Rapid identification and quantitation of small numbers of microorganisms by a chemiluminescent immunoreaction.

A method (patent pending) for rapidly identifying and quantitating small numbers of microorganisms was developed based on the specific immunoreaction of microorganisms with homologous antibodies linked by conjugation to peroxidase. The high sensitivity of the method is due to the use of a chemiluminescent reaction for the determination of the enzyme. The reaction was performed on Alcar supports with low nonspecific adsorption. The very low noise achieved permitted the detection of as few as 30 to 300 bacterial cells.     

Survival conditions of microorganisms under extremely severe environment]

The survival conditions of microorganisms under extremely severe environment are of interest in various areas of biology, sterilization, and space engineering, especially where resistance to microorganisms is concerned. Despite the interest, the resistance to microorganisms under extremely severe environment such as space environment or other planetary environment is not known well. In order to investigate survival conditions of microorganisms under extremely severe environment, surviving fractions for spores and vegetative cells of Bacillus subtilis were surveyed in various chemical species of atmosphere at various pressures and various temperatures, and the dependence on time for surviving fractions was examined. The results show: (i) Surviving fractions depend on chemical species of atmosphere. (ii) At high pressure and high temperature, surviving fractions are low and the resistance of spores is stronger than that of vegetative cells. (iii) Surviving fractions decrease as first-order reaction along with time elapsed.     

红枣贮藏期果面微生物对碳源的利用及主成分分析

Biolog方法就是微生物在利用碳源过程中产生的自由电子, 与四唑盐染料发生还原显色反应, 颜色的深浅可以反映微生物对碳源的利用程度。采用Biolog方法, 研究红枣贮藏期果面微生物对FF和ECO微孔板上碳源的利用情况, 进行主成分分析(Principal component analysis, PCA)。羧酸类、吐温类、碳水化合物、酯类、氨基酸类及胺类碳源是红枣贮藏期果面微生物群落在FF和ECO微孔板上利用的主要碳源。随着贮藏时间的延长, 红枣果面微生物对碳源的利用情况差异较大, 用保鲜剂处理过的红枣果面微生物对碳源的利用远远低于未处理的红枣果面微生物, 而且贮藏时间越长, 果面微生物对碳源的利用程度越高。利用ECO微孔板上31种碳源作PCA, 第一主成分特征值的贡献率为78.54%, 第二主成分特征值的贡献率为19.06%。     

食品中低温微生物的适冷机制研究进展

低温贮藏是延长食品货架期、维持食品鲜度和质量安全的重要方法,然而仍有部分微生物能适应低温环境,使食品发生腐败变质。主要从细胞膜、适冷酶、冷休克蛋白、冷适应蛋白、代谢水平及低温防护剂等角度阐述国内外食品中低温微生物适冷机制的研究进展,为低温微生物在食品领域的应用与防护提供参考。     

Immunocytoadherence indices in evaluating the specificity and activity of an inflammatory process

A method for determination of specificity and activity of the inflammatory process is described. The method is based on the reaction of immunocytoadherence between granulocytes with the round nucleus (nuclei) and microorganisms. The method is sensitive, as the phagocytic reaction is inhibited and the patient's blood cells interact with microbial cells due to the presence of immunoglobulins or receptors of the immunoglobulin nature, active against the causative agent of the disease, on the blood cell surface. The values of immunocytoadherence in tuberculosis, brucellosis and pneumonia are presented.     

Assessment of antimicrobial activity of nanosized Ag doped TiO2 colloids

In the present research, the antimicrobial effects of nanosized silver (Ag) doped TiO(2) colloidal solutions prepared using a sol-gel technique were investigated. In order to determine the solution characteristics, the turbidity, viscosity and pH of the colloidal solutions were measured. Differential thermal analysis-thermogravimetry equipment was used to determine the chemical structures and reaction types of the films formed from these solutions. The morphology of Ag doped TiO(2) nanoparticles was evaluated by atomic force microscopy. The disc diffusion method was employed to explore antimicrobial activity, and the Broth Microdilution method was used to obtain MIC values of nanosized Ag doped TiO(2) colloidal solutions against the test microorganisms Escherichia coli, Staphylococcus aureus, Candida albicans, Bacillus subtilis, and Salmonella typhimurium. It was found that the silver doped TiO(2) nanoparticles inhibited the growth and multiplication of the test microorganisms, including the fungus C. albicans. Antimicrobial activity was observed against all tested microorganisms at a very low concentration of 1.125-2.81 μg/ml of nano silver in 1-25 % Ag-TiO(2) solutions.     

