Evaluation of a method for the determination of antibacterial activity of chitosan

A method for the determination of the antimicrobial activity of chitosan with the use of organic salts for the production of pH in the range of 5.5–8.2 was studied. The double-dilution method demonstrated the effectiveness of the determination of the antimicrobial activity of chitosan samples with different molecular weights and solubilities. It was found that the antibacterial activity increased at low pH values with increasing molecular weight, but chitosans with a molecular weight of 5–6 kDa showed higher activity at neutral and slightly alkaline pH levels. Determination of the antimicrobial activity of various chitosan samples at different pH values allowed a more reliable assessment of the potential biological activity of chitosan.     

The origins of the back-mutation assay method: a personal recollection

The back-mutation assay method for determining the mutagenicity of various treatments was first developed a little over 50 years ago and has been in continuous use ever since. Shortly after the method was first used it became evident that certain factors of cell density, composition of media, etc., had to be carefully controlled to preserve an acceptable reliability of the method. A factor of particular importance was the suppression of growth of back-mutant prototrophic cells by the large number of auxotrophic cells present, a phenomenon which later became known as the "Grigg Effect." This review describes the origins of the back-mutation method and of the confounding competitive suppression phenomenon, the cause of competitive suppression, methods of diagnosing whether it is likely to bias the interpretation of a particular back-mutation experiment, and an experimental design which removes it entirely as a possible source of error. A number of other phenomena, such as phenotypic lag and coincident mutation associated with back-mutation, are also discussed as possible sources of error.     

Proposal of a method for the genetic transformation of Gordonia jacobaea

AIMS: Gordonia jacobaea is a recently isolated bacterial species with potential industrial application on account of its ability to store large quantities of trans-canthaxanthin. Its genetic manipulation is, however, difficult and cumbersome owing to the presence of mycolic acids in the cell wall and, especially, because of current lack of knowledge about its basic genetics. The present work describes a method for the genetic transformation of G. jacobaea. METHODS AND RESULTS: Gordonia jacobaea was grown in media supplemented with different glycine, penicillin G and isoniazid concentrations. The temperature, carbon source, growth phase and ultrasounds were analyzed for improving the method efficiency. The cells were finally transformed by electroporation. Finally, the method was applied to Brevibacteriumlactofermentum and Gordonia bronchialis. CONCLUSIONS: The growth of G. jacobaea in the presence of glycine and isoniazid is essential for obtaining electrocompetents cells. The temperature, growth phase and ultrasounds appeared as the main factors for increasing the transformation efficiency. The use of shuttle plasmids became necessary. The method described can be used with other Corynebacteria species. SIGNIFICANCE AND IMPACT OF THE STUDY: Because of the importance of the CNM group (Corynebacteria, Nocardia and Mycobacteria genera) in different areas such as industry, bioremediation improve the knowledge of their molecular mechanisms are becoming essential. The method described here improves the genetic manipulation of this group of bacteria.     

An immunoglobulin preparation as a calibrating agent for the gel filtration method

The fractional composition of commercial immunoglobulin was studied by the method of gel filtration on ultragel AcA-34 and the possibility of its use for the calibration of a chromatographic column was shown. The fractionation of a specimen of IgG revealed the presence of 4 fractions. Their molecular weights corresponded to dimers, monomers, Fab fragments and low-molecular peptides. The qualitative fractional composition of the preparation was shown to be stable during 3 years of storage, as well as after keeping the preparation at 37 degrees C for 1 month. The attested characteristic of each IgG fraction, the distribution coefficient (Kav), was established. The use of a specimen of immunoglobulin obtained with the use of the modified Cohn's method--as calibrant will make it possible to calibrate the columns for a shorter period and to control the correctness of the analysis.     

Evaluation of a radiometric method for studying bacterial activity in the presence of antimicrobial agents

In a study involving 2760 tests, the BACTEC semi-automatic radiometric method which measures bacterial metabolic activity and produces a BACTEC growth index, was compared with two conventional methods commonly used for determining growth, absorbance and viable counts. In 92% of radiometry tests the suppression of growth was inversely related to the antibiotic concentration. This compared with 83% for absorbance and 63% for viable counts. The radiometric method was found to be more rapid, easier to use and more reproducible in determining the effect of antibiotics on the activity of bacteria than viable counting or absorbance methods.     

