EVALUATION OF BACTERIAL GROWTH ON VARIOUS MATERIALS
Eckhard Voß¹, Christian Störch^2
1 Wendel GmbH, Am Güterbahnhof, 35683 Dillenburg, Germany
²Waldstraße 28, 35745 Herborn, Germany

Introduction
In this lecture, the impact of silver containing enamel on bacterial survival rates is reported. The influence of silver containing enamel is compared with the analogous characteristics of stainless steel, ceramics and thermoplastic materials. Furthermore, the impact of nano-coating (Crystal Guard as produced by the company Chemetall) onto enamel has been evaluated. These materials are mainly used for sink units in private household and public buildings. Enamel is also often applied in hot water boilers used for the heating of drinking water supplies. Particularly in this area of application, the inhibition of bacterial growth can be of great advantage. Although, the water that is fed into the drinking water supply network is generally not contaminated with any harmful micro-organisms, an increase in the number of organisms can occur as a result of water remaining in the tubing and the boilers over a longer period of time. If water remains in the pipelines and boilers of such systems, micro-organisms construct a bio-film in which they may be capable to resist the actions of commonly used disinfectant agents. For our experiments, two-indicator bacteria - Enterococcus faecium and Escherichia coli – and a typical water inhabitant germ – Pseudomonas aeruginosa – were used as indicators

Germ reduction
Basically, the methods are classified according to the degree of their detrimental impact on the micro-organisms. If a method is suited to obtain total destruction of all micro-organisms, the method is designated as "sterilisation." A disinfect ion is a method which results in the reduction of the number of germs to a level which is no longer capable of causing infections in humans. Furthermore, we differentiate between methods that show a bactericide action, meaning that by these methods the bacteria are actually destroyed, and those that are bacteriostatic. In the latter cases, "merely" the proliferation of the bacteria is inhibited. The latter type of methods is mainly used for conservation purposed in food technology and the cosmetics industry [15].
The effectiveness of a method is indicated the abbreviation "RF." This indication refers to the number of logarithmic levels by which the number of the micro-organisms has been decreased in comparison to their initial concentration [7, 32]. Cleaning procedures can result in a diminution of 0.4 to 1 RF [7]. In some cases, such cleaning procedures prove to be quite adequate to obtain the set goals. Before applying a disinfecting procedure or a sterilisation procedure, simple cleaning procedures are always indicated [14].
Physical influences can be detrimental to micro-organisms to such an extent, that they are no longer capable of reproducing or simply die.
Among the thermal procedures, the application of dry heat in a hot-air cupboard or the application of heat under pressure in an autoclave are well-known methods. These procedures are extremely effective, and the amount of reduction in respect to the number of germs is high enough to consider these methods comparable to sterilisation. If disinfecting procedures satisfy the requirements, the product is boiled or submitted to water vapour at 100 degrees Centigrade. In connection with food materials, a method designated as "Pasteurisation" is usually applied. Depending on what variation of this method is used, food is subjected to temperatures between 62 and 150 oC for various lengths of time. This method really is a compromise between the need for the elimination of bacteria and the requirement to retain the original characteristics of the food materials that are to be treated.
Another method consists of eliminating the micro-organisms by submitting them to high-energy radiation. This can be achieved with UV-Radiation from a special lamp or by the application of ionised radiation from radioactive substances.
In recent times microwaves have been also been used, especially for the decontamination of waste materials [7].
On the other hand, there are a number of chemical disinfecting procedures. In this group, one can use oxidants such as Ozone, potassium permanganate and hydrogen peroxide. These chemicals act through the oxidation of the structural proteins and the enzymes, thereby being toxic to the cells.
Especially the SH-Groups of the enzyme proteins are denaturised through oxidation, and after that, they are no longer available for the metabolism of the cell [7]. Another important group of oxidants consist of Chlorine and the other halogens. Yet another group is made up out of Potassium hydroxide and sodium hydroxide. These two chemicals act as disinfecting agents, whereby the OH- group is the active component of the molecules.
For the purpose of disinfecting surfaces and instruments, aldehydes can be used. Alcohols, on the other hand, are applied when disinfecting of skin areas and other surfaces [4, 7].

