A simple and rapid method for screening amylolytic bacteria

A laboratory class was designed for the study of the ecology of amylolytic bacteria in soil, although other sources may be equally suitable for this purpose. Groups of three students carried out the following: (a) preparation and sterilization of medium and plates, (b) collection and preparation of soil samples, spreading the samples on the plates, (c) incubation of the plates at 37 degrees C overnight, a further 1 h incubation at 60 degrees C to observe amylolytic activity due to thermophilic bacteria, and (d) interpretation and discussion of the results. These tasks are accomplished in two periods of 4h on consecutive days. No sophisticated instruments are required for these experiments, which can be carried out in three classes of 4h each. On the first day the students prepare culture media, buffers and reagents, as well as collect and grow soil samples. The second day is spent for both taxonomic identification of colonies and the HAI determination.     

A simple and rapid test for differentiation of aerobic from anaerobic bacteria

A rapid qualitative test is proposed for bacterial respiratory type based on 24 h culturing of bacteria in liquid medium supplemented with a redox indicator: methylene blue or resazurin. Five reference bacterial strains with definite respiratory type as well as nine bacterial isolates from a laboratory digester for methane fermentation were used. Results obtained showed that both indicators can be used for distinction of strict aerobes from other bacterial representatives with definite respiration. In addition, the resazurin is able to differentiate strict anaerobes from microaerophiles and other anaerobes. The main advantages of the methylene blue is that it is a cheap, easily accessible dye, wide-used in microbiological practice and the results obtained with it are more stable over time. It was also noticed that the test with both indicators gave reliable results for the bacterial respiration only when an inoculum up to 48 h old was used.     

A simple and rapid method for preparing genomic DNA from gram-positive bacteria

Molecular epidemiologic and other studies may require preparation of genomic DNA from large numbers of bacteria in sufficiently pure form for restriction endonuclease digestion, cloning, RAPD-PCR, Southern hybridization, and so on.Staphylococcus and other Gram-positive bacteria have a rigid cell wall and can be difficult to lyse. Here, a simple and rapid method for the preparation of genomic DNA from multiple samples is reported. This method produces clean DNA for use in most molecular biology methods in <90 min.     

A rapid method for the identification of plasmid desoxyribonucleic acid in bacteria

A fast and very sensitive procedure is described for detecting plasmids in bacterial strains. The size of plasmids is determined by agarose gel electrophoresis. Plasmids present in one or more copies per cell with a molecular mass ranging from 2 to over 150 megadaltons may be identified.     

A simple and rapid method for the elimination of R plasmids from enteric bacteria

A rapid, simple, and effective method for the curing of a wide range ofEscherichia coli antibiotic resistance plasmids is described. Treatment with acridine orange followed by growth in sublethal concentration of antibiotics and penicillin selection under such bacteriostatic conditions resulted in a curing efficiency of more than 98% in all cases tested. The method is equally applicable, with modifications, to other enteric bacteria such asKlebsiella pneumoniae. It is also equally applicable to nutritional markers for which toxic analogues exist, and to elimination of recombinant bacteriophages containing antibiotic resistance transposons.     

A simple PCR-RFLP method for identification and differentiation of 11 Malassezia species

A PCR-RFLP method targeted toward 26S rDNA and with 2 restriction enzymes, CfoI and BstF51, was developed to identify 11 Malassezia species. Not only type and standard strains but also 13 clinical isolates were identified successfully in this study. The results of identifications were confirmed by DNA sequencing.     

A rapid and simple method for extracting intracellular metabolites from Escherichia coli bacteria

The article deals with the development of a new method for the extraction of intracellular glycolytic metabolites from bacterial cells. The study has been made on the culture of E. coli B/r CSH. In accordance with this method, the same bacterial filter is used for both filtration (the removal of the culture fluid) and the extraction of low-molecular components of the cells with perchloric acid. The advantage of this method is the absence of unnecessary operations due to the use of a filter installation designed by the author. Quantitatively, this method yields better and reproducible results. The filtration capacity of different types of filters has been analyzed. The optimal time for the extraction of low-molecular cell components has been determined. A change in the concentration of pyruvate in the process of the cellular cycle of E. coli synchronous culture grown in the presence of glucose has been shown to occur. The newly developed method of extraction can be used not only for E. coli, but also for cells of other types.     