Development of a robust microtiter plate-based assay method for assessment of bioactivity

A microtiter plate-based assay was developed for the quantitative monitoring of bioactive compound production in Streptomyces hygroscopicus fermentation samples. The method reported demonstrates the successful application of the theories of disk diffusion based methods of bioactivity assessment, to a microtiter assay for high throughput analysis. The assay method facilitates the generation of the dose-response curve of test organisms (Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae) to a bioactive compound. Using this dose-response curve, the method facilitates definition of three distinct Minimum Inhibitory Concentration (MIC) values for use in the characterisation of the bioactive attributes of a sample. The assay uses established standard procedures to facilitate adaptation of the assay for use with a wider range of test microorganisms. Errors due to the assumption of a linear relationship between turbidity and biomass concentration are also reduced, due to incorporation of a step to convert turbidity to biomass concentration, for use in the calculation of bioactivity.     

Rapid synthesis of oligodeoxyribonucleotides: a new solid-phase method.

A method is described that makes use of a new polyamide resin for the rapid synthesis of short oligodeoxyribonucleotides. The method is illustrated by the preparation of two heptadeoxyribonucleotides, d(pT6-C) and d(pC-A-G-T-G-A-T) using a phosphodiester approach. A further development involved use of phenyl isocyanate as an in situ drying agent, which obviated the need for solvent co-evaporation prior tothe internucleotidic coupling steps. Improved fractionation of thymidyl oligonucleotides was obtained by use of a new microparticulate, silica-based anion-exchanger.     

Development of a new method for the preparation of an acellular allodermis,quality control and cytotoxicity testing

Demand for use of acellular allodermis is high but commercially appropriate products are not used routinely because of very high price and limited availability. These facts did motivate us to prepare acellular allodermis using a new, simple and less expensive method. We have developed a original method for preparation of acellular allogeneic dermis based on action of a proteolytic enzyme in combination with distilled water. Hypotonic environment in comparison with SDS or Triton ansure no toxicity of the final product. Trials for determination of optimal trypsin concentrations, temperature and time of action were performed. According to our results, the use of 2.5% trypsin/EDTA solution overnight at +4 °C was proving to be optimal. The histology confirmed absence of cells in the prepared dermis. No toxicity of final acellular dermis was confirmed by three independent tests (agar diffusion test contact cytotoxicity test and grow curve). The prepared acellular dermis seems to be suitable not only for direct clinical use, but it can be used as a scaffold for cell cultivation as well.     

Measurement of KLa by a gassing-in method with oxygen-enriched air

The dynamic gassing-out method using nitrogen for gas-liquid K(L)a measurements has been modified so that gassing-out is performed with air and gassing-in with oxygen-enriched air. This new method was proven theoretically valid for use in inert model systems and in actual fermentation systems. The K(L)a values were measured in a 1-m-high bubble column and compared with those obtained from the traditional gassing-in method for three different mixing models in batchwise contacting: mixed gas and liquid (MMB); plug-flow gas and mixed liquid (PMB); and plug-flow gas and plug-flow liquid (PPB). The K(L)a values obtained from the new method were consistent with those of the method of Bartholomew et al. Discrimination between the models appeared to be not important for the 1-m-high column, but the theoretical analysis revealed that it would become necessary for columns about 10 m and higher.     

Improved method for the assay of phenylglycosidase activity with a 4-aminoantipyrine reagent

An improved method for the assay of nmole quantities of phenol with a 4-aminoantipyrine reagent is reported and the use of the method for phenylglycosidase assay is shown. Tissue homogenate, serum, and different buffers did not interfere with the assay, thus making protein precipitation unnecessary.     

A rapid immunofluorescent method of determining the antibiotic sensitivity of brucella

Optimal conditions for rapid assay of Brucella antibiotic sensitivity with the immunofluorescent method were developed. With this method high sensitivity of the main Brucella species to tetracycline, doxycycline and rifampicin was confirmed. It was found actually possible to use the immunofluorescent method for rapid assay of Brucella antibiotic sensitivity in practice.     