Silver
The use of silver against diseases and for the preservation of drinking water is nothing new. The application of silver has a tradition of about 2500 years. It is known that the Persian King Cyrus had his stock of boiled drinking water transported in silver containers to protect it from turning foul. About 100 years ago, in Europe, the bacteriological treatment of drinking water with silver started. However, the method is very expensive and for that reason it is no longer used in larger water purification plants [15, 24]. Today, the addition of silver to water is used only for the preservation of drinking water when travelling in tropical areas or for the conservation of drinking water in containers in trains and on ships [4, 5, 15]. In addition, methods have been developed that combine the fast action of chlorine with the conserving capacities of silver.

The bactericidal properties of silver are referred to as oligo-dynamic. This term indicates that silver is active even in very low concentrations [4, 5, 15, 17]. The inhibition or the destruction of the microorganisms has its cause in the formation of free ions which absorb at the cell surface and react with the SH groups of the enzymes and proteins. As a result, the proteins are destroyed [10, 14].
Through the disturbed enzymatic action, metabolism is impeded, and irreversible damage of the germs may occur.
Feng et al. [21] have impressively shown that silver has another effect of silver on Escherichia coli and Staphylococcus aureus. Silver ions lead to the detachment of the membrane between cytoplasm and cell-wall from the latter organelle, resulting in a distinct and visible gap between the two organelles.
The centre of the cell becomes nearly transparent and the clustered DNA becomes visible. The bacterial cell obviously clusters its genetic material. At this stage, however the reproductive capabilities of the bacterium are no longer available. Silver is not the only metal that has been found to deploy this capacity. Copper, mercury and cadmium have shown to possess similar properties. The latter three elements, however, have at least two great disadvantages. First of all they are toxic for human beings, and secondly, they are not as effective as silver when it comes to their bactericidal properties [15]. Silver ions, on the other hand, in concentrations usually applied for antibacterial purposes, have hitherto not deployed any known harmful effect on human health [15, 24, 26].
In comparison to chlorine, silver had the advantage that it does not change the smell or the taste of water. Furthermore, it does not react with the water, and therefore it does not give rise to any salt that could possibly have a disadvantageous effect on human health [15].
For usage in water treatment procedures, silver can be added to ion exchangers or to the filter materials. This has, for example be practices in procedures using Katadyn® filters [15, 17].
In addition to what has been said above, it is possible to produce free silver ions by means of a tension source. For this purpose, a silver electrode is connected to a tension source, and as a result, silver ions are freed into the water. This procedure is mainly used to fight Legionella, a type of bacterium that proliferates freely in water in parts of water networks that have not been properly heated [15].
The effectiveness of silver is controlled by the following factors, some of them indicating the possibly necessity of an initial treatment of the water supply.
Very important is the initial concentration of micro-organisms because each and every individual bacterium binds silver, which is then no longer available. Furthermore, other organic substances, such as carbohydrates, proteins, and urea also bind silver. Therefore, the presence of these molecules lower the free concentration of the silver available for disinfecting purposes.
Similar to other chemical processes, the activity of silver is temperature dependent. A higher temperature augments the effectiveness of the silver ions. On the other hand, the effectiveness of the silver is impaired by organic contamination of the water. The amount of free ions available depends on the solubility of the silver salt that is used. When using silver chloride a concentration of 1200 μg/l is can be obtained. Silver carbonate, on the other hand, yields a concentration of only 500 μg/l [15]. An increased salt content also can influence the solubility adversely; the application therefore confines itself on water with a low content of salts and/or organic loads [15]. Salts of substances other than silver also lower the effectiveness of the latter. For that reason, the application of silver is only useful for the treatment of water with a low salt content and minimal organic pollution [15].

General microbiology
Bacteria are single-celled organisms. They are 0,5-5 μm large. Bacteria belong to the Prokaryotes.
These are organisms that have no real nucleus that is separated from the other cell contents by a membrane. Instead they have a so-called nucleoid. This nucleoid lies free within the cytoplasm and contains the genetic information. A number of organelles that are found the cells of higher organisms, such as human beings are not present in bacterial cells. They miss, for example, mitochondria. Also the bacterial ribosomes are smaller than those of eukaryontic organisms (organisms with a real nucleus). Ribosomes are protein factories. Mitochondria are the power plants of the eukarytiontic cells.
In the mitochondria chemical energy from food is transformed into the energy carrier ATP.

