Study Of Effects Of Silver Ions And Silver Nanoparticles On Bacteria Based On Microscopy Imaging Techniques

It is well known that silver has antibacterial effects. Silver ions and silver nanopaticles as antibacterial components have extensive applications in the fields of clinical medicine and daily life. In recent years, with the emergence of multidrug resistance in many bacterial strains because of antibiotic misuse, silver has gained attention once again. However, the antibacterial mechanisms of silver ions and silver nanoparticles have not been well-established so far. Though biochemistry techniques have provided some useful informations for understanding their antibacterial mechanism, the direct observation of morphological alterations would help to clarify their antimicrobial mechanism by imaging techniques. In this thesis, the effects of silver ions and silver nanoparticles on bacteria were studied using atomic force microscopy (AFM) and darkfield microscopy, which are particularly well suited for imaging of biological samples. The changes of bacterial-surface ultrastructures were investigated on nanoscale. The results provided important morphologic informations for understanding the antibacterial mechanism of silver ions and silver nanoparticles. The main researches are summarized as follows:1. The effect of silver ions on bacteria was investigated using AFM imaging technique. Gram-negative Escherichia coli and Gram-positive Staphylococcus epidermidis cells after treatment with silver ions were imaged in ambient air with a tapping mode AFM. The results showed that in the case of E. coli, vesicles appeared on the cell walls, and the size of vesicles became larger with the increase of the incubation time. However, in the case of S. epidermidis, irregular and deep grooves appeared on the cell walls, and the cytoplasmic membrane shrank and became separated from the cell wall. The significant differences in cell wall changes between E. coli and S. epidermidis after treatment with silver ions were related to the differences of their structural characters. The above investigation of AFM imaging is important in understanding the relationship between structure and function of microbial cell, and provides useful morphologic informations for understanding the antibacterial mechanism of silver ions.2. The effect of silver nanoparticles on bacteria was studied by the combination of AFM and darkfield microscopy. Firstly, silver nanoparticles with an average size of 10.3 nm were synthesized by reducing silver ions with NaBH4. E. coli cells were imaged in ambient air with a tapping mode AFM and darkfield microscopy after treatment with silver nanoparticles. The results indicated that silver nanoparticles were attached to the outer membrane of E. coli, especially located at the two ends of the cells. The E. coli cells were damaged with the collapse of the two ends and formation of"pits"in the outer membrane. Moreover, silver nanoparticles also penetrated into the cells. The outer membrane was damaged by silver nanoparticles, which was further confirmed by a control experiment of lysozyme treatment. The above results showed changes of bacterial-surface ultrastructures after treatment with silver nanoparticles at nanometer scale, and distribution of silver nanoparticles on live bacteria, which offered important informations for the further antibacterial mechanism study of silver nanoparticles.3. Antibacterial activity of the silver nanoparticles biosynthesized by Morus alba L. leaf was investigated using AFM imaging technique. Firstly, silver nanoparticles with an average size of 32.2 nm were synthesized by reducing silver ions with Morus alba L. leaf. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) analysis showed a narrow and uniform distribution of the as-prepared silver nanoparticles, and its corresponding electron diffraction pattern confirmed the face-centered cubic crystalline of metallic silver. From the results of Fourier transform infrared (FTIR) spectroscopy, it was confirmed that the polyol components and protein residues were mainly responsible for the reduction of silver ions and stabilization of silver nanoparticles, respectively. The silver nanoparticles were found to be stable in the high salt media. Moreover, the excellent antibacterial activity of the silver nanoparticles against E. coli were investigated using turbidimetry and high resolution AFM imaging technique at the macro- and micro-level, respectively.