The present study gives evidence that silver nanoparticles (AgNPs) are spontaneously formed from Ag⁺ ions in Mueller-Hinton broth, which is frequently used as a standard cultivation medium for many types of bacteria. Silver ions often serve as a reference in the determination of minimum inhibitory concentration (MIC) values of engineered AgNPs. It is thus a question if the MIC values determined for engineered AgNPs are not influenced by the presence of spontaneously formed AgNPs. Furthermore, as shown here, the addition of augmented concentrations of selected amino acids, namely glutamic acid and glutamine, can change the growth and characteristic features of spontaneously formed AgNPs. For the sake of a direct comparison, the influence of the two selected amino acids on characteristics and MIC values determination of engineered AgNPs has been also investigated. The determined MIC values of all investigated systems (i.e., with and without the presence of engineered AgNPs) and their mutual comparison demonstrated that MIC values are slightly influenced by the actual composition of a cultivation medium for bacterial growth. On the other hand, the actual composition of a cultivation medium is crucial for the final characteristics of AgNPs. The changes in characteristic features of spontaneously formed as well as engineered AgNPs are most probably induced by the covalent bonding of amino acids to AgNPs surface which is proven by vibrational spectroscopic techniques.

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