Morphology
Bacterial cells occur in various forms. Some are spherical, in which case the bacteria are referred to as cocci. Some Bacteria have rod-shaped cells, in which case one speaks of bacilli. Some bacteria have a spiral form. These are referred to as spirochetes. Bacteria with a comma-shaped cell form are simply called comma bacteria. Mycoplasms differ from bacteria only by lacking a rigid cell wall. They occur only as intercellular organisms.
Spherical bacteria can occur in clumps. In that case, they are referred to as staphylococci.
Streptococci, on the other hand are spherical bacteria that occur in chains. Cocci that occur in pairs are called diplococci, if they occur in distinct groups of four; they are referred to as tetracocci.
Bacteria can basically classified as Gram-positive or Gram-negative. This differentiation is based on a staining technique developed by the Danish scientist Hans Christian Gram back in 1884.
In the case of the Gram-positive bacteria, the initial stain (crystal violet) cannot be removed by the decolourant (Acetone-Alcohol mixture), and the secondary stain (usually safranin) has no effect. The bacterial cell wall remains dark violet. Gram-negative bacteria, on the other hand do not retain the crystal violet colour and take up the red colour of the secondary dye [1, 2, 9, 10, 12].
The different bacterial species have adapted to various defined temperature conditions. If the bacteria show a preference for temperatures between 30 and 40 degrees Centigrade, they are considered to belong to the mesophile species. If, on the other hand, the germ prefers temperatures between 0 and 20 degrees centigrade, it is considered to be psychrophile. Thermophile bacteria grow at temperatures between 50 and 70 degrees centigrade whereby some species tolerate temperatures as high as 100 degrees centigrade.
Bacteria can also be classified according to the acidity of their environment. Many bacteria prefer a neutral environment; some few specialised species tolerate an acid, respectively an alkaline environment. Most free-living species require water for their metabolism, and the salt concentration of the water should be close to the physiological value of 0.9 % [1, 10].

Fig. 1 Basic morphological shapes of bacteria and their position to each other. From: Steuer et al., Leitfaden der Desinfektion, Sterilisation und Entwesung [Guide to disinfection, sterilization, and disinfestations] Gustav Fischer Publishing house, Stuttgart 1998. a) Spheroid bacteria = cocci, in pairs, (Diplococci), in clumps (staphylococci), in chains (streptococci) b) Rod-shaped bacteria, short forms, long forms, with flagella, with spores, c) spiral bacteria, spiral-shaped, screw-shaped, comma-shaped.

Micro-organisms in connection with drinking water
Through the secretions of humans and animals, pathogens (bacteria that invoke illnesses) can be released into our environment. Depending on their degree of resistance, they survive to a smaller or larger extend the influences of the acidity (pH) of the surrounding medium, the prevailing temperatures, and the UV radiation [7]. If the filtering action of the soil is not efficiently high, germs can penetrate to the ground water level. If that happens, the bacterial may well end up in our drinking water preparation plants. Water that is micro-biologically contaminated can potentially lead to a number of very serious and potentially lethal diseases such as cholera, typhoid fever. Also Escherichia coli, a bacterium necessarily present in our intestines, can cause a number of potentially dangerous conditions [11, 18].
According to the laws and regulations controlling the quality of our drinking water, which de facto is our No. 1 food, stipulate that it may not contain any amount of bacteria that could possibly be detrimental to human health.

As long as the amount of bacteria in our water is below the 100 KBE/ml, one does not assume that any health hazard may arise from the physical quality of the water. For this knowledge, mankind must be indebted to Robert Koch, the German bacteriologist who carried out the pertinent experiments with drinking water about a century ago [5, 6].
As a detailed search for pathogens is too complicated and very expensive, this study is limited to bacteria that show a distribution pattern similar to the pathogens, but that are much easier to cultivate.
For that reason, the following bacteria were used as indicators for faecal pollution: Escherichia coli, coliform bacteria, and enterococci. The examination criterion was that these bacteria were not to be proven present in 100 ml of the sample [18].

