A rapid screening method for the isolation of metal-accumulating microorganisms

Summary An agar plate screening method was developed for the rapid isolation of heavy metal-accumulating microorganisms and preliminary estimation of their biosorption capacity. The test is based on the visulaization and interpretation of the metal distribution between agar and colonies by chemical preciptitation with hydrogen sulphide or ammonium sulphide. The heavy metals silver, thallium, lead, copper, nickel and cadmium have been tested successfully. The efficiency of the method is demonstrated for isolating silver-accumulating bacterian and estimating silver biosorption capacity.     

A rapid colony screening method for the detection of arsenate-reducing bacteria

A rapid and simple method has been developed for the detection of arsenate reducing bacteria based on the presence of arsenite [As (III)], the end product of anaerobic arsenate [As (V)] respiration. Confirmation of As (III) product is made by the reduction of starch-iodine complex. The method can be used over a large pH range (5.5–9.0) and can easily be determined at arsenite concentration as low as 0.025 mM. Major advantages of this technique are that a large number of samples can be analyzed easily at a time.     

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 rapid method for screening vaccinia virus recombinants.

A rapid and small-scale method for screening vaccinia virus recombinants employing micrococcal nuclease is described. This protocol utilizes the differential sensitivity of cellular and viral DNA to the nuclease, which can be selectively activated by addition of Ca2+ and inactivated by elimination of Ca2+. Two to five micrograms of viral DNA can be obtained from one infected L cell plate (50 mm) after overnight incubation.     

琥珀酸产生菌的快速选育

以牛瘤胃内容物为菌源,在高浓度CO2下向培养基额外添加莫能菌素和富马酸钠实现选择性富集培养.溴甲酚绿中性平板变色圈作为筛选标记,以变色区域大小初步筛选得到500株产酸菌株.结合TLC法快速地确定了其中含有28株产琥珀酸菌株.在HPLC和HPCE对发酵液检测的对比过程中优选HPCE法,检测发酵液得到6株优良菌株,其中发现15号菌株琥珀酸产量达11.5g/L,有较少的甲酸和乙酸产生.验证试验表明:该方法对牛瘤胃中的琥珀酸产生菌可以实现快速、有效的分离筛选,具有很好的重现性.     

A rapid and sensitive method for the screening of DNA intercalating antibiotics

An improved, rapid and inexpensive gel mobility shift assay was developed for the screening of anthracycline antibiotics. The assay based on the intercalation activity of these molecules into dsDNA was used to assess the activity of partially purified antibiotics. Detection limits were of 0.1 ng ml^–1 with an average run time of 2 h. The assay is potentially useful for high throughput screening in bioprospecting, for monitoring fermentation production phases and downstream purification process.     

A rapid method for the screening of plasmids in transformed yeast strains

A method for the rapid screening of plasmids in yeast cells has been developed. The method is an adaptation of the currently used alkaline lysis methods forEscherichia coli plasmids. Following the conditions described, several dozen ofSaccharomyces cerevisiae-transformed clones can be analyzed for their plasmid content in less than 2 h. The plasmids obtained by this procedure are suitable for restriction analysis or forE. coli andS. cerevisiae transformation.     

A rapid screening method for bacteria degrading polycyclic aromatic hydrocarbons

Aims:  A rapid procedure was developed to screen for bacteria that are able to grow on polycyclic aromatic hydrocarbons (PAHs).
Methods and Results:  A drop of ethyl ether-dissolved PAH is spread on a sterilized cellulose acetate/nitrate filter lying on the top of a mineral salts agarose plate. After the evaporation of ethyl ether, a serially diluted sample is spread over the filter and incubated. Subsequently, the PAH degrading bacteria can be counted and isolated.
Conclusions:  This procedure is a simple method for screening bacterial isolates for the ability to grow with PAHs.
Significance and Impact of the Study:  This technique is rapid to screen and/or count PAH-degrading bacteria and is also used to streak cultivation without disrupting the PAH layer on plate.     

A rapid bactometer method for screening of biocides against sulfate-reducing bacteria

A rapid and efficient bactometer method was developed for screening biocides against sulfate-reducing bacteria. The method is based on impedance microbiology principles and uses double-layer API (American Petroleum Institute) agar medium supplemented with 0.1% sodium thioglycolate as a reducing agent. Compared to the conventional API procedure, which requires 28 days, the present technique takes only 1 day to obtain test results. Excellent linear correlation (r=–0.98) was found between the impedance detection time and log initial cell concentration. The results of the bactometer test were comparable to that of the API bottle test.     

A rapid plate culture method for screening of amylase producing micro-organisms

A simple and rapid method is described for the detection of amylase-producing micro-organisms on solid media using Remazol Brilliant Blue-starch as substrate. The blue starch is incorporated into nutrient agar and amylase production is detected by the disappearance of the colour around the colonies. The method which is non-destructive, allows for direct visualization and isolation of amylolytic micro-organisms from the environment without prior replication of colonies.     

A rapid fluorescent screening method for cellular sensitivity to anti-cancer compound

The tetrazolium salts (MTT, XTT, MTS, WST) based colorimetric assay or resazurin based fluorimetric assay are currently typical methods for cell sensitivity determination to anticancer compounds. We presented here a new rapid method for this purpose. This method uses a fluorescent dye named DCFH-DA which is previously taken as a intracellular probe for measurement of H(2)O(2) levels within a cell. The application basis for this method lies in two facts: the membrane permeable feature of the final metabolite of DCFH-DA inside a cell, and the linearity relationship between cell number and H(2)O(2) level. The results showed that there was a perfect association between cell number and fluorescent intensity determined by the DCFH-DA method, no matter whether using resuspended or adherent cells, and further 50% concentration of inhibition (IC(50)) comparison between data obtained by DCFH-DA method or MTT method using a positive known anticancer compound Baicalin showed that there were no significant differences in cellular sensitivity determination to compound Baicalin though there existed a relatively higher coefficient of variation of IC(50) by the DCFH-DA method than that by the MTT method. Thus our data indicate that DCFH-DA might not only be a fine reagent for determination of H(2)O(2) levels in cells but also an ideal fluorescent dye for cellular sensitivity test of anti-cancer compounds, and may be suitable for primary high-throughput drugs screening.     

