Abstract. We developed the new automatic method that combines the method of forced luminescence and stimulated Brillouin scattering. This method is used for monitoring pathogens, genetically modified products and nanostructured materials in colloidal solution. We carried out the statistical spectral analysis of pathogens, genetically modified soy and nano-particles of silver in water from different regions in order to determine the statistical errors of the method. We studied spectral characteristics of these objects in water to perform the initial identification with 95% probability. These results were used for creation of the model of the device for monitor of pathogenic organisms and working model of the device to determine the genetically modified soy in meat.We developed the coherent spectroscopic method for the monitoring of the pathogenic organisms directly in water pipe-line, genetically modified products and nanostructured materials in colloidal solution. The method is based on an analysis of spectral characteristics of stimulated radiation, passed through the solution.We studied transmission IR spectra of a number of pathogens (salmonella, viruses of herpes genitaler, hepatitis A and C, grippe A and B ecd) and nanosilver solutions and luminescence of nanomarkers. In our experiments, the laser radiation passed through a quartz cell with water solutions of the pathogens, nanosilver or nanomarkers. Spectra were analyzed by the spectrum analyzer "Agilent" (USA) with spectral resolution 0.5 nm. The solid DPSS lasers ("Laser-export Co. Ltd.", Russia) with wavelengths 1017, 810, 670, 532, 480 nm were used as the sources of exciting radiation. The input/output waveguides bring the exciting and passing radiations from laser to the cell and from cell to the analyzer, respectively.We found that exciting radiations with wavelengths 1017 and 810 nm induce the stimulated Brillouin scattering in spectra of the water containing pathogen DNAs. We revealed that peak positions and widths of "fingerprints" for pathogens under study, and optical densities of these bands were proportional pathogen content, if their content was
The aim of the work is monitoring the formation of biofilms by opportunistic and pathogenic microorganisms. Materials and methods. The cultures of the genera Salmonella, Escherichia, Yersinia, Proteus, Citrobacter, Enterobacter, Prtovidenzia, Morganella, Klebsiella, Cronobacter, Pseudomonas, Bacillus, Staphylococcus were used in the work. The studied microorganisms were cultured in polystyrene 96-well plates. For this purpose, a daily culture of microorganisms was introduced into the wells with meat-peptone broth, having previously established a concentration of 104 mc / ml, and incubated for 24...96 hours at temperature of 37 °C. Then the medium with plankton cells was removed from the wells. 200 μl of filtered 0,1% crystal violet solution was poured into the wells of the plate and the plates were kept for 10...15 min at room temperature. Then dye was removed from the wells. Unbound dye was thoroughly washed with saline or distilled water. The plates were turned over on filter paper and dried. The presence and density of biomatrix (biofilm) was determined visually by the intensity of staining the surfaces of plates. Then, for the extraction of paint from the film, 200 μl of 96% ethanol was added to the wells and the optical density was measured on KFK-3KM spectrophotometer at a wavelength of 590 nm. Results of research. The results of the experiments allowed us to assert that within 48 hours of cultivation microorganisms form a mature biofilm, which can serve as a model for studying the process of biofilm formation. Biofilm of microorganisms of different taxonomic groups differs in density. In addition, even bacteria belonging to the same genus, under the same conditions, can form a biofilm, the density of which differs by 30...60%.
To study the process of biofilm formation, microorganisms were cultured in 96-well plates, on meat-peptone broth, stained with a 0,1% solution of crystalline violet for 10...15 minutes, after which the unbound dye was washed off. The quantitative accounting of the bound dye was carried out by spectrophotometry at a wavelength of 490 nm. The technique for making bacterial preparations for light and scanning electron microscopy on dodged glasses immersed in Petri dishes with a liquid nutrient medium is proposed. A suspension of bacteria at a concentration of 105 m.k/ml in a volume of 5 ml was shaken on Vortex apparatus and introduced into Petri dishes with 20 ml of meat-peptone broth. Sterile non-greased cover glasses were placed on sterile object glasses and immersed in a liquid nutrient medium in Petri dishes. The material was incubated for 18...24 hours at 37 °C. Then the cover slips were removed with tweezers and some of them were stained with 1% aqueous solution of methylene blue (for light microscopy), and some were placed in Petri dishes with bottomed filters (for electron microscopy). The latter, in order to preserve natural architectonics, were fixed in vivo by pairs of 25% glutaraldehyde for 3...5 hours. Vapors of 2...4% osmic acid solution were used for 2...3-minutes to contrast the preparations. After treatment with vapors of osmic acid, biofilms with included bacteria acquired yellowish or brown color. The obtained preparations after dehydration with propylene oxide vapors and spraying with gold ions were examined in a scanning electron microscope (SEM). The technique allows us to study the phases of development of biofilms and obtain objective data on the morphology of populations of pathogenic and conditionally pathogenic bacteria without disturbing natural architectonics. It is shown that the intensity of biofilm formation by pathogenic microorganisms, such as salmonella, Yersinia, Staphylococcus aureus was slightly higher than that of non-pathogenic: Escherichia, Proteus, Citrobacter, Enterobacter.
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