Graphic method of assessing the maximum cumulative excretion of a drug

Inaccuracy in estimation of the maximum excretion of drugs (M infinity e) is the main cause of errors in determination of their pharmacokinetic parameters by the data of the cumulative excretion (Me(t)). It was shown that the M infinity e value could be determined analytically with the following equation: (formula; see text) where S0Me leads to t is the area under the cumulative excretion curve and ke1 is the elimination constant. The equation was derived from integration of the equation of the cumulative excretion of drugs, the pharmacokinetics of which could be formalized with the linear one-compartmental model. When the data were linearized on the coordinates S0Me leads to t/t - Me(t)/t, the M infinity e value was determined by the portion of the curve on the ordinate and the ke1 value was calculated by the negative reverse value of the regression coefficient. The advantage of the method is that collection of the excrete samples is not limited by the use of the method. The calculations may be performed with a calculator or manually. The possibilities of the method are illustrated with reference to an analysis of the simulated and experimental data.     

Indicator method of determining the beta-lactamase-inhibiting activity of various compounds

An indicator for determination of beta-lactamase inhibitory activity of various compounds was developed. The method is based on the direct contact of beta-lactamase with the compounds tested. It excludes the use of test-bacteria and provides recording in the data on the day of the experiment. The indicator method enables the detection of the beta-lactamase inhibitory properties of both beta-lactamase inhibitors and beta-lactam antibiotics, not subjected to destruction by beta-lactamases. The method is likely to be fit for detection of atypical beta-lactams having beta-lactam groups in their molecules (bleomycin group). Antibiotics not belonging to the group of beta-lactams, such as gentamicin, sisomicin, lincomycin and fusidin showed no beta-lactamase inhibitory activity under the conditions of the indicator method. The use of the indicator method provided determination of the inhibitory activity with respect to penicillinase of Bac. licheniformis 749/C in 30 (8.5 per cent) out of 350 fermentation broths of actinomycetes.     

Use of a cryoultramicrotomy method for the purpose of studying chlamydial ultrastructure]

The method of cryoultramicrotomy was adapted for the study of the ultrastructure of HeLa and McCoy cells in monolayer cultures infected with Chlamydia, obligatory intracellular procaryotic parasites, the causative agents of ornithosis (strain Loth) and paratrachoma (strain LB 1). The cryosections were obtained by the fixation of the monolayer with 2.5% glutaraldehyde, by the gradual infiltration of precipitated cells with sucrose (0.6--1.2--1.8--2.3 M) prior to freezing in liquid nitrogen, and by the treatment of sections with 1% aqueous methyl cellulose solution before drying. This method ensured good preservation of both Chlamydia, in intracytoplasmic inclusions and host cells, as well as regular reproducibility of the results. Ultrathin sections showed a considerable polymorphism in the vegetative forms of Chlamydia, which was probably due to the structure of their cell walls. Chlamydia, were found to form small vesicle-like structures in the cavities of inclusions. The cell walls and granules inside the elementary bodies of the causative agent of ornithosis were stained with the use of phosphotungstic acid--HCl, pH 0.5.     

Development of a heat-mediated protein blotting method

Western blotting is a significant tool employed for the detection of cell proteins. High-molecular-weight proteins have proven a challenge to detect by western blotting, but proteins even of 100 KDa can still present difficulties in detection. This work reports the development of a heat transfer method that is suitable for both low- and high-molecular-weight proteins. The procedure involves the use of a constant temperature at 78 °C in a dedicated heat transfer module. Through the use of this protocol the neuronal adaptor protein X11α (120 KDa), which prior to this methodology was undetectable endogenously in the neuroblastoma cell line (N2a), was successfully detected in the N2a cell line. The procedure provides a reproducible protocol that can be adapted for other high-molecular-weight proteins, and it provides the advantage that low-molecular-weight proteins are not sacrificed by the methodology.     

A novel method for estimating the number of species within a region

Ecologists are often required to estimate the number of species in a region or designated area. A number of diversity indices are available for this purpose and are based on sampling the area using quadrats or other means, and estimating the total number of species from these samples. In this paper, a novel theory and method for estimating the number of species is developed. The theory involves the use of the Laplace method for approximating asymptotic integrals. The method is shown to be successful by testing random simulated datasets. In addition, several real survey datasets are tested, including forests that contain a large number (tens to hundreds) of tree species, and an aquatic system with a large number of fish species. The method is shown to give accurate results, and in almost all cases found to be superior to existing tools for estimating diversity.     