Solution hybridization assay for detecting genetically engineered microorganisms in environmental samples

A solution hybridization method was developed for detecting genetically engineered microorganisms in environmental samples. The detection method involves recovery of DNA from the microbial community of an environmental sample followed by hybridization in solution with a radiolabeled RNA gene probe. After nuclease digestion of non-hybridized probe RNA, the DNA-RNA hybrids formed in the solution hybridization reaction are separated by sephadex or hydroxyapatite column chromatography and detected by liquid scintillation counting. Using solution hybridization-gene probe detection, as few as 100-1000 target cells per gram sediment sample of a 2,4,5-T-degrading genetically engineered microorganisms could be detected.     

The appropriateness of swab cultures for the release of human allograft tissue

Surgeries utilizing human allograft tissues have increased dramatically in recent years. With this increase has come a greater reliance on the use of swab culturing to assess allograft tissues for microbial contamination prior to distribution. In contrast to the typical industrial microbiological uses for swabs, the tissue banking industry has relied on swab cultures as a sterility release method for allograft tissues. It has been reported in the literature that swabs have limitations, both in sensitivity and reproducibility, so their suitability as a final sterility release method was evaluated in this study. Two different swab-culturing systems were evaluated (COPAN, EZ Culturette) using human allograft tissues spiked with low levels of multiple bacterial and fungal microorganisms. The average microbial recoveries for all challenge microorganisms for each tissue type and each swab system were calculated. Percent recoveries for each challenge microorganism were also calculated and reported. The results indicated that both swab systems exhibited low and highly variable recoveries from the seeded allograft tissues. Further analysis indicated there was no statistical difference (∝=0.05) between the two swab systems. It is the recommendation of the authors that swab culturing not be used to assess relatively low levels of microbial contamination on allografts. Instead, alternative validated microbial detection methods with improved sensitivity and reproducibility should be employed and validated for this critical task.     

Mining of genomic databases to identify novel biodesulfurizing microorganisms

The commercialization of the biocatalytic desulfurization process does not seem to be realistic in the near future because of the low desulfurization rate of the known microorganisms. Hence, the future development will depend on either genetically modifying the currently available bacteria or identifying novel biodesulfurizers. In this study an in silico method to identify new biodesulfurizing microorganisms was adopted. By screening the available genomic databases, 13 novel desulfurizing microorganisms belonging to 12 genera were identified. Several of these could be of immense utility as they have both environment pollutant and industrial waste degrading capability.     

Demonstration of microorganisms in tissues by the ABT and KOH-ABT topo-optical reactions.

The aldehyde-bisulphite-toluidine blue (ABT) reaction, as a selective topo-optical test of vicinal OH and amino-OH groups is suited for the selective demonstration in tissues of microorganisms of polysaccharide containing cells. Alkaline pretreatment of the polysaccharide cell walls, releases, by splitting the O-acyl radicals, further vicinal OH groups for the ABT reaction, thus actually increases the sensitivity of the method. The topo-optical reactions are characterized by a strong birefringence induced by oriented dye-binding, due to the linear arrangement of polysaccharides composing the cell wall. Differences in the character of birefringence have made it possible to work out a new method for the analysis of the cell wall ultrastructure as well as to demonstrate microorganisms in tissues. The practical value of the reactions is illustrated by examples.     

PCR-DGGE as a supplemental method verifying dominance of culturable microorganisms from activated sludge

To verify the dominance of microorganisms in wastewater biological treatment, PCR-DGGE (denaturing gradient gel electrophoresis) was performed as a supplementary support method for screening of the dominant microorganisms from activated sludge. Results suggest that the dominant microorganisms in activated sludge are primarily responsible for strengthening its effectiveness as a biological treatment system, followed by the non-main dominant microorganisms, whereas the non-dominant microorganisms showed no effects. The degree of microbial abundance present on the profile of PCR-DGGE was in line with the treatment efficiency of augmented activated sludge with isolated cultures, suggesting that PCR-DGGE can be used as an effective supplementary method for verifying culturable dominant microorganisms in activated sludge of coking wastewater.     