Bio-films
Today, it is generally accepted that very few water-dwelling bacteria, occur as single individuals. In general they unite to form to what is referred to as a "Bio-film" [18, 29, 30].
Such a "union" to a conglomerate brings many advantages for the bacteria. Through the production of extra-cellular polymer substances, the bacteria can put themselves beyond the reach and effectiveness of antibiotics and disinfectants. Otherwise effective agents can prove to be ineffective under such circumstances. This explains, for example, why approximately one hundred people were killed after applying an inhalation chemical that was contaminated with Pseudomonas aeruginosa in 1993/1994 [29, 30].
A study revealed that within 10 minutes, a concentration of 2 mg/l chlorine is capable of reducing the initial numbers of free living bacterial cells by a factor of 3 logarithmic steps. The number of bacteria within the bio-film, however, was nearly unchanged when evaluated after one hour of chlorine treatment [3].
Cells that are embedded within matter can survive long periods of dryness. In this way, the settlement in other locations becomes possible.

Materials and methods
Bacteria used in this study
Escherichia coli
Escherichia coli is classified as a Gram-negative bacterium. This organism has peritichous flagellation.
Therefore, it can move actively. Escherichia coli is a normal component part of the physiological intestinal flora of humans and other warm-blooded animals and is excreted with the faeces. As this
germ shows a low natural occurrence in water, and proves to be easily cultivated in vitro, it has been used as an indicator for faecal water pollution in many drinking water analyses. Escherichia coli is facultative pathogenic and can cause infections through contact with susceptible body regions such as the urinary-genital tracts. If Escherichia coli finds its way into the circulatory system, it can cause blood poisoning. In this context it is interesting to note that 15% of the hospital-acquired (nosocomial) infections are caused by Escherichia coli [9, 10, 12].
Beside the non-pathogenic and facultative pathogenic forms, some strains of Escherichia coli are intestinal pathogens. The intake of these forms may lead to diarrhoea. Very often they are the cause for travel diarrhoea, also known as "Montezuma's Revenge."

Enterococcus faecium
Enterococcus faecium belongs to the Gram-positive cocci. They appear in winding chains. These bacteria belong to the normal flora of the human large intestine. If, however, these organisms manage to reach other parts of the human body, they can cause severe and potentially dangerous infections.
This pathogenic germ is responsible for various diseases such as wound infections, infections of the urinary-genital tract and Endocarditis, a condition in which the bacteria attack the heart valves.
Enterococcus faecium ranks second after Escherichia coli in respect to causing urinary-genital tract infection [9, 10, 12, 18].

Pseudomonas aeruginosa
Pseudomonas aeruginosa belongs to the Gram-negative bacteria. It causes turquoise pus and is dreaded in hospital-acquired (nosocomial) infections. In the USA, 11 % of all nosocomial infections are caused by this pathogen. This germ is linked to the highest percentage of lethal bacterial infections [18].
This rod-shaped bacterium is 2 to 4 μm long and has polar flagellation. They are also ciliated. With the help of the cilia, Pseudomonas aeruginosa can adhere to surfaces. This organism occurs single, in pairs or in short chains. It is a human pathogen and is capable of producing extra-cellular polymeric compounds that play an important role in the generation of bio-films. The bacteria of the genus Pseudomonas prefer aerobe environmental conditions and temperatures between 20-30 degrees Centigrade [9, 10, 12].
Unlike the two aforementioned bacteria, this micro-organism is widely distributed in our environment. It can also be isolated from water and soil samples. It can grow on plants as well as in the intestines of humans and animals [10]. In hospitals, this pathogen can be transmitted through the use of basins, humidifiers, endoscopes, artificial respiration equipment and the tubing thereof, kitchen utensils and various cleaning equipments.

Test vessels
Silver-containing white enamel
The vessels were produced out of sheet steel with a thickness of 1 mm. They have an inside diameter of Di = 73 mm. This results in a bottom surface area of 4,190 mm2. With a filling of 20 ml, the total surface in contact with the bacterial suspension is 5,290 mm2. The test vessels have a white enamelled surface.

White enamel without silver
The dimensions of these test vessels are identical to those described under the title of silver-containing white enamel. Therefore, the contact area is also 5,290 mm2. The enamel is the white enamel that is commonly used for sinks.

Fig. 2 Test vessel coated with silver-containing white enamel.

These vessels cannot be differentiated by any visual means from the vessels described above. For that reason, an additional illustration does not seem to be indicated. The white enamel is intended for use for the coating of sink surfaces.

