibacterial agents in cleaning products include didecyldimethyl ammonium chloride (DDAC), alkyl dimethyl benzyl ammonium chloride (ADBAC), benzalkonium chloride (BAG), hydrogen peroxide, phenoxyethanol, chlorhexidine, triclosan, lactic acid, and formic acid and other agents, for example as listed in https://www.intechopen.com/chapters/55303 and https://www.sciencedirect.com/topics/chemistrv/antibacterial -aqent.

Certain microbes produce a component which may be secreted to bind a metal ion for use in biological processes in the microbe, thereby removing the metal ion from its environment. For example, gram positive and gram negative bacterial strains, plants, cyanobacteria and some fungal species excrete siderophores to sequester iron ions, typically Iron III (Fe ³⁺ ), for use in metabolic processes within the bacteria. A wide range of siderophores are known, including catecholates and phenolates, hydroxamates and hydroxycarboxylates. The affinity of a siderophore for iron III is dependent on pH with different siderophores binding more or less strongly at a higher or lower pH.

There are several technologies for detecting microbes, for example bacteria, however these are often strain specific, require specific chemical environments, require incubation high concentrations of microbes or are fluorescent and so require a UV light source for detection. Such systems may be sensitive to the presence of certain components, for example chelating agents, and be sensitive or rendered inactive at certain pHs. Known products for providing a visual indication of microbes accordingly have certain drawbacks limitations or complexity.

Some compositions are known which claim to provide an indication of the presence of microbes and a cleaning effect in a single product, employing a surfactant. There remains a need to provide an indicator composition for visually showing the presence of microbes on a surface and providing an antibacterial effect to kill the microbes rather than merely a cleaning effect, without compromising the indicator and which is stable during storage.

We have now devised an antimicrobial indicator composition which has an antibacterial effect and provides a visual indication of the presence or absence of microbes, especially bacteria.

In a first aspect, the invention provides an antimicrobial visual-indicator composition comprising a colour-change component, a metal ion and an antimicrobial component wherein the molar ratio of the metal ion to antimicrobial component is 1 to greater than 7.

Advantageously, the antimicrobial indicator composition according to the invention enables the detection of microbes by providing a visual indication by changing colour upon contact with live microbes. The antibacterial indicator composition may then be removed and a further quantity of the composition applied to the surface. A change of colour is indicative of live microbes being present on the surface. By repeating this process until there is no change of colour, the surface may then be considered as substantially free of live microbes at a level which would trigger a change of colour.

Accordingly, in another aspect, the invention provides a method of determining the presence or absence of live microbes comprising: i) applying an antimicrobial indicator composition according to the invention to a surface and observing the composition and upon observing a colour-change in the composition, removing the composition from the surface; ii) applying a further quantity of the composition to the same surface, observing the composition and: a. in the event of colour change, removing the composition from the surface and repeating step i); and b. In the event of no colour change, concluding that the surface is substantially free of live microbes.

The antimicrobial indicator composition of the invention suitably is effective against a broad spectrum of microbes, especially against a broad spectrum of bacteria.

The composition of the invention is suitably able to detect and visualise the presence of the main bacteria found in the particular environment such as in a domestic, commercial, industrial, medical and manufacturing environment, for example, at home in kitchens, bathrooms, lavatories and other functional areas such as utility or washing rooms, and in clinics, hospitals, commercial or industrial kitchens and the like.

Advantageously, the composition provides a real time indicator of the antimicrobial agent action on the surface to which it is applied and allows the user to observe the presence and/or absence of living microbes, while not affecting the antimicrobial performance of the antimicrobial agent. The composition of the invention avoids the need for additional packaging or complex further steps such as the use of fluorescence or magnification means to determine the presence of live microbes.

The composition may be used on any inorganic surface, for example a hard surface in any type of environment where absence of live microbes is important or an organic surface for example human or animal skin, to provide an antimicrobial effect with a colour-change to indicate the presence of live microbes. The composition of the invention is especially suited for use on a hard surface. Upon application, the composition of the invention indicates the presence of microbes by a colour change of the composition on the surface. A further quantity of the composition may then be applied to the surface until no colour-change is observed, indicating the absence of live microbes. The user may then be confident that the surface has been effectively cleaned and sterilised of microbes. This is particularly important in areas of food preparation, hygiene, health care, clean rooms and other environments where microbiological control or sterility is important, for example in hospitals.

