O. V. Ignatov

Acrylamide and acrylic acid determination using respiratory activity of microbial cells

Abstract The possibility of acrylamide and acrylic acid determination using respiratory activity of microbial cells of the strain Brevibacterium sp. has been investigated. The conditions to obtain specific respiratory activity (SRA) of the cells for both substrates have been examined. The substrate concentration dependences of SRA have been obtained; they exhibited linearity in the range of 0.01–0.1 g/l acrylic acid and 0.01–0.075 g/l acrylamide. The selectivity of the cell respiratory activity for various organic compounds and storage conditions of the active biomass have been studied. Acrylamide quantification in the real waste waters has been made.

Obtaining phage mini-antibodies and using them for detection of microbial cells with an electroacoustic sensor

Phage mini-antibodies to bacterial cells of strain Azospirillum brasilense Sp245 were obtained, and the possibility of using them for detection of microbial cells with a lateral field excited piezoelectric resonator was studied. It has been found that the frequency dependences of the real and imaginary parts of electrical impedance of such a resonator loaded with a suspension of A. brasilense Sp245 cells with the mini-antibodies differ significantly from the dependences of the resonator with a control cell suspension without mini-anti-bodies. The limit of possible determination of the concentration of microbial cells is found to be 103 cells/mL upon interaction with mini-antibodies. It has been ascertained that detection of A. brasilense Sp245 cells with the aid of mini-antibodies is possible even in the presence of other cultures, for example, E. coli BL-Ril and A. brasilense Sp7. Therefore, it has been shown for the first time that detection of microbial cells with an electroacoustic sensor is feasible.

Determination of organophosphorus aromatic nitro insecticides and p-nitrophenol by microbial-cell respiratory activity

We examined the possibility of measuring the organophosphorus aromatic nitro insecticides metaphos and sumithion as well as their hydrolysis product p-nitrophenol (PNP) by the specific respiratory activity (SRA) of Pseudomonas putida C-11, P. putida BA-11, and Acinetobacter calcoaceticum A-122. The plots of cellular SRA against the two insecticides and PNP were linear over the ranges of 0.5-2.5 microM for P. putida C-11 and BA-11 and 0.5-1.0 microM for A. calcoaceticum A-122. P. putida BA-11 showed the greatest respiratory-response selectivity in the determination of the test substrates. We made comparison studies of the SRA of cells immobilised by two methods: carrier-surface adsorption and inclusion in various gels. We discuss the feasibility of developing a microbial sensor system for the determination of metaphos, sumithion, and PNP in aqueous media.

Electro-optical properties of microbial cells as affected by acrylamide metabolism

Copyright (c) 1997 Elsevier Science B.V. All rights reserved.The electro-optical (EO) properties of a microbial cell suspension of a Brevibacterium strain were studied during the process of metabolism of acrylamide and certain other amides. Upon optimisation of cell growth conditions, followed by EO measurements, we found that acrylamide produced a specific effect on the cell orientational spectra (OS) over the frequency range 10 to 502 kHz. The other amides used also affected the EO properties of the cells.

Investigation of specific interactions between microbial cells and polyclonal antibodies using a resonator with lateral electric field

The interaction between polyclonal antibodies and Azospirillum brasilense Sp7 cells was studied using a resonator with lateral electric field. To this end, specific polyclonal rabbit antibodies against the O-antigen epitopes of the strain A. brasilense Sp7 were obtained and the possibility of their application for detection of microbial cells using a piezoelectric resonator with lateral electric field was shown. It was established that frequency dependences of the real and imaginary parts of electrical impedance of such a resonator loaded with the suspension of A. brasilense Sp7 cells and antibodies substantially differed from those of the resonator with the control suspension of cells without antibodies. It was shown that the obtained antibodies interacted with azospirilla cells, and the marker was accumulated all over the cell surface. The limit of possible detection of microbial cells during their interaction with antibodies was found to be 104 cells/mL. Detection of A. brasilense Sp7 cells using antibodies proved to be possible in the presence of foreign bacteria. The presented results demonstrate the possibility of recording the interaction between microbial cells and antibodies and developing a biosensor for quantitative detection of microbial cells.