Anammox bacteria enrichment in upflow anaerobic sludge blanket (UASB) reactor

We investigated the anaerobic ammonium oxidation (anammox) reaction in a labscale upflow anaerobic sludge blanket (UASB) reactor. Our aim was to detect and enrich the organisms responsible for the anammox reaction using a synthetic medium that contained low concentrations of substrates (ammonium and nitrite). The reactor was inoculated with granular sludge collected from a full-scale anaerobic digestor used for treating brewery wastewater. The experiment was performed during 260 days under conditions of constant ammonium concentration (50 mg NH₄/⁺-N/L) and different nitrite concentrations (50∼150 mg NO₂-N/L). After 200 days, anammox activity was observed in the system. The microorganisms involved in this anammox reaction were identified as CandidatusB. Anammoxidans andK. Stuttgartiensis using fluorescencein situ hybridization (FISH) method.     

Optical method for the determination of the oxygen-transfer capacity of small bioreactors based on sulfite oxidation.

The growth of microorganisms may be limited by operating conditions which provide an inadequate supply of oxygen. To determine the oxygen-transfer capacities of small-scale bioreactors such as shaking flasks, test tubes, and microtiter plates, a noninvasive easy-to-use optical method based on sulfite oxidation has been developed. The model system of sodium sulfite was first optimized in shaking-flask experiments for this special application. The reaction conditions (pH, buffer, and catalyst concentration) were adjusted to obtain a constant oxygen transfer rate for the whole period of the sulfite oxidation reaction. The sharp decrease of the pH at the end of the oxidation, which is typical for this reaction, is visualized by adding a pH dye and used to measure the length of the reaction period. The oxygen-transfer capacity can then be calculated by the oxygen consumed during the complete stoichiometric transformation of sodium sulfite and the visually determined reaction time. The suitability of this optical measuring method for the determination of oxygen-transfer capacities in small-scale bioreactors was confirmed with an independent physical method applying an oxygen electrode. The correlation factor for the maximum oxygen-transfer capacity between the chemical model system and a culture of Pseudomonas putida CA-3 was determined in shaking flasks. The newly developed optical measuring method was finally used for the determination of oxygen-transfer capacities of different types of transparent small-scale bioreactors.     

Screening for lectin-like protein-producing microorganisms based on cell surface proteins

A method for screening lectin-producing microorganisms was developed. The presence of lectin on microbial cell surfaces was used as an index for their selective isolation. The lectin-producing microorganisms adhered to sugar-modified agarose beads and were selectively eluted with specific saccharide solutions. Spin columns were an effective tool for excluding non-lectin producers. Eighty-seven percent of the microorganisms that were eluted from the beads showed hemagglutination. The results of sequence analysis indicated that some of the eluted microorganisms have not been previously identified as lectin-producing microorganisms.     

Qualitative and quantitative methodologies for determination of airborne microorganisms at concentrated animal-feeding operations

The generation of airborne microorganisms from concentrated animal-feeding operations (CAFOs) is a concern from a human and animal health perspective. To better understand the airborne microorganisms found in these environments, a number of collection and analytical techniques have been utilized and will be discussed in this review. The most commonly used bioaerosol collection method is the liquid impingement format, which is suitable with a number of culture-based and non-culture molecular-based approaches, such as polymerase chain reaction. However, the vast majority of airborne microorganism studies conducted at CAFOs utilize culture-based analyses. Because of the limitations often associated with culture-based analyses, we focused our discussion on the application of molecular-based techniques to identify and/or quantify microorganisms, as they have promising application in bioaerosol research. The ability to rapidly characterize airborne microorganisms will help to ensure protection of public and environmental health. The use or mention of any commercial products does not imply any endorsement of that product by either the authors or the US Department of Agriculture.     

A nonradioactive method for the assay of polyphosphate kinase activity and its application in the study of polyphosphate metabolism in Burkholderia cepacia

Studies of polyphosphate (polyP) metabolism in microorganisms have been hampered by the lack of a convenient method for the assay in cell extracts of the activity of polyphosphate kinase (PPK), the enzyme principally responsible for microbial polyP biosynthesis. We report the development of such an assay, based on the well-established metachromatic reaction, with toluidine blue, of the polyP formed during the PPK-catalyzed reaction. The method was successfully used in the characterization of PPK activity in crude extracts of an environmental Burkholderia cepacia isolate. The development of a protocol for the physical recovery of polyP from solution is also reported.     

Method for recovery of intact DNA for community analysis of marine intertidal microbial biofilms

A protocol is described for rapid DNA isolation from marine biofilm microorganisms embedded in large amounts of exopolysaccharides. The method is a modification of the hot phenol protocol used for plants tissues, where nonexpensive and easily available enzymes were used. The method is based on the incubation of biofilm biomass samples in an extraction buffer mixed with phenol preheated at 65°C. The procedure can be completed in 2 h and up to 20 samples can be processed simultaneously with ease and DNA of excellent quality, as shown by successfully amplification of polymerase chain reaction (PCR) products. DNA was recovered from a range of intertidal marine biofilms with varying amounts of exopolysaccharides.     