White enamel without silver and "Crystal Guard" coating
This material is also used for the coating of sink surfaces. Optically, this material cannot be differentiated from the previously described white enamel.

High-grade steel
The high-grade steel vessels have almost the same dimensions as those described above except for the insignificant deviations caused by the absence of an enamel layer. The material designation is ST4301. For this type of steel, the descriptions indicate a content of 18 % chromium, 8.9 % nickel and 1 % manganese.

Plastics material
This dark grey material is produced and sold under the name "Kerrock" by a Croatian manufacturer. According to an expertise from the Bavarian State Trading Authority, this material is suited for use in connection with food products. The inner diameter is comparable to those of the vessels described above.

Ceramics
As a fourth basic material to be used in this study we have selected small vessels made out ceramics. They have an inner diameter that is comparable to those of the vessels described above. The vessels are glazed with stoneware glazing.

Methods
Preparation of the test vessels
Before the test series were started, the test vessels were repeatedly rinsed with hot water and finally rinsed with distilled water. In order to ascertain sterility at the time the test is started, the vessels made out of steel and ceramics were heated at 180 degrees centigrade for two hours in a hot-air incubator.
Because of their limited thermal stability, the vessels made out of synthetic materials were sterilised in an autoclave at 121 degrees centigrade and a pressure of 1 bar for 30 minutes. The vessels survived this procedure without damage.

Fig. 3 Test vessel made out of stainless steel ST4301.

Fig. 4 Test vessel coated with plastics material.

In order to ascertain that airborne germs could not corrupt the experimental results, each vessel was covered with a glass Petri dish. These Petri dishes were washed after each application and sterilised by 180 oC. The Petri dishes were stored protected against germs and dust.

Fig. 5 Test vessel made out of ceramics.

Filling the vessels with the test suspension
To obtain the same initial concentration for each series of experiments, a colony of bacteria is taken from the growth medium by means of a sterile swab and transferred to a test tube containing 4.5 ml water of standardized hardness. The suspension is homogenized using a Vortex-shaking device and, if required, thinned down with WSH to adjust the liquid to the desired Mc Farland standard of 0.5.
Adjustments are made using a dulling gauge produced by Becton Dickenson. The necessary test concentration was determined in a series of preliminary experiments. This necessary concentration was obtained by further diluting the suspension by means of WSH. The final suspension is filled into a screw-cap glass bottle. Depending on the number of vessels that are to be tested, there is need for 600 to 900 ml of the bacterium suspension. In order to ascertain, that the bacterial suspension in each of the test vessels contains the same concentration of germs, the glass bottle is shaken thoroughly before each removal of suspension. By means of a sterile 20 ml pipette, the test suspension is taken out of the bottle. The Petri dish covering the test vessel is slightly lifted, allowing insertion of the pipette and deposition of the test suspension. After the test vessels have been filled with the bacterial suspension, they are brought into an incubator where they remain for 24 hours at a temperature of 20 oC.

Sampling After the test vessels have been incubated for 24 hours under constant conditions, they are removed
from the incubator and transferred to a shaking platform in order to homogenise the suspension. The shaking platform moves the test vessels with a rotation of 125 min–1. During this procedure a definite wave movement of the suspension in each test vessel can be observed. In order to ascertain that the UV Radiation of daylight does not corrupt the results, the test vessels are covered with cellulose sheets during the homogenising procedure. After exactly 10 minutes, the first test vessel is removed from the shaking platform and put onto the laboratory bench. Immediately thereafter, the glass lid of the vessel is slightly lifted. With the sterile tip of a 200 μl Eppendorf pipette, bacteria that may sticking to the vessel surface are loosened. For that purpose, the suspension is taken up with the pipette and released again. This is repeated three or four times. After this procedure, two sample of 200 μl each of the suspension is taken and each sample is brought onto a Petri dish filled with DST-Agar. The sample is evenly distributed onto the agar surface by means of a sterile stainless steel Drigalski-Spatula. The Petri dishes with the sample are now incubated at 37 degrees centigrade for 24 hours.
After the samples have been taken, the remaining test suspension is transferred from the test vessels to an Erlenmeyer flask containing a disinfectant in order to eliminate test organisms. In order to make sure that no contamination of the test array or the personnel occurs, the samples for the AAS are sterilised by means of adding 100 μl chlorine bleach.
The decision to use the spatula procedure was taken because this method, compared to the glass plate method, makes the counting of the bacterial colonies that grow on the DST agar easier, as they are on a single plane. The second advantage of this method consists in the fact that the germs or not submitted to thermal stress from hot agar [13]. The membrane filter technique was not chosen because this latter method would have required much more material resources and would have taken much longer to carry through.