Determination of the spectrum of lytic activity of bacteriophages by the method of acoustic analysis

The changes in the electroacoustic parameters of a cell suspension due to the interaction between cells and bacteriophages were studied both in pure culture and in the presence of extraneous microflora. It was shown that the specific changes in the electroacoustic parameters of a cell suspension under the influence of bacteriophage occur only in the microbial cells that are sensitive to this bacteriophage. A sensor unit allowed us to distinguish the situation where bacterial cells are infected with specific bacteriophages of the control experiments from the situation without such infection. An approximate criterion for specific interactions between bacteriophages and cells in the suspension was developed. According to this criterion, the change in electrical impedance of a sensor unit must not be less than ~1%. The standard microbiological technique of sowing the cells infected with bacteriophages on solid nutrient media was used as a control. The possibility of using the method of electroacoustic analysis to determine the spectrum of lytic activity of bacteriophages has been shown for the first time. The results that we obtained can be used to develop a new express method for determining the sensitivity of microbial cells to bacteriophages.

Action of ampicillin and kanamicin on the electrophysical characteristics of Escherichia coli cells

We examined the effect of ampicillin and kanamycin on the electrophysical characteristics of ampicillin- and kanamycin-sensitive and ampicillin- and kanamycin-resistant Escherichia coli cells. Substantial changes in the orientational spectra (OS) of suspensions of cells incubated with various ampicillin and kanamycin concentrations took place only at the first five frequencies of the orienting electric field (10–1000 kHz). The maximal change in the magnitude of the electro-optical signal occurred at 50 µg mL−1 of ampicillin and 10 µg mL−1 of kanamycin. The suspension-OS changes did not depend on the antibiotic-action period. Under the action of ampicillin and kanamycin, sensitive and resistant E. coli strains gave different electro-optical (EO) effects. It follows that the sensitive and resistant E. coli strains exhibit different of the EO effect on the action of ampicillin and kanamycin. Thus, the suspension-OS changes occurring under the effect of ampicillin and kanamycin may be used as a test for resistance to this antibiotic in the cells studied.

An acoustic method for the analysis of bacterial cells

The application of a biological electroacoustic sensor based on a lateral electric-field-excited piezoelectric resonator for the study of bacterial cells that interact with specific bacteriophages, mini-antibodies, and polyclonal antibodies was successfully demonstrated. The determined lower limit of microbialcell detection was approximately of 103 to 104 cells/mL for the duration of the assay of 10 min. The possibility of bacterial-cell detection via interaction with specific agents in the presence of extraneous microbiota was shown. The method allowed us to determine the spectrum of lytic activity of bacteriophages and the sensitivity of microbial cells to bacteriophages. The results of the study showed that application of a sensor piezoelectric lateral-field resonator is a promising technique for the detection and identification of microbial cells and determination of their phage resistance in microbiology, medicine, and veterinary medicine. Furthermore, the results of the experiments made it possible to understand the mechanisms of the processes that occur in a suspension of bacterial cells in the presence of various biological agents. The method also may provide useful information regarding biophysical mechanisms of interactions that occur in microbial populations.

Electrooptical parameters of kanamycin-treated E. coli cell suspensions

The effect of kanamycin on the electrophysical parameters of cell suspensions of Escherichia coli K-12 and pMMB33 was investigated. Incubation of the sensitive K-12 strain with kanamycin resulted in significant changes in the orientation spectra (OS) of the cell suspensions; these changes were not revealed in the case of the resistant pMMB33 strain. In the case of the sensitive K-12 strain incubated with different kanamycin concentrations, changes in the OS of the cell suspensions occurred within the 10–1000 kHz frequency range of the orienting electrical field. The most pronounced change in the electrooptical signal was observed at 10 μg/ml of kanamycin. Control experiments were carried out by standard plating on nutrient media. Thus, the OS changes of suspensions in the presence of antibiotics may be used as a test for microbial resistance to such antibiotics.

Electrophysical analysis of microbial cells and biosensor technology

A new kind of biosensor system may be developed by using an electrophysical technique for measurement of electrophysical properties of microbial cells. The electro-optical (EO) analysis of cell suspensions was used as the basis for our work. It is based on the recording of changes in the optical characteristics of cell suspensions under the orienting effect of an electric field. Promising fields of use of microbial cell suspensions for EO analysis were developed. One of the promising fields is the development of biosensor systems for analysis of low-molecular-weight substances. Another field is the investigation of cellular metabolism. The most recent direction of our work is the application of EO analysis to cell detection. We showed that the determination of bacteria may be achieved by selection and matching of antibodies specific to individual bacterium types and by comparing spectra of bacteria in the presence and absence of specific antibodies. The same principles were used for investigations of the bacteria–phage interaction. Thus electro-optical analysis of cell suspensions opens new opportunities for creation of new biosensor methods in biotechnology, environmental control and medicine.