The composition suitably also does not stain a surface to which it is applied and is stable during storage. Preferably, the antimicrobial indicator composition is homogeneous. Suitably, the composition provides a large specific absorptivity and a low detection limit to provide optimal sensitivity and response enabling low levels of microbes to be detected and for the visual indication to become apparent, preferably readily apparent under ambient lighting, to the human eye relatively rapidly through a colour-change. In an alternative embodiment, the colour-change component may provide a change in the ultra-violet spectrum, detectable by application of ultra-violet light to the surface being treated.

The antimicrobial indicator composition may comprise any metal ion which is used in a biological process of a microbe such that upon contact with the microbe the metal ion is capable of release from the composition for uptake in the microbe. Suitably, the metal ion is present in the composition as a metal ion complex or upon binding to the component from the microbe is capable of forming a complex such that appearance, disappearance or a change in colour occurs when the composition is applied to a surface.

The component of the microbe may comprise a range of functional groups, or be of a particular class of compound, for example carboxylate groups or carboxylic acids. The metal ion is suitably selected according to the anticipated microbes with which the composition is to be used. The composition may comprise a plurality of different metal ions for interaction with different components from different or the same microbes.

There are many siderophores present in a wide range of bacteria and some fungi, for example enterobactin, aerobactin, salmochelin, staphyloferrin A, desferrioxamine B, pyoverdine. Siderophores may be analysed using the method described at pages 48-49 in ANALYTICAL BIOCHEMISTRY 160,47-56 (1987) and may be employed to check the presence or level of siderophores.

Preferably, the composition comprises Iron III (Fe ³⁺ ) ions.

The metal ion is suitably provided as a salt, optionally a hydrated salt. Iron III (Fe ³⁺ ) ions are suitably provided as Iron (III) chloride, preferably as the hexahydrate.

The colour-change component is capable of binding the metal ion in the antimicrobial indicator composition. Preferably, the colour-change component comprises a polybasic acid, more preferably a polybasic carboxylic acid, especially C.l. Mordant Blue 29, known as Chrome Azurol S or CAS. Suitably, the colour change component may exist in a plurality of prototropic forms, such that the colour of the composition may differ according to pH. CAS is a polybasic acid and has six different structural forms according to the pH value of the solution. The colour of these charged species changes with variation in hydrogen-ion concentration. CAS may be used for the quantitative and qualitative determination of a variety of metal ions. CAS is sensitive but nonselective and reacts to form red, blue, or green complexes with many metal ions according to the pH value of the solution.

The composition is suitably formulated such that the composition has a pH of 2 to 5, preferably 3 to 5. The composition may be formulated as an alkaline composition provided the metal ion is stabilised such that it does not precipitate, suitably having a pH of 10 to 13, preferably 11 to 12, for example for use in medical, kitchen and food preparation areas. As iron ions may precipitate to form iron hydroxide at an alkaline around pH, where the metal ion comprises Iron III (Fe ³⁺ ), the composition suitably has a pH of under 8 and especially 3 to 5.

The antibacterial indicator composition of the invention may comprise a buffer system to control the pH of the composition to provide a stable formulation.

Suitably, the composition comprises a coloured complex which changes colour on removal of a component of the complex. Suitably, the complex comprises the metal ion, the colour-change component and the antimicrobial component and optionally, one or more other components of the composition, for example non-ionic surfactants, Examples of suitable non-ionic surfactants include polysorbates, glucosides, particularly alkyl polyglucosides, alcohol initiated alkylene oxides particularly fatty alcohols with 9 to 18 carbon atoms and 5 to 20 alkylene oxide, especially ethylene oxide, units. Removal of the metal ion for example Iron III (Fe ³⁺ ) ions, by preferential binding to a component of the microbe for example siderophore causes a colour change, for example from blue to pink, blue to green, blue to yellow and purple or pink to yellow, depending on the pH, other components in the composition and the component of the microbe, for example a siderophore.

For the metal ion to be available for transfer to the component of the microbe, the affinity constant for the metal ion and the component of the microbe is greater than the affinity constant of the complex of the colour-change component, antimicrobial component and the metal ion in the particular conditions for example the pH of use.