A rapid method for differentiation of dairy lactic acid bacteria by enzyme systems

Summary A rapid and simple technique utilizing the APIZYM enzymatic patterns complemented with arginine dihydrolase and citratase was developed for species differentiation of 40 lactic acid bacteria relevant to the dairy industry.Streptococcus species in general produced no -galactosidase, except forStreptococcus thermophilus. Lactobacillus species showed strong aminopeptidases and galactosidases but contained no arginine dihydrolase and citratase. Among the group N-streptococci,Streptococcus diacetylactis produced citratase, whereasStreptococcus cremoris differed by the production of butyrate esterase.Streptococcus faecalis was readily distinguishable fromStreptococcus lactis by butyrate esterase activity that was the basis of the differential agar developed. Heterofermentative lactobacilli differed from homofermentative lactobacilli in possessing arginine dihydrolase and citratase but by not producing leucine-aminopeptidase.     

A simple method for detecting heparinase-producing bacteria

A simple method for detecting heparinase-producing bacteria is described. The method is based on the metachromatic reaction between heparin and toluidine blue. Though developed primarily for Bacteroides spp., the method should find application in the detection of other aerobic and anaerobic heparinase-producing bacteria, without the need for large amounts of media and expensive spectrophotometric equipment.     

A rapid and simple PCR method for identifying isolates of the genus Azospirillum within populations of rhizosphere bacteria

Aims: Escherichia coli is the pre‐eminent microbiological indicator used to assess safety of drinking water globally. The cost and equipment requirements for processing samples by standard methods may limit the scale of water quality testing in technologically less developed countries and other resource‐limited settings, however. We evaluate here the use of ambient‐temperature incubation in detection of E. coli in drinking water samples as a potential cost‐saving and convenience measure with applications in regions with high (>25°C) mean ambient temperatures. Methods and Results: This study includes data from three separate water quality assessments: two in Cambodia and one in the Dominican Republic. Field samples of household drinking water were processed in duplicate by membrane filtration (Cambodia), Petrifilm? (Cambodia) or Colilert^® (Dominican Republic) on selective media at both standard incubation temperature (35–37°C) and ambient temperature, using up to three dilutions and three replicates at each dilution. Matched sample sets were well correlated with 80% of samples (n = 1037) within risk‐based microbial count strata (E. coli CFU 100 ml^?1 counts of <1, 1–10, 11–100, 101–1000, >1000), and a pooled coefficient of variation of 17% (95% CI 15–20%) for paired sample sets across all methods. Conclusions: These results suggest that ambient‐temperature incubation of E. coli in at least some settings may yield sufficiently robust data for water safety monitoring where laboratory or incubator access is limited. Significance and Impact of the Study: Ambient‐temperature incubation of E. coli may be a promising option for reducing the complexity and costs associated with water safety monitoring for faecal indicator bacteria such as E. coli in a field context in resource‐limited settings, as are often encountered in developing countries and after disasters.     