Thermodynamic analysis of trinitrotoluene biodegradation and mineralization pathways

Biodegradation of 2,4,6-trinitrotoluene (TNT) proceeds through several different metabolic pathways. However, the reaction steps which are considered rate-controlling have not been fully determined. Glycolysis and other biological pathways contain biochemical reactions which are acutely rate-limiting due to enzyme control. These rate-limiting steps also have large negative Gibbs free energy changes. Because xenobiotic compounds such as TNT can be used by biological systems as nitrogen, carbon, and energy sources, it is likely that their degradation pathways also contain acutely rate-limiting steps. Identification of these rate-controlling reactions will enhance and better direct genetic engineering techniques to increase specific enzyme levels.This article identifies likely rate-controlling steps (or sets of steps) in reported TNT biodegradation pathways by estimating the Gibbs free energy change for each step and for the overall pathways. The biological standard Gibbs free energy change of reaction was calculated for each pathway step using a group contribution method specifically tailored for biomolecules. The method was also applied to hypothetical "pathways" constructed to mineralize TNT using several different microorganisms. Pathways steps that have large negative Gibbs free energy changes are postulated to be potentially rate-controlling. The microorganisms which utilize degradation pathways with the largest overall (from TNT to citrate) negatiave Gibbs free energy changes were also determined. Such microorganisms can extract more energy from the starting substrate and are thus assumed to have a competitive advantage over other microorganisms. Results from this modeling-based research are consistent with much experimental work available in the literature. (c) 1996 John Wiley & Sons, Inc.     

Microbiological Aspects of Ethylene Oxide Sterilization: I. Experimental Apparatus and Methods

A specially built thermochemical death-rate apparatus is described which can be used to determine the resistance of microorganisms to ethylene oxide under controlled conditions. The apparatus was designed to provide instantaneous exposure of microorganisms to ethylene oxide and to eliminate variables that could result in errors when death kinetic reaction rates are calculated. The apparatus is used to obtain ethylene oxide resistance data which are useful in evaluating and developing sterilizing cycles for materials with known bacterial concentrations, as well as for calculating probability factors on which a given test condition can be expected to provide sterilization.     

Reaction of microorganisms to the digestive fluid of the earthworms

The reaction of soil bacteria and fungi to the digestive fluid of the earthworm Aporrectodea caliginosa was studied. The fluid was obtained by centrifugation of the native enzymes of the digestive tract. The inhibition of growth of certain bacteria, spores, and fungal hyphae under the effect of extracts from the anterior and middle sections of the digestive tract of A. caliginosa was discovered for the first time. In bacteria, microcolony formation was inhibited as early as 20-30 s after the application of the gut extracts, which may indicate the nonenzymatic nature of the effect. The digestive fluid exhibited the same microbicidal activity whether the earthworms were feeding on soil or sterile sand. This indicates that the microbicidal agents are formed within the earthworm's body, rather than by soil microorganisms. The effect of the digestive fluid from the anterior and middle divisions is selective in relation to different microorganisms. Of 42 strains of soil bacteria, seven were susceptible to the microbicidal action of the fluid (Alcaligenes.faecalis 345-1, Microbacterium sp. 423-1, Arthrobacter sp. 430-1, Bacillus megaterium 401-1, B. megaterium 413-1, Kluyvera ascorbata 301-1, Pseudomonas reactans 387-2). The remaining bacteria did not die in the digestive fluid. Of 13 micromycetes, the digestive fluid inhibited spore germination in Aspergillus terreus and Paecilomyces lilacinus and the growth of hyphae in Trichoderma harzianum and Penicillium decumbens. The digestive fluid stimulated spore germination in Alternaria alternata and the growth of hyphae in Penicillium chrysogenum. The reaction of the remaining micromycetes was neutral. The gut fluid from the posterior division of the abdominal tract did not possess microbicidal activity. No relation was found between the reaction of microorganisms to the effects of the digestive fluid and the taxonomic position of the microorganisms. The effects revealed are similar to those shown earlier for millipedes and wood lice in the following parameters: quick action of the digestive fluid on microorganisms, and the selectivity of the action on microorganisms revealed at the strain level. The selective effect of the digestive gut fluid of the earthworms on soil microorganisms is important for animal feeding, maintaining the homeostasis of the gut microbial community, and the formation of microbial communities in soils.