European Standards EN 1276 (Quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants & antiseptics used in food, industrial, domestic and institutional areas) and EN 13697 (Quantitative non-porous surface test for the evaluation of bactericidal and/or fungicidal activity of chemical disinfectants used in the food, industrial and institutional areas) provide standard tests for the assessment of antibacterial and antifungal activity which a component or composition must meet to be considered for use in a food, industrial, domestic and institutional area. Compositions according to the invention suitably meet the standard required to be classified as a cleaner or disinfectant, by killing 99.999% of microbes, within 5 minutes. Similar regulations applicable in USA or other countries for defining antibacterial and/or disinfectant efficacy may also provide a standard test for determining antibacterial or antifungal efficacy. Regulations also exist for other product forms, for example hand wash and sanitiser and the like and their effect, in a range of countries.

The antibacterial component is present in the composition at a level to provide an antibacterial effect and preferably a disinfectant effect. The antimicrobial indicator composition of the present invention comprises the antibacterial component at such a level that the composition may be classed as providing an antibacterial effect and preferably a disinfectant effect.

Suitably the antibacterial component comprises one or more antibacterial components capable of forming a complex with the colour-change component and the metal ion. The antimicrobial component preferably comprises one or more of a quaternary ammonium compound, formic acid, phenoxyethanol, DDAC, BAG, ADBAC, chlorhexidine, triclosan, hydrogen peroxide and lactic acid which has an antimicrobial effect. Suitably, the antimicrobial component provides an antibacterial or antifungal effect sufficient to be considered as an antibacterial and more preferably, a disinfectant, in accordance with regulations such as European Standard EN 1276 and EN 13697 or similar regulations applicable in the USA or other countries for defining antibacterial and/or disinfectant efficacy.

Preferably, the antimicrobial component comprises one or more of a tetraalkyl quaternary ammonium compound, for example a tetraalkyl ammonium halide and an aryl trialkyl ammonium halide. Preferably the alkyl and aryl groups are independently selected from -CH ₂ R where, for each substituent, R is independently selected from H, aryl, for example phenyl, and alkyl. Where -CH ₂ R is an alkyl group, R is suitably selected from H and Ci to C ₄₀ , more preferably Ci to C ₂₂ alkyl group, for example methyl, decyl dodecyl and hexadecyl alkyl groups.

Preferably the tetraalkyl ammonium compound comprises 2 or 3 methyl groups, and at least 1 alkyl group having 10 or more carbon atoms and preferably at least 2 alkyl group having 10 or more carbon atoms, for example didecyldimethyl ammonium species. An example of a preferred aryltrialkyl ammonium compound is a benzalkonium species, for example benzalkonium chloride.

The quaternary ammonium compound suitably has a halide counterion, preferably a chloride or bromide ion, especially chloride.

DDAC and BAC are especially preferred as the antimicrobial agent as they are nonstrain specific and thus will act on a wide range of common microbes.

The composition may comprise one or more antibacterial components including a quaternary ammonium compound as defined above, formic acid, lactic acid, DDAC, BAC, ADBAC, hydrogen peroxide and phenoxyethanol provided they do not materially adversely interact with the colour-change component and metal ion. In one embodiment, the antimicrobial component comprises DDAC and one or more of BAC, lactic acid and formic acid, preferably DDAC and BAC. Preferably, the antibacterial component does not form a precipitate with the any other components in the composition.

Suitably, the antimicrobial component(s) is present at a level of 0.001 to less than 10%, preferably less than 7% of the composition, more preferably 0.1 to 5%, especially 0.1 to 5%, for example 1 to 5%.

Suitably the metal ion is present at a level of 0.0001 to 2% of the composition, preferably 0.0005 to 1%, more preferably 0.001 to 0.1% of the composition.

Suitably the colour-change component is present at a level of 0.001 to 2% of the composition, preferably 0.005 to 1%, more preferably 0.01 to 0.1% of the composition. The molar ratio of the colour-change component to the antimicrobial component is suitably from 1 :1 to 1:500, preferably from 1:10 to 1:300, for example 1 :220.

The molar ratio of the metal ion to the colour-change component is suitably from 1:20 to 5:1, preferably from 1 :10 to 1:1, more preferably from 1 :5 to 1:1 , for example 1 :5, 1 :4, 1 :3, 1:2 and 1 :1.