病死猪堆肥高温降解菌的筛选、鉴定及堆肥效果

摘要:【目的】为分离得到高温高效降解菌,加快病死猪的降解。【方法】本研究通过稀释平板法和选择培养基初筛及酶活性复筛的方法,从锯末和病死猪(粉碎)好氧堆肥样品中筛选获得两株能分别高效降解蛋白质和脂肪的高温菌株N-3和Y-3。通过16S rDNA对两菌株进行鉴定,并采用L9(34)正交设计对菌株培养条件进行优化。再利用10 L全自动发酵罐按优化后的培养条件对两菌进行发酵生产(菌数达到108CFU /mL)并等体积混合制备成液体菌剂,分别按发酵物料湿重的0%、0.3%、0.6%、0.9%接种至锯末+病死猪(粉碎)堆肥中进行堆肥效果验证。【结果】共分离得到两株高效降解菌,N-3为芽孢杆菌(Bacillus aestuarii),可高效降解蛋白质,其最适生长温度55 ℃,pH为7.2,转速200 r/min,通气量4 L/min;Y-3为嗜热脱氮芽孢杆菌(Geobacillus thermodenitrificans),能高效降解脂肪,其最适生长温度60 ℃,pH为7.2,转速300 r/min,通气量3 L/min。堆肥过程中对照组和接菌各组(0.3%、0.6%、0.9%)最高温度分别为58.3、69.0、68.9、66.3 ℃,各接菌组间无显著差异(P＞0.05),但均极显著高于对照组(P＜0.01),且各接菌组堆肥温度达到60 ℃以上天数分别为8、10、9 d,极显著高于对照组的0 d(P＜0.01)。至堆肥结束时,对照组和接菌各组的病死猪降解率分别为71.2%、75.7%、96.7%、97.1%。接菌各组(0.3% 接菌组除外)病死猪降解率均极显著高于对照组(P＜0.01),0.3%接菌组与对照组间无显著差异(P＞ 0.05)。【结论】筛选获得的高温腐熟菌N-3和Y-3为能高效降解蛋白质和脂肪的高温菌株,可以用于病死猪腐熟堆肥,且两菌等体积混合后按0.6% 添加量接种至病死猪堆肥中,能提高堆肥温度,维持高温时间,加快病死猪的降解,从而有效杀灭病原微生物,达到无害化要求。     

Random amplified ribosomal DNA restriction analysis for rapid identification of thermophilic Actinomycete-like bacteria involved in hypersensitivity pneumonitis

Hypersensitivity pneumonitis (HP) is a pulmonary disease characterised by inflammation that can be caused by, amongst other substances, a subset of 4 thermophilic mycelial bacteria: Saccharopolyspora rectivirgula, Saccharomonospora viridis, Thermoactinomyces sacchari, and Thermoactinomyces vulgaris. Air sampling analyses in highly contaminated environments are often performed to evaluate exposure to these species which are difficult and fastidious to identify by conventional techniques. The aim of this study was to use amplified ribosomal DNA restriction analysis (ARDRA) to develop a method of identification for those thermophilic organisms that would be more rapid and simple. Strains of these 4 species were obtained from the American type culture collection (ATCC) and were characterized using biochemical tests and ARDRA patterns obtained on their partial-lenght amplified 16S rDNAs. To validate this approach, ARDRA with two restriction enzymes, TaqI and HhaI, was applied to 49 thermophilic actinomycete-like strains from environmental samples (sawmills). The results obtained show that combining some cultural characteristics and biochemical tests, such as xanthine or hypoxanthine decomposition, growth in the presence of NaCl, lysozyme or novobiocin, and spore resistance over 100 degrees C provide a rough identification and selection of the genera of interest. Consequently, target species could be confirmed by digestion of partial-lenght 16S rDNA with the use of Taql and HhaI restriction enzymes that gave specific restriction patterns. ARDRA analyses on the 49 environmental actinomycete-like organisms revealed the presence of 8 Saccharopolyspora rectivirgula, 2 Saccharomonospora viridis, and 15 Thermoactinomyces vulgaris strains, the other strains had restriction patterns different than those of the species of interest. Results of the present study will be applicable to other potential HP environments such as dairy barns, peat bogs and compost plants.     

A simple and rapid method for the preparation of plasma membranes

A simple and rapid method for preparing plasma membranes from isolated cells or tissues is described. The membranes were characterised (a) biochemically by an analysis of specific marker enzymes, (b) by quantitation of cell surface receptors, and (c) immunologically by their ability to elicit specific allogeneic responses from cytotoxic T cells in secondary in vitro stimulations. Based on both biochemical and immunologic criteria, plasma membranes prepared by the method described here are of equal or greater 'purity' compared to those prepared by two other methods that are most widely used to date and the yields are several-fold higher.