Counting the colonies that have developed on the growth media
After the inoculated Petri dishes with DST growth media have been kept for 24 hours at 37 degrees centigrade in the incubator, they are removed and placed onto a dark surface. In this way, recognition and counting of the colonies that have developed on the growth media in the Petri dishes is facilitated.
If the amount of colonies that has developed on the plates is lower than about 300, all colonies are counted. In the case of test series, where more than 300 colonies grow on the media in each Petri dish, a representative portion of the plate is chosen, and only the colonies that have developed on that section are counting. By way of mathematics, one simply computes the total amount of colonies for the entire growth media plate. For this purpose, the division of the plates as drawn on the background is of great assistance.
At least three or four plates are counted for each tested material. From the counts, the mean value is calculated. Such a mean value is computed and recorded for each and every tested material type.
The amount of bacteria is put in relation to the initial concentration of the bacterial suspension. The reduction in number of bacteria is given as a percentage. The mean values obtained in the various test series are used for the final evaluation.

Fig. 6 Various growth media.

Results
Escherichia coli after incubation for 48 hours
After incubation for 2 days, an elimination of 99.8 % of Escherichia coli can be observed in test vessels coated with silver-containing enamel. For vessels coated with simple white enamel without addition of silver, the reduction is 44.2 %. After 48 hours incubation in the stainless steel vessels, there is a reduction of 93 % of bacterial content. The lowering of bacterial content in the plastic vessels lies by 39 %.
The tests with the second silver-containing material - "Crystal Guard" - show a reduction of the initial concentration of the bacteria by 100 %. Without addition of silver, the germ reduction is merely 22.0 %. In ceramics vessels, a germ reduction of 48.8 % is obtained.

Fig. 7 Reduction of the numbers of Escherichia coli after 48 hours.

Enterococcus faecium after incubation for 48 hours
After the incubation for two days, the tested vessels coated with silver-containing white enamel show a reduction of 88.1 % of Enterococcus faecium. The analogous reduction in the case of vessels coated with white enamel without any silver is 86.8 %. The stainless steel vessels managed to eliminate 88.5 % of the bacteria. The reduction of the germs in the case of the vessels made out of plastics materials was 87 %. The vessels with silver-containing "Crystal Guard" enamel coating were equally capable of neutralising 87 % of the germs. In the case of test vessels made out of ceramic materials, the reduction in number of bacteria remained just about the same. Enterococcus faecium shows a low survival rate on all tested surfaces. Silver-containing enamel does not show any stronger reduction in numbers as compared to the other tested surfaces.

Summary
Notwithstanding the fact, that one may assume that the water that is initially fed into our drinking water reservoirs corresponds to all values set by the state health authorities, a certain amount of "ageing" occurs until the water is taken from the taps by the final user. By means of stagnation and/or through loosening of bacteria and other organic materials from a bio-film that settles on the surfaces within the water network, a serious hazard can occur in connection with our drinking water, which is, as we have already said, the No. 1 food. As even the slightest amounts of nourishing materials can give rise to the formation of bio-films, it may very well be indicated to use, under these circumstances, work materials that do at least inhibit the adhesion of bacteria to the walls of the tubing and boilers.

Fig. 8 Reduction of numbers of Enterococcus faecium after 48 hours.

Fig. 9 Reduction of numbers of Pseudomonas aeruginosa after 48 hours.