The molar ratio of the metal ion to the antibacterial component is suitably 1 : greater than 7, preferably 1 :to greater than 50, especially 1 to greater than 300, preferably from 1 : to greater than 500, particularly from 1:500 to 1 :5000, more preferably from 1 :500 to 1 :2000, especially 1:600 to 1 :1200, for example 1 :700 to 1000 and especially 1 :750 to 1000. Lower ratios, for example 1 to greater than 7 and less than 500 may be preferred provided the colour-change complex is stabilised at a high pH where the metal ion may otherwise precipitate, for example by encapsulation.

The invention provides in another aspect, an antimicrobial visual-indicator composition comprising a colour-change component, a metal ion and an antimicrobial component wherein the molar ratio of the metal ion to antimicrobial component is 1 to greater than 7 and less than 500 and the colour-change complex is stabilised at a pH where the metal ion otherwise would precipitate.

In a preferred embodiment, the invention provides an antimicrobial visual-indicator composition comprising a colour-change component, a metal ion and an antimicrobial component wherein the molar ratio of the metal ion and antimicrobial component is 1 :greater than 500, the molar ratio of the colour-change component and the antimicrobial component is from 1 :1 to 500, the molar ratio of the metal ion and the colour-change component is from 1 :20 to 5:1 , the antimicrobial component is selected from a didecyldimethyl ammonium salt, a benzalkonium salt, phenoxyethanol, formic acid, chlorhexidine, triclosan and lactic acid, the colour-change component comprises Chrome Azurol S and the metal ion comprises Iron III (Fe ³⁺ ).

The level of the components in this preferred composition is suitably in the range for these components recited above.

In addition to the components of the antimicrobial composition listed in the first aspect of the invention, the composition may further comprise one or more cleaning components provided the component or the level of the component does not adversely interact with the metal ion, colour-change component and anti-bacterial component or are otherwise not compatible with the composition. The cleaning component may provide a detergency effect, a degreasing effect, a limescale removal function or other effects according to the intended use of the composition. The composition may comprise one or more of a surfactant, soap, chelating agent and/or a rheology modifier.

Where present and provided it does not adversely affect the antimicrobial component, the colour-change component and the metal ions especially metal ion mobility, such as to render the composition ineffective as an indicator or antimicrobial composition, the surfactant is present at a level of about 0.1 to about 40%, preferably from about 1 to about 20%, optimally from about 1 to about 5% by weight of the composition. The surfactant may be selected from the group consisting of anionic, non-ionic, cationic and amphoteric surfactants and alcohols.

Particularly preferred non-ionic surfactants are those with a C10-C20 fatty alcohol or acid hydrophobe condensed with from 2 to 100 moles of ethylene oxide or propylene oxide per mole of hydrophobe; C2-C10 alkyl phenols condensed with from 2 to 20 moles of alkylene oxide; mono- and di-fatty acid esters of ethylene glycol; fatty acid monoglyceride; sorbitan, mono- and di- C8-C20 fatty acids; and polyoxyethylene sorbitan as well as combinations thereof. Alkyl polyglycosides and saccharide fatty amides (e.g. methyl gluconamides) are also suitable non-ionic surfactants.

Preferred anionic surfactants include soap, alkyl ether sulfates and sulfonates, alkyl sulfates and sulfonates, alkylbenzene sulfonates, alkyl and dialkyl sulfosuccinates, C8- C20 acyl isethionate, C8-C20 alkyl ether phosphates, C8-C20 sarcosinates and combinations thereof.

Any known rheology modifier may be employed in the composition. Examples of suitable rheology modifiers include a non-ionic surfactant, glycol, a polyalkylene glycol such as polyethylene glycol and polypropylene glycol, polymer such as hydroxypropyl methyl cellulose, and xanthan gum.

Suitably, the metal ion may be present in the composition at a weight ratio relative to the surfactant and rheology modifier of 1 to up to 20000, preferably 1 to less than 10000, Preferably, the weight ratio of the metal ion relative to the surfactant and rheology modifier is 1 to at least 100, preferably 1 to at least 1000.

Suitably the surfactant is present in the composition at a level up to 10% by weight, preferably 5% or less and especially 1 to less than 5%.

The rheology modifier is suitably present in the composition at a level of 0.05 to 1% by weight of the composition, preferably from 0.1 to 0.5%.

The composition may comprise further functional components provided they do not adversely interact with the metal ion, colour-change component and antibacterial component. Further functional components may be selected from known components for antimicrobial or cleaning products and include colourant, perfume, solubiliser, for example to aid solubility or homogeneity of the composition.

