## 3 Materials and Methodology
### Materials Used in the Research
An EM product manufactured by Greenland Technologia was used in the research. The company deals with the production and sale of products based on the technology of Effective Microorganisms(r), which are widely used in agriculture and horticulture, animal husbandry, or in biological environmental treatment (municipal and home sewage treatment plants, regeneration of soil and water reservoirs). Greenland Technologia EM Ltd. is the sole licensee of EM Research Organization Inc. (EMRO) for Poland and Central and Eastern Europe. The Japanese company EMRO is also a licensor for producers and distributors of products in several dozen countries on all continents. The second additive used in the research is colloidal silver, also known as nanosilver and silver solution. Ion silver is as transparent as water, is stored in glass bottles, and consists of 90% silver ions and only about 10% silver particles. This type of silver is called \"silver solution\". After entering the body, silver ions immediately combine with it, creating an insoluble salt-silver chloride. Such a property is not exhibited by electrically neutral silver particles. This salt does not have any properties that are attributed to colloidal silver. Nanosilver consists of microscopic particles and silver ions that can only be observed through an electron microscope. Due to the high concentration of silver nanoparticles, colloidal silver does not resemble water. Its characteristic feature is a clear color, which can range from yellow to amber. Colloidal silver has a darker color because the silver particles dispersed in the water block the light passing through them with a specific wavelength, which is approximately 400 nm. Additionally, colloidal silver can be stored in plastic bottles because silver nanoparticles are chemically insensitive and completely resistant to the damaging effects of UV radiation. Colloidal silver with silver nanoparticles is the least common type of silver commercially available due to its complicated production process and high production costs. However, it is the only effective silver with scientifically proven properties. Real colloidal silver consists of 80% silver particles, with the remaining 20% made up of silver ions. The decisive factors for the quality of real colloidal silver are the content of silver particles and their very active total surface. In colloidal silver, nanometer-sized particles form a colloid, so it is not necessary to use a protein substrate to suspend them in water. Due to their electrokinetic potential, nanoparticles repel each other, keeping the colloid stable. Silver suppressed to the form of nanoparticles has a large active surface, and enormous biocidal potential. The productive value of nanosilver includes the eradication of over 99.99% of bacteria, viruses, mold, and fungi. Good carriers for nanosilver are activated carbon fibers (ACFs), commonly used in sewage treatment plants for removing various types of contamination [44]. Figure 1: The commercial forms of effective microorganisms [42; 43]. Experimental data from studies [45] show that silver nanoparticles at a concentration of 4 mg/mL completely inhibit the growth of _Staphylococcus aureus_ bacteria. The results of transmission electron microscopy confirmed the damage to the cell walls caused by silver nanoparticles and the accumulation of these particles in the bacterial cell membrane [46; 47]. Silver solution and colloidal nanosilver was used for the tests in the commercial form shown in Figure 2. Silver solution (transparent), sold as Silver Klar Ag+, was created on the basis of demineralized water and ionic silver (25 ppm), and was produced by the Naoliwieni company located in Rosanow (Poland). Colloidal silver (amber), sold as ARGENTUM200, was created on the basis of demineralized water with a colloidal non-ionic silver content of 25 ppm, and was manufactured by Aura Herbals Ltd in Sopot (Poland).