In our experiments we have simulated the effects of stagnating drinking water onto the surfaces of the various work materials. The results are interesting indeed. We were able to establish that some materials have a distinct impact onto the survival capacity of bacteria. These results are important for all enamel-coated products.
In the case of the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, a strong inhibition of bacterial growth could be observed when using silver-containing enamel. In the case of the Gram-positive germ Enterococcus faecium, the survival potential of the germs is low on all surfaces tested, and an addition of silver does not show any significant additional reductive action.
Stainless steel 4301 does show a bactericide action, which, however, is lower than that of silver-containing enamel. Plastic surfaces considerably enhance the growth of Pseudomonas aeruginosa, a germ definitely pathogenic to humans. Ceramics with a normal titanium-white glazing have no recognisable impact onto the growth of bacteria. The results obtained with hydrophobic coatings are non-consistent and therefore inconclusive, and no bactericide action or inhibition of germs could be detected.
By means of this study, which was carried out in the premises of a state laboratory, the germ-reducing action of silver-containing enamel, in respect to the gram-negative testing organisms could be clearly established. On the basis of these experimental results, the positive characteristics of enamelled surfaces can thus be improved and the corresponding coating materials can be applied to a large number of products.

References
[1] Hirsch-Kaufmann, Monica, Schweiger, Manfred Biologie für Mediziner und Naturwissenschaftler, 4. Edition, Thieme Publishing House, Stuttgart & New York (2000).
[2] Koecke, Hans Ulrich, Emschermann, Peter, Härle, Eckhart Biologie; Lehrbuch der allgemeinen Biologie für Mediziner und Naturwissenschaftler, 4. Edition, Schattauer Publishing House, Stuttgart & New York (2000).
[3] Lengeler, Joseph W., Drews, Gerhart, Schlegel, Hans G. Biology of the prokaryotes, Thieme Publishing House, Stuttgart & New York (1999).
[4] Beck, Ernst G., Schmidt, Pavel, Hygiene: Umweltmedizin; 100 Tabellen 6. Edition, Ferdinand Enke Publishing House, Stuttgart (1996).
[5] Borneff, Joachim, Borneff, Marianne, Hygiene: Ein Leitfaden für Studenten und Ärzte, 5. Auflage, Thieme Publishing House, Stuttgart & New York (1991).
[6] Gundermann, K. O., Rüden, H., Sonntag, H. G. Lehrbuch der Hygiene;G. Fischer Publishing House, Stuttgart & New York (1991).
[7] Steuer, Walter, Lutz-Dettinger, Ursula, Schubert, Friedemann Leitfaden der Desinfektion, Sterilisation und Entwesung; mit Grundlagen der Mikrobiologie, Infektionslehre, Epidemiologie und der tierischen Schädlingen, 7. Edition, G. Fischer Publishing House, Stuttgart, Jena, Lübeck & Ulm (1998).
[8] Askeland, Donald R. Materialwissenschaften: Grundlagen, Übungen, Lösungen, übersetzt von Wolfgang Fahland und Wilfried Holzhäuser, Spektrum Akad. Verlag, Heidelberg, Berlin & Oxford; (1996).
[9] Klein, P. (Begr.), Hahn, Helmut (Hrsg.), Falke, Dietrich, Kaufmann, Stefan H.E., Ullmann, Uwe Medizinische Mikrobiologie und Infektiologie 4. Edition, Springer Publishing House, Berlin, Heidelberg & New York (2001).
[10] Köhler, W., Eggers, H. J., Fleischer, B., Marre, R., Pfister, H., Pulverer, G. (Hrsg.) Medizinische Mikrobiologie, 8. Edition, Urban & Fischer Publishing House, München &⋅Jena (2001).