The composition according to the invention suitably comprises a solvent. The solvent may comprise water, alcohol, for example ethanol and/or other solvents conventionally employed in home care, personal care, hygiene applications or other antimicrobial applications. In one embodiment, the solvent comprises water as the major component and, preferably is solely water whereby the composition is an aqueous solution. In another embodiment, the solvent may comprise an alcohol as the major component, for example in a hand sanitiser or hand wash product. The solvent may comprise water and ethanol, preferably in a ratio by volume of 1 :99 to 99:1 , for example 70:30 to 30:70. The composition may be diluted with water at the point of use, as desired.

Suitably, the composition is stable at a temperature of 4°C, preferably at a temperature of 0°C, and more preferably at a temperature of -5°C. The composition is suitably stable at a temperature of 40°C and more preferably at 50°C. The composition suitably is stable and does not change for at least 7 days, preferably 28 days and more preferably 3 months or more. Stability may be determined by measuring the absorption spectrum of the composition on formation and periodically checking the absorption spectrum to determine any change in absorptivity and lambda max.

The antimicrobial indicator composition according to the invention may be employed in identifying and killing microbes of any type which excrete a component to bind to the metal ion of the composition and, in so doing, remove the metal ion from the composition and cause a colour-change. In a preferred embodiment, the antimicrobial indicator composition of the invention is suitable for use in indicating the presence of and killing bacteria which excrete siderophores to attract Iron III, for example Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus hirae, Enterococcus faecium, Salmonella typhimurium, Lactobacillus brevis and Enterobacter cloacae, methicillin-resistant Staphylococcus aureus among other siderophore-producing bacteria.

The composition is preferably non-toxic to humans and animals. Suitably, upon application of the composition to a surface, the surface is ready for use or human contact with no or minimal further cleaning within a relatively short period of time. Upon application to the surface, the composition is suitably left on the surface for a period of time to allow colour-change to occur if microbes are present on the surface. Suitably, colour-change occurs within 10 minutes, more preferably within 5 minutes, especially within 1 minute and optimally within 20 seconds of application to a surface, indicating the presence of microbes.

The antimicrobial indicator composition of the invention may be applied directly to a surface in any suitable form, for example as a liquid, a foam and as a spray, or applied to the surface indirectly, for example by application of the antimicrobial indicator composition to a cloth and wiping the surface with the cloth to transfer the composition to the substrate. Preferably, the antimicrobial indicator composition of the invention is sprayable. The composition may be capable of being formed into a spray or mist, for example using a conventional spray container with a nozzle or may be in the form of an aerosol.

In another embodiment, the antimicrobial indicator composition may be present on or impregnated in a substrate and the substrate used to apply the composition to the surface. The composition may be applied neat or dilute or applied using a substrate which has been pre-wetted with water or another solvent to aid application of the composition to the surface.

The substrate may comprise a natural or man-made fabric, fibre or other non-woven material. Suitably, the substrate comprises a paper or fabric comprising any known material used in surface wipes or cloths. Suitably, the substrate comprises one or more of cellulose, lignin, protein, alginate, chitosan, polyester, acrylic, nylon, aramid, polyurethane, poly-e-lysine, cross-linked poly-e-lysine and/or mixtures thereof, for example lignocellulosic fibres. The substrate may comprise pulp, wool, silk, jute, linen, ramie, sisal, bagasse, banana fibres, hemp, flax, camel hair, kenaf and/or mixtures thereof.

Suitably, the substrate is biodegradable. The substrate may comprise an antimicrobial component for example poly-e-lysine, cross-linked poly-s-lysine, in addition to the antimicrobial indicator composition.

The substrate comprising the antimicrobial indicator composition is suitably in the form of a cloth, a “wipe”, a towel, a tissue or other hand-held product or as a component of a cleaning implement, for example a floor mop. The substrate is suitably adapted for usage on a surface, for example a hard surface or on the body of a human or animal as a cleaning, antiseptic or disinfectant product such as a skin wash or sanitiser, soap product or the like.

The invention also provides the use of a colour-change component, for example CAS, in an antimicrobial composition to provide a colour-change effect to indicate the presence of microbes, for example bacteria, and the absence of live microbes wherein the antibacterial composition comprises an antibacterial component and a metal ion wherein the molar ratio of the metal to antibacterial component is greater than 1 :500. The colourchange component may be mixed with the antimicrobial composition prior to application to a surface or may be mixed in-situ on the surface by application of the colour-change component to the surface and the antimicrobial composition to the surface whereupon, if microbes are present on the surface, the mixture of the colour-change component and the antimicrobial composition changes colour.

All percentages and parts are parts by weight and all measurements made are at 25° C, unless otherwise stated or made clear by the context. Ratios are molar ratios unless otherwise stated or made clear by the context.

The invention is illustrated by the following non-limiting examples.

EXAMPLES

Example 1

A composition according to the invention was prepared by forming a CAS-Fe complex as set out below. All equipment glassware and containers used in producing the composition was washed with detergent, rinsed several times with deionised water, soaked for 24 hours in a solution containing 1% HCI (37%) and rinsed again multiple times with deionised water.

A solution of the complex CAS-Fe was prepared by forming Solutions A and B to the following formulations:

1. Solution A: A solution containing 1.78 g/L CAS in deionised water.

2. Solution B: A solution containing 0.24 g/L FeCI ₃ .6H ₂ O in 12mM HCI.

The CAS-Fe complex was prepared by slowly adding Solution A to Solution B with constant agitation in a 1 :1 weight ratio. This solution is referred to as Solution C.

An aqueous solution containing 6.0% (w/w) Benzalkonium Chloride (BAC) was prepared (Solution D). The antibacterial colour change composition according to the invention was prepared by mixing in a 1 :2 weight ratio of Solution C and Solution D to provide a final composition as set out in Table 1..

Table 1

The ratio of components in the composition is set out in Table 2.

Table 2

The solution is a purple/blue colour with (l _max =630nm. The absorption spectrum of the composition is shown in Figure 1. Upon contact with bacteria, this solution changes colour to green and yellow indicating the presence of bacteria. Upon wiping the composition from the surface, a further quantity of the composition is applied and a colour change indicates the presence of live bacteria. The surface may be repeatedly cleaned until the colour change is reduced or ceases, indicating the absence of live bacteria.

Example 2

An aqueous solution containing 4.8% (w/w) Benzalkonium Chloride (BAC) and 3.0% (w/w) didecyldimethyl ammonium chloride (DDAC) was prepared (Solution E). An antibacterial colour change composition according to the invention was prepared by mixing in a 1:2 weight ratio of Solution C in Example 1 and Solution E to provide a final composition as set out in Table 3.

Table 3

The ratio of components in the composition is set out in Table 4.

Table 4

The solution is a deep blue colour.

Upon contact with bacteria, this solution changes colour to green, yellow and pink indicating the presence of bacteria. Upon wiping the composition from the surface, a further quantity of the composition is applied and a colour change indicates the presence of live bacteria. The surface may be repeatedly cleaned until the colour change is reduced or ceases, indicating the absence of live bacteria.

Comparative Example 3

An aqueous solution containing 6.33% (w/w) Polysorbate 80 and 0.05% hexadecyl trimethyl ammonium bromide was prepared (Solution F).

An antibacterial colour change composition was prepared by mixing in a 1 :2 weight ratio Solution C in Example 1 and Solution F to provide a final composition as set out in Table 5.

Table 5

The ratio of components in the composition is set out in Table 6.

Table 6

The solution is a deep blue colour. Upon contact with bacteria, this solution changes colour to green, yellow and pink indicating the presence of bacteria. Upon wiping the composition from the surface, a further quantity of the composition is applied and a colour change indicates the presence of live bacteria. Upon repeated application, live bacteria remain. The HDTMA does not provide an adequate antimicrobial effect at the concentration in Comparative Example 3. Example 4

An aqueous solution containing 6.0% (w/w) Benzalkonium Chloride (BAC) and 7.5% non-ionic surfactant, a C alcohol initiated ethyoxylate with 9 ethylene oxide units, was prepared (Solution G). An antibacterial colour change composition according to the invention was prepared by mixing in a 1 :2 weight ratio of Solution C in Example 1 and Solution G to provide a final composition as set out in Table 7.

Table 7

The ratio of components in the composition is set out in Table 8.

Table 8

The solution is a purple/ blue colour. Upon contact with bacteria, this solution changes colour to green and yellow indicating the presence of bacteria. Upon wiping the composition from the surface, a further quantity of the composition is applied and a colour change indicates the presence of live bacteria. The surface may be repeatedly cleaned until the colour change is reduced or ceases, indicating the absence of live bacteria,