An impedimetric method for rapid screening of cosmetic preservatives

An efficient impedance method was developed for rapid evaluation of cosmetic preservatives. The method used decimal reduction time or D-value to assess preservative efficacies. The D-value, which was calculated from the plot of Log CFU ml^–1 versus time by linear regression analysis, could be obtained within 48 h. Thus, the time required for the challenge test was reduced from 4–8 weeks with the standard procedures (eg US Pharmacopeia), to 2 days with the current method. A calibration curve (r=-0.95) was established by plotting the Log CFU ml^–1 versus capacitance detection time (DT) of 108 samples. With the calibration, CFU can be estimated directly from the impedance test without plating. Two commercial biocides and several other chemicals were evaluated in a shampoo by the impedance procedure againstPseudomonas aeruginosa. The D-values obtained from the impedance test were not significantly different from those produced by the conventional plate count method. The technique was found to be particularly useful when screening a large number of compounds to find novel preservatives and synergistic preservative combinations.     

A rapid screening method for micro-organisms degrading polycyclic aromatic hydrocarbons in microplates

A method for enumerating micro-organisms degrading polycyclic aromatic hydrocarbons (PAHs) was developed. The micro-organisms present in water samples are incubated in 96-well microplates, in which the desired PAHs are available as sole carbon source in a liquid mineral-salts medium (MSM). Walls and bottoms of the wells in the microplates are covered with PAHs by dissolving them in a non-polar solvent and pipetting this solution into the wells. After solvent elimination under vacuum, the PAHs remain on the surface of the wells. The formation of coloured products during microbial degradation of PAHs causes colouring of the MSM, thus allowing evaluation of the cell titre by determining the most probable number. Usage of an electronic multichannel pipette makes the work faster and more effective. This allows the inoculation of several microplates pre-treated with different PAHs out of one serial dilution. On the one hand, this method is very effective in screening the usability spectrum of different PAHs microorganisms; on the other hand it allows the additional employment of other sources of hydrocarbons. Correspondence to: M. Stieber     

A new method for rapid screening of bacterial species- or subspecies-specific DNA probes

A simple assay for the rapid screening of bacterial species- or subspecies-specific DNA probes for the random cloning method is presented, involving the use of genomic DNAs as probes and recombinant plasmid DNAs containing genomic DNA digested with HindIII as targets. The optimal amount of target DNAs and the concentration of digoxigenin-labeled genomic DNA probes were 20 ng and 100 ng ml(-1) (or 10 ng and 200 ng ml(-1)), respectively. The method was applied to the development of Fusobacterium nucleatum subspecies-specific probes. Our results showed that four out of 96 probes were F. nucleatum subspecies-specific, which was confirmed by Southern blot analysis. Our results indicate that the new method can be used for the rapid screening of species- or subspecies-specific probes.     

A rapid and simple method for screening large numbers of recombinant DNA clones

A simple and rapid method has been described for the isolation of plasmid, phagemid and phage DNAs. Hundreds of recombinant clones can be screened in one day employing this method. It takes half an hour to prepare plasmid DNA from ten clones, and the DNA prepared from a single colony using this method is of sufficient quality and in sufficient amount to perform at least five restriction digestions. This method eliminates the need for RNase treatment and phenol chloroform extraction if the plasmids are needed only for the restriction digestion. If needed, RNAs can be removed after restriction digestion by adding RNase and incubating for two minutes at room temperature. After RNase treatment and phenol/chloroform extraction, the plasmid DNA serves as a good template for sequencing. The DNA can be stored at -20 degrees C for over eight weeks.     

一种筛选胞外纤维素酶产生菌的快捷、灵敏、有效的方法

【目的】建立一种能快捷、灵敏、有效筛选高β-葡萄糖苷酶活性的胞外纤维素酶产生菌的方法,用于胞外纤维素酶产生菌检测筛选。【方法】将以微晶纤维素(Avicel)或羧甲基纤维素(CMC)为底物的胞外纤维素酶产生菌平板筛选法常用的刚果红或碘液浸泡染色改为碘液熏染,减少碘液的消耗和对环境的污染;建立以对硝基苯酚-β-1,4-葡萄糖苷(pNPG)为底物的β-葡萄糖苷酶发色底物平板筛选法;将两方法串联用于高β-葡萄糖苷酶活性的胞外纤维素酶产生菌的筛选。【结果】建立了分别以CMC和Avicel为底物结合碘液熏染的胞外纤维素酶产生菌平板筛选法和以pNPG为底物的β-葡萄糖苷酶发色底物平板筛选法,从56株真菌中筛选出了8株纤维素酶活性水平为++++的胞外纤维素酶产生菌,从后者筛选出4株高β-葡萄糖苷酶活性的胞外纤维素酶产生菌。【结论】将以CMC和Avicel为底物结合碘液熏染的平板筛选法和以pNPG为底物的发色底物平板筛选法串联,可快捷、灵敏、有效地用于高β-葡萄糖苷酶活性的胞外纤维素酶产生菌的筛选。