Rapid tests for esculin hydrolysis by anaerobic bacteria

Esculin hydrolysis is one of the biochemical tests used in the identification of anaerobic microorganisms. The conventional method by use of growing microbial cells requires 24–48 hours of incubation. On the other hand, growth independent methods like the buffered esculin test, the spot test, and the PathoTec strip test utilize the presence of constitutive enzymes and, therefore, yield results in 1–4 hours. A total of 817 anaerobic organisms were used in this study to determine the sensitivity and specificity of the three rapid methods. All three rapid methods gave excellent correlation with the standard conventional method. Over 99% of the organisms gave comparable results with the spot test and the buffered esculin test within one hour; the PathoTec strip test required up to 4 hours. The former two were not only more rapid but also more economical than the PathoTec strip test.     

Rapid screening of lactic acid bacteria for restriction endonuclease activity

Summary A rapid microscale heparin Sepharose CL-6B affinity gel procedure was developed for detecting restriction endonuclease (RE-Nase) activity in a variety of lactic acid bacteria. RE-Nase-containing extracts free of DNA, RNA and nonspecific nuclease activity can be produced for forty or more strains daily and only 10–12 ml of each log phase culture was required for screening. RE-Nase activity was detected in several streptococci and lactobacilli. With appropriate modifications, this procedure should allow rapid detection of RE-Nase activity in other bacterial species.     

Database of natural luminescent bacteria

The database of luminescent bacteria stored in the IBSO collection is one of the metasections of BIOLUMBASE. A logical schema of the metasection “Natural luminescent organisms”, classification of entities, and methods of attribute presentation have been developed. The database of luminescent bacteria maintained in the IBSO collection is being widened by findings of the collection staff as well as by information from scientific literature. The expectant contents of the database will be useful for resolving various problems of microbial ecology and biotechnology which deal with luminescent bacteria, luminescent system derived from them, and lux-genes cloned to other organisms. A potential user would be able not only to access cataloged data on strains but also to get information on properties, functions, use, and bibliography and to perform an attribute-match search of a strain.     

Rapid test for indole formation with non-proliferating bacteria

A rapid indole test is described which distinguishes within 5 to 120 minutes between organisms that can convert tryptophan to indole and those that cannot.     

Definition of the physiological condition of bacteria in soil by means of luminescent dye L7012

By means of dye L7012, the number and physiological condition (damage rate of membranes) of bacterial cells is defined. The results testify to considerable physiological heterogeneity of bacteria cells in soils. In fresh samples of soil, the percentage of intact cells reached 60–70%. Damaged membranes occurred in 30–40% of cells. The number of damaged cells dramatically increased downwards through the soil profile. Drying and freezing of soil samples considerably reduced the quantity of intact cells and increased the percentage of cells with damaged membranes; the number of intact cells was 10–20%. Treatment with biocide agents resulted in lysis of the majority of cells and cells injuries. However, some of the cells kept an intact cellular membrane, which testifies to the high stability of bacteria in soil. These data allow us to offer a method of staining a soil suspension with the use of luminescent dye L7012 with the quality of an express method that gives the chance to monitor the number and physiological condition of the bacterial complex of soils.     

Carbohydrate specificity of lectins from luminescent bacteria

The presence of lectins on a cell surface was demonstrated for 70 cultures of luminescent bacteria using hemagglutination reactions. It was shown that hemagglutination of luminescent bacteria is inhibited by glucose, maltose, fructose, mannose, and N-acetyl-D-glucosamine. The differences in the inhibition of hemagglutination of luminescent and nonluminescent (spontaneous mutants) symbiotic cultures by N-acetyl-D-galactosamine were revealed. The fact that N-acetyl-D-galactosamine inhibits hemagglutination of the luminescent symbiotic bacteria but does not inhibit hemagglutination of the symbiotic cultures lacking luminescence suggests that lectins with N-acetyl-D-galactosamine specificity are possibly involved in the formation and functioning of the symbiosis of luminescent bacteria with marine animals possessing luminous organs.     

Synthesis of reserve polyhydroxyalkanoates by luminescent bacteria

The ability of marine luminescent bacteria to synthesize polyesters of hydroxycarboxylic acids (polyhydroxyalkanoates, PHA) as reserve macromolecules was studied. Twenty strains from the collection of the luminescent bacteria CCIBSO (WDCM839) of the Institute of Biophysics, Siberian Branch, Russian Academy of Sciences, assigned to different taxa (Photobacterium leiognathi, Ph. phosphoreum, Vibrio harveyi, and V. fischeri) were analyzed. The most productive strains were identified, and the conditions ensuring high polymer yields in batch culture (40–70% of the cell dry mass weight) were determined. The capacity for synthesizing two-and three-component polymers containing hydroxybutyric acid as the main monomer and hydroxyvaleric and hydroxyhexanoic acids was revealed in Ph. leiognathi and V. harveyi strains. The results allow luminescent microorganisms to be regarded as new producers of multicomponent polyhydroxyalkanoates.     

Rapid screening for plasmid DNA

Summary A procedure is described for demonstrating plasmid DNA and its molecular weight, based on rate zonal centrifugation of unlabelled DNA in neutral sucrose gradients containing a low concentration of ethidium bromide. Each DNA species is then visualized as a discrete fluorescent band when the centrifuge tube is illuminated with ultra-violet light. Plasmids exist as closed circular and as relaxed circular molecules, which sediment separately, but during preparation of lysates, closed circular molecules are nicked so that each plasmid forms only a single band of relaxed circles within the gradient.     

Recombinant luminescent bacteria for measuring bioavailable arsenite and antimonite.

Luminescent bacterial strains for the measurement of bioavailable arsenite and antimony were constructed. The expression of firefly luciferase was controlled by the regulatory unit of the ars operon of Staphylococcus aureus plasmid pI258 in recombinant plasmid pTOO21, with S. aureus RN4220, Bacillus subtilis BR151, and Escherichia coli MC1061 as host strains. Strain RN4220(pTOO21) was found to be the most sensitive for metal detection responding to arsenite, antimonite, and cadmium, the lowest detectable concentrations being 100, 33, and 330 nM, respectively. Strains BR151(pTOO21) and MC1061(pTOO21) responded to arsenite, arsenate, antimonite, and cadmium, the lowest detectable concentrations being 3.3 and 330 microM and 330 and 330 nM with BR151(pTOO21), respectively, and 3.3, 33, 3.3, and 33 microM with MC1061(pTOO21), respectively. In the absence of the mentioned ions, the expression of luciferase was repressed and only a small amount of background light was emitted. Other ions did not notably interfere with the measurement in any of the strains tested. Freeze-drying of the cells did not decrease the sensitivity of the detection of arsenite; however, the induction coefficients were somewhat lower.     

Development of a baseball-specific battery of tests and a testing protocol for college baseball players

In this study, the relationship between the physical fitness of college baseball players found from 6 field tests and a performance evaluation by coaches was investigated. The purpose was to ascertain whether the results would be similar to those obtained in a previous study. The subjects of the study were 43 college baseball players (mean age, 20.7 +/- 1.4 years; mean athletic career, 10.9 +/- 2.6 years). Referring to the previous study, the field tests of physical fitness were composed of 6 items: throwing distance, back strength, medicine ball throwing, standing long jump, T-test, and base running. For capabilities in batting, fielding, and running, the coach's evaluation was expressed by T scores. The results of the analysis indicated that those players with high evaluation scores had significantly better test results in comparison with those players who were rated low in the evaluation. Although the multiple regression models of the previous study were associated with a middle goodness of fit, a significant correlation was found between physical fitness found in the field tests and performance. The results from a partial correlation analysis indicated a significant correlation between the following: batting evaluation with back strength (p < 0.01) and medicine ball throwing (p <0.01); fielding evaluation with throwing distance (p < 0.05); and running evaluation with medicine ball throwing (p < 0.01), standing long jump (p < 0.05), T-test (p < 0.01), and base running (p < 0.01). It is certain that the performance of college baseball players is related to their physical fitness.     

Rapid screening: a comparative study

Although rapid screening of negative and inadequate cervical smears is a quality assurance requirement for all UK laboratories, there has been little attempt to standardize the method and laboratories make use of a number of different techniques and times. The aim of this study was to assess the sensitivity of these various techniques by measuring their ability to pick out known false-negative smears. Completed questionnaires from 123 laboratories across England revealed that 52% of laboratories use a "step" technique, 19% use "turret", 15% use random paths and 34% attempt to rescreen the whole slide quickly. Twenty-two percent of laboratories use a mixture of techniques. Timings are also variable, with the majority of laboratories allowing screeners to review slides at a pace decided by themselves but usually between 1 and 2 min. The study involved 120 participants who performed a total of 24 000 rapid screens. The results showed that, of the 90 abnormal slides used in the study, 62 cases (69%) were identified as abnormal or needing review by more than 50% of participants. Overall rapid screening picked out 58% of high-grade squamous abnormalities, 59% of low-grade abnormalities and 72% of glandular lesions. Step screening performed best, followed by whole slide/random and then turret. One minute was the optimum time and there was a significant fall in performance once individuals attempted to rescreen large numbers (>50). The most significant finding was the marked variation in the performance of individuals using the same slide sets.     

New biosensors for assessment of environmental toxicity based on marine luminescent bacteria

Sixteen strains of luminescent bacteria of Vibrio and Photobacterium genera were isolated from water of the Azov and Black seas. Two strains prospective for biotesting were genetically identified as Vibrio fisheri V-9579 and Vibrio fisheri V-9580 according to Russian Industrial Microorganism Collection (VKMP) classification and accepted for depositing. The isolated luminescent strains exhibited high individual sensitivity to oil derived products, heavy metal salts, sodium dodecyl sulfate (SDS) and phenol (up to the maximum concentration limit for fishery impoundments). According to EC₅₀, they are ten times more sensitive to heavy metal salts and potassium dichromate and 2–6 times more sensitive to SDS and phenol compared to P. phosphoreum (Cohn) Ford and Escherichia coli C600 (pPLS-5) strains. Using Vibrio fisheri VKMP V-9579 and Vibrio fisheri VKMP V-9580 as biosensors, we have shown their high sensitivity and efficacy to marine ecosystem toxicity assessment.     

Toward a census of bacteria in soil

For more than a century, microbiologists have sought to determine the species richness of bacteria in soil, but the extreme complexity and unknown structure of soil microbial communities have obscured the answer. We developed a statistical model that makes the problem of estimating richness statistically accessible by evaluating the characteristics of samples drawn from simulated communities with parametric community distributions. We identified simulated communities with rank-abundance distributions that followed a truncated lognormal distribution whose samples resembled the structure of 16S rRNA gene sequence collections made using Alaskan and Minnesotan soils. The simulated communities constructed based on the distribution of 16S rRNA gene sequences sampled from the Alaskan and Minnesotan soils had a richness of 5,000 and 2,000 operational taxonomic units (OTUs), respectively, where an OTU represents a collection of sequences not more than 3% distant from each other. To sample each of these OTUs in the Alaskan 16S rRNA gene library at least twice, 480,000 sequences would be required; however, to estimate the richness of the simulated communities using nonparametric richness estimators would require only 18,000 sequences. Quantifying the richness of complex environments such as soil is an important step in building an ecological framework. We have shown that generating sufficient sequence data to do so requires less sequencing effort than completely sequencing a bacterial genome.