Development of a PCR-based method for diagnostics of mycoses

Since the express-diagnostics of mycoses in immune-deficit patients still remains an acute problem, we developed an effective test system (Kan-Am) to detect DNA Candida albicans, which is a leader in the list of causative agents of candidosis. A comparison study of three PCR-systems used to detect a broad spectrum of fungoid pathogens was carried out, and a universal system (FungAm), which ensures the detection of DNAs of above 78 strains of 25 types of pathogenic fungi, was selected. The results of clinical testing of the species-specific and universal PCR-systems are well confirmed by the culture method, and they are indicative of the efficacy of applying them for the diagnostics of mycoses in neonatology. The use of the mentioned systems is a promising factor for the express-diagnostics of mycoses in immunodeficiency patients. The high sensitivity of the method makes it possible to detect 10 to 100 cells of a causative agent in 100 mcl of the examined biological material, which is compatible with the culture method. A kit of dry reagents (IonoMix) designed for an accelerated sample preparation and isolation, from them, of DNAs on the basis of Chelex-100 and of proteinase K was worked out; the kit is portable and meant for a long-term storing.     

lpp deletion as a permeabilization method

Our earlier studies with outer membrane permeability in E. coli showed that an insertion mutation in lpp gene (encoding Braun's lipoprotein) drastically changed the outer membrane permeability, resulting in significant acceleration of whole-cell catalyzed reactions. In order to gain a mechanistic understanding of the nature of permeability change, the lpp region was sequenced. The results revealed that Lpp was not expressed in the insertion mutant, suggesting that the absence, rather than the alteration, of Lpp is responsible for the observed permeability change. This surprising result prompts us to investigate the possibility of establishing lpp deletion as a general permeabilization method. Two lpp deletion mutants were generated from strains with different genetic background and the effect of lpp deletion on cell physiology was investigated. While lpp deletion had no significant effect on cell growth, carbon metabolism, and fatty acid compositions, it enhanced permeability of various small molecules, consistent with the results with the insertion mutant. This phenotype is useful in a wide range of biotechnological applications. We illustrate here the use of the mutant with organophosphate hydrolysis and L-carnitine synthesis, where permeability is known to be a limiting factor. Both processes were significantly improved with the mutant because of enhanced permeability through the outer membrane. Therefore, this study has established an easy yet generally applicable method for permeabilizing E. coli cells without significant adverse effects. Further, as lpp homolog is known to exist in gram-negative bacteria, we expect that this method will be applicable to other gram-negative bacteria.     

Use of the protoplast fusion method in the selection of Streptomyces griseus--the producer of the streptothricin antibiotic grisin

An effective method for protoplast fusion in S. griseus producing grisin was developed. The method requires the use of polyethylene glycol with a molecular weight of 1000. It was demonstrated that protoplasts formed most effectively in this organism, when the mycelium of the streptomycete previously treated with ultrasound in the process of its growth was used for the treatment with lysozyme. The efficacy of protoplast regeneration in the strains with the use of the modified hypertonic medium R2MD was 25-75 per cent. The possibility of using the protoplast fusion method for constructing phage resistant strains producing kormogrisin was shown.     

Development of a one-step ELISA method using an affinity peptide tag specific to a hydrophilic polystyrene surface

Glutathione S-transferase genetically fused with an affinity peptide tag, PS19 (RAFIASRRIKRP) having a specific affinity for a hydrophilic polystyrene (PS) surface, was preferentially immobilized on a hydrophilic PS (phi-PS) plate without suffering from interference by coexisting protein molecules. Furthermore, rabbit IgG chemically conjugated with a peptide, KPS19R10, in which (10)Lys in PS19 was replaced with Arg and one Lys residue was added at the N-terminus as a coupling site for glutaraldehyde, showed a higher immobilization affinity to the phi-PS plate than that conjugated with the PS19 peptide. On the basis of these findings, the use of a phi-PS plate and peptide tag-linked ligand proteins permitted a one-step or two-step enzyme-linked immunosorbent assay (ELISA) to be achieved, resulting in a substantial reduction in operational time compared with the conventional ELISA method using a hydrophobic PS (pho-PS) plate, while maintaining a high sensitivity. Furthermore, the sensitivity was increased to a greater extent compared to the conventional ELISA meihod when the one-step ELISA was applied to the detection of bovine insulin in a sandwich mode, due to the reduced number of washing and incubation steps. The method proposed here would be a versatile method for use in various ELISA techniques such as sandwich and competitive ELISAs using an antigen, an antibody and streptavidin that are genetically fused or chemically conjugated with the PS-specific affinity peptide as the ligand protein.