Membrane filter staining method: bacterial plate counts in 24 H

We describe a technique to stain bacterial colonies on membrane filters. The procedure yielded reliable and reproducible bacterial plate counts in 24 h. The procedure can be applied to treated and untreated water samples requiring prompt analysis.     

The Analysis of Bacterial Results in the Kidney Perfusion Fluid and Bone Allografts in Human Multiorgan Donors

In 19 young human multiorgan donors, we simultaneously analyzed the bacterial contamination of the kidney perfusion fluid and all retrieved bone allografts. Donor exclusion criteria were done according to the American and European Association of Tissue Banks excluding all patients with perforating wounds. The kidney perfusate revealed a contamination in 17 of 19 (89.5%) donors. Allograft testing demonstrated positive bacterial growth in 34 of 76 allografts (44.7%). Microorganisms originated from the normal skin flora and could be related to contamination during the harvesting procedure. In 5 cases we cultured identical bacterial subspecies in both cultures as a possible sign for systemic bacterial spreading during the multiorgan harvesting procedure.     

Improved Direct Viable Count Procedure for Quantitative Estimation of Bacterial Viability in Freshwater Environments

A direct viable count (DVC) procedure was developed which clearly and easily discriminates the viability of bacterial cells. In this quantitative DVC (qDVC) procedure, viable cells are selectively lysed by spheroplast formation caused by incubation with antibiotics and glycine. This glycine effect leads to swollen cells with a very loose cell wall. The viable cells then are lysed easily by a single freeze-thaw treatment. The number of viable cells was obtained by subtracting the number of remaining cells after the qDVC procedure from the total cell number before the qDVC incubation. This improved procedure should provide useful information about the metabolic potential of natural bacterial communities.     

Improved direct viable count procedure for quantitative estimation of bacterial viability in freshwater environments

A direct viable count (DVC) procedure was developed which clearly and easily discriminates the viability of bacterial cells. In this quantitative DVC (qDVC) procedure, viable cells are selectively lysed by spheroplast formation caused by incubation with antibiotics and glycine. This glycine effect leads to swollen cells with a very loose cell wall. The viable cells then are lysed easily by a single freeze-thaw treatment. The number of viable cells was obtained by subtracting the number of remaining cells after the qDVC procedure from the total cell number before the qDVC incubation. This improved procedure should provide useful information about the metabolic potential of natural bacterial communities.     

Community level physiological profile of soil bacteria unaffected by extraction method.

Extraction and purification of bacteria from soil by the Nycodenz gradient centrifugation procedure described by Bakken and Lindahl (1995; Recovery of bacterial cells from soil. In: van Elsas, J.D., Trevors, J.T. (Eds.), Nucleic Acids in the Environment: Methods and Applications. Springer Verlag, Berlin, pp. 9-27) were compared to soil slurry extractions. Bacterial communities from four different soils were described by the bacterial abundance, CTC-reducing capacity, culturability and the community level physiological profiles (CLPP) in BIOLOG GN plates. A significant loss of both total and culturable number of bacteria g(-1) soil dry weight were found after extraction and purification of cells. The origin of soil influenced the yield of cells and a difference between the four soils and an interaction between the soils and extraction procedure were found. The culturability and the CLPP were different between the four soils but were unaffected by the extraction procedure. The bacterial community obtained after extraction and purification thus represented the same fraction of the indigenous bacterial community.     

A method for the estimation of bacterial biomass and community structure in mangrove-associated sediments

Many recent reports have proposed that certain monocarboxylic fatty acids found in sediments originate in the in situ bacterial population. In this study we have divided the acids derived from bacteria into nine subgroups, each characteristic of a distinct compositional group of bacteria. It is proposed that the abundance of selected marker acids from each bacterial subgroup (chemotype) can be used to estimate the biomass of that chemotype. Conversion factors from acid abundance to bacterial biomass have been estimated using literature data. Since this procedure results in nine biomass parameters, bacterial communities can be compared in terms of both total biomass and chemotype distribution, that is, biomass and community structure. The ability of this procedure to resolve community structure variations is illustrated with the interpretation of the fatty acid profiles of a spatially distributed set of mangrove-associated sediments.     

A Quantitative Real-time PCR Assay for Quantification of Viable Listeria Monocytogenes Cells After Bacteriocin Injury in Food-First Insights

Quantitative real-time PCR may be a rapid and automated procedure for detection of bacterial pathogens from food samples. Nevertheless, when testing the effects of antimicrobials on the viability of bacterial pathogens in foods, we found that DNA from dead cells interfered greatly in the detection of viable Listeria monocytogenes after treatment with the broad-spectrum bacteriocin enterocin AS-48. To overcome this problem, a quantitative real-time PCR (qRT-PCR) assay based on bacterial mRNA was adapted to quantify viable L. monocytogenes in food after bacteriocin treatments. The procedure allowed a better and faster estimation of viable cells compared to PALCAM viable cell counts when the threshold level was 2 log units/g of food, while PALCAM viable count allowed detection of one log unit/g. This procedure may be useful to verify the efficacy of bacteriocins against L. monocytogenes in foods.     

Leukocyte agglomeration test to reveal bacterial infections in mice

A simple and rapid procedure was used to detecting covert bacterial infections in mice. The procedure was based on observations that bacterial infections were associated with clumping of leukocytes. A large drop of citrated venous blood was placed on a slide and allowed to spread. After fixation and staining the percentage of agglomerated leukocytes was determined by counting. Experimental urinary tract infections caused by either Escherichia coli or Proteus mirabilis served as a model to test the efficacy of the method. Elevation of leukocyte agglomeration was observed in these localized infections.     

The use of non-denaturing Deriphat-polyacrylamide gel electrophoresis to fractionate pigment-protein complexes of purple bacteria

The suitability of Deriphat-polyacrylamide gel electrophoresis as a method for separating purple bacterial pigment-protein complexes has been tested. When appropriate non-denaturing detergents are used to solubilize chromatophores, this method provides a rapid, easy and microscale procedure for analyzing the composition of the bacterial photosynthetic apparatus with minimal disruption of individual pigment-proteins. Its usefulness is further illustrated by employing it to test for suitable detergents with which to solubilize purple bacterial chromatophores, and as an assay to study variation in the composition of the photosynthetic unit of bacterial cultures grown under different conditions.     

Bacterial Density in Water Determined by Poisson or Negative Binomial Distributions

The question of how to characterize the bacterial density in a body of water when data are available as counts from a number of small-volume samples was examined for cases where either the Poisson or negative binomial probability distributions could be used to describe the bacteriological data. The suitability of the Poisson distribution when replicate analyses were performed under carefully controlled conditions and of the negative binomial distribution for samples collected from different locations and over time were illustrated by two examples. In cases where the negative binomial distribution was appropriate, a procedure was given for characterizing the variability by dividing the bacterial counts into homogeneous groups. The usefulness of this procedure was illustrated for the second example based on survey data for Lake Erie. A further illustration of the difference between results based on the Poisson and negative binomial distributions was given by calculating the probability of obtaining all samples sterile, assuming various bacterial densities and sample sizes.     

Permanent Stained Agar Preparation of Mycoplasma and of L Forms of Bacteria

A procedure is described that permits the preparation of permanent stained mounts of Mycoplasma and of bacterial L forms grown on the surface of and within agar media. These preparations are especially useful for making representative photographs. The cultures are fixed with Formalin vapor. Thin slices of agar are stained at elevated temperature between 50 and 60 C and at a low pH, are dried rapidly, and are mounted in Canada balsam. The results with this staining procedure are illustrated by photographs of various strains of Mycoplasma and of bacterial L forms.     

毛细管电泳在细菌分离分析中的应用

介绍了近年来毛细管电泳技术在细菌分离分析方面的研究进展。毛细管电泳以细菌表面的特征信息为分离的基础,可以快速鉴定相应的菌株,可以对微生物进行快速定量,可以反映细菌特殊时期的生理特征,也可以研究微生物与分子之间的相互作用。同时应用该技术可分离分析自然界不能纯培养的微生物。因而毛细管电泳分离与检测细菌方法的建立及其应用在分离科学和微生物学方面都有很大的实际意义。     

The suboptimal structures find the optimal RNAs: homology search for bacterial non-coding RNAs using suboptimal RNA structures

Non-coding RNAs (ncRNAs) are regulatory molecules encoded in the intergenic or intragenic regions of the genome. In prokaryotes, biocomputational identification of homologs of known ncRNAs in other species often fails due to weakly evolutionarily conserved sequences, structures, synteny and genome localization, except in the case of evolutionarily closely related species. To eliminate results from weak conservation, we focused on RNA structure, which is the most conserved ncRNA property. Analysis of the structure of one of the few well-studied bacterial ncRNAs, 6S RNA, demonstrated that unlike optimal and consensus structures, suboptimal structures are capable of capturing RNA homology even in divergent bacterial species. A computational procedure for the identification of homologous ncRNAs using suboptimal structures was created. The suggested procedure was applied to strongly divergent bacterial species and was capable of identifying homologous ncRNAs.     

A rapid plate assay for screening l-asparaginase producing micro-organisms

A pH and dye-based fast procedure for screening l -asparaginase-producing micro-organisms is reported. The procedure is suitable for bacterial and fungal screening. The results are obtained within 24 and 48 h for bacteria and fungi, respectively. The results correlate with quantitative estimations in culture broths.     

Evaluation of membrane adsorption-epifluorescence microscopy for the enumeration of bacteria in coastal surface films

We have evaluated a method for enumerating surface slick bacteria by combining a membrane adsorption procedure with epifluorescence microscopy. Various chemicals were investigated for their ability to enhance bacterial elution from the membrane filters. The results of the elution-epifluorescence method were compared to plate counts and to direct epifluorescence counts of the sampling membrane filters. In all tests, the elution-epifluorescence technique yielded significantly higher bacterial concentrations.     

Crystallizable HIV-1 protease derived from expression of the viral pol gene in Escherichia coli

A plasmid vector was used to express the HIV-1 pol open reading frame under the regulation of the bacterial trp promoter in Escherichia coli. This expression system has been used as a source of recombinant viral protease. The self-processed active enzyme was recovered from a soluble fraction of a bacterial cell lysate and purified by a procedure involving four steps of chromatography. The protocol yielded 0.3 mg of protease for each liter of bacterial culture. The protease formed tetragonal bipyramidal crystals which have been used in high-resolution X-ray diffraction studies.     

Meta‐barcoded evaluation of the ISO standard 11063 DNA extraction procedure to characterize soil bacterial and fungal community diversity and composition

This study was designed to assess the influence of three soil DNA extraction procedures, namely the International Organization for Standardization (ISO‐11063, GnS‐GII and modified ISO procedure (ISOm), on the taxonomic diversity and composition of soil bacterial and fungal communities. The efficacy of each soil DNA extraction method was assessed on five soils, differing in their physico‐chemical characteristics and land use. A meta‐barcoded pyrosequencing approach targeting 16S and 18S rRNA genes was applied to characterize soil microbial communities. We first observed that the GnS‐GII introduced some heterogeneity in bacterial composition between replicates. Then, although no major difference was observed between extraction procedures for soil bacterial diversity, we saw that the number of fungal genera could be underestimated by the ISO‐11063. In particular, this procedure underestimated the detection in several soils of the genera Cryptococcus, Pseudallescheria, Hypocrea and Plectosphaerella, which are of ecological interest. Based on these results, we recommend using the ISOm method for studies focusing on both the bacterial and fungal communities. Indeed, the ISOm procedure provides a better evaluation of bacterial and fungal communities and is limited to the modification of the mechanical lysis step of the existing ISO‐11063 standard.     

Analysis of surface receptor expression on bacteria isolated from patients with endocarditis

Fifteen bacterial isolates (12 streptococcal and 3 staphylococcal strains) from patients with bacterial endocarditis were screened for a variety of surface receptors, in an attempt to identify a common feature that might contribute to their ability to attach to and colonize damaged heart tissue. The bacterial receptors screened for, using a dot-blot autoradiographic procedure, included those for the Fc region of human IgG, fibrinogen, fibronectin and human C1q. Bacteria with receptors for each of the probes used could be identified, but no common receptor was present on all endocarditis-causing strains.