[11] Madian, Michael T., Martinko, John M., Parker, Jack, Brock, Thomas D. (Begr.) [Brook biology of microorganisms] Mikrobiologie. Translated from the English by Kurt Beginnen, Julia Karow, Brigitte Pakendorf, Lothar Seidler, Olaf Werner, German Translation, Edited by Werner Goebel, Spektrum Akad. Publishing House, Berlin (2000).
[12] Kayser, Fritz H., Bienz, Kurt A., Eckert, Johannes, Zinkernagel, Rolf M. Medizinische Mikrobiologie, verstehen, lernen, nachschlagen, 10. Edition, Thieme Publishing House, Stuttgart & New York (2001).
[13] Bast, Eckhard Mikrobiologische Arbeitsmethoden: eine Einführung in grundlegende Arbeitstechniken; 2. Edition, Spektrum Akadem. Publishing House, Heidelberg & Berlin (2001).
[14] Kappstein, Ines, Nosokomiale Infektionen; Prävention, Labor-Diagnostik, Antimikrobielle Therapie, 2. Edition, W. Zuckschwerdt, Münich, Bern, Vienna & New York (2002).
[15] Wallhäußer, Karl Heinz Praxis der Sterilisation-Desinfektion-Konservierung-Keimidentifizierung- Betriebshygiene, 4. Edition, Thieme Publishing House, Stuttgart & New York (1985).
[16] Schoenen, Dirk, Schöler, H.F. Trinkwasser und Werkstoffe: Praxisbeobachtungen und Untersuchungsverfahren, Fischer Publishing House, Stuttgart & New York (1983).
[17] Schmidt, Joachim, Naumann, Günter, Horsch, Wolfgang, Fickweiler, Eckart, Sterilisation, Desinfektion, Konservierung, und Entwesung in der med. u. pharmazeut. Praxis, 2. Edition, Thieme Publishing House, Leipzig (1990).
[18] Höll, Karl, Grohmann, Andreas (Hrsg.) Wasser: Nutzung im Kreislauf, Hygiene, Analyse und Bewertung, 8. Edition, de Gruyter Publishing House, Berlin & New York (2002).
[19] Kölle, Walter Wasseranalysen - richtig beurteilt; Grundlagen, Parameter, Wassertypen, Inhaltsstoffe, Grenzwerte nach Trinkwasserverordnung und EU-Richtlinie; Wiley-VCH Publishing House, Weinheim (2001).
[20] Flemming, Hans Curt Biofilme, Biofouling und mikrobielle Schädigung von Werkstoffen /H.-C. Flemming. Forschungs- und Entwicklungsinstitut für Industrie- und Siedlungswasserwirtschaft, sowie Abfallwirtschaft e. V., Stuttgart – München : Oldenburg Stuttgarter Berichte zur Siedlungswasserwirtschaft; 129 Zugl.: Stuttgarter, Univ. Habil.-Schrift., R. Oldenburg Publishing House, München 1994.
[21] Feng, Q.L., Wu, J., Chen, G.Q., Cui, F.Z., Kim, T.N., Kim, J.O Journal Biomed. Material Res, 52 (2000) 662-668.
[22] Cowan, Marjore M., Abshire, Kelly Z., Houk, Stephanie L., Evans, Suzanne M. Journal of industrial Microbiology & Biotechnology, 30 (2003), 102-106.
[23] Multanen, Markku, Talja, Martti, Hallanvuo, Saija, Siitonen, Anja, Välimaa, Tero, Tammela, Teuvo L. J., Seppälä, Jukka, Törmälä, Pertti Urol Res, 28 3(2000).,27-331.
[24] Silver, Simon Microbiology Reviews, 27 (2003), 341-353.
[25] Bellantone, Maria, Williams, Huw D., Hench, Larry LBroad; Antimicrobial Agents and Chemotherapy, 46 ,(2002),1940-1945.
[26] Davies, Richard L., Etris, Samuel F. Catalysis Today, 36, (1997), 107-114.
[27] Rusin, P., Bright, K., Gerba, C. Letters in Applied Microbiology, 36, (2003), 69-72.
[28] Rivera-Garza, M., Olguin, M.T., Garcia-Sosa, I., Alcantara, D., Rodriguez-Fuentes, G. Microporos and Mesoporous Materials, 39; (2000). 431-444.
[29] Anonym, Der Biofilm-Bildung, Eigenschaften und Wirkungen; Der Hygieneinspektor, 12: (2002), 21-28.
[30] Costerton, J.W., Stewart, Philip S. Bekämpfung bakterieller Biofilme, Spektrum der Wissenschaft; 11: (2001), 58 ff.
[31] Bicanova M, Thinschmidt G. Oligodynamische Wirkung der Silberionen auf Mikroorganismen, Pharmazie, 40: (1985), 736.
[32] Gebel, J., Werner, H.-P., Kirsch-Altena, A., Bansemir, K. Standardmethoden der DGHM zur Prüfung chemischer Desinfektionsverfahren, mhp-Verlag GmbH, Wiesbaden, Stand: 1. September 2001.
[33] OXOID Handbuch (1993), 5. Auflage, Unipath GmbH Wesel.