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.     

Rapid screening for bacteriuria with a laser nephelometer

Detection of significant bacteriuria with a laser nephelometer was evaluated in this study and compared with the results obtained by the quantitative loop method. We screened 1002 urine specimens and 220 (21.95%) were found to be positive at greater than or equal to 10(5) colony-forming units (CFU)/mL of urine by the standard method. Of the 220 positive specimens, 210 (95.4%) were detected in 6 h or less and 177 (80.4%) were detected within 3 h. The false-positive rate was 2.3% at 3 h and 19.7% at 6 h. These findings suggest that a 6-h procedure is necessary to detect 95% or more of significant bacteriuria. Laser nephelometer is versatile and can be used for rapid screening of bacteriuria.     

Rapid spectrofluorometric screening of poly-hydroxyalkanoate-producing bacteria from microbial mats.

Microbial mat ecosystems are characterized by both seasonal and diel fluctuations in several physicochemical variables, so that resident microorganisms must frequently adapt to the changing conditions of their environment. It has been pointed out that, under stress conditions, bacterial cells with higher contents of poly-hydroxyalkanoates (PHA) survive longer than those with lower PHA content. In the present study, PHA-producing strains from Ebro Delta microbial mats were selected using the Nile red dying technique and the relative accumulation of PHA was monitored during further laboratory cultivation. The number of heterotrophic isolates in trypticase soy agar (TSA) was ca. 107 colony-forming units/g microbial mat. Of these, 100 randomly chosen colonies were replicated on mineral salt agar limited in nitrogen, and Nile red was added to the medium to detect PHA. Orange fluorescence, produced upon binding of the dye to polymer granules in the cell, was detected in approximately 10% of the replicated heterotrophic isolates. The kinetics of PHA accumulation in Pseudomonas putida, and P. oleovorans were compared with those of several of the environmental isolates spectrofluorometry. PHA accumulation, measured as relative fluorescence intensity, resulted in a steady-state concentration after 48 h of incubation in all strains assayed. At 72 h, the maximum fluorescence intensity of each strain incubated with glucose and fructose was usually similar. MAT-28 strain accumulated more PHA than the other isolates. The results show that data obtained from environmental isolates can highly improve studies based on modeling-simulation programs, and that microbial mats constitute an excellent source for the isolation of PHA-producing strains with industrial applications.     

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.     

The classification of luminescent bacteria

Summary The luminescent bacteria are logically placed in two genera. The common coccoid and frequently non-motile species placed byBeije-rinck first in his genusPhotobacterium, 1889 under the namePhotobacterium phosphorescens syn.Bacterium phosphorescens Fischer, should be recognized as the type species ofPhotobacterium. Other characters indicate that this genus should be placed in the FamilyPseudomonadaceae Winslowet al.. and should include other straight, rod-shaped, luminescent, polar flagellate bacteria that ferment glucose without, however, necessarily producing gas (H₂ and CO₂) as does the type species. The species that have the form of vibrios should be accepted as members of the genusVibrio as suggested by several previous investigators. They have characters much like those ofVibrio comma, the type species of the genusVibrio.     

Fraser river sediments and waters evaluated by the battery of screening tests technique

The suitability of a variety of microbiological, biochemical and toxicant screening tests to become part of a battery of test procedures to identify degraded or degrading water bodies are evaluated in this report. Data were collected from 40 sampling sites within the Fraser River Basin in British Columbia. These data re-emphasize that individual toxicant, biochemical or microbiological screening tests do not provide a sufficient data base upon which realistic management decisions can be made. This study also confirms that the fecal sterol tests do not seem amenable to a'battery of tests' approach and that the Daphnia magna test continues to be the most sensitive procedure for indicating the presence of contaminants with toxicant activity.     

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.     

荧光法快速检测活菌数的方法研究及应用

【背景】Calcein UltraGreen~(TM)AM是一种新型荧光染料,用于标记和监测活细胞。【目的】基于该荧光染料的荧光特性及其在活细胞内的稳定特性,建立一种荧光定量快速检测活细菌总数的方法,并在实际样品中应用校正。【方法】通过应用荧光染料对细菌进行染色,再进行荧光强度检测,同时以平板计数法作平行对照,建立荧光强度值-活菌数标准曲线。【结果】确定了染色细菌的最佳pH值为8.0。该检测方法仅需固定染色温度,染色时间在20-30min范围即可快速检测。建立了革兰氏阴性菌铜绿假单胞菌NY3、大肠杆菌和革兰氏阳性菌芽孢杆菌、红平红球菌FF、金黄色葡萄球菌和枯草芽孢杆菌的细菌总数与相对荧光强度值标准曲线。当菌悬液OD600值在0.01-0.30范围内时,上述6种细菌与荧光信号强度呈良好的线性关系(R20.99)。【结论】当样品菌悬液浓度范围控制在105-109CFU/mL时,建立的荧光检测方法快速便捷,精密度、重复性、稳定性、回收率和准确度均较好,可应用于微生物实验、固体菌剂发酵、食品卫生与安全、环境检测等领域的活细菌总数现场快速检测。     

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.     

Use of luminescent bacteria for rapid screening and characterization of short cationic antimicrobial peptides synthesized on cellulose using peptide array technology

The increasing multi-resistance of pathogenic bacteria requires the development of novel classes of antibiotics. Antimicrobial host defense peptides represent one promising class. Here we describe a protocol for screening large numbers of peptides against any microbe of interest. Peptides synthesized on a cellulose support by peptide array technology can be added to a microbe that expresses the luxCDABE (luciferase) gene cassette. Any substance that decreases the energy level within the microbe will cause a quantifiable decrease in light production. The potency of the compound, at different concentrations, is reflected by the rate of decrease in luminescence. In conjunction with peptide array technology, the screening assay is rapid and high throughput and demonstrates good correlation with conventional killing or minimal inhibitory concentration assays performed with the same peptides synthesized by standard solid-phase peptide synthesis. The protocol can be completed in 3 d.     

Significance tests for cancer screening trials

The goal of a cancer screening program is to reduce cancer mortality by detecting tumors at earlier stages of their development. For some types of cancer, screening tests may allow the preclinical detection of benign precursors of a tumor, and thus a screening program could result in reductions in both cancer incidence and mortality. For other types of cancer, a screening program will not reduce cancer incidence, and thus the expected outcome in a randomized cancer screening trial would be equal cancer incidence rates in control and study groups, but reduced cancer mortality in the study group. For the latter situation, we employ a variety of Poisson models for cancer incidence and mortality to derive optimal tests for equality of cancer mortality rates in a cancer screening trial, and we compare the asymptotic relative efficiencies of the test statistics under various alternatives. We demonstrate that testing equality of case mortality rates using Fisher's exact test or its Pearson chi-square approximation is nearly optimal when cancer incidence rates are equal and is fully efficient when cancer incidence rates are unequal. When valid, this comparison of case mortality rates in the study and control groups can be considerably more powerful than the standard comparison of population mortality rates. We illustrate the results using data from a clinical trial of a